CELL TRANSPLANTATION FOR MYOCARDIAL REPAIR
A Thesis Submitted to the Faculty of Graduate Studies
and Research in partial fuJfiJment of the
requirements for the Degree of
Master of Science
Daniel Marelli,
Department of Surgery,
Division of Surgical Research,
McGiJl University, Montreal,
March 1992
PREFACE:
The experiments leading to this thesis were performed in the University Surgical
Clinic of the Montreal General Hospital under the direction of Dr. Ray C.-J. Chiu and
Dr. David S. Mulder. This ongoing research in myocardial regeneration is funded by the
Quehec Heart Foundation.
The protocol used was developed in order to crea te a new avenue of research for the
cardinvascular surgery laboratory.
The cell culture methods were set up in order to
complete this project. Equipment for these was donated by Dr. Gustavo Bounous.
The proposed concept was formulated by Dr. Race Kao of the Allegheny-Singer
Research Institute in Pittsburgh. His collaboration was essential for the undertaking of these
experiments. Dr. Kao's preliminary results show that skeletal muscle satellite cells can be
transformed into cardiac muscle when implanted into acutely injured myocardium. Ta the
'"
,tl
'"
knowledge of my supervisors and myself, this is an original observation that has never been
reported by any other research group. One of the objectives of this study was ta confirm
or refute Dr. Kao's results. One week was spent in Pittsburgh Jearning the methodology for
satellite ccII isolation, multipJication, labelling and implantation. Embedding and sectioning
of the specImens for histology was carried out under the supelvision of Dr. John Lough of
the pathology department at the Montreal General Hospital. Radioautography was carried
out at McGiII University within the department of anatomy under the direction of Dr.
Leblond
~lI1d
Dr. M. EI-Alfy.
c.P.
ii
ACKNOWLEDGEMENTS:
1 am grateful for the leadership of the director of the Montœal General Hospital
University Surgical Clinie, Dr. David S. Mülder, who has supported my rescarch effort with
great enthusiasm. My immediate supervisor, Dr. Ray
c.-J. Chiu,
has guidcd me as weil as
my career in a manner that a parent guides a child. 1 wOllld like to thank him for this
exceptional patience and cOllnse1. Ms. Carolyne Desrosiers assisted in every experiment as
weil as in the preparation of this thesis. Her partnership and friendship extended heyond
the call of duty. Dr. Peter Metrakos provided conceptual and technical advlce tor many of
the methods used to carry out this research proJect. 1 am also indehted to Mf. Danny Yee
and Dr. Dominique Shum-Tim for technical assistance in the pertormance of the
experiments. Mrs. Emma Lisi cuntributed great moral support and professional skill to the
accomplishment of this the sis. Finally, 1 owe a debt of gratitude to my hest friends,
Dr. Maurice Tapiero and Dr. Maurice Suissa, who have constantly cncouraged and
motivated me throughout the course of the experiments leading to this thesis.
1
III
ABSTRACI':
Myocardium lacks the ability to regenerate following injury. This is in contrast ta
skeletal muscle (SKM), in which capacity for tissue repair is attributed to the presence of
satellite cells. ft was hypothesized that SKM satellite cells multiplied in vitro could be used
to repair injured heart muscle. The purpose of this s\udy was to test this hypothesis byautotransplanting SKM satellite cells to a myocardial injury site. Fourteen dogs underwent
explantation of the anterior tibialis muscle. Satellite ceUs were multiplied in vitro and their
nuclei were labelled with tritiated thymidine 24 hours prior to implantation. The same dogs
were then suh]ected to a myocardial injury by the application of a cryoprobe. The cells were
suspended in serum free growth medium and implanted within the damaged muscle.
Medium without cells was inJected into an adjacent site to serve as a control. Endpoints
compnsed histology using standard stains as weil as Masson trichrome (specific for
connective tissue), and radio-autography. The
MuJ~'
was aborted in nine of the fourteen
dogs because of technical error or peri-operative death. In four additional contrais, cryoinjured skeletal muscle was electrically stimulated with a pacemaker.
ln 3 dogs, specimens were taken within 6-8 weeks. There was persistence of the
implantation channels in the experimental sites wiïcn cQmpared to the controls.
Macroscopically, muscle tissue completely surrounded by scar could be seen.
Masson
trichrome staining showed homogeneous scar in the control site, but not in the test site
where a patch of muscle fihres containing intercalated dises (characteristic of myocardial
tissue) was ohserved. In 2 other dogs, specimens were taken at 14 weeks post-implantation.
Muscle tissue cou Id not be found. Electrically stimulated skeletal muscle regenerates did not
show histological evidence of cardiac transformation.
These results support the hypothesis that SKM satellite cells can farm neomyocardium within an appropria te environment. The specimens faHed to demonstrate the
presence of myocyte nuclei. It can be further hypothesised that in the late post-operative
period, the muscle regenerate failed to survive within the scar tissue because of inadequate
'1
micro-circulation.
iv
RESUMÉ:
Le muscle cardiaque est incapable de régénération suite fi une blessure. Par contre,
le muscle squelettique a un potentiel de renouvellement qui est attnbué il la presence de
cellules satellites. La possibilité d'utiliser les cellules satellites du muscle squelettique pour
la
~éparation
du myocarde endommagé a été envisagée dans un cadre d'auto-greffe. Suite
à une série d'études préliminaires, le muscle tibiale a été retranché chez quatorze chiens.
Les cellules satellites on été, multipliées, et marquées avec de la thymidine radioactive. Dix
Jours plus tard, les chiens ont subi une blessure au myocarde à J'aide d'une sonde
cryogenique.
Les cellules on en!iuite été implantées dans la lesion myocardique.
Une
implantation de milieu de culture liquide sans cellules dans une région endommagée
adjacente a servi de contrôle.
Neuf implantations n'ont pas réussi il cause d'erreur
technique ou ùe mort périopératoire. Chez quatre autres contrôles, un muscle squelettique
cryoblessé en phase de régénération a été induit à se contracter de façon rythmique il l'aide
d'un stimulateur électrique.
Chez trois chiens, des spécimens ont été obtenus dans un délai de six il huite
semaines après l'implantation des cellules.
Il y avait une démarcation du canal
d'implantation dans la région experimentale mais pas dans la région de contrôle.
A
l'inspection macroscopique, le tissue musculaire était entouré de tissue cicatriciel. Une
coloration tri chromée de Masson a démontré une cicatrice homogène à la région de contrôle
mais non à la région experimentale.
Dans celle-ci, il y avait la présence de fibres
musculaires dans lesquelles il y avait des disques ifltercalés caractèristiques du myocarde.
Chez deux autres chiens, des spécimens on été obtenus après quatorze semaines. Il n'y avait
pas de tissue musculaire dans ces cicatrices.
La stimulation électrique d'un muscle
squeletique en phase de régénération n'a pas occasionné une transformation cardiaque
reconnaissable à l'examen histologique.
Les résultats appuient l'hypothèse que les cellules satellites du muscle squelettique
f
peuvent être transformées en néomyocarde selon un stimulus approprié.
Le~
spéCimens
n'ont pas révélé la présence de noyaux dans le néo myocarde. On peut donc émettre
l'hypothèse que dans la période post-opératoire tardive, le muscle régénéré n'a pus survivre
à cause d'une mlcrocirculation inadéquate dans la cicatrice environante.
v
TABLE OF CONTENTS
1. INTRODUcrlON
Clinical Perspective ............................................. 1
Muscle Regeneration
.......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 2
i.
Skeletal Muscle
..................................... 2
i i.
Cardiac Muscle
..................................... 3
The Skeletal Muscle Satellite Cell .................................. 5
J.
Definition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 5
ii.
Anatomy
m.
Biolob'Y
.......................................... 6
........................................... 7
Can Terminal Differentiation of Muscle Cells Be Intluenced'?
SkcletaJ Muscle Satellite Cell Transplantation
Hypothesis and Purpose
............. 8
......................... 9
........................................ 10
Il. MATERIALS AND METHODS
Study Design
......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Il
Operative Procedures
.......................................... Il
i.
Muscle Biopsy
..................................... Il
Il.
CryoinJury to the Myocardium
iii.
Cell Implantation ................................... 14
IV.
Skeletal Muscle Injury and Electrical Stimulation
......................... 12
........... 18
CcII Culture Methodology ...................................... 18
i.
Isolation and Platmg of Satellite Cells .................... 18
iL
ln Vitro CcII Multiplication
iii.
Cell Lahelling
iVe
Cell Preparation for Implantation ....................... 20
.................•... . . . . .. 19
..................................... 20
Specimen Preparation for Histology
............................... 22
VI
III. RESULTS
Operative Results ............................................. 25
In Vitro Studies
Histology
.............................................. 26
................................................... 30
i.
Ce}} Implantation Within Acutely InJured Mym:arùium
ii.
Electrically Stimulated lnjured Skeletal Musde ............. 30
....... 30
IV. DISCUSSION ................................................ 39
APPENDIX 1 ..................................................... 43
APPENDIX II
REFERENCES
.................................................... 46
........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 4X
Chapter 1
INTRODUCfION
1
CLINICAL PERSPECfIVE:
It is estimated that 9 percent of the general population !lutters hom heart di!lea~l.·177).
This Iimlts actlvlty m about one thml of the se people. Cardiomyopathy rders to the tmal
corn mon pathway ot ail heart disease whlch is !lpeclflc muscle cell death. healt fallure. ami
its eventual lethal complicatIOns.
Once patients hecome
!lympt~)l11atll·.
one hait of the
persans with heart failure are expected to die within fivc year!l reganJless of the
call~c.
Thi!l
is generally attributed to the inability of mammalian cardlac mU!lcle 10 rcgenerate fllllowmg
injury(81). Currently, treatment for cardiomyopathy consists of medlcalmanagcmcnt of pump
failure, heart transplantation and artlflcial circuléltory support.
Medical management invlllve!l relievmg the work of the heart pUlllp Itselt throllgh
manipulatIon of filling volume!l and afterload reductlon. To thc!lc modahtle~. one can adl!
agents that augment myocardial contractility.
Heart tran!lplantatlon
I~
currcntly gamlllg
widespread use(7R). Unfortunately, even though the ~ucce!lS rate I~ HO perrent .,lIrvlval at 1
year follow up, this treatment IS limlted hy don or aVaIlahJllty and there1me cannot he oltcred
ta ail those that require It. Mechanical clrculatory support remam!l largely cxpcfll11ental(7").
but is an expanding field of research that holds promi!le for the future.
More recently, cardlOmyoplasty u!ling skeletal muscle ti!lMIC to support ventncular
function has been developed(l), In the last ten years, the !lurgery la bora tory at The Montreal
General Hospital has heen involved in such experimental and c1imcal manipulation of
skeletal muscle for cardiac assist(2). Cardiomyoplasty mvolve~ tran~f()rméltl()n ot mature
skeletal muscle from a fatiguahle metaholism. to a fatigue re!ll!ltant can.lJac hkc .,tatc usmg
electrical stimulation and then wrapping the muscle tl~sue élround the heart. ThlJ~, u~mg the
appropriate stimulus, phenotypical expression of skeletal muscle gcnes l'an he altcred and
skeletal muscle can then be used for cardiac work. The
pre~ent
proJcct l'an he viewcd a!l
repre!lenting an exten!lion of this type ot manipulation at the cellular Icvcl. The !luhJect ot
this thesis is to test the teaslhlhty of using skeletal mu~c1e lo.atelhte cclls ln rcpair damaged
heart muscle. Ultimately, this may lead to a new form of treatmcnt tor cardlomyopathy.
.'
The intended approach consists of applymg the physiologie mechalllsm!l undcrlymg skeletal
muscle regeneration to mYOl:ardial regeneration hy auto-transplanting ~atcllite cells trom
2
~kclctal mu~ch~
into acutely injured myocardium.
MUSCLE REGENERATION:
i. Skeletal Muscle:
Dc~cfJptions of ~keletal mu~c1e regeneration date back ta the turn of the century(4,5.6).
Early ohserva!J(lnS concerned themselves with trymg ta identify the process of regencration
lI~clf and the ~cquence of events involved in muscle repair(97). In 1934, Millar described
complete ~keletal mu~cJe regeneration following injury using an animal model(98). In the
deSCription of the events he observe d, he identified prohferating nuclei but he could not
c1early determme the source of the regenerate. Twelve years later, Le Gros Clark described
a model of regeneration in whlch pieces of skeletal muscle were resected and then grafted
m ~ltU(II'). The advantage of thi~ model was that it produced isolated ischemic necrosis of
a muscle secti<'n. He observed the presence of signs of regeneration as early as one week
followmg implantation and the
pre~enee
of young fibres undergoing maturation 2-3 weeks
later. He speclflcd that the regenerated fibres keep their orientation with respect ta the
dying flbre~ sllggesting a directive action of endomysial tubes surviving inJury. This has been
confirmed
ln
more recent studies(28.35.IOI). Finally, he suspected that myoblasts were derived
l'rom lIisintegrated fragments of muscle but was not able to demonstrate this.
Gay and
Hunt, in 1954(1\ descnhed the maturation of regenerating skeletal muscle fibres as observed
m phase microscopy. They compared regeneration to ontogenic development anù described
centrally placed nllcJei moving to the periphery of fibres as they mature. They alluded to
smgle cells fusmg tn form a new fiore.
ln 1961 Mauro described the skeletal muscle "satellite" cell and suggested that these
eells may he the source of nuclei for muscle regeneration. This process mimics embryonic
muscle development in which individual mononucleated undifferentiated, uncommitted
myohlasts undergo multiple cell divisions and then fuse ta form muscle fibres in which the
post-mitotic nuclei are exclusively conœrned with protein synthesis.(4,12,14,82,102,103). Growth
then depends on further replicatlOn of the satellite cells which persist into adult life(24). In
muscle regeneration. fihres first undergo degeneration. The post-mitotic nuclei, responsible
for ITlctaholic activity, do not contribute to the regenerative response(lOS).
In contrast,
3
satellite cells survive injury and hecome myogenic(I()(),IOI.It'). The newly formeù muscle then
undergoes a maturation proeess(HK1). Biochemically, the lactate dchydrogel1HSe and creatine
kinase isozyme profiles share similarities hut are not exact Juplicates of the ones ohscrved
in embryonie differentiatlon(84.101) This has heen interpreled as betng due
metabolic environment of regenerating muscle
lf1
10
the har~îlL'1
comparisol1 to developing muscle. The
proeess of regen~ration Itself h':1s been c1asslfled mto two types(tl'i l .
In continuous regeneration, new fibres grow in the l'orm ot huds l'rom ùamaged preexisting ones that retain a healthy portion. In the more eommon discontinuous regeneratlon,
new muscle is formed from myogenic ce Ils. Such Iegeneration of skeleml muscle has heen
confirmed in human studies(86).
ii. Cardiac Muscle
Cardiac muscle resembles skeletal muscle in that it is striated(14.1 "i). In contrast, the
ce Ils are mononucleated and they form a syncytium in which they are connected hy
specialized ccli junctions referrec! to as intercalated
from that of skeletal muscle
lf1
di:~cs
(Figure 1). hs metaholism differs
that it is fatigue resl~tant('i). Thus, myocardllll11 is more
differentiated théln skeletal muscle and is hlghly adapted tn tunction.
Emhryogencsis nt
heart muscle occurs earlier than that of skeletal muscle. Il11tially, myocardllll11 consists of
developing muscle cells that origll1ate from splanchnic mesoderm.
A~
carùmc
myobla~ts
mature, they synthesize myofibrils which align tltem!lelve~ progressivelyCH7). The ce Ils then
elongate and intercalated dises form a right angle with respect lo the tibrils.
Carùiac
myoblasts are able to hoth synthesize myofibrillar proteins and divide(l('). This is in contrast
to skeleta\ muscle in which muscle specittc gene expression precJudes mitotic cycIing(HO,HR).
Lastly, mitotic activity is lost in the neonatal period, and following birth, the heart enlarges
only by hypertrophy of its myoeytes.
4
FIGURE 1
Normal cardiae muscle showing eentrally located nuclei and
interealated dises at cell junctions (x400, toluidine blue stain)
5
Regeneration of myocardium following injury is poorly defined(lb 21.60,61). Cytokinesis
has not yet been described. Following cell death, myocardial repair essentially consists of
replacement with connective tissue and inevitably, loss of function ensues. This inahility for
repair or "regeneration" following injury may be circumstantially attributed to the absence
of satellite ce Ils in mammalian cardiac muscle(8). Such cells have been observed in cardiac
muscles of decapod crustace ans whieh consist of multinucleated fibres joined together by
intercalated discS(89).
Several studies have attempted to characterize the potential for regeneration in
myocardium. Long term culture of adult mammalian cardiac muscle cells have revealed that
these cells undergo biosynthetic and morphological changes to eventually resemble
embryonic cardiac cells(91.93,109). They resume DNA synthesis and about 90% of these cells
contain more than one nucIeus(92).
Looking at DNA synthesis studies in response to
experimental injury carried out on neonatal rats, Nag et al showed an age dcpendent
potential for regeneration that becomcs attenuated by ahout 4 weeks of age(l(}). In this
study, the regenerating cardiac myocyte was characterized as a cell with many polysomes
presumably synthesizing new myofibrils. StiJl, mitotie division was not observed. Oron and
Mandelberg reported similar intracellular regeneration following cryoinjury. Thcir findings
were Iimited ta the perinecrotie area(94). Such forms of regeneration have been confirmed
in human autopsy studies(96). Others have tried to focus on the transplantation of cardiac
tissue into ectopie sites. Jockusch et al showed that xenografts of ventricular tissue from
newborn rats implanted inta an athymie host were able to regenerate(95). Thus, the common
denominator Iinking these studies is that although newborn heart muscle rnay have sorne
capacity for limited regeneration, mature heart muscle cannot repair itself following injury.
THE SKELETAL MUSCLE SATELLITE CELL:
i. Definition
Originally described in frog muscle(7), these cells are found in aIl known mammalian
voluntary musc1e. Their creatine kinase (CK) enzyme prafi1e resembles that of embryonic
myoblasts(28). They are absent in cardiac and smooth muscle(7). PhysiologiciJlly, they have
6
the ability to divide and give rise ta differentiating cells as weil as new satellite ceUs. Their
nuclei constitute the only ones among striated muscle nuclei that have the capacity for
mitosis(21,24,28,29). As weil, they have the ability to cycle in vitro(27). Thus, these ceUs can be
defined as stem cells of embryonic origin(41).
ii. Anatomy
Using electron microscopy, satellite cells are distinguishable by their position beneath
the basal lamina(24). The plasma membrane of these ce Ils is separated from that of the
"parent" fibre by a gap 15-20#-, wide.
The celI nucleus itself is slightly smaller than a
myofibre nucleus. In general, human satellite ceUs measure 301-' in length(8). They make up
about 5% of nuclei in muscle fibres. This has been confirmed in laboratory animaIs as weIl
as in humans(JO,31).
ln mature muscle, satellite cells appear to be clustered near the motor end-plate(105).
(
They are not, however, evenly distributed among the fibre types and trends have not been
identified according to muscle fibre type composition(l06). Su ch proportion al differences
between various muscle groups appear to be related to nerve supply(107).
The morphology of satellite cells was described in the mid 1960'S(25). In mature
muscle, few organelles are seen. The width of the cell itself is slightly greater th an that of
the nucleus and the shape is fusiform. Occasion al Golgi apparatus can be found near the
nucleus and small, poorly developed mitochondria can be seen at the tape ring ends of the
cell. Free ribosomes can be found within the limited cytoplasm. FinaIly, centrioles are
present at the side of the nucleus thus reflecting the ability that these ceUs have for mitotic
cycling.
ln growing muscle, satellite cells appear to be more metabolicallyactive(26). The ceUs
lie beneath the basal lamina of muscle fibres but are more conspicuous in that the plasma
membranes of the satellite ceUs and the muscle fibres are not parallel to each other and are
:(
'i.
separated by a gap up to 60 nm wide. Ribosomes are numerous and are both free and
7
bound to endoplasmic reticulum. The Golgi apparatus is weil devc1oped. Finally, sorne
satellite ceUs can be observed to be in the process of fusion with a muscle fibre. Eventually,
with age, the eells evolve to take on a morpholobYf that retlects their quiescent nature.
iii. Biology
Laboratory animal and human studies have confirmed that adult satellite ceUs revert
to their active form after being stimulated by stresses such as dlsease, direct tréluma,
ischemia or exercise(8,30,31).
Regenerating skeletal muscle is solely dependent upon the
intrinsic satellite cell population that survives injury(34,3~,36).
Furthermore, the myogenk
potential of satellite cells decreases with age and multiplicity of regenerating cycles( U,ll).
Within the basal lamina, satellite cells have the ability to migrate toward the injury sitc(IJ).
This suggests the possible role of muscle injury in releasing factors that can activate satellite
cells and guide them to the injury site(37).
One question that arises in discussing satellite cells is whether or not these cells are
identical to embryonic myoblasts. Much of the evidence availahle suggests that although
satellite cells resemble embryonic muscle cells, they are most Iikely a suhpopulation of
these(9,38-42).
As stated above, adult satellite ce lis have the same levels and CK isozyme
profiles as embryonic myoblasts. The predominant farm
IS
BB and as in emhryonic muscle,
the MM isozyme accounts for the increase in CK activity ohserved during fusion leading to
formation of multinuc1eated fibres. Analysis of muscle regeneration has shown that muscle
satellite cells synthesize embryonic isoforms of myosin and tropomyosin. This suggests that
in muscle regeneration originating from satellite ce Ils, the dlfferentiation sequence is similar
to that of embryonic muscle(42). In vitro, satellite cell derived myotuhes l'rom fast muscle
synthesize only fast myosin light chains, while myotuhes derived from slow muscle satellite
cells synthesize both fast and slow Iight chains(41). This indicate!> Hwt satellite ce Ils are to
sorne extent more differentiated than primitive emhryonic myoblasts. Such a concept of
heterogeneity among myogenic cells is consistent with the observation that acetylcholine
receptors are expressed at ail stages by satellite eclls but appear in embryonic cells only at
the onset of terminal differentiation(40.9). Moreover, 12-0·tetradecanoylphorhol-] 3-acetate
8
(TPA), a tumor promoter, can be used to suppress myotube formation in embryonic
myohlasts.
When the sa me treatment is applied ta cultures of adult satellite ceUs or
myohlasts from nider embryos, it doesn't suppress myotube formation. Thus, embryonic
muscle histogenesis appears ta be a sequence of distinct phases that are reflected in the
consecutive appearance of various subpopulations of myogenic ce]]s, one of which is
represented hy satellite cells.
Satellite and embryonic muscle cells can be further distinguished from each other by
their morpholob'Y upon withdrawal from mitotic cyc1ing(38). In culture, the transition from
the proliferatlve stage to the post-mitotic state is associated with a change from a round
shape tn a spindle shape in satellite cells. Embryonic myoblasts, on the other hand, always
appear spindle shaped.
Interestingly, using tritiated thymidine labelling and radio-
élutography, it was found that myoblasts and satellite cells were able to recognize each other
and fuse to fmm hybrid myotubes when co-cultured together. Finally, at the onset of
(
myogenesis in regenerating muscle, satellite ceUs divide as little as once before fusing inta
multinucleated myofibres(43-45). This is in contrast to embryonic myoblasts which divide at
least four times before terminally differentiating.
CAN TERMINAL DIFFERENTIATION OF MUSCLE CELLS BE INFLUENCED?:
The proposed hypothesis (vida infra) suggests that satellite ceHs, which are relatively
uncommiUed skeletal muscle cells, can be influenced to differentiate into striated cardiac
muscle. in general, myogenic differentiation depends mainly on growth factors and neural
control(4fl,47,411).
In the model, satellite ce)]s, displaced from their normal physiological
environmellt, may he able to alter their normal differcntiation sequence as a result of a
change in availahle growth factor and neural control.
Myogenic differentiation can be defined as the process by which myoblasts become
post-mitotic and subsequently fuse ta form myotubes which express muscle specifie genes.
G rowth factors are able ta interact with specifie receptors on the cell surface and by tbis
mechanism induce illtra-cellular signalling whieh can affect both proliferation rate and
')
differentiation. These effects can be induced in many œil types and various cmnhinations
of growth factors may have additive effects. Although discussion of specitk growth factors
is beyond the scope of this proposai, a rccent article hy Eghali et al is rcpresentative of this
growing field of study(76). These authors descrihe a series of experiments in which cultured
cardiac fibroblasts treated with transforming growth factor B, can display
li
myocytc
phenotype and express sarcomeric actin mRNA. One cao only speculate on the implications
of such an observation, but this may mean that future stlldies cOllld identify ncw rcgulatory
factors for muscle cells that may be different l'rom those that are alreaùy known(4" 'i \). SlIch
regulatory factors are described as having distinct biolngical roles in controlhng muscle
development
The final stage of maturation depends on innervation of the deve\oping myotuhes.
This has also been confirmed for skeletal muscle regeneration(S5,'i(,).
Experiments have
shown that fast twitch muscles could be converted to slow twitch muscle by cross-innervation
with a slow twitch nerve(54). Such a transformation is accompanied hy changes in regulatory
proteins of the muscle cells, isozyme profiles, and histochemlcal charactenstics.
aJteration of innervation leads to alterations
III
Thus,
the translation or transcnptlon actlvities of
the cell. Ongoing studies in the surgical laboratory of the Montreal General Hospital have
involved the transformation of mature skeletal muscle using electrical stimulation.
This
results in conversion of the contractile and calcium regulating systems tn profiles
approaching those of cardiac musdes(57). It is not known if ske\etal muscle satellite cells can
be transformed to cardiac muscle. The objective of this proposai
IS
tn investigate thls
question. Another outcome would be that the satellite cdb implanted mto the myocardium
would fuse to form skeletal muscle with a cardiac muscle like metabolism and in this way
may contribute to myocardial function.
SKELETAL MUSCLE SATELLITE CELL TRANSPLANTATION:
Studies from the 1960's and early 1970's, establishcd that skeletal muscle fihres are
formed via fusion of mononucIeated myoblasts(12,24). In 1965, Yaffe and Feldman examined
the possibiJity of fusion of cells of different genotypes using radio-autographie celliahelliog
10
techniques(f11). They were able ta show in vitro production of hybrid muscle fibres using
myoblasts l'rom calf fetuses and newborn rabbits and rats.
These findings suggest the
possibility of using cell transplantation in a way that may alter genetie make-up of muscles.
ln 1979, Jones as weil as Lipton and Shultz developed experimental models whieh
combined in vitro and in vivo methods(I 10.29). Isolated myogenic cells from regenerating adult
rat skeletal muscle, were grown in culture, labelled with tritiated thymidine and grafted back
into adult
~keletal
muscle. Using sueh a mode l, Jones was able to show limited survival of
homografts (implantatIOn into a dil'l'erent hast of the same species) and long term survival
of autografts. This model suggested that cells rather than tissue could be transplanted for
organ repair and that immune response might be expeeted with such manipulations. Indeed,
Karpati et al have shown that major histocompatibility complex gene products are expressed
in regenerating human skeletal museles(lll), In the la st twelve years, others have confirmed
that implanted satellite cells multiplied in vitro retain their myogenic potential, participate
in regeneration of injured skeletal muscle and could co-express at least sorne of their genes
with host nuclei within the same muscle fibre(27.64-67), Law et al have shown that myoblasts
l'rom embryos cou Id he used to improve structure and function of dystrophie muscles(68-70),
Furthermore, satellite cells multiplied in vitro have been used to regenerate skeletal muscle
following experimental injury<27.34). Clmically, although the results are not known, trials have
hecn undcrtaken in humans to use satellite cell transplantation in the treatment of museular
dystrophy(7I.72) .
HYPOTHESIS AND PURPOSE:
Appropriate stimulation can aetivate dormant skeleta 1 muscle satellite eells and this
leads to skeletal muscle regeneration.
It is hypothesized that satellite cells, which are
relatively undifferentiated, can he transplanted into acutely injured heart muscle and
eonscquently differentiate into myoeardial eells refleeting their acquired environment.
The purpose of this study is to test this hypothesis by multiplying canine skeletal
muscle satellite eclls in vitro and grafting them into injured heart muscle tissue.
Chapter II
MATERIALS AND METHODS
11
.
STUDY DESIGN:
Twcnty mongrcl dogs agcd 1-2 years old were divided into 2 groups representing
sequential in vitro and in vivo series of expcriments.
In group A, 6 dogs underwent excisional biopsy of the anterior tibial muscle of the
hmd limb. These first studies were carricd out in order to set up a functional cell culture
facllity and to learn the technical aspects of manipulating and labelling cells in vitro. These
cxperiments were also used to verity myogenicity of the isolated cells.
In group B, 14 dogs were subjected to a cryoinjury of the heart muscle followed by
skeletal muscle satellite ccII implantation into the acutely injured myocardium.
These
implanted cells were obtained from the same dog about 10 days prior ta the time of injury.
Preparation of the cells consisted of in vitro multiplication and labelling with tritiated
thymidine
III
order tn trace their evolution at the time of sacrifice several weeks after
implantation. Control consisted of injection of serum-free growth medium without cells into
an adjacent implantation channel within the injury site. Additionally, cryoinjury of the left
latissimus dorsi muscle was carried out in 4 of these do&s. This skeletal muscle was then
electrically stimulated with a pacemaker in the postoperative period in order ta transform
it to a fatigue reslstant cardiac-Iike state. The purpose of this control was to determine if
electrical stimulation alone could transform replicating satellite ce Ils within a muscle
regenerate into cardiac muscle cells.
Endpoints consisted of post mortem histological and radioautographical studies
carried out at 6 to 14 weeks post operatively. AIl experiments were performed according
to guidelines of the Canadian Council on Animal Care.
OPERATIVE PROCEDURES:
i. Muscle Binpsy
General
anaesthesia with pentobarbital (30 mglkg) was induced following
estahlishment of intravenous access. The airway was secured with endotracheal intubation
12
and anaesthesia maintained with 1% Halothane administered hy inhalation. Mechanical
ventilation was pressure regulated to a maximum of 25 cm H 20 and set al a raft' of 10-15
breaths per minute. The dog was placed
ln
the right lateral decuhitus po~ition and the left
hind Iimb was shaved and prepared with iodinc solution. A longitudlllai incision was llsed
to expose the anterior tibial muscle. The dissection nt the muscle was carried nut l'rom ils
tendinous insertion distally to ItS ongin on the tihia proximally (Figure 2). Fcedmg vessels
were doubly Iigated using 3-0 silk ties and dlvided hetween the tics.
Once the muscle was excised, il was placed in co Id
~aline
solution (ahout 10°) llntil
it could be processed for tissue culture one hour later (vida mfra). The incision was c10sed
using interrupted absorbable suture (2-0 Vicryl) for the fascial layer~ and a continuous
subcuticular absorbable suture for the skin (4-0 Dexon). A small drain
wa~
leU in the wound
and removed 24 hours later. Extubation was usually possible one h(llIr post-operalivdy. No
appreciable handicap was observed 3 days atter the dog regained consCÎousness.
ii. Cryoinjury to the Myocardium
The same dog used for ccII preparation was re-operated for œil implantation.
Induction and maintenance of general anaesthesia was as descrihed above. Peri-operative
antibiotic prophylaxis conslsted of one gram of Cetazolin.
Sy~temic
hlood pre~sure was
mopitored using an arterial cannula placed percutaneou~ly in the lemoral artery and
connected to a pressure transducer adapted tn produce a tracing.
A standard left anterolateral thoracotomy was performed through the sixth intercostal
spa ce and the lung was retracted posteriorly. The lateral wall of the pericardium was
opened longitudinally and its edges suspended to the che st wall. Care was taken to preserve
the phrenic nerve. The prospective injury site was idemified on the lateral wall of the left
ventricle between the left anterior descending and circumtlex arteries. Syslemic heparin was
given (100 lU/kg) in arder to prevent intra-cavitary thrombus formation dunng the time of
cryoinjury and a lidocaine drip at 35 J,Lg!kglmin was initiated and continued for ] 2 hours
post-operatively for prevention of arrhythmias.
13
FIGURE 2
Excision of the anterior tibial muscle of the canine hind Iimb
l
14
A cryoinjury lesion ta the myocardium was achieved using a copper disc of either 2.5 or 5.0
cm in diameter depending on the size of the heart. The dise was mounted on an adaptor
that was placed on a probe in which IiqUid nitrogen was perfused (Fndgitronics, Connecticut,
USA). The probe was cooled to -160°C and the frosted dise was firmly applicd to the
surface of the left ventricle. In order 10 ensure an adcquate inJury, If the 5 cm diameta disc
was used, injury time was 2 cycles of 3 minutes each. For the smaller disc (2.5 cm dJamder),
injury time consisted of two cycles of 12 mmutes each. This estimation was arrived at hy
calculating the disc area reduction (proportional to 1rr2) and assuming that the additional
time needed for an injury of similar depth using the smaller disc was inverscly proportiona\.
If cardiac output was perceived to he depressed during the tlme ot InJury, either hy detceting
a faH in systemic blond pressure or hy ohservmg depressed contractility and hradycardia,
Epinephrine (0.1 - 0.2 mg) was given intravenously for tem ,1orary inotroplc support.
AdditionaHy, the pressure apphed on the disc hy the operator was reduced
minimize deformation of the heart cavltles and the hemodynamic
to this. The frozen dise was not withdrawn l'rom the
cycle.
~urtace
consequence~
III
order ln
attrihutahle
of the ventnc\e dunng an inJury
This was necessary in order tn ensure a homngeneolls quasI transmural inJury.
Following complet ion of the injury, the pro he was warmed to 40°C, removed, and the area
of injury was allowed ta thaw (Figure 3).
iii. Cell Implantation
While the heart recovered from inJury, cell preparation for implantation, whlch had
been initiated earlier on the same day prior to inJury, was completed (vida infra). Ahout 0.5
- 1.5 million cells were collected l'rom the culture dishes in Vitro and suspended
ln
1-2 cc of
serum-free growth medium within a Hamilton glass syringe. A 2.5 cm linear pouch was then
created within the damaged tissue in the suhepicardial myocardium using a Tru-cut hlopsy
needle (Figure 4). The distal end of the pouch was marked with a 5-0 polypropylcne suture.
A similar suture was placed at the proximal end of the pOllch and left untied. Next, the cell
implantation was carried out by adapting a 14-gauge Tetlon catheter to the ~yringe
containing the cells. Excess air was evacuated t'rom the syringe and thè catheter was then
introduced into the implantation channel. A half hitch was then placed on the proximal
1
15
suture and the channel opening was tightened over the catheter in order ta prevent baek
leak of the eells during infusion. Thus, with tension being maintained on the proximal suture
hy the operator, ilnother operator injected the cells within the channel over a period of
ahout 15 to 30 seconds. Immediately thereafter, the catheter was rapidly withdrawn and the
channel opening cIosed hy completing the hiteh on the suture. As stated above, control
consisted of inject mg serum-frec growth medium without cells in an adjacent site within the
inJured area using an identleal protoeol.
The pericardium was left open, and the chest was c10sed in layers using 1-0
polyglactin ahsorhable suture. A drain was left in the chest cavity for 24 hours thereafter.
Extuhation was usually possible within two hours post-operatively.
J
16
FIGURE 3
Lateral wall of the left ventricle following cryoinjury.
The thawed muscle appears purple.
17
FIGURE 4
f
\
Creation of a channel within the acutely injured heart
muscle using a Tru-cut biopsy needJe.
18
Ïv.
Skeletal Muscle Injul)' and Electrical Stimulation
The left latissimus dorsi muscle was already exposed at the time of thoracotomy.
Cryoinjury was carried out using the same protocol as the one ùescribed above and the
injury site was marked with circumferential polypropylene stay sutures. Transformation was
achieved by using a programmable pulse generator (Itrel, Medtronic Inc.) 10 stimulate the
muscle by connecting the electrical le ad to the motor nerve of the latissimus muscle
(thoracodorsal nerve). The transformation parameters used were: pulse amplitude of 1 v,
36 pulses per second burst frequency and a pulse width of 21O#-,sec. Stimulation was carried
out in discontinuous fashion with an "off' period lasting 0.4 seconds and "on" perioù of 0.1
sec (2 Hz burst stimulation). This proto col for muscle transformation to a fatiguc-resistant
cardiac-Iike metabolism had been described previously(2) and routinely used in the surgical
laboratory at the Montreal General Hospital.
CELL CULTURE MElliODOLOGY:
i. Isolation and Initial Platini of Satellite Cells
Once the muscle tissue was obtained it was weighed and rinsed in 70% ethanol for
about 30 seconds. The 50-60 grams of muscle obtained were then dissected in order to c1ear
tendon and cartilage tissue from the muscle tissue. The muscle was then submerged in 100
cc of Hank's balanced salt solution with 1% penicillin-streptomycin and was findy minced.
Using a modification of a method described by Cossu et al(38,39,40) the tissue was incuhated
in M199 solution (Appendix 1) with 1% collagenase, 0.2% hyaluronidase (filtered, sterilized
solution) for 17 minutes at 37°C. The released material from this first digestion consisted
mostly of connective tissue cells(27). Muscle fragments were then sedimented at 540 g x 5
minutes and incubated again this time, in M199 with 1% protease (filtered, sterilized
solution) for 12 minutes at 37°C. The released cells were then collected hy sedimcnting the
digested muscle tissue and the enzyme reaction was arrested by adding 10% horse serum
(Gibco, New York) to the cell suspension. The cells were then collected in a pellet by
sedimenting the suspension at 775 g x 10 minutes. The supernatant was discarded, and the
cells were re-suspended in M199 solution with 1% penicillin-streptomycin and 0.25%
Fungizone.
The purpose of this step was to rinse away debris from the ccII pellet.
19
Following re-sedimentation and suspension for a total of four wash cycles. The cells were
counted on a hemocytometer using the trypan blue dye exclusion test. This was achieved
hy adding a drop of cell suspension to a drop of 0.1 % trypan blue. Using a Pasteur pipette,
the counting chamber was filled, and the ce Us counted. The trypan blue was taken up by
the dead cells and these were excluded. Final counts were then multiplied by 2 to correct
for the trypan blue dilution.
Initial plating was at a density of about 6-8 x
lOS ceUs per 60 mm culture treated dish
in 3 cc of medium consisting of M199 supplemented with 10% horse serum, 10% Lglutamine (Gibco, New York) and 7.4% minimum essential medium (Appendix 1). The
antibiotics added consisted of 1% penicillin-streptomycin, and 0.25% Fungizone. The dishes
were placed in an incubator in which the atmosphere comprised 5% CO2, air and humidity.
Medium changes were carried out every 48 hours.
iL ln Vitro Cell Multiplication
Cells were grown and passaged in order to prevent muscle fibre formation in vitro.
At confluence, the cells were split into an increasing number of dishes. Thus over the 10
day period following the initial plating prior ta implantation, passaging was carried out at
day 4 and day 7 (Figure 5), celllabelling at day 9 (vida infra) and implantation at day 10.
The protocol used for passaging the cells consisted of first aspirating the growth medium off
the dishes. Next, 0.5 cc of 0.05% trypsin was added to each 60 mm dish. The trypsin was
removed 30 seconds thereafter and the dishes were left untouched for Il minutes according
to a protocol described by Kaighn et al(74).
Following this, the dishes were rinsed by
pi petting M 199 sol utinn onto the ceUs thus enhancing their detachment from the dish surface
and causing a stream effect. The ce)) sUl)pension was then poured without pipetting into a
conical 50 cc polypropylene tube containing 5 cc of horse serum in arder ta inactivate the
trypsin. This rinsing was repeated three times. In between rinses, the dishes were inspected
using a phase contrast microscope ta verify cell detachment. If detachment was poor, gentle
traction with a cell scraper was used. This was avoided on the first passage (day 4) in arder
to minimize detachment of non myogenic cells(74). The cell suspension was then spun at 773
20
g for 10 minutes and the cells were re-suspended in M 199, counted as described above and
plated at a lesser density (6 x
Hr ceUs in 3 cc of medium per 60 mm dish).
üie Cell Labelling
Nine days following initial plating, one day prior to cell implantation, the cells were
labelled with tritiated thymidine (activity 70-80 Ci per mmol, Amersham, USA). This was
done by adding the radioactive thymidine to the growth medium at a concentration of JO
#,Ci/ml(113). The "co Id" medium was removed and the cells were then grown in "hot" medium
for 20 min. Thereafter, they were rinsed with M199 twice (3 cc per 60 mm dish) and
regrown in "cold" medium. This 20 minute pulse was repeated every 5 hours over a period
of 15 hours for a total of 4 pulses.
ive Cell Preparation for Implantation
On the day of implantation, the labelled cells were harvested from the equivalent of
one hundred 60 mm dishes (thirty six 100 mm dishes were used in order tn facilitate the
manipulations). This process was initiated by another operator at the sa me time that the
thoracotomy was carried out on the redpient dog. Briefly, the ce Ils were detached fmm the
dishes with trypsinization as described above, and placed within about twelve conical
polypropylene tubes. These were then spun at 773 g x 10 minutes and the combined pellets
(0.5-1.5 X 106 ceUs) were then re-susp~ftded in one tube, counted, re-centrifuged and resuspended in 1-2 cc of serum-free growth medium thus completing the cell preparation for
the ensuing implantation.
21
FIGURES
t
~
,r.~
~
8.
,
,
.1
,.
"
J~
1
-'li.
1
"\~ ,
~
"
Cultured skeletal muscle satellite ce Ils at confluence prior to passaging.
l
(Fixed in vitro with 10% formaldehyde, toluidine blue stain, x400)
22
SPECIMEN PREPARATION FOR HISTOLOGY:
Preparation of the specimen for histological analysis consisted of fifst excising a
bloodless, perfused fixed heart at the time of animal sacrifice. Under general anaesthesia.
with the animal mechanically ventilated, a standard median sternotomy was performed using
an electrical saw. The anterior wall of the pericardium was incised longitudinally and its
edges were suspended to the chest wall. The left laleral wall of the pericardium and the Idt
lung, usually adherent to the previous injury site, were gently peeled off the epicardium with
blunt dissection. Mobilization of the superior vena cava, inferior vena cava and aortic arch
was then carried out with sharp dissection and each of the se structures were encircled with
umbilical tape. A 14-gauge Teflon cannula was then inserted into the aortic root proximal
to the vessel tape and special care was taken so that the aortic valvular mechanism remained
intact. Cardioplegia solution (made by adding 40 mEq of potassium chloride to one litre of
normal saline) was then connected to the cannula in preparation for infusion. The
inft~rior
and superior vena cava were then ligated and with the volume depleted heart still beating,
the aorta was ligated at the arch. Immediately thereafter, cardioplegia was administered at
150 mmHg using a pressure infuser. The aortic root could be seen to distend, retlecting an
intact aortic valve, and shortly after, the heart appeared pale and flaccid indicating adequate
coronary perfusion. The heart was vented to prevent over distention by incising the right
and the left atrial appendages. This ensures adequate coronary perfusion.
Mechanical
ventilation was stopped and the entire litre of solution was infused in order to c1ear the
myocardium of blood.
Next, perfusion fixation was accomplished by using the same apparatus to infuse 7()()
cc of Karnovsky fixative solution (Appendix
2)(75)
into the aortic root cannula. The heart,
now firm due to the calcium in the Karnovsky solution, was excised by first dividing the
inferior and superior vena cava at their respective entry points into the nght atrium. Next,
while lifting the ventricles out the pericardium, the pulmonary veins were divided posteriorly
and the pulmonary artery and aorta eut anteriorly. The heart was then stored in 300 cc of
Karnovsky solution for about four hours. Following this, the injury site was carved out of
the left ventricle wall and placed in a small container with Karnovsky fix solution for another
23
4 hours. The specimen was then stored overnight in sodium cacodylate buffer solution
(Appendix 2).
The next day, specimen cutting was carried out under the dissecting
microscope (Figure 6).
Tissue samples for paraffin embedding were stored in 10%
formaldehyde solution and samples for epon embedding were stored in sodium cacodylate
huffer solution. Processing was carried by the department of pathology. Radioautography
was carried out at McGiII University in the department of anatomy. Briefly, unstained,
uncovered sections were dipped in photographie emulsion(l13). The B particIes released from
the radioactive tritium produce an image in the emulsion which can be seen after treatment
with developer. Finally, the slides were stained according to standard protocols. For the
paraffin sections, this consisted of hematoxylin and eosin as weB as Masson trichrome blue
specifie for connective tissue(1l4). Toluidine blue was used for epon sections.
24
t
FIGURE 6
III,
Excised lateral wall of the left ventricle following perfusion-fixation 8 weeks post-cryoinjury.
Red ink delineates the left ante ri or descending and circumflex coronary arteries. The scar
tissue appears discolored and is clearly distinct from the surrounding normal cardiac muscle.
There are 3 implantation channels irradiating from a common point and extending towards
the coronary arteries shown. Polypropylene sutures were used tn close the channels and to
mark out their respective locations.
l
Chapter DI
RESULTS
r
,
25
OPERATIVE RESULTS:
A total of 20 dogs were operated (Table 1). Six dogs (group A) underwent
explantation of the anterior tibial muscle in order ta develop a working knowledge of cell
culture methods needed to isolate and grow skeletal muscle satellite ceUs. Fourteen other
dogs (group B) underwent attempts at cell implantation into acutely inJured myocardlllm
after tibial muscle excision. Overall, of the 20 leg dissections carried out, there was one
wound infection treated with revision of clos ure.
Of the 14 attempts al myocardial
cryoinjury, there were five intra-operative deaths due to irreversible cardiac arrest.
Table 1
Operative Results
n
Group A
(anterior tibialis
muscle excision)
Group B
(myocardial cryoinjury with
attempted satellite
cell implantation)
Morbidity
Mortality
6
1
()
14
0
5
Of the nine dogs in group B that underwent successful cryoinjury, four dogs were
exc1uded from the results. In three of these, there was technical difficulty in creating the
implantation channel. Two had uncontrollable back bleeding form an injured vessel within
the channelleading ta leakage of the cells out of the implantation site. In the other, the
channel was made too superficially and the ce lis leaked out during the implantation
reflecting an inadequate channel roof. Another dog underwent sacrifice at eight weeks postcell implantation, but was found to have an unsatisfactory inJury at post-mortem studies.
The scar resulting from the cryoinjury was limited to the epicardium and the implantation
channel could not be identified with certainty in the normal myocardium. Thus, tive cell
.'~
implantations are inc1uded in the histology results (vida infra); one dog was sacrificed at 6
26
weeks post-ccII implantation, 2 at eight weeks, and another 2 after fourteen weeks.
IN VITRO STUDIFS (Group A):
Initial in vitro studies consisted of excision of the anterior tibial muscle of the hind
hmh, qualification of ccII yield and confirmation of a muscle forming cell population. Mean
muscle weight was 52.5+ 11.6g. Cell yield was 2.3+ 1.0 x
Hf
cells per gram of muscle
tissue. Viahility of initial cell populations was about 60-80 percent (trypan blue exclusion
test). Myogenic cells appeared fusiform as compared to non myogenic cells which were
ahout the same size hut rather circular(llO). Myogenicity of the cell cultures was confirmed
hy ohserving muJtinucleated muscle fibre formation using phase contrast microscopy (Figure
7). Further studies revealed that such myogenicity was persistent following passaging of
cultures (Figure 8). The cell populations observed were not composed entirely of muscle
forming cells. Background cells were non-muscle forming and varied from 20 to 40 per cent
of the total cell population observed.
(
Finally, cell labelling with tritiated thymidine was confirmed (Figure 9). These
lahelled cells were also tested for myogenicity and it was found that cell labelling did not
affect the ability of the cultured satellite cells to fuse and form muscle fibres.
(...
27
FIGURE 7
;"'. '
"; ~
•
Il
,;
•
t
"
,-'"
il,"
..
j
•
"
Phase contrast view of unpassaged cultured satellite ce Us. A multinucleated muscle fibre is
se en to have formed from fusion of the cells confirming the myogenicity of the cultured cell
'"'
~">
population. Non myogenic cells can be observed in the background. (x100)
28
FIGURE 8
•,
Muscle fibre originating form passaged satellite cell culture.
Note multiple nuclei within the maturing fibre (fixed in vitro with
10% formaldehyde, phosphotungstinic acid hematoxylin stain, xIOOO).
29
FIGURE 9
•
Epon black section of a pellet of satellite cells (775g x 10 min.)
showing radioautograph of nuclei labelled with tritiated thymidine.
(Toluidine blue stain. x1000)
~-----------------
-------
30
HISfOLOGY (Group D):
i. Cell Implantation within Acutely Injured Myocardium
Results for the sacrifices carried out at 6 and 8 weeks post~operatively were identical.
Macroscopically, quasi transmural inju!)' was verified and the implantation channel was
confirrned tn be weil within the scar a few millimetres beneath the epicardium (Figure 10).
The channel itself was then cut longitudinally and opened in the same way that a. book
opens. This revealed an area of red-brown tissue entirely surrounded by scar tissue (Figure
]
]).
Microscopically, this area of tissue was observed to be striated muscle surrounded by
dense connective tissue (Figure 12 and Figure 13). Further studies of paraffin black sections
using trichrome blue staining for connective tissue showed that there was inhomogeneous
staining in the experimental site when compared to the control site reflecting the presence
of muscle tissue within the satellite cell implant (Figure 14 and Figure 15). Finally, using
epon blocks and toluidine blue staining, it was found that the muscle tissue observed lacked
nuclei but did reveal the presence of intercalated discs typical of cardiac muscle (Figure 16).
Analysis of specimens from sacrifice at 14 weeks post-implantation faited to show muscle
tissue within the scar tissue that had formed.
ii. Electrically Stimulated Regenerating Skeletal Muscle
These studies were carried out in order to determine if electrical stimulation al one
could transform regenerating skeletal muscle into morphologically recognizable cardiac
muscle. Histolob'Y results Îrom these experiments showed that 8 weeks following cryoinjury,
ck,,,,ârically stimulated latissimus dorsi muscle regenerated into skeletal muscle and faHed to
reveal élny transformation into cardiac muscle (Figure 17).
31
FIGURE 10
CENTU.
Myocardial scar 8 weeks post-cryoinjury. Ink is used to mark the specimen to demonstrate
that the scar is transmural at its center. The implantation channel is seen marked at its
extremities by polypropylene sutures. Note that the scar tissue is more dense and contracted
in comparison to the surrounding heart muscle.
32
FIGURE Il
. - - - .. .
~
.
1
2
CENTIMETRES
Muscle tissue within the cell implantation channel which has been cut horizontally along its
length. The proximity of the polypropylene suture confirms that site as the one which
satellite cells have been transplanted at the time of cryoinjury 8 weeks previously.
.'
33
FIGURE 12
Paraffin block section of the tissue seen in Figure 11. Muscle tissue is
surrounded by scar tissue. (Hematoxylin and Eosin, x4(0)
...
----------------------------~~---
t
34
FIGURE 13
High power magnification of the tissue seen in Figure 12.
Note striations confirming the nature of the tissue observed.
(Hematoxylin and Eosin, xlO00)
35
1
FIGURE 14
1
Masson trichrome blue stain of a cell implantation channel 8 weeks
post-cryoinjury showing inhomogeneous red staining reflecting the
presence of muscle tissue.
36
1
FIGURE 15
Masson trichrome blue stain of a control site within a specimen 8 weeks
post-cryoinjury. Note the homogeneous blue eolour reflecting the uniform
nature of the scar.
•
37
FIGURE 16
.
•
1
Epon block section of a specimen originating t'rom the cell implantation
site within an 8 week old cryoinjury. Note the presence of intercalated
dises suggesting that the tissue seen is myocardial syncytium. (Toluidine
blue stain, x10(0)
38
FIGURE 17
Latissimus Dorsi muscle regenerate 8 weeks following cryo-injury and electrical
1
stimulation. Note the presence of nuclei located peripherally and the absence of
intercalated discs. (Hematoxylin and Eosin stain, x1000)
Chapter IV
DISCUSSION
.'
1
39
Heart muscle lacks the ability for regeneration following injury(16-21,60,61). It has been
suggested that this is because unlike skeletal muscle, there are no satellite ce Ils associated
with myocardial syncytium(8). These ce Ils, normally dormant in mature skeletal muscle, have
the role of initiating a regenerative response following muscle damage(8,30,31). Furthermore,
studies have shown that skeletal muscle satellite cells resemble embryonic myoblasts and are
not yet fully differentiated(9,38-42). It has also been observed that muscle in jury can release
factors that can activate satellite cells(37). It was therefore hypothesised that if myocardium
could acquire satellite ce lis, it may gain the capacity for repair foHowing injury and that tt. ~
stimulus for the activation and differentiation of the satellite cells would be the damaged
heart muscle itself.
The purpose of the present study was to test this hypothesis by
multiplying skeletal muscle satellite cells in vitro and implanting them into acutely injured
myocardium.
:1
The study groups chosen represent the various phases of the experiments leading to
this thesis. A cell culture facility was estab1ished and the biology of the satellite ce Ils in vitro
was investigated. ft was necessary to multiply the satellite cells in vitro prior to using them
for myocardial repair in order to get an adequate number of eells for implantation. This
experimental method is weil described and is used in the treatment of muscular
dystrophy(27,34,64,67,71.72,llO). Such experiments have led ta the conclusion that adequate cell
implantation could be achieved with a minimum of 0.3 x 106 cells(66). This number was
respected throughout the course of the present study.
Further refinement of this
manipulation would foc us on separating the cultured satellite cells from non-myogenic cells
using cell separation techniques thereby creating a more homogeneous cell population at the
time of cell implantation. The trypsinization protocol used in the present study achieves this
to sorne extent by not giving enough time for fibroblasts to dissociate form the cell culture
surface thus purifying the cell culture with every passage(74).
Sorne researchers have
observed that non-myogenic cells are necessary in order to adequately grow satellite cells(72).
Others have used preplating technique in which initial culture and replated aCter 30 minutes
1
in order to allow attachment of non-myogenic cells which is usually faster(llO). Ideally, cell
fractionation on
il
Pereoll gradient would achieve nearly 100% purity at the time of
1
40
implantation(67).
A method for myocardial injury that lends itself to anatomical studies was used. The
cryoinjury model has been weil described and is known to produce a homogeneous sharply
defined scar(58,59). Thus, folJowing ce)) implantation within the injury site, any muscle tissue
seen in the post-operative period, once scar has forme d, could be attributed to the implanted
cells. It follows that, a minimum of 6 weeks was chosen in order to allow scar formation
following myocardial cryoinjury. Long term studies whereby specimens were examined at
14 weeks post-operatively were carried out in order to see if any newly formed myocardium
(neomyocardium) could survive. Control consisted of injection of serum-free growth medium
into acutely injured myocardium so that the possibility of surviving myocardium within the
cryoinjury scar could be excluded. Lastly, an additional set of experiments were carried out
to see if electrical stimulation alone, reproducing the mechanical environ ment of eontracting
1
heart muscle, is enough to cause satellite cells to transform into morphologically recognizable
myocardium.
The technique of autotransplantation used to implant the satellite cells into the
injured myocardium was chosen because of its simplicity and in anticipation of the expected
endpoint. The model used in the present set of experiments does not represent a c1inical
simulation where scarring is often inhomogeneous and diffuse. Clinical application of the
work presented would require scattered injections of satellite ce Ils in order tn strengthen
large areas of damaged myocardium characteristic of end-stage cardiomyopathy. Bcfore
designing an experimental model to test this, it is first necessary to c1early show anatomieal
proof of the hypothesis proposed. Further stlldies would then be aimed at analyzing the
biochemistry of the implanted cells to verify metabolie transformation. The final phase
would be to determine if indeed implanted satellite cells could contrihute tn myocardial
function. If this is established, it would then be necessary to study the immlinolob'Y of
allogenic cell transplantation.
The study protocol used consisted of macroscopy and histological studies. These were
41
undertaken under the guidance of the department of anatomy. Standard paraffin block
sections were used to study large areas of collected specimens. Sm aller sections of tissue
(~2mm ") were embedded within epon blocks to allow for higher resolution microscopy and
radioautography. Preparation of such blocks requires precise dissection of the specimen
which was carried out using microsurgical techniques. Prior immersion into cacodylate buffer
solution facilitated this dissection by softening the tissue samples and making them easier
to cut. Three different types of stains were used. Hematoxylin and eosin standard staining
for paraffin sections provided contrast for gross identification of muscle tissue within scar
tissue. Such findings were confirmed using Masson trichrome blue stain which is specifie for
connective tissue(114). Toluidine blue was used for epon section staining. This stain has the
advantage of contrast for radioautography. Also, it was discovered during the course of the
prdiminary experiments that it enhances contrast for identification of intercalated discs
typical of cardiac muscle cell junctions (Figure 1).
Several findings resulted from this study. There was presence of mm'de tissue within
the myocardial scar at 6 and 8 weeks post-satellite cell implantation but not at 14 weeks.
Further analysis failed to reveal the presence of nuclei in the observed specimens and th us
radioactive œil label cou Id not he identified. These findings were not present in the control
site within the cryo-injured myocardium. Moreover, Masson trichrome staining was markedly
inhomogeneous at sites where satellite cells had been implanted indicating the presence of
muscle tissue mixed with scar tissue. Toluidine blue staining of epon block sections of the
muscle tissue found at cell implantation sites within scars revealed the presence of
intercalated discs. Interpretation of these data therefore suggest the hypothesis that skeletaJ
muscle satellite cells implanted within acutely injured heart muscle can give rise to
neomyocardium which itseIf then t'ails to survive. This delayed cell death is reflected by the
absence of nuclei in the observed muscle tissue. Thus the evidence present for the proposed
hypothesis remains circumstantial since absence of nuclei precluded radioautographic
identification of the source of the presumed neomyocardium. In future studies, an additionaJ
cytoplasmic label would have ta be used. One such method is ta use fluorescent beads(27).
1
42
Various reasons may é!ccount for the death of the newly formed myocardium with the
cryoinjury scar. Firstly, scar tissue has a different density when compared to muscle tissue.
It is contracted and this may not permit for adequate micro-vascularization which is needed
for muscle survival. Another possibility is that the neomyocardium within the scar tissue was
not adequately innervated. Such innervation is needed for proper maturation and survival
of developing skeletal muscle and it may be that a similar requirement is necessary for
cardiac muscle(55,56).
Further studies are needed to provide conclusive evidence for the hypothcsis that
skeletal muscle satellite ce Ils can be transformed into cardiac muscle cells. If indeed this
evidence is found as was seen from the studies using electrical stmmlation of inJured
latissimus dorsi muscle it can already be concluded that electrical stimulation leatling to
alteration of the mechanical environment of satellite cells is probahly not enough to
1
transform these into cardiac muscle ce Ils.
This points to the speculation that injuretl
myocardium provides growth factors necessary for the transformation of the rclatively
undifferentiated skeletal muscle satellite cell into neomyocardium. Current ongoing stutlies
at the Montreal General Hospital Surgical Research Lahoratory are focusing on trackmg
labelled satellite cells implanted into acutely injured myocardium.
In
the~e
sets of
experiments, specimens will be studied at 1 to 6 weeks post-operatively in order tn follow
the evolution of the transplanted skeletal muscle satellite cells. Other ongoing studies are
focusing on demonstrating thclt cytosol extract from myocardial tissue can enhance gmwth
of skeletal muscle satellite ce Ils in vitro. These studies may lead tn identification of growth
factors that my transform satellite cells into cardiac muscle cells. The molecular hiology of
such cell manipulation is beyond the scope of this thesis but this avenue would have to he
explored in future studies. The model descrihed in this thesis would be ideally suited to
study the molecular ontogeny of cardiac muscle.
The concept of cell transplantation
introduced an application of cell biology that can eventually complement organ
transplantation in the treatment of heart disease.
43
APPENDIXI
r
44
COMPOSmON OF Ml99 SOLUTION (commerciaUy available from Gihco, New York)
AMlNO ACIDS:
mgIL
VITAMINS: (Conl'd)
DL-A1amne
5000
Choline Chlonde
050
Adenine sulfate
1000
L-Argmme HCL
7000
Folle aCld
0.01
AdcnoslnClnphol>phalc
(dlsodlUm sail)
100
DL-Aspartlc 8cid
6000
1-ln05llol
005
Adenyhc dCld
020
Menadlone
001
Choll'Sterol
020
Nlaem
0125
Deoxynbose
OSO
Nlaemamlde
0025
D-Glucose
Para-amlnobenzolc aeld
005
GIUI.llhlone (rcdutcd)
oOS
OJO
L-Cysleme HCL H 20
L-Cysleme
L-Cyslem 2HCL
mg/L
O11mR COMPONI!.NTS
0.11
2600
DL-Glulamlc
aeld H 20
15000
L-Glulamme
10000
Pyndoxal HeL
0025
GUdmllc IICL
Glycme
5000
Pyndoxme HCL
0025
IIEPES
L-Hlslldme
HCLH 20
Rloonavln
001
Ilypoxanthmc
2188
L-Hydroxyprohne
10.00
llllamme HCL
001
Hypoxanlhmc (Nd
DL-lsoleucme
4000
Vltamm A (aeetate)
014
Phenol red
DL-Leucme
120.00
INORGANIC SALTS:
L-Lysme HCL
7000
CaCI 2 (anhyd )
DL-Methlonme
3000
Fe(NO J )3-9l!ZO
DL-Phenylalanme
5000
KCL
L-Prohne
4000
KH2 P04
DL-Senne
5000
MgS04 (anhyd)
DL-Threomne
6000
MgS0 4 711zO
DL-Tryptophan
2000
NaCl
L-Tryosme
mg/l
-
L-Tryosme
(dlsodlUm sail)
5766
DL-Vabne
50.00
VITAMINS:
Ascorblc aCld
005
cr-Tocopherol
phosphate
(dlsodlUm sail)
0.01
d-blotm
0.01
Calclfcrol
010
D-Ca pantothenate
001
RIbose
~II)
OJ<;4
2000
o~o
SodIum acelalc
SOOO
072
Thymine
OJO
400.00
Tween 80
2000
UracIl
OlO
200.00
9767
Xanthine
Xanthine (Na sail)
6800000
NaHC03
NaH 2 P04 11 Z0
100000
14000
Na2HP04 (anhyd)
-
Na2HP04·711Z0
-
0144
l
45
COMPOSITION OF MINIMUM ESSENTIAL MEDIUM (commercially available (rom Gibco,
New York)
AMINO A(:II>S:
mgIL
INORGANIC SAL1'S:
L·Tryplophan
1000
CaC/ 2 (anhyd.)
1.·Tynll>mc
3600
CaCI 2 1-:12 O
1.·Tymsmc
(dl,ndlUm roidi)
Fe(N03
D·Valme
KCI
mg/L
200.00
°
h -91-:12
400.00
AMlNO ACIDS: (cont'd)
L-Aspartie aeld
L-Cyslme
mg/L
2400
L-Cystme 2HCI
-
L-Cysteme HCI HZO
-
KH 2P04
-
L-Glulamlc aCld
VrI'AMINS:
MgCI 2 (anhyd)
-
L-Glutamme
1.·Â'>Corblc aeld
MgCI 2 GH2 O
-
G1ycme
-
L-Hlstldme
-
L-Hlslldme (free base)
-
L·Vahne
4600
BlOlm
-
MgS0 4 (anhyd)
D·Ca panlolhenalc
100
MgS04 71'!20
Chlomc bltartralc
20000
292.00
NaCI
6800.00
L-Hlstldme HCI H 2 0
42.00
Chollnc chlonde
100
NaHC0 3
220000
L-lsoleueme
5200
Fohe aeld
100
NaH 2PO4 ·H 2 O
14000
L-Leueme
5200
1·lnol>l101
2.00
Na2HP04(anhyd.)
Nlcollnamldc
100
Na211PO4 '7H 2O
l'yndnxal IICI
100
O'OIER COMPONBNTS:
Rloonavm
0.10
D·Glueose
l111ammc IICl
1.00
HEPES
Vllamm "12
-
KIRONUL'.JOClSII)ES:
Adenosmc
-
L-Methlonine
15,00
-
L-Phenylalanme
32,00
Llpolc aCld
-
L-Prohne
Phenol red
1000
L-Senne
1000.00
Potassium pcmellhn G
(iuanosmc
SodIUm succmale
1)I~OXYRIII()NIJCU~OSI()f~:
2'Dl'OX}'ddenO!.mc
Streplomycm sulfate
SUCCIRIe aeld
-
L-Lysmc (free base)
72.50
SodIUm Pyruvate
-
L-Lysme
L-Lysme HCI
Cylldme
Undme
-
-
AMINO ACIDS:
2'I>coxycylldmc 1IC1
L-A1anmc
-
Z'IK'OXygudnosmc
L-Argmmc
-
Z'lkI\\)'thymuhnc
L-Argmme HCI
12600
°
L-Asparagme 1-:12
-
L-Threonme
4800
46
APPENDIX II
l
47
1
'"
A
Protocol for Makinll Karnoysky Solution:
For every 100 ml:
Two grams of paraformaldehyde (FW=(30.03)n) in 25 cc distilled H 20
are dissolved by bringing the suspension to near boiling using a hot
plate in a fume hood.
A few drops of 0.1 N NaOH are then added to the mixture to make
it c1ear, and the solution is then cooled to room temperature.
Next, 50 ml of 0.2 M sodium cacodylate solution, 10 ml of 25%
glutaraldehyde solution, and 2 ml of 0.1 M calcium chloride solution are
added, and the pH is adjusted to 7.3.
Finally, the resulting solution is made up to 100 ml by adding distilled water.
B.
Protocol for Making Cacodylate Buffer Solution:
Thirty-five grams of sucrose (FW=342,3) are added to 250 cc of
distilled H 20 and this solution is mixed with 250 cc of 0.2 M sodium
cacodylate solution.
N.B.
The above protocols were obtaineù from the Department of Anatomy at
McGiII University
48
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~
99.
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60
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