Extra- and intrafusal muscle fibre type compositions
of the human masseter at young age
In perspective of growth and functional maturation
of the jaw-face motor system
Catharina Österlund
Departments of Odontology, Clinical Oral Physiology, and
Integrative Medical Biology, Section for Anatomy, Umeå
University, SE-901 87 Umeå, Sweden
Umeå 2011
Responsible publisher under swedish law: the Dean of the Medical Faculty
This work is protected by the Swedish Copyright Legislation (Act 1960:729)
ISBN: 978-91-7459-285-6. ISSN: 0345-7532. Series No 120.
Cover image: Muscle cross-section from young masseter stained by monoclonal antibody
MHCn against myosin heavy chain-fetal, showing extrafusal muscle fibres, and intrafusal
muscle fibres within a muscle spindle. Note above, muscle spindle containing numerous
intrafusal fibres. Variability in staining intesity reflect variable physiological properties of
fibres.
Elektronisk version tillgänglig på http://umu.diva-portal.org/
Tryck/Printed by: Print & Media, Umeå Universitet Umeå, Sweden, 2011
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TABLE OF CONTENTS
Table of contents
i
Abstract
Abbreviations
Svensk sammanfattning
Original papers
iii
iv
v
vi
BACKGROUND
1
INTRODUCTION
1
Muscles and muscle fibres
Sarcomeric myosin
Muscle plasticity
Muscle development
Jaw functions
Jaw-face growth
Masseter muscle
Biceps brachii muscle
Muscle spindles
Myofascial pain
Purpose of the thesis
1
5
6
7
8
8
10
13
14
18
19
AIMS & HYPOTHESES
20
Questions to be answered
21
MATERIALS & METHODS
22
Materials
Methods
Statistics
22
23
26
REUSLTS
28
Extrafusal fibre type composition (Paper I)
MyHC content and distribution of extrafusal fibres (Paper III)
Comparison of extrafusal muscle fibres of young masseter
with young biceps
Comparison of extrafusal muscle fibres of young masseter
with adults and elderly
Comparison of extrafusal muscle fibres of young biceps
with adults and elderly
Muscle spindles (Paper II)
Intrafusal fibre type composition (Paper II)
MyHC composition of intrafusal fibres (Paper IV)
Comparison of muscle spindles and intrafusal fibres of
young masseter with young biceps
Comparisonof of muscle spindles and intrafusal fibres of
young masseter and biceps with adults
i
28
29
31
31
37
39
40
41
43
43
DISCUSSION
45
Extrafusal fibre types (Papers I and III)
Muscle spindles and intrafusal fibre types (Papers II and IV)
Clinical implications
Future perspective
45
51
54
55
SUMMARY
57
CONCLUSIONS
60
ACKNOWLEDGEMENTS
62
REFERENCES
65
PAPERS I-IV
ii
Organization
Departments of Odontology
and Integrative Medical Biology,
Umeå University
Document type
Doctoral thesis
Date of publication
9 sptember 2011
Author
Catharina Österlund
ABSTRACT
Muscles control body posture and movement by extrafusal and intrafusal
(muscle spindle) fibres. The purpose of this thesis was to provide insight into
the muscular basis for human jaw function at young age. Extrafusal and
intrafusal fibres in the young masseter, and for comparison young biceps,
were examined for composition of fibre types and myosin heavy chain
(MyHC) isoforms by means of morphological, enzyme-histochemical,
biochemical and immuno-histochemical techniques. For evaluation of
plasticity during life span the data for young muscles were compared with
previous reported data for adult and elderly muscles.
The results showed significant differences in extrafusal fibre types and
MyHC expression between young masseter and young biceps and between
young masseter and masseter in adults and elderly. Compared with young
biceps, young masseter was more intricate in composition of extrafusal
MyHC expression. Muscle spindles were larger and more frequent in the
masseter than in the biceps. Masseter and biceps muscle spindles showed
fundamental similarities but also marked differences in MyHC expression.
The results suggest that the young masseter is specialized in fibre types
already at young age and shows a unique fibre type growth pattern. Whereas
masseter extrafusal fibres display marked plasticity in fibre types and MyHC
isoforms during life span muscle spindles/intrafusal fibres are
morphologically mature already at young age and precede extrafusal fibres in
growth and maturation. Results showed similarities in intrafusal MyHC
expression between young masseter and biceps, but also differences
implying muscle specific proprioceptive control. Differences in fibre types
and MyHC expression between young masseter and young biceps extrafusal
fibres are proposed to reflect diverse evolutionary and developmental origins
and accord with the masseter and biceps being separate allotypes of muscle.
Keywords
Jaw, limb, human, muscle, morphology, fibre type, myosin heavy chain, muscle spindle
Language
English
ISBN
978-91-7459-285-6
ISSN
0345-7532
iii
Number of pages
86 + 4 papers
ABBREVIATIONS AND DEFINITIONS
Adult masseter
Biceps
Childhood
Deep
Development
EF
Elderly masseter
GE
Growth
IF
IHC
mAb
mATPase
Maturation
Muscle allotype
MYH
MyHC
MyHC-I
MyHC-α c
MyHC-emb
MyHC-fet
MyHC-sto
MS
PAP
PBS
Sup ant
Sup post
Young masseter
Masseter muscle samples from subjects mean
age 28 years
Short, inner, head of the biceps brachii muscle
Age 3-7 years
Deep region of the masseter
Changing from an immature to a mature stage
Extrafusal fibres
Masseter muscle samples from subjects mean
age 74 years
Gel elctrophoresis
Change in size with age
Intrafusal fibres
Immuno-histochemistry
Monoclonal antibody
Myosin adenosine triphosphatase
The process of becoming mature
Muscles wich are developmentally, structurally
and functionally distinct (e.g. limb/trunk, jaw,
extra ocular)
Sarcomeric myosin heavy chain gene
Myosin heavy chain
MyHC- slow twitch, MyHC-β cardiac
MyHC-α cardiac
MyHC-embryonic
MyHC-fetal
MyHC-slow tonic
muscle spindles
Peroxidase-antiperoxidase
Phosphate buffer saline
Superficial masseter, anterior region
Superficial masseter, posterior region
Masseter muscle samples from subjects aged 37 years, mean age 5 years
iv
SVENSK SAMMANFATTNING
Skelettmuskulaturen kontrollerar kroppens hållning och rörelser med två
muskelfibersystem extrafusala och intrafusala muskelfibrer. Extrafusala
muskelfibrer bygger upp muskelmassan och genererar muskelkraft och
rörelser. Intrafusala muskelfibrer är en del av muskelpolarna, som är
sinnesorgan inuti musklerna för muskelkontroll och rörelser.
Avhandlingens syfte var att undersöka muskelfibertypsammansättningen i
käkmuskulatur och som jämförelse armmuskulatur vid tidig ålder.
Muskelprover från masseter och biceps muskulatur undersöktes med
avseende på muskelfibertyper och kontraktila proteiner (myosin heavy chain
MyHC) i extrafusala- och intrafusala muskelfibrer. För att undersöka
plasticitet, det vill säga förändringsmönster för muskelfibertyper under
livscykeln, jämfördes resultaten med tidigare publicerade data för vuxna och
äldre individer. Ett flertal tekniker användes för muskelfiberanalys;
morfologiska, enzym- och immun-histokemiska samt biokemiska metoder.
Resultaten visade att masseter är speciell i sammansättning av muskelfibrer
och kontraktila proteiner i jämförelse med biceps. Jämförelse med data från
muskler hos vuxna individer visade att masseter har ett annat
tillväxtmönster än biceps. Muskelspolar i masseter är betydligt fler och
större än i biceps och med flest sammansatta spolar i dess djupa portion.
Jämförelse med data från vuxna individer visade att både i masseter och
biceps är muskelspolarna morfologiskt mogna redan i tidig ålder.
Extrafusala fibrerenas sammansättningen av MyHC i masseter muskeln
visade mer komplex sammansättning med flera olika isomyosiner och med
blandformer, i jämförelse med biceps muskulatur. Jämförelse med data från
vuxna individer tyder stora skillnader på ett uttalat förändringsmönster
under livscykeln. Intrafusala fibrernas MyHC sammansättning visade på
likheter men även skillnader mellan masseter och biceps.
Sammanfattningsvis tyder resultaten på att masseter muskeln redan i tidig
ålder är specialiserad vad gäller fibertyper samt uppvisar ett unikt
tillväxtmönster. Medan extrafusala fibrer uppvisar åldersrelaterade
förändringar i sammansättning av muskelfibertyper och kontraktila
proteiner är muskelspolar med dess intrafusala fibrer morfologiskt mogna
redan i tidig ålder. Muskelspolar tillväxer och mognar före det extrafusala
muskelfibersystemet, vilket tyder på behov av reflexstyrd kontroll av käkrespektive armrörelser. Resultaten tyder på att masseter muskeln
utvecklingsmässigt, strukturellt och funktionellt är skild från biceps.
v
ORIGINAL PAPERS
This thesis is based on the following papers, which will be referred to in the
text by their roman numerals:
I.
Differences in fibre type composition between human masseter and
biceps muscles in young and adults reveal unique masseter fibre
type growth pattern
Österlund C, Thornell L-E and Eriksson P-O
The Anatomical Record, 2011: 294;1158-1169.
II.
Muscle spindle composition and distribution in human young
masseter and biceps brachii muscles reveal early growth and
maturation
Österlund C, Liu J-X, Thornell L-E and Eriksson P-O
The Anatomical Record, 2011: 294;683-693.
III.
Remarkable heterogeneity in myosin heavy chain composition of the
human young masseter compared with young biceps brachii
Österlund C, Lindström M, Thornell L-E and Eriksson P-O
Submitted, 2011
IV.
Intrafusal myosin heavy chain expression of human masseter and
biceps muscles at young age shows fundamental similarities but also
marked differences
Österlund C, Liu J-X, Thornell L-E and Eriksson P-O
Manuscript
Reprints were made with kind permission from the publisher
vi
BACKGROUND
The muscles control body movements by virtue of two muscle fibre systems,
the extrafusal fibres and the intrafusal fibres. These systems have quite
different functional roles. The extrafusal fibres build up the muscle mass,
generate force and execute movements. The intrafusal fibres are part of the
muscle spindles- sensory receptors for muscle control and movements. This
thesis investigates the structural basis for jaw sensory-motor control at
young age. Specifically, the masseter muscle in childhood was examined
for compositions of fibre types and contractile proteins, myosin heavy chain
(MyHC) isoforms, of the extrafusal and intrafusal fibres, i.e. the basis for
power, velocity and endurance of muscle contraction, and the finely tuned
adjustments of tension and length of muscle. Determination of composition
and distribution of muscle fibre types and MyHC isoforms in a given muscle
is critical for a better understanding of its physiological properties. In this
context, there is a gap of in-depth knowledge for the human young masseter
muscle. To elucidate the possible influence of differences in genetic and
embryological origins, innervation and functional context on extra- and
intrafusal fibre types and expression of MyHC isoforms, a spinal nerve
innervated muscle, the young biceps brachii, was included in the study.
Finally, the masseter and biceps muscles were evaluated for age-related
plasticity of fibre types and MyHC expression during the life span by
comparing the results with previously reported data for adults (Eriksson,
1982; Stål, 1994; Liu, 2004) and elderly (Monemi, 1999).
INTRODUCTION
Muscles and muscle fibres
Skeletal muscles
Humans have about 640 skeletal muscles (Grounds and Shavlakadze, 2011)
of which numerous jaw, face, tongue and neck muscles are activated in
natural jaw function, i.e integrative jaw-neck motor control. Skeletal muscles
are made up of individual cells known as muscle fibres. Muscle fibres
contain myofibrils, the actual force generators. The myofibrils are composed
of series of sarcomeres, the functional units of muscle contraction. The
sarcomere consists of thick filaments that are mainly composed of myosin
and thin filaments composed of actin, troponin and tropomyosin. Interaction
between the filaments is the basis for muscle contraction (Fig. 1).
1
Figure 1: Schematic illustration of components of skeletal muscle. Whole muscle and
a bundle of muscle fibres. Muscle fibres are composed of myofibrils, which contain
myofilaments. The myofibrils have distinct repeating microanatomical units termed
sarcomeres, which represent the basic contractile units of the muscle. The sarcomere
is composed of thick and thin filaments, myosin and actin, respectively. Together
with regulatory proteins, troponin and tropomyosin, chemical and physical
interactions between the actin and myosin cause the sarcomere length to shorten.
Figure published with kind permission from Per Stål, Umeå University.
2
Muscles in human adults
Each human muscle is unique in fibre type- and myosin compositions. The
muscles in the jaw-face region are further specially designed in comparison
with spinal nerve innervated muscles such as leg, arm, hand and neck
muscles (Fig. 2). Cranial nerve innervated muscles, e.g. extra ocular, jaw,
face and tongue muscles, differ in composition of fibre types and contractile
proteins. Therefore, by mapping the fibre type and MyHC composition of
muscles, a better understanding of its physiological properties for sensorymotor control can be achieved.
Figure 2: Schematic illustration of muscles in human adults in relation to the cortical
representation. Note that the jaw, face, tongue and hand regions have large
representation in the cortex, which means that more brainpower is dedicated to
controlling these body parts. Figure published with kind permission from Per Stål,
Umeå University.
3
Muscle fibre types
Skeletal muscle fibres can be characterized qualitatively by enzymehistochemical and immuno-histochemical (IHC) techniques. Enzymehistochemistry has been generally used to classify muscle fibres on the basis
of reactions at alkaline and acid pH. The fibres are classified into type I fibres
and type II fibres with subtypes IIA, IIB and IIC (Brooke and Kaiser, 1970).
Human adult skeletal type I fibres have weak mATPase staining at alkaline
pH and strong staining at acid pH. They have low glycolytic activity and high
oxidative capacity and are resistant to fatigue and belong to slow twitch
motor units that have relatively long contraction time. Type IIA fibres have
strong mATPase staining at alkaline pH and weak staining at acid pH, belong
to fast twitch motor units that contract fast and are relatively resistant to
fatigue. Type IIB fibres have strong mATPase staining at alkaline pH and
weak staining at pH 4.3, have high glycolytic activity and low oxidative
capacity, are fatigable and belong to fast twitch motor units. Type IIC fibres
show strong mATPase staining at alkaline pH and intermediate staining at
acid pH (Dubowitz, 1985, 2007). Type mATPase-intermediate fibre type,
type IM, is a typical fibre type in human jaw muscles, but normally not
present in limb and trunk muscles. (Ringqvist, 1973).
An advancement in fibre typing was the generation of monoclonal antibodies
(mAbs) to different MyHC isoforms. (Schiaffino, 2010). By IHC technique
the mAbs reveal MyHC composition of individual fibres. In adult human
muscles, three MyHC isoforms have been identified, which are correlated to
the myosin ATPase based classification system. The slow twitch MyHC
isoform I/β-cardiac is present in type I fibres. The fast-twitch myosins,
MyHC-IIa is present in type IIA fibres, and MyHC-IIx is present in type IIB
fibres. The unique expression of any one of these MyHC isoforms in a single
muscle fibre is responsible for the distinct fibre types observed in skeletal
muscles after staining for myofibrillar ATPase following acid or alkaline
preincubations (Termin et al., 1989). Type IIC fibres which in general are
rare, appear during devlopment and in pathological conditions can contain a
mixture of slow and fast isoforms (Pette and Staron, 2000; Dubowitz, 2007)
and isoforms related to muscle development or muscle fibre regeneration i.e.
MyHC-embryonic and MyHC-fetal isoforms (Schantz and Dhoot, 1987;
Monemi et al., 1999a; Pette and Staron, 2000; Schiaffino, 2010). Both the
myofibrillar ATPase activites and the MyHC based fibre types composition of
a muscle can be used to predict the contractile properties of the muscle. The
mAbs make it possible to detect fibres expressing a single MyHC isoform
(“pure” fibres) or two or more mixed isoforms (“hybrid” fibres).
4
Electrophoretic separation of MyHC isoforms by sodium dodecyl sulphate
polyacrylamide gel electrophoresis (SDS-PAGE GE) procedure (Bar and
Pette, 1988) allows quantification of separate isoforms.
What decides the characteristic properties of a muscle?
Muscles differ in contraction speed, force generation, fatigability and
endurance. Numerous different properties decide the characteristics of
muscle fibres and whole muscles such as the content of various sarcomeric
proteins, enzyme activity, capillarisation, fibre cross-sectional area, muscle
architecture, and influence of genetic programs, motorneurones and
different hormones.
Sarcomeric myosin
A sarcomere is the basic contractile unit of a muscle (Fig. 1). Sarcomeric
myosin, the most abundant protein in skeletal muscle, is a conventional class
II myosin (Pette and Staron, 1990; Schiaffino and Reggiani, 1994; Bottinelli
and Reggiani, 2000).
The myosin molecule is an important motor protein in the sarcomere. It
powers muscle contraction and determines the contractile speed and thus
belongs to the foundation of muscle structure and function. It is a complex of
two heavy chains and four light chains that exist in multiple isoforms. An
isoform is a particular protein with slight variations in amino acid
composition (Schiaffino and Reggiani, 1994). In muscle cells, MyHC
isoforms contain both the ATPase and the actin-binding site- determinants
of speed and force of contraction (Pette and Staron, 1990; Bottinelli et al.,
1991). Slow MyHC isoforms predominate in slow-muscles, fast MyHC
isoforms in fast-muscles. The MyHC composition is regarded as the best
marker of the functional heterogeneity among muscle fibres (Larsson and
Moss, 1993).
Genome
A multigene family, formed by gene duplications approximately 75-110
million years ago encodes the MyHC isoforms (Fig.3). The gene organization
is highly conserved through evolution in terms of genomic organization and
the order of the MyHC genes (MYH) on the chromosomes (Schiaffino and
Reggiani, 1996; Weiss et al., 1999; Berg et al., 2001). Eleven MYH genes have
been detected in the mammalian genome and expressed in striated muscles
in a tissue-specific and developmental stage-specific manner (Schiaffino and
5
!
!
!
!
Reggiani, 1996; Weiss and Leinwand, 1996; Weiss et al., 1999; Sciote and
Morris, 2000).
!
MYH13
MYH8
MyHC-extra ocular
MyHC-fetal
!
MYH4
MyHC-IIb
MYH1
MyHC-IIx
MYH2
MYH3
MyHC-IIa
MyHC-embryonic
MYH6
MyHC-! cardiac
MYH7
MyHC-" cardiac
MYH14
MyHC-slow tonic
MYH15
MYH16
MyHC-masticatory
Figure 3: Scheme illustrating the evolutionary relationships among sarcomeric MYH
genes in mammals with the corresponding MyHC proteins. Spacing and length of the
branches are not consistent with actual scale in this simplified scheme. Figure is
redrawn from Rossi et al (Rossi et al., 2010). For scheme with more detailed branch
length, see c.f. Stedman et al (Stedman et al., 2004).
Muscle plasticity
The adaptive potential of muscle tissue is an important evolutionary
achievement because it improves the ability of survival. Both jaw and limb
muscles are highly adaptive to physiological demands. This adaptive
responsiveness has been termed “plasticity of muscle” (Pette and Staron,
2001). Muscle plasticity is the ability of a muscle cell to alter either the
quantity of protein and/or the type of protein (i.e., isoform), in response to
any stimulus (use or inactivity) (Baldwin and Haddad, 2002). A given
protein can be replaced by another protein, which is better suited for a
specific physiological or pathological state. The need for new motor skills as
the child grows and develops will place extra demands on the muscle
plasticity during these formative years.
6
Muscle development
Embryology
Muscles of different anatomical origins also have different embryological
origins. Skeletal muscle cells derive from the paraxial mesoderm. Jaw
muscles (masseter, temporalis, the lateral and medial pterygoids and the
anterior digastric) originate from the head paraxial mesoderm
(somitomeres) and migrate into the first branchial arch, whereas muscles of
facial expression and the posterior digastric migrate into the second
branchial arch (Noden, 1983; Noden and Francis-West, 2006). Tongue,
cervical and limb muscles are derived from the somitic mesoderm.
Myogenesis
Muscle fibres are multinucleated cells formed as a result of myogenesis.
During myogenesis mononucleated muscle precursor cells (myoblasts)
proliferate and then differentiate and fuse to form thin multinucleated
muscle cells (myotubes). Primary myotubes form around 12 weeks of
gestation for the masseter muscle and around 8-10 weeks for the biceps
(Barbet et al., 1991). A second generation of myotubes forms around the
primary fibres. The muscle spindles and the extrafusal fibres arise from
different myogenic lineages. There are special myotubes during development
that are destined to be muscle spindles (Pedrosa and Thornell, 1990; Soukup
et al., 1995). However, the precise origin of a spindle precursor is unknown.
Skeletal muscles develop initially without innervation; myoblasts proliferate
and differentiate and fuse to form primary myotubes. Motor nerves establish
contact to the myotubes accompanied by sensory contacts, which trigger
divergent differentiation of primary myotubes into extrafusal fibres and
intrafusal fibres, respectivey (Zelená, 1994). The differentiation into
extrafusal slow-twitch fibres are motor nerve dependent, whereas fast-twitch
fibres can differentiate in the absence of nerves. The differentiation into
intrafusal fibres is dependent of sensory innervation to develop MyHC-slow
tonic. The myotubes mature and grow in width (hypertrophy) and in length
into multinucleated muscle cells or fibres. Failure of nerve contact or
inactivity results in decrease in muscle mass (atrophy).
The muscle fibres stabilise in length as skeletal bone growth ceases. An
average adult human extrafusal muscle fibre reaches about 30-60µm in
diameter and 20-30 mm in length (range about 2-600 mm) (Grounds and
Shavlakadze, 2011), whereas intrafusal fibres diameter are about 10-20µm
and 3-4 mm length (Eriksson and Thornell, 1990). Some myoblasts persist
between the sarcolemma (muscle cell membrane) and the overlying
7
basement membrane. These cells are called satellite cells (Mauro, 1961) and
are responsible for generation of new myoblasts for muscle regeneration in
muscle repair, and providing nuclei to growing muscle fibres.
Jaw functions
In natural jaw function, the jaw muscles interact with other muscle groups in
order to achive specific tasks. The jaw and neck muscles act as an integrative
sensory-motor system during jaw actions by simultaneous recruitment of
jaw and neck muscles and concomitant movements in the
temporomandibular, the atlanto-occipital and the cervical spine joints.
Natural jaw function is by definition “integrated jaw-neck function”
(Eriksson et al., 1998; Eriksson et al., 2000). The involved muscles act as
synergies, i.e. coordinated activations of groups of muscles for a specific task
or function. The central nervous system selects the appropriate muscle
activity patterns to achieve a behavioural goal (d'Avella et al., 2003). Jaw
functions like mouth opening, suckling, swallowing, gaping and breathing
are based on innate reflexes necessary for surviving. During maturation,
eating behaviour (mastication and swallowing) and communication (speech
and mimic) activate by learning. Additional jaw muscle functions are
positioning the mandible, posture control, and mediation and execution of
jaw reflexes (jaw opening and closing reflexes).
Jaw muscles and limb muscles perform different types of functions and thus
have evolved different fibre types to match these specific functional
demands. Jaw and limb muscles, respectively, are said to belong to distinct
higher order classes called allotypes, developed from different lineages of
myoblasts, committed to form a specific subset of myofibrillar proteins
(Hoh, 1993; Hoh, 2002). Satellite cells in jaw muscles are pre-programmed
to express masticatory myosin even when re-innervated by a limb fast nerve
(Hoh and Hughes, 1988).
Jaw-face growth
Combination of genetic and epigenetic (regulated gene expression by
response to the environment) is responsible for the adult jaw-face
morphology. Development of the jaw-face region takes place early in foetal
life, but is underdimensioned in favor of development and growth of the
cranium (Fig. 4). The muscles growth and attachment are closely associated
8
with the jaw-facial skeleton morphology, which undergoes growth and
remodelling throughout childhood. The muscles must readjust their
insertion and site of origin. The mandible and the maxilla grow and remodel
in a posterior-superior manner leading to jaw displacement in forward and
downward directions. As a consequence, the lower face (below the eyes)
becomes longer and larger to accommodate the expansion of nasal region
and the eruption of permanent teeth. The cranium, instead, grows much less
than the lower part of the face after the first year of life, because most of the
cranium growth, takes place in the neonatal and infancy period. At childhood
(the age of 3-7 years), the cranium has already reached about 80-90 % of its
final size (Mann, 1984). The lower face grows (condylar grow) between 20 to
60 % of the total size, and reaches its full size at approximately 14 years of
age (Buschang et al., 1999). A pronounced shift in motor behaviour occurs
during teeth eruption when mastication succeeds suckling and develops and
improves throughout early childhood (Green et al., 1997). The jaw muscle
capacity varies between growing individuals as measured by bite force
(Kiliaridis et al., 1993) and muscle thickness (Raadsheer et al., 1996). At the
age of 6 years maximum bite force is about 50 % (85 Newton) that of the
maximum bite force at the age of 18-20 years (176 Newton) (Braun et al.,
1996). It can be assumed that changes in skeletal morphology and
transitions in functions during growth and maturation from childhood to
adulthood will be reflected in the muscle morphology.
Figure 4: Schematic illustration of the relative growth of the skull (blue) and lower
face (beige) of neonate and adulthood skulls. The figure is redrawn from Dal Martello
and Maloney (Dal Martello and Maloney, 2006). At birth the skull has reached about
60-65% of its final size and in childhood nearly full size. The size of the face (below
the eyes) at birth is around one eight of the total size of the head, whereas in adult, it
is half of it.
9
Masseter muscle
The masseter muscle, which is the strongest jaw closing muscle, is essential
in eating and communication behaviours. The muscle grows in parallel with
the jaw-face skeletal growth and remodelling and with teeth eruption. The
masseter muscle morpholgy is previously studied in prenatal (Barbet et al.,
1992), postnatal (Vignon et al., 1980; Soussi-Yanicostas et al., 1990; Barbet
et al., 1992; Bontemps et al., 2002), puberty (Vignon et al., 1980), adult
(Ringqvist, 1973, 1974; Eriksson, 1982; Stål, 1994; Korfage et al., 2000) and
in elderly (Monemi, 1999) ages.
Architecture
The human masseter muscle comprises of two main muscle portions, a
superficial portion with vertically-posteriorly directed fibres and a deep
portion with vertically oriented fibres (Ebert, 1938/39; Schumacher, 1961)
(Fig. 5). The muscle portions are divided by internal tendinous or connective
tissue septa, which run in parallel alternatively from the zygomatic arch and
the mandible. Between these tendon layers, numerous short muscle fibres
are arranged in a multipennate form. This intricate anatomy suggests that
the masseter is organized into functionally separate compartments
(Schumacher, 1961; Widmer et al., 2007), activated as synergic groups.
Masseter deep
Masseter sup post
Masseter sup ant
Figure 5: Schematic illustration of the masseter muscle portions; masseter
superficialis anterior (sup ant), masseter superficial posterior (sup post) and
masseter deep (deep).
10
Innervation
Sensory and motor branches of the trigeminal nerve innervate the jaw
muscles. The trigeminal motor nucleus lies in the mid pons level in the
brainstem and the component of the trigeminal sensory system that relay
information from muscle spindles is located in the mesencephalic trigeminal
nucleus at the mid brain level of the brainstem.
The motor unit
The central nervous system does not operate “in muscles”, i.e. a muscle is
never activated as a whole, but instead activates individual motor-units.
Thus, the motor unit is the basic unit of motor activity. It consists of a single
α-motor neuron and the set of muscle fibres innervated by this neuron (first
described by Sherrington, 1929). A given muscle fibre belongs to only one
motor unit. When a motor unit is activated, all of its fibres are activated and
produce a force. Muscles that require precision and fine movement control
usually have many motor units with a small number of fibres in each unit.
The masseter motor units contain fewer muscle fibres than those in limb
muscles (Stålberg et al., 1986). There is an orderly recruitment of motor
units. Small, slow contracting, fatigue resistant motor units, produce small
forces. With increasing force demands, large, fast-contracting fatigable
motor units join in. Slower fibre types/motor units are more frequently used
than faster ones. When more force is needed, the larger motor units are
recruited. Control of muscles, including jaw muscle function, relies on
selective recruitment of motor units.
The masseter muscle from prenatal age to puberty
The human masseter muscle morphology at prenatal (28 foetal weeks) and
postnatal (1.5 years) stages is characterised by presence of two distinct fibre
populations of different fibre size (Barbet et al., 1992). Large diameter fibres
express MyHC-I exclusively or in association with MyHC-embryonic and
MyHC-fetal isoforms. They give rise to adult type I fibres. Small diameter
fibres express MyHC-embryonic, MyHC-fetal and MyHC-II isoforms and
give rise to adult fibre types IIA, IIB and IIC (Soussi-Yanicostas et al., 1990;
Barbet et al., 1992; Bontemps et al., 2002). In puberty (10-13 years), the type
II fibre diameter is about half of that of type I fibres (Vignon et al., 1980).
The adult and elderly human masseter muscle
The adult human masseter muscle, like other jaw closing muscles, is distinct
from limb muscles due to smaller fibre diameter and differences in enzymehistochemical fibre types and MyHC isoform expression. In addition to type
I fibres and small type II fibres with subtypes IIA, IAB, IIB and IIC, the
11
masseter typically contains fibres which are intermediate to type I and type
II in mATPase staining and diameter, termed mATPase-IM fibres, IM
(Ringqvist, 1973; Eriksson, 1982; Eriksson and Thornell, 1983).
The adult human masseter shows regional differences in fibre type
composition (Eriksson, 1982; Eriksson and Thornell, 1983; Stål, 1994a;
Monemi et al., 1998; Korfage et al., 2000; Korfage et al., 2005a). The deep
masseter portion contains the highest proportion of type I fibres and in the
superficial masseter portion, the posterior part contains more type IIB fibres
than the anterior part (Eriksson, 1982; Stål et al., 1994a; Korfage et al.,
2000). The elderly masseter contains a larger proportion of type IIB fibres,
type IIA fibres and a larger amount of fibres expressing developmental
MyHC (Monemi et al., 1996).
The regional specialization and heterogeneity in composition and
distribution of fibre types, suggest that the human masseter is genetically
destined to develop, grow and mature into not one muscle, but an entity of
functionally different muscle portions acting in a task-related synergy
(Eriksson and Thornell, 1983). Moreover, aging is associated with significant
phenotypic alterations in muscle structure (Monemi et al., 1998)
GE of adult and elderly masseter muscle extract shows content of MyHC-I,
MyHC-IIa, MyHC-IIx and MyHC-fetal (Butler-Browne et al., 1988; Monemi,
1999a). IHC studies have demonstrated that in contrast to type I fibres
containing MyHC-I and type II fibres containing MyHC-II, the type IM fibres
express both MyHC-I and MyHC-II isoforms (Eriksson, 1982; Thornell et al.,
1984). Later studies confirmed and extended this finding of a heterogeneous
MyHC composition of the adult masseter fibres, and presented evidence of
proteins characteristic of developing muscles, MyHC-neonatal/fetal and
MyHC-embryonic isoforms (Butler-Browne et al., 1988; Soussi-Yanicostas et
al., 1990). Other special features of the adult masseter are the presence of
MyHC isoform originally described in the heart, MyHC-α cardiac (Bredman
et al., 1991; Pedrosa-Domellöf et al., 1992; Sciote et al., 1994; Stål et al.,
1994a), and that the type IM fibres typically contain mixtures of two or more
MyHC isoforms (Stål et al., 1994a; Monemi et al., 1999a; Korfage et al.,
2005b). Interestingly, a transition in masseter contractile proteins during
aging opposite to that of limb muscle, has been identified by relatively more
MyHC-II and MyHC-fetal isoforms and more fibres with a mixture of MyHC
isoforms in the masseter of elderly (Monemi et al., 1999a).
12
Other specialized muscles
Diversity in MyHC expression is also seen in extra ocular (Kjellgren et al.,
2003) and laryngeal (Hoh, 2005) muscles, and mirrors evolutionary
development into allotypes which are genetically different from that of limb
and trunk muscles (Hoh, 1993; Hoh, 2002). Thus, some cranial muscles have
emerged with multiple MYH genes and MyHC isoforms reflecting a
considerable phylogenetic plasticity in relation to functional demands (Hoh,
2002). The craniofacial muscles are evolutionary, morphologically and
molecularly distinct from trunk muscles, and the craniofacial muscles
myogenic gene network probably arises independently of the trunk muscle
regulatory program (Sambasivan et al., 2011).
Biceps brachii muscle
The biceps brachii muscle, which in this thesis is included for comparison, is
of different embryonic origin, innervation, of simpler architecture and
involved in more gross movements than the masseter muscle (Fig. 6). The
human biceps brachii muscle fibre type composition has been described in
all age groups including prenatal, postnatal, childhood, adulthood and in
elderly (Brooke and Engel, 1969; Colling-Saltin, 1978).
Figure 6: Schematic illustration of the biceps brachii muscle.
In limb and trunk muscles the type I and type II fibres are of about the same
diameter and homogeneously distributed (Dubowitz, 2007). The adult biceps
muscle is made up of types I, IIA and IIB fibres of about equal proportions
and diameters evenly distributed over the muscle cross-section (Eriksson,
1982; Stål, 1994a), and type IIC fibres which in limb and trunk muscles
appear only during devlopment and in pathological conditions (Pette and
Staron, 2000; Dubowitz, 2007).
13
Muscle spindles
Muscle spindle structure
Muscle spindles are muscle mecanoreceptors sensitive to muscle length and
change in muscle length. Because they respond to stimuli from within the
muscle they belong to so called proprioceptors, which provide information
about position of body parts in postures and movements to the central
nervous system. They consist of a bundle of small muscle fibres, intrafusal
fibres, located in parallel with extrafusal fibres, partly surrounded by a
fusiform fluid-filled connective tissue capsule. They are unique by receiving
both a motor and a sensory innervation. Due to regional differences in
morphology the muscle spindle has been separated into the A-regionconstituting its central equatorial, and the B-region- bilateral polar regions
reaching to the capsule end, and the C-region- extracapsular, where some
intrafusal fibres extend beyond the capsule to fuse with extrafusal fibre
tissue (Barker and Banks, 1994) (Fig. 7).
!" - motor innervation
!
Extrafusal
muscle fibre
Sensory innervation
Ia II
!
!- motor innervation
Capsule
Bag1
Bag2
Chain fibres
!!
0,5-1mm
"!
C
!!
B!
A
!!
!B
C
Figure 7: Schematic illustration of a muscle spindle, showing different intrafusal
fibres, afferent sensory innervation and efferent motor innervation. Furthermore, the
muscle spindle capsular region A (equatorial region), B (polar region), and C
(extracapsular region). The figure is redrawn from Bradley (Bradley, 1995).
Intrafusal fibre types
Mammalian intrafusal fibres differentiate into three fibre types. Two “bag”
fibres develop from primary and secondary myotubes, and “chain” fibres
differentiate from secondary myotubes (Kucera and Walro, 1989, 1990;
Pedrosa-Domellöf et al., 1991; Pedrosa-Domellöf and Thornell, 1994). Figure
8 shows masseter muscle spindle with bag and chain fibres.
14
Figure 8: Cross-section of a masseter muscle spindle, sample aged 6 years, stained
with antibody ALD19 against MyHC-slow tonic. Dark stained fibres are bag fibres.
Unstained fibres within the capsule are chain fibres. Outside the capsule, unstained
extrafusal fibres. Bar 50µm.
Nuclear bag1 (dynamic bag1) and nuclear bag2 (static bag2), with bag of
nuclei, and nuclear chain (static chain) fibres, with a single row of nuclei,
have been classified on basis of morphology, myosin ATPase activity (Ovalle
and Smith, 1972) and physiological properties (Matthews, 1971, 1981; Banks,
1994). In addition, an acid stable bag1 fibre (AS-bag1) has been distinguished
in human masseter muscle spindles (Eriksson and Thornell, 1985, 1990;
Eriksson et al., 1994)
Innervation
Intrafusal fibres both send afferent innervation to the central nervous system
and receive efferent innervation from the central nervous system. Initially
the dual innervation is established with the primary myotubes (Zelená,
1994). Two types of afferents, the primary (Ia) and the secondary (II), and in
addition two types of efferents, dynamic and static ϒ- motoneurons
innervate the intrafusal fibres. Also β-motoneurons, connect to muscle
spindles (Boyd and Smith, 1984; Bradley, 1995), as well as to extrafusal
fibres. Afferent nerves contact the central equatorial muscle spindle region.
The afferents have responsible for supplying the central nervous system with
information on muscle length and velocity changes of length. The efferent γnerve fibres contact the polar ends. By γ-nerve fibres length and velocity
changes causes intrafusal muscle fibres to regulate the muscle length. The
muscle spindle afference from the masseter muscle is relayed to the
mesencephalic trigeminal nucleus in the brainstem. The fact that more than
two-thirds of the myelinated nerve fibres in a muscle nerve innervate the
15
muscle spindles (Boyd and Smith, 1984) implies strong functional impact of
the very small intrafusal muscle fibre population (Fig. 9).
Figure 9: Cross-sections from masseter muscle spindles in childhood sample 6 years
(left) and in adulthood sample 58 years (right). Note the peripheral nerves (arrows)
in near relation to the muscle spindles. Bar 50 µm.
MyHC isoforms
In human muscles, intrafusal bag fibres express MyHC-slow tonic, MyHC-I
(slow-twitch), MyHC-embryonic and MyHC-fetal and MyHC-α cardiac
isoforms. Chain fibres express MyHC-embryonic, MyHC- fetal and MyHC-II
(fast-twitch).
Muscle spindle density
Muscle spindle density varies significantly between different muscles (Boyd
and Smith, 1984) and can be regarded as an indicator of functional
differences. High muscle spindle density characterizes muscles initiating fine
movements (e.g. lumbrical muscles and extra ocular muscles) (Bruenech and
Ruskell, 2001; Boyd-Clark et al., 2002; Soukup et al., 2003) or maintaining
posture (e.g. neck muscles) (Boyd-Clark et al., 2002; Liu et al., 2003),
whereas low spindle density is characteristic of muscles initiating gross
movements (e.g. biceps brachii).
Muscle spindle functions
Muscle spindles play multifunctional roles contributing in proprioception,
postural and movement control, motor learning and in plasticity of motor
behaviors (Windhorst, 2007, 2008), in predicting future kinematic states by
acting as forward sensory model (Dimitriou and Edin, 2010), and as
16
suggested, in the genesis and spread of chronic muscle pain (Johansson et
al., 1999; Blair et al., 2003; Johansson et al., 2003).
Muscle spindles in the masseter muscle regulate jaw position and
movements during chewing and speech (Smith et al., 1991; Scutter and
Turker, 2001). Excitation of muscle spindle afferent fibres monosynaptically
activates jaw-closing α-motoneuron in the trigeminal motor nucleus, causing
jaw-closing or jaw stretch reflex. In childhood, the proprioceptive
information from the masseter muscle spindles is highly important for
developing fine movement and postural control of the jaw for eating and
speech behaviours and for posturing the mandible during brisk
walking/running (Smith et al., 1991; Miles et al., 2004; Miles, 2007). While
children are developing jaw motor skills, the reflex development occurs
coincidently (Smith et al., 1991). The masseter muscle spindles activity
regulates the jaw motor pattern to smooth and relatively stable jaw positions
and movements with impact from the proprioceptive reflex (Jaaskelainen,
1993; Scutter and Turker, 2001; Miles et al., 2004; Miles, 2007) and
information from other peripheral receptors (periodontal receptors, golgitendon organs).
Adult masseter and biceps muscle spindles
The human adult masseter muscle (Voss, 1971) compared with the biceps
muscle shows higher spindle density and larger and more complex spindles,
especially in the deep masseter portion. The spindles are located closely
together in clusters, sharing the same capsule and described as compound in
arrangement. Similar to the adult biceps (Liu et al., 2002), the muscle
spindles in the adult masseter (Eriksson and Thornell, 1985, 1987, 1990;
Eriksson et al., 1995) show large variations in fibre type composition with
heterogeneous intrafusal fibre type composition. In the adult masseter
(Eriksson et al., 1994) and biceps (Liu et al., 2002), bag1 fibres mainly
express MyHCs- slow-tonic and I; AS-bag1 fibres MyHCs- slow-tonic, fetal
and embryonic; bag2 fibres MyHCs- slow-tonic, I and fetal, and chain fibres
MyHC- II, fetal and embryonic.
17
Myofascial pain
Myofascial pain in the jaw system is characterized by pain at rest and
function, tiredness/stiffness, weakness and tenderness of jaw muscles.
Diagnosis is based on history and clinical examination (Dworkin and
LeResche, 1992; Okeson, 2008). Examination may reveals limited mouth
opening. Women report pain more frequently, severely and of longer
duration than men. The reason for this difference is unclear and in what way
pain modulation is influenced by sex hormones have gained increasing
interest (Dao and LeResche, 2000; LeResche, 2000; Ernberg, 2004; Shinal
and Fillingim, 2007).
The mechanisms behind muscle pain are not fully understood. Both
peripheral and central mechanisms are involved. Nociceptive afferent, Aδand C fibres mediate pain. In the masseter, the nociceptive afferent fibres
from the trigeminal nerve have their cellbodies in the trigeminal ganglion
and terminate in the subnucleus caudalis in the brain stem. The nociceptive
activity from muscle tissue is sensitive to stimuli that can modulate the pain
transmission. One molecule in the pain transmission is serotonin (5-HT).
Studies suggest that serotonin in the central nervous system can reduce pain,
whereas serotonin in blood contributes to local progression of muscle pain.
Serotonin in the human masseter has been studied by means of
microdialysis technique (Ernberg et al., 1999) and intramuscular injection of
granisetron, a selective 5-HT receptor antagonist, into the masseter muscle
has been found to reduce myofascial pain (Christidis et al., 2007).
Myofascial pain in the jaw-face can be paralleled by pain in the neck,
shoulders and back, and also by generally spread pain. Recent findings show
that pain and dysfunction in trigeminally, as well as spinally, innervated
areas mutually predict the onset of new symptoms in the other region,
indicating common pathophysiological mechanisms and individual
vulnerability (Marklund et al., 2010).
In the national guidelines issued by the National Board of Health and
Welfare in Sweden, myofascial pain can have a major impact on oral health
including impaired ability to eat, chew and speak and influence the
individual´s mental and social well-being. Effort to identify, assess and
reduce myofascial pain are therefore of importance.
18
Purpose of the thesis
The general aim of this thesis was to increase the knowledge about the
structure and function of the human jaw system at young age. Information
on the structural basis for human masseter function at young age, in terms of
composition and distribution of extrafusal muscle fibre types and contractile
proteins/MyHC isoforms, is currently lacking. Likewise, there is no
knowledge for the human young masseter on the morphology and
distribution of muscle spindles and the expression of MyHC isoforms in the
intrafusal fibre population. Such knowledge would present a more profound
understanding of natural masseter function and therefore jaw motor control,
as well as assessment of dysfunction at young age. Furthermore, data from
this age group are essential for evaluation of age-related plasticity of the
human masseter extra- and intrafusal fibres during the life span, knowledge
that would mirror adaptation of motor and sensory control during growth
and aging.
19
AIMS & HYPOTHESES
Aims
This thesis is part of a larger project focused on increasing the knowledge on
the structural basis for sensori-motor control of human jaw function
- The specific aim was to examine the extra- and intrafusal muscle fibres of
the human young masseter muscle for composition and distribution of fibre
types and for expression of MyHC isoforms.
- For comparison and to elucidate the influence on muscle growth of genetic
and embryologic origins, innervation and functional context, the young
biceps brachii muscle was included in the study.
- Possible age related changes from childhood to adulthood and old age, i.e.
muscle plasticity during life span, was evaluated by comparing the results
with previously reported data on the human masseter and biceps muscle in
adults and elderly.
Hypotheses
Changes in jaw-face skeletal morphology and jaw function from childhood to
adulthood
are paralleled by alterations of fibre types and MyHC expression
- in masseter extrafusal fibres and
- in masseter intrafusal fibres (muscle spindles)
The masseter muscle is divergent from limb muscle in growth pattern.
20
Questions to be answered for the young masseter and biceps
brachii muscles
1. What is the extrafusal muscle fibre type composition in childhood?
2. Are there any intra-muscular regional differences in distribution and
size of fibre types in the masseter muscle for this age group?
3. What is the MyHC composition in the extrafusal muscle fibres?
4. What are the morphological characteristics of muscle spindles and the
composition of intrafusal fibre types and MyHC isoforms in childhood?
5. What similarities and differences are there between the young masseter
and biceps muscles in their extra- and intrafusal musclefibre
populations?
6. What similarities and differences are there between the young masseter
and biceps muscles vs. muscles in adults and elderly in their extra- and
intrafusal musclefibre populations?
21
MATERIALS & METHODS
Materials
Muscle samples
Subjects and muscle samples included in this thesis are shown in table 1.
Table 1: Subjects and muscle samples. M= Morphological analysis, EHC= enzymehistochemistry, IHC= immuno-histochemistry, GE= SDS-Page gel electrophoresis,
WB= Western blot technique, f= female subject, m= male subject.
Age
Sex
Masseter
sup ant
Masseter
sup post
Masseter
deep
3 years
f
M
EHC
IHC
M
EHC
IHC
3 years
m
4 years
m
M
EHC
IHC
GE
M
EHC
IHC
GE
WB
4 years
m
M
EHC
IHC
GE
M
EHC
IHC
GE
WB
M
EHC
IHC
GE
M
EHC
IHC
GE
M
EHC
IHC
GE
M
EHC
IHC
GE
WB
M
EHC
IHC
GE
6 years
m
7 years
f
7 years
f
M
EHC
IHC
GE
M
EHC
IHC
GE
M
EHC
IHC
GE
WB
WB
M
EHC
IHC
GE
M
EHC
IHC
GE
M
EHC
IHC
GE
WB
M
EHC
IHC
GE
M
EHC
IHC
GE
M
EHC
IHC
GE
WB
WB
WB
22
weeks
3
months
f
WB
22
Biceps
Paper
M
EHC
IHC
GE
M
EHC
IHC
GE
M
EHC
IHC
I, II, III,
IV
M
EHC
IHC
GE
WB
M
EHC
IHC
GE
I, II, III,
IV (biceps)
I, II, III,
IV (biceps)
I, II, III,
IV
I, II, III
I, II, III
M
EHC
IHC
GE
WB
WB
I, II, III,
IV
WB
III
III
18 years
m
GE
GE
GE
22 years
83 years
m
WB
WB
WB
WB
GE
WB
III
III
III
The muscle samples were obtained one to three days post mortem, a delay
which does not hamper reliable fibre typing (Eriksson et al., 1980). The
muscle samples were rapidly frozen in propane chilled with liquid nitrogen
and stored at -80°C until analysis.
Ethic approval
All human muscle tissue samples in this thesis were collected before 1990 in
accordance with the National Board of Health and Welfare in Sweden,
regulation 1975:190, concerning the exploitation of biological material for
research purposes (Socialstyrelsens föreskrifer 1975:190 rörande
tillvaratagande av biologiskt material för forskningsändamål)
Methods
Morphology
Serial muscle cross-sections, stained for enzyme-histochemistry and IHC
were analyzed using a light microscope (Leica DMR) connected with an
image analyses system (Leica QWin), and then photographed (Leica DC 220)
at x2.5, x10 and x40 magnification. Two masseter samples from each of the
anterior (sup ant) and posterior (sup post) superficial portions and the deep
(deep) portion were examined. Each sample was explored in six to eight
randomly selected fields (Fig. 10). On average 1500 individual fibres/subject
were examined in the masseter. In the biceps, from six subjects, one muscle
sample was analysed in seven to eight random fields and on average 650
individual fibres/subject. Classification of fibre types was performed at x40
magnification. The diameter of each fibre was calculated by measuring the
circumference of each fibre directly on the computer screen.
23
Figure 10: Sampling for identification of individual extrafusal fibres. In the light
microscope, overview at x2.5 magnification magnification, random fields were
marked (left). Each field was photographed at a higher magnification, x40 (middle)
for serial cross-sections with three enzyme-histochemical stainings and ten mAbs
stainings (middle). Classified fibres were marked with its circumference on the
computer screen at x40 magnification (right).
Muscle spindles
Distribution and composition of muscls spindles including muscle spindle
density, single and compound spindles, muscle spindle capsule diameters,
relative frequency of intrafusal fibre types, intrafusal fibre type diameters,
number of intrafusal fibres per muscle spindle and MyHC expression were
examined. For morphological analysis the serial cross-sections of the muscle
spindles were devided into localisation of muscle spindle regions. Muscle
spindle region-A included, the central part of the spindle capsule, the
equator and the juxta-equatorial part. This part contains ”paraxial fluid
space”. Muscle spindle region-B was from the end of the fluid space to the
capsular end. Muscle spindle region-AB was defined as the region in between
A and B. Muscle spindle region-C was extracapsular.
Examination of fibre types and MyHC isoforms content and distribution
Enzyme-histochemistry and IHC methods were used for classification of
muscle fibre types. Quantification of MyHC isoforms was performed by gel
electrophoresis (GE). These three methods enabled comparison with
previously reported data from our laboratory. For detailed description of
fibre typing of extra- and intrafusal fibres see papers I and II, and for
evaluating MyHC content and distribution of extra- and intrafusal fibres
with IHC see papers III and IV. Details concerning quantification of MyHC
isoforms from whole muscle extracts determined by GE separation are
described in paper III. Primary antibodies for IHC staining are listed in
(Table 2).
24
Table 2: MyHC monoclonal antibodies (mAbs) used for immuno-histochemistry and
the corresponding MYH genes, short name and references.
Antibodies
MyHC
Genes1
Short
names
References
MyHC mAb
ALD19a
MyHC-slow tonic
MYH14
MyHC-sto
(Sawchak et al., 1985)
(Pedrosa et al., 1989)
(Cho et al., 1993;
Hughes et al., 1993)
(Cho et al., 1993;
Hughes et al., 1993)
(Hughes et al., 1993; Liu
et al., 2002)
A4840b
A474b
First
developmental
MyHC-I
Second developmental
MyHC-I
Third developmental
MyHC-I
MyHC-IIa
MyHC-fetal/perinatal
MyHC-extra ocular
MyHC-α cardiac
MyHC-IIa
SC71c
MyHC-IIa
MYH2
MyHC-IIa
BF35c
MyHC-I
MyHC-IIa
MyHC-embryonic
MyHC-fetal/perinatal
MyHC-extra ocular
MyHC-α cardiac
MyHC-slow tonic
MyHC-fetal/perinatal
MYH7
MYH2
MYH3
MYH8
MYH13
MYH6
MYH14
MYH8
MyHC-“all
except
IIx”
MyHC-embryonic
MyHC-α cardiac
MYH3
MYH6
MyH-emb
MyHC-α c
(Ecob-Prince et al.,
1989; Weiss et al., 1999)
(Silberstein et al., 1986)
(Léger, 1985)
Laminin-α2 chain
LAMA2
Ln α2
(Sewry et al., 1998)
A4951b
N2261b
NCLMHCnd
F1652a
F88e
Laminin
mAb
5H2d
MYH7
MYH7
MYH7
MYH2
MYH8
MYH13
MYH6
MYH2
MyHC-I/β
cardiac
MyHC-I/β
cardiac
MyHCI+IIa
MyHC-IIa
MyHC-fet
(Hughes et al., 1993; Liu
et al., 2002)
(Schiaffino et al., 1989;
Liu et al., 2002)
(Schiaffino et al., 1989;
Liu et al., 2002)
Official gene nomenclature according to OMIM (http://www.ncbi.nlm.nih.gov/omim/)
Gift from Donald A. Fischman (Cornell University; New York, NY)
b Purchased from Developmental Studies Hybridoma Bank (Department of Biological Sciences,
University of Iowa; Iowa City, IA, USA)
c Gift from Prof. S. Schiaffino (University of Padova; Padova, Italy)
d Purchased from Novocastra Laboratories (Newcastle upon Tyne, UK)
e Gift from Dr. Jean J. Leger (Institut de la Sante et de la Recherche Meicale, Unite 249;
Montpellier, France)
1
a
25
Western blot analysis
The presence of MyHC-fetal was determined using Western blot analysis and
the mAb NCL-MHCn (Novocastra Laboratories Ltd).
Relative cross-sectional area (RCA)
To evaluate the relative muscle mass of each fibre type, the relative crosssectional area (RCA) was calculated by the formula: total cross-sectional area
of each fibre type/total cross-sectional area of all fibres x100.
Coefficient of variation (CV)
In addition to mean fibre diameter, the variability in fibre type diameter was
assessed with the coefficient of variation (CV) by the formula: (Standard
deviation x1000/Mean fibre diameter). In normal limb and trunk muscles
the CV is less than 250 and any sample with CV greater than this indicate
abnormal appearance (Dubowitz, 1985, 2007).
Statistics
Means and standard deviations (SD) were used for descriptive statistics. The
precision in the estimation of mean was calculated by the formula: SD / √n,
expressed by the standard error of the mean (SE). As the number of subjects
was low, the study population could not be assumed to be normal distributed
and consequently non-parametric statistics were applied. Statistical
calculations were performed in computer programs (InStat 3.0 and Prism
5.0 for Macintosh). The null hypothesis was rejected at the 0.05 level of
significance.
Wilcoxon signed rank test, a paired non-parametric test, was used to check if
there were any significant differences in relative frequency of fibre types and
fibre type diameter between fibre types, masseter portions and the masseter
and biceps muscles (I).
Paired t-tests, were used to test if there were any significant differences in
muscle spindle density, muscle spindle capsule diameter, intrafusal fibres
per muscle spindle, relative frequency of intrafusal fibre types, and intrafusal
fibre diameters between masseter portions, masseter and biceps brachii (II).
Paired t-tests were used to test if there were any significant differences in
relative content of MyHC isoforms between masseter regions and between
masseter and biceps muscles (III).
26
Mann-Whitney test, an unpaired non-parametric test, was used to calculate
whether two independent groups had equally large values. Mann-Whitney
tests were used to test for any significant differences between the different
age groups, young vs. adult and young vs. elderly (I, II).
The relationship between mean values from GE and IHC analyses was
evaluated by Pearson correlation coefficient (r).
27
RESULTS
Extrafusal muscle fibre type composition (Paper I)
Fibre types
In the young masseter types I, IM, IIC, IIAB, IIB, and rarely type IIA fibres
were distinguished. The masseter muscle fibres were generally rounded in
shape. The young masseter muscle showed marked intra-muscular
variability in fibre type composition (Fig. 11) as well as inter-individual
variation.
Figure 11: Muscle cross-section from young masseter sample, stained with mAb A474
(MyHC-IIa). Note intra-muscular variability. Bar 300µm
In the young biceps types I, IIA, IIAB, IIB, and rarely types IM and IIC fibres
were found. The biceps brachii fibre types were more angular and evenly
distributed over the muscle cross-section.
Relative frequency of muscle fibre types
In the young masseter the mean values of fibre type proportions were, type I
47.7 %, IM 21.2 %, IIC 7.3 %, IIA 0.1 %, IIAB 4.8 % and IIB 18.8 %. The
young masseter showed regional differences in fibre type proportions. The
type I fibre proportion was larger in the deep region, 53.9 %, than in the sup
ant, 45 %, and sup post, 43.8 %, regions (p= 0.03, both comparisons). The
type IM fibre proportion was smaller in the deep region, 14.9 %, than in the
sup ant, 28.4%, and sup post, 21.4 %, regions (p=0.03 and 0.05,
respectively).
28
In the young biceps the mean values of fibre type proportions were for types
I 54.5 %, IM 0.2 IIC 0 %, IIA 24.7 %, IIAB 18.8 % and IIB 1.8 %. The type I
fibre proportion was larger than those of the types IIA and IIB fibres (p=0.03
for both comparisons). The types IIA and IIAB proportions were larger than
that of type IIB (p=0.03 for both comparisons).
Muscle fibre type diameters
In the young masseter, the order of mean values of fibre diameters were,
types I 25.4µm >IM 20.8µm >IIC 16.2µm >IIB 14.6µm > IIAB 14µm. The
type II fibre diameter (mean fibre diameter for types IIA, IIAB and IIB) was
significantly smaller than type I (p= 0.02).
In the young biceps, the order of mean values of fibre diameters were type I
26.1µm >IIB 24.6µm>IIA / IIAB 22.5µm.
Variability in fibre type diameter (CV value)
The CV values for young masseter and biceps brachii muscles were (mean,
SD) 179 (20) and 140 (17), respectively.
Relative cross-sectional area (RCA) of fibre types
Percentage of RCA formed by different fibre types in different masseter
portions and in the biceps at young age, showed that the type I fibre RCA
values in all young masseter regions were about the same as for the
corresponding value for young biceps brachii.
MyHC content and distribution of extrafusal muscle fibres (Paper
III)
MyHC content
In the young masseter the results from GE showed an apparent interindividual variability in density of the different MyHC bands. Three bands
were identified in the gels loaded with 2.0µl muscle extract as MyHC-I,
MyHC-IIa and MyHC-IIx, and an additional faint band above the MyHC-I
corresponding to MyHC-α cardiac (Liu et al., 2002). The MyHC-I band, with
the highest mobility, was the most prominent. It was larger in the sup ant
masseter than in the sup post masseter (p=0.032). No band representing
29
MyHC-fetal was detected but this could be expected since this developmental
MyHC isoform is known to be present in the masseter of young adults and
elderly (Monemi et al., 1996). Therefore, the GE analysis was extended to
include gels loaded with 3.0µl muscle extract to allow low content isoforms
to appear. The 3.0µl gels revealed more details for the low mobility bands
with three low mobility bands including a band inbetween the MyHC-IIa and
the MyHC-IIx bands. This band corresponded to MyHC-fetal (Liu et al.,
2002). Also, a faint band was seen with lower mobility than that of the
MyHC-IIx, corresponding to MyHC-embryonic (Liu et al., 2002).
In the young biceps the results showed an apparent inter-individual
variability in density of the different MyHC bands. Three bands were
identified in the gels loaded with 2.0 µl and 3.0 µl muscle extract as MyHC-I,
MyHC-IIa and MyHC-IIx (Liu et al., 2002). The MyHC-I band was the most
prominent.
To confirm the presence of MyHC-fetal a Western blot analysis using the
mAb MHCn was performed. In the fetal samples, the result showed one
band approximately at the level of MyHC-IIa. Notably, two bands were
detected in the masseter samples aged 3 months and 4 and 7 years. One
band appeared at the level of the band in the fetal samples. This band was
also seen in the elderly masseter. The other,”extra”, band was located in
between the MyHC-IIa and MyHC-IIx bands. This “extra” band was also
seen in the adult masseter (sup ant). No reaction of bound anti-MHCn was
observed in the young and adult biceps specimens.
MyHC distribution
In the young masseter analysis with IHC showed a marked variability in
staining with all mAbs and of all enzyme-histochemical fibre types. A MyHC
isoform not previously described, tentatively termed IIx´, was detected in
the masseter. Based on pooled data for all subjects in the masseter, 44 % of
the fibres contained one MyHC isoform, either MyHCs- I, IIa, IIx or IIx´.
The rest of the fibres, 56%, showed mixtures of two to four isoforms in
twenty-two combinations, fifteen when less frequent combinations, <1 %,
were excluded. MyHC-fetal and MyHC-α cardiac isoforms always occurred
in combinations with other isoforms. Matching the mATPase fibre types with
MyHC isoforms revealed a systematic continuum of the MyHC isoforms.
Analysis of masseter regional differences for slow and fast MyHCs showed a
larger proportion of fibres containing fast MyHC in the sup ant and sup post
masseter than in the deep masseter (p= 0.026 and p=0.012, respectively).
The proportion of MyHC-IIx was larger in the sup post than in the deep
masseter (p=0.039).
30
In the young biceps, the staining pattern with mAbs was simpler than in
young masseter with no stained fibres with mAbs MHCn, F88, F1652 and
ALD19. Based on pooled data 99 % of the fibres contained only one isoform.
Correlation between GE and IHC analysis
In the young masseter, there was a strong correlation between the GE data
and the IHC data for MyHC-I (r=0.895, p=<0.0001), moderate correlation
for MyHC-IIx (r= 0.540, p=0.021) and no correlation for MyHC-IIa
(r=0.184 p > 0.05).
Comparison of extrafusal muscle fibres of young masseter with
young biceps
Young masseter and biceps were similar in that type I fibres outnumbered
other fibre types and were of the same diameter. The results showed
significant differences in composition and distribution of fibre types between
young masseter and young biceps. The type IIB fibre proportions in the sup
post portion were larger than in the biceps (p=0.03). Young masseter
differed from biceps by smaller mean fibre diameter for type II fibre
diameters (i.e. types IIA, IIAB and IIB) in the masseter than in the biceps
(p=0.03). Both muscles showed a predominant content of MyHC-I.
Differences were seen between the young masseter and biceps with higher
MyHC-IIa content in the biceps (p=0.017). A fundamental difference was no
expression of MyHC-fetal and MyHC-α cardiac isoforms in the biceps
muscle. Young masseter extrafusal fibres showed a much more intricate
MyHC distribution including unique MyHC isoforms compared with biceps
fibres. Heterogeneous IHC staining, and fibres co-expressing MyHC
isoforms were less pronounced in the biceps.
Comparison of extrafusal muscle fibres of young masseter with
adults and elderly
Fibre type proportion
The young masseter differed in age-related changes in composition and
distribution of fibre types compared with adults (Eriksson, 1982; Eriksson
and Thornell, 1983) and elderly (Monemi et al., 1998). Young and adult
masseter muscles showed an overwhelming proportion of type I fibres (48%
and 62%, respectively), with a lower proportion in elderly muscles (33%),
31
whereas the proportion of type IIB fibres was larger with age (young 19%,
adult 27%, and elderly 37%, respectively). The type IM proportion was
largest in young masseter (young 21%, adult 6%, and elderly 13%,
respectively) (Fig. 12). The different age groups of the masseter showed
regional differences in relative frequency of fibre types but in different ways.
The young masseter, compared with data from the adult masseter (Eriksson,
1982; Eriksson and Thornell, 1983), showed larger type IM proportion in the
sup ant and sup post portions (p=0.04, p=0.002, respectively), smaller type
IIB proportion in the sup post (p=0.002) and smaller type I fibre population
in the deep portion (p=0.03). The young masseter, compared with data from
elderly masseter (Monemi et al., 1998a), showed larger type I proportion in
sup ant and sup post portions (p=0.009, p=0.002, respectively), larger type
IM proportions in sup ant and sup post portions (p=0.015, p=0.018,
respectively), smaller type IIA proportion in sup ant, sup post and deep
masseter portions (p=0.009, p=0.012, p=0.005, respectively), smaller type
IIAB proportion in sup post portion (p=0.005) and smaller type IIB
proportions in sup ant and sup post portions (p=0.015,
p=0.005,
respectively).
Masseter
Relative frequency (%)
80
70
I
60
50
IM
40
IIC
30
IIA
20
IIAB
10
IIB
0
Young
Adult
Elderly
Figure 12: Relative frequency (%) of fibre types in young masseter compared with
data from adult (Eriksson, 1982; Eriksson and Thornell, 1983) and elderly (Monemi
et al., 1998) masseter muscles. Mean and SD.
Fibre type diameter
In the young masseter, type I fibre diameter was smaller compared with
adult and elderly (p=0.002, p=0.001, respectively), type IM fibre diameter
was smaller compared with adult and elderly (p=0.002, p=0.001,
32
respectively), type IIC fibre diameter was smaller compared with adult and
elderly (p=0.002, p=0.014, respectively), type IIAB fibre diameter was
smaller compared with elderly (p=0.045), and type IIB fibre diameter was
smaller compared with adult (p=0.005) (Eriksson, 1982; Eriksson and
Thornell, 1983; Monemi et al., 1998). Comparison of mean fibre diameter for
young masseter fibres with adult and elderly showed 1.8 times larger
diameter in the adult masseter and 1.4 times larger in the elderly masseter
(Eriksson, 1982; Eriksson and Thornell, 1983; Monemi et al., 1998). No agerelated difference was found for masseter type II fibre diameter (Fig.13).
Figure 13: Illustration of fibre type diameter in young, adult (Eriksson, 1982;
Eriksson and Thornell, 1983) and elderly (Monemi et al., 1998) masseter and biceps
muscles. Every fibre type is labelled for its absolute diameter.
33
Relative cross-sectional area (RCA)
Percentage of overall muscle fibre cross-sectional area formed by different
fibre types of masseter regions and biceps in young, adult (Eriksson, 1982;
Eriksson and Thornell, 1983) and elderly (Monemi et al., 1998) muscles (Fig.
14).
Figure 14. RCA (%) of different fibre types in different masseter regions and in biceps
of young, adult (Eriksson, 1982; Eriksson and Thornell, 1983) and elderly (Monemi et
al., 1998) muscles. Note the predominance of type I fibre muscle mass.
34
MyHC content
Comparison MyHC content between young masseter and adult and elderly
muscles showed smaller proportion of MyHC-I with age accompanied by
larger proportions of MyHC-IIa, MyHC-IIx (Fig. 15). The MyHC-fetal
content was largest in elderly masseter, especially in the sup ant region
(Monemi et al., 1999a). The deep masseter showed no major age-related
differences in MyHC content.
Masseter
100
90
Relative content (%)
80
70
60
I
50
IIa
40
IIx
30
fet
20
10
0
0 1 Young
2 3 4 Adult
Elderly
Figure 15: Relative content (%) of MyHC-I, MyHC-IIa, MyHC-IIx and MyHC-fetal in
young, adult and elderly masseter (Monemi et al., 1999a). Mean and SD.
MyHC distribution
IHC showed higher proportion of MyHC-I, MyHC-fetal and MyHC-α cardiac
in young masseter vs. smaller proportion in adult and elderly (Stål et al.,
1994a; Monemi et al., 1999a). Young masseter individual fibres showed four
single and fifteen mixed ”MyHC based fibre types” and in total twenty-six
when occasional combinations were included (<1%). Adult masseter,
individual fibres show eight ”MyHC based fibre types”, three single and five
mixed and nine when occasional combinations included (<1%) (Stål et al.,
1994a). Elderly masseter individual fibres show eleven ”MyHC based fibre
types”, three single and eight mixed, and sixteen when occasional
combinations included (<1%) (Monemi et al., 1999a) (Fig. 16).
35
Masseter young
Biceps young
IIx´+fet+! car IIx+fet
1.5%
1% IIx+! car
IIx´
1%
IIa+IIx
1% IIx´+fet
IIx
4%
0.5%
6%
IIa+fet+! car
IIa+! car 4%
I
2%
IIa
33%
IIa+fet
4%
1%
IIx
0.5%
IIa+IIx
1%
IIa
40%
I
58.5%
I+IIa+fet+! car
I+fet
10%
9%
I+IIa+! car
I+! car
6% I+IIa+fet
I+fet+! car 3%
4% I+IIa
7%
2%
Masseter adult
IIa+IIx
3%
IIa
2%
I+IIx+fet+! car
2%I+IIx+fet
IIx+fet
11%
IIx
13%
Biceps adult
IIx
6%
IIa+IIx
35%
I
62%
IIa
16%
4% I+ IIx
3%
Biceps elderly
Masseter elderly
IIx+fet
8%
IIx
24%
IIa+IIx+fet
2%
I
43%
IIa+IIx
8% IIa
4%
IIx
1%
IIa+IIx
29%
I
31%
I+IIa+fet
I+IIx+fet I+IIx 5%
3%
4%
I+fet
1%
I
48%
IIa
21%
I+IIa
10%
I+IIa
1%
Figure 16: Proportion (%) of single (shaded) and combinations of MyCH isoforms in
extrafusal fibres in the young masseter and biceps muscles and for comparison adult
(Stål et al., 1994a) and elderly (Monemi et al., 1999a). Note apparent differences
between masseter and biceps.
36
Comparison of extrafusal muscle fibres of young biceps with
adults and elderly
The young biceps differed in age-related changes of relative frequency of
fibre types between young age, adulthood and elderly with larger type IIB
proportion in elderly (p=0.004)(Monemi et al., 1998) (Fig. 17).
Biceps
Relative frequency (%)
80
70
60
I
50
IIC
40
IIA
30
IIAB
20
IIB
10
0
Young
Adult
Elderly
Figure 17: Relative frequency (%) of fibre types in young biceps compared with adult
(Eriksson, 1982; Eriksson and Thornell, 1983) and elderly (Monemi et al., 1998)
biceps. Mean and SD.
The mean muscle fibre diameter was 3.0 times larger in the adult biceps than
in young biceps and 2.2 times larger in elderly biceps (Eriksson, 1982;
Eriksson and Thornell, 1983; Monemi et al., 1998) (Fig. 13).
RCA (%) of different fibre types showed a predominant area of type I fibres
in young biceps with a smaller area in adult and elderly biceps in favour of
larger type II fibres area (Eriksson, 1982; Eriksson and Thornell, 1983;
Monemi et al., 1998) (Fig. 14).
37
In the biceps GE showed the content of MyHC-I largest in young and elderly
biceps and smallest in adult accompanied by large amounts of MyHC-IIa and
MyHC-IIx in adult biceps (Monemi, 1999) (Fig. 18).
Biceps
100
90
Relative content (%)
80
70
60
I
50
IIa
40
IIx
30
fet
20
10
0
0 1 Young
2 3 4 Adult
Elderly
Figure 18: Relative content (%) of MyHC-I, MyHC-IIa, MyHC-IIx and MyHC-fetal in
young, adult and elderly biceps (Monemi et al., 1999a). Mean and SD.
Detected MyHC isoforms with IHC showed for the biceps no major agerelated differences in proportions for MyHC-I and MyHC-IIa. The
proportion of MyHC-IIx isoform was larger in elderly (Stål, 1994a; Monemi,
1999a). Young biceps fibres showed four ”MyHC based fibre types”, two
single and two mixed and six when occasional combinations were included
(<1 %). Adult biceps individual fibres showed four ”MyHC based fibre types”,
three single and one mixed and five when occasional combinations were
included (<1 %). Elderly biceps individual fibres showed five ”MyHC based
fibre types”, three single and two mixed (Fig. 16).
38
Muscle spindles (Paper II)
Muscle spindle density
In the young masseter the muscle spindle density, MS/cm2, was higher
(mean, SD) in the deep 43 (16) than in the sup ant 27 (11) and sup post 4 (6)
masseter portions, and higher in the sup ant than in the sup post. In the
young biceps the density was 20 (13), which was lower than that of the deep
masseter (Fig. 19).
60
Muscle spindle density (MS/cm2)
p=0.045
50
p=0.044
p=0.001
40
p=0.002
30
20
10
0
Sup ant
Sup post
Deep
Biceps
Figure 19: Muscle spindle density (MS/cm2) in the anterior (Sup ant) and posterior
(Sup post) superficial and deep (Deep) masseter and biceps muscles. Mean and SD.
Compound muscle spindles
In the young masseter compound muscle spindles were most frequent in the
deep portion, 24 %, compared with the sup ant, 10 %, and sup post, 0 %
portions. In the young biceps 4 % of the spindles were compound.
Capsule diameter
For both the masseter and biceps, the diameter of muscle spindle regions- A
and AB were significantly larger than that of region-B. The diameter of
region-A was significantly larger than that of region-AB. The mean capsule
diameter (µm, mean SD) of pooled data for single muscle spindles was 96
(37) in the masseter and 76 (28) in the biceps. There were no significant
differences in capsule diameter between masseter portions or between
masseter and biceps (Fig. 20).
39
250
Capsule diameter (µm)
p=0.035
p=0.010
p=0.024
200
150
Masseter
Biceps
100
p=0.035
50
0
p=0.013
p=0.041
A
AB
B
Figure 20: The mean muscle spindle capsule diameter (µm, mean SD) of single
spindles in muscle spindle regions A, AB and B in young masseter and young biceps
muscles.
Intrafusal fibre type composition (Paper II)
Intrafusal fibre types
In the young masseter the number of intrafusal fibres per muscle spindle was
on average 8.3 (min-max 1-23) in single and 18.8 (min-max 7-38) in
compound muscle spindles. Of the examined spindles, 90 % contained bag1,
15 % AS-bag1, 76 % bag2, and 96 % chain fibres. The average composition of
intrafusal fibre types in single muscle spindles was 2 bag1, 1 bag2 and 4 chain
fibres. Single muscle spindles contained 67 combinations of intrafusal fibre
types, whereas compound muscle spindles contained 22 combinations. A
unique allotment of number of intrafusal fibres was presented in 70 % of the
muscle spindles.
In young biceps there were on average 7.4 (min-max 3-19) intrafusal
fibres in single muscle spindle and 22 (min-max 22-23) in compound
muscle spindles. Of the examined spindles, 96 % contained bag1, 45 %
AS-bag1, 28 % bag2 and 100 % chain fibres. The average composition of
intrafusal fibre types in single muscle spindles was 2 bag1 and 4 chain
fibres. Single muscle spindles contained 39 combinations of intrafusal
fibre types, whereas compound muscle spindles contained 2
combinations. A unique allotment of number of intrafusal fibres was
present in 79 % of the muscle spindles.
40
Intrafusal fibre type diameter
The intrafusal fibre type diameter in young masseter and biceps muscles are
seen in (Fig. 21). The intrafusal fibre diameter did not differ between the
masseter muscle portions, but did between the masseter and biceps
(p=0.045).
%#"
Intrafusal fibre diameter (µm)
p=0.028
p=0.013"
"
%!"
$#"
Masseter
$!"
Biceps
p=0.002
"
p=0.002
"
p=0.001
#"
!"
"
Bag1
AS-bag1
Bag2
Chain
Figure 21: Intrafusal fibre type diameter in young masseter and biceps muscles. In
the masseter, bag2 fibres were significantly larger than both bag1 and chain fibres.
Also in the biceps, bag1, AS-bag1 and bag2 fibres were significantly larger than chain
fibres. Masseter bag1 fibres were significantly larger than those of the biceps
(p=0.049). The mean intrafusal fibre diameter for pooled data of bag1, AS-bag1, bag2
and chain fibres (µm, mean SD) was significantly larger in the masseter, 13.5 (2),
than in the biceps, 10.0 (1) (p =0.045).
MyHC composition of intrafusal fibres (Paper IV)
The mAb staining pattern of the young biceps intrafusal fibres was similar to
that of the masseter for mAbs ALD19, N2261 and F1652. For the other mAbs
some differences were seen. Details are presented in paper IV. In both
muscles eight MyHC isoforms- slow tonic, I, IIa, IIx´, IIx, α-cardiac, fetal
and embryonic were detected.
In the young masseter bag1 fibres contained predominantly MyHC isoforms
slow-tonic, α-cardiac and I and substantial proportions of IIa, IIx´, IIx and
fetal; AS-bag1 contained predominantly slow-tonic, α-cardiac, I, IIa and
substantial proportions of fetal, IIx´ and embryonic; bag2 contained
predominantly slow-tonic, I, α-cardiac, fetal, IIa and substantial proportions
of IIx´; chain contained predominantly IIa and fetal and substantial
proportions of embryonic, α-cardiac and I.
41
Comparison between the three muscle spindle regions for differences in
MyHC content showed for the masseter bag1 fibres smaller proportion of
MyHC-I in the A+AB region, and for masseter bag1 and bag2 fibres larger
proportion of MyHC- fetal in the A+AB region than in the B region. There
were 56 combinations of MyHC isoforms, 24 when less frequent
combinations, <1 %, were excluded (Fig.22). The most common MyHC
combination for bag1 and AS-bag1 fibres was slow tonic + I + IIa + α-cardiac,
for bag2 fibres was slow tonic + I + IIa + fetal + cardiac and for chain fibres
was IIa + fetal.
In the young biceps bag1 fibres contained predominantly MyHC isoforms
slow-tonic and I and substantial proportions of IIx, IIa and α-cardiac, IIx
and embryonic; AS-bag1 contained predominantly slow-tonic, I and IIx´ and
substantial proportions of IIa and fetal embryonic; bag2 contained
predominantly slow-tonic, I, IIa, fetal and α-cardiac and substantial
proportions of embryonic; chain contained predominantly fetal, IIa and I
and substantial proportions of embryonic and α-cardiac. In young biceps
bag1 fibres showed larger proportion of MyHC-IIx ́ in the A+AB region, and
bag2 fibres showed larger proportion of MyHC-fetal in the A+AB region than
in the B region. There were 38 combinations of MyHC isoforms, 26 when
less frequent combinations, <1 %, were excluded (Fig. 22). The most
common MyHC combinations for bag1 fibres was slow tonic + I + IIx´, for
AS-bag1 fibres was slow tonic + I + IIa, for bag2 fibres was slow tonic + I + IIa
+ fetal and for chain fibres was IIa + fetal.
Masseter
Biceps
Figure 22: Proportion (%) of single (shaded) and combinations of MyHC isoforms in
intrafusal fibres in the young masseter (left) and young biceps (right) muscles. The
pie diagrams show for the young masseter 24 MyHC isoform combinations
represented with ≥1%, and for the young biceps 26. Note co-expression
(combinations) of MyHC isoforms in generally all fibres.
42
Comparison of muscle spindles and intrafusal fibres of young
masseter with young biceps
The muscle spindle density in the young deep masseter portion was higher
than in the biceps (p=0.044). There were no significant differences in
capsule diameter between young masseter and biceps muscle spindles. The
masseter contained significantly more bag2 fibres, 16 % versus 4 %
(p=0.008) and less chain fibres, 52 % versus 64 % (p=0.024), than the
biceps. Masseter bag1 fibres were significantly larger in diameter than those
of the biceps (p=0.049). The mean intrafusal fibre diameter for pooled data
of bag1, AS-bag1, bag2 and chain fibres (µm, mean SD) was significantly larger
in the masseter, 13.5 (2), than in the biceps, 10.0 (1) (p=0.045).
There were notable similarities and marked differences in MyHC
distribution of intrafusal fibres for young masseter and biceps. SimilaritiesEight MyHC isoforms. Mixtures of two to six isoforms in all intrafusal fibres.
Numerous of combinations of MyHC isoforms. Bag1, AS-bag1 and bag2 fibres
showed MyHC-slow tonic, and bag1 also MyHC-I. Chain fibres showed
MyHC-IIa and MyHC-fetal. Some combinations were seen in both muscles.
In chain fibres, the two most frequent MyHC isoform combinations were
seen in both muscles. Differences- In the young masseter, 98 % of bag1 fibres
showed MyHC-α cardiac vs. 30 % in the young biceps, all AS-Bag1 showed
MyHC-α cardiac vs. 10 % in the the biceps, 35 % of bag2 fibres showed
MyHC-IIx ́ vs. none in the biceps, 17 % of chain fibres showed MyHC-I vs. 61
% in the biceps. The most frequent MyHC isoform combinations in bag1 and
bag2 fibres differed between the muscles.
Comparison of muscle spindles and intrafusal fibres of young
masseter and biceps with adults
Comparison of young masseter muscle spindles with muscle spindles in
adult muscles showed that the muscle spindle density was significantly
higher (p=0.005) in young masseter, 25, than in adult (Eriksson and
Thornell, 1987) masseter, 7. In both young and adult masseter the deep
portion showed the majority of spindles (young 61 % and adult 74 %), and
compound muscle spindles occured in a significant amount. Young masseter
did not differ from the adult (Eriksson and Thornell, 1990) in number of
intrafusal fibres of single spindles (young 8.3, adult 7.3), capsule diameter of
single spindles (young 96 µm, adult 98 µm) or intrafusal fibre diameter
(young 13.5 µm, adult 16 µm). The ratio between the mean bag fibre
43
diameter (pooled data for bag1, AS-bag1 and bag2 fibres, n=439) and the mean
diameter of extrafusal type I fibres (Österlund et al., 2011a) was 0.8 (16.5 µm
/21.7 µm) for the young masseter versus 0.4 (18.1 µm/43.9 µm) for the adult
(Eriksson and Thornell, 1990).
Comparison of muscle spindles and intrafusal fibres of young biceps with
muscle spindles in adult (Liu et al., 2002) muscles showed a similarity in
enzyme-histochemicl staining pattern of intrafusal fibre types, mean number
of intrafusal fibres per muscle spindle heterogeneity in combinations of
intrafusal fibre types and lack of bag2 fibres.
MyHC-I was identified in chain fibres in both the masseter and biceps,
although in diverse proportions, about one-fifth in masseter and two-thirds
in young biceps. These fibres were in contrast to reports of lack of MyHC-I in
chain fibres in both the masseter (Eriksson et al., 1994) and biceps (Liu et al.,
2002) of adults. Likewise, whereas we could detect MyHC-α cardiac in about
one-fifth of the chain fibres of both the young masseter and biceps, chain
fibres in the adult biceps lack this isoform (Liu et al., 2002).
44
DISCUSSION
The main findings in this thesis were, firstly, that the young masseter
differed profoundly from the young biceps muscle in composition and
distribution of extrafusal fibre types and MyHC isoforms, and in age-related
plasticity from childhood to adulthood. This suggests that the human
masseter is specialized in fibre types already at young age and has a unique
pattern of growth of fibre types and contractile proteins. Secondly, findings
of fundamental similarities in intrafusal MyHC expression between young
masseter and biceps, but also marked differences implying muscle specific
proprioceptive control. Findings of similarities in mATPase fibre types and
MyHC expression of intrafusal fibres between young masseter and biceps
muscle spindles and spindles in adult muscles suggest early maturation of
muscle spindles and that muscle spindles/intrafusal fibres precede
extrafusal fibres in growth and maturation. This in turn implies early need
and capacity of reflex, proprioceptive control in learning, performing and
improving jaw skills and arm behaviour. Thus, the hypothesis of masseter
extrafusal muscle plasticity during life span in parallel with changes in jawface skeletal morphology and jaw function, and the hypothesis that the
masseter is divergent from limb muscle in growth pattern could be verified.
However, the hypothesis of changes in masseter intrafusal (muscle spindle)
fibre types and MyHC expression from childhood to adulthood was rejected.
Extrafusal fibre types (Papers I and III)
Extrafusal fibres in perspective of evolution and development
To explain the findings of fundamental differences between the masseter and
biceps in extrafusal fibre types and MyHC expression, and in muscular
plasticity during life span, they can be viewed in perspective of evolution of
the MyHC genes and current knowledge on the diversity in evolution and
development between the craniofacial and the limb and trunk muscles.
Firstly, in the last decade, all the genes in the human genome has been
characterized and an evolutionary tree has been created, which shows that in
human muscles eleven genes are linked to the expression of MyHC isoforms
and have over the years evolved for specific purposes. Hoh has studied in
detail the evolution of jaw closing muscles and limb and trunk muscles in
vertebrates (Hoh, 2002). He refers the phenomenon of phylogenetic muscle
plasticity to adaptive changes in muscle properties in response to changes in
functional load during phylogeny. The functional properties of jaw closing
45
muscles across species are complex and varied. Jaw muscles show a much
greater phylogenetic plasticity than limb and trunk muscles. In terms of
myosin expression it fits with the extensive repertoire of myosin expression
in jaw closers across species. In contrast, limb muscles show a minimal
degree of phylogenetic plasticity and manage to optimize contractile
functions by means of a limited number of MyHC isoforms.
Secondly, recent studies have provided evidence that the skeletal muscles of
the head that control mastication, facial expression, and eye movements are
evolutionary, morphologically and molecularly distinct from those of the
trunk and limbs (Sambasivan et al., 2011). In evolutionary terms the head of
vertebrates is thought to be a novel structure and some muscles; extra
ocular, jaw and facial muscles are also a vertebrate novelty. They all derive
from the cranial mesoderm, an embryonic tissue that is also unique to
vertebrates. It has been suggested that the head muscles have arisen
independently of trunk muscles, which means that the cranial mesoderm
derived progenitors are evolutionary distinct from the somatic muscle
progenitor pool (Sambasivan et al., 2011). Furthermore, it is now known that
different genetic regulatory cascades operate within the individual
craniofacial muscle groups, and that there is a remarkable relationship with
cardiomyogenesis (Sambasivan et al., 2011).
Finally, Hoh introduced the term allotype for muscles, which were
developmentally, structurally and functionally distinct. The first group of
muscles to be an allotype separated from limb and trunk muscles was the jaw
muscles (Hoh, 2002). The results in this thesis concord with the masseter
and biceps being different allotypes of muscles (Hoh, 1993; Hoh, 2002), and
with the divergence between jaw and limb muscles in growth patterns and
regulatory proteins (Pavlath et al., 1998; Rios and Marcelle, 2009). The
findings are in line with results from previous studies on human jaw opening
and jaw closing (Eriksson, 1982; Eriksson and Thornell, 1983; Stål, 1994;
Monemi, 1999) facial (Stål et al., 1990), tongue (Stål et al., 2003; Granberg et
al., 2010) and extra ocular muscles (Kjellgren et al., 2003) as well as small
and large limb muscles (Eriksson, 1982; Stål et al., 1987) showing that each
muscle has its special fibre type composition.
Thus, the present results of key differences in structure and growth pattern
between the human masseter and biceps most probably reflect diversity in
evolutionary and developmental origins for jaw and limb muscles.
46
Fibre type composition
The findings in the young masseter of specialized composition and
distribution of fibre types already at three years of age suggests early
diversified and fine-tuned internal motor control for specific tasks. The deep
masseter with vertically oriented fibres and slow twitch motor units
(Stålberg et al., 1986; Stålberg and Eriksson, 1987) and a predominance of
type I fibres and slow MyHC and high muscle spindle density, probably is of
special importance for anti-gravity function in jaw positioning. More type IM
fibres composed of slow and fast MyHCs in mixture in the anterior
superficial masseter, and more type IIB fibres and fast MyHCs and few
spindles in the posterior portion near the fulcrum imply regional
specialization of the superficial masseter in force development and
endurance during biting, chewing, swallowing and speech. The internal
diversity tells that the superficial masseter is genetically destined to develop,
grow, and mature into not one muscle, but an entity of different muscle
portions acting in a task-related synergy. Moreover, marked inter-individual
heterogeneity in MyHC expression of the young masseter, compared with the
young biceps, reflects individual characteristics in functional properties.
The young masseter and young biceps muscles were similar in that type I
fibres, with slow MyHC, outnumbered other fibre types and were of similar
diameter. This suggests that at young age both muscles are composed mainly
of slow-twitch, fatigue resistant motor units and have high oxidative
capacity. However, differences in composition for the other fibre types reflect
basic diversity in morphology and function.
Significant age-related muscular plasticity from childhood to adulthood was
revealed when comparing the young masseter with data from the adult
(Eriksson, 1982; Eriksson and Thornell, 1983) and elderly (Monemi et al.,
1998) masseter. Thus, young masseter contains more type IM and less type
IIB fibres in the superficial portion, and less type I fibres in the deep portion
than the adult (Eriksson, 1982; Eriksson and Thornell, 1983) masseter. The
young masseter has more types I and IM proportions and less types IIA and
IIAB proportion in superficial portion than elderly masseter (Monemi et al.,
1998).
Fibre type growth pattern
Smaller type II fibre diameter in the young masseter than in young biceps
reflects diverse fibre type growth patterns. Generally, small diameter fibres
in the masseter together with multipennate muscle architecture (Ebert,
1938/39; Schumacher, 1961; Gaspard, 1987) should allow a relatively high
number of motor units. In fact, there is evidence from electrophysiological
47
studies of fewer muscle fibres per motor unit in the masseter than in large
limb muscles but about the same number as in the small first dorsal
interosseus muscle (FDI) (Stålberg et al., 1986). The FDI, like the masseter,
has a large cortical representation and is used in finely graded movements.
The average muscle fibre diameter of the FDI is about the same as in other
limb muscles (Polgar et al., 1973) and thus much larger than that of the
masseter. Therefore, given that the FDI is of equal volume as the masseter,
or rather smaller, the masseter should contain a higher number of motor
units than the FDI. High number of motor units, in turn, implies a wide span
of continuous contractile properties in the masseter as suggested by EMG
studies (Yemm, 1977).
Our comparison between young and adult (Eriksson, 1982; Eriksson and
Thornell, 1983) and elderly (Monemi et al., 1998) masseter and biceps
muscles showed significant differences between their fibre type growth
patterns. The difference in fibre type growth pattern between the masseter
and biceps reflects differences in development and maturation of fibres,
optimally designed to meet special force and speed demands in a cost and
energy effective manner. The small type II fibres can be the result of both
genetic program and phenotypic adaptation.
Extrafusal MyHC expression
The young masseter differed from the young biceps in MyHC expression of
the extrafusal fibres, the masseter being more complex by containing six
isomyosins including exclusive MyHCs- fetal and α-cardiac, in addition to
slow and three fast MyHC isoforms. Typically, the young masseter displayed
a more variable mAbs-staining pattern, and a major proportion of fibres
composed of mixtures of different MyHC isoforms resulting in a higher
number of “MyHC-fibre types”. Notably, there was a continuum of single and
combinations of MyHC isoforms systematically related to enzymehistochemical fibre types promoting correlation between mATPase fibre
types and MyHC isoform expression. The heterogeneous MyHC composition
in the young masseter may reflect an immature composition of MyHCs at
young age, which during growth and maturation, in parallel with craniofacial
changes, teeth eruption and improvement of jaw functions, will evolve and
adapt to functional demands.
The result provides evidence that the young masseter muscle is unique in
MyHC expression, including unconventional MyHC isoforms as well as
hitherto unrecognized spliced isoforms of MyHC-fetal and MyHC-IIx.
Differences in MyHC expression vs. young biceps are proposed to reflect
diverse evolutionary and developmental origins and accord with the
masseter and biceps being separate allotypes of muscle. Comparison with
48
MyHC expression in adults and elderly revealed differences between
masseter and biceps in MyHC plasticity, and therefore alterations in
contractile properties, during life span.
Slow- and fast MyHC expression
For slow MyHC in the young masseter, there was generally good agreement
between the IHC and GE results (73 % and 86 %, respectively) and the
proportion of enzyme-histochemical fibres expressing slow MyHC (83%
types I, IM and IIC). Similar congruence was not seen for the fast MyHCs.
IHC stained 45 % of the young masseter fibres for fast MyHC in accordance
with the proportion of enzyme-histochemical fibre types expressing fast
MyHC (55 % types II and IM). However, fast MyHC content in the GE was
only 13 %. This seemingly disparity in results of the two methods can be
explained. Firstly, the mAbs against MyHCs used have very high sensitivity.
Thus individual fibres, which contain relatively small amounts of fast
isomyosins, as reflected in the gels, will nevertheless appear as stained fibres
by IHC. Secondly, in young masseter, the type II fibre diameter is smaller
than the type I type diameter (Österlund et al., 2011b), which means that a
given proportion of fibres detected with mAbs will correspond to a relatively
smaller muscle mass quantified by GE.
The findings for fast MyHC distribution may be due to high mAb sensitivity.
With respect to specificity of the used mAbs, we were aware that Western
blots of the used mAbs have revealed that some have broader reactivity
pattern than originally published or proposed by manufacturer. This holds
for the mAbs SC71, A474 and N2261. The first two are reported to identify
MyHC-IIa, but show affinity, albeit weaker, also for MyHC-IIx (Soukup &
Thornell, unpublished). The mAb N2261 showing very strong affinity for
MyHC-IIa, also detects MyHC-I and MyHC-extra ocular, whereas the mAb
BF35 reacts with MyHCs I, α-cardiac, IIa, slow-tonic and extra ocular (Liu et
al., 2002). This taken into account when classifying MyHC staining in
relation to ATPase fibre typing, nevertheless we had to introduce a fibre type
MyHC-IIx´ as the staining pattern of these fibres did not accord with
presumptive staining patterns of used mAbs. The mAb BF35 negative fibres
are supposed to reflect fibres expressing MyHC-IIx, being the only MyHC
which they do not detect. In the present study however, a proportion of the
mAb BF35 unstained fibres were also unstained for the mAb N2261,
indicating lack of MyHC-IIa. On the other hand, the fibres stained with the
mAbs SC71 and A474, which preferentially detect MyHC-IIa according to the
manufacturer and researchers introducing them (Schiaffino et al., 1989;
Hughes et al., 1993; Liu et al., 2002). However, these antibodies also have
been shown to have affinity for MyHC-IIx, albeit clearly weaker than for
49
MyHC-IIa. Thus, unfortunately these mAbs do not reliably separate fibres
containing MyHCs- IIa and IIx (Smerdu and Soukup, 2008). Until further
information is available, we tentatively interpret the MyHC-IIx´ fibre to
reflect the occurrence of a spliced isoform of MyHC-IIx. Interestingly, the
young biceps lacked this isoform, which may reflect some disparity in
molecular structure between cranial and limb fast MyHC isoforms. This may
be related to divergence between the masseter and biceps in evolutionary
and developmental origins (Sambasivan et al., 2011).
Actually, existences of spliced MyHC isoforms are not unique. Indeed
isoforms of MyHC-I have been presented in human muscle related to
development (Hughes et al., 1993). However, using the same mAbs, we did
not observe distinct evidence for spliced variant of MyHC-I in childhood.
MyHC-fetal and MyHC-α cardiac expression
A fundamental difference between young masseter and young biceps was the
presence of MyHC-fetal and MyHC-α cardiac isoforms in the masseter, but
not in the biceps. The results of the two methods with respect to amount and
occurrence of MyHC-fetal and MyHC-α cardiac in the young masseter also
seemed to be at variance. By IHC, both isoforms were detected in about half
of the young masseter fibre population, dispersed over the complete fibre
type population though preferentially expressed in IM and IIC fibres. By GE,
they were detectible in the overloaded gels but faintly visible in the regular
mini-gels. Like the result for fast MyHC content, the finding for fetal and αcardiac isoforms may be due to high mAb sensitivity. The result confirms
that these isoforms, which in limb muscles are expressed only during muscle
development, are constituent contractile proteins in the human masseter
muscle during the life span (Butler-Browne et al., 1988; Soussi-Yanicostas et
al., 1990; Bredman et al., 1991; Barbet et al., 1992; Pedrosa-Domellöf et al.,
1992; Eriksson et al., 1994; Sciote et al., 1994; Stål et al., 1994a; Monemi et
al., 1996; Monemi et al., 1999a; Korfage et al., 2000; Bontemps et al., 2002).
Our result of two bands stained for MyHC-fetal with Western blot technique
was unexpected but might be of considerable interest in terms of MyHC
expression and phylogenetic regulation. One band corresponded in mobility
with a band in the fetal sample known to reflect the MyHC-fetal isoform
(Butler-Browne et al., 1988). This band co-migrated with the MyHC-IIa band
in our gels. The other band had slower mobility and co-migrated with the
MyHC-IIx band. We could exclude that the extra band was related to MyHCembryonic, both on the basis of known mobility of MyHC-embryonic in our
gel system (Kjellgren et al., 2003), and the fact that the mAb F1652, specific
50
for human MyHC-embryonic (Karsch-Mizrachi et al., 1989), stained our fetal
control samples but left young masseter samples unstained. Further studies
are needed to ascertain the existence of the two fetal isoforms and their
functional significance, but our results indicate the existence of a so far
unrecognized spliced variant of MyHC-fetal in human skeletal muscles.
The special MyHC expression in the masseter reflects evolutionary aspects
on the jaw-closing muscles. The significance of small proportions of fetal and
α-cardiac contractile proteins in the masseter and for jaw function during
growth and maturation has not clearly been revealed. Recent molecular
studies by mRNA techniques indicate adaptive changes in MyHC-fetal
expression in the human muscle in response to altered biomechanics from
dental and surgical treatments (Harzer et al., 2010; Oukhai et al., 2010).
Experimental results in rat cardiac myocytes indicate that even small
amounts of MyHC-α cardiac significantly augment myocyte power output
(Herron and McDonald, 2002). An age-related decrease of this isoform is
suggested when the present result in young masseter is compared with data
from adult and elderly masseter.
Muscle spindles and intrafusal fibre types (Papers II and IV)
The results revealed both differences and similarities between the young
masseter and the young biceps in muscle spindle morphology. The masseter
showed higher spindle density, larger number of intrafusal fibres per
spindle, larger average fibre diameter and more compound muscle spindles
than young biceps. The masseter spindles contained more bag2 fibres and
less chain fibres than the biceps spindles. The young masseter and young
biceps were similar in their strikingly heterogeneous allotment of types and
number of intrafusal fibres. Therefore, the ‘‘average’’ composition of
intrafusal fibre types should not be taken as ‘‘the typical’’ muscle spindle
appearance. Rather, most spindles were unique in the combination of
intrafusal fibre types. This variability in morphology between spindles and
muscles suggest highly differentiated proprioceptive abilities allocated in
governing mandibular and arm positions and movements. Furthermore,
young masseter and biceps muscle spindles expressed a broad repertoire of
MyHC isoforms in a complex pattern and still use genes that appeared early
in evolution. The findings of fundamental similarities in intrafusal MyHC
expression between young masseter and biceps, but also marked differences
implying muscle specific proprioceptive control, for jaw- as well as arm
motor skills.
51
Muscle spindle density
The present finding of almost three times higher muscle spindle density in
the deep than in the superficial masseter, accords with findings in the adult
masseter (Eriksson and Thornell, 1987). From experience that smaller
muscles have higher muscle spindle densities than larger muscles it was
suggested that higher density is appropriate for small muscles involved in
fine adjustments (Barker and Banks, 1994; Banks, 2006). Comparison of
human small and large muscles acting in parallel demonstrated that smaller
muscles contained about 3.7 times the spindle density of larger muscles. This
difference was related to greater relative excursions of the smaller muscles,
which therefore were appropriate for length sensors (Peck et al., 1984).
Lengh changes of spindles depend on features such as extrafusal fibre length,
degree of pinnation and moment-arm length (Gans and Bock, 1965;
McClearn, 1985). The masseter is multipennate with relatively short muscle
fibres. The deep masseter, with vertically oriented fibres and a
predominance of type I fibres/slow motor units (Stålberg and Eriksson,
1987), seems to be appropriate to house length sensors involved in fine
position control of the mandible. Studies indicate that this regional
specialization is early evolved. The number of spindles supplying the jaw
muscles seems to become larger from the lower primates towards man. This
increasing jaw muscle spindle density accords with increasing complexity of
jaw skills along the evolutionary line of the primates (Kubota and Masegi,
1977), indicating impact on mandibular movement control in chewing and
speech, a function evolved only in man.
Intrafusal fibre type composition
Bag2 fibres in young biceps muscle spindles were relatively scarce. There was
a lack of bag2 fibres in the young masseter and biceps muscle spindles.
Spindles lacking bag2 fibres have previously been reported for the human
adult biceps (Liu et al., 2002) and deep neck (Liu et al., 2003) muscles. Bag1
fibres are known to play a major role in the production of the velocitysensitive (dynamic) response in the primary endings whereas bag2 fibres
mediate the length sensitivity (static) along with chain fibres (Boyd, 1980;
Matthews, 1981; Barker and Banks, 1994). Furthermore, five of the young
masseter spindles lacked chain fibres, but of these, all contained bag1 and
four contained bag2 fibres, suggesting ability for both dynamic and static
sensitivity. Lack of bag2 and chain fibres would again reflect diversity in
physiological qualities between spindles and muscles, preferentially in static
length sensitivity properties. One could speculate that the proportions of
MyHCs- slow-tonic, I and α-cardiac in bag1 is correlated with “dynamic”
properties, and that MyHCs- IIa and fetal in bag2 and chain fibres are
significant myosin isoforms for “static” actions.
52
In line with classification of the “intermediate” AS-bag1 fibre on basis of its
mATPase staining profile (Eriksson and Thornell, 1985, 1987, 1990; Eriksson
et al., 1994), IHC placed this fibre type in between bag1 and bag2 fibres by
showing MyHCs- I, α-cardiac and IIa, isoforms present in both bag1 and
bag2. Its physiological characteristics and role in proprioceptive control
remains to be examined.
Only one intrafusal fibre type combination overlapped all three masseter
portions, and few combinations overlapped the young masseter and biceps.
Also different subjects were unique in fibre type composition of muscle
spindles, with hardly any overlapping combinations between subjects.
Although noted in a limited number of subjects, this variability in
morphology between spindles, between muscle portions and between
muscles and subjects suggest highly differentiated proprioceptive abilities
allocated in governing jaw and arm positions and movements. The finding of
a resemblance in muscle spindle morphology between young and adult
(Eriksson and Thornell, 1987) masseter and between young and adult (Liu et
al., 2002) biceps, suggest that muscle spindles in these muscles are
morphologically mature already at young age and precede the extrafusal
fibres in growth and maturation.
Intrafusal MyHC expression
Precise determination of muscle fibre types and their composition of
contractile proteins in a given muscle are critical for in-depth knowledge of
muscle diversity and better understanding of physiological properties and
pathophysiological behaviour. The MyHC isoforms detected in the intrafusal
fibres are coded by separate genes. The fast skeletal gene cluster codes for
MyHC-IIa (MYH2), MyHC-IIx (MYH1). MYH3 and MYH8 genes code for the
developmental MyHC-embryonic and MyHC-fetal isoforms. Two cardiac
myosin genes MYH6 and MYH7 code for MyHC-α cardiac and MyHC-β
cardiac/slow-twitch. Furthermore, unlike extrafusal fibres the intrafusal
fibres contained MyHC-slow tonic, the evolutionary second most ancient
MyHC isoform, coded by MYH14 (Rossi et al., 2010), after the most ancient
masticatory IIM isoform, codoed by MYH16 (Hoh, 2002; Stedman et al.,
2004). MyHC-slow tonic was first recognized in presumptive muscle spindle
fibres in human 10-11 weeks of gestation old foetuses (Thornell et al., 1988;
Pedrosa-Domellöf and Thornell, 1994). Thus, the broad repertoire of MyHC
isoforms in the intrafusal fibres still uses genes appeared early in evolution.
Interestingly, the number of muscle spindles in the jaw muscles seems to
increase in the evolutionary series from lower primates towards man
(Kubota and Masegi, 1975, 1977), with the highest spindle density in the jaw-
53
closing muscles. The intricate MyHC expression seen in human muscle
spindles probably reflects the higher complexity of their sensory and motor
innervation (Kucera, 1986). The sensory and motor innervations are well
known factors influencing the expression of MyHC isoforms in intrafusal
fibres (Pedrosa et al., 1989; Soukup et al., 1995; Walro and Kucera, 1999).
Clinical implications
This thesis will contribute to a better understanding of growth, maturation
and aging of human skeletal muscle fibres and muscle spindles with special
focus on the jaw sensory-motor system. The findings that the masseter has a
complicated
structure
with
intra-muscular
variability,
regional
differentiation and fibre type diameter differences that change and adapt
over the life span, will be of value in examination by means of, e.g. EMG,
muscle biopsy and microdialysis techniques.
Training
In response to load, muscle fibres can alter in MyHC isoforms and crosssectional area, which determine their contraction velocity and maximum
force generation. Reduced muscular activity induces transition towards
faster more fatigable fibre types and a decrease in fibre cross-sectional area,
whereas increased muscular activity elicits transition towards slower more
fatigue-resistant fibre types and enlargement of fibre cross-sectional area (c.f
Grunheid et al., 2009; Vreeke et al., 2011). Soft food diet reduces the capacity
of the masseter muscle in animals (Kiliaridis et al., 1988) with decrease in
cross-sectional area of fibres co-expressing MyHCs- I and α-cardiac (Vreeke
et al., 2011). The finding that jaw muscles can adapt structurally to reduced
or increased load has implications for selection of rehabilitation program.
Muscle pain
Pain influences jaw muscle proprioception and deteriorate motor
performance. The effect of muscle pain on motor control is, however, not
fully understood. Experimentally induced jaw muscle pain causes transient
decrease in the maximum voluntary contraction resulting in lower bite force
(Svensson et al., 1998a; Svensson et al., 1998b; Svensson et al., 2004).
Moreover, muscle pain causes a change in coordination and decrease in
movement amplitude and velocity (Lund, 1991; Stohler, 1999; Svensson and
Graven-Nielsen, 2001). It has been shown experimentally that pain will via
the ϒ-muscle spindle system, increase the sensitivity of the muscle spindles,
54
resulting in increased reflex-mediated muscle stiffness (Johansson et al.,
1999). Likewise, incorrect information from the jaw muscle spindle system
may cause a sensory mismatch, with disturbed proprioceptive information,
which may cause disturbance of the postural and movement system of the
jaw (Ro and Capra, 2001; Capra et al., 2007).
Finally, it is intriguing to speculate that the evolutionary and developmental
relations between the heart and jaw muscle as reflected by MyHC-α cardiac
expression also in jaw muscle may have some bearing for the clinical
experience that jaw-face pain can sometimes be referred heart pain (Kreiner
et al., 2007).
Disease
Muscle diseases like hereditary myosin myopathies, are caused by mutation
in skeletal MyHC genes, which may affect MYH8 (codes for MyHC-fetal),
and can give clinical signs as jaw trismus and problems with mastication.
Two another diseases affects MYH3 (codes for MyHC-embryonic) or MYH7
(codes for MyHC-I), which can give rise to facial anomalies and jaw muscle
weakness (Oldfors, 2007).
Future perspective
From structure to function- Study of integrative jaw-neck motor control in
childhood.
Previous findings of coordinated mandibular and head-neck movements
during natural jaw-opening closing tasks suggest a functional linkage
between the jaw and the neck sensori-motor systems. Studies also indicate
that this functional connection between the jaw and neck is innate (Zafar et
al., 2000; Häggman-Henrikson and Eriksson, 2004; Eriksson et al., 2007).
Studies of comorbidity of jaw and neck myofascial pain have tried to explain
amplification and spread of muscle pain, by pain modulating mechanisms
via central sensitization (Arendt-Nielsen and Henriksson, 2007).
Furthermore, there is experimental evidence of intersegmental reflex
connections between the masseter and neck muscles (Hellström et al., 2000;
Hellström et al., 2002). To evaluate the functional maturation of the jawneck motor coupling in childhood, a pilot study examined concomitant
mandibular and head-neck movements during jaw opening-closing motor
activities in 5-year-old children (Fig. 23). The result demonstrated that the
movement pattern in children was irregular and variable compared with the
well-coordinated movement patterns seen in adults. This suggests an
55
immature reflex motor programming of jaw actions in children, although as
demonstrated in the present thesis, the muscle spindle system may be
morphologically well differentiated and matured.
Figure 23: Movement patterns of concomitment mandibular (blue) and head-neck
(red) movements during maximal jaw-opening-closing movements in a child aged 5years (left panel) and young adult 23-years (right panel). Note irregular and variable
mandibular and head-neck movements for the child (left panel) and regular and wellcoordinated movements for the adult (right panel). (Zafar, Österlund, Eriksson, pilot
data).
56
SUMMARY
Extrafusal fibre types
Young masseter
- Young masseter contained fibre types I, IM, IIC, IIAB, IIB and scarce
IIA. Type I fibres predominated. The inter-individual variation was
marked.
-
The deep portion showed more type I, and less type IM than the
superficial portion.
-
Type II fibres were smaller in diameter than type I, and smaller than
biceps type II.
-
Compared with adult masseter, young masseter showed larger
proportion of type IM and smaller proportion of type IIB in the
superficial portion, and smaller proportion of type I fibres in the
deep portion.
-
Compared with elderly masseter, young masseter showed larger
proportions of types I and IM, and smaller proportions of types IIA,
IIAB and IIB in the superficial portion.
-
The fibre diameters of types I, IM and IIC diameters were smaller in
the young masseter than in the adult and elderly masseter. The type
IIB diameter was smaller than in adults and type IIAB diameter
smaller than in elderly.
-
The average fibre diameter was 1.8 times larger in adult and 1,4
times larger in elderly masseter.
Young biceps
- Young bicep contained types I, IIA, IIAB and few IIB.
-
Compared with adult and elderly biceps, young biceps showed
smaller proportion of type IIB.
-
The average fibre diameter was 3.0 times larger in adult and 2.2
times larger in elderly masseter.
57
Extrafusal fibre MyHC expression
Young masseter
- GE identified MyHCs- I, IIa, IIx and small proportions of fetal and
α-cardiac isoforms.
-
MyHC-I predominated with larger content in the sup ant than in the
sup post masseter.
-
Two MyHC-fetal bands were identified.
-
Young masseter showed six isomyosins, MyHCs- I, IIa, IIx´, IIx,
fetal and α-cardiac.
-
The content of MyHC-IIa and IIx was larger in the superficial than in
the deep masseter.
-
The majority of fibres, 56%, co-expressed two to four MyHC
isoforms, i.e. were mixed in MyHC composition.
-
Compared with adult and elderly masseter, young masseter showed
more MyHC-I, less MyHCs- IIa, IIx and fetal isoforms and more
mixed fibres.
Young biceps
- GE and IHC showed MyHCs- I, IIa and IIx. MyHC-I predominated.
-
Only occational fibres contained mixtures of two MyHC isoforms.
-
Young biceps showed more MyHC-I and less fast isoforms MyHCsIIa, IIx and fetal than the adult and elderly biceps.
Muscle spindles
- Muscle spindle density was higher in the deep masseter than in the
superficial and in the biceps, and higher in the sup ant than in the
sup post masseter.
-
Compound muscle spindles were frequent in the deep masseter, and
few in the biceps.
-
Muscle spindle diameter was larger in the A and AB-regions than in
the B-region.
58
-
Muscle spindles were very heterogenous in allotment of types and
number of intrafusal fibres.
-
Intrafusal fibre number was larger in the deep masseter than in the
biceps.
-
Young masseter muscle spindles contained more bag2 fibres and less
chain fibres than the biceps spindles.
-
The mean intrafusal fibre diameter was larger in the young masseter
than in the young biceps.
-
Bag2 fibres were larger in diameter than bag1 and chain fibres.
-
No major differences in spindle morphology were found in
comparison with spindles in adult masseter and biceps, respectively.
Intrafusal fibre MyHC expression
Similarities between young masseter and young biceps in intrafusal MyHC
expression
- Eight MyHCs- slow-tonic, I, IIa, IIx´, IIx, fetal embryonic and αcardiac.
- Co-expression, mixtures of two to six isoforms in all intrafusal fibres.
- Numerous of combinations of MyHC isoforms. Bag1, AS-bag1 and
bag2 fibres showed MyHC-slow tonic, and bag1 also MyHC-I. Chain
fibres showed MyHCs- IIa and fetal.
- In chain fibres, the two most frequent MyHC isoform combinations
were seen in both muscles.
Differences between young masseter and young biceps in intrafusal MyHC
expression
- In the young masseter, 98 % of bag1 fibres showed MyHC-α
cardiac vs. 30 % in the young biceps, all AS-Bag1 fibres showed
MyHC-α cardiac vs. 10 % in the biceps, 35 % of bag2 fibres
showed MyHC-IIx ́ vs. none in the biceps, 17 % of chain fibres
showed MyHC-I vs. 61 % in the biceps.
- The most frequent MyHC isoform combinations in bag1 and bag2
fibres differed between the muscles.
59
CONCLUSIONS
Extrafusal fibres
-
The results suggest that the human masseter is specialized in
composition of muscle fibre types already at young age and shows a
unique fibre type growth pattern, including relatively small type II
fibres.
-
The findings of unconventional MyHC isoforms as well as hitherto
unrecognized spliced isoforms of MyHC-fetal and MyHC-IIx provide
evidence that the young masseter muscle has a unique MyHC
composition.
-
The finding of differences in fibre types and MyHC expression
between young masseter and the masseter in adults and elderly
suggests marked plasticity of extrafusal contractile proteins during
growth and aging.
-
Differences between masseter and biceps in composition and
distribution of fibre types and MyHC isoforms, and in muscular
plasticity during life span are probably related to divergent
evolutionary and developmental origins, resulting in fibres optimally
designed to meet special force and speed demands in a cost and
energy effective manner. The differences accord with the masseter
and biceps being separate allotypes of muscle.
Muscle spindles/Intrafusal fibres
- The MyHC-IIx´ isoform demonstrated in masseter and biceps
intrafusal fibres as well as in masseter extrafusal fibres has not
previously been reported in human intra- or extrafusal fibres.
-
Findings of fundamental similarities in intrafusal MyHC expression
between young masseter and biceps, but also marked differences
implying muscle specific proprioceptive control, probably related to
diverse evolutionary and developmental origins.
-
Findings of similarities in mATPase fibre types and MyHC
expression of intrafusal fibres between young masseter and biceps
muscle spindles and spindles in adult muscles suggest early
maturation of muscle spindles. This in turn implies early need and
60
capacity of reflex, proprioceptive control in learning, performing and
improving jaw skills and arm behaviour.
General conclusion
- This thesis will contribute to a better understanding of growth,
maturation and aging of human skeletal muscle fibres and muscle
spindles, with special focus on the jaw sensory-motor system. The
results will also be of scientific and clinical value in examination by
means of e.g. EMG, muscle biopsy and microdialysis techniques.
Verified hypotheses
- The hypothesis of masseter extrafusal muscle plasticity during life
span in parallel with changes in jaw-face skeletal morphology and
jaw function.
- The hypothesis that the masseter is divergent from limb muscle in
growth pattern.
Rejected hypothesis
- The hypothesis of changes in masseter intrafusal (muscle spindle)
fibre types and MyHC expression from childhood to adulthood was
rejected.
61
ACKNOWLEDGEMENTS
Of all my heart I would like to express my gratitude to all of you who have
helped me and been involved in this project. Over the years, this work has
given me the chance to get to know people who have inspired me by their
wisdom, helpfulness and good humour and altogether made the time spent
on this project worthwhile. In particular I would like to acknowledge:
My supervisor professor emeritus Per-Olof Eriksson, you have guided me
through this thesis. You gave me the opportunity to learn from your
profound knowledge of muscle morphology and jaw-neck sensory-motor
system. At the very beginning, you were a newly installed professor and I was
an under-graduate student, and barking on PhD and ST-studies in clinical
oral physiology. With the experiments being done, we had to decide on an
interpretation of the results that we eventually could agree upon. Thank you
PO for being my mentor. I hope we can meet and have a good cup of coffee,
the best kind of “kanelbulle” and discuss the news in DN.
My assistant supervisor professor emeritus Lars-Eric Thornell, you are clearsighted and kind-hearted. Thank you for sharing your unique and extensive
knowledge about muscles. I am grateful that you shared this knowledge with
me so that I could finish this thesis.
Assistant supervisor associated professor Per Stål, you generously jumped on
the train on time. Lucky me!
I would also like to thank the following persons:
•
Dr. Jin-Xia Liu and Dr. Mona Lindström, thank you for contributing
to paper III and IV and for your moral support and many laughs.
•
Anders Wänman, innovative, skilful and successful professor of
clinical oral physiology. You convey the attitude that nothing is
impossible. I am so proud of being one in your KOF-team.
•
Dr. Birgitta Häggman-Henrikson, my friend and invaluable advisor.
You stand out as an example for me. You taught me how to allocate
my priorities. “The rest is just pebbles and sand”.
62
•
Dr. Susanna Marklund, my friend, colleague, and my VLL-boss. You
have inspired me with your excellent ability to see the individual in a
context, meet the person and give a gentle push forward if needed.
•
Ewa Lampa, Christina Storm-Mienna, Susanne Eriksson and Dr.
Birgitta Wiesinger, for sharing your clinical skills, scientific
knowledge and good company.
•
Inga Johansson, thank you for providing excellent down-to-earth
technical assistance.
•
Agnetha Lundström, I appreciate your friendship and sincerity. It is
always nice to talk with you and put things in perspective.
•
Dr. Hamayun Zafar for your knowledge in integrative jaw-neck
motor behaviour.
•
Dr. Homa Tajsharghi, for generously providing test with mRNAanalysis.
•
Ann-Sofie Strandberg, Hjördis Olsson and Lillemor Hägglund, for
administrative support and academic logistic.
•
Dr. Albert Crenshaw for linguistic revision of the English text.
•
Colleagues and workmates at the Department of Odontology and the
muscle research group at the Department of Integrative Medical
Biology for support and encouragement.
•
Colleagues and workmates at utbildningstandvården
specialistttandvården for support and encouragement.
63
and
Min familj
Min familj är skogens träd, starka med årsringar många
Doftande blommor växer bland mossa och knotig gren i den rikaste mull
Här finns fåglar som kvittrar, stigar som letar sig till drömmar och mål att fånga
Bland brokiga stammar drar vinden ostyrig och varm och viskar tillitsfull
Min familj är sanning och verklighet i all sin härlighet
Mamma Barbro och pappa Kurt, ni ger mig kloka ord och kärleksfull
omtanke varje dag. En uppmuntran att lyssna inåt och våga ett kliv till. Den
grund ni har lagt för mig bär jag med mig genom livet. Min käre storebror
Michael, du är mitt stora stöd. Du hjälper mig alltid, utan krångel ställer du
upp. Tillsammans med Eva finner du nya dörrar att glänta på. Jag finns för
dig närhelst du behöver. Min kära lillasyster Åsa, du är unik med ditt mod
och din ärlighet. Du inspirerar mig i livet. Tillsammans med Eric, Sara, Lowe
och Miro, ger ni mig så mycket glädje. Mina älskade barn; Maria, klok,
insiktsfull, kärleksfull supertjej. Magnus, förnuftig, målmedveten,
omtänksam superkille. Hela ert liv har ni levt med en mamma som jobbat,
jäktat, förmanat och försakat men som i livets villervalla för alltid älskar er.
Man måste ha mod att gå vidare dit möjligheterna finns. Mina svärföräldrar
Nanna och Runo, ni har följt mig i många, tack för ert stöd och er omtanke.
Min älskade levnadskamrat Jan, vi lever ett liv ihop med kärlek mellan oss.
Du är mitt hjärtas kär.
This work was supported by grants from the Department of Odontology at
Umeå University, Västerbotten County Council and the Swedish dental
society.
64
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