Diss. ETH no. 22743
The role of microRNA-29a in the regulation of
myogenic progenitor proliferation and adult skeletal
muscle insulin sensitivity
A thesis submitted to attain the degree of
DOCTOR OF SCIENCES OF ETH ZURICH
(Dr.Sc. ETH Zurich)
presented by
Artur Galimov
M.Sc. in bioengineering, Lomonosov Moscow State University
born on October 15, 1984
citizen of Russian Federation
Accepted on the recommendation of
Prof. Dr. Christian Wolfrum
Prof. Dr. med. Jan Krützfeldt
Prof. Dr. med. Michael Ristow
Prof. Dr. med. Daniel Konrad
May, 2015
Table of contents
SUMMARY ............................................................................................................................................. III
SOMMARIO............................................................................................................................................. V
ACKNOWLEDGEMENTS ..................................................................................................................... VII
ABBREVIATIONS ................................................................................................................................ VIII
CHAPTER I ............................................................................................................................................. 1
THE REGULATION OF SKELETAL MUSCLE BY MICRORNAS......................................................... 1
1.1 SKELETAL MUSCLE PHYSIOLOGY........................................................................................................ 1
1.1.1 Skeletal muscle regeneration.................................................................................................. 1
1.1.1.1 Skeletal muscle structure................................................................................................................. 1
1.1.1.2 Morphological changes during skeletal muscle regeneration .......................................................... 2
1.1.1.3 Satellite cells.................................................................................................................................... 4
1.1.1.4 Satellite cell self-renewal ................................................................................................................. 5
1.1.1.5 Myogenic regulatory factors............................................................................................................. 6
1.1.1.6 Secreted factors in regulation of muscle regeneration ..................................................................... 8
1.1.1.7 A role for basement membrane in skeletal muscle regeneration ................................................... 10
1.1.2 Skeletal muscle metabolism and insulin resistance.............................................................. 13
1.1.2.1 Skeletal muscle as a metabolic tissue ........................................................................................... 13
1.1.2.2 Skeletal muscle and type 2 diabetes ............................................................................................. 14
1.1.2.3 The regulation of adult skeletal muscle metabolism by growth hormone ....................................... 15
1.1.2.4 GH deficiency in humans and GH replacement therapy ................................................................ 16
1.2 MICRORNA BIOLOGY AND ITS ROLE IN SKELETAL MUSCLE METABOLISM AND REGENERATION .............. 18
1.2.1 MicroRNA biology.................................................................................................................. 18
1.2.2 The involvement of microRNAs in muscle differentiation / regeneration .............................. 20
1.2.3 The role of microRNA in the regulation of muscle metabolism and insulin resistance. ........ 21
1.2.4 MicroRNA-29 family, its regulation and cellular functions..................................................... 21
CHAPTER II .......................................................................................................................................... 23
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE ...................................................................................................... 23
2.1 OUTLINE OF THE WORK ................................................................................................................... 24
2.2 INTRODUCTION ............................................................................................................................... 25
2.3 RESULTS........................................................................................................................................ 27
2.3.1 MicroRNA-29a is downregulated by serum starvation in primary myoblasts........................ 27
2.3.2 FGF-2 induces mir-29 expression......................................................................................... 27
2.3.3 miR-29 inhibition/deletion affects myoblast proliferation and differentiation ......................... 28
2.3.4 Inhibition/deletion of miR-29 derepresses targets related to basement membrane and
downregulates cell cycle genes ..................................................................................................... 28
2.3.5 miR-29a is induced during the proliferative phase of muscle regeneration and regulates
basement membrane-related targets in vivo.................................................................................. 29
2.3.6 MP-specific miR-29a deletion results in decreased myofiber formation, reduced muscle
mass, and fibrotic degeneration in regenerating muscle ............................................................... 29
2.3.7 Exercise-induced muscle regeneration is blunted in muscles with miR-29a-depleted MPs. 30
2.4 DISCUSSION AND OUTLOOK ............................................................................................................. 31
i
2.4.1 miRNA-29a and basement membrane remodeling during early events of muscle
regeneration ................................................................................................................................... 31
2.4.2 MicroRNA-29 and the regulation of muscle regeneration after exercise .............................. 32
2.4.3 miRNA-29a and myogenesis ................................................................................................ 32
2.4.4 miRNA-29a and FGF signalling ............................................................................................ 34
2.4.5 Basement membrane components and human myopathies ................................................ 34
2.5 FIGURES ........................................................................................................................................ 36
CHAPTER III ......................................................................................................................................... 51
GROWTH HORMONE REPLACEMENT THERAPY REGULATES MICRORNA-29A AND TARGETS
INVOLVED IN INSULIN RESISTANCE ................................................................................................ 51
3.1 OUTLINE OF THE WORK ................................................................................................................... 52
3.2 INTRODUCTION ............................................................................................................................... 53
3.3 RESULTS........................................................................................................................................ 55
3.3.1 GHRT induces insulin resistance in mice and stimulates extracellular matrix gene
expression ...................................................................................................................................... 55
3.3.2 miRNA-29a and its ECM-related targets are reciprocally regulated by GHRT ..................... 55
3.3.3 IGF1 downregulates miR-29a in human myotubes............................................................... 56
3.3.4 IGF1 and miR-29a are reciprocally regulated in insulin resistant patients............................ 56
3.3.5 The Regulation of miRNA-29a targets in GHRT is associated with insulin resistance......... 57
3.3.6 miRNA-29a and its targets are not regulated in obesity associated insulin resistance ........ 57
3.4 DISCUSSION AND OUTLOOK ............................................................................................................. 58
3.4.1 The regulation of gene expression during GHRT ................................................................. 58
3.4.2 IGF1 regulation during GHRT and its relation to miRNA-29a levels and insulin resistance. 59
3.4.3 miRNA-29a in GHRT-induced and obesity-induced insulin resistance................................. 60
3.5 FIGURES ........................................................................................................................................ 61
CONCLUDING REMARKS ................................................................................................................... 72
CHAPTER IV ......................................................................................................................................... 75
MATERIALS AND METHODS.............................................................................................................. 75
4.1 ANIMALS ........................................................................................................................................ 75
4.2 PARTICIPANTS ................................................................................................................................ 77
4.3 SKELETAL MUSCLE HISTOLOGY AND IMMUNOFLUORESCENCE ............................................................ 78
4.4 RNA ISOLATION, QRT-PCR, MIRNA MICROARRAY, AND MRNA SEQUENCING.................................... 78
4.5 NORTHERN BLOTTING ..................................................................................................................... 79
4.6 PRIMARY MOUSE AND HUMAN MYOBLAST CULTURES ......................................................................... 79
4.7 W ESTERN BLOTTING ....................................................................................................................... 81
4.8 PROLIFERATION RATE AND MITOCHONDRIAL ACTIVITY IN PRIMARY MYOBLASTS ................................... 82
4.9 LUCIFERASE PLASMID, ANTAGOMIR, MIRNA MIMIC AND SIRNA TRANSFECTION INTO PRIMARY MOUSE
MYOBLASTS.......................................................................................................................................... 82
4.10 STATISTICAL ANALYSES ................................................................................................................ 83
CURRICULUM VITAE........................................................................................................................... 85
REFERENCES ...................................................................................................................................... 87
ii
SUMMARY
SUMMARY
Skeletal muscle represents one of the key tissues responsible for metabolic, cardiovascular and
overall health. Abnormalities in skeletal muscle metabolism are considered as a primary event in the
development of type 2 diabetes. At the same time muscle possesses a prominent capacity to regenerate
and adapt to required loads. The regeneration process largely relies on muscle stem cells, which are
activated in response to injury and exercise, massively proliferate and rebuild new myofibers, as well
as provide nuclei for regenerating ones. Growth factors, such as growth hormone (GH), insulin growth
factor 1 (IGF1) and fibroblast growth factor (FGF) have been repeatedly reported to have an important
role in different aspects of muscle physiology, including both muscle regeneration and metabolism.
MicroRNAs have been demonstrated to be crucial regulators evolved to fine-tune gene
expression and to have an established role in tissue development and regeneration. Many microRNAs,
including muscle specific microRNAs, so called myomiRs, have been shown to regulate muscle
function.
The work presented in this thesis focuses on the role of the microRNA pathway, namely
microRNA-29a, in mediation of physiologic functions of soluble growth factors: growth hormone and
fibroblast growth factor.
Chapter I provides broad introduction into skeletal muscle physiology (section 1.1) and
microRNA function in relation to skeletal muscle (section 1.2). Within this chapter, different relevant
aspects of skeletal muscle biology are discussed, including satellite cell biology, markers of muscle
development, role of growth factors, aspects of muscle metabolism, and pathophysiology of type 2
diabetes.
Chapter II presents our study on the role of the microRNA pathway in mediation of instructing
signal of FGF for satellite cells to proliferate and maintain uncommitted state. MiR-29a was observed
to be upregulated by FGF in myogenic progenitors both in vitro and in vivo. We demonstrated that
deletion of miR-29 in primary myoblasts (in vitro) or myogenic progenitors (in vivo) decreased their
proliferation and led to premature differentiation. In vivo miR-29a deletion blunted both injury and
exercise-induced muscle regeneration. Regenerating muscles in miR-29a knocked-out (KO) animals
were characterized with increased fibrosis and reduced muscle mass, and this phenotype was present
after 4 weeks of regeneration, when muscle regenerates completely in control animals.
Chapter III presents our study on the role of microRNAs in GH mediated skeletal muscle
insulin resistance. We have demonstrated that GH downregulates miR-29a in skeletal muscle of
human patients and GH deficient mice, and that this regulation is mediated by IGF1. Furthermore,
changes in miR-29a expression in muscle during GHRT correlated with changes in insulin sensitivity
iii
SUMMARY
in human patients. Besides, miRNA-29a downregulation reciprocally correlated with induction of its
targets, associated with tissue inflammation and insulin resistance.
Chapter IV provides information about material and methods used
Overall, our findings highlight the importance of miR-29a for different aspects of muscle
physiology, such as myogenic progenitor function during muscle regeneration and control over insulin
resistance in adult tissue. Further research is needed in order to uncover details of miR-29a functioning
in skeletal muscle and develop therapeutic approaches.
iv
SOMMARIO
SOMMARIO
ll muscolo scheletrico rappresenta uno dei tessuti principali responsabili per la salute
metabolica, la salute cardiovascolare e la salute in generale. Anomalie nel metabolismo del muscolo
scheletrico sono considerate come l’evento primario nello sviluppo del diabete di tipo 2. Allo stesso
tempo, il muscolo scheletrico possiede una prominente capacità di rigenerarsi ed ad adattarsi ai carichi
richiesti. Il processo di rigenerazione si affida principalmente sulle cellule staminali muscolari, che si
attivano in risposta a lesioni ed esercizio fisico. In seguito, esse proliferano massicciamente andando a
ricostruire nuove fibre muscolari. I fattori di crescita, come l'ormone della crescita (GH), il fattore di
crescita insulino-simile (IGF1) ed il fattore di crescita dei fibroblasti (FGF) sono conosciuti per avere
un ruolo importante in diversi aspetti della fisiologia muscolare, tra cui la rigenerazione ed il
metabolismo muscolare. I microRNA giocano un importante ruolo nella regolazione dell’espressione
genica, inoltre contribuiscono significativamente allo sviluppo e nella rigenerazione dei tessuti. Molti
microRNA, inclusi microRNA specifici muscolari, i cosiddetti myomeRs, hanno dimostrato di
regolare
la
funzione
muscolare.
Il lavoro presentato in questa tesi si concentra sul ruolo del microRNA-29a, nella mediazione
delle funzioni fisiologiche di fattori di crescita solubili, come l’ormone della crescita (GH) ed il fattore
di crescita dei fibroblasti (FGF).
Il Capitolo I fornisce un'ampia introduzione sulla fisiologia del muscolo scheletrico (sezione
1) e la funzione dei microRNA in relazione al muscolo scheletrico (sezione 2). All'interno di questo
capitolo sono trattati diversi aspetti riguardanti la biologia del muscolo scheletrico: la biologia delle
cellule satelliti, i marcatori di sviluppo muscolare, il ruolo dei fattori di crescita, aspetti del
metabolismo muscolare e la fisiopatologia del diabete di tipo 2.
Il Capitolo II presenta il nostro studio sul ruolo dei microRNA nella mediazione di segnale
FGF per istruire le cellule satelliti a proliferare e mantenere il loro stato uncommitted . Si è potuto
dimostrare che MiR-29a viene upregulated da FGF nei progenitori miogenici sia in vitro che in vivo.
Abbiamo dimostrato che la delezione di miR-29 in mioblasti primari (in vitro) o progenitori miogenici
(in vivo) diminuisce la proliferazione e porta a differenziazione prematura. La delezione in vivo di
miR-29a diminuisce la rigenerazione muscolare, indotta da lesioni ed esercizio fisico. I muscoli
rigeneranti di topi Knock out per miR-29a sono caratterizzati da una maggiore fibrosi e da una massa
muscolare ridotta. Questo fenotipo era presente ancora dopo 4 settimane di rigenerazione, ossia ad un
punto dove il muscolo di topi normali è già rigenerato completamente.
Il capitolo III presenta il nostro studio sul ruolo dei microRNA sulla resistenza all'insulina del
muscolo scheletrico mediata dall’ormone della crescita (GH). Abbiamo dimostrato, che l’ormone della
v
SOMMARIO
crescita (GH)” downregulates” miR-29a nel muscolo scheletrico in pazienti umani e topi che non
producono l’ormone della crescita. Questo effetto è mediato da IGF1. Abbiamo inoltre scoperto che
miR-29a è l'unico miRNA a correlare con la sensibilità all'insulina in pazienti umani. Inoltre, la
downregualtion di miRNA-29° causa l'induzione dei suoi “targets”, associati a fibrosi dei tessuti e la
resistenza all'insulina.
Nel capitolo IV sono descritti i materiali ed i metodi utilizzati.
Nel complesso, i nostri risultati rilevano l’importanza di miR-29a per diversi aspetti della
fisiologia muscolare, come il controllo della funzione delle cellule miogeniche progenitrici durante la
rigenerazione muscolare ed il controllo sulla resistenza all'insulina nel tessuto muscolare adulto.
Tuttavia, sono necessarie nuove ricerche per scoprire ulteriori dettagli riguardante il funzionamento di
miR-29a nel muscolo scheletrico, in modo da poter sviluppare approcci terapeutici.
vi
ACKNOWLEDGEMENTS
ACKNOWLEDGEMENTS
I would like thank all the people that have helped me throughout my PhD project.
I wish to express my great appreciation to Prof. Jan Krützfeldt for giving me the opportunity to work
on these projects, ideas and guidance he provided along these 4.5 years, as well as for enthusiastic
encouragement. I would also like to express my gratitude to Prof. Christian Wolfrum, Prof. Michael
Ristow, and Prof. Daniel Konrad for generously agreeing to take part in my PhD committee, valuable
suggestions, useful critiques, and productive collaboration.
I would like to specially thank my collaborators, in particular, Edlira Luca, Angelika Hartung, Troy
Merry and Elisabeth Rushing. Without your input these projects would not have been completed.
Furthermore, I wish to acknowledge all the people in the department. In particular, Giatgen Spinas for
his continuous interest to the projects as well as Fabrizio Lucchini, Richard Züllig and Maren Dietrich
for their helpfulness. I’m also thankful to my student colleagues for providing stimulating and fun
environment. I’ve learned a lot from you. Many thanks in particular to Katarina Turcekova, Tatiane
Gorsky and Amir Mizbani.
I would like to thank my friends who made these years enjoyable, entertaining and so fun, in particular
Arkadiy Simonov, Nikolay Komarevsky, Aleksander Dorodniy and Vardan Andriasyan.
And finally, my deepest thanks to my family: my parents, Ljudmila and Rafael, my wife Gulnara, and
my brother Eugeniy for your love, invaluable support, constant help, your trust in me, and your
encouraging.
vii
ABBREVIATIONS
ABBREVIATIONS
ACC-2
Acetyl-coA carboxylase-2
AGO2
Protein argonaute-2
AKT
Protein kinase B
AMP
Adenosine monophosphate
AMP-PK
AMP-activated protein kinase
ATP
Adenosine triphosphate
B-CLL
B-cell chronic lymphocytic leukemia
BM
Basement membrane
BMI
Body mass index
B-Myb
Myb-related protein B
CD31
Cluster of differentiation 31
CD45
Cluster of differentiation 45
CD56
Cluster of differentiation 56
Cdc6
Cell division cycle 6
Cdc45
Cell division cycle 45
CEBP
CCAAT/enhancer-binding protein
COL
Collagen
CTX
Cardiotoxin
DMEM
Dulbecco’s modified Eagle's medium
DNA
Deoxyribonucleic acid
ECM
Extracellular matrix
EDTA
Ethylenediaminetetraacetic acid
EdU
5-Ethynyl-2´-deoxyuridine
ELISA
Enzyme-linked immunosorbent assay
eMHC
Embryonic myosin heavy chain
ERK
Extracellular-signal-regulated kinase
Ezh2
Enhancer of zeste homolog 2
FAK
Focal Adhesion Kinase
Fbn1
Fibrillin-1
FFA
Free fatty acid
FGF
Fibroblast growth factors
FAPs
Fibroadipogenic progenitors
FPKM
Fragment per kilobase of exons per million reads
viii
ABBREVIATIONS
Fstl1
Follistatin-related protein 1
GH
Growth hormone
GHD
Growth hormone deficiency
GHR
Growth hormone receptor
GHRT
Growth hormone replacement therapy
GO
Gene ontology
GTT
Glucose tolerance test
GW182
Trinucleotide repeat containing 6A
H&E
Hematoxylin and eosin
HbA1C
Glycated hemoglobin A1C
HDAC4
Histone deacetylase 4
HFD
High fat diet
HGF
Hepatocyte growth factor
HOMA-IR
homeostatic model assessment of insulin resistance
HSPG2
Heparan sulfate proteoglycan 2
IFN
Interferon
IFNAR1
Interferon-alpha/beta receptor alpha chain
IGF1
Insulin-like growth factor 1
IPG
Impaired glucose tolerance
IRS
Insulin receptor substrate
ISI
Insulin sensitivity index
KO
Knock-out
LamC1
Laminin subunit gamma-1
MAPK
Mitogen-activated protein kinase
MB
Myoblast
MCK
Muscle creatine kinase
Mcm2
Minichromosome maintenance complex component 2
Mcm7
Minichromosome maintenance complex component 7
MCR
Metabolic clearance rate
MEF2
Myocyte enhancer factor 2
MHC
Myosin heavy chain
miRISC
miRNA-mediated silencing complex
miRNA, miR
MicroRNA
MMP2
Matrix metalloproteinase-2
MP
Myogenic progenitor
MRF
Myogenic regulatory factor
ix
ABBREVIATIONS
MRTF
Myocardin-related transcription factor
MSTN
Myostatin
MTK
Mitotracker green
Myc
Myelocytomatosis oncogene
MYH6
Myosin, heavy polypeptide 6
MyHC
Myosin heavy chain
MyoD
Myogenic differentiation 1
NGT
Normal glucose tolerance
Nid2
Nidogen 2
NO
Nitric oxide
OGTT
Oral glucose tolerance test
Pax3
Paired box 3
Pax7
Paired box 7
PBS
Phosphate-buffered saline
PCNA
Proliferating cell nuclear antigen
PDK
Phosphoinositide-dependent protein kinase
PI-3
Phosphatidylinositol-3
PIK3R1
Phosphatidylinositol 3-kinase regulatory subunit alpha p85
PKC
Protein kinase C
qRT-PCR
Real-time polymerase chain reaction
RNA
Ribonucleic acid
Rybp
RING1 and YY1 binding protein
SDS
Sodium dodecyl sulfate
SerpinH1
Serpin peptidase inhibitor, clade H member 1
Six1
Sine oculis-related homeobox 1
SMAD3
SMAD family member 3
Sparc
Secreted acidic cysteine rich glycoprotein
T2D
Type 2 diabetes
TA muscle
Tibialis anterior muscle
TCF/LEF
TCF/LEF family of transcription factors
TMRE
Tetramethylrhodamine, ethyl ester
Tnni
Troponin I
TRBP
Trans-activation response (TAR) RNA binding protein
WT
Wild type
x
CHAPTER I
CHAPTER I
THE REGULATION OF SKELETAL MUSCLE BY
MICRORNAs
1.1 Skeletal muscle physiology
Skeletal muscle is an organ that constitutes up to 40% of body mass and accounts
not only for body movements, but also represents a key metabolic tissue. The
following section reviews the literature on muscle regeneration as well as aspects
of metabolic dysregulation of the tissue, known as insulin resistance.
1.1.1 Skeletal muscle regeneration
High capacity for regeneration is an intrinsic attribute of skeletal muscle. Susceptibility of
skeletal muscle to injury due to either direct trauma or as a result of overload during physical activity
resulted in the development of remarkable ability to regenerate along the evolution. Regeneration of
skeletal muscle is a highly synchronized process, which involves activation of various cellular
responses.
1.1.1.1 Skeletal muscle structure
Skeletal muscle is a highly structured tissue, in which motor units have three levels of
organization, and on each level structures are separated by respective connective tissue sheath (fig.
1.1A)(1). Every muscle is encapsulated inside a sheath, termed epimysium. Within epimysium,
smaller structural units, so-called muscle fascicles are aligned along each other and separated by a
sheath, termed perimysium. The fascicle is a bunch of muscle cells, myofibers, placed within elastic
and flexible third connective tissue layer, endomysium. Besides capillary network and nerve fibers,
this layer encompasses satellite cells, muscle stem cells, which are activated upon muscle damage. At
each end of the muscle, the collagen fibers of the epimysium, perimysium, and endomysium come
close to form a tendon, a connective bunch linking a muscle to a bone.
Every myofiber contains a bunch of molecular contraction rods, termed myofibrils, organized
into longitudinally repeated motor units, called sarcomeres. Each sarcomere represents a basic
1
THE REGULATION OF SKELETAL MUSCLE BY MICRORNAs
molecular unit of fibre contraction and contains aligned thin (actin) filaments, which can slide along
thick (myosin) filaments, transforming energy of ATP into motor force (Fig. 1.1B). The smaller
protein components involved in the sarcomere contraction include troponin, tropomyosin, nebulin,
actinin, and titin.
Fig. 1.1 The Organization of Skeletal Muscles. A) The epimysium, a connective tissue layer that surrounds the
entire muscle. The further connective sheath, perimysium, divides the muscle into compartments, termed
fascicles. Within a fascicle, single cell muscle fibers are enclosed by the third connective layer, endomysium.
Close to sarcolemma and within endomysium there is a niche for muscle stem cells, myosatellite cells. A muscle
fiber contains aligned rods of contractile fibers, called myofibrils. B) Myofibers are composed of repeated
motor units, sarcomeres. As a major structural components, sarcomere comprises thick (myosin) and thin
(actin) filaments, with the latter capable of sliding along the former ones. This sliding requires energy of ATP
and ends up with contraction. Modified from (1)
1.1.1.2 Morphological changes during skeletal muscle regeneration
Adult skeletal muscle is a stable tissue characterized with a moderate turnover of nuclei (2).
Day-to-day physical activity requires not more then 1-2% of myonuclei replacement every week (2).
Nevertheless, skeletal muscle is capable to accomplish rapid and extensive regeneration in response to
injury. Regeneration in skeletal muscle is characterized, basically, by two phases: a degenerative
phase, accompanied by muscle fiber necrosis, and regenerative phase, with massive progenitor cell
proliferation and differentiation (reviewed in (3)).
2
THE REGULATION OF SKELETAL MUSCLE BY MICRORNAs
It is assumed that in the first phase, muscle fiber injury leads to a loss of sarcoplasmic
reticulum integrity and results in a calcium homeostasis dysregulation. This, in turn, activates calciumdependent proteases and drives tissue degeneration (4,5). Factors released by myofiber rapture activate
muscle resident immune cells which, in turn, provide chemotactic signals for an amplification of the
inflammatory response (reviewed in (6)). As early as 1-6h post-
Fig. 1.2 The Regeneration of a Multinucleate Skeletal Muscle Fiber. A) In response to myotrauma satellite cells
get activated and massively proliferate. Some portion of cells restores a pool of quiescent satellite cells via selfrenewal process. Chemotaxis drives migration of activated satellite cells to the injury site, where cells either
fuse to regenerating myofiber or to each other to form new myofiber. Newly formed myofiber contains
discretely distributed centrally localized nuclei. Once fusion of myoblasts is completed, myofiber grows in size
and nuclei migrate to the periphery. B) The diagram schematically represents temporal pattern of proliferation
and differentiation of myogenic progenitors in the process of cardiotoxin induced muscle regeneration.
Modified from (7).
injury neutrophils infiltrate regenerating muscle (8). By 48 hours postinjection, macrophages are the
predominant immune cell type in the damaged region, which is attracted to phagocytose cellular debris
and might be involved in the activation of myogenic progenitors (9,10).
3
THE REGULATION OF SKELETAL MUSCLE BY MICRORNAs
Degenerative phase is followed by muscle regeneration process. Proliferation of myogenic
progenitors is a necessary part of muscle repair which provides sufficient number of myonuclei
(reviewed in (7)). Following proliferation, myogenic cells migrate to the injury site to differentiate and
either fuse to regenerating fiber or to each other to form a new myofiber (11,12). Some proportion of
myogenic progenitors downregulate differentiation program, exit cell cycle and become quiescent in
order to restore a satellite cell pool in a process called self-renewal (13,14). Newly formed myofibers
are characterized by the expression of embryonic myosin heavy chain isoform and have centrally
located nuclei. In a separately isolated regenerating myofiber, nuclei are observed to be discretely and
evenly distributed along the fiber, suggesting that the fusion occurs rather focal to the injury site (15).
As soon as fusion is completed, newly formed muscle fibers start to enlarge, and nuclei are moved to
the periphery of the fiber (Fig 1.2A).
Although muscle regeneration is similar for different muscles and for varying injury types, the
magnitude and kinetics can vary significantly. In order to study muscle regeneration in a reproducible
way, several mouse models have been developed, including the use of myotoxins, such as cardiotoxin
(CTX) or bupivacaine (marcaine), as well as ischemia- or freeze-induced injury (16-18). The most
widespread way of muscle regeneration induction is the injection of CTX, peptide isolated from snake
venoms which a has protein kinase-C inhibitor activity. CTX injection leads to the depolarization of
sarcolemma, contraction of muscle cells, and disruption of membrane structures. CTX-induced
damage is followed by a boost of myogenic progenitor proliferation, which peaks on day 3. Myoblast
differentiation is evident on day 5-6 after injection and, by day10, the overall muscle architecture is
recovered (fig. 1.2B, (7)). The complete recovery is evident at 3-4 weeks postinjection.
1.1.1.3 Satellite cells
In the postnatal period, aspects of skeletal muscle such as myofiber growth and repair depend
on satellite cells, muscle stem cells, which reside in a niche located between sarcolemma and basal
lamina, surrounding individual myofiber (Fig. 1.1A, 1.2A)(7,19). These cells are characterized by the
increased nuclear-to-cytoplasm ratio, elevated heterochromatin, and lowered amount of organelles,
which reflects satellite cells’ mitotic quiescence (20). Unless stimulated by the myofiber injury, the
niche allows satellite cells to stay in a dormant, non-proliferative state, which is necessary for their
maintenance throughout lifespan (20,21). The satellite cell population is distributed unequally among
muscles with different fiber types (with prevalence in slow myofibers)(22) and also varies with age
(22-24). If at birth satellite cells constitute 30% of myofiber nuclei, in two-month old mice this
number is decreased to less than 5%. With sexual maturity this decline, although slowly, continues.
4
THE REGULATION OF SKELETAL MUSCLE BY MICRORNAs
One molecular marker of satellite cells is a transcription factor Pax7, which is specifically
expressed in these cells (25,26). Analysis of Pax7 KO mice revealed a striking absence of satellite
cells. Primary cultures derived from muscles of KO mice failed to generate myoblasts, instead giving
rise to fibroblasts and adipocytes. Pax7 was shown to be expressed both in dormant and activated
proliferating satellite cells (26).
In order to isolate satellite cells, Sacco and colleagues (27) utilized FACS with markers for
negative selection, including CD31 (to exclude endothelial cells), CD45 (for hematopoietic cell
exclusion) and Sca1 (marker of progenitor populations from multiple tissues, but not muscle). At the
same time, CD31,CD45, and Sca1 negative population was sorted for integrin α7 positive cells, as this
marker is restricted to skeletal muscle lineage (28).
1.1.1.4 Satellite cell self-renewal
Skeletal muscle has a prominent regenerative capacity and even after multiple cycles of
injury/reparation the satellite cell pool is maintained at a steady size. This observation indicates the
existence of a mechanism to restore the population of stem cells. Self-renewal process can be
mediated by either stochastic differentiation or asymmetric division (29). Stochastic differentiation
implies that symmetrically dividing satellite cells give rise to two undetermined daughter cells, and the
cell fate of each of them is further determined by stochastic process. It has been reported by (29,30),
that a proportion of satellite cells, around 10%, apply asymmetric division, which gives rise to a father
cell (is identical to original satellite cell) and a daughter cell (is committed to myogenesis). In this
process, the location of the satellite cell between sarcolemma, on the one hand, and basal lamina, on
the other hand, predisposes asymmetry in signaling keys received from each side and, finally,
asymmetry of the satellite cell division. The dependence of asymmetrical division and, therefore, selfrenewal on stem cell niche can explain difficulties of ex-vivo cultivation and maintenance of
uncommitted state in muscle derived satellite cells (31). Another evidence of asymmetrical division
within stem cell population comes from experiments with muscle fiber cultures, which allow to
monitor both MyoD and Pax7 expression. In these experiments asymmetric divisions of a subset of
proliferating committed Pax7 / MyoD double positive cells have been shown to give rise to satellite
Pax7-pos / MyoD-neg cells (32). This observation suggests a flexible mechanism of protection of stem
cell pool from terminal differentiation.
5
THE REGULATION OF SKELETAL MUSCLE BY MICRORNAs
1.1.1.5 Myogenic regulatory factors
Myofiber damage leads to the exit of myofiber’s satellite cells from dormant state, their
activation, massive proliferation, and migration to the site of injury (33). Following the rapid
proliferation period, the majority of satellite cells differentiate and fuse to repair damaged myofiber or
to each other to form new one. The process of satellite cell activation and differentiation in
regeneration of skeletal muscle resembles embryonic muscle development. The same as in
embryogenesis, the critical role in adult muscle repair belongs to myogenic regulatory factors (MRFs,
Fig. 1.3).
MRFs family comprises four highly conserved helix-loop-helix transcription factors: MyoD,
Myf5, Myogenin, and MRF4, all of which possess myogenic potential and are able to induce myoblast
traits in non-muscle cells (34-37). Helix-loop-helix-domain mediates heterodimerization of MRF with
E-proteins, which can recognize E-boxes (DNA motif detected in the promoter regions of many
muscle-specific genes). The second domain of the MRFs - basic domain is responsible for DNA
binding (38).
At the gene expression level, activation of satellite cells is characterized by the upregulation of
two MRFs, Myf5 and MyoD (39-41). Both these factors have been revealed to be redundant, as in
contrast to complete lack of skeletal muscle in double knockout (KO), single KOs of either MyoD or
Myf5 have moderate abnormalities in muscle development (42-44). When proliferation phase is over,
expression of two other MRFs – Myogenin and Mrf4 is induced in order to allow for terminal
differentiation (41,45,46). Summarized, these studies provide a model, which describes MyoD and
Myf5 as redundant factors acting upstream of Mrf4 and myogenin that direct myoblasts to terminal
differentiation.
The next, higher level of genetic hierarchy that governs myogenesis is determined by the
paired-homeobox transcription factors, Pax3 and Pax7. These factors are expressed in the
dermomyotome, a region of the somite in an embryo that gives rise to skeletal muscle (47).
However, while Pax7 knockout seems to be dispensable for embryonic development, Pax3 mutated
embryos do not develop limb and diaphragm muscles (26,48). Pax3, but not Pax7 was reported to be
expressed in cells migrating from dermomyotome to form limb muscles (49). Given that no MyoD is
detected in limbs of Pax3 mutated embryo, Pax3 is placed upstream of MyoD.
On the other hand, deletion of Pax7-expressing cells led to abnormalities in later stages of
development, such as smaller muscles and reduced number of myofibers in limbs in newborns (26,50).
These observations allow to consider a model of embryonic development, in which Pax3 positive cells
are primary cells that form a template for initial fibers in the limb, and Pax7 expressing cells further
contribute to form secondary fibers and generate satellite cell pool (51).
6
THE REGULATION OF SKELETAL MUSCLE BY MICRORNAs
Fig. 1.3 Hierarchy of transcription factors governing myogenesis. Myogenic progenitors in early embryogenesis
directly become myoblasts, omitting quiescent stage. In postnatal muscle, a portion of progenitors remain as
satellite cells, while another part represents committed progenitors. Some activated committed myoblasts still
can revert to dormant state. Six1/4 and Pax3/7 are embryonic master regulators that are responsible for early
lineage specification. Myf5 and MyoD instruct progenitors to myogenic program. Finally Myogenin and Mrf4
take part in order to direct myoblasts to fusion and myotube formation (terminal differentiation). Modified
from (52).
The sine oculis-related homeobox 1 (Six1) and Six4 are believed to be transcription factors
placed at the apical end of the regulatory hierarchy that turn embryonic progenitors toward the
myogenic lineage. Six proteins act to activate transcription of target genes, including Pax3, MyoD,
Mrf4, and myogenin (53). The overexpression of Six1 in somite explants induces expression of Pax3,
and Six1/Six4 double KO embryos have no expression of Pax3 in hypaxial deromyotome (54).
7
THE REGULATION OF SKELETAL MUSCLE BY MICRORNAs
1.1.1.6 Secreted factors in regulation of muscle regeneration
Hepatocyte growth factor
Skeletal muscle regeneration is a highly regulated process, which involves several levels of
regulation, including contact signals from basement membrane and myofiber as well as signals from
secreted factors. Muscle injury has been shown to cause a release of soluble factors, responsible for
activation and proliferation of satellite cells. For example, extracts from crushed muscle demonstrated
proactive and mitogenic effect on satellite cells in vitro (55). Recent studies indicated that mechanical
stress or damage in muscle fibers activates nitric oxide (NO) signaling, which, in turn, leads to a
release of hepatocyte grows factor (HGF) from extracellular matrix (ECM) of BM (56). HGF is now
considered as a primary growth factor capable of induction of quiescent satellite cell activation (57).
Besides progenitor activation, HGF might be also involved in the satellite cell migration to the site of
injury through activation of Ras/Ral pathway (58). HGF role in muscle repair is more important in the
early phase as evidenced from immunostaining studies and an absence of the effect of HGF on
terminal differentiation (57).
Fibroblast growth factors
In addition to HGF, several fibroblast growth factors (FGF) have been shown to be important
mitogenic factors, also playing a role in inhibition of satellite cell differentiation during proliferative
phase (59). FGF6 was shown to be muscle specific FGF, which expression is upregulated in the early
phase of muscle regeneration (60). Studies of crush-induced injury in FGF6 KO mice and FGF6 KO
mice crossed to mdx background (mutation in dystrophin gene leading to a chronic muscle damage,
regeneration cycles and, eventually, to muscle dystrophy) have shown reduced muscle regenerative
capacity characterized by a decreased number of myoD- and myogenin-positive cells as well as
increased fibrosis. Then, following study by Flore et al (61) reported no phenotype in FGF6 KO mice.
This discrepancy might be explained by redundancy of FGFs in skeletal muscle, since different FGFs,
including 2, 5, 6 and 7 have been reported to be expressed in skeletal muscle (62). Further
investigation compared phenotypes of different combinations of FGF KOs crossed to mdx background
and revealed severe regeneration inhibition in FGF2 / FGF6 double KO / mdx mice, characterized by a
prominent fibrosis as well as reduced differentiation and proliferation markers (63). In addition to
genetic studies, experiments with injection of FGF into muscle of mdx mice and FGF neutralizing
antibodies into muscle lesions further confirmed a role for FGF in muscle regeneration (59,64).
8
THE REGULATION OF SKELETAL MUSCLE BY MICRORNAs
Four receptors for FGF, FGFR1-4 have been identified, with FGFR1 and 4 being expressed in
satellite cells. FGF2 and FGF6 have the highest affinity to FGFR1 and 4 (65). FGFR1 is dramatically
upregulated in the early, proliferative phase of myogenesis and this effect is augmented by HGF (66).
FGFR4, in contrast to FGFR1, is expressed in differentiating myoblasts and is downregulated during
active proliferation (67). The availability of FGFR1 is critical for satellite cell proliferation, as full
length FGFR1 overexpression promotes cell replication and decreases differentiation, while truncated
FGFR1 expression has reverse effects. FGFRs are transmembrane receptor tyrosine kinases. Upon
ligand binding, the receptor dimers autophosphorylate each other and induce several signaling
branches, including MAPK-ERK, AKT and phospholipase C mediated pathways (68). Both FGF and
HGF are dependent on heparan sulphate proteoglycans, ECM proteins, which could serve as depots for
growth factors in close proximity to target cells and are involved in regulation of receptor signaling
(69).
Insulin-like growth factors
The role of insulin growth factors (IGFs) in regulation of growth and developmental program
in many tissues is well documented. Not surprisingly, the involvement of IGFs in skeletal muscle
development and regeneration has been also reported. IGFs have been shown to regulate MRFs and
promote both proliferation and differentiation of myoblasts in cell culture experiments (70,71). In-vivo
elevated IGF1 leads to hypertrophic effects that is attributed to both stimulation of satellite cell
proliferation, providing more myonuclei, as well as an anabolic action on existing myofibers (72-74).
Levels of IGFs are also induced during muscle regeneration (75). IGF1 and 2 have been reported to
improve pathophysiology phenotype of aged muscle and dystrophic changes in mdx mice (76,77). This
amelioration is likely due to induced proliferation and hypertrophic effect described for normal
muscle, though IGF1 might also promote muscle regeneration via stimulation of cell survival (78)
Myostatin
Since its discovery in 1997, myostatin (MSTN) attracted significant attention. MSTN
expression during development and in postnatal growth is restricted to skeletal muscle and MSTN KO
mice have been shown to have profoundly elevated muscle mass (79). This property of MSTN has
been also demonstrated for other species, including homo sapiens (80,81). For a long time MSTN KO
phenotype had been explained by its inhibitory effect on satellite cell activation and proliferation.
MSTN levels correlate with satellite cell numbers if fast contracting muscles (high MSTN, low
number of progenitors) are compared to slow ones (low MSTN, high number of progenitors) or during
muscle unloading that suppresses satellite cell proliferation while leading to elevated levels of MSTN
9
THE REGULATION OF SKELETAL MUSCLE BY MICRORNAs
(82). The inhibitory effect was further reported from studies analyzing an effect of high MSTN doses
in cell culture proliferation assays (83,84). However, recent studies revealed that myostatin’s action is
mostly catabolic and controls muscle size by signaling via Activin receptor type 2, highly expressed
on muscle fibers. No significant increase in the number of satellite cells or myofibers have been
reported in muscles of MSTN KO mice or mice treated with MSTN inhibitor (85,86). Following study
of mice having genetically inhibited MSTN and no functional satellite cells demonstrated that muscle
fiber hypertrophy induced by MSTN deletion did not rely on progenitor cells (87). These results
encourage the application of anti-MSTN treatments to myopathies, as in this approach muscle fiber
hypertrophy can compensate for myofiber loss without high risk of exhaustion for satellite cell pool
(87).
1.1.1.7 A role for basement membrane in skeletal muscle regeneration
Every skeletal muscle fiber is structurally supported by a surrounding ECM membrane, called
basement membrane (BM) (88). BM was first discovered in skeletal muscle in 19th century. When
fibers were dissected and the cell content was shrinked and lysed, transparent and elastic sheath
remained visible (88). The BM is composed of two layers: internal – basal lamina, which is directly
connected to sarcolemma and external one, fibrillary reticular lamina (Fig. 1.4). Basal lamina is a
highly structured sheath, which can be divided into two layers: the lamina lucida and the lamina
densa. The first one, lamina lucida, is a lattice network of self-assembled laminins (most abundant in
muscle is laminin-2, composed of α2β1γ1 subunits), which interact with integrins (in muscle it is
basically integrin α7β1) and dystroglycans (89). This laminin grid is covered by a more dense lattice
network of self-assembled nonfibrillar collagen 4 (most abundant in muscle is α1α1α2 combination of
subunits). And these two aligned layers are interconnected by crosslinking structural components,
nidogens and perlecans (90)(fig. 1.4).
The reticular lamina, more external layer of BM, is a more cluttered and loose stratum, which
is composed mainly of branched collagen I and III fibers with presence of other fibrillary components,
including fibrillin, fibronectin, and proteoglycans. Reticular lamina is anchored to basal lamina via
collagen 6 (88).
Besides structural functions, basal lamina provides cells with signals in two ways. First of all,
interaction of collagens, laminins, and fibronectin with integrins, which bridge structural ECM
proteins to cytoskeleton, leads to formation of focal adhesion complexes, followed by an induction of
focal adhesion kinase activity (FAK) (69). From the other hand, heparin and heparin sulfate branches
of several proteoglycans, such as perlecans (in basal lamina) and syndecans (cellular transmembrane
proteins), can bind growth factors (including HGF and FGF) and mediate their delivery, as well as
10
THE REGULATION OF SKELETAL MUSCLE BY MICRORNAs
directly participate in extracellular signaling. For example, perlecan has been shown to be a necessary
component of FGFR signaling (91) and syndecan 4 oligomerization was shown to be inhibited by
FGFR signaling in order to prevent focal adhesion coordinated cytoskeletal changes (92). Heparan
sulphate proteoglycans represent a depot for growth factors and can modulate stable gradients,
directing cell migration and development. Growth factors can be released from this depot by
metalloprotease mediated degradation of ECM proteins or as a result of glycosaminoglycan turnover
(69).
Mutations in structural components of basal lamina are often associated with myopathies.
Around half of hereditary muscular dystrophies is caused by mutations in laminin-2. Collagen 4
mutations have a broader spectrum of symptoms, including some forms of myopathy, and abnormal
Collagen 6 leads to Ulrich myopathy (93). Mutations in perlecan have been reported to cause
Schwartz–Jampel syndrome associated with muscle weakness and stiffness, growth retardation, as
well as abnormalities in bone and cartilage development (94).
BM integrity is critical for muscle regeneration. It has been reported that if the basement
membrane is not damaged, muscle injury is followed by almost complete restoration of structure and
function. On the contrary, rupture of myofiber’s BM led to formation of scar tissue in between the
myofiber stumps (95). BM is considered to remain unchanged along the process of myofiber
degeneration and represent a tubular scaffold within which the myofiber repair or formation of new
myofiber occurs (18,96). In this process, new myofiber produces its own basal lamina of normal size
during growth, which substitutes the old one with time (18).
11
THE REGULATION OF SKELETAL MUSCLE BY MICRORNAs
Fig. 1.4 Structure of Masement membrane. Basement membrane is composed of two layers, dense
and structured basal lamina and more cluttered and loose lamina reticularis. Basal lamina is further
devided into lamina lucida and lamina densa. Lamina lucida comrises highly structured planar laminin
network and laminin interacting intgrines and dystroglycans, anchored in sarcolemma. This layer is
covered by lamina dense, representing more dense planar collagen 4 network. Both laminin and
collagen 4 networkes are crosslinked by nidogens and perlecans. Nidogen and perlecan provide basal
lamina with rigidity. Besides that perlecan is involved in deposition of growth factors and participate
in extracellular signalling. Adopted and modified from (97) and http://meddic.jp/lamina_lucida.
Skeletal muscle stem cell niche is located in between sarcolemma and basal lamina. Basal
lamina plays a critical role in maintanance of satellite cells quiescence and also takes part in the
activation of these cells after muscle injury. Mechanical stretch of sarcolemma or basal lamina leads to
stimulation of NO synthase, activation of metalloproteases (including Mmp2), and consecutive release
of HGF from ECM (56). Following satellite cell activation, FGF-release from the basal lamina’s
glycoprotein matrix (including perlecans) leads to massive cell proliferation. Besides that, the basal
lamina provides instructions for asymmetric division during satellite cell proliferation in the niche
(31). Thus, BM plays versatile roles in skeletal muscle ranging from stem cell niche homeostasis and
structural support in intact muscle to repair guidence and satellite cell activation during muscle
regeneration.
12
THE REGULATION OF SKELETAL MUSCLE BY MICRORNAs
1.1.2 Skeletal muscle metabolism and insulin resistance
Skeletal muscle is one of the key metabolic tissues that consumes and stores nutrients in a
highly regulated manner. Skeletal muscle insulin resistance, a condition of poor responsiveness of the
tissue to insulin, often precedes the development of type 2 diabetes. This section reviews literature on
muscle glucose and lipid metabolism, its hormonal regulation, as well as development of insulin
resistance.
1.1.2.1 Skeletal muscle as a metabolic tissue
Healthy skeletal muscle carefully balances between utilization of glucose and fatty acids,
depending on fed state and muscle activity. Skeletal muscle is the largest insulin responsive tissue in
the body and accounts for up to 80% of post-prandial glucose disposal in humans (98). Under fed
state, increasing levels of insulin allow skeletal muscle to uptake glucose and store it as glycogen. At
this state, a decision which energy source to use is determined by cytoplasmic citrate levels. Citrate
activates acetyl-coA carboxylase-2 (ACC-2) that produces malonyl-coA, an inhibitor of carnitinepalmitoyltransferase and, thereby, restricts an influx of activated fatty acids into mitochondria, and
consecutive β-oxidation. During fasting, when glucose and insulin levels fall down, AMP / ATP ratio
increases and leads to the activation of AMP-activated protein kinase (AMP-PK). Both elevated AMP
and AMP-PK inhibit ACC-2 as well as activate decarboxylase which results in elimination of
malonyl-coA and promotion of fatty acid influx into mitochondria (99).
Insulin mediates glucose uptake through a set of highly regulated signalling events. Binding of
insulin to insulin receptor leads to its autophosphorylation and consecutive phosphorylation of insulin
receptor substrates 1 and 2 (IRS1,2). Activated IRS binds to regulatory subunit of PI3 kinase that
activates its catalytic subunit, producing phosphoinositides, including 3,4,5-triphosphate. This, in turn,
promotes an activation of phosphoinositide-dependent protein kinase (PDK) and downstream kinases
– protein kinase B (Akt) and protein kinase C (PKC). Akt substrate, AS160, accordingly mediates the
translocation of glucose transporter to sarcolemma that allows for glucose uptake. Inside a cell glucose
is rapidly phosphorylated by hexokinase II and directed to either oxidative (glycolysis and oxidative
phosphorylation) or non-oxidative pathway (mainly glycogen synthesis) (100). In a fed state, most of
glucose (~70%) is converted into glycogen, predominantly in skeletal muscle.
During exercise skeletal muscle might require up to 100 times more ATP than at rest. ATP
itself is not suitable for storage of energy reserves as it is involved in allosteric regulation of many
regulatory enzymes. Instead, muscle uses creatine phosphate to store high-energy phosphate bond,
13
THE REGULATION OF SKELETAL MUSCLE BY MICRORNAs
which can be easily converted into ATP by enzyme creatine kinase (99). At the same time, creatine
kinase serves as a good marker of myoblast differentiation (101) and muscle damage (102).
1.1.2.2 Skeletal muscle and type 2 diabetes
Type 2 diabetes (T2D) is a metabolic disease characterized by a poor responsiveness of insulin
dependent tissues, basically, skeletal muscle, liver, and fat to the action of insulin, hyperglycemia and
hyperinsulinemia, corresponding pathological changes in metabolism, as well as the development of
complications due to toxic effects of elevated lipid and glucose blood levels. Insulin resistance
gradually progresses from normal state through impared glucose tolerance (delayed normalization of
glucose levels after meal, but normal fasting glucose levels) to T2D (elevated fasting glucsose). While
hyperglycemia comes from hepatic insulin resistance that accounts for an inability of liver to stop
glucose release in response to insulin, the initial defect leading to T2D - skeletal muscle insulin
resistance, is evident years before the disease manifestation (103,104). It has been repeatedly reported
that genetically predisposed individuals, for example, lean glucose tolerant offspring of T2D patiants,
exhibit skeletal muscle insulin resistance (105-108). As disease progresses from normal to impaired
glucose tolerance (IGT), skeletal muscle insulin sensitivity decreases substantially, while glucose
tolerance is affected moderatelly due to a compensatory increase in insulin production (109). The
earliest metabolic defect in skeletal muscle associated with insulin resistance is reduced glycogen
synthesis and glycogen synthase activity (98). Importantly, this defect in glycogen synthesis is
observed before the insulin mediated supression of hepatic glucose production gets deteriorated and βcell function is compromised (108).
Along the disease progression, insulin resitance increases and require more and more insulin
to compensate elevated glucose levels. At certain stage β-cells fail to produce insulin amounts needed
to control glucose, and hyperglycemia manifests T2D. Interestingly, insulin resistance itself can
further promote the decrease in insulin sensitivity via elevated insulin levels. Infusions of
physiological concentrations of insulin for 72h in healthy individuals resulted in a decreased glucose
uptake, glycogen synthesis, and glycogen synthase activity (110).
At the molecular level, key intracellular proteins mediating insulin signaling have been shown
to be disregulated in diabetic skeletal muscle. Reduced IRS-1 tyrosine phosphorylation as well as a
decrease in PI-3 kinase and Akt kinase activity have been reported in skeletal muscle biopsies of T2D
patients (111). At the same time, an increased phosphorylation of a serine residue in IRS-1 has been
shown to be associated with decreased Akt activity in normal glucose tolerant (NGT) offspring of T2D
patients (112).
14
THE REGULATION OF SKELETAL MUSCLE BY MICRORNAs
Hyperinsulinemia in NGT patients can also contribute to the skeletal muscle insulin resistance
via defects in insulin mediated control of lipolysis in adipocytes. NGT offspring of T2D patients have
elevated levels of fasting plasma free fatty acids (FFA) and demonstrate impaired insulin mediated
suppression of lipid oxidation, as well as markedly reduced capacity of insulin to inhibit lypolysis
(108). This might explain increased intramyocellular lipid content in NGT offspring of T2D
individuals (113). Elevated levels of lipids and their derivatives (such as diacylglycerol and ceramide)
have been shown to interfere with insulin IRS-1 phosphorylation and might be important contributors
to skeletal muscle insulin resistance (114). Importantly, 4 days of lipid infusions at physiologic
concentrations led to decreased glucose uptake and deteriorated insulin signaling in normal individuals
(106). Furthermore, abdominal obesity, which is characterized by elevated blood triglycerides, have
been recognized as a strong risk factor of T2D (115).
At the same time, intramuscular lipid accumulation might be caused by decreased muscle
mitochondria content and function. Independent studies reported a decreased expression of genes
implicated in mitochodrial oxidative metabolism and a markedly reduced ATP production in skeletal
muscle of NGT offspring of T2D patients (116-118). And another group has shown reduced
mitochondria density in obese and T2D individuals (119). Impairment in insulin signaling can further
contribute to a decrease in mitochondria function, since insulin is a known inducer of many
mitochondria genes (120).
Overall, mitochondrial dysfunction of the skeletal muscle and elevated intramuscular lipid
content might promote each other and together contribute to muscle insulin resistance. As a result,
elevated insulin might further exacerbate muscle insulin resistance as well as promote hepatic and
adipocyte insulin resistance. This developing syndrom, involving several mutual contributors, ends up
with an inability of β-cells to compensate for the increasing glucose levels and T2D manifestation.
1.1.2.3 The regulation of adult skeletal muscle metabolism by growth hormone
One of the major hormones involved in the regulation of metabolism at organismal level and,
particularly, adipose and muscle metabolism is pituitary gland produced growth hormone (GH). Its
production is mainly regulated by hormones: positively, by GH releasing hormone and negatively, by
somatostatin, both released from hypothalamus (121). GH exerts its functions primarily to promote
skeletal muscle mass and bone density during periods of positive energy balance as well as to protect
these tissues from catabolism during starvation. The main function of growth hormone in adults is to
preserve muscle and bone during fasting. GH can directly (or acting synergistically with IGF1)
preserve nitrogen by decreasing urea blood levels and its excretion via acceleration of protein
synthesis without a change in protein degradation (122). In the night, low nutrient levels in concert
15
THE REGULATION OF SKELETAL MUSCLE BY MICRORNAs
with sleep rhythms promote GH release, which acute action is to induce lipolysis in adipose tissue (in
order to raise FFA blood levels and supply muscle) and gluconeogenesis in liver. FFA release is a very
rapid response, since FFA level reaches its peak 2-3h after GH pulses and lasts for 8-9h (123). During
the day, elevated blood levels of glucose and lipids supress GH production (124). In childhood and
puberty, prolonged production of GH in concert with nutrient supply and insulin promotes hepatic (as
well as local) IGF1 production that is responsible for body growth (125).
It has been reported that GH treatment leads to a marked decrease in glucose uptake and
oxidation in skeletal muscle as well as inhibition of glycogen synthesis (126,127). GH also promotes
intramuscular lipid accumulation and oxidation (128). Negative effect of GH on muscle glucose
metabolism is mainly mediated by elevated FFA serum levels and intamuscular lipid accumulation
(FFA converted into acetil-CoA can block pyruvate dehydrogenase complex and thus glucose
oxidation (129) ), though some studies argue for an involvement of direct effects of GH (126). Besides
its effects on muscle, GH has been reported to decrease hepatic insulin sensitivity as well as abrogate
the antilipolytic effect of mild hyperinsulinemia (130,131). Overall, elevated GH production or GH
replacement therapy may induce both hepatic and muscle insulin resistance, and glucose and lipid
profiles in such patients must be controlled.
1.1.2.4 GH deficiency in humans and GH replacement therapy
Adult onset GH deficiency is a rare endocrinological disorder (~0.01% of population in USA,
http://hgfound.org) that mainly results from pituitary tumors, surgical or radiology treatment of such
tumors, or traumatic brain injuries (132). GH- deficient patents are characterized with increased
visceral adipose tissue and reduced lean body mass, decreased skeletal muscle strength, exercise
performance and cardiac capacity, as well as decreased bone density and increased risk of fracture
(133). Children with GH deficiency have decreased fasting glucose and delayed recovery from
hypoglycemia (due to over-responsiveness of liver to insulin and decreased glucose production) (134).
From the other hand, adult GH deficient patients demonstrate normal fasting glucose and impaired
insulin sensitivity characterized by a decrease in glycogen synthase activity. The mechanism
responsible for glucose intolerance in adults with GH deficiency is not clear, although the level of
visceral adiposity (and therefore elevated FFA release) correlates with the severity of insulin resistance
in these patients and is considered as the main cause (135). Similar to humans, GH receptor-deficient
(GHR-KO) mice have been reported to have decreased fasting glucose and insulin levels in young
animals and normalization of glucose with age (136). At the same time, in contrast to humans, the
insulin level is decreased along the life span in GHR KO mice, which results in increased insulin
sensitivity and longevity (136,137). GHR KO mice also exhibit decreased pancreas and β-cell mass
16
THE REGULATION OF SKELETAL MUSCLE BY MICRORNAs
that lead to reduced insulin production and glucose intolerance during GTT (137,138). Nevertheless,
secreted insulin amount is enough to control glucose, which, together with high insulin sensitivity,
suggests careful attribution of the glucose intolerance term to these animals.
GH replacement therapy (GHRT) has a positive effect on body composition leading to a
marked decrease in visceral fat, an increase in lean mass, muscle mass and strength, as well as bone
density (139). An effect of GHRT on glucose metabolism is individual and might be either positive
(due to reduction of visceral fat) or negative (due to elevated levels of FFA and direct opposing of
insulin signaling by GH), and may also depend on the pre-treatment glucose status. Nevertheless, on
average, short-term effect of GHRT is associated with an increased insulin resistance, which resolves
gradually along the therapy timeline, and long-term effects are ameliorated (140,141). One study
emphasizes primary detrimental effect of GHRT on muscle glycogen synthase activity as well as
glycogen, and glucose content after 24 month of GHRT while no change in hepatic insulin resistance
(142). The same study reported skeletal muscle insulin resistance in GH deficiency patients before
GHRT. Deteriorated insulin sensitivity in skeletal muscle after GHRT was further demonstrated in a
separate study (143). Interestingly, interruption of GHRT for one year led to an improvement in
insulin sensitivity in a placebo controlled study, in spite of accumulation of fat mass (144). This
implies that more detailed understanding of mechanisms underlying glucose homeostasis is needed to
minimize risk factors for metabolic syndrome during long-term GHRT in patients with different
metabolic status.
17
THE REGULATION OF SKELETAL MUSCLE BY MICRORNAs
1.2 MicroRNA biology and its role in skeletal muscle
metabolism and regeneration
MicroRNAs (miRNAs) are a large family of non-coding posttranscriptional
regulators of only 20-24 nucleotides in length. miRNAs are involved in the
regulation of almost all intracellular processes in all mammalian tissues. This
section will introduce you to principles of miRNA biology and review miRNA
functions in the regulation of muscle regeneration and metabolism.
1.2.1 MicroRNA biology
MiRNAs are important posttranscriptional regulators predicted to control ~50% of all proteincoding genes (145). MiRNA family comprises several hundreds of members in humans, the amount
comparable to those of transcription factors. Many of miRNAs are expressed in tissue-specific manner
or at distinct developmental stages, thereby playing a role in cell specification. Each miRNA targets
multiple genes and for some of them more than a thousand targets are predicted. At the same time,
miRNAs realize a gentle mode of regulation working to fine tune the translation of a pool of targets
(146).
Most of microRNAs come from independent miRNA coding genes (canonical pathway)
regulated similar to protein coding genes. The rest miRNAs are processed from introns of proteincoding genes (147). The processing of miRNAs precursor in canonical pathway is performed in two
steps (Fig 1.5). In the first one, primary transcribed by RNA-polimerase II precursor (pri-miRNA) is
processed with RNase III family enzyme, Drosha, in complex with dsRNA binding protein DGCR8.
This event takes place in the nucleus and produces 70nt miRNA precursor (pre-miRNA). Some other
pre-miRNAs are the result of splicing of very short introns (called mirtrons). In either case, premiRNAs are exported to the cytoplasm with the help of Exportin 5, where the second processing event
takes place. Another RNase III family enzyme, Dicer, in complex with dsRNA binding proteins,
TRBP and AGO2 (possesses RNaseH-like activity), processes pre-miRNA in a ~20bp
miRNA/miRNA* duplex. Afterwards, one strand of the duplex (the guide strand) is incorporated into
miRNA induced silencing complex (miRISC), while the other one is degraded. As a part of miRISC
complex, miRNA can base-pair mRNA targets with its seed region (nucleotides from 2 to 8), which
leads to either endonuclease cleavage and following degradation (rare in animals), inhibition of
translation (via attraction of translation repressors) or deadenylation (148). Since seed regions
18
THE REGULATION OF SKELETAL MUSCLE BY MICRORNAs
Fig 1.5. MicroRNA biogenesis. Adopted from (146). See explanation in the text.
can be found in multiple mRNA untranslated regions, each miRNA potentially targets a pool of
mRNAs. The mechanisms of miRNA-mediated translation repression are currently not well studied,
while it is established that miRISC can induce mRNA deadenylation via GW182 protein that attracts
deadenylation factors (149).
19
THE REGULATION OF SKELETAL MUSCLE BY MICRORNAs
1.2.2 The involvement of microRNAs in muscle differentiation /
regeneration
MicroRNAs have been widely acknowledged as the key players in tissue development.
Severely reduced muscle mass and deteriorated myofiber morphology was reported in muscle-specific
Dicer KO (150). In a separate mouse model which allows for skeletal muscle specific tamoxifeninduced Dicer deletion, muscle regeneration was severely blunted after muscle injury (151). Several
miRNAs demonstrated skeletal muscle- (or in some cases skeletal and cardiac muscle) specific
expression, including miR-1, miR-206, miR-208, miR-133a/b and miR-499 (152). MiR-1, miR-206,
and miR-133 have been reported to have the strongest induction during myoblast differentiation
(153,154) and were also highly upregulated in muscle regeneration (155,156). Genes of miR-1/miR206 and miR-133 families are controlled by myogenic transcription factors: MyoD, myocyte enhancer
factor-2 (MEF2), and serum response factor (SRF) (157-160). miR-1 and miR-206 as well as miR-486
are involved in downregulation of Pax3 and Pax7 transcription factors in order to allow myoblast
differentiation (161). MiR-1 also targets histone deacetylase 4 (HDAC4) which blunts differentiation,
partly, by downregulation of MEF2 (153). MiR-133a can repress SRF representing negative feedback
loop (153). At the same time miR-133 directs progenitors towards myogenesis by targeting PRDM16,
transcription factor, which otherwise promotes cell to brown fat lineage (162).
Another set of microRNAs, which are expressed within introns of myosin heavy chain genes
control myosin content regulate fiber type switch in skeletal muscle (163). MiR-208a is expressed
within cardiac-specific MYH6 (encodes fast myosin), and promotes slow myosin genes. Slow Myosin
genes, MYH7 and MYH7b, co-express in their introns miR-208b and mir-499 respectively, which
acting redundantly, further reinforce slow fiber type maintenance / formation by targeting MYH7 and
MYH7b inhibitors.
Another microRNA, miR-486, though not muscle specific, has been reported to be highly
enriched in skeletal muscle. This miRNA is expressed within an intron of Ankyrin-1 gene and is
upregulated by myocardin-related transcription factor-A (MRTF-A), SRF and MyoD (164). MiR-486
was reported to activate PI3K/Akt signaling by targeting its inhibitors, PTEN and FOXO-1a, and is
highly upregulated during myoblast differentiation (165).
Several other miRNAs that are expressed ubiquitously have been reported to be upregulated
during myoblast differentiation and target several negative regulators of myogenesis: polycomb
protein Ezh2 (miR-214 (166) and mir-26a (167)), Myc (niR-24 (168)), homeobox protein Hox-A11
(miR-181 (169)), Pax3 (miR-27a (170)) and cdc25 (miR-322 and miR-503 (171)).
20
THE REGULATION OF SKELETAL MUSCLE BY MICRORNAs
1.2.3 The role of microRNA in the regulation of muscle metabolism and
insulin resistance.
A number of miRNAs have been reported to have a role in the regulation of metabolism and
potential involvement in pathogenesis of T2D (172). For example, miR-375 has been demonstrated to
regulate insulin secretion via targeting a component of exocytosis pathway, myotrophin (173). In
another study, miR-103/107 cluster have been shown to regulate blood glucose levels by upregulating
gluconeogenesis and glucose production in liver as well as adipocyte size and insulin resistance in fat
tissue (174). The authors have also demonstrated that mir-107 inhibition can ameliorate glucose
intolerance in obese mice.
Only few studies so far investigated an involvement of miRNAs in skeletal muscle insulin
resistance. Granjon and colleagues profiled miRNAs in healthy individuals before and after 3-hourhyperinsulinemic euglycemic clamp and observed 39 miRNAs to be significantly downregulated,
including miR-1, miR-206, miR-133 and miR-29a,c (175). The authors highlighted that transcription
factors MEF2 and (SREBP)-1c mediated insulin-induced regulation of miR-1 and miR-133. In a
separate study, miRNAs were profiled in NGT, IGT and T2D cohorts of individuals and one third of
detected miRNAs (n=62) were regulated (176). Interestingly, a cohort of miRNAs was regulated in
both IGT and T2D groups suggesting a role for these miRNAs in muscle insulin resistance before T2D
manifestation. The most downregulated (5 fold), miR-133 expression was also negatively correlated
with fasting glucose levels and levels of glycated serum hemoglobin (HbA1c).
In animal models of T2D (goto-kakizaki rats) and IGT (Wistar Kyoto rats), several miRNAs
were reported to correlate with glycemia level: miR-10b in muscle, miR-195 and miR-103 in liver,
miR-222 and miR-27a in adipose tissue (177).
Overall, miRNAs proved to be involved in regulation of skeletal muscle metabolsim as well as
glycemic control, and represent an attractive targets for therapeutic approach to T2D.
1.2.4 MicroRNA-29 family, its regulation and cellular functions
MicroRNA-29 family comprises four members, miR-29a, miR-29b1, miR-29b2, and miR-29c.
The pairs of miRNAs, miR-29a / miR-29b1 and miR-29b2 / miR-29c are expressed tandemly as
bicistronic genes. MiR-29a/b1 gene is located on Chr. 7 in humans and Chr. 6 in mice, while miR29b2/c gene is located on Chr. 1 in both species. Sequences of all three members are highly conserved
in mouse, human and rat, and share the same seed regions, which imply that targets within the miRNA
family largely overlap.
21
THE REGULATION OF SKELETAL MUSCLE BY MICRORNAs
MiR-29a/b1 and miR-29b2/c genes might be differently regulated at both transcriptional and
the translational levels. miR-29b2 and c are processed from last exon of the primary transcript, while
MiR-29a and b1 might be processed either from last intron or last exon of two different primary
transcript, depending on alternative splicing (178,179). This implies that MiR-29a/b1 transcriptional
regulation may depend on cell type or developmental stage.
MiR-29a/b1 and miR-29b2/c genes have overlapping, but largely different set of transcription
binding sites in their promoters. C-Myc has been identified with chromatin precipitation in promoters
of both miR-29a/b1 and miR-29b2/c genes (178,179). Other studies revealed YY1(180) and SMAD3
(181) in promoter region of miR-29b2 / c as well as NFkB (179), Gli (179), TCF/LEF(182) and CEBP
(183) in miR-29a / b1 promoter.
MiR-29 family has around 1000 of conserved predicted targets in mouse and human.
Importantly, miR-29 has been reported to target a group of genes related to extracellular matrix,
including both structural (several collagen genes, elastin, integrin-β1, and laminin) and ECMmodifying (Mmp2 and Sparc) genes, which indicates its role in fibrosis control in many tissues, such
as bone, heart, kidney and lung (184-187).
Besides its role in fibrosis, miR-29 has been implicated in regulation of immune response and
cancerogenesis. MiR-29a/b1 has been reported to target interferon receptor (IFNAR1) in thymic
stromal cells and mediate their insensitivity to normal basal IFN level needed for thymocyte
development. However without miR-29, stromal cells become oversensitive to IFN (like in periods of
infection, when IFN levels are much higher than basal) and drive thymus involution (188). MiR29a/b1 also plays a role in determination of T-helper specification which can lead to the development
of two major subtypes, Th1-cells, mediating immunity against intracellular bacteria and protozoa, and
Th2-cells, which drive immune response to extracellular parasites. MiR-29 has been reported to target
the key determinants of Th1-cells, IFN as well as transcription factors T-bet and Eomes, and, thereby,
drive maturation to Th2 direction (189).
MiR-29 is also implicated in cancer development. Its role is complicated as it has been
reported to have both pro- and anti-cancerogenous properties, depending on the cancer cell type and
stage of cancer progression. MiR-29 is an important trigger of indolent B-cell chronic lymphocytic
leukemia (B-CLL) (190). Upregulation of miR-29 has been also reported to induce malignancy in
breast cancer by targeting tristetraprolin (191). At the same time miR-29 has been shown to target
several important oncogenes, such as coactivator of Act, Tcl1 (192), antiapoptotic Bcl-2 member,
Mcl1 (193) or p53 inhibitors, p85α and CDC42 (194). That is why it is not surprising, that progression
of many cancers, including B-CLL is characterized with decreased levels of miR-29 and miR-29
mimetic treatment is beneficial in some cancers (195).
22
CHAPTER II
CHAPTER II
FGF MODULATES MICRORNA-29A TO CONTROL
SKELETAL MUSCLE REGENERATION DURING
INJURY AND EXERCISE
This chapter contains unpublished data as well as fragments and figures adopted or reproduced
from the following manuscript, currently submitted for publication:
microRNA-29a controls skeletal muscle regeneration during injury and exercise
Artur Galimov1,5, Troy Merry2, Edlira Luca1, Elisabeth J. Rushing3, Carlo M. Croce4, Michael Ristow2,
and Jan Krützfeldt1,5,6.
1
Division of Endocrinology, Diabetes, and Clinical Nutrition, University Zurich and University Hospital
Zurich, Switzerland, 2Department of Health Sciences and Technology, ETH Zurich, Switzerland,
3
Institute of Neuropathology, University Zurich and University Hospital Zurich, Switzerland,
4
Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University, USA,
5
Competence Center Personalized Medicine, ETH Zurich and University of Zurich, Switzerland, 6Zurich
Center for Integrative Human Physiology, University of Zurich, Switzerland
Corresponding author and person to whom reprint requests should be addressed to:
Prof. Jan Krützfeldt, MD, University Hospital Zurich, Division of Endocrinology, Diabetes, and Clinical
Nutrition, Rämistrasse 100, 8091 Zurich, Switzerland. Phone: +41 (0)44 255 36 27, Fax: +41 (0)44 255
9741, E-mail: [email protected]
23
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
2.1 Outline of the work
Growth factors play a critical role in the expansion of myogenic progenitors (MPs) during
muscle regeneration. Access of MPs to growth factors, including mitogen fibroblast growth factor 2
(FGF-2), is mediated by myofiber basement membrane integrity and turnover. We studied a role of
microRNAs in the promotion of MP proliferation by FGF-2 and identified microRNA-29 to be
specifically upregulated by FGF in cultured myoblasts. Functional analysis utilizing both
pharmacological and genetic deletion of this miRNA revealed that miRNA-29a modulation is critical
for myoblast proliferation and maintenance of undifferentiated state. Furthermore, screening of
miRNA targets in miRNA-29a KO myoblasts revealed upregulation of many extracellular matrix
proteins, with the most abundant subgroup representing all major structural components of basement
membrane (Col4a1, Lamc1, Nid2, Hspg2 and Fbn1) as well as its modifiers (Sparc).
Using miR-29a inhibition and overexpression, we confirmed basement membrane related
genes as targets of miR-29a in vitro and have also demonstrated their upregulation in miR-29adepleted MPs during cardiotoxin- (CTX) induced muscle regeneration. Further analysis of CTXinduced regeneration in mice exhibited that miR-29a deletion in MPs leads to prominent decrease in
mass of regenerating muscle and consecutive fibrotic degeneration. This phenotype was due to
decreased proliferation of MPs and a blunted ability to form new myofibers. Dysregulated MP
proliferation and fiber formation was also evident from the more physiologic model of muscle
regeneration employing eccentric exercise.
Overall our results identify miRNAs-29a as a novel checkpoint of basement membrane in the
skeletal muscle stem cell niche that provides mitogenic signals from FGF-2 and preserves progenitors
from premature differentiation.
24
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
2.2 Introduction
It is widely accepted that skeletal muscle mass and function are critical for the maintenance of
overall health. Recently, exercise capacity has been identified as a powerful predictor of survival in
men (196). Furthermore a decline in muscle mass due to ageing has been reversely correlated with
mortality (197). That is why preservation of muscle mass as well as physical fitness (198) could delay
senescence and prolong lifespan. Adult skeletal muscle has a remarkable capacity to regenerate and
strategies that improve muscle regeneration could be the basis for the treatment of muscle-wasting
diseases (199).
Complete destruction of myofibers induced via intramuscular (i.m.) injection of cardiotoxin
(CTX) can be completely repaired within four weeks (see section 1.1.1.2). This regeneration process is
driven by the adult muscle stem cells, also termed as satellite cells or myogenic progenitors (MPs).
These cells are quiescent in an intact muscle, but can be rapidly activated upon injury and display a
high proliferation rate (see section 1.1.1.2-3). MPs are located between the plasma membrane of
muscle fibers and the basement membrane, an environment known as the adult muscle stem cell niche
(see section 1.1.1.4). The basement membrane consists of the structured basal lamina, which is
directly connected to sarcolemma, and reticular lamina, composed of more cluttered fibrils of collagen
I and III as well as microfibrills of fibrillin-1 (Fbn-1) and fibronectin. Basal lamina comprises two
aligned lattice networks of self-assembled laminins and collagen 4, which are crosslinked by nidogens
and heparin sulfate proteoglycans (HSPGs) (section 1.1.1.7). HSPGs are required for growth factor
binding, such as hepatocyte growth factor (HGF) and fibroblast growth factor (FGF). Laminin
mediates contact of basement membrane to MPs and myofibers by binding of cell surface components,
mainly integrins and -dystroglycan. Upon injury, components of the basement membrane become
degraded by proteinases (200), and important members of the basement membrane, such as collagenIV (Col4a1), heparan sulfate proteoglycan-2 (HSPG2, perlecan), laminin gamma-1 (Lamc1) and
nidogen-1 (NID1, entactin-1), are downregulated at the transcriptional level (201,202). This
dismantling of the basement membrane is important for the release of growth factors that drive
expansion of MPs. The proper coordination of breakdown and rebuilding of the basement membrane
in muscle regeneration provides an efficient muscle recovery and prevents fibrotic degeneration.
However, the molecular mechanisms regulating basement membrane in skeletal muscle are poorly
understood.
Growth factors are critical for activation and expansion of dormant MPs during muscle
regeneration. One of the key growth factors released from the extracellular matrix as a result of muscle
injury is FGF2 (see section 1.1.1.6). FGF2 is a potent mitogen that induces proliferation of myoblasts
in vitro and is a key component of growth media for cultivation of primary MP cultures. In vivo, FGF2
25
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
is expressed in the developing (203) and adult skeletal muscle where its expression correlates with
muscle regeneration activity (204). FGF2 is induced in skeletal muscle during exercise, chronic nerve
stimulation as well as muscle injury, and has been associated with MP proliferation and fast to slow
fiber type conversion (205-209). Furthermore, exogenous application of FGF2 improves muscle
regeneration and MP proliferation in vivo (see section 1.1.1.6). In cultured muscle cells as well as in
MPs in vivo, the FGF receptor-1 (Fgfr1) is responsible for the effects of FGF2 on proliferation via
activation of ERK1/2 signaling (210,211).
MicroRNAs (miRNAs), a family of non-coding RNAs, have been reported to have an
important role in muscle development and regeneration (see sections 1.2.2). MiRNAs are short RNA
molecules of 20-24 nucleotides in length that, as a part of complex RNA-protein machinery, basepair
to the 3`UTR of their target mRNAs and direct them to degradation or inhibit their translation (see
section 1.2.1). A single miRNA can have important biological effects by regulating a large number of
target genes that are acting in a similar pathway (212,213). Several mouse models harboring genetic
miRNA deletion have demonstrated that miRNAs have an important role for skeletal muscle
regeneration and function (see section 1.2.2).
Here, we hypothesized that growth factors, and specifically, FGF2 act through miRNAs to
govern MPs expansion. We report that FGF2 induces miR-29a (see section 1.2.4) expression, and
miR-29a and its targets, related to basement membrane, are reciprocally regulated in activated MPs
during muscle regeneration in vivo. Consequently, pharmacologically induced deletion of miR-29a in
MPs prevented efficient muscle regeneration after injury and exercise. Our results indicate that miR29a participates in the dismantling of the basement membrane of the adult muscle stem cell niche
during regeneration of skeletal muscle.
26
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
2.3 Results
2.3.1 MicroRNA-29a is downregulated by serum starvation in primary
myoblasts
To identify miRNAs that are regulated by growth factors, and specifically by FGF2, in proliferating
myoblasts, we used two independent approaches. First, we cultured primary mouse myoblasts under
growth conditions and compared their miRNA profile to cells incubated for 48 h under serum
starvation and without growth factors using microRNA microarrays. Second, we performed miRNA
profiling in primary human myoblasts incubated for 48 h under growth conditions with or without
exogenous FGF2 using next generation RNA sequencing.
miRNA microarrays identified only 3 miRNAs that were differentially regulated with a pvalue of < 0.05 after 48 h of serum starvation in mouse myoblasts (Fig.2.1A). MiR-29a was
downregulated by 58.5%, p-value 0.0393. The signals for the two other miRNAs obtained in the
arrays were very low and therefore we focused on miR29a. Downregulation of miR-29a was
confirmed by northern blotting (Fig.2.1B) and was detectable by qRT-PCR already within the first 16
h of serum starvation (Fig.2.1C). To understand which member of the miR-29 family is the most
relevant for myoblasts, we assessed their expression using deep sequencing of small RNAs in
myoblasts and differentiated myotubes from both mice and humans. Importantly, miR-29a was by far
the most abundant miR-29 member in both cell types and species, while miR-29b and –c were almost
undetectable. The sequencing results also reaffirmed that miR-29a levels were lower in differentiated
myotubes as compared to myoblasts (Fig.2.1D). The primary transcript of the gene encoding for miR29a/b1, but not miR-29b2/c, was decreased by serum starvation, indicating that the regulation of miR29a under these conditions occurs mainly at the transcriptional level (Fig.2.1E).
2.3.2 FGF-2 induces mir-29 expression
Consistent with the results obtained in mouse myoblasts, miR-29a was induced >1.5-fold in
human myoblasts cultured in the presence of FGF2 as determined by next generation RNA sequencing
(Fig.2.2A). Although under these conditions miR-29b was the highest induced miRNA, it was also
100-fold less abundant than miR-29a (in spite of miR-29b derives from the same primary transcript as
miR-29a). The induction of miR-29a by FGF2 was again confirmed by qRT-PCR in mouse and human
myoblasts.
Furthermore, this upregulation was suppressed by PD173074, an inhibitor of FGFR1, thus
indicating specificity for FGF signalling in modulation of miR-29a expression (Fig.2.2B). Importantly,
27
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
regulation of miR-29a by FGF2 was also observed for another distinct muscle derived lineage,
fibroadipogenic progenitors (Fig.2.2B), suggesting that FGF-miR-29a regulatory axis is not restricted
to MPs and might involve progenitor lineages from other tissues. Together, our results demonstrate
that FGF2 regulates miR-29a in various cell types from mice and humans.
2.3.3 miR-29 inhibition/deletion affects myoblast proliferation and
differentiation
To test whether inhibition of miR-29a averts FGF2 function in primary myoblasts, we
performed loss of function assays using antagomirs, specific inhibitors of micrornas (214). Myoblasts
cultured with growth media containing FGF2 display a round morphology, while FGF2 withdrawal
causes them to elongate and differentiate (Fig.2.3A). Inhibition of miR-29a in myoblasts grown in the
presence of FGF2 leads to the similar morphological phenotype as myoblasts grown in the absence of
FGF2. Consistently, antagomir-29a decreased proliferation and increased the differentiation of
myoblasts as measured by muscle differentiation markers and mitochondrial function (Fig.2.3B,C).
Since pharmacological inhibition of miRNAs can yield contrasting results to a genetic deletion
(215,216), we generated transgenic mice containing the floxed miR-29a/b1 gene and a Pax7CE
transgene that allows for Pax7 promoter-dependent expression of a tamoxifen-inducible Cre
recombinase (Fig.2.4A). Tamoxifen incubation reduced miR-29a by > 90% in myoblasts isolated from
miR-29a floxed mice, but not from control mice (Fig.2.4B). Importantly, genetic deletion of miR-29a
confirmed the results obtained with the use of pharmacological inhibitors on myoblast morphology,
proliferation and differentiation (Fig.2.4C,D,E). Together, our results demonstrate that loss of miR29a abrogates the effects of FGF2, leading to suppressed proliferation and induction of differentiation
in primary myoblasts.
2.3.4 Inhibition/deletion of miR-29 derepresses targets related to basement
membrane and downregulates cell cycle genes
To identify the relevant targets of miR-29a underlying its effects on myoblast proliferation and
differentiation, we performed deep sequencing of RNAs obtained from myoblasts with or without
genetic deletion of miR-29a. We compared the group of genes from the RNA sequencing data
upregulated in miR-29 KO to a list of predicted miR-29a targets in mouse, generated by the online
prediction service Targetscan (145). 47 genes common to both lists were upregulated at least 1.5-fold
after the miRNA-29a knockout and were therefore identified as predicted miR-29a targets (Fig.2.5).
GO term clustering revealed that 50% of these genes (26/47) belong to the functional cluster
28
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
“extracellular region”. Importantly, the most abundant candidate targets within this cluster are
associated with basement membrane, and 2 of them (Nid2, Hspg2) represent previously unknown
targets of miR-29a. At the same time, according to GO term clustering, the most numerous group of
downregulated genes belongs to the cluster “cell cycle”. Within this group the most prominent was a
subgroup of genes related to replication: PCNA, Mcm2-7, Cdc6, and Cdc45. Targets attributed to the
basement membrane and several regulated cell cycle genes were confirmed and validated using qRTPCR in myoblasts after genetic (Fig.2.6A) or antagomir-mediated inhibition of miR-29a, and
conversely, after overexpression of miR-29a using miRNA mimics (Fig.2.6B). Together, these results
identify the major components of the basement membrane as the most abundant targets of miR-29a in
myoblasts.
2.3.5 miR-29a is induced during the proliferative phase of muscle
regeneration and regulates basement membrane-related targets in vivo
Next, we investigated the relevance of miR-29a for MP proliferation in vivo using CTXinduced muscle regeneration, where maximal proliferation occurs three days after CTX administration
(217). CTX was injected into the tibialis anterior muscle, MPs were isolated using FACS and directly
processed for RNA isolation without intermittent cell culture. MiR-29a expression in activated MPs
was 2.5 fold higher than in quiescent MPs (Fig.2.7B, left). Conversely, the confirmed targets of miR29a representing components of the basement membrane were downregulated (Fig.2.7B, right). These
results demonstrate that miR-29a and its targets are reciprocally regulated during muscle regeneration.
To investigate if miR-29a is necessary for the formation of new skeletal muscle during
regeneration, we deleted miR-29a specifically in MPs and injected CTX into the tibialis anterior
muscle of control and knockout mice (Fig.2.7A). Tamoxifen-induced Cre induction efficiently reduced
miR-29a levels in activated MPs after CTX injection by >90% (Fig.2.7C, left). Conversely, the
expression of its basement membrane targets significantly increased in the miR-29a knockout MPs
confirming that they are miR-29a targets in vivo (Fig.2.7C, right).
2.3.6 MP-specific miR-29a deletion results in decreased myofiber
formation, reduced muscle mass, and fibrotic degeneration in regenerating
muscle
Then, we assessed an impact of miR-29a deletion in MPs in vivo. We characterized new
myofiber formation by eMHC and EdU immunostaining on cryosections of CTX-injected TA
muscles, TA muscle mass recovery after CTX injection, as well as assessed fibrosis score. Consistent
29
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
with the role of miR-29a in myoblast proliferation, deletion of miR-29a in MPs decreased the total
number of newly formed embryonic myosin heavy chain (eMHC) positive fibers (Fig.2.8A and B,
left) as well as the proliferation rate of MPs merging to these newly formed fibers as measured by the
percent of myofibers double positive for both (eMHC) and EdU 4 days after CTX injection (Fig.2.8A
and B, right). Furthermore, skeletal muscle mass was reduced in miR-29a knockout animals 9 days
after CTX. This reduction persisted for at least 30 days after CTX injection and was not observed in
control mice that contained Pax7CE transgene and WT miR-29a locus (Fig. 2.9). At day 30, fibrotic
areas between fibers became frequently apparent in muscle tissue from the miR-29a knockout mice
(Fig.2.10). We, therefore, conclude that miR-29a is critical for the proliferation rate of MPs and that
loss of miR-29a leads to decreased myofiber regeneration / formation and fibrosis development
following muscle injury.
2.3.7 Exercise-induced muscle regeneration is blunted in muscles with miR29a-depleted MPs
To determine whether miR-29a is involved in a more physiologic stimulation of muscle
regeneration as well as to extend our studies to a different muscle group, we subjected the floxed miR29a and Pax7CE+ mice to a single bout of eccentric exercise (downhill running)(Fig. 2.11A, left).
Tamoxifen-induced MP-specific deletion of miR-29a did not affect the aerobic capacity in mice one
week after the exercise bout (Fig. 2.11A, right). The unchanged aerobic capacity was expected, since
downhill running induced only small areas of muscle regeneration (Fig. 2.11B, left). Importantly,
myofiber formation was significantly reduced in mice depleted for miR-29a (Fig. 2.11B). EdUpositive fibers were observed in muscles from 4 out of 14 control mice and from none out of 13 miR29a KO mice. Together, we conclude that miR-29a is necessary for muscle regeneration not only after
CTX-induced injury, but also in response to eccentric exercise.
30
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
2.4 Discussion and outlook
2.4.1 miRNA-29a and basement membrane remodeling during early events
of muscle regeneration
The results of this study reveal that miR-29a expression in MPs is necessary for muscle
regeneration. We propose that during proliferative phase of muscle regeneration FGF induces miR29a, which leads to inhibition of gene expression for key basement membrane
components and, thereby, dismantling of surrounding basement membrane structure. This, in turn, is
followed by further release of growth factors from the depot of degrading basement membrane and
increase in satellite cell niche elasticity, thus providing conditions for MP proliferation (Fig. 2.12).
The dismantling of the basement membrane is critical for MP expansion and maintenance of
skeletal muscle mass during muscle regeneration, but its regulation is incompletely understood. We
demonstrate that miR-29a targets the major structural components of the basement membrane,
including Col4a, lamc1, Nid2 and Hspg2, and that upregulation of miR-29a and downregulation of its
targets in MPs within the adult muscle stem cell niche precedes muscle regeneration in vivo. This
novel layer of regulation of the basement membrane by a microRNA provides a mechanism for the
previously described changes in gene expression in MPs during muscle regeneration. Quiescent MPs
express higher levels of the components of the basement membrane compared to activated MPs,
including the miR-29a targets Col4a1, Lamc1 and Hspg2 that we identified in our study (201,202).
From the other hand, importance of structural integrity of myofiber’s basement membrane for satellite
cell function becomes evident from studies of Collagen 6 KO. These KO mice, although having
elevated numbers of satellite cells before injury, demonstrated impaired muscle regeneration and
reduced ability of stem cells to self-renew (218). Following muscle injury, interference with normal
dismantling of basement membrane and its glycoprotein components in satellite cell niche could
decrease MP and myoblast proliferation by changing the substrate elasticity of the niche (219,220) or
by decreasing the access of cellular receptors to growth factors (221-223). Genetic deletion of miR29a resulted in increased expression of ECM proteins, decreased proliferation of MPs in vivo and in
vitro, and promoted differentiation in cultured myoblasts. Inhibited proliferation and premature
differentiation in miR-29a KO MPs, in turn, led to failure in myofiber regeneration, consecutive
fibrotic replacement and reduction in muscle mass. Importantly, this phenotype was evident even 30
days after injury, while normal muscle regenerates completely within 3-4 weeks, indicating
importance of miR-29 for long-term muscle recovery.
Elevation of basement membrane components in miR-29 KO MPs might be implicated in
premature differentiation. It has been reported that basement membrane members, including laminin,
31
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
nidogen and collagen 4, are elevated in premuscle masses during embryonic development (222). In
another study, myoblasts cultured on plastic coated with laminin, nidogen, HSPG2 and collagen 4
demonstrated higher differentiation (224). Given the large number of predicted and confirmed targets
of miR-29a, we cannot exclude that the role of miR-29a for MP proliferation involves also targets
other than the basement membrane. However, since we identified these targets based on their high
abundance in proliferating myoblasts and considering the consistent regulation of miR-29a and this
group of targets in activated MPs in vivo, there is strong evidence for their involvement in miR-29a
function.
2.4.2 MicroRNA-29 and the regulation of muscle regeneration after exercise
Transgenic mice overexpressing miR-499 have increased numbers of myofibers of slowtwitch fiber type and a longer mean running time on a treadmill (225), but the impact of genetically
deleting a miRNA on the response of skeletal muscle to exercise has not been demonstrated yet.
Downhill running has been used as a physiological model for the stimulation of muscle regeneration
and it is well established that exercise can cause muscle damage especially when it involves eccentric
components in both mice (226) and humans (227). The activation of MPs is known to be involved in
muscle hypertrophy after repeated eccentric exercise interventions (228,229). The mechanisms that
regulate MP proliferation during eccentric exercise could involve ECM modulation. For example, it
has been reported that following a single bout of eccentric exercise collagen synthesis and degradation
were elevated (230), and transgenic induction of skeletal muscle specific α7β1-integrin accelerated
myogenesis (226). Our results show that muscle regeneration after exercise depends on miR-29a and
intact MP proliferation, similarly to CTX-induced muscle injury. The deletion of miR-29a in adult
skeletal muscle stem cells blunted the formation of new myofibers after a single bout of exercise.
Although we did not detect changes in aerobic capacity after a single bout of downhill running, this
was to be expected, given that changes might require several repeated running bouts or running tests
after a longer period of time (229).
2.4.3 miRNA-29a and myogenesis
We did not follow up on the regulation of miR-29b and miR-29c in myoblasts based on their
very low abundance and since serum starvation regulated only the primary transcript of miR-29a/b,
but not miR-29b/c. Contrary to our results, miR-29a-c levels were reported to be induced in the C2C12
cell line during serum starvation and differentiation, and miR-29b expression was reduced in human
rhabdomyosarcoma primary cells (180). In this study authors concentrated on mir-29b/c and
32
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
transcription factor YY1, which binding sites were found in mir-29b/c, but not mir-29a/b gene
promoter, and later on, a role for mir-29b/c in C2C12 myoblast differentiation was also reported in the
following publications (180,231-233). Importantly, in most of these studies miR-29 regulation was
concluded based only on qRT-PCR data using single normalizer gene, U6. Furthermore, in contrast to
our study, none of the works utilized miR-29 inhibition / KO approach in order to provide functional
data for miR-29 role in myoblast differentiation (Wang et. al, 2008 (180) showed that inhibition of
miR-29c can decrease differentiation in C2C12 myotubes, but no role in differentiation was reported
for proliferating myoblasts) (Table 2.1).
Another explanation for the discrepancy is that all miR-29 studies mentioned above used
cancer cell lines (mostly C2C12 cell line, see table 2.1), which are cultured without FGF and are wellknown to have dysregulated signaling pathways implicated in metabolism, proliferation and
differentiation. This is furthermore plausible, since in cancers, the miR-29 family plays a dual role
depending on the type of cancer it is expressed (see also section 1.2.4). In hematopoietic or cervical
cells, for example, miR-29 acts as a tumor suppressor by targeting apoptosis, cell cycle and
proliferation pathways (195,234). On the other hand, miR-29a stimulates proliferation in breast cancer
or mouse B cells (190,191). Therefore, we suspect that the miR-29 family might be differentially
regulated in cancerous cells and cancer cell lines compared to miR-29a in MPs in vitro and in vivo. In
addition, differences in miRNA expression based on qRT-PCR could also be caused by the
normalization method applied. Indeed, we noticed that in contrast to sno234 and let-7a (which we
proved to be stable during myoblast differentiation), the RNA transcripts of U6 are two fold
downregulated in differentiated vs proliferating primary myoblasts (Fig 2.13). In our study, we have
several independent approaches to confirm downregulation of miR-29a in MP differentiation,
including microarrays, northern blotting as well as next generation RNA sequencing and qRT-PCR, in
which normalization to sno234 was also confirmed by another normalizer – let7a (Fig 2.13).
Consistently, a previous microarray analysis on C2C12 differentiation failed to detect induction of
miR-29a (153).
In contrast to previous works, we have also used two independent approaches – genetic
deletion and pharmacologic inhibition, for functional analysis of MP proliferation and differentiation
(see Table 2.1). We investigated miR-29a function at early stages of muscle differentiation, based on
the high expression levels of miR-29a in proliferating myoblasts and MPs. Deletion of miR-29a might
reveal different effects during later stages of muscle differentiation as miR-29 has been assigned a promyogenic function in terminal differentiation of C2C12 myotubes by targeting YY1 (180), Rybp, and
Akt3 (232,233). However, none of these genes were regulated in our miR-29a knockout myoblasts
(data not shown). Attempts to use overexpression of miR-29a in order to increase MP proliferation
have to consider the level of overexpression and the regulation of false positive targets. Indeed,
adenovirus-mediated overexpression of miR-29a decreased cell proliferation in primary murine
33
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
myoblasts by targeting IGF1, phosphatidylinositol 3-kinase regulatory subunit alpha p85 (PIK3R1)
and B-Myb (235), which were also not regulated in our miR-29a knockout myoblasts (data not
shown).
2.4.4 miRNA-29a and FGF signalling
We identified miR-29a as a part of the screen for miRNAs regulated by FGF2 signaling in
myoblasts. Inhibition of miR-29a attenuated the stimulation of myoblast proliferation by FGF2,
indicating that miR-29a could be involved in mediating downstream effects of this growth factor.
Since the regulation of miR-29a by FGF2 was also observed in fibroadipogenic progenitors, miR-29a
might also be implicated in FGF2 signaling in other tissues. Correction of miR-29a expression might
offer therapeutic opportunities for pathologic conditions characterized with overly activated FGF2
signaling in the adult muscle stem cell niche, particularly, during ageing. FGF2 is the highest induced
FGF ligand in aged muscle fibers and its protein content was increased specifically in the adult muscle
stem cell niche under the basal lamina of muscle fibers (211). Aberrant stimulation of the FGF
pathway leads to increased activation and depletion of quiescent satellite cells, and provides a
mechanism for the decreased number of MPs in aged skeletal muscle (211). Correction of miR-29a
under these conditions would be expected to reverse some of the negative effects of increased FGF2
signaling and preserve quiescent MPs. Intriguingly, a microarray screen in skeletal muscle from 4
months old rat versus 28 months old rats revealed a 13-fold induction of miR-29a (235). From the
other hand, our data propose that complete miR-29a inhibition might lead to decrease in proliferation
of activated satellite cells, their premature differentiation and, consequently, defects in regeneration.
2.4.5 Basement membrane components and human myopathies
An improved understanding of the regulation of the adult muscle stem cell niche provides
opportunities for the development of therapeutic strategies relevant to the disease states where the
basement membrane is involved. Several human diseases are linked to muscle weakness and mutations
in the basement membrane. Around half of hereditary muscular dystrophies are caused by mutations in
laminin-2. Collagen 4 mutations have a broader spectrum of symptoms, including myopathy, and
abnormal Collagen 6 leads to Ulrich myopathy (93). Mutations in Hspg2 have been reported to cause
Schwartz–Jampel syndrome associated with muscle weakness and stiffness, growth retardation as well
as abnormalities in bone and cartilage development (94). Mutations in the miR-29a target fibrillin-1
(Fbn1) can result in muscle weakness and decreased muscle mass, presumably by poor anchoring of
the myofiber basal lamina to surrounding ECM within endomysium (236). We identified a novel role
34
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
of miR-29a as a regulator of the basement membrane during muscle regeneration. Normalization of
the miRNA levels by targeting miR-29a might improve disease states where FGF2 signaling and miR29a are induced, and the expression of basement membrane components is defective.
35
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
2.5 Figures
A
B
C
miR-29a
relative levels
1.2
1.0
**
0.8
***
**
***
0.6
0.4
0.2
0.0
0
8
16
24
32
40
Serum starvation
(h)
E
RNAseq (mouse)
miR-29a
miR-29b
miR-29c
1.0
0.8
0.6
0.4
0.2
0.0
2.0
% of hsa-miRNAs
miR-29a
miR-29b
miR-29c
1.6
1.2
0.8
0.4
T
Pri-miR-29a / b1
Pri-miR-29b2 / c
1.5
1.0
0.5
0.0
***
CTR
serum starvation
M
B
T
M
M
B
0.0
M
% of mmu-miRNAs
1.2
RNAseq (human)
Gene expression (rel U)
D
Fig 2.1. Serum starvation decreases miR-29a expression in primary myoblasts.
Primary mouse myoblasts were grown for 48 h under growth conditions (ctrl) or serum starvation
and RNA was isolated for gene expression analysis. A) miRNA expression was analyzed using miRNA
microarrays, n=2. B) Confirmation of miR-29a decrease and upregulation of differentiation marker
miR-1 detected by northern blotting. C) Time-dependent downregulation of miR-29a by serum
starvation as shown by qRT-PCR. n=4. D) results of RNA sequencing for members of miR-29 family in
mouse and human myoblasts (MB) and myotubes (after 4 days of serum starvation, MT), n=2. E) qRTPCR for the two primary transcripts of the miR-29 family as normalized to 18s RNA. n=10. *, p<0.05,
**, p<0.01, *** compared to control.
36
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
A
human myoblasts
Fold regulation
(FGF / no FGF)
3,0
miR-29b
2,5
miR-29a
2,0
1,5
1,0
0,5
0,0
100
1 000
10 000
100 000
1 000 000
miRNA reads
B
0.5
1.0
1.5
**
0.5
***
1.0
0.5
0.0
FG
FG F
F
in
h
CT
R
0.0
FG
FG F
F
in
h
0.0
CT
R
1.5
miR expression (rel U)
1.0
fibroadipogenic
progenitors
FG
FG F
F
in
h
**
miR expression (rel U)
miR expression (rel U)
1.5
human
Myoblasts
CT
R
mouse
Myoblasts
Figure 2.2. FGF2 activates miR-29a expression in primary myoblasts.
A) RNA sequencing data for miRNA expression in human myoblasts incubated for 48 h with or
without FGF2, n=2. B) Upregulation of miR-29a by FGF2 incubation for 48 h in primary mouse
myoblasts, human myoblasts and fibroadipogenic progenitors as measured by qRT-PCR. Fgf1r was
inhibited by incubations in the presence of PD173074. n=4. *, p<0.05, **, p<0.01, ***, p<0.001
compared to control.
37
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
B
A
Proliferation
EDU+ cells (%)
100
Antagomir-ctr
60
40
20
C
an
t-2
9a
0
TR
Antagomir-29a
**
80
Mitochondria
function
1.4
Fold change
1.2
FGF
CTR
TMRE
MT Green
1.0
0.8
0.6
0.4
0.0
Differentiation
markers
Gene expression
MCK luc
2.0
*
**
1.5
Fold change
Fold change
2.0
myogenin
*
0.2
C
MyHC
**
1.0
0.5
1.5
*
1.0
0.5
α-tubulin
0.0
TnnI
Myoglobin
0.0
Ant-29a
Figure 2.3. Loss of miR-29a decreases myoblast proliferation.
A) Morphological changes of mouse myoblasts after FGF2 withdrawal for 48 h (w/o FGF) or 48 h after
transfection with antagomir-29a (compared to antagomir-ctrl) shown by light microscopy. B) Effect
of antagomir-29a compared to control antagomirs on proliferation rate in primary myoblasts as
measured by EdU incorporation as well as on mitochondria mass (MTG) and membrane potential
(TMRE), measured using flow cytometry. n=3-5. C) Differentiation markers were measured after
antagomir-29a treatment for 48h: protein levels of MyHC and myogenin were assessed by westernblot, gene expression levels of TnnI and Mb were measured by qRT-PCR, induction of MCK promoter
activity was measured in luciferase assay. *, p<0.05, **, p<0.01.
38
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
Wt /CreERT2
Pax7
Wt /CreERT2
Pax7
D
fl/fl
/ miR-29a/b1
Tmx / Eth
1
= Ctrl
Analysis
2
3
**
60
40
20
0
Ctrl miR-29a
KO
E
(Tamoxifen / vehicle ratio)
1.2
Mitochondria function markers
1.0
(Tamoxifen / vehicle ratio)
*
0.8
0.6
Ctrl
miR-29a
KO
**
2.0
0.4
Fold change
Fold change
80
4 days
miR-29
B
(Tamoxifen / vehicle
difference)
100
wt/wt
/ miR-29a/b1
0
Proliferation
= miR-29a KO
Proliferation rate, %
A
**
0.2
0.0
Ctrl miR-29a
KO
C
1.5
1.0
0.5
M
TM
R
E
TG
0.0
Differentiation markers
(Tamoxifen / vehicle ratio)
Ctrl
miR-29a
Pax7 Wt/Cre
KO/ miR-29ab wt/wt
2.5
**
miR-29a KO
Fold change
2.0
Ctrl
Pax7 Wt/Cre / miR-29ab fl/fl
*
1.5
1.0
0.5
Tn
nI
M
B
0.0
Figure 2.4. Loss of miR-29a decreases myoblast proliferation and induces differentiation
A) Strategy for genetic deletion of miR-29a in primary myoblasts isolated from 2 different transgenic
mouse lines, miR-29a KO and control (ctrl). B) miR-29a expression levels 4 days after the start of Tmx
incubations as measured by qRT-PCR. Ctrl and miR-29a KO refer to myoblasts isolated from mice with
genotype as depicted in A). Fold changes are ratios of values obtained from myoblasts treated with
Tmx vs ethanol. C) Morphological changes 4 days after the start of Tmx shown by light microscopy.
D) Proliferation rate and E) induction of differentiation, measured as in B) 4 days after the start of
Tmx. n=3-6. *, p<0.05, **, p<0.01, ***, p<0.001.
39
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
A
CTR
miRNA-29a targets
1 800
Tamoxifen
1 600
1 400
1 200
FPKM
1 000
800
600
400
200
Slc16a2
Mxd1
Ypel2
Sh3rf3
Lif
Gxylt2
Col11a1
Col4a5
Loxl4
Adamts5
Dnmt3a
Per1
Purg
Cilp2
C1qtnf6
Tet3
Zfp36l1
Ubtd2
Fam167a
Tgfb2
Hbegf
Mmp2
Kdm5b
Col2a1
Col1a1
Lox
Ifi30
Adam19
Ing4
Cd276
Hexa
Klhdc3
Fbn1
Pxdn
Ppic
Lamc1
Cx3cl1
Col5a1
Erp29
Nid2
Fstl1
Col4a2
Col4a1
Col3a1
Hspg2
Serpinh1
Sparc
0
-200
Etracellular
matrix
+
Basement
membrane
+ +++
+ +
++ +
+
B
Cell cycle genes
500
+ + ++
+ +
+ + +
+ +++ + +
+
+
+
+
+
+
+
++
+
CTR
Tamoxifen
450
400
FPKM
350
300
250
200
150
100
50
0
Figure 2.5. mRNA screening of miR-29a targets and cell cycle genes in miR-29-KO myoblasts
RNA sequencing data for cultured myoblasts isolated from Pax7CE x miR-29a flox/flox mice and
harvested 4 days after incubation with Tmx. Results are average values within Tmx and ethanol
control (ctrl) groups . n=3. A) regulation of miR-29a targets and their allocation to ECM and basement
membrane groups B) regulation of cell cycle genes.
40
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
A
Basement membrane
Cell cycle
*
*
**
**
1.5
2.0
**
**
1.5
Fold change
1.0
0.5
1.0
1.0
0.8
0.6
***
***
0.2
0.5
0.0
mimic-29a
Ant-29a
**
1.5
**
**
**
0.5
-7
0.6
0.4
0.2
0.5
0.0
Fb
n1
Fb
Lan1
m
La c1
m
c1
Ni
d2
N
id
C 2
C ol4
ol a
4a 1
1
HS
H P
SP G
G 2
2
SpSp
ar arc
c
0.0
-7
*
cm
***
*
**
M
***
1.0
***
Cd
c6
1.0
0.8
**
Cd
c45
p<0.1
1.0
**
p<0.1
Fold change
Fold change
Fold change
1.5
**
**
2.0
Ant-29a
1.2
PC
NA
2.0
Sp M
ar c
c m
HS
PG c d
2 c-6
c
dc
Co
-4
l4
5
a1
PC
NA
Ni
d2
mimic-29a
Ant-29a
c1
0.0
Fb
n1
Sp
ar
c
2
HS
PG
Co
l4
a1
Ni
d2
c1
La
m
Fb
n1
0.0
B
0.0
***
***
0.4
La
m
Fold change
2.0
1.2
Fold change
2.5
ctrl
mimic-29a
miR-29a
Ant-29a KO
Fig 2.6 Gene expression of miR-29a targets and cell cycle genes in miR-29-KO and antagomir-29a /
miR-29a mimic treated myoblasts.
A) Regulation of components of the basement membrane and cell cycle genes as measured by qRTPCR in control myoblasts (n=2) and myoblasts from Pax7CE x miR-29a flox/flox mice (n=3). The fold
change is the effect of tmx compared to incubations with ethanol. Cells were harvested on day 4
after tmx treatment. B) Regulation of components of the basement membrane and cell cycle genes
as measured by qRT-PCR 72 h after transfection of myoblasts with either mimic-29a (n=5) or
antagomir-29a (n=9), respectively. *, p<0.05, **, p<0.01, ***, p<0.001.
41
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
A
CTX i.m.
Day: 0 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9
Tmx / veh i.p injections
CTX i.m.
Day: 0 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10...30
Tmx / veh i.p injections
B
Day3-ctrl
Day3-CTX
1.4
3
***
1.2
Fold change
2
1
1.0
0.8
0.6
0.4
*
0.2
**
***
*
***
***
Sp
ar
c
H
C
SP
G
2
ol
4a
1
id
2
N
Fb
n1
TX
C
TR
C
C
c1
0.0
0
La
m
Gene expression (rel U)
miR-29a
Pax7 wt / miR-29 fl/fl
miR-29a
Pax7 CreER / miR-29 fl/fl
Gene expression (rel U)
0.4
0.2
***
1
-
42
Sp
ar
c
2
SP
G
H
ol
4a
1
C
N
f/f
id
2
0
CreER/wt CreER/wt
+
**
***
c1
f/f
**
2
La
m
miR-29ab
f/f
Pax7
wt/wt
Tamoxifen
+
***
*** ***
3
1
0.0
4
FB
N
miR expression (rel U)
0.6
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
Fig. 2.7. Regulation of miR-29a and its targets in activated MPs during muscle regeneration after
CTX-induced injury.
A) Strategy for the induction of Cre recombinase in Pax7+ MPs using Tmx injections during CTXinduced muscle regeneration and time indication for sample harvesting. B) Activated MPs were
isolated from the TA muscle 3 days after cardiotoxin injection using FACS and directly used for RNA
isolation. miR-29a levels (left, n=4) and expression of components of the basement membrane (right,
n=4) were analysed using qRT-PCR and compared to MPs isolated from uninjected muscle. C)
Myogenic progenitors were isolated like in B) from mice harbouring inducible Cre-gene under control
of Pax7 with either floxed or Wt miR-29a/b1 gene. miR-29a levels and its basement membrane
related targets were quantified using qRT-PCR and compared to genetic and vehicle controls. n=4.
*,p<0.05; **, p<0.01; ***, p<0.001.
43
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
A wt; miR29fl/fl
B
Pax7CE/+; miR29fl/fl
Day 4
Day 4
100
60
80
40
60
**
**
40
20
20
0
Pa
x7
C
E/
w
t;
t;
m
w
m
iR
R
29
fl/
fl
29
fl/
fl
fl/
fl
29
m
R
Pa
x7
C
E/
w
t;
w
t;
m
iR
29
fl/
fl
0
Fig. 2.8. Impairment of myofiber formation and MP proliferation in regenerating skeletal muscle
with miR-29a deficient MPs.
A) eMHC positive (red) and EdU positive (green) myofibers on frozen sections of TA muscles from
tmx-treated Pax7 Cre positive and negative miR-29a floxed mice 4 days after CTX injection. B) eMHC+
fibers (left) as well as EdU+/eMHC+ fibers (right) were quantified n=4-6. **, p<0.01. Preparation of
tissue sections, immunohistochemical staining and analysis were done by Edlira Luca, PhD
44
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
Skeletal muscle mass
Day 9
Day 9
1.0
Day 30
1.4
1.0
1.2
**
0.2
0.6
0.4
***
1.0
CTX inj/ uninj
0.4
Muscle mass ratio
0.6
CTX inj/ uninj
0.8
Muscle mass ratio
CTX inj/ uninj
Muscle mass ratio
0.8
0.8
0.6
0.4
0.2
0.2
29
fl/
fl
29
fl/
fl
R
iR
Pa
x7
C
E/
w
t;
m
t;
m
Pa
x7
C
E/
w
t
t
w
fl/
fl
29
C
E/
w
t;
m
R
Pa
x7
Pa
x7
C
E/
w
t;
m
R
29
fl/
fl
29
fl/
fl
iR
t;
m
w
0.0
0.0
w
0.0
Fig. 2.9. MiR-29a deletion in MPs abrogates muscle mass recovery in CTX-induced muscle
regeneration.
At D9 or D30 after CTX-injection TA muscles were dissected from miR-29a floxed mice with or
without Pax7 Cre transgene having vehicle or tmx treatment. Skeletal muscle mass recovery is shown
as ratio of the CTX-injected TA-muscle mass compared to the uninjected one of the same mouse.
Grey and black bars indicate mice that received Tmx, white bars indicate mice that received corn oil
(vehicle). n=5-8 (D9), n=8-11 (D30). **, p<0.01; ***, p<0.001.
45
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
A
H&E stain
Pax7CE/wt; miR29fl/fl
Pax7CE/wt; miR29fl/fl
B
D30
D30
3
Fibrosis Score
3
Fibrosis Score
Trichrome stain
2
1
0
2
1
0
wt; miR29fl/fl
Pax7CE; miR29fl/fl
wt; miR29fl/fl
Pax7CE; miR29fl/fl
Fig. 2.10. miR-29a deletion promotes fibrosis during CTX-induced muscle regeneration.
A) H&E staining (left )and Trichrome staining (right) for frozen sections from Tmx treated Pax7CE/wt;
miR29fl/fl mice at D30 after CTX injection. B) semiquantitative estimate of fibrosis score ranging from
0-3 in H&E staining (left ) and Trichrome staining (right) is provided for each individual mouse.
Cryosectioning, H&E as well as trichrome stainings and score analysis was done by Elisabeth Rushing,
MD.
46
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
A
Single downhill
exercise bout:
-20°, 90 min
Aerobic capacity test
Aerobic capacity test
IF: EdU / eMHC
40
30
Day:0 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7
20
10
EdU injection
0
Pa
x7
C
E/
w
t;
m
R2
9
fl/
f
l
w
t;
m
iR
29
fl/
fl
Tmx / veh i.p injections
B
Fiber formation
6
4
*
2
fl/
fl
29
m
R
t;
E/
w
Pa
x7
C
w
t;
m
iR
29
fl/
fl
0
Fig. 2.11. Tamoxifen-induced deletion of miR-29a in Pax7+ MPs in vivo decreases muscle fiber
formation after a single bout of eccentric exercise.
A) Strategy for the induction of Cre recombinase from Pax7 Cre transgene in MPs using Tmx
injections during exercise induced muscle regeneration and time indication for sample harvesting /
aerobic test performance (left) and time to exhaustion (TTE) in aerobic capacity test 7 days after the
downhill running exercise. n=15 for controls and n=14 for miR-29a KO mice B) New myofiber
formation 7 days after the downhill running exercise as determined by eMHC and EdU
immunostaining on frozen sections from gastrocnemius muscle, n=14 for controls and n=13 for miR29a KO mice. *, p<0.05. Preparation of tissue sections, immunohistochemical staining and analysis
were done by Edlira Luca, PhD. Downhill exercise experiment and aerobic capacity test was done
with the help of Troy Merry, PhD
47
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
EXERCISE / MUSCLE INJURY
FGF-2
miR-29a
HSPG2
NID2
SPARC
COL4a1
LAMC1
FBN1
Dismantling of the
basement membrane
Release of
growth factors
Modulation of
niche elasticity
Adult muscle stem
cell proliferation
Fig 2.12 Model for the regulation of adult muscle stem cell proliferation by miR-29a
48
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
U6 levels normalized
for sno234
2.0
2.0
1.5
1.5
0.5
20% FBS
U6 levels
*
0.5
20% FBS
Let-7a levels
Let7a levels
1.0
1.0
0.5
0.5
0.0
0.0
20% FBS
0.5% FBS
20% FBS
0.5% FBS
0.5% FBS
15
20% FBS
0.5% FBS
1.5
U6 levels
1.5
1.0
0.0
0.5% FBS
U6 levels normalized
for let-7a
sno234 levels
2.0
**
1.0
0.0
B
U6 levels (AU)
U6 levels (AU)
A
Sno234 levels
20% FBS
0.5% FBS
20% FBS
0.5% FBS
10
5
0
20% FBS
0.5% FBS
Fig. 2.13. Regulation of common RNAs species used for normalization in qRT-PCR in mouse
myoblasts during serum starvation.
Primary myoblasts were cultured under normal growth conditions (20% FBS+FGF-2) and serum
starvation (0.5% FBS), and RNA was isolated after 48h. A) Levels of U6 normalized for sno234 (left)
and let-7a (right) as determined by RT-qPCR. n=3. B) Levels of U6, let-7a and sno234 as determined by
qRT-PCR without further normalization.
49
FGF MODULATES MICRORNA-29A TO CONTROL SKELETAL MUSCLE REGENERATION
DURING INJURY AND EXERCISE
miR-29
overexpression
approach
Wang H et al Cancer Cell
2008
(180)
Zhou L et al PLoS one 2012
(237)
Wang XH et al J Am Soc
Neph 2011
(238)
Winbanks CE et al JBC
2011
(231)
Functional analysis
qPCR normalization
to determine miR29 regulation
Cell type used for
functional
experiments
Accelerated
differentiation
U6
C2C12
miR-29a (was only
measured in C2C12
differentiation)
Not done
Accelerated
differentiation
U6
C2C12
Not specified
Not done
Accelerated
differentiation
U6
C2C12
miR-29a,b and c
Proliferating
myoblasts
myotubes
No change in
differentiation status
miR-29b and c
miR-29a, b, c (regulation
only)
Not done
Accelerated
differentiation
Sno135
C2C12
Not done
Accelerated
differentiation
U6
primary myoblasts,
C2C12
Not done
Accelerated
differentiation
U6
Wang H et al Cancer Cell
2008
(180)
No change
Decreased
differentiation
(antagomir-29C only
was used)
C2C12
Wang L et al Mol ther, 2012
(239)
Not done
Not done
(only used for
verification of targets)
C2C12
Zhou L et al PLoS one 2012
(237)
Not done
Not done
(only used for
verification of targets)
C2C12
Wang L et al Mol ther, 2012
(239)
Not done
Not done
(only used for
verification of targets)
C2C12
Wang L et al Mol ther, 2012
(239)
Wei W et al Cell Death and
Disease 2013
(233)
miR-29 members
studied
C2C12
for functional experiments,
combination of miR29a,b,c, mimics was used
miR-29c was used for
functional experiments
Antagomirs used are not
specified
Mainly miR-29b and c
were studied
miR-29 inhibition
approach
Table 2.1. Summary of literature on miR-29 studies, related to skeletal muscle and myogenesis.
50
CHAPTER III
CHAPTER III
GROWTH HORMONE REPLACEMENT THERAPY
REGULATES MICRORNA-29A AND TARGETS
INVOLVED IN INSULIN RESISTANCE
This chapter contains unpublished data as well as fragments and figures adopted or reproduced
from the following manuscript, currently submitted for publication:
GH replacement therapy regulates microRNA-29a and targets involved in insulin resistance
Artur Galimov1,8, Angelika Hartung1, Roman Trepp2, Alexander Ushmaev3, Martin Flück4, Hans
Hoppeler5, Axel Linke6, Matthias Blüher7, Emanuel Christ2, and Jan Krützfeldt1,8,9.
1
Division of Endocrinology, Diabetes, and Clinical Nutrition, University Hospital Zurich, Switzerland,
2
Division of Endocrinology, Diabetes and Clinical Nutrition, University Hospital Bern, Inselspital,
Switzerland, 3Division of Trauma Surgery, University Hospital Zurich, Switzerland, 4Department of
Orthopedics, University Hospital Balgrist, Zurich, Switzerland, 5Department of Systematic Anatomy,
University of Bern, Switzerland, 6Heart Center Leipzig, University of Leipzig, Germany, 7Department of
Medicine, University of Leipzig, Germany, 8Competence Center for Systems Physiology and Metabolic
Diseases, ETH Zurich and University of Zurich, Switzerland, 9Zurich Center for Integrative Human
Physiology, University of Zurich, Switzerland
Corresponding author and person to whom reprint requests should be addressed to:
Prof. Jan Krützfeldt, MD, University Hospital Zurich, Division of Endocrinology, Diabetes, and Clinical
Nutrition, Rämistrasse 100, 8091 Zurich, Switzerland. Phone: +41 (0)44 255 36 27, Fax: +41 (0)44 255
9741, E-mail: [email protected]
51
GROWTH HORMONE REPLACEMENT THERAPY REGULATES MICRORNA-29A AND TARGETS
INVOLVED IN INSULIN RESISTANCE
3.1 Outline of the work
Growth hormone (GH) replacement therapy (GHRT) improves many clinical symptoms in
GH-deficient patients, including elevated adiposity, muscle weakness and osteoporosis. However,
GHRT is associated with a risk of metabolic syndrome which might involve development of skeletal
muscle insulin resistance. GH-deficient mice (Ghrhr
lit/lit
) developed glucose intolerance and
hyperinsulinemia as a result of GH treatment. mRNA deep sequencing in skeletal muscles of these
mice before and after GH treatment revealed upregulation of the extracellular matrix (ECM) and
downregulation of miRNA-29a. Based on the results of this screening we selected several upregulated
genes, which were predicted as miR-29a targets and known to be either negative regulators of insulin
signaling or profibrotic/proinflammatory components of the ECM. Using gain- and loss- of function
studies, five of these genes were confirmed as endogenous targets of miR-29a in human myotubes
(PTEN, COL3A1, FSTL1, SERPINH1, SPARC). These targets were also upregulated in muscle
biopsies from a cohort of GH-deficient patients as a result of GHRT.
Then, with the use of in vitro incubations of human myotubes, we revealed that IGF1, but not
GH is responsible for downregulation of miR-29a. These results were supported by analysis of
patients before and after GHRT received for four months. Patients whose insulin sensitivity (IS)
decreased after GHRT, in contrast to a group with improved IS, had significantly elevated IGF1 and
decreased miRNA-29a levels in muscle biopsies. Furthermore, the decrease in miRNA-29a levels
from muscle biopsies correlated with aggravation of insulin resistance in patients received GHRT.
Overall, our results provide a link between induction of IGF1, miRNA-29a downregulation
and respective upregulation of insulin resistance related miRNA targets in skeletal muscle during
GHRT.
52
GROWTH HORMONE REPLACEMENT THERAPY REGULATES MICRORNA-29A AND TARGETS
INVOLVED IN INSULIN RESISTANCE
3.2 Introduction
GH is an anabolic hormone that promotes skeletal muscle mass and bone density during
periods of energy surplus and prevents their catabolism during starvation. In adults, GH preserves
muscle and bone during fasting by switching metabolism from carbohydrate utilization to
consumption of lipids via direct stimulation of lipolysis in adipose tissue and acceleration of
gluconeogenesis in the liver (see section 1.1.2.3). GH deficiency is a rare endocrinological disorder
(see section 1.1.2.4 for details) that is characterized with increased adiposity, decreased skeletal
muscle and cardiac capacity, as well as reduced bone density. In case of severe GH deficiency with the
presense of osteopenia and high risk of cardiovascular disease, a course of GH replacement therapy
(GHRT) might be prescribed (see section 1.1.2.4). However, together with its beneficial effects,
GHRT has been reported to have an avdverse effect on glucose metabolism, mainly due to increased
lipolysis (although direct interference of GH with insulin signaling might be involved). GHRTinduced insulin resistance is more prominent in short-term treatments and gradually ameliorates in
case of longer treatment periods (140,141). Besides, temporal changes in insulin sensitivity during
GHRT are individual and some patients may demonstrate certain improvement, which might depend
on pre-treatment glucose status, and results probably due to decrease in visceral fat. Recently, longterm GHRT has been reported to increase the prevalence of the metabolic syndrome, despite certain
improvements of LDL cholesterol levels (240), and inversely, discontinuing of a long-term GHRT for
one year has resulted in improved insulin sensitivity (241). This implies that more detailed
understanding of mechanisms underlying glucose homeostasis durng GHRT is needed to minimize
risk factors for metabolic syndrome in patients with different metabolic status.
Skeletal muscle is an important metabolic tissue, mainly responsible for postprandial glucose
disposal, which has been reported to develop insulin resistance before the onset of type 2 diabetes (see
section 1.1.2.2). Furthermore, elevation of lipids level, which is an inevitable consequence of GHRT,
has been proposed as a strong contributor to skeletal muscle insulin resistance (106,114). Moreover,
skeletal muscle insulin resistance has been reported for GH-deficient patients, and long-term GHRT
could further deteriorated insulin sensitivity in muscle (143).
MicroRNAs (miRNAs), a family of non-coding RNAs, have been identified as important
regulators in metabolic tissues, including skeletal muscle. A single miRNA can potentially regulate
large number of genes attributed to the same biological function, and miRNAs may therefore be
involved in pathogenesis of complex diseases, such as type 2 diabetes. Several miRNAs have been
reported to be regulated in skeletal muscles of glucose intolerant or type 2 diabetic patients as well as
during hyperinsulinemic clamp (see section 1.2.3). We therefore hypothesized that miRNA pathway in
skeletal muscle is involved in the mechanisms that contribute to increased insulin resistance after
GHRT.
53
GROWTH HORMONE REPLACEMENT THERAPY REGULATES MICRORNA-29A AND TARGETS
INVOLVED IN INSULIN RESISTANCE
Here we report that miR-29a is downregulated during GHRT in skeletal muscle both in mice
and humans. We have also revealed that changes in miR-29a expression correlate with changes in
insulin sensitivity in human patients, and a set of miR-29a targets involved in insulin resistance are
reciprocally regulated during GHRT. Besides, we have shown that miRNA-29a regulation is not a
direct effect of GH and is mediated through the action of IGF1.
54
GROWTH HORMONE REPLACEMENT THERAPY REGULATES MICRORNA-29A AND TARGETS
INVOLVED IN INSULIN RESISTANCE
3.3 Results
3.3.1 GHRT induces insulin resistance in mice and stimulates extracellular
matrix gene expression
To elucidate the mechanisms that regulate muscle metabolism during GHRT we chose Ghrhrlit/lit mice
as a model for GH deficiency (GHD). Ghrhrlit/lit mice are homozygous for a missense mutation in the
growth hormone-releasing hormone receptor (Ghrhr) resulting in pituitary and serum GH levels of less
than 5%, and serum IGF1 levels of 15-20% of normal (242). As expected from previous protocols
(243), 3 weeks of GHRT induced insulin resistance in the GH-deficient mice (compared to saline
injections) as demonstrated by increased fasting and random glucose values and higher insulin levels
(Fig.3.1A). GHRT also increased bodyweight (data not shown). To identify relevant changes in gene
expression that could affect insulin sensitivity in skeletal muscle, we performed next generation
sequencing on muscle tissue from GHD mice with or without GHRT. The GH treatment significantly
regulated 1422 transcripts (1063 upregulated and 360 downregulated). Strikingly, the 3 most
significantly induced functional clusters in the GHRT group involved extracellular matrix (ECM)
organization, while the three functional clusters with the most significant downregulation included
fatty acid metabolism (Fig.3.1B).
3.3.2 miRNA-29a and its ECM-related targets are reciprocally regulated by
GHRT
To identify novel mechanisms for the regulation of ECM during GHRT we turned to the miR-29
microRNA family (miR-29a, -29b and -29c) which is a well-known regulator of ECM (244). Small
RNA sequencing identified miR-29a as the most abundant member of the miR-29 family in skeletal
muscle from both mice and humans (see section 2.3.1). Importantly, miR-29a was significantly
decreased in skeletal muscles (both fast-twitch enriched, TA, and slow-twitch enriched, SOL), but not
the liver of GHRT treated mice (Fig.3.2). We then searched for all predicted miR-29a targets in the
group of upregulated genes from our sequencing data that are conserved between mice and humans
using the online prediction service Targetscan (145). Importantly, the list of predicted targets that were
≥2-fold upregulated was enriched for profibrotic and proinflammatory genes that belong to ECM
genes (Fig.3.3A) and we chose 5 candidates from the 10 highest expressed genes (Col3a1, Fstl1,
Col6a3, Serpinh1, and Sparc). From the list of miR-29a targets that were upregulated less than 2-fold
we selected Pten since it is a well-established inhibitor of the intracellular insulin signaling (245). The
regulation of these selected predicted miR-29a targets during GHRT was confirmed using qRT-PCR
55
GROWTH HORMONE REPLACEMENT THERAPY REGULATES MICRORNA-29A AND TARGETS
INVOLVED IN INSULIN RESISTANCE
(Fig. 3.3B). Together these data identify miR-29a and a set of its targets that could contribute to the
changes in glucose tolerance during GHRT. To test whether the confirmed targets of miR-29a could
also be relevant for human skeletal muscle, we transfected miR-29a mimics or inhibitors (antagomirs)
into human myotubes (Fig. 3.4). As expected, the effect of miR-29a inhibition was in general weaker
than that of the overexpression, given that endogenous miR-29a levels are lower than the
supraphysiological overexpression with the mimics. Overall, our approach validated 5 of the 6
predicted miR-29a targets as endogenous targets in differentiated human skeletal muscle cells: PTEN,
COl3A1, FSTL1, SERPINH1, and SPARC.
3.3.3 IGF1 downregulates miR-29a in human myotubes
To test whether the regulation of miR-29a in skeletal muscle was directly related to the
GHRT, we incubated human myotubes with either GH or IGF1. IGF1, but not GH, gradually
decreased miR-29a levels over the course of 48 hours (Fig.3.5A,B). We conclude that downregulation
of miR-29a in skeletal muscle during GHRT could be a direct consequence of increased IGF1 levels.
3.3.4 IGF1 and miR-29a are reciprocally regulated in insulin resistant
patients
To understand whether the regulation of miR-29a and its targets is important for the change in
insulin sensitivity during GHRT in humans, we studied a cohort of GHD patients that received GHRT
for four months. We used HOMA-IR to allocate the patients to a group with increased or decreased
insulin sensitivity (IS) after GHRT. As expected, insulin levels negatively correlated with insulin
sensitivity (IS) and was significantly up- and downregulated in IS-decreased and IS-increased groups
respectively (Table 3.1). Furthermore, triglyceride levels were significantly higher in the insulin
resistant group after GHRT compared to patients with improved insulin sensitivity. Importantly, a
significant induction of IGF1 and downregulation of miR-29a was only observed in the group of
patients with increased HOMA-IR (Fig.3.6A). In addition, we found a significant correlation between
the change in miR-29a levels and the change in insulin sensitivity expressed as HOMA-IR during
GHRT (Fig.3.6B). This observation argues that downregulation of miR-29a in skeletal muscle after
GHRT is due to the increase in serum IGF1 and correlates with the change in insulin sensitivity.
56
GROWTH HORMONE REPLACEMENT THERAPY REGULATES MICRORNA-29A AND TARGETS
INVOLVED IN INSULIN RESISTANCE
3.3.5 The Regulation of miRNA-29a targets in GHRT is associated with
insulin resistance
Next, we analyzed the five validated miR-29a targets in the GHRT human cohort. We
observed a significant induction of these target genes after GHRT, confirming a conserved role of
these genes in our animal model of GHRT and the patients (Fig.3.7A). To assess the contribution of
miR-29a to the regulation of these genes we separately analyzed their regulation in individuals with
improved insulin sensitivity (HOMA-IR decreased) or decreased insulin sensitivity (HOMA-IR
increased). Importantly, a significant miR-29a target upregulation was only observed in subjects with
increased HOMA-IR (Fig.3.7B), consistent with the significant downregulation of miR-29a in this
group. We conclude that miR-29a contributes to the effects of GHRT on skeletal muscle insulin
resistance by participating in the regulation of the ECM and PTEN.
3.3.6 miRNA-29a and its targets are not regulated in obesity associated
insulin resistance
To test whether regulation of miR-29a in skeletal muscle also occurs in insulin resistance
associated with obesity, we investigated muscle biopsies from both a cohort of prediabetic patients
with abnormal OGTT and an average BMI of 30.4±0.7 kg/m², as well as in mice fed with a high fat
diet (HFD). However, miR-29a did not correlate with the metabolic clearance rate (MCR) or insulin
sensitivity index (ISI) in the 33 insulin resistant subjects (Fig.3.8). In mice, HFD led to an increase in
fasted and random blood glucose, but miR-29a levels were not altered in skeletal muscle (Fig.3.9A).
We also did not detect any differences in miR-29a target gene expression in skeletal muscle from HFD
mice (Fig.3.9B). These data show that miRNA regulation and gene expression in skeletal muscle after
GHRT differs from the obesity-associated insulin resistance.
57
GROWTH HORMONE REPLACEMENT THERAPY REGULATES MICRORNA-29A AND TARGETS
INVOLVED IN INSULIN RESISTANCE
3.4 Discussion and outlook
3.4.1 The regulation of gene expression during GHRT
Our study reports for the first time about the regulation of miRNA expression in skeletal
muscle by GHRT and provides evidence from analysis of both human and mouse samples. With the
help of next generation sequencing, we identify the ECM cluster is the most significant upregulated
group of genes in skeletal muscle following GH treatment of Ghrhr lit/lit mice. Next, we demonstrate
that miR-29a, a known regulator of ECM genes, is decreased during GHRT in two distinct skeletal
muscle groups. Furthermore, we validate five genes as targets of miR-29a in both mice and human
myotubes. One of these genes, PTEN, is a known suppressor of insulin signalling, while the remaining
four genes are all involved in ECM remodelling. These results are corroborated in human patients who
received GHRT for four months. Furthermore, we revealed that miRNA-29a decrease in skeletal
muscle during GHRT correlates with the increase in insulin resistance in a group of patients with
deteriorated insulin sensitivity. Besides, we demonstrated that IGF1 could be responsible for
downregulation of miR-29a, since IGF1, but not GH decreased miR-29a levels in human myotubes,
and IGF1 and miRNA-29a levels were significantly and reciprocally regulated in human patients with
decreased insulin sensitivity. Our data, therefore, provide evidence that miR-29a along with its targets
could promote skeletal muscle insulin resistance following GHRT (Fig 3.10).
GH is known to have a profound effect on the ECM and has been shown to induce collagen
synthesis in skeletal muscle (246). It has been reported that induction of the ECM in human skeletal
muscle is associated with insulin resistance (247,248). Our data support this notion since GHRTmediated induction of hyperinsulinemia and deterioration of glucose tolerance in mice were
accompanied with induced ECM gene expression in skeletal muscle (fig. 3.1). Furthermore, some of
those ECM-related genes have been previously validated as endogenous targets of miR-29a, and are
implicated in the processes of fibrosis and inflammation (Col3a1 (247,249), Fstl1 (250), SerpinH1
(heat shock protein 47) (251) and Sparc (252,253)). We propose that disinhibition of these ECMspecific target genes of miR-29a could contribute to impaired insulin signaling following GH
treatment.
58
GROWTH HORMONE REPLACEMENT THERAPY REGULATES MICRORNA-29A AND TARGETS
INVOLVED IN INSULIN RESISTANCE
3.4.2 IGF1 regulation during GHRT and its relation to miRNA-29a levels
and insulin resistance
A possible mechanism by which GH might affect ECM and miR-29a expression is through
IGF1. IGF1 has been reported as a major mediator of GH effects and is mostly produced systemically
from liver as a response to GH (99), though IGF1 might also be induced locally to act in
paracrine/autocrine manner (254). Several reports link GH treatment to muscle ECM synthesis and
IGF1 serum elevation (255,256) and, besides that, IGF1 levels are positively correlated or reported to
directly regulate collagen gene expression in skeletal muscle (255,257,258). In our study, not only did
IGF1 decrease miR-29a levels in human myotubes, but a significant reduction of miR-29a in skeletal
muscle was only observed in the subgroup of insulin resistant patients with a significant increase in
serum IGF1 (fig. 3.5-6). Our results support the hypothesis that GH, through IGF1 signaling, could
induce expression of ECM components partly by decreasing the expression of miR-29a. The
contribution of miR-29a to the regulation of its targets during GHRT is best reflected by the divergent
regulation of these targets between the group of insulin resistant patients (with decreased miR-29a
levels) and the group of patients with improved insulin sensitivity (having no change in miR-29a
levels) following GHRT (fig. 3.7B). Although miR-29a downregulation and the effect on its targets
are mild, the target regulation would be expected to occur over a long period of time and, thereby,
slowly contribute to the deterioration of insulin signaling in skeletal muscle. Moreover, we cannot rule
out the possibility that miR-29a could target additional mRNAs related to ECM during GHRT.
Downregulation of miR-29a could also impact the insulin response in skeletal muscle by
targeting intracellular regulators of insulin signaling. Indeed, we identified PTEN as an endogenous
target of miR-29a in mouse and human muscle biopsies, and confirmed it in human myotubes. PTEN
is an inhibitor of insulin signaling and its upregulation following GHRT would expect to decrease the
response to insulin in muscle tissue (245).
Our results in human myotubes (fig. 3.4) indicate that overexpression of miRNA inherently
results in false positives since we identified more targets of miR-29a with this approach than with the
loss of function experiments. These results provide a caveat for the interpretation of miR-29a
overexpression experiments in the literature. Indeed, overexpression of miR-29a in cell lines
antagonizes insulin signaling (259-261) contrary to our data in mice and human subjects, and
inhibition of miR-29a levels in 3T3-L1 adipocytes failed to reverse the effect of overexpression of
miR-29a on insulin-induced glucose uptake (261).
59
GROWTH HORMONE REPLACEMENT THERAPY REGULATES MICRORNA-29A AND TARGETS
INVOLVED IN INSULIN RESISTANCE
3.4.3 miRNA-29a in GHRT-induced and obesity-induced insulin resistance
We did not observe any correlation between miR-29a levels and insulin sensitivity in obese
humans or mice with diet-induced obesity (Fig. 3.8-9), which indicates differences in mechanisms of
insulin resistance in skeletal muscle during GHRT and obesity. However, the average fasting insulin
level in the prediabetic subjects (0.141 nmol/l) might not have been high enough to activate the IGF1
receptor and affect miR-29a expression. In this regard, it would be interesting to assess miR-29a
regulation in skeletal muscle from insulin resistant or diabetic subjects with a more pronounced
hyperinsulinemia. Recently, miR-29a has been reported to be upregulated in skeletal muscles from
insulin resistant mice fed with HFD (259). We and other groups (262,263) did not find such an
induction. Overall, we exclude a relevant regulation of miR-29a in skeletal muscle of HFD fed mice
not only by miRNA qRT-PCR, but also by the absence of any changes in the expression of miR-29a
target genes (Fig 3.9B). Potential explanations for the discrepant results include the different response
of C57Bl6 substrains to HFD (264) or differences in the HFD itself. Intriguingly, miR-29a was among
the most downregulated miRNAs in human skeletal muscle during a 3-h hyperinsulinemic-euglycemic
clamp (175). The average insulin concentration achieved during this clamp was slightly above 1 nM
and could have been sufficient to activate the IGF1 receptor in skeletal muscle. Due to technical
limitations with the glucose clamp technique, we used HOMA-IR as a surrogate to determine insulin
sensitivity. However, the significantly higher triglyceride levels in a group of patients with increased
HOMA-IR support a clinically meaningful separation of the metabolic state in our study. Compared to
patients with improved insulin sensitivity following GHRT, miR-29a levels decreased in patients with
deteriorated insulin sensitivity by 30%. The impact of such miRNA regulation that persists over long
periods of time, e.g. months, in vivo has not been experimentally validated yet. However, regulation of
less than 2-fold of miR-499 is associated with the induction of the type 1 muscle program in active
compared to sedentary human subjects (225), and a 40% induction of miR-375 is associated with
altered pancreatic beta cell proliferation in obese mice (212). Therefore, we believe that our data
provide relevant evidence for implication of miR-29a and its targets into development of skeletal
muscle insulin resistance during GHRT.
60
GROWTH HORMONE REPLACEMENT THERAPY REGULATES MICRORNA-29A AND TARGETS
INVOLVED IN INSULIN RESISTANCE
3.5 Figures
A
Glucose, fasted
Glucose, random
3
*
6
1
1.5
Insulin, ng/ml
Glucose, mM
Glucose, mM
*
2
Insulin, random
4
2
*
1.2
0.9
0.6
0.3
H
G
G
PB
S
H
0.0
PB
S
G
H
0
PB
S
0
B
GHT > PBS
Category
Term
Count
%
PValue
GOTERM_BP_FAT GO:0030198~extracellular matrix organization
28
3.73
1.73E-015
GOTERM_BP_FAT GO:0007155~cell adhesion
62
8.26
8.92E-013
GOTERM_BP_FAT GO:0022610~biological adhesion
62
8.26
9.57E-013
Count
%
PValue
GOTERM_BP_FAT GO:0016053~organic acid biosynthetic process
7
3.29
4.44E-003
GOTERM_BP_FAT GO:0046394~carboxylic acid biosynthetic process
7
3.29
4.44E-003
GOTERM_BP_FAT GO:0006631~fatty acid metabolic process
7
3.29
1.54E-002
GHT < PBS
Category
Term
Figure 3.1. GHRT induces glucose intolerance and hyperinsulinemia in GHD mice and upregulates
ECM genes in skeletal muscle from.
A) Fasted and random blood glucose and serum insulin levels in GHD mice after 3 weeks of GH PBS
treatment. B) RNA samples from tibialis anterior muscle from GHD mice treated with PBS or GH were
analyzed using RNA deep sequencing. The three most significantly regulated functional clusters are
depicted for the group of genes that are increased after GHRT (GHT>PBS) or decreased (GHT<PBS).
Results in (A) show combined data for male and female mice (n=7 for GH and n=8 for PBS) and were
analyzed using student`s t test. *, p<0.05
61
GROWTH HORMONE REPLACEMENT THERAPY REGULATES MICRORNA-29A AND TARGETS
INVOLVED IN INSULIN RESISTANCE
PBS
GH
1.2
1.0
***
0.8
***
0.6
0.4
0.2
0.0
1.5
miRNA levels, rel U
miRNA-29a
in liver
1.2
0.9
0.6
0.3
H
PB
S
L
SO
TA
0.0
G
miRNA levels, rel U
1.4
miRNA-29a in
sk. muscle
Figure 3.2. GHRT dowregulates miRNA-29a in both fast and low-twitch enriched skeletal muscle
types, but not in liver.
qRT-PCR analysis of miR-29a in RNA from tibialis anterior and soleus muscles normalized to miR-let7a and shown as relative units (rel. U). Analysis was performed in the same mice as described in
Fig.3.1. Results show combined data for male and female mice (n=7 for GH and n=8 for PBS) and
were analyzed using student`s t test. ***, p<0.001.
62
GROWTH HORMONE REPLACEMENT THERAPY REGULATES MICRORNA-29A AND TARGETS
INVOLVED IN INSULIN RESISTANCE
A
miRNA-29a targets
1 400
PBS
GH
1 200
FPKM
1 000
800
600
400
200
Col11a1
Pthlh
Mest
Lox
Etv4
Cilp2
Fos
C1qtnf6
Hmgn3
Fbxl18
Col16a1
Mfap2
Zhx3
Nid2
Col5a2
Col5a3
Col5a1
Pxdn
Fbn1
Mmp2
Col6a2
Col3a1
Fstl1
Col15a1
Col6a3
Col1a1
Serpinh1
Col4a2
Col4a1
Col1a2
Sparc
0
+ PTEN (1.3-fold upregulation)
9
8
7
6
5
4
3
2
1
0
PBS
GH
**
***
Sp
ar
c
1
*
Se
rp
in
H
ol
6a
3
C
ol
3a
1
C
Fs
tl1
*** ***
*
Pt
en
Gene expression, rel U
B
Figure 3.3. miRNA-29a targets are upregulated in GHRT in mice.
A) All predicted miR-29a targets that are conserved between mice and humans and upregulated by
GH ≥ 2 -fold in the RNA deep sequencing screen described in Fig.3. FPKM means “fragments per
kilobase of exon per million fragments mapped”. Selected targets associated with fibrosis and
inflammation, as described in the text, are pointed out by arrows. B) qRT-PCR analysis of gene
expression in skeletal muscle after GH- (grey) or PBS-treatment (white). mRNA levels were
normalized to 18S RNA amounts and shown as relative units (rel. U). Results show combined data for
male and female mice (n=7 for GH and n=8 for PBS) and were analyzed using student`s t test. *,
p<0.05, **, p<0.01, ***, p<0.001.
63
GROWTH HORMONE REPLACEMENT THERAPY REGULATES MICRORNA-29A AND TARGETS
INVOLVED IN INSULIN RESISTANCE
mimic-ctrl
mimic-29a
antagomir-ctrl
antagomir-29a
2.0
1.0
*
***
0.5
**
***
***
***
Gene expression, rel U
1.5
*
1.5
*
*
*
**
1.0
0.5
ar
c
Sp
1
in
H
Se
rp
C
ol
6a
3
Fs
tl1
Pt
en
rc
Sp
a
Se
rp
in
H
1
C
ol
6a
3
Fs
tl1
C
ol
3a
1
Pt
en
C
ol
3a
1
0.0
0.0
Figure 3.4. Target gene regulation in human myotubes by miR-29a inhibition and overexpression.
qRT-PCR analysis in human myotubes after miR-29a mimic transfection (grey) compared to control
(white), n=4, or after antagomir-29a transfection (grey) compared to control (white), n=4-5. Results
are shown for myotubes harvested 48 h after transfection. mRNA levels were normalized to 18S RNA
amounts and shown as relative units (rel. U). Student`s t test was used to compare the 2 groups. *,
p<0.05, **, p<0.01, ***, p<0.001. Myoblast cell culturing, differentiation, and transfection was done
by Angelika Hartung, PhD
64
GROWTH HORMONE REPLACEMENT THERAPY REGULATES MICRORNA-29A AND TARGETS
INVOLVED IN INSULIN RESISTANCE
A
GH 1ug / ml
IGF1 10nM
***
***
B
Ctrl
Treatment
miRNA levels, rel U
1.5
1.0
*
0.5
F
IG
G
H
0.0
Figure 3.5. IGF1, but not GH, downregulates miR-29a levels in human myotubes.
A) Human myotubes were incubated for the indicated time intervals with either 1 ug/ml GH or 76
ng/ml (10 nM) IGF1 before RNA was isolated. miR-29a levels were analyzed using qRT-PCR and
normalized to miR-let-7a. n=4 for GH and n=10 for IGF1. B) Direct comparison of the effects of GH
and IGF1 on miR-29a levels shown after 48 h incubation in an independent set of experiments, n= 35. Results are shown relative units (rel U). Changes in (A) were analyzed using ANOVA and Dunnett`s
post-test comparing all the samples to the control samples (at 0 h). The effects of GH and IGF1 on
miR-29a levels in (B) were analyzed using student`s t test. *, p<0.05. ***, p<0.001. Myoblast cell
culturing, differentiation, and incubations with GH / IGF1 was done by Angelika Hartung, PhD
65
GROWTH HORMONE REPLACEMENT THERAPY REGULATES MICRORNA-29A AND TARGETS
INVOLVED IN INSULIN RESISTANCE
A
HOMA-IR increased
2,5
2,5
2,0
2,0
fold change
fold change
HOMA-IR decreased
1,5
1,0
0,5
*
1,5
1,0
*
0,5
0,0
0,0
IGF1
miR-29a
IGF1
miR-29a
B
4
p = 0.011
R2 = 0.577
3
2
1
0
0.4
0.6
0.8
1.0
1.2
1.4
miR-29a levels
Figure 3.6. Increased serum IGF1 and decreased miR-29a levels in skeletal muscle from GHD
patients with decreased insulin sensitivity after GHRT.
A) Serum IGF1 and miR-29a expression in skeletal muscle in GHD patients before or after four
months of GHRT. Values were separately analyzed in individuals with improved IS (HOMA-IR
decreased), n=5, or worsened IS (HOMA-IR increased), n=5, after GHRT. B) Relationship between the
change in insulin sensitivity (ΔHOMA-IR) induced by four months of GHRT and the change in miR-29a
levels in skeletal muscle biopsies. MiRNA levels were analyzed by qRT-PCR and normalized to miR-let7a. Results are shown as fold change of values after GHRT compared to values before GHRT. *,
p<0.05 (student`s t test).
66
GROWTH HORMONE REPLACEMENT THERAPY REGULATES MICRORNA-29A AND TARGETS
INVOLVED IN INSULIN RESISTANCE
A
Human SM in GHRT
*
3.0
Gene expression, rel U
before GHRT
after GHRT
2.5
*
2.0
*
1.5
*
*
1.0
0.5
pi
c
ar
Se
r
HOMA-IR decreased
HOMA-IR increased
4
4
mRNA levels (AU)
mRNA levels (AU)
Sp
nH
1
tl1
1
3a
Fs
B
C
ol
Pt
en
0.0
3
2
1
0
3
*
2
*
*
1
0
Figure 3.7. Regulation of miR-29a targets in human skeletal muscle by GHRT.
A) qRT-PCR analysis of selected miRNA-29a targets in skeletal muscle biopsies before (white) or after
(grey) four months of GHRT, n=9 B)
RT-PCR analysis as described in A), but analysed separately
for HOMA-IR decreased, n=5, and HOMA-IR increased groups, n=4. Results were normalized to 18S
RNA and shown as relative units (rel. U). Student`s t test was used to compare the 2 groups. *,
p<0.05.
67
GROWTH HORMONE REPLACEMENT THERAPY REGULATES MICRORNA-29A AND TARGETS
INVOLVED IN INSULIN RESISTANCE
miR-29a
miR-29a
30
p = 0.907
R2 = 0.0004
miRNA levels (AU)
miRNA levels (AU)
30
25
20
15
10
5
1
2
3
4
5
6
7
8
9
25
20
15
10
5
0
0.00
0
0
p = 0.651
R2 = 0.007
10
0.05
0.10
calculated ISI (umol*kg-1*min-1*pmol/l)
calculated MCR (ml*kg-1*min-1)
Figure 3.8 miR-29a levels in skeletal muscle do not correlate with insulin sensitivity in patients with
prediabetes.
OGTTs were performed in 33 subjects with prediabetes and the metabolic clearance rate (MCR) or
the insulin sensitivity index (IS) calculated as described in Methods. miR-29a levels were measured in
muscle biopsies and normalized to miR-let-7a and shown as arbitrary units (AU). miRNA levels were
plotted against MCR or ISI, respectively.
68
GROWTH HORMONE REPLACEMENT THERAPY REGULATES MICRORNA-29A AND TARGETS
INVOLVED IN INSULIN RESISTANCE
A
Glucose, fasted
6
Glucose, random
8
***
Mir-29a
1.5
*
6
4
1.0
4
2
0.5
2
F
H
TR
C
H
F
0.0
C
tr
l
H
F
0
C
tr
l
0
Sp
ar
c
a3
C
ol
6
Fs
tl1
a1
C
ol
3
Pt
en
Gene expression, rel U
B
Figure 3.9. Unchanged miR-29a levels and miR-29a target gene expression in skeletal muscle after
HFD in mice.
A) C57Bl6 mice were subjected to HFD diet for over 20 weeks and glucose values were measured in
the fasted or non-fasted (random) state. qRT-PCR analysis of miR-29a levels in tibialis anterior muscle
were normalized to miR-let-7a levels and shown as relative units (rel. U). B) qRT-PCR analysis of
selected miR-29a target genes (see text) in skeletal muscle after HFD (grey) or normal chow (white).
Results for mice after HFD and normal chow (control) are each n=7 (male mice). mRNA levels were
normalized to 18S RNA amount. Results for HFD versus control were analyzed using student`s t test.
69
GROWTH HORMONE REPLACEMENT THERAPY REGULATES MICRORNA-29A AND TARGETS
INVOLVED IN INSULIN RESISTANCE
Growth
hormone
IGF1
miR-29a
SPARC
FSTL1
COL3a1
SerpinH1
PTEN
Induction of
extracellular matrix
Inhibition of
insulin signaling
Inflammation
Skeletal muscle
insulin resistance
Fig 3.10 Model for the regulation of skeletal muscle insulin resistance by GHRT
70
GROWTH HORMONE REPLACEMENT THERAPY REGULATES MICRORNA-29A AND TARGETS
INVOLVED IN INSULIN RESISTANCE
IS increased
IS decreased
GHRT
4 months
p values
start vs
4 months
p values
IS decreased
vs. increased
Sex (n)
Male
Female
Age (years)
BMI (kg/m2)
Lean mass (kg)
Waist (cm)
Fasted glucose (mM)
Fasted Insulin (mU/l)
HOMA-IR
IGF1 (ug/l)
Triglycerides (mM)
NEFA (mM)
Total cholesterol (mM)
LDL (mM)
HDL (mM)
GHRT
start
5
4
1
35.0±3.7
27.1±1.3
55.7±5.0
93.8±3.3
4.6±0.3
4.8±1.5
1.0±0.4
104.8±20.9
1.7±1.0
0.5±0.1
4.9±0.5
3.4±0.3
1.2±0.1
27.4±1.5
57.8±5.4
93.1±3.9
4.6±0.3
3.0±1.4
0.7±0.4
175.2±23.6
0.9±0.3
0.6±0.1
4.8±0.9
3.3±0.8
1.4±0.1
0.63
0.13
0.53
0.56
0.01
0.01
0.06
0.26
0.78
0.92
0.86
0.18
0.10
0.30
0.79
0.88
0.07
0.03
0.04
0.88
0.01
0.12
0.83
0.85
0.25
Sex (n)
Male
Female
Age (years)
BMI (kg/m2)
Lean mass (kg)
Waist (cm)
Fasted glucose (mM)
Fasted Insulin (mU/l)
HOMA-IR
IGF1 (ug/l)
Triglycerides (mM)
NEFA (mM)
Total cholesterol (mM)
LDL (mM)
HDL (mM)
5
3
2
47.6±6.5
25.5±0.8
56.1±7.8
95.9±4.5
4.6±0.3
5.0±1.1
1.1±0.3
93.0±24.3
1.8±0.2
0.7±0.1
5.5±0.4
3.9±0.4
1.2±0.1
25.6±1.0
60.3±8.8
94.0±4.9
5.1±0.1
7.8±1.6
1.8±0.3
180.4±29.0
2.1±0.3
0.7±0.0
5.0±0.4
3.4±0.4
1.2±0.1
0.70
0.09
0.29
0.13
0.02
0.01
0.01
0.16
0.58
0.07
0.01
0.86
Table 3.1. Characteristics at GHRT start and at 4 months. Values shown are means ± SEM.
71
CONCLUDING REMARKS
CONCLUDING REMARKS
Recent studies highlight the importance of skeletal muscle for health and longevity (196-198).
Sarcopenia characterized with decreased skeletal muscle regenerative capacity and reduced muscle
mass, from the one hand, and type 2 diabetes, which initiation depends on skeletal muscle insulin
resistance, from the other one, both represent important aging-associated pathophysiological
conditions. This thesis describes miRNA-29a as an important factor, involved both in the
physiological proliferative response of myogenic progenitors during skeletal muscle regeneration and
regulation of muscle insulin resistance in growth hormone replacement therapy.
The second chapter of this thesis provides evidence that miRNA-29a acts downstream from
FGF-2 to mediate dismantling of basement membrane in satellite cell niche and promote MP
proliferation during muscle regeneration. Induced miRNA-29a deletion in satellite cells inhibited MP
proliferation, promoted fibrosis and blunted both injury- and exercise-induced muscle regeneration.
Importantly, FGF-2 has been previously reported to be induced in the satellite cell niche of aged mice
(211). The age-related elevation of FGF in the niche is associated with hyperactivation of dormant
satellite cells and gradual depletion of quiescent satellite cell pool. miRNA-29a has also been observed
to be highly elevated in aged muscle (235). The relevance of the miRNA-29a elevation specifically in
the aged satellite cell niche though still needs to be elucidated. Although in this paper authors report
about Wnt-3a signalling as an extracellular regulator, our data imply that FGF might be involved.
While the role of miRNA-29a as a regulator of ECM is established in many tissues, our study is the
first to demonstrate that miRNA-29a controls all major structural components of basement membrane
in skeletal muscle progenitors, such as Col4a1, Nid2, Hspg2, Fbn1, and Lamc1 as well as its highly
abundant regulators, including Sparc. Homeostasis of these key ECM proteins is pivotal for normal
functioning of the niche since their remodelling triggers the activation of satellite cells via both
changing the stiffness and releasing growth factors to the surrounding media (265). In vitro
experiments demonstrate that both satellite cell stemness and differentiation potential depend on
matrix composition and stiffness (219,266-268). Besides, extracellular matrix components, such as
perlecan and decorin can also influence the rate of symmetric/ assimmetric devisions within the
satellite cells pool via binding (and therefore immobilization) of Notch and Wnt ligands (269). Our
data imply that miRNA-29a is critically involved in the regulation of ECM homeostasis of satellite
cell niche, although the mechanism still remains to be elucidated
Although, we did not identify what transcription factor is responsible for miRNA-29a
induction by FGF-2, one recent paper might give a clue. A study done in keratinocytes reports that
miRNA-29a might be induced by Nrf2 binding an enhancer element upstream of miRNA-29ab1
transcription start (270). Interestingly, FGF-1 was shown to activate Nrf2 in astrocytes (271), and,
72
CONCLUDING REMARKS
given that FGF-1 and FGF-2 target the same receptors (65), it is tempting to speculate that FGF2 –
Nrf2 – miRNA-29a axis might take place in skeletal muscle progenitor cells.
Our data also suggest that FGF-miRNA-29a axis might be relevant for other progenitor cell
types, since a link between FGF and the miRNA was confirmed for distinct muscle derived lineage,
fibroadipogenic progenitors. Given an established role of different FGFs in embryogenesis and
development of many tissues as well as ubiquitous pattern of miRNA-29a expression, we propose that
FGF – miR-29a axis might also be important in the development of many tissues, other than skeletal
muscle. Besides the role in development, this axis might be also relevant for the function of endocrine
FGFs. It would be interesting to verify whether miRNA-29a is involved in the regulation of hepatic
metabolism by FGF-21 or implicated into phosphate / vitamin D metabolism regulation by FGF-23.
We have also carefully studied an effect of deletion / inhibition of miRNA-29a on early
myoblast differentiation. Importantly, this is the first careful functional analysis of myoblasts without
miRNA-29a, as previous reports mostly relied on overexpression approaches, which are more prone to
artefacts (see section 2.4.3). Clear negative effect of miRNA-29a inhibition / deletion on myoblast
proliferation and corresponding stimulatory effect on differentiation imply that this approach might be
potentially useful for the treatment of rhabdomyosarcoma.
In the third chapter we report that IGF1 induced during GHRT might be responsible for
miRNA-29a downregulation in adult skeletal muscle, and the changes in miRNA-29a expression
correlate with changes in insulin sensitivity in human patients. We also demonstrated that the miRNA29a decrease leads to induction of both targets involved in insulin signalling as well as targets related
to extracellular matrix, which upregulation is a hallmark of insulin resistant skeletal muscle (247,248).
Increased deposition of collagen was well documented in skeletal muscle biopsies of obese and
diabetic patients (247). It is proposed that elevated ECM content results (at least partially) from
obesity driven chronic inflammation of the muscle tissue and might affect the sensing of
mechanoreceptors, which link muscle contraction to mitochondria biogenesis and tissue metabolism
(272). Thus miRNA-29a downregulation by IGF1 might represent another cause for the increase of
ECM deposition in insulin resistant skeletal muscle in the context of GH therapy.
The risk of T2D increases with age and although IGF1 levels decline in aging, the observed
IGF1-miR29a regulation still might be relevant for prediabetic patients with hyperinsulinemia, since
IGF1 and insulin signalling overlap. Furthermore, we observed that, similar to IGF1, insulin can
downregulate miRNA-29a in human myotubes and miRNA-29a was also shown to be decreased in
human skeletal muscle as a result of 3-hour hyperinsulinemic clamp (175).
Interestingly, our deep sequencing RNA screens in myoblasts with deleted miRNA-29ab1
gene as well as in muscle biopsies with decreased miRNA-29a levels derived from GH-treated mice
indicate that miRNA-29a regulates overlapping sets of targets in progenitors and in adult tissue. These
targets include all major structural basement membrane components (Col4a1, Nid2, Hspg 2, Fbn1, and
73
CONCLUDING REMARKS
Lamc1) as well as other targets, such as Sparc, SerpinH1, and Fstl1 (data for some targets are not
shown). We also demonstrated that miRNA-29a is a predominant member of the miRNA family both
in myoblasts and in adult muscle tissue.
Reported elevation of miRNA-29a in aged muscle and pathophysiology phenotypes associated
with the decrease of miRNA-29a level either in adult tissue or progenitor cells imply that the levels of
this miRNA are tightly regulated in a healthy muscle. Our data warns about potential risks of crude
inhibitory approaches to treat elevated miRNA-29a in aged skeletal muscle and call on for the
development of new methods to more carefully adjust the miRNA levels in the tissue. Since miRNA29a targets a wide range of ECM genes and the therapy of ECM pathophysiology might require
targeted manipulations of its composition, one therapeutic approach might be the use of microRNA
Target Site Blockers (273). These antisense oligonucleotides can specifically protect discrete ECM
mRNAs from degradation by miRNA-29a and, by this means, allow targeted and gentle manipulation
of ECM composition in the tissue.
Altogether, our data provide evidence that miRNA-29a plays an important role both in skeletal
muscle stem cell niche as well as in adult tissue. miRNA-29a implements a gentle regulation of ECM
during muscle regeneration and is involved in ECM homeostasis, critical for healthy muscle
metabolism. Dysregulation or inappropriate inhibition of this miRNA both in MP or adult tissue might
have adverse effects.
74
CHAPTER IV
CHAPTER IV
MATERIALS AND METHODS
4.1 Animals
All mice were maintained in a 12 hr. light / dark cycle in a pathogen-free animal facility.
C57Bl6/6J mice were purchased from Harlan Laboratories. To induce muscle regeneration,
cardiotoxin (CTX, Sigma) was injected into the tibialis anterior muscle (50ul / muscle, final conc.
10mM reconstituted in PBS).
Satellite specific-inducible miR-29ab1 KO mice were obtained by crossing Pax7tm2.1
(cre/ERT2) mice to mice, bearing floxed miR-29ab1 gene. Pax7tm2.1(cre/ERT2) mice were obtained
from Jackson Laboratories. Genomic DNA was isolated from ear biopsies. Biopsies were lysed in the
following buffer (100mM Tris, 10mM EDTA, 100mM NaCl, 0.2% SDS and 0.2mg/ml proteinase K)
for 2-3 hours at 56°C and proteinase inactivated at 72°C for 30 min. Then cellular debris and
undigested material were spun by centrifugation at 14000 rpm for 5 min., and 700ul of supernatant
were precipitated with isopropanol in the presence of 0.15M NaOAc (final conc. 3M stock solution
had pH 5.2). Samples were incubated at -20°C for 1 hour, spun down at 14000 rpm for 30 min at 4°C,
and pellets reconstituted in water.
Pax7Cre
knock-in
was
genotyped
using
following
primers:
Pax_Cre_fw
(ACTAGGCTCCACTCTGTTCCTTC) and PaxCre_rev (GCAGATGTAGGGACATTCCAGTG).
From WT animals a 725nt band was produced, while analysis of samples from knock-in mice resulted
in 250nt band. PCR cycling program was as following: initial denaturation at 94°C-9 min, 42 cycles
with denaturation at 95°C for 30 sec., annealing at 65°C for 30 sec. and elongation at 72°C for 1 min.
Cycles were followed by terminal elongation at 72°C for 10 min.
Homozygous floxed miR-29ab1
mice (C57BL6 strain) were genotyped using PCR as previously described (274). Primer sequences
were
29ab1
forward
(TGTGTTGCTTTGCCTTTGAG),
29ab1
LOXP
reverse
(Rev)
(CCACCAAGAACACTGATTTCAA). From WT animals a 420nt band was produced, while analysis
of samples from floxed mice resulted in 600nt band. PCR cycling program was as following: initial
denaturation at 94°C-9 min, 42 cycles with denaturation at 95°C for 1 min., annealing at 60°C for 1
min. and elongation at 72°C for 2 min. Cycles were followed by terminal elongation at 72°C for 10
min.
Tamoxifen (Sigma) was dissolved in corn oil (Sigma) at 20 mg/ml in a 37°C water bath for 24 h. The samples were shaken every 5-10 min. This solution was applied by intraperitoneal
administration as indicated or stored frozen in aliquots at -80°C. An aliquote of tamoxifen was thawed
75
MATERIALS AND METHODS
for 5-10 min. at 37°C just before the injection. One day before sacrificing the mice, 5`-ethynyl-2´deoxyuridine (EdU) was injected intraperitoneally at 5 ug/g body in 300ul PBS.
In exercise experiments mice preformed a single 90 min bout of downhill running (-20°
decline) on a treadmill (Panlab) with the following protocol: at a speed of 15 m/min from 0-60 min, 18
m/min from 60-75 min and 21 m/min from 75-90 min. A mild electrical stimulus was provided as
motivation via a shock-grid located at the end of the treadmill belt. To avoid any muscular priming
adaptations mice were familiarized to treadmill running immediately prior to completing downhill
running. This consisted of 5 min running on the treadmill at zero incline with speed gradually
increasing to 15 m/min. Exercise tolerance was determined using an incremental treadmill test to
exhaustion. The treadmill was set at a 5° incline and speed was increased by 2 m/min every 2 min until
the mouse spent >5 s on the shock grid without attempting to continue running.
C57BL/6J117 Ghrhrlit/J mice were purchased from Jackson Laboratories. Homozygous females
were bred to heterozygous males and homozygous pups were distinguished from heterozygous
littermates by size at the age of four weeks. Between 10 and 14 weeks of age, mice were daily injected
with recombinant GH subcutaneously (Genotropin, Pfizer) at a dose of 6 ug per g bodyweight per day.
We chose this dosage since 6 ug per g bodyweight induced insulin resistance in GHD mice in a
previous report (243). After 21 days, mice were sacrificed and the tibialis anterior muscle and liver
tissue removed, snap frozen in liquid nitrogen and stored at -80°C. Serum was collected from tail-vein
blood and analyzed for glucose using FreeStyle Lite (Abbott) and for insulin using the ultrasensitive
mouse insulin ELISA kit (Crystal Chem). C57Bl6/6J mice were purchased from Harlan Laboratories.
Starting at 4 weeks of age, mice were fed a 58% fat and sucrose diet (D12331, Research Diets, USA).
After 20 weeks mice were sacrificed and the tibialis anterior muscle removed and processed as
described above. All animal studies were approved by the ethics committee of the Kantonale
Veterinäramt Zürich and the principles of laboratory animal care were followed.
76
MATERIALS AND METHODS
4.2 Participants
Muscle biopsies from 10 patients with adult onset GHD for at least 12 months (3 women, 7
men, and age range 24 to 62 years) were recruited from a previously reported trial at the University of
Bern, Switzerland (275). 3 patients had craniopharyngeomas, 3 patients had hormone-inactive
pituitary adenomas, 1 patient had a Rathke`s cleft cyst, 1 patient had traumatic hypopituitarism, 1
patient had a macroprolactinoma and 1 patient had hypophysitis. Inclusion and exclusion criteria have
been described in detail previously (275). Biopsies were harvested from the middle portion of the right
tibialis anterior muscle under local anesthesia before and after a total of 4 months of GHRT (275).
HOMA-IR was calculated as fasted insulin (μU/ml) x fasted glucose (mmol/l) / 22.5. A second cohort
of participants was recruited from the University of Leipzig, Germany (276). Patients of either sex
referred to the University of Leipzig Heart Center, Germany, were invited to participate if they
displayed impaired fasting glucose (>6.0 and <7.0 mmol/l with HbA1C <6%) or impaired glucose
tolerance (>7.8 and <11.1 mmol/l) 2 h after oral intake of 75 g glucose and angiographic evidence of
coronary artery disease (>50% stenosis diameter in at least one major epicardial artery). Vastus
lateralis muscle biopsies were obtained under local anaesthesia and immediately snap frozen in liquid
nitrogen. 75-g oral glucose tolerance tests (OGTT) were performed under standard conditions. In total,
muscle biopsies from 33 patients (27 males, 6 females) were analyzed. We calculated the metabolic
clearance rate (MCR) (19.240-0.281*BMI-0.00498*Ins120-0.333*Gluc120) and the insulin sensitivity
index (ISI) (0.222-0.00333*BMI-0.0000779*Ins120-0.000422*Age) from the OGTT since they
correlate well with insulin sensitivity as measured by the glucose clamp technique (r values of 0.79 for
MCR and ISI, respectively (277)). Average age was 62.1±1.1 years, BMI 30.4±0.7 kg/m², glucose
during OGTT at 0 h was 6.1±0.1, at 1 h 11.7±0.4 and at 2h 9.1±0.3 mM. All values are means with
SEM.
77
MATERIALS AND METHODS
4.3 Skeletal muscle histology and immunofluorescence
For immunofluorescence, skeletal muscle was dissected and flash frozen in isopentane / liquid
nitrogen. Frozen sections of 10 μm were prepared either from 3 different areas of the tibialis anterior,
500 μm apart, or from the whole gastrocnemius muscle. Sections were then processed for
immunofluorescence using the Vector M.O.M. Immunodetection kit and protocol (Vector
Laboratories). Slides were mounted with Fluoroshield with DAPI (Sigma). Antibodies against eMyHC
(BF-G6-s) were obtained from the Developmental Studies Hybridoma Bank, University of Iowa. Cell
proliferation was detected as EdU incorporation into DNA using Click-iT Plus Edu imaging kit
(Molecular Probes).
Images for each section were obtained using Leica confocal laser scanning microscope SP5
Mid UV356 VIS and evaluated for fiber and cell counts. For routine histochemistry, skeletal muscle
was flash frozen in isopentane/liquid nitrogen. Consecutive 10 μm cryostat sections were prepared and
stained with hematoxylin and eosin (H&E) or modified Gomori trichrome, according to standard
procedures. The stained preparations were evaluated for variation in myofiber diameter, the presence
of atrophic and hypertrophic fibers, internalized nuclei, regenerating and degenerating fibers,
myophagocytosis, inflammation and fibrosis.
4.4 RNA isolation, qRT-PCR, miRNA microarray, and mRNA
sequencing
Total RNA was isolated using the Trizol reagent (Invitrogen) according to manufacturer’s
instructions. For qRT-PCR, total RNA was subjected to DNAse digestion with the DNA-free DNA
Removal Kit (Life technologies) for 1 hour and precipitated with 2.5 volumes of absolute EtOH in
presence of 0.1M ammonium acetate (Gene link). RNA concentration and purity was analyzed with
Nanodrop Lite (Thermo scientific). Small quantities of RNA were analyzed for integrity with Agilent
RNA 6000 Pico Kit (Agilent Technologies) according to manufacturer’s instructions. In brief, samples
with RNA conc. 50–5000 pg/μl as well as RNA ladder were denatured at 72C for 2 min. and loaded
(1ul each sample) into a beforehand prepared agilent pico chip. A chip was then shaken in a Vortex
Mixer Adapter for 1 min. and analyzed using 2100 Bioanalyzer Instrument.
Reconstituted DNA free RNA was then used for cDNA preparation with the Super Script III
Reverse Transcriptase (Life technologies), employing random hexamer primers. mRNAs were
analyzed by quantitative real-time PCR using the FastStart Universal SYBR Green Master Mix
(Roche) with a 7500 Fast Start Real-Time PCR system (Applied Biosystems). Transcript levels were
normalized to 18S ribosomal mRNA levels. Primer sequences are available upon request. miRNA
78
MATERIALS AND METHODS
levels were measured using qRT-PCR with the TaqMan miRNA assays (Applied Biosystems), and
normalized to sno234 and/or let7a. In short, for the reverse transcription reaction, 1.5ul of 3ng/ml
RNA sample were added to a 8.5ul of mastermix, containing 0.4ul of primer stock, 1.2ul of enzyme,
as well as 0.25ul of dNTP mix. For the qPCR reaction, 1.5ul of RT reaction were added to a
mastermix containing 0.3ul of labeled primer to end up with 10ul of total volume.
For miRNA microarrays and Illumina deep sequencing, RNA was isolated using RNA cleanup and concentration kit (Norgen), according to manufacturer’s instructions. Further, RNA was
processed and analyzed at LC Sciences (Houston, TX, USA). The deep-sequencing results were
obtained as FPKM (fragment per kilobase of exons per million reads) for each transcript.
4.5 Northern blotting
Northern blotting was performed as previously described in non-reducing polyacrylamide gels (214).
In brief: 5ug of RNA in each sample was denatured in 2xRNA-Loading buffer (8 M Urea, 50 mM
EDTA pH 8.0, 0.4mg/ml Bromphenol blue) for 1 min. at 95°C and 5 min. at 65°C, and loaded on nonreducing polyacrylamide gel (12% gel in 1x TBE, formed in presence of 0.1% Ammonium Persulfate
and 20ul of TEMED / 50ml of gel). The RNAs were run in 0.5x TBE buffer at 45mA, ~20W and
stained in 100ml 0.5xTBE with 30ul 0.5% EtBr2. Then, the RNA was transferred to Hybond N+
membrane (Amersham) at 50V for 2h in Running buffer. The chamber was chilled with the ice
buckets. Afterward membrane was dried, UV cross-linked (1200J, 30sec), and baked for 1h at 80°C.
DNA antisense probes were labelled with 30μCi of [γ-32P] ATP (3000Cimmol-1;
PerkinElmer) by T4 polynucleotide kinase (NEB) for 15 min, and residual radiolabeled ATP was
removed by filtration through illustra MicroSpin G-25 Columns (GE Healthcare) during centrifugation
for 1 min at 735g. Then probes were denaturated for 1 min. at 95°C, and ½ of the probe was
hybridized with membrane at 50°C overnight in 15ml of hybridization buffer (for 1 blot: 35ml
SDS10%, 12.5ml SSC20x, 1ml 1M Na2HPO4, pH 7.2, 1ml 50x Denhardt`s solution and 0.5ml
10mg/ml DNA). The membrane was washed for 10 min twice in 100ml of washing buffer 1 (125ml
20xSSC, 250ml 10%SDS and125ml H20) and ones in 100ml of washing buffer 2 (50ml SDS10%,
25ml 20xSSC, 425ml H20), and, finally, exposed to Hyperfilm MP (Amersham). Following recipes
were used for SSC20x: (175g NaCl/l, 88g/l NaCitratex2H20, pH 7.0) and Denhardt`s solution: (1%
Albumin fraction V, 1% Polyvinylpyrrolidon K30, 1% Ficoll 400).
4.6 Primary mouse and human myoblast cultures
79
MATERIALS AND METHODS
Human skeletal muscle was obtained from elective surgery of the lower limb performed on
two healthy donors (males, age 20 and 22 years, BMI 22 kg/m2) at the University Hospital of Zurich.
The study protocol was approved by the local ethics committees. All subjects gave written informed
consent before enrollment.
Sorting of human myogenic progenitors was based on presence of CD56 expression and
absence of CD15, CD31 and CD45 staining, while mouse MP isolation was based on the presence of
α7-integrin and absence of Sca1, CD31 and CD45 staining. Mouse fibroadipogenic progenitors
(FAPs) were isolated based on the presence of Sca1 and absence of a7-integrin, CD31 and CD45.
In order to isolate progenitor populations, muscles from mice or human muscle biopsies were
minced and incubated consecutively with three fresh 5ml portions of 2mg/ml of collagenase II (Life
technologies) solution (prepared in 1x HBSS, 1.5% BSA) for 20 min. at 37°C in a water bath shaker.
After each 20 min. digestion, samples were briefly span (700rpm or 80g for 30 sec.) and the
supernatant with digested tissue was transferred into a tube with 5 ml of 5% FBS / DMEM solution for
collagenase neutralization, while the pellet was reconstituted in a next fresh portion of collagenase
solution. Cell samples were then filtered consecutively with 100um and 40um Falcon Nylon Mesh
Cell Strainers (BD Biocsiences). Erythrocytes were removed by 1 min incubation at 4°C with
erythrocyte lysis buffer (154 mMNH4Cl, 10mM KHCO3, 0.1mM EDTA). After each step (filtration,
erythrocyte lysis), cells were span at 1400rpm (350g) for 5 min. Then cells were stained with
antibodies for 45 min. in a rotating chamber at 4°C, washed three times, stained with 7AAD (Sigma,
1ul of 0.33mg/ml per 500ul of a sample) to exclude dead cells and sorted in a FACS facility. For
FACS we used following antibodies from Biolegend: PE anti-human CD56 (5ul / 500ul sample), APC
anti-human CD15 (0.2ul / 500ul sample), FITC anti-human CD31 (0.5ul / 500ul sample), alexa488
anti-human CD45 (0.5ul / 500ul sample), Alexa 488 anti-mouse CD45 (0.5ul / 500ul sample), Alexa
488 anti-mouse CD31 (0.5ul / 500ul sample), PE anti-mouse a7-integrin (5ul / 500ul sample), APC
anti-mouse Sca1 (0.5ul / 500ul sample)). Cells were sorted either directly into TRIzol® Reagent (Life
Technologies) for RNA extraction or into growth media for further culturing.
Cells were grown on collagen-coated plates (incubated for 24 hours in 0.1mg/ml Collagen I
(Life Technologies) water solution) in growth medium (1:1 v/v mix of F10 nutrient mixture and low
glucose DMEM, containing 20% fetal bovine serum (FBS), 1% Penicillin/Streptomycin solution and 5
ng/ml basic FGF, all from Life Technologies). Differentiation was initiated when myoblasts reached
subconfluency and induced by incubation with media containing 2% Horse Serum (Life technologies),
low glucose DMEM and 1% Penicillin/Streptomycin solution. Serum starvation was induced by
similar media, in which 2% Horse serum was replaced with 0.5% FBS.
FGFR1 inhibitor, PD173074 (Selleckchem) was used at 100nM conc. and incubated with cells
for 48 hours before harvesting. The media was changed after first 24 hours. 50mM stock solution of
80
MATERIALS AND METHODS
PD173074 prepared in DMSO was stored at -80°C. Each aliquot was first dissolved in 2ml of growth
media (1:1000) which then was used as a 500x stock for preparation final media solution.
Tamoxifen was dissolved in 100% ethanol and diluted 1:5000 in growth media (final
concentration was 20 nM) or stored in aliquots at -80°C. Cells were incubated with tamoxifen for a 48
h and then media was changed.
Human myotubes were incubated for the indicated times points with IGF1 or GH. We chose 1
ug/ml GH according to previous reports that demonstrated 500 – 1000 ng/ml GH to have maximal
effects on GH signaling in myoblasts and myotubes (278-280). For IGF1 incubations, we chose 76
ng/ml (10 nM), a concentration for which Baudry et al (281) demonstrated maximal effects on glucose
uptake and IGF1 signalling in myotubes.
4.7 Western blotting
Cell and tissue samples were lysed in RIPA buffer (25 mM Tris HCl pH7.6, 150 mM NaCl,
1% NP- 40, 1% sodium deoxycholate, 0,1% SDS, 1x cOmplete EDTA-free protease inhibitor cocktail
and 1x PhosSTOP phosphatase inhibitor cocktail, both from Roche) on ice, the cellular debris was
removed by centrifugation at max speed for 20 min. at 4°C. Then samples were sonicated for 20 sec.,
denaturated at 70°C for 10 min. and loaded to pre-cast gel (NuPage 4-12% Bis Tris gel, Life
technologies) in loading buffer (1x NuPage LDS Sample Buffer, 2.5 ul of NuPage Sample reducing
agent for 25ul of total volume, all from Life technologies). Protein concentration was determined
using Pierce BCA Protein Assay (thermos Scientific) in Biotek PowerWave 340 plate reader. Samples
were run in 1x NuPage MOPS SDS buffer (Life technologies) with 2.5% of NuPage antioxidant (Life
technologies) for 50 min. at 200 V and starting current at 100-125mA. Then proteins were transferred
to PVDF WB membrane in 1x NuPage Transfer Buffer (Life technologies), containing 10% of
methanol (Sigma) and 1% of NuPage antioxidant overnight at 10V.
Membrane was incubated with PONSO (1g Poinceau S, 50 ml acetic acid, glacial in 1L) for
protein staining, washed and blocked in 5% milk / TBST (100mM Tris, 1.5M NaCl, 0.1% Tween,
pH7.5) for 30 min at room temp. The membranes were stained with primary antibodies diluted in 5%
milk / TBST overnight at 4°C, washed three times for 10 min. with TBST and stained with secondary
antibodies in TBST. After washing three times, membranes were incubated with a 1:1 mix of Lumilight WB substrates 1 and 2 (Roche) and exposed in Luminiscent image analyser LAS-3000
(Fujifilm). Following primary antibodies were used: anti-alpha-tubulin (DSHB, 12G10, 1:500), antimyogenin (Santa Cruz, sc-12732, 1:200) and anti-adult myosin heavy chain (DSHB, MF-20, 1:100).
81
MATERIALS AND METHODS
4.8 Proliferation rate and mitochondrial activity in primary
myoblasts
Proliferation rate was measured using Click-iT® EdU Alexa Fluor® 647 Flow Cytometry Assay Kit
(Life technologies) according to manufacturer’s instructions. In brief: EdU was added at conc. 2.5uM
at 12-18 hours before harvesting. Cells were detached by incubation in 0.025% trypsin (Life
technologies) for 2 min., washed with 0.5% BSA/PBS and fixed in 3.7% Formaldehyde / PBS
(provided by the kit), then washed with saponin-containing buffer for membrane permeabilization and
incubated in buffer allowing conjugation of EdU and Alexa-647 in click reaction. Afterwards cells
were washed in saponin-containing buffer and analyzed with flow cytometry.
Mitochondria mass and mitochondrial membrane potential were measured using MitoTracker® Green
FM (MTK, Life technologies) and Tetramethylrhodamine (TMRE, Life technologies) respectively. In
brief, cells were trypsinized, washed with 0.5% BSA/PBS and counted. 200-300 thousands of cells in
each sample were incubated for 20 min. at 37°C with either 100 nM TMRE or 20 nM MTK solution.
Cells were span at 200g for 4 min and staining solutions were removed and replaced with 0.5% BSA
in PBS. Then cells were kept at +4°C and cell fluorescence was analyzed using flow cytometry.
4.9 Luciferase plasmid, antagomir, miRNA mimic and siRNA
transfection into primary mouse myoblasts
For MCK luciferase assay, primary myoblasts were transfected with MCK-luciferase plasmid (gift
from Robert Benezra (Addgene plasmid # 16062, 59) and pRL-TK plasmid (Promega, #E2241) using
lipofectamin 2000 (Life technologies, #11668-027), according to manufacturer’s instructions. Growth
media was exchanged after 24 h and analysis performed at 72 h after transfection using the DualLuciferase Reporter Assay (Promega, #1960). Primary myoblasts were transfected with small RNAs
using Lipofectamin RNAimax (Life technoges), according to manufacturer’s instructions. In brief, in
each well of 6-well plate small RNAs (1.3ul of 18pM/ul antagomir solution or 5ul of 20 pM/ul mimics
solution) were mixed with 5ul of lipofectamine RNAiMax in 400ul of low glucose DMEM (Life
Technologies) and incubated for 30 min. In the meantime, cells were trypsinized, washed and counted,
and finally 1600ul of cell suspension in growth media were added to a lipofectamine RNAiMAX/
RNA mixture. Growth media was exchanged after 24 h and analysis performed after the indicated time
periods. Human miRNA mimics (Mission, Sigma-Aldrich) were used at conc. 38 nM, while miRNA
inhibitors, antagomirs (Mission, Sigma-Aldrich, (214)) were used at 12nM conc. 1mg/ml antagomir
stock solutions were prepared by dissolving antagomirs in H2O at 37°C for 30 min.
82
MATERIALS AND METHODS
4.10 Statistical analyses
Data shown as relative units (rel U) are normalized to the average value of the control group. Groups
were compared using Student’s t-test in Excel software. Correlation analysis was performed using the
GraphPad Prism software. Fold-change regulations were analyzed using one-sample t-test with the
hypothetical means of 1 using the GraphPad Prism software. P-values smaller than 0.05 were
considered significant. Results are shown as means ± SEM.
83
CONTRIBUTIONS
CONTRIBUTIONS
Most of the experiments were done by Artur Galimov. Preparation of tissue sections,
immunohistochemical staining and analysis were done by Dr. Edlira Luca. Cryosectioning,
H&E, as well as trichrome stainings for fibrosis score analysis of miR-29a KO mice were
done by Dr. Elisabeth Rushing. Downhill exercise experiment and aerobic capacity test were
done with the help of Dr. Troy Merry. Human myoblast cell culturing, differentiation,
transfection, and treatment with growth factors were done by Dr. Angelika Hartung.
84
CURRICULUM VITAE
CURRICULUM VITAE
Artur R. Galimov
Weinbergstrasse 91,
Zürich 8006,
Date of birth: October 15, 1984
Nationality: Russian
Phone: +41-76-77-55-881
E-mail: [email protected]
EDUCATION
12.2010 – 05.2015
Doctorate in molecular biology,
Eidgenössische Technische Hochschule Zürich
Department of Health sciences and technology
PhD thesis: “The role of miR-29a in the regulation of
myogenic progenitor proliferation and adult skeletal muscle
insulin sensitivity”
09.2002 – 07.2007
MA (honors degree) in Bioengineering and bioinformatics,
Lomonosov Moscow State University,
Department of Bioengineering and Bioinformatics
 Diploma thesis: “Study of Chromosomal localization and
molecular organization of human genomic fragment
containing TNF/LT locus, in transgenic mice”
 GPA 4.7 out of 5
06.2005 – 07.2005
Summer student, Leiden University Medical Center, Leiden
Development of a method for computer search of
conserved regulatory motifs in untranslated regions of
genes in chromosome area linked to osteoarthritis
TECHNICAL
09.1992
– 06.2002
SKILLS
TEACHING
ACTIVITIES
09.2007 – 05.2008
Certificate of Secondary Education (honors degree),
Dmitrov, Moscow region
 Advanced in mathematics and physics
 GPA 4.8 out of 5
Supervision of one year-long student course project
Lomonosov Moscow State University,
Department of Bioengineering and Bioinformatics
85
CURRICULUM VITAE
 English (fluent)
 Russian (native)
LANGUAGES
PUBLICATIONS
1.
Galimov A. et al. “GH replacement therapy regulates
microRNA-29 and targets involved in insulin resistance”, J
Mol Med (Berl). 2015 Jul 23. [Epub ahead of print]
2.
Galimov A.R. et al “microRNA-29a controls skeletal muscle
regeneration during injury and exercise”, submitted to Stem
Cells
3.
Kuchmiy AA, Kruglov AA, Galimov AR et al. New TNF
reporter mouse to study TNF expression. 2011; Russ J
Immunol 5:205–214 (in russian, not represented in pubmed)
Liepinsh D.J., Kruglov A.A., Galimov A.R. et al.
4.
“Accelerated thymic atrophy as a result of elevated
homeostatic expression of the genes encoded by the
TNF/lymphotoxin cytokine locus” 2009; Eur J Immunol;
Oct;39(10):2906-15.
5.
SELECTED
CONFERENCES
Galimov A.R. et al, “Chromosomal localization and
molecular organization of human genomic fragment
containing TNF/LT locus in transgenic mice”. 2008; Mol Biol
(Mosk). 42(4):629-38.
1. Galimov A, Turcekova K, Krützfeldt J. “A novel FGFmicroRNA pathway provides a link between mitochondrial
activity and muscle differentiation”, 2013 Schweizerische
Gesellschaft für Endokrinologie und Diabetologie Tagung,
oral presentation
2. Efimov G.A., Galimov A.R., Kruglov A.A., Vakhrusheva
O.A., Sazykin A.Y., Nedospasov S.A. “Whole-Body
Molecular Imaging of Tumor Necrosis Factor in
Autoimmune and Infectious Disease Models”, 2009, II
International Symposium TOPICAL PROBLEMS OF
BIOPHOTONICS – 2009, poster presentation
3. Galimov A.R., Kruglov A.A., Kuprash D.V. and
Nedospasov S.A. “Analysis of engineered mice with
ectopic human TNF/LT loci”, 2008, XX international
congress in Genetics, session of immunogenetics, oral
presentation
86
REFERENCES
REFERENCES
1.
Frederic H. Martini JLN, Edwin F. Bartholomew. Fundamentals of Anatomy and Physiology.
Ninth Edition ed. San Francisco, CA 94111: Pearson Education, Inc.; 2012.
2.
Schmalbruch H, Lewis DM. Dynamics of nuclei of muscle fibers and connective tissue cells
in normal and denervated rat muscles. Muscle & nerve. 2000;23(4):617-626.
3.
Charge SB, Rudnicki MA. Cellular and molecular regulation of muscle regeneration.
Physiological reviews. 2004;84(1):209-238.
4.
Alderton JM, Steinhardt RA. How calcium influx through calcium leak channels is
responsible for the elevated levels of calcium-dependent proteolysis in dystrophic myotubes.
Trends in cardiovascular medicine. 2000;10(6):268-272.
5.
Belcastro AN, Shewchuk LD, Raj DA. Exercise-induced muscle injury: a calpain hypothesis.
Molecular and cellular biochemistry. 1998;179(1-2):135-145.
6.
Tidball JG. Inflammatory cell response to acute muscle injury. Medicine and science in sports
and exercise. 1995;27(7):1022-1032.
7.
Hawke TJ, Garry DJ. Myogenic satellite cells: physiology to molecular biology. Journal of
applied physiology. 2001;91(2):534-551.
8.
Fielding RA, Manfredi TJ, Ding W, Fiatarone MA, Evans WJ, Cannon JG. Acute phase
response in exercise. III. Neutrophil and IL-1 beta accumulation in skeletal muscle. The
American journal of physiology. 1993;265(1 Pt 2):R166-172.
9.
Lescaudron L, Peltekian E, Fontaine-Perus J, Paulin D, Zampieri M, Garcia L, Parrish E.
Blood borne macrophages are essential for the triggering of muscle regeneration following
muscle transplant. Neuromuscular disorders : NMD. 1999;9(2):72-80.
10.
Merly F, Lescaudron L, Rouaud T, Crossin F, Gardahaut MF. Macrophages enhance muscle
satellite cell proliferation and delay their differentiation. Muscle & nerve. 1999;22(6):724-732.
11.
Darr KC, Schultz E. Exercise-induced satellite cell activation in growing and mature skeletal
muscle. Journal of applied physiology. 1987;63(5):1816-1821.
12.
Snow MH. An autoradiographic study of satellite cell differentiation into regenerating
myotubes following transplantation of muscles in young rats. Cell and tissue research.
1978;186(3):535-540.
13.
Halevy O, Piestun Y, Allouh MZ, Rosser BW, Rinkevich Y, Reshef R, Rozenboim I,
Wleklinski-Lee M, Yablonka-Reuveni Z. Pattern of Pax7 expression during myogenesis in the
posthatch chicken establishes a model for satellite cell differentiation and renewal.
Developmental dynamics : an official publication of the American Association of Anatomists.
2004;231(3):489-502.
14.
Zammit PS, Partridge TA, Yablonka-Reuveni Z. The skeletal muscle satellite cell: the stem
cell that came in from the cold. The journal of histochemistry and cytochemistry : official
journal of the Histochemistry Society. 2006;54(11):1177-1191.
15.
Blaveri K, Heslop L, Yu DS, Rosenblatt JD, Gross JG, Partridge TA, Morgan JE. Patterns of
repair of dystrophic mouse muscle: studies on isolated fibers. Developmental dynamics : an
official publication of the American Association of Anatomists. 1999;216(3):244-256.
87
REFERENCES
16.
d'Albis A, Couteaux R, Janmot C, Roulet A, Mira JC. Regeneration after cardiotoxin injury of
innervated and denervated slow and fast muscles of mammals. Myosin isoform analysis.
European journal of biochemistry / FEBS. 1988;174(1):103-110.
17.
Hall-Craggs EC. Rapid degeneration and regeneration of a whole skeletal muscle following
treatment with bupivacaine (Marcain). Experimental neurology. 1974;43(2):349-358.
18.
Vracko R, Benditt EP. Basal lamina: the scaffold for orderly cell replacement. Observations
on regeneration of injured skeletal muscle fibers and capillaries. The Journal of cell biology.
1972;55(2):406-419.
19.
Mauro A. Satellite cell of skeletal muscle fibers. The Journal of biophysical and biochemical
cytology. 1961;9:493-495.
20.
Schultz E, Gibson MC, Champion T. Satellite cells are mitotically quiescent in mature mouse
muscle: an EM and radioautographic study. The Journal of experimental zoology.
1978;206(3):451-456.
21.
Abou-Khalil R, Le Grand F, Pallafacchina G, Valable S, Authier FJ, Rudnicki MA, Gherardi
RK, Germain S, Chretien F, Sotiropoulos A, Lafuste P, Montarras D, Chazaud B. Autocrine
and paracrine angiopoietin 1/Tie-2 signaling promotes muscle satellite cell self-renewal. Cell
stem cell. 2009;5(3):298-309.
22.
Gibson MC, Schultz E. Age-related differences in absolute numbers of skeletal muscle
satellite cells. Muscle & nerve. 1983;6(8):574-580.
23.
Bockhold KJ, Rosenblatt JD, Partridge TA. Aging normal and dystrophic mouse muscle:
analysis of myogenicity in cultures of living single fibers. Muscle & nerve. 1998;21(2):173183.
24.
Charge SB, Brack AS, Hughes SM. Aging-related satellite cell differentiation defect occurs
prematurely after Ski-induced muscle hypertrophy. American journal of physiology Cell
physiology. 2002;283(4):C1228-1241.
25.
Hubank M, Schatz DG. Identifying differences in mRNA expression by representational
difference analysis of cDNA. Nucleic Acids Res. 1994;22(25):5640-5648.
26.
Seale P, Sabourin LA, Girgis-Gabardo A, Mansouri A, Gruss P, Rudnicki MA. Pax7 is
required for the specification of myogenic satellite cells. Cell. 2000;102(6):777-786.
27.
Sacco A, Doyonnas R, Kraft P, Vitorovic S, Blau HM. Self-renewal and expansion of single
transplanted muscle stem cells. Nature. 2008;456(7221):502-506.
28.
Blanco-Bose WE, Yao CC, Kramer RH, Blau HM. Purification of mouse primary myoblasts
based on alpha 7 integrin expression. Experimental cell research. 2001;265(2):212-220.
29.
Kuang S, Gillespie MA, Rudnicki MA. Niche regulation of muscle satellite cell self-renewal
and differentiation. Cell stem cell. 2008;2(1):22-31.
30.
Shinin V, Gayraud-Morel B, Gomes D, Tajbakhsh S. Asymmetric division and cosegregation
of template DNA strands in adult muscle satellite cells. Nature cell biology. 2006;8(7):677687.
31.
Peault B, Rudnicki M, Torrente Y, Cossu G, Tremblay JP, Partridge T, Gussoni E, Kunkel
LM, Huard J. Stem and progenitor cells in skeletal muscle development, maintenance, and
therapy. Molecular therapy : the journal of the American Society of Gene Therapy.
2007;15(5):867-877.
88
REFERENCES
32.
Zammit PS, Golding JP, Nagata Y, Hudon V, Partridge TA, Beauchamp JR. Muscle satellite
cells adopt divergent fates: a mechanism for self-renewal? The Journal of cell biology.
2004;166(3):347-357.
33.
Schultz E, Jaryszak DL, Valliere CR. Response of satellite cells to focal skeletal muscle
injury. Muscle & nerve. 1985;8(3):217-222.
34.
Braun T, Bober E, Winter B, Rosenthal N, Arnold HH. Myf-6, a new member of the human
gene family of myogenic determination factors: evidence for a gene cluster on chromosome
12. The EMBO journal. 1990;9(3):821-831.
35.
Edmondson DG, Olson EN. A gene with homology to the myc similarity region of MyoD1 is
expressed during myogenesis and is sufficient to activate the muscle differentiation program.
Genes & development. 1989;3(5):628-640.
36.
Braun T, Buschhausen-Denker G, Bober E, Tannich E, Arnold HH. A novel human muscle
factor related to but distinct from MyoD1 induces myogenic conversion in 10T1/2 fibroblasts.
The EMBO journal. 1989;8(3):701-709.
37.
Rhodes SJ, Konieczny SF. Identification of MRF4: a new member of the muscle regulatory
factor gene family. Genes & development. 1989;3(12B):2050-2061.
38.
Massari ME, Murre C. Helix-loop-helix proteins: regulators of transcription in eucaryotic
organisms. Molecular and cellular biology. 2000;20(2):429-440.
39.
Cornelison DD, Wold BJ. Single-cell analysis of regulatory gene expression in quiescent and
activated mouse skeletal muscle satellite cells. Developmental biology. 1997;191(2):270-283.
40.
Cooper RN, Tajbakhsh S, Mouly V, Cossu G, Buckingham M, Butler-Browne GS. In vivo
satellite cell activation via Myf5 and MyoD in regenerating mouse skeletal muscle. Journal of
cell science. 1999;112 ( Pt 17):2895-2901.
41.
Smith CK, 2nd, Janney MJ, Allen RE. Temporal expression of myogenic regulatory genes
during activation, proliferation, and differentiation of rat skeletal muscle satellite cells.
Journal of cellular physiology. 1994;159(2):379-385.
42.
Rudnicki MA, Braun T, Hinuma S, Jaenisch R. Inactivation of MyoD in mice leads to upregulation of the myogenic HLH gene Myf-5 and results in apparently normal muscle
development. Cell. 1992;71(3):383-390.
43.
Braun T, Rudnicki MA, Arnold HH, Jaenisch R. Targeted inactivation of the muscle
regulatory gene Myf-5 results in abnormal rib development and perinatal death. Cell.
1992;71(3):369-382.
44.
Rudnicki MA, Schnegelsberg PN, Stead RH, Braun T, Arnold HH, Jaenisch R. MyoD or Myf5 is required for the formation of skeletal muscle. Cell. 1993;75(7):1351-1359.
45.
Hasty P, Bradley A, Morris JH, Edmondson DG, Venuti JM, Olson EN, Klein WH. Muscle
deficiency and neonatal death in mice with a targeted mutation in the myogenin gene. Nature.
1993;364(6437):501-506.
46.
Zhou Z, Bornemann A. MRF4 protein expression in regenerating rat muscle. Journal of
muscle research and cell motility. 2001;22(4):311-316.
47.
Kassar-Duchossoy L, Giacone E, Gayraud-Morel B, Jory A, Gomes D, Tajbakhsh S.
Pax3/Pax7 mark a novel population of primitive myogenic cells during development. Genes &
development. 2005;19(12):1426-1431.
89
REFERENCES
48.
Bober E, Franz T, Arnold HH, Gruss P, Tremblay P. Pax-3 is required for the development of
limb muscles: a possible role for the migration of dermomyotomal muscle progenitor cells.
Development. 1994;120(3):603-612.
49.
Daston G, Lamar E, Olivier M, Goulding M. Pax-3 is necessary for migration but not
differentiation of limb muscle precursors in the mouse. Development. 1996;122(3):1017-1027.
50.
Hutcheson DA, Zhao J, Merrell A, Haldar M, Kardon G. Embryonic and fetal limb myogenic
cells are derived from developmentally distinct progenitors and have different requirements
for beta-catenin. Genes & development. 2009;23(8):997-1013.
51.
Maqbool T, Jagla K. Genetic control of muscle development: learning from Drosophila.
Journal of muscle research and cell motility. 2007;28(7-8):397-407.
52.
Bentzinger CF, Wang YX, Rudnicki MA. Building muscle: molecular regulation of
myogenesis. Cold Spring Harbor perspectives in biology. 2012;4(2).
53.
Grifone R, Demignon J, Houbron C, Souil E, Niro C, Seller MJ, Hamard G, Maire P. Six1 and
Six4 homeoproteins are required for Pax3 and Mrf expression during myogenesis in the
mouse embryo. Development. 2005;132(9):2235-2249.
54.
Grifone R, Demignon J, Giordani J, Niro C, Souil E, Bertin F, Laclef C, Xu PX, Maire P.
Eya1 and Eya2 proteins are required for hypaxial somitic myogenesis in the mouse embryo.
Developmental biology. 2007;302(2):602-616.
55.
Bischoff R. A satellite cell mitogen from crushed adult muscle. Developmental biology.
1986;115(1):140-147.
56.
Tatsumi R, Liu X, Pulido A, Morales M, Sakata T, Dial S, Hattori A, Ikeuchi Y, Allen RE.
Satellite cell activation in stretched skeletal muscle and the role of nitric oxide and hepatocyte
growth factor. American journal of physiology Cell physiology. 2006;290(6):C1487-1494.
57.
Tatsumi R, Anderson JE, Nevoret CJ, Halevy O, Allen RE. HGF/SF is present in normal adult
skeletal muscle and is capable of activating satellite cells. Developmental biology.
1998;194(1):114-128.
58.
Suzuki J, Yamazaki Y, Li G, Kaziro Y, Koide H. Involvement of Ras and Ral in chemotactic
migration of skeletal myoblasts. Molecular and cellular biology. 2000;20(13):4658-4665.
59.
Lefaucheur JP, Sebille A. Basic fibroblast growth factor promotes in vivo muscle regeneration
in murine muscular dystrophy. Neuroscience letters. 1995;202(1-2):121-124.
60.
Floss T, Arnold HH, Braun T. A role for FGF-6 in skeletal muscle regeneration. Genes &
development. 1997;11(16):2040-2051.
61.
Fiore F, Sebille A, Birnbaum D. Skeletal muscle regeneration is not impaired in Fgf6 -/mutant mice. Biochem Biophys Res Commun. 2000;272(1):138-143.
62.
Kastner S, Elias MC, Rivera AJ, Yablonka-Reuveni Z. Gene expression patterns of the
fibroblast growth factors and their receptors during myogenesis of rat satellite cells. The
journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.
2000;48(8):1079-1096.
63.
Neuhaus P, Oustanina S, Loch T, Kruger M, Bober E, Dono R, Zeller R, Braun T. Reduced
mobility of fibroblast growth factor (FGF)-deficient myoblasts might contribute to dystrophic
changes in the musculature of FGF2/FGF6/mdx triple-mutant mice. Molecular and cellular
biology. 2003;23(17):6037-6048.
90
REFERENCES
64.
Lefaucheur JP, Sebille A. Muscle regeneration following injury can be modified in vivo by
immune neutralization of basic fibroblast growth factor, transforming growth factor beta 1 or
insulin-like growth factor I. Journal of neuroimmunology. 1995;57(1-2):85-91.
65.
Ornitz DM, Xu J, Colvin JS, McEwen DG, MacArthur CA, Coulier F, Gao G, Goldfarb M.
Receptor specificity of the fibroblast growth factor family. The Journal of biological
chemistry. 1996;271(25):15292-15297.
66.
Sheehan SM, Allen RE. Skeletal muscle satellite cell proliferation in response to members of
the fibroblast growth factor family and hepatocyte growth factor. Journal of cellular
physiology. 1999;181(3):499-506.
67.
Zhao P, Hoffman EP. Embryonic myogenesis pathways in muscle regeneration.
Developmental dynamics : an official publication of the American Association of Anatomists.
2004;229(2):380-392.
68.
Turner N, Grose R. Fibroblast growth factor signalling: from development to cancer. Nature
reviews Cancer. 2010;10(2):116-129.
69.
Sarrazin S, Lamanna WC, Esko JD. Heparan sulfate proteoglycans. Cold Spring Harbor
perspectives in biology. 2011;3(7).
70.
Coleman ME, DeMayo F, Yin KC, Lee HM, Geske R, Montgomery C, Schwartz RJ.
Myogenic vector expression of insulin-like growth factor I stimulates muscle cell
differentiation and myofiber hypertrophy in transgenic mice. The Journal of biological
chemistry. 1995;270(20):12109-12116.
71.
Florini JR, Ewton DZ, Roof SL. Insulin-like growth factor-I stimulates terminal myogenic
differentiation by induction of myogenin gene expression. Molecular endocrinology.
1991;5(5):718-724.
72.
Barton-Davis ER, Shoturma DI, Sweeney HL. Contribution of satellite cells to IGF-I induced
hypertrophy of skeletal muscle. Acta physiologica Scandinavica. 1999;167(4):301-305.
73.
Musaro A, McCullagh KJ, Naya FJ, Olson EN, Rosenthal N. IGF-1 induces skeletal myocyte
hypertrophy through calcineurin in association with GATA-2 and NF-ATc1. Nature.
1999;400(6744):581-585.
74.
Bark TH, McNurlan MA, Lang CH, Garlick PJ. Increased protein synthesis after acute IGF-I
or insulin infusion is localized to muscle in mice. The American journal of physiology.
1998;275(1 Pt 1):E118-123.
75.
Krishan K, Dhoot GK. Changes in some troponin and insulin-like growth factor messenger
ribonucleic acids in regenerating and denervated skeletal muscles. Journal of muscle research
and cell motility. 1996;17(5):513-521.
76.
Musaro A, McCullagh K, Paul A, Houghton L, Dobrowolny G, Molinaro M, Barton ER,
Sweeney HL, Rosenthal N. Localized Igf-1 transgene expression sustains hypertrophy and
regeneration in senescent skeletal muscle. Nature genetics. 2001;27(2):195-200.
77.
Barton ER, Morris L, Musaro A, Rosenthal N, Sweeney HL. Muscle-specific expression of
insulin-like growth factor I counters muscle decline in mdx mice. The Journal of cell biology.
2002;157(1):137-148.
78.
Lawlor MA, Rotwein P. Insulin-like growth factor-mediated muscle cell survival: central roles
for Akt and cyclin-dependent kinase inhibitor p21. Molecular and cellular biology.
2000;20(23):8983-8995.
91
REFERENCES
79.
McPherron AC, Lawler AM, Lee SJ. Regulation of skeletal muscle mass in mice by a new
TGF-beta superfamily member. Nature. 1997;387(6628):83-90.
80.
Grobet L, Martin LJ, Poncelet D, Pirottin D, Brouwers B, Riquet J, Schoeberlein A, Dunner S,
Menissier F, Massabanda J, Fries R, Hanset R, Georges M. A deletion in the bovine myostatin
gene causes the double-muscled phenotype in cattle. Nature genetics. 1997;17(1):71-74.
81.
Schuelke M, Wagner KR, Stolz LE, Hubner C, Riebel T, Komen W, Braun T, Tobin JF, Lee
SJ. Myostatin mutation associated with gross muscle hypertrophy in a child. The New England
journal of medicine. 2004;350(26):2682-2688.
82.
Carlson CJ, Booth FW, Gordon SE. Skeletal muscle myostatin mRNA expression is fiber-type
specific and increases during hindlimb unloading. The American journal of physiology.
1999;277(2 Pt 2):R601-606.
83.
Thomas M, Langley B, Berry C, Sharma M, Kirk S, Bass J, Kambadur R. Myostatin, a
negative regulator of muscle growth, functions by inhibiting myoblast proliferation. The
Journal of biological chemistry. 2000;275(51):40235-40243.
84.
Taylor WE, Bhasin S, Artaza J, Byhower F, Azam M, Willard DH, Jr., Kull FC, Jr., GonzalezCadavid N. Myostatin inhibits cell proliferation and protein synthesis in C2C12 muscle cells.
American journal of physiology Endocrinology and metabolism. 2001;280(2):E221-228.
85.
Amthor H, Otto A, Vulin A, Rochat A, Dumonceaux J, Garcia L, Mouisel E, Hourde C,
Macharia R, Friedrichs M, Relaix F, Zammit PS, Matsakas A, Patel K, Partridge T. Muscle
hypertrophy driven by myostatin blockade does not require stem/precursor-cell activity. Proc
Natl Acad Sci U S A. 2009;106(18):7479-7484.
86.
Wang Q, McPherron AC. Myostatin inhibition induces muscle fibre hypertrophy prior to
satellite cell activation. The Journal of physiology. 2012;590(Pt 9):2151-2165.
87.
Lee SJ, Huynh TV, Lee YS, Sebald SM, Wilcox-Adelman SA, Iwamori N, Lepper C, Matzuk
MM, Fan CM. Role of satellite cells versus myofibers in muscle hypertrophy induced by
inhibition of the myostatin/activin signaling pathway. Proc Natl Acad Sci U S A.
2012;109(35):E2353-2360.
88.
Sanes JR. The basement membrane/basal lamina of skeletal muscle. The Journal of biological
chemistry. 2003;278(15):12601-12604.
89.
Mayer U. Integrins: redundant or important players in skeletal muscle? The Journal of
biological chemistry. 2003;278(17):14587-14590.
90.
LeBleu VS, Macdonald B, Kalluri R. Structure and function of basement membranes.
Experimental biology and medicine. 2007;232(9):1121-1129.
91.
Aviezer D, Hecht D, Safran M, Eisinger M, David G, Yayon A. Perlecan, basal lamina
proteoglycan, promotes basic fibroblast growth factor-receptor binding, mitogenesis, and
angiogenesis. Cell. 1994;79(6):1005-1013.
92.
Murakami M, Horowitz A, Tang S, Ware JA, Simons M. Protein kinase C (PKC) delta
regulates PKCalpha activity in a Syndecan-4-dependent manner. The Journal of biological
chemistry. 2002;277(23):20367-20371.
93.
Carmignac V, Durbeej M. Cell-matrix interactions in muscle disease. The Journal of
pathology. 2012;226(2):200-218.
94.
Arikawa-Hirasawa E, Le AH, Nishino I, Nonaka I, Ho NC, Francomano CA, Govindraj P,
Hassell JR, Devaney JM, Spranger J, Stevenson RE, Iannaccone S, Dalakas MC, Yamada Y.
92
REFERENCES
Structural and functional mutations of the perlecan gene cause Schwartz-Jampel syndrome,
with myotonic myopathy and chondrodysplasia. American journal of human genetics.
2002;70(5):1368-1375.
95.
Ciciliot S, Schiaffino S. Regeneration of mammalian skeletal muscle. Basic mechanisms and
clinical implications. Current pharmaceutical design. 2010;16(8):906-914.
96.
Sanes JR, Marshall LM, McMahan UJ. Reinnervation of muscle fiber basal lamina after
removal of myofibers. Differentiation of regenerating axons at original synaptic sites. The
Journal of cell biology. 1978;78(1):176-198.
97.
Breitkreutz D, Koxholt I, Thiemann K, Nischt R. Skin basement membrane: the foundation of
epidermal integrity--BM functions and diverse roles of bridging molecules nidogen and
perlecan. BioMed research international. 2013;2013:179784.
98.
Shulman GI, Rothman DL, Jue T, Stein P, DeFronzo RA, Shulman RG. Quantitation of
muscle glycogen synthesis in normal subjects and subjects with non-insulin-dependent
diabetes by 13C nuclear magnetic resonance spectroscopy. The New England journal of
medicine. 1990;322(4):223-228.
99.
Smith C. Marks Basic Medical Biochemistry A Clinical Approach. Second edition ed:
Lippincott Williams & Wilkins; 2003.
100.
Stump CS, Henriksen EJ, Wei Y, Sowers JR. The metabolic syndrome: role of skeletal muscle
metabolism. Annals of medicine. 2006;38(6):389-402.
101.
Zalin R. Creatine kinase activity in cultures of differentiating myoblasts. The Biochemical
journal. 1972;130(2):79P.
102.
Baird MF, Graham SM, Baker JS, Bickerstaff GF. Creatine-kinase- and exercise-related
muscle damage implications for muscle performance and recovery. Journal of nutrition and
metabolism. 2012;2012:960363.
103.
Lillioja S, Mott DM, Howard BV, Bennett PH, Yki-Jarvinen H, Freymond D, Nyomba BL,
Zurlo F, Swinburn B, Bogardus C. Impaired glucose tolerance as a disorder of insulin action.
Longitudinal and cross-sectional studies in Pima Indians. The New England journal of
medicine. 1988;318(19):1217-1225.
104.
Warram JH, Martin BC, Krolewski AS, Soeldner JS, Kahn CR. Slow glucose removal rate
and hyperinsulinemia precede the development of type II diabetes in the offspring of diabetic
parents. Annals of internal medicine. 1990;113(12):909-915.
105.
Perseghin G, Ghosh S, Gerow K, Shulman GI. Metabolic defects in lean nondiabetic offspring
of NIDDM parents: a cross-sectional study. Diabetes. 1997;46(6):1001-1009.
106.
Kashyap SR, Belfort R, Berria R, Suraamornkul S, Pratipranawatr T, Finlayson J, Barrentine
A, Bajaj M, Mandarino L, DeFronzo R, Cusi K. Discordant effects of a chronic physiological
increase in plasma FFA on insulin signaling in healthy subjects with or without a family
history of type 2 diabetes. American journal of physiology Endocrinology and metabolism.
2004;287(3):E537-546.
107.
Vaag A, Henriksen JE, Beck-Nielsen H. Decreased insulin activation of glycogen synthase in
skeletal muscles in young nonobese Caucasian first-degree relatives of patients with noninsulin-dependent diabetes mellitus. The Journal of clinical investigation. 1992;89(3):782788.
93
REFERENCES
108.
Gulli G, Ferrannini E, Stern M, Haffner S, DeFronzo RA. The metabolic profile of NIDDM is
fully established in glucose-tolerant offspring of two Mexican-American NIDDM parents.
Diabetes. 1992;41(12):1575-1586.
109.
Weyer C, Bogardus C, Mott DM, Pratley RE. The natural history of insulin secretory
dysfunction and insulin resistance in the pathogenesis of type 2 diabetes mellitus. The Journal
of clinical investigation. 1999;104(6):787-794.
110.
Iozzo P, Pratipanawatr T, Pijl H, Vogt C, Kumar V, Pipek R, Matsuda M, Mandarino LJ, Cusi
KJ, DeFronzo RA. Physiological hyperinsulinemia impairs insulin-stimulated glycogen
synthase activity and glycogen synthesis. American journal of physiology Endocrinology and
metabolism. 2001;280(5):E712-719.
111.
Krook A, Bjornholm M, Galuska D, Jiang XJ, Fahlman R, Myers MG, Jr., WallbergHenriksson H, Zierath JR. Characterization of signal transduction and glucose transport in
skeletal muscle from type 2 diabetic patients. Diabetes. 2000;49(2):284-292.
112.
Morino K, Petersen KF, Dufour S, Befroy D, Frattini J, Shatzkes N, Neschen S, White MF,
Bilz S, Sono S, Pypaert M, Shulman GI. Reduced mitochondrial density and increased IRS-1
serine phosphorylation in muscle of insulin-resistant offspring of type 2 diabetic parents. The
Journal of clinical investigation. 2005;115(12):3587-3593.
113.
Petersen KF, Dufour S, Shulman GI. Decreased insulin-stimulated ATP synthesis and
phosphate transport in muscle of insulin-resistant offspring of type 2 diabetic parents. PLoS
medicine. 2005;2(9):e233.
114.
Hotamisligil GS, Peraldi P, Budavari A, Ellis R, White MF, Spiegelman BM. IRS-1-mediated
inhibition of insulin receptor tyrosine kinase activity in TNF-alpha- and obesity-induced
insulin resistance. Science. 1996;271(5249):665-668.
115.
Pouliot MC, Despres JP, Nadeau A, Moorjani S, Prud'Homme D, Lupien PJ, Tremblay A,
Bouchard C. Visceral obesity in men. Associations with glucose tolerance, plasma insulin, and
lipoprotein levels. Diabetes. 1992;41(7):826-834.
116.
Mootha VK, Lindgren CM, Eriksson KF, Subramanian A, Sihag S, Lehar J, Puigserver P,
Carlsson E, Ridderstrale M, Laurila E, Houstis N, Daly MJ, Patterson N, Mesirov JP, Golub
TR, Tamayo P, Spiegelman B, Lander ES, Hirschhorn JN, Altshuler D, Groop LC. PGC1alpha-responsive genes involved in oxidative phosphorylation are coordinately
downregulated in human diabetes. Nature genetics. 2003;34(3):267-273.
117.
Patti ME, Butte AJ, Crunkhorn S, Cusi K, Berria R, Kashyap S, Miyazaki Y, Kohane I,
Costello M, Saccone R, Landaker EJ, Goldfine AB, Mun E, DeFronzo R, Finlayson J, Kahn
CR, Mandarino LJ. Coordinated reduction of genes of oxidative metabolism in humans with
insulin resistance and diabetes: Potential role of PGC1 and NRF1. Proceedings of the National
Academy of Sciences of the United States of America. 2003;100(14):8466-8471.
118.
Petersen KF, Dufour S, Befroy D, Garcia R, Shulman GI. Impaired mitochondrial activity in
the insulin-resistant offspring of patients with type 2 diabetes. The New England journal of
medicine. 2004;350(7):664-671.
119.
Kelley DE, He J, Menshikova EV, Ritov VB. Dysfunction of mitochondria in human skeletal
muscle in type 2 diabetes. Diabetes. 2002;51(10):2944-2950.
120.
Stump CS, Short KR, Bigelow ML, Schimke JM, Nair KS. Effect of insulin on human skeletal
muscle mitochondrial ATP production, protein synthesis, and mRNA transcripts. Proceedings
of the National Academy of Sciences of the United States of America. 2003;100(13):79968001.
94
REFERENCES
121.
Raben MS. Growth hormone. 1. Physiologic aspects. The New England journal of medicine.
1962;266:31-35.
122.
Henneman PH, Forbes AP, Moldawer M, Dempsey EF, Carroll EL. Effects of human growth
hormone in man. The Journal of clinical investigation. 1960;39:1223-1238.
123.
Djurhuus CB, Gravholt CH, Nielsen S, Pedersen SB, Moller N, Schmitz O. Additive effects of
cortisol and growth hormone on regional and systemic lipolysis in humans. American journal
of physiology Endocrinology and metabolism. 2004;286(3):E488-494.
124.
Ho KY, Veldhuis JD, Johnson ML, Furlanetto R, Evans WS, Alberti KG, Thorner MO.
Fasting enhances growth hormone secretion and amplifies the complex rhythms of growth
hormone secretion in man. The Journal of clinical investigation. 1988;81(4):968-975.
125.
Wurzburger MI, Prelevic GM, Sonksen PH, Balint-Peric LA, Wheeler M. The effect of
recombinant human growth hormone on regulation of growth hormone secretion and blood
glucose in insulin-dependent diabetes. The Journal of clinical endocrinology and metabolism.
1993;77(1):267-272.
126.
Moller N, Jorgensen JO, Alberti KG, Flyvbjerg A, Schmitz O. Short-term effects of growth
hormone on fuel oxidation and regional substrate metabolism in normal man. The Journal of
clinical endocrinology and metabolism. 1990;70(4):1179-1186.
127.
Bak JF, Moller N, Schmitz O. Effects of growth hormone on fuel utilization and muscle
glycogen synthase activity in normal humans. The American journal of physiology.
1991;260(5 Pt 1):E736-742.
128.
Krag MB, Gormsen LC, Guo Z, Christiansen JS, Jensen MD, Nielsen S, Jorgensen JO.
Growth hormone-induced insulin resistance is associated with increased intramyocellular
triglyceride content but unaltered VLDL-triglyceride kinetics. American journal of physiology
Endocrinology and metabolism. 2007;292(3):E920-927.
129.
Roden M, Price TB, Perseghin G, Petersen KF, Rothman DL, Cline GW, Shulman GI.
Mechanism of free fatty acid-induced insulin resistance in humans. The Journal of clinical
investigation. 1996;97(12):2859-2865.
130.
Rizza RA, Mandarino LJ, Gerich JE. Effects of growth hormone on insulin action in man.
Mechanisms of insulin resistance, impaired suppression of glucose production, and impaired
stimulation of glucose utilization. Diabetes. 1982;31(8 Pt 1):663-669.
131.
Moller N, Butler PC, Antsiferov MA, Alberti KG. Effects of growth hormone on insulin
sensitivity and forearm metabolism in normal man. Diabetologia. 1989;32(2):105-110.
132.
Stochholm K, Gravholt CH, Laursen T, Jorgensen JO, Laurberg P, Andersen M, Kristensen
LO, Feldt-Rasmussen U, Christiansen JS, Frydenberg M, Green A. Incidence of GH
deficiency - a nationwide study. European journal of endocrinology / European Federation of
Endocrine Societies. 2006;155(1):61-71.
133.
Carroll PV, Christ ER, Bengtsson BA, Carlsson L, Christiansen JS, Clemmons D, Hintz R, Ho
K, Laron Z, Sizonenko P, Sonksen PH, Tanaka T, Thorne M. Growth hormone deficiency in
adulthood and the effects of growth hormone replacement: a review. Growth Hormone
Research Society Scientific Committee. The Journal of clinical endocrinology and
metabolism. 1998;83(2):382-395.
134.
Bougneres PF, Artavia-Loria E, Ferre P, Chaussain JL, Job JC. Effects of hypopituitarism and
growth hormone replacement therapy on the production and utilization of glucose in
childhood. The Journal of clinical endocrinology and metabolism. 1985;61(6):1152-1157.
95
REFERENCES
135.
Johansson JO, Fowelin J, Landin K, Lager I, Bengtsson BA. Growth hormone-deficient adults
are insulin-resistant. Metabolism: clinical and experimental. 1995;44(9):1126-1129.
136.
Panici JA, Wang F, Bonkowski MS, Spong A, Bartke A, Pawlikowska L, Kwok PY,
Masternak MM. Is altered expression of hepatic insulin-related genes in growth hormone
receptor knockout mice due to GH resistance or a difference in biological life spans? The
journals of gerontology Series A, Biological sciences and medical sciences.
2009;64(11):1126-1133.
137.
Liu JL, Coschigano KT, Robertson K, Lipsett M, Guo Y, Kopchick JJ, Kumar U, Liu YL.
Disruption of growth hormone receptor gene causes diminished pancreatic islet size and
increased insulin sensitivity in mice. American journal of physiology Endocrinology and
metabolism. 2004;287(3):E405-413.
138.
Guo Y, Lu Y, Houle D, Robertson K, Tang Z, Kopchick JJ, Liu YL, Liu JL. Pancreatic isletspecific expression of an insulin-like growth factor-I transgene compensates islet cell growth
in growth hormone receptor gene-deficient mice. Endocrinology. 2005;146(6):2602-2609.
139.
Hoffman AR, Kuntze JE, Baptista J, Baum HB, Baumann GP, Biller BM, Clark RV, Cook D,
Inzucchi SE, Kleinberg D, Klibanski A, Phillips LS, Ridgway EC, Robbins RJ, Schlechte J,
Sharma M, Thorner MO, Vance ML. Growth hormone (GH) replacement therapy in adultonset gh deficiency: effects on body composition in men and women in a double-blind,
randomized, placebo-controlled trial. The Journal of clinical endocrinology and metabolism.
2004;89(5):2048-2056.
140.
O'Neal DN, Kalfas A, Dunning PL, Christopher MJ, Sawyer SD, Ward GM, Alford FP. The
effect of 3 months of recombinant human growth hormone (GH) therapy on insulin and
glucose-mediated glucose disposal and insulin secretion in GH-deficient adults: a minimal
model analysis. The Journal of clinical endocrinology and metabolism. 1994;79(4):975-983.
141.
Svensson J, Fowelin J, Landin K, Bengtsson BA, Johansson JO. Effects of seven years of GHreplacement therapy on insulin sensitivity in GH-deficient adults. The Journal of clinical
endocrinology and metabolism. 2002;87(5):2121-2127.
142.
Christopher M, Hew FL, Oakley M, Rantzau C, Alford F. Defects of insulin action and
skeletal muscle glucose metabolism in growth hormone-deficient adults persist after 24
months of recombinant human growth hormone therapy. The Journal of clinical
endocrinology and metabolism. 1998;83(5):1668-1681.
143.
Bramnert M, Segerlantz M, Laurila E, Daugaard JR, Manhem P, Groop L. Growth hormone
replacement therapy induces insulin resistance by activating the glucose-fatty acid cycle. The
Journal of clinical endocrinology and metabolism. 2003;88(4):1455-1463.
144.
Norrelund H, Vahl N, Juul A, Moller N, Alberti KG, Skakkebaek NE, Christiansen JS,
Jorgensen JO. Continuation of growth hormone (GH) therapy in GH-deficient patients during
transition from childhood to adulthood: impact on insulin sensitivity and substrate
metabolism. The Journal of clinical endocrinology and metabolism. 2000;85(5):1912-1917.
145.
Friedman RC, Farh KK, Burge CB, Bartel DP. Most mammalian mRNAs are conserved
targets of microRNAs. Genome research. 2009;19(1):92-105.
146.
Krol J, Loedige I, Filipowicz W. The widespread regulation of microRNA biogenesis,
function and decay. Nature reviews Genetics. 2010;11(9):597-610.
147.
Carthew RW, Sontheimer EJ. Origins and Mechanisms of miRNAs and siRNAs. Cell.
2009;136(4):642-655.
96
REFERENCES
148.
Ameres SL, Zamore PD. Diversifying microRNA sequence and function. Nature reviews
Molecular cell biology. 2013;14(8):475-488.
149.
Fabian MR, Sonenberg N, Filipowicz W. Regulation of mRNA translation and stability by
microRNAs. Annual review of biochemistry. 2010;79:351-379.
150.
O'Rourke JR, Georges SA, Seay HR, Tapscott SJ, McManus MT, Goldhamer DJ, Swanson
MS, Harfe BD. Essential role for Dicer during skeletal muscle development. Developmental
biology. 2007;311(2):359-368.
151.
Cheung TH, Quach NL, Charville GW, Liu L, Park L, Edalati A, Yoo B, Hoang P, Rando TA.
Maintenance of muscle stem-cell quiescence by microRNA-489. Nature.
2012;482(7386):524-528.
152.
Kirby TJ, McCarthy JJ. MicroRNAs in skeletal muscle biology and exercise adaptation. Free
radical biology & medicine. 2013;64:95-105.
153.
Chen JF, Mandel EM, Thomson JM, Wu Q, Callis TE, Hammond SM, Conlon FL, Wang DZ.
The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and
differentiation. Nature genetics. 2006;38(2):228-233.
154.
Kim HK, Lee YS, Sivaprasad U, Malhotra A, Dutta A. Muscle-specific microRNA miR-206
promotes muscle differentiation. The Journal of cell biology. 2006;174(5):677-687.
155.
Greco S, De Simone M, Colussi C, Zaccagnini G, Fasanaro P, Pescatori M, Cardani R,
Perbellini R, Isaia E, Sale P, Meola G, Capogrossi MC, Gaetano C, Martelli F. Common
micro-RNA signature in skeletal muscle damage and regeneration induced by Duchenne
muscular dystrophy and acute ischemia. FASEB journal : official publication of the
Federation of American Societies for Experimental Biology. 2009;23(10):3335-3346.
156.
Jeng SF, Rau CS, Liliang PC, Wu CJ, Lu TH, Chen YC, Lin CJ, Hsieh CH. Profiling musclespecific microRNA expression after peripheral denervation and reinnervation in a rat model.
Journal of neurotrauma. 2009;26(12):2345-2353.
157.
Zhao Y, Samal E, Srivastava D. Serum response factor regulates a muscle-specific microRNA
that targets Hand2 during cardiogenesis. Nature. 2005;436(7048):214-220.
158.
Rosenberg MI, Georges SA, Asawachaicharn A, Analau E, Tapscott SJ. MyoD inhibits Fstl1
and Utrn expression by inducing transcription of miR-206. The Journal of cell biology.
2006;175(1):77-85.
159.
Rao PK, Kumar RM, Farkhondeh M, Baskerville S, Lodish HF. Myogenic factors that
regulate expression of muscle-specific microRNAs. Proc Natl Acad Sci U S A.
2006;103(23):8721-8726.
160.
Liu N, Williams AH, Kim Y, McAnally J, Bezprozvannaya S, Sutherland LB, Richardson JA,
Bassel-Duby R, Olson EN. An intragenic MEF2-dependent enhancer directs muscle-specific
expression of microRNAs 1 and 133. Proc Natl Acad Sci U S A. 2007;104(52):20844-20849.
161.
Gagan J, Dey BK, Dutta A. MicroRNAs regulate and provide robustness to the myogenic
transcriptional network. Current opinion in pharmacology. 2012;12(3):383-388.
162.
Yin H, Pasut A, Soleimani VD, Bentzinger CF, Antoun G, Thorn S, Seale P, Fernando P, van
Ijcken W, Grosveld F, Dekemp RA, Boushel R, Harper ME, Rudnicki MA. MicroRNA-133
controls brown adipose determination in skeletal muscle satellite cells by targeting Prdm16.
Cell metabolism. 2013;17(2):210-224.
97
REFERENCES
163.
van Rooij E, Quiat D, Johnson BA, Sutherland LB, Qi X, Richardson JA, Kelm RJ, Jr., Olson
EN. A family of microRNAs encoded by myosin genes governs myosin expression and
muscle performance. Developmental cell. 2009;17(5):662-673.
164.
Small EM, O'Rourke JR, Moresi V, Sutherland LB, McAnally J, Gerard RD, Richardson JA,
Olson EN. Regulation of PI3-kinase/Akt signaling by muscle-enriched microRNA-486. Proc
Natl Acad Sci U S A. 2010;107(9):4218-4223.
165.
Dey BK, Gagan J, Dutta A. miR-206 and -486 induce myoblast differentiation by
downregulating Pax7. Molecular and cellular biology. 2011;31(1):203-214.
166.
Juan AH, Kumar RM, Marx JG, Young RA, Sartorelli V. Mir-214-dependent regulation of the
polycomb protein Ezh2 in skeletal muscle and embryonic stem cells. Molecular cell.
2009;36(1):61-74.
167.
Wong CF, Tellam RL. MicroRNA-26a targets the histone methyltransferase Enhancer of
Zeste homolog 2 during myogenesis. The Journal of biological chemistry. 2008;283(15):98369843.
168.
Lal A, Navarro F, Maher CA, Maliszewski LE, Yan N, O'Day E, Chowdhury D, Dykxhoorn
DM, Tsai P, Hofmann O, Becker KG, Gorospe M, Hide W, Lieberman J. miR-24 Inhibits cell
proliferation by targeting E2F2, MYC, and other cell-cycle genes via binding to "seedless"
3'UTR microRNA recognition elements. Molecular cell. 2009;35(5):610-625.
169.
Yamamoto M, Kuroiwa A. Hoxa-11 and Hoxa-13 are involved in repression of MyoD during
limb muscle development. Development, growth & differentiation. 2003;45(5-6):485-498.
170.
Crist CG, Montarras D, Pallafacchina G, Rocancourt D, Cumano A, Conway SJ, Buckingham
M. Muscle stem cell behavior is modified by microRNA-27 regulation of Pax3 expression.
Proc Natl Acad Sci U S A. 2009;106(32):13383-13387.
171.
Sarkar S, Dey BK, Dutta A. MiR-322/424 and -503 are induced during muscle differentiation
and promote cell cycle quiescence and differentiation by down-regulation of Cdc25A.
Molecular biology of the cell. 2010;21(13):2138-2149.
172.
Guay C, Roggli E, Nesca V, Jacovetti C, Regazzi R. Diabetes mellitus, a microRNA-related
disease? Translational research : the journal of laboratory and clinical medicine.
2011;157(4):253-264.
173.
Poy MN, Eliasson L, Krutzfeldt J, Kuwajima S, Ma X, Macdonald PE, Pfeffer S, Tuschl T,
Rajewsky N, Rorsman P, Stoffel M. A pancreatic islet-specific microRNA regulates insulin
secretion. Nature. 2004;432(7014):226-230.
174.
Trajkovski M, Hausser J, Soutschek J, Bhat B, Akin A, Zavolan M, Heim MH, Stoffel M.
MicroRNAs 103 and 107 regulate insulin sensitivity. Nature. 2011;474(7353):649-653.
175.
Granjon A, Gustin MP, Rieusset J, Lefai E, Meugnier E, Guller I, Cerutti C, Paultre C, Disse
E, Rabasa-Lhoret R, Laville M, Vidal H, Rome S. The microRNA signature in response to
insulin reveals its implication in the transcriptional action of insulin in human skeletal muscle
and the role of a sterol regulatory element-binding protein-1c/myocyte enhancer factor 2C
pathway. Diabetes. 2009;58(11):2555-2564.
176.
Gallagher IJ, Scheele C, Keller P, Nielsen AR, Remenyi J, Fischer CP, Roder K, Babraj J,
Wahlestedt C, Hutvagner G, Pedersen BK, Timmons JA. Integration of microRNA changes in
vivo identifies novel molecular features of muscle insulin resistance in type 2 diabetes.
Genome medicine. 2010;2(2):9.
98
REFERENCES
177.
Herrera BM, Lockstone HE, Taylor JM, Ria M, Barrett A, Collins S, Kaisaki P, Argoud K,
Fernandez C, Travers ME, Grew JP, Randall JC, Gloyn AL, Gauguier D, McCarthy MI,
Lindgren CM. Global microRNA expression profiles in insulin target tissues in a spontaneous
rat model of type 2 diabetes. Diabetologia. 2010;53(6):1099-1109.
178.
Chang TC, Yu D, Lee YS, Wentzel EA, Arking DE, West KM, Dang CV, ThomasTikhonenko A, Mendell JT. Widespread microRNA repression by Myc contributes to
tumorigenesis. Nature genetics. 2008;40(1):43-50.
179.
Mott JL, Kurita S, Cazanave SC, Bronk SF, Werneburg NW, Fernandez-Zapico ME.
Transcriptional suppression of mir-29b-1/mir-29a promoter by c-Myc, hedgehog, and NFkappaB. Journal of cellular biochemistry. 2010;110(5):1155-1164.
180.
Wang H, Garzon R, Sun H, Ladner KJ, Singh R, Dahlman J, Cheng A, Hall BM, Qualman SJ,
Chandler DS, Croce CM, Guttridge DC. NF-kappaB-YY1-miR-29 regulatory circuitry in
skeletal myogenesis and rhabdomyosarcoma. Cancer cell. 2008;14(5):369-381.
181.
Qin W, Chung AC, Huang XR, Meng XM, Hui DS, Yu CM, Sung JJ, Lan HY. TGFbeta/Smad3 signaling promotes renal fibrosis by inhibiting miR-29. Journal of the American
Society of Nephrology : JASN. 2011;22(8):1462-1474.
182.
Kapinas K, Kessler C, Ricks T, Gronowicz G, Delany AM. miR-29 modulates Wnt signaling
in human osteoblasts through a positive feedback loop. The Journal of biological chemistry.
2010;285(33):25221-25231.
183.
Eyholzer M, Schmid S, Wilkens L, Mueller BU, Pabst T. The tumour-suppressive miR-29a/b1
cluster is regulated by CEBPA and blocked in human AML. British journal of cancer.
2010;103(2):275-284.
184.
Cushing L, Kuang PP, Qian J, Shao F, Wu J, Little F, Thannickal VJ, Cardoso WV, Lu J.
miR-29 is a major regulator of genes associated with pulmonary fibrosis. American journal of
respiratory cell and molecular biology. 2011;45(2):287-294.
185.
Li Z, Hassan MQ, Jafferji M, Aqeilan RI, Garzon R, Croce CM, van Wijnen AJ, Stein JL,
Stein GS, Lian JB. Biological functions of miR-29b contribute to positive regulation of
osteoblast differentiation. The Journal of biological chemistry. 2009;284(23):15676-15684.
186.
Ramdas V, McBride M, Denby L, Baker AH. Canonical transforming growth factor-beta
signaling regulates disintegrin metalloprotease expression in experimental renal fibrosis via
miR-29. The American journal of pathology. 2013;183(6):1885-1896.
187.
van Rooij E, Sutherland LB, Thatcher JE, DiMaio JM, Naseem RH, Marshall WS, Hill JA,
Olson EN. Dysregulation of microRNAs after myocardial infarction reveals a role of miR-29
in cardiac fibrosis. Proc Natl Acad Sci U S A. 2008;105(35):13027-13032.
188.
Papadopoulou AS, Dooley J, Linterman MA, Pierson W, Ucar O, Kyewski B, Zuklys S,
Hollander GA, Matthys P, Gray DH, De Strooper B, Liston A. The thymic epithelial
microRNA network elevates the threshold for infection-associated thymic involution via miR29a mediated suppression of the IFN-alpha receptor. Nature immunology. 2012;13(2):181187.
189.
Ma F, Xu S, Liu X, Zhang Q, Xu X, Liu M, Hua M, Li N, Yao H, Cao X. The microRNA
miR-29 controls innate and adaptive immune responses to intracellular bacterial infection by
targeting interferon-gamma. Nature immunology. 2011;12(9):861-869.
190.
Santanam U, Zanesi N, Efanov A, Costinean S, Palamarchuk A, Hagan JP, Volinia S, Alder
H, Rassenti L, Kipps T, Croce CM, Pekarsky Y. Chronic lymphocytic leukemia modeled in
mouse by targeted miR-29 expression. Proc Natl Acad Sci U S A. 2010;107(27):12210-12215.
99
REFERENCES
191.
Gebeshuber CA, Zatloukal K, Martinez J. miR-29a suppresses tristetraprolin, which is a
regulator of epithelial polarity and metastasis. EMBO reports. 2009;10(4):400-405.
192.
Pekarsky Y, Santanam U, Cimmino A, Palamarchuk A, Efanov A, Maximov V, Volinia S,
Alder H, Liu CG, Rassenti L, Calin GA, Hagan JP, Kipps T, Croce CM. Tcl1 expression in
chronic lymphocytic leukemia is regulated by miR-29 and miR-181. Cancer research.
2006;66(24):11590-11593.
193.
Mott JL, Kobayashi S, Bronk SF, Gores GJ. mir-29 regulates Mcl-1 protein expression and
apoptosis. Oncogene. 2007;26(42):6133-6140.
194.
Park SY, Lee JH, Ha M, Nam JW, Kim VN. miR-29 miRNAs activate p53 by targeting p85
alpha and CDC42. Nature structural & molecular biology. 2009;16(1):23-29.
195.
Garzon R, Heaphy CE, Havelange V, Fabbri M, Volinia S, Tsao T, Zanesi N, Kornblau SM,
Marcucci G, Calin GA, Andreeff M, Croce CM. MicroRNA 29b functions in acute myeloid
leukemia. Blood. 2009;114(26):5331-5341.
196.
Myers J, Prakash M, Froelicher V, Do D, Partington S, Atwood JE. Exercise capacity and
mortality among men referred for exercise testing. N Engl J Med. 2002;346(11):793-801.
197.
Srikanthan P, Karlamangla AS. Muscle mass index as a predictor of longevity in older adults.
The American journal of medicine. 2014;127(6):547-553.
198.
Blair SN, Kohl HW, 3rd, Barlow CE, Paffenbarger RS, Jr., Gibbons LW, Macera CA.
Changes in physical fitness and all-cause mortality. A prospective study of healthy and
unhealthy men. Jama. 1995;273(14):1093-1098.
199.
Price FD, von Maltzahn J, Bentzinger CF, Dumont NA, Yin H, Chang NC, Wilson DH,
Frenette J, Rudnicki MA. Inhibition of JAK-STAT signaling stimulates adult satellite cell
function. Nat Med. 2014;20(10):1174-1181.
200.
Pallafacchina G, Francois S, Regnault B, Czarny B, Dive V, Cumano A, Montarras D,
Buckingham M. An adult tissue-specific stem cell in its niche: a gene profiling analysis of in
vivo quiescent and activated muscle satellite cells. Stem cell research. 2010;4(2):77-91.
201.
Bentzinger CF, Wang YX, von Maltzahn J, Soleimani VD, Yin H, Rudnicki MA. Fibronectin
regulates Wnt7a signaling and satellite cell expansion. Cell stem cell. 2013;12(1):75-87.
202.
Fukada S, Uezumi A, Ikemoto M, Masuda S, Segawa M, Tanimura N, Yamamoto H,
Miyagoe-Suzuki Y, Takeda S. Molecular signature of quiescent satellite cells in adult skeletal
muscle. Stem cells. 2007;25(10):2448-2459.
203.
Joseph-Silverstein J, Consigli SA, Lyser KM, Ver Pault C. Basic fibroblast growth factor in
the chick embryo: immunolocalization to striated muscle cells and their precursors. J Cell
Biol. 1989;108(6):2459-2466.
204.
DiMario J, Buffinger N, Yamada S, Strohman RC. Fibroblast growth factor in the
extracellular matrix of dystrophic (mdx) mouse muscle. Science. 1989;244(4905):688-690.
205.
Dusterhoft S, Putman CT, Pette D. Changes in FGF and FGF receptor expression in lowfrequency-stimulated rat muscles and rat satellite cell cultures. Differentiation; research in
biological diversity. 1999;65(4):203-208.
206.
Morrow NG, Kraus WE, Moore JW, Williams RS, Swain JL. Increased expression of
fibroblast growth factors in a rabbit skeletal muscle model of exercise conditioning. J Clin
Invest. 1990;85(6):1816-1820.
100
REFERENCES
207.
Breen EC, Johnson EC, Wagner H, Tseng HM, Sung LA, Wagner PD. Angiogenic growth
factor mRNA responses in muscle to a single bout of exercise. J Appl Physiol (1985).
1996;81(1):355-361.
208.
Do MK, Suzuki T, Gerelt B, Sato Y, Mizunoya W, Nakamura M, Ikeuchi Y, Anderson JE,
Tatsumi R. Time-coordinated prevalence of extracellular HGF, FGF2 and TGF-beta3 in crushinjured skeletal muscle. Animal science journal = Nihon chikusan Gakkaiho.
2012;83(10):712-717.
209.
Anderson JE, Mitchell CM, McGeachie JK, Grounds MD. The time course of basic fibroblast
growth factor expression in crush-injured skeletal muscles of SJL/J and BALB/c mice. Exp
Cell Res. 1995;216(2):325-334.
210.
Jones NC, Fedorov YV, Rosenthal RS, Olwin BB. ERK1/2 is required for myoblast
proliferation but is dispensable for muscle gene expression and cell fusion. Journal of cellular
physiology. 2001;186(1):104-115.
211.
Chakkalakal JV, Jones KM, Basson MA, Brack AS. The aged niche disrupts muscle stem cell
quiescence. Nature. 2012;490(7420):355-360.
212.
Poy MN, Hausser J, Trajkovski M, Braun M, Collins S, Rorsman P, Zavolan M, Stoffel M.
miR-375 maintains normal pancreatic alpha- and beta-cell mass. Proc Natl Acad Sci U S A.
2009;106(14):5813-5818.
213.
Zhu H, Shyh-Chang N, Segre AV, Shinoda G, Shah SP, Einhorn WS, Takeuchi A, Engreitz
JM, Hagan JP, Kharas MG, Urbach A, Thornton JE, Triboulet R, Gregory RI, Consortium D,
Investigators M, Altshuler D, Daley GQ. The Lin28/let-7 axis regulates glucose metabolism.
Cell. 2011;147(1):81-94.
214.
Krutzfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, Manoharan M, Stoffel M.
Silencing of microRNAs in vivo with 'antagomirs'. Nature. 2005;438(7068):685-689.
215.
Liu N, Williams AH, Maxeiner JM, Bezprozvannaya S, Shelton JM, Richardson JA, BasselDuby R, Olson EN. microRNA-206 promotes skeletal muscle regeneration and delays
progression of Duchenne muscular dystrophy in mice. The Journal of clinical investigation.
2012;122(6):2054-2065.
216.
Chen JF, Tao Y, Li J, Deng Z, Yan Z, Xiao X, Wang DZ. microRNA-1 and microRNA-206
regulate skeletal muscle satellite cell proliferation and differentiation by repressing Pax7. The
Journal of cell biology. 2010;190(5):867-879.
217.
Joe AW, Yi L, Natarajan A, Le Grand F, So L, Wang J, Rudnicki MA, Rossi FM. Muscle
injury activates resident fibro/adipogenic progenitors that facilitate myogenesis. Nat Cell Biol.
2010;12(2):153-163.
218.
Urciuolo A, Quarta M, Morbidoni V, Gattazzo F, Molon S, Grumati P, Montemurro F,
Tedesco FS, Blaauw B, Cossu G, Vozzi G, Rando TA, Bonaldo P. Collagen VI regulates
satellite cell self-renewal and muscle regeneration. Nature communications. 2013;4:1964.
219.
Gilbert PM, Havenstrite KL, Magnusson KE, Sacco A, Leonardi NA, Kraft P, Nguyen NK,
Thrun S, Lutolf MP, Blau HM. Substrate elasticity regulates skeletal muscle stem cell selfrenewal in culture. Science. 2010;329(5995):1078-1081.
220.
Cosgrove BD, Gilbert PM, Porpiglia E, Mourkioti F, Lee SP, Corbel SY, Llewellyn ME, Delp
SL, Blau HM. Rejuvenation of the muscle stem cell population restores strength to injured
aged muscles. Nature medicine. 2014;20(3):255-264.
101
REFERENCES
221.
Gutierrez J, Brandan E. A novel mechanism of sequestering fibroblast growth factor 2 by
glypican in lipid rafts, allowing skeletal muscle differentiation. Mol Cell Biol.
2010;30(7):1634-1649.
222.
Neu R, Adams S, Munz B. Differential expression of entactin-1/nidogen-1 and entactin2/nidogen-2 in myogenic differentiation. Differentiation. 2006;74(9-10):573-582.
223.
Knox S, Merry C, Stringer S, Melrose J, Whitelock J. Not all perlecans are created equal:
interactions with fibroblast growth factor (FGF) 2 and FGF receptors. J Biol Chem.
2002;277(17):14657-14665.
224.
Langen RC, Schols AM, Kelders MC, Wouters EF, Janssen-Heininger YM. Enhanced
myogenic differentiation by extracellular matrix is regulated at the early stages of myogenesis.
In vitro cellular & developmental biology Animal. 2003;39(3-4):163-169.
225.
Gan Z, Rumsey J, Hazen BC, Lai L, Leone TC, Vega RB, Xie H, Conley KE, Auwerx J,
Smith SR, Olson EN, Kralli A, Kelly DP. Nuclear receptor/microRNA circuitry links muscle
fiber type to energy metabolism. The Journal of clinical investigation. 2013;123(6):25642575.
226.
Lueders TN, Zou K, Huntsman HD, Meador B, Mahmassani Z, Abel M, Valero MC, Huey
KA, Boppart MD. The alpha7beta1-integrin accelerates fiber hypertrophy and myogenesis
following a single bout of eccentric exercise. American journal of physiology Cell physiology.
2011;301(4):C938-946.
227.
Kadi F, Thornell LE. Training affects myosin heavy chain phenotype in the trapezius muscle
of women. Histochemistry and cell biology. 1999;112(1):73-78.
228.
Bruusgaard JC, Johansen IB, Egner IM, Rana ZA, Gundersen K. Myonuclei acquired by
overload exercise precede hypertrophy and are not lost on detraining. Proc Natl Acad Sci U S
A. 2010;107(34):15111-15116.
229.
Rathbone CR, Wenke JC, Warren GL, Armstrong RB. Importance of satellite cells in the
strength recovery after eccentric contraction-induced muscle injury. American journal of
physiology Regulatory, integrative and comparative physiology. 2003;285(6):R1490-1495.
230.
Koskinen SO, Ahtikoski AM, Komulainen J, Hesselink MK, Drost MR, Takala TE. Shortterm effects of forced eccentric contractions on collagen synthesis and degradation in rat
skeletal muscle. Pflugers Archiv : European journal of physiology. 2002;444(1-2):59-72.
231.
Winbanks CE, Wang B, Beyer C, Koh P, White L, Kantharidis P, Gregorevic P. TGF-beta
regulates miR-206 and miR-29 to control myogenic differentiation through regulation of
HDAC4. J Biol Chem. 2011;286(16):13805-13814.
232.
Zhou L, Wang L, Lu L, Jiang P, Sun H, Wang H. A novel target of microRNA-29, Ring1 and
YY1-binding protein (Rybp), negatively regulates skeletal myogenesis. J Biol Chem.
2012;287(30):25255-25265.
233.
Wei W, He HB, Zhang WY, Zhang HX, Bai JB, Liu HZ, Cao JH, Chang KC, Li XY, Zhao
SH. miR-29 targets Akt3 to reduce proliferation and facilitate differentiation of myoblasts in
skeletal muscle development. Cell Death Dis. 2013;4:e668.
234.
Li Y, Wang F, Xu J, Ye F, Shen Y, Zhou J, Lu W, Wan X, Ma D, Xie X. Progressive miRNA
expression profiles in cervical carcinogenesis and identification of HPV-related target genes
for miR-29. J Pathol. 2011;224(4):484-495.
235.
Hu Z, Klein JD, Mitch WE, Zhang L, Martinez I, Wang XH. MicroRNA-29 induces cellular
senescence in aging muscle through multiple signaling pathways. Aging. 2014;6(3):160-175.
102
REFERENCES
236.
Percheron G, Fayet G, Ningler T, Le Parc JM, Denot-Ledunois S, Leroy M, Raffestin B,
Jondeau G. Muscle strength and body composition in adult women with Marfan syndrome.
Rheumatology. 2007;46(6):957-962.
237.
Zhou L, Wang L, Lu L, Jiang P, Sun H, Wang H. Inhibition of miR-29 by TGF-beta-Smad3
signaling through dual mechanisms promotes transdifferentiation of mouse myoblasts into
myofibroblasts. PloS one. 2012;7(3):e33766.
238.
Wang XH, Hu Z, Klein JD, Zhang L, Fang F, Mitch WE. Decreased miR-29 suppresses
myogenesis in CKD. Journal of the American Society of Nephrology : JASN.
2011;22(11):2068-2076.
239.
Wang L, Zhou L, Jiang P, Lu L, Chen X, Lan H, Guttridge DC, Sun H, Wang H. Loss of miR29 in myoblasts contributes to dystrophic muscle pathogenesis. Molecular therapy : the
journal of the American Society of Gene Therapy. 2012;20(6):1222-1233.
240.
Claessen KM, Appelman-Dijkstra NM, Adoptie DM, Roelfsema F, Smit JW, Biermasz NR,
Pereira AM. Metabolic profile in growth hormone-deficient (GHD) adults after long-term
recombinant human growth hormone (rhGH) therapy. J Clin Endocrinol Metab.
2013;98(1):352-361.
241.
Filipsson Nystrom H, Barbosa EJ, Nilsson AG, Norrman LL, Ragnarsson O, Johannsson G.
Discontinuing long-term GH replacement therapy--a randomized, placebo-controlled
crossover trial in adult GH deficiency. J Clin Endocrinol Metab. 2012;97(9):3185-3195.
242.
Donahue LR, Beamer WG. Growth hormone deficiency in 'little' mice results in aberrant body
composition, reduced insulin-like growth factor-I and insulin-like growth factor-binding
protein-3 (IGFBP-3), but does not affect IGFBP-2, -1 or -4. The Journal of endocrinology.
1993;136(1):91-104.
243.
Masternak MM, Panici JA, Wang F, Wang Z, Spong A. The effects of growth hormone (GH)
treatment on GH and insulin/IGF-1 signaling in long-lived Ames dwarf mice. The journals of
gerontology Series A, Biological sciences and medical sciences. 2010;65(1):24-30.
244.
Hubmacher D, Apte SS. The biology of the extracellular matrix: novel insights. Current
opinion in rheumatology. 2013;25(1):65-70.
245.
Pal A, Barber TM, Van de Bunt M, Rudge SA, Zhang Q, Lachlan KL, Cooper NS, Linden H,
Levy JC, Wakelam MJ, Walker L, Karpe F, Gloyn AL. PTEN mutations as a cause of
constitutive insulin sensitivity and obesity. The New England journal of medicine.
2012;367(11):1002-1011.
246.
Doessing S, Heinemeier KM, Holm L, Mackey AL, Schjerling P, Rennie M, Smith K,
Reitelseder S, Kappelgaard AM, Rasmussen MH, Flyvbjerg A, Kjaer M. Growth hormone
stimulates the collagen synthesis in human tendon and skeletal muscle without affecting
myofibrillar protein synthesis. The Journal of physiology. 2010;588(Pt 2):341-351.
247.
Berria R, Wang L, Richardson DK, Finlayson J, Belfort R, Pratipanawatr T, De Filippis EA,
Kashyap S, Mandarino LJ. Increased collagen content in insulin-resistant skeletal muscle.
American journal of physiology Endocrinology and metabolism. 2006;290(3):E560-565.
248.
Tam CS, Covington JD, Bajpeyi S, Tchoukalova Y, Burk D, Johannsen DL, Zingaretti CM,
Cinti S, Ravussin E. Weight gain reveals dramatic increases in skeletal muscle extracellular
matrix remodeling. The Journal of clinical endocrinology and metabolism. 2014;99(5):17491757.
249.
Richardson DK, Kashyap S, Bajaj M, Cusi K, Mandarino SJ, Finlayson J, DeFronzo RA,
Jenkinson CP, Mandarino LJ. Lipid infusion decreases the expression of nuclear encoded
103
REFERENCES
mitochondrial genes and increases the expression of extracellular matrix genes in human
skeletal muscle. The Journal of biological chemistry. 2005;280(11):10290-10297.
250.
Fan N, Sun H, Wang Y, Wang Y, Zhang L, Xia Z, Peng L, Hou Y, Shen W, Liu R, Yin J,
Peng Y. Follistatin-like 1: a potential mediator of inflammation in obesity. Mediators of
inflammation. 2013;2013:752519.
251.
Zha Y, Le VT, Higami Y, Shimokawa I, Taguchi T, Razzaque MS. Life-long suppression of
growth hormone-insulin-like growth factor I activity in genetically altered rats could prevent
age-related renal damage. Endocrinology. 2006;147(12):5690-5698.
252.
Nie J, Sage EH. SPARC inhibits adipogenesis by its enhancement of beta-catenin signaling.
The Journal of biological chemistry. 2009;284(2):1279-1290.
253.
Kos K, Wilding JP. SPARC: a key player in the pathologies associated with obesity and
diabetes. Nature reviews Endocrinology. 2010;6(4):225-235.
254.
Hameed M, Lange KH, Andersen JL, Schjerling P, Kjaer M, Harridge SD, Goldspink G. The
effect of recombinant human growth hormone and resistance training on IGF-I mRNA
expression in the muscles of elderly men. The Journal of physiology. 2004;555(Pt 1):231-240.
255.
Doessing S, Holm L, Heinemeier KM, Feldt-Rasmussen U, Schjerling P, Qvortrup K, Larsen
JO, Nielsen RH, Flyvbjerg A, Kjaer M. GH and IGF1 levels are positively associated with
musculotendinous collagen expression: experiments in acromegalic and GH deficiency
patients. European journal of endocrinology / European Federation of Endocrine Societies.
2010;163(6):853-862.
256.
Sjogren K, Leung KC, Kaplan W, Gardiner-Garden M, Gibney J, Ho KK. Growth hormone
regulation of metabolic gene expression in muscle: a microarray study in hypopituitary men.
American journal of physiology Endocrinology and metabolism. 2007;293(1):E364-371.
257.
Hansen M, Boesen A, Holm L, Flyvbjerg A, Langberg H, Kjaer M. Local administration of
insulin-like growth factor-I (IGF-I) stimulates tendon collagen synthesis in humans.
Scandinavian journal of medicine & science in sports. 2013;23(5):614-619.
258.
Reiser K, Summers P, Medrano JF, Rucker R, Last J, McDonald R. Effects of elevated
circulating IGF-1 on the extracellular matrix in "high-growth" C57BL/6J mice. The American
journal of physiology. 1996;271(3 Pt 2):R696-703.
259.
Yang WM, Jeong HJ, Park SY, Lee W. Induction of miR-29a by saturated fatty acids impairs
insulin signaling and glucose uptake through translational repression of IRS-1 in myocytes.
FEBS letters. 2014;588(13):2170-2176.
260.
Pandey AK, Verma G, Vig S, Srivastava S, Srivastava AK, Datta M. miR-29a levels are
elevated in the db/db mice liver and its overexpression leads to attenuation of insulin action on
PEPCK gene expression in HepG2 cells. Molecular and cellular endocrinology. 2011;332(12):125-133.
261.
He A, Zhu L, Gupta N, Chang Y, Fang F. Overexpression of micro ribonucleic acid 29, highly
up-regulated in diabetic rats, leads to insulin resistance in 3T3-L1 adipocytes. Molecular
endocrinology. 2007;21(11):2785-2794.
262.
Chen GQ, Lian WJ, Wang GM, Wang S, Yang YQ, Zhao ZW. Altered microRNA expression
in skeletal muscle results from high-fat diet-induced insulin resistance in mice. Molecular
medicine reports. 2012;5(5):1362-1368.
104
REFERENCES
263.
Mohamed JS, Hajira A, Pardo PS, Boriek AM. MicroRNA-149 inhibits PARP-2 and promotes
mitochondrial biogenesis via SIRT-1/PGC-1alpha network in skeletal muscle. Diabetes.
2014;63(5):1546-1559.
264.
Harley IT, Giles DA, Pfluger PT, Burgess SL, Walters S, Hembree J, Raver C, Rewerts CL,
Downey J, Flick LM, Stankiewicz TE, McAlees JW, Wills-Karp M, Balfour Sartor R,
Divanovic S, Tschop MH, Karp CL. Differential colonization with segmented filamentous
bacteria and Lactobacillus murinus do not drive divergent development of diet-induced
obesity in C57BL/6 mice. Molecular metabolism. 2013;2(3):171-183.
265.
Thomas K, Engler AJ, Meyer GA. Extracellular matrix regulation in the muscle satellite cell
niche. Connective tissue research. 2015;56(1):1-8.
266.
Grefte S, Vullinghs S, Kuijpers-Jagtman AM, Torensma R, Von den Hoff JW. Matrigel, but
not collagen I, maintains the differentiation capacity of muscle derived cells in vitro.
Biomedical materials. 2012;7(5):055004.
267.
Wilschut KJ, Haagsman HP, Roelen BA. Extracellular matrix components direct porcine
muscle stem cell behavior. Experimental cell research. 2010;316(3):341-352.
268.
Engler AJ, Griffin MA, Sen S, Bonnemann CG, Sweeney HL, Discher DE. Myotubes
differentiate optimally on substrates with tissue-like stiffness: pathological implications for
soft or stiff microenvironments. The Journal of cell biology. 2004;166(6):877-887.
269.
Brack AS, Conboy IM, Conboy MJ, Shen J, Rando TA. A temporal switch from notch to Wnt
signaling in muscle stem cells is necessary for normal adult myogenesis. Cell stem cell.
2008;2(1):50-59.
270.
Kurinna S, Schafer M, Ostano P, Karouzakis E, Chiorino G, Bloch W, Bachmann A, Gay S,
Garrod D, Lefort K, Dotto GP, Beer HD, Werner S. A novel Nrf2-miR-29-desmocollin-2 axis
regulates desmosome function in keratinocytes. Nature communications. 2014;5:5099.
271.
Vargas MR, Pehar M, Cassina P, Martinez-Palma L, Thompson JA, Beckman JS, Barbeito L.
Fibroblast growth factor-1 induces heme oxygenase-1 via nuclear factor erythroid 2-related
factor 2 (Nrf2) in spinal cord astrocytes: consequences for motor neuron survival. The Journal
of biological chemistry. 2005;280(27):25571-25579.
272.
Coletta DK, Mandarino LJ. Mitochondrial dysfunction and insulin resistance from the outside
in: extracellular matrix, the cytoskeleton, and mitochondria. American journal of physiology
Endocrinology and metabolism. 2011;301(5):E749-755.
273.
Lino Cardenas CL, Henaoui IS, Courcot E, Roderburg C, Cauffiez C, Aubert S, Copin MC,
Wallaert B, Glowacki F, Dewaeles E, Milosevic J, Maurizio J, Tedrow J, Marcet B, LoGuidice JM, Kaminski N, Barbry P, Luedde T, Perrais M, Mari B, Pottier N. miR-199a-5p Is
upregulated during fibrogenic response to tissue injury and mediates TGFbeta-induced lung
fibroblast activation by targeting caveolin-1. PLoS genetics. 2013;9(2):e1003291.
274.
Smith KM, Guerau-de-Arellano M, Costinean S, Williams JL, Bottoni A, Mavrikis Cox G,
Satoskar AR, Croce CM, Racke MK, Lovett-Racke AE, Whitacre CC. miR-29ab1 deficiency
identifies a negative feedback loop controlling Th1 bias that is dysregulated in multiple
sclerosis. Journal of immunology. 2012;189(4):1567-1576.
275.
Trepp R, Fluck M, Stettler C, Boesch C, Ith M, Kreis R, Hoppeler H, Howald H, Schmid JP,
Diem P, Christ ER. Effect of GH on human skeletal muscle lipid metabolism in GH
deficiency. American journal of physiology Endocrinology and metabolism.
2008;294(6):E1127-1134.
105
REFERENCES
276.
Moreno-Navarrete JM, Martinez-Barricarte R, Catalan V, Sabater M, Gomez-Ambrosi J,
Ortega FJ, Ricart W, Bluher M, Fruhbeck G, Rodriguez de Cordoba S, Fernandez-Real JM.
Complement factor H is expressed in adipose tissue in association with insulin resistance.
Diabetes. 2010;59(1):200-209.
277.
Stumvoll M, Van Haeften T, Fritsche A, Gerich J. Oral glucose tolerance test indexes for
insulin sensitivity and secretion based on various availabilities of sampling times. Diabetes
Care. 2001;24(4):796-797.
278.
Sotiropoulos A, Ohanna M, Kedzia C, Menon RK, Kopchick JJ, Kelly PA, Pende M. Growth
hormone promotes skeletal muscle cell fusion independent of insulin-like growth factor 1 upregulation. Proc Natl Acad Sci U S A. 2006;103(19):7315-7320.
279.
Mavalli MD, DiGirolamo DJ, Fan Y, Riddle RC, Campbell KS, van Groen T, Frank SJ,
Sperling MA, Esser KA, Bamman MM, Clemens TL. Distinct growth hormone receptor
signaling modes regulate skeletal muscle development and insulin sensitivity in mice. The
Journal of clinical investigation. 2010;120(11):4007-4020.
280.
Resmini E, Morte B, Sorianello E, Gallardo E, de Luna N, Illa I, Zorzano A, Bernal J, Webb
SM. Identification of novel GH-regulated genes in C2C12 cells. Hormone and metabolic
research = Hormon- und Stoffwechselforschung = Hormones et metabolisme.
2011;43(13):919-930.
281.
Baudry A, Lamothe B, Bucchini D, Jami J, Montarras D, Pinset C, Joshi RL. IGF-1 receptor
as an alternative receptor for metabolic signaling in insulin receptor-deficient muscle cells.
FEBS letters. 2001;488(3):174-178.
106