Umeå University Medical Dissertations
From the Department of Clinical Sciences, Ophthalmology
and the Department of Integrative Medical Biology, Section for Anatomy,
Umeå University, Sweden
HUMAN EXTRAOCULAR MUSCLES
Molecular diversity of a unique muscle allotype
Daniel Kjellgren
Umeå 2004
Copyright by Daniel Kjellgren, 2004
Illustrations by the author
ISBN 91-7305-638-3
Printed in Umeå, Sweden by
Solfjädern Offset AB
Cover picture: Photo of a cross-section from a rectus superior muscle,
showing the heterogeneity in MyHCIIa content of the fibers
2
To my wife Åsa,
my daughter Rebecka
and my mother Catharina
”Behind every successful man is a surprised woman”
Maryon Pearson
“in your eyes
the resolution to all the fruitless searches”
Peter Gabriel
”Det tog sin tid, sade Toke; men nu har han pissat färdigt”
Frans G Bengtsson
3
Table of contents
Abbreviations .................................................................... 6
Abstract ............................................................................. 7
Original papers.................................................................. 9
Introduction..................................................................... 11
Anatomy__________________________________________ 11
Histology _________________________________________ 12
Fiber types________________________________________ 12
Innervation _______________________________________ 12
Physiology ________________________________________ 13
Muscle contraction _________________________________ 13
Myosin ___________________________________________ 14
SERCA ___________________________________________ 15
Myosin binding protein C ____________________________ 15
Basement membrane ________________________________ 16
Laminin __________________________________________ 16
Clinical implications ________________________________ 17
Aims of the study............................................................. 19
Materials and methods.................................................... 20
Histochemistry_____________________________________ 20
Immunocytochemistry _______________________________ 20
Fiber typing _______________________________________ 21
Fiber area ________________________________________ 21
Biochemistry ______________________________________ 21
Capillaries________________________________________ 22
Statistics _________________________________________ 22
4
Results.............................................................................. 23
Biochemistry ______________________________________ 23
Morphology _______________________________________ 23
Enzyme histochemistry ______________________________ 23
Immunocytochemistry _______________________________ 24
Tables............................................................................... 27
Discussion ........................................................................ 31
Validity___________________________________________ 31
EOM fiber type composition __________________________ 31
Extracellular matrix ________________________________ 34
Levator palpebrae __________________________________ 34
Human EOM versus EOM of other species_______________ 35
Human EOM versus human limb muscle ________________ 36
Complexity and function _____________________________ 36
Future directions ___________________________________ 37
Conclusions...................................................................... 38
Acknowledgments ........................................................... 39
References........................................................................ 42
Original papers ............................................................... 53
Paper I
Paper II
Paper III
Paper IV
5
Abbreviations
ATPase
BM
CMD
EOM
ECM
GL
Ln
LP
Lu
MAb
MIF
MTJ
MyBP-C
MyBP-Cfast
MyBP-Cslow
cMyBP-C
MyHC
MyHC-cardiac
MyHCemb
MyHCeom
MyHCI
MyHCIIa
MyHCIIb
MyHCIIx
MyHCsto
NMJ
OL
SDS-PAGE
SERCA
SERCA1
SERCA2
SIF
SR
Tn-C
T-tubules
6
Adenosine triphosphatase
Basement membrane
Congenital muscular dystrophy
Extraocular muscle
Extracellular matrix
Global layer
Laminin
Levator palpebrae
Lutheran glycoprotein
Monoclonal antibody
Multiply innervated fiber
Myotendineous junction
Myosin binding protein C
Fast isoform of MyBP-C
Slow isoform of MyBP-C
Cardiac isoform of MyBP-C
Myosin heavy chain
-cardiac isoform of MyHC
Embryonic isoform of MyHC
Extraocular isoform of MyHC
Slow isoform of MyHC
Fast isoform IIa of MyHC
Fast isoform IIb of MyHC
Fast isoform IIx of MyHC
Slow tonic isoform of MyHC
Neuromuscular junction
Orbital layer
Sodium dodecyl sulfate polyacrylamide gel
electrophoresis
Sarco(endo)plasmic reticulum Ca2+ATPase
SERCA fast isoform
SERCA slow isoform
Singly innervated fiber
Sarcoplasmic reticulum
Tenascin-C
Transverse tubules
Abstract
Introduction: The extraocular muscles (EOMs) are highly specialized and differ
so significantly from other muscles in the body that they are considered a
separate class of skeletal muscle, allotype. A thorough characterization of the
molecular and cellular basis of the human EOM allotype has not been presented
although it is expected to contribute to a better understanding of the functional
properties of these muscles and the reasons why they are selectively spared in
some neuromuscular disorders.
Myosin is the major contractile protein in muscle and consists of two heavy and
four light chains. The myosin heavy chain (MyHC) isoforms contain both the
ATPase and the actin binding site and are therefore the best molecular markers of
functional heterogeneity of muscle fibers.
The relaxation rate, another important physiological parameter of muscle fibers,
reflects the rate at which Ca2+ is transported back from the myofibrilar space into
the sarcoplasmic reticulum (SR) mostly by SR Ca2+ATPase (SERCA). The major
SERCA isoforms in muscle are SERCA1 (fast) and SERCA2 (slow).
Myosin binding protein C (MyBP-C), the second most abundant thick filament
protein in striated muscle, plays a physiological role in regulating contraction.
There are 3 major isoforms of human MyBP-C: MyBP-Cfast, MyBP-Cslow and
cardiac MyBP-C.
The laminins (Ln) are the major non-collagenous components of the basement
membrane (BM) surrounding muscle fibers and are important for muscle fiber
integrity. They are composed of an -, a - and a -chain, all of which exist in
multiple isoforms.
Methods: Adult human EOMs were studied with SDS-PAGE, immunoblots and
immunocytochemistry, the latter with antibodies against six MyHC isoforms,
SERCA1 and 2, MyBP-Cslow, MyBP-Cfast and eight laminin chain isoforms.
The myofibrillar ATPase, NADH-TR activity, fiber area and capillary density
were also determined.
Results: Important heterogeneity in fiber composition was the hallmark of the
human EOMs. Three major groups of fibers could be distinguished. Fast fibers
that stained with anti-MyHCIIa, slow fibers that stained with anti-MyHCI and
MyHCeompos/MyHCIIaneg-fibers that stained with neither of these antibodies but
with anti-MyHCI+IIa+eom and anti-MyHCeom. Approximately 62% of the
sampled fibers in the global layer and 81% in the orbital layer were MyHCIIa
positive fibers and 14% of the fibers in the global layer and 16% in the orbital
7
layer were slow fibers. The remaining were MyHCeompos/MyHCIIaneg-fibers.
The vast majority of the slow fibers also stained with anti-MyHCsto.
Anti-MyHC-cardiac stained approximately 26% of the slow fibers in the orbital
and 7% in the global layer. MyHCeom was also present in some of the MyHCIIa
positive fibers and slow fibers. MyHCemb was co-expressed in some of the fibers
of all groups. The staining levels of each antibody varied among fibers.
Almost all MyHCIIa positive fibers contained SERCA1 and 86% of them also
contained SERCA2, whereas <1% were unlabeled with both antibodies. Thirteen
percent of the slow fibers contained SERCA2 only, 86% contained both SERCA1
and 2 and 1% were unstained. Biochemically SERCA2 was more abundant than
SERCA1.
MyBP-Cfast was not present in the EOMs and MyBP-Cslow was only detected
immunocytochemically. Interestingly, two unrecognized bands were seen in the
MyBP-C region in SDS-PAGE of whole muscle extracts of EOM but not of limb
muscle.
The extrasynaptical BM of the EOM muscle fibers contained Ln2, 1, 2, 1,
and to some extent also Ln4 and 5 chains. The capillary density in the EOMs
was very high (1050 +/-190 capillaries/mm2) and significantly (p<0.05) higher in
the orbital than in the global layer.
Conclusions: The human EOMs have a very complex fiber type composition
with respect to the major proteins regulating fiber contraction velocity and force
(MyHCs, MyBP-Cs) and fiber relaxation rate (SERCAs). There are also
important differences in laminin isoform composition and capillary density when
compared to human limb muscle. The presence of additional laminin isoforms
other than laminin-2 in the extrasynaptic BM, could partly explain the sparing of
the EOMs in Ln2 deficient congenital muscle dystrophy.
The co-existence of complex mixtures of several crucial protein isoforms provide
the human EOMs with a unique molecular portfolio that a) allows a highly
specific fine-tuning regime of contraction and relaxation, and b) imparts
structural properties that are likely to contribute to protection against certain
neuromuscular diseases.
8
Original papers
This thesis is based on the following original papers, which are referred to in the
text by their Roman numerals:
I. Kjellgren D, Thornell L-E, Andersen J, Pedrosa-Domellöf F. Myosin heavy
chain isoforms in human extraocular muscles. Invest Ophthalmol Vis Sci.
2003;44:1419-25.
II. Kjellgren D, Ryan M, Ohlendieck K, Thornell L-E, Pedrosa-Domellöf F.
Sarco(endo)plasmic reticulum Ca2+ ATPases (SERCA1 and -2) in human
extraocular muscles. Invest Ophthalmol Vis Sci. 2003;44:5057-62.
III. Kjellgren D, Stål P, Larsson L, Fürst D, Pedrosa-Domellöf F Uncoordinated
expression of myosin heavy chains and myosin binding protein C isoforms in
human extraocular muscles, Manuscript.
IV. Kjellgren D, Thornell L-E, Virtanen I, Pedrosa-Domellöf F. Laminin isoforms
in human extraocular muscles. Submitted.
9
10
Introduction
Vision is by far the most important of our senses for communication with the
world around us. The process of imaging is complex and includes the optics of the
eye, the photoreceptors, the optical tract and the occipital cortex. In order to
optimize perception and maintain stereo vision in every gaze position, it is
necessary to perfectly control and co-ordinate the delicate movements of the
eyeballs. The eyes are capable of highly specialized movements such as slow
pursuit movements as well as rapid, instant change from one point of fixation to
another (saccades). Eye movements are the result of the action of the extraocular
muscles (EOMs).
Anatomy
There are six EOMs (fig 1): the
medial, lateral, superior and inferior
recti, the superior and inferior
oblique. The four recti originate
from the tendinous ring around the
optic canal, run forward in the orbit
and insert into the eyeball at the
medial, lateral, superior and inferior
side respectively. The obliquus
superior arises from the sphenoid
bone just superior to the tendinous
ring, runs forward to the trochlea, a
fibrous pulley located at the
superior and medial angle of the
orbital anterior margin. Here it
becomes tendinous and runs backwards
Figure 1: Extraocular muscles and
and laterally, passing below the rectus
levator palpebrae of the left orbit
superior to be inserted into the superior
surface of the eyeball. The obliquus
inferior originates from the orbital floor, just lateral to the lacrimal fossa, runs
laterally and posteriorly, between the rectus inferior and the orbit floor and inserts
into the inferior side of the eyeball (fig 1).
11
Histology
The fibers of the EOMs are organized into two separate layers. The thin orbital
layer faces the orbital wall. The global layer consists of the central part of the
muscle.1 This organization into layers is most clearly seen in the midbelly part of
the muscles. In the oblique muscles the orbital layer may totally surround the
global layer.2 A third layer – the marginal zone – covering parts of the outer
surface has been described in human EOMs.3
Fiber types
Human skeletal muscle fibers are usually divided into three major types depending
on their physiological, biochemical and histochemical characteristics: 1) slow
twitch, fatigue resistant (type I fibers), 2) fast-twitch, fatigue resistant (type IIA
fibers) and 3) fast-twitch fatigable (type IIB fibers). However, this general
categorization into fiber types does not apply to more specialized muscles4 and
especially not to the EOMs.5 Six fiber types have been described in the EOMs of
several species on the basis of fiber location, innervation and color: 1) orbital
multiply innervated, 2) orbital singly innervated, 3) global multiply innervated, 4)
global red singly innervated, 5) global intermediate singly innervated and 6) global
pale singly innervated fibers.6 However, when studying the original papers2, 5, 7 it is
clear that the urge to categorize has led to an oversimplification. In particular, the
original data show that the human EOMs are far more complex and that the
properties of their fibers vary in a continuum.5, 7
Innervation
The EOMs are richly innervated and their ocular motor units are very small,
involving approximately 10 muscle fibers per neuron.8 There are differences in the
innervation of the fibers in the orbital and global layers. The singly innervated
fibers (SIF) have a single synaptic site, or neuromuscular junction (NMJ), in the
middle region of the muscle fiber and have a twitch mode of contraction.9 The
SIFs contain a great amount of mitochondria throughout their length. In the global
layer the red SIFs have more mitochondria and the pale SIFs have fewer.10 The
multiply innervated fibers (MIF) of the global layer have a tonic mode of
contraction, multiple innervation points along the fiber length and contain only
few mitochondria. In contrast, the multiple innervated fibers of the orbital layer
have a twitch mode of contraction and an endplate-like neuromuscular junction in
the midbelly part,1, 6 but multiple innervation points and tonic mode of contraction
in the peripheral parts of the fiber. Accordingly, the midbelly part of the orbital
MIFs is also rich in mitochondria, as the SIFs.10 The peripheral parts of the MIFs
of the orbital layer and all of the global MIFs contain fewer mitochondria. At the
distal end of the muscle, at the myotendinous junctions (MTJs), the global layer is
12
enclosed by a tangle of nerve endings called palisade endings,11 supposed to have a
proprioceptive function.
Physiology
The extraocular muscles
(EOMs) are unique in
many ways, as shown by
data collected from
several species. The
EOMs are among the
fastest muscles in the
body1 and yet they are
extremely fatigue
resistant,12 as reviewed by
Porter.9 The nontwitch
motor units, consisting of
MIFs, respond to stimulation
Figure 2: Role of Ca2+ in contraction and relaxation of
muscle fibers
with slow tonic contraction
and are highly fatigue
resistant.13 In monkey EOMs, the twitch motor units, consisting of SIFs have been
divided into fast and slow according to their fusion frequency, although the
distribution of this parameter is unimodal without an apparent dividing
frequency.14 When tested for fatigue resistance, the twitch motor units have a
bimodal distribution of fatigable and fatigue resistant units.14 So depending on
their physiological properties a system of five different types of EOM motor units
have been proposed: 1) nontwitch (NT), 2) fast fatigable (FF), 3) fast fatigue
resistant (FR), 4) slow fatigable (SF) and 5) slow fatigue resistant (S).15 A
correlation between this division and the six histochemical fiber types described in
the EOMs (see previous section on fiber types) has not been made.
Muscle contraction
When a muscle fiber is
activated, the action potential
stimulus spreads along the
sarcolemma and reaches the
depth of the muscle fiber
through the transverse
tubules (t-tubules) (fig 2, 3).
The T-tubules form junctions
with the terminal cisterna of
the sarcoplasmatic reticulum
Figure 3: Organisation of the SR and the T-tubules
13
(SR) and depolarization triggers the release of Ca2+ from the SR into the
sarcoplasm. The raised Ca2+ concentration alters the structure of the actin bound
proteins troponin and tropomyosin, moving them to the side making it possible for
the head of the myosin heavy chain (MyHC) to bind to actin. Then the myosin
filaments slide along the actin filaments and the muscle fiber contracts (fig 2).
During relaxation, Ca2+ ions are actively pumped from the myofibrilar space into
the lumen of the SR and the Ca2+ gradient between the SR and the cytosol is
restored. This is accomplished mostly by sarco(endo)plasmatic reticulum
Ca2+ATPases (SERCA) using ATP as the source of energy, as reviewed by Dux.16
Myosin
Myosin, class II sarcomeric, is the major contractile protein in skeletal muscle17
and it is a multimeric complex of two heavy and four light chains (fig 4). Both
heavy and light chains exist in multiple isoforms.18, 19 The sarcomeric myosin
heavy chains are coded by 8 genes located on human chromosomes 14 and 17.20
The MyHCs are expressed in a tissue- and developmental stage specific manner
and their expression is
regulated by not fully
understood physiological,
hormonal and tissue
dependent factors. Since the
MyHC contains both the
ATPase and the actinbinding site, it thereby
determines the speed of
contraction and the
contraction force.18, 21, 22
Hence, the MyHC
4: Schematic illustration of myosin II
composition is regarded as the Figure
sarcomeric
best marker of the functional
heterogeneity among muscle
fibers.23
The MyHC isoforms can be classified into fast (MyHCIIa, IIx, IIb, eom), slow
(MyHCI, -cardiac, slow tonic) and developmental (MyHCembryonic, fetal).24 In
human limb muscle the slow twitch, type I fibers, contain MyHCI; the fast twitch,
so called types IIA and IIB fibers, contain MyHCIIa or IIx respectively.25
The MyHC content of the EOMs is more complex. In addition to MyHCIIa and
MyHCI, the mammalian EOMs express a specific fast isoform, MyHCeom,26-28
embryonic and fetal MyHC,26, 29, 30 slow tonic MyHC (MyHCsto)31, 32 and MyHC cardiac.33 A possible correspondence between the previously described six EOM
14
fiber types, in the rat, and the predominant expression of a given MyHC has
recently been suggested, although not really investigated.28 In addition, the MyHC
isoform content is not uniform along the length of some EOM fibers.3, 28, 34, 35
SERCA
In addition to contraction velocity and force, another important physiological
property of muscle fibers is their relaxation rate. The relaxation rate reflects the
rate at which Ca2+ is transported back from the myofibrilar space into the lumen of
the sarcoplasmic reticulum (SR). The major determinant of relaxation rate is the
SERCA isoform.16
SERCAs are large integral proteins and the major protein components of the SR.
Three differentially expressed genes encode SERCA proteins in human: SERCA1,
SERCA2 and SERCA3.36, 37 Differential splicing of the primary transcripts results
in further variations and at present at least seven SERCA isoforms have been
described in human.38 SERCA1a is present in fast-twitch skeletal muscle fibers
and SERCA2a is found in cardiac and slow-twitch muscle fibers. SERCA1b is
present in neonatal muscles, SERCA2b in almost all nucleate cells and
SERCA3a/b/c in several types of non-muscle cells. Mutations of the three SERCA
genes have been associated with disease. For example, mutation of the SERCA1
gene is associated with Brody's disease, a condition characterized by impairment
of skeletal muscle relaxation after exercise, stiffness and cramps.39 The distribution
of SERCA1 has been reported as rather complex in rat and rabbit EOMs40 but data
on human EOMs are lacking.
Myosin binding protein C
Myosin binding protein C (MyBP-C) is, after myosin, the second most abundant
thick filament protein in striated muscle41 and it has a regulatory role in sarcomere
assembly42, 43 MyBP-C is composed of a single subunit of 130 kDa and both its Cand the N-terminals bind to myosin.44, 45 MyBP-C has an essential physiological
role in regulating contraction by modulating maximal shortening velocity.46
There are 3 major isoforms of MyBP-C in human muscle: fast skeletal (MyBPCfast), slow skeletal (MyBP-Cslow) and cardiac (cMyBP-C).47 MyBP-Cfast,
detected both with in situ hybridization and immunocytochemistry, is present in
type II fibers, whereas MyBP-Cslow is present in both type I (slow) and type II
(fast), in human skeletal muscle.48 The cardiac isoform is restricted to the heart.48
Recent analysis of single fibers by SDS-PAGE, revealed the coordinated
expression of MyBP-Cslow in fibers containing MyHCI (slow), MyBP-Cfast in
fibers with MyHCIIx and an additional isoform, intermediate MyBP-C in fibers
containing MyHCIIa in human limb muscle.49 Coordinated isoform changes
indicating that MyBP-C expression is linked to MyHC expression have been
15
reported during skeletal muscle hypertrophy in the rat.50 However, in the human
masseter, a masticatory muscle with very special properties,4, 51, 52 the very complex
MyHC composition of its fibers was not paralleled by an intricate MyBP-C
pattern.49
Basement membrane
The muscle fibers are surrounded and supported by a layer of specialized
extracellular matrix (ECM) called the basement membrane (BM). BMs are present
under all epithelial cells, in blood vessels and surrounding many cells including
muscle cells and Schwann cells.53 The BM provides strength and elasticity, serves
as a selective filter and is necessary for muscle fiber maintenance and
regeneration.
At the neuromuscular junction (NMJ), where the peripheral nerve connects with
the muscle fiber, a specialized BM continues between nerve and muscle,
delineating the postsynaptic membrane.53 In the same way, the BM extends into
the deep junctional folds of the myotendineous junction (MTJ), where force is
transmitted from muscle to tendon.54
Laminin
The major non-collagenous protein of the
basement membrane is laminin.55 Laminins
(Ln) adhere to collagen and are essential for
the assembly of the BMs and appear very
early in development.56 Laminins strengthen
the BM, connect the BM to the cells and
furthermore, interact with cells via integrin
receptors57 and -dystroglycan.58 Laminins
are glycoproteins composed of three subunits
(, and chains). Presently there are 5
different -chains (1-5), 3 -chains (1-3)
and 3 -chains (1-3) known. Different
combinations of these chains assembly into
at least 14 laminin isoforms.55, 59 The
predominant laminin in the BM of muscle
and peripheral nerve is Ln-2 (211).60 At
the NMJ the composition of the BM changes
from Ln-2 to Ln-4 (221), Ln-9 (421)
and Ln-11 (521) and the juxtapositioned
Schwann cells are covered with a BM
containing Ln-2 and Ln-8 (411).61
16
Figure: 5 Laminin
The BM at the MTJ is also specialized and contains Ln-4 (221),62, 63 but there
is also expression of Ln5 and possibly Ln1.62
Clinical implications
The EOMs differ from other
skeletal muscles in that they are
often spared in systemic
neuromuscular diseases such as
Duchenne muscle dystrophy
(DMD), merosin deficient
congenital muscular dystrophy
(CMD) and other muscular
dystrophies.64
DMD, a recessive x-linked
disease, is caused by a defect in
the DMD gene at Xp21.2
coding for dystrophin.
Dystrophin is a cytoskeletal
protein located in close
association with the
sarcolemma. The lack of
dystrophin leads to progressive
Figure 6: Dystrophin associated protein
muscle fiber destruction, with
complex
devastating consequences and
typical symptoms, e.g. weakness,
developmental delay, enlargement of the calf usually present at 3-5 years of age,
progress of loss of ambulation, cardiac involvement and respiratory problems.64, 65
Merosin (laminin-2) deficient CMD accounts for approximately half of the CMD
cases and is caused by a gene defect in LAMA2 affecting the Ln2 chain.64, 66, 67
The clinical picture includes postnatal hypotonia with contractions, weakness and
delayed motor development. Death in the first year as a result of respiratory failure
is not uncommon. Variants of CMD with partial laminin-2 deficiency often have a
less severe phenotype.
Both laminin-2 and dystrophin are needed for an intact link between the ECM and
the cytoskeleton of muscle fibers, through the dystrophin associated protein
complex (DAPC).64 DAPC includes many proteins e.g. dystroglycans,
sarcoglycans and syntropin. It links to the BM and the ECM by laminin-2 and to
the intracellular actin cytoskeleton by dystrophin (fig 6). Defects involving the link
between ECM and the cytoskeleton (e.g. collagen, laminin, dystrophin, desmin)
cause muscle dystrophy.64, 68
17
The EOMs are remarkably unaffected in both DMD64 and merosin deficient
CMD69 in spite of severe general muscle fiber degeneration in other muscles of the
body. The reason for this is still essentially unknown, and the thought that this
should be simply due to the small size of the EOM fibers and lack of mechanical
stress64 or as a result of a disturbed calcium homeostasis, has been surpassed by a
more wide approach where the unique properties and molecular diversity of the
EOMs must be taken in consideration.70, 71
The EOMs are preferentially involved in Graves’ disease, myasthenia gravis and
Miller-Fisher. Graves’ disease is an autoimmune disorder following
hyperthyreosis, with myosit, swelling of the EOMs and the optic nerve.72
Myasthenia gravis is caused by an autoimmune attack against the
acetylcholinesterase receptors in all neuromuscular junctions. It results in
tiredness, ptosis and dysfunction of the EOMs. The ocular symptoms often
precede the general symptoms, but not always. Miller-Fisher syndrome is a rare
variant of Guillian-Barré syndrom, characterized by ophthalmoplegia, ataxia and
areflexia. The cause is believed to be inflammatory and the prognosis is good.72, 73
From an ophthalmologist’s point of view, the investigation of the molecular basis
of the unique phenotype of the EOMs is expected to provide further understanding
of their properties and behavior in disease and thereby open the opportunity for
new therapeutical strategies.
18
Aims of the study
The aims of the present investigation were to characterize the human EOMs with
respect to fundamental contractile and structural proteins, in particular:
•
to investigate the MyHC composition of the fibers and to determine
whether the previous fiber type classifications correlate to the MyHC
composition
•
to investigate the SERCA composition of the fibers and to determine
whether there is a correlation between the MyHC and SERCA isoform
content
•
to investigate the MyBP-C composition of the fibers and to determine
whether there is a coordinated expression of MyHC and MyBP-C
•
to investigate the composition of the basement membranes with respect to
laminin chains and to compare them with those of human limb muscle
19
Materials and methods
Human EOMs (paper I-IV) and also the levator palpebrae (paper I and III) were
collected at autopsy or following enucleation. Samples were also taken from
biceps brachii, first lumbrical, quadriceps femoris, psoas and heart muscle for
comparison. All samples were collected from subjects with no previously known
neuromuscular disease. The study followed the Swedish Transplantation Law and
had the approval of the Medical Ethical Committee, Umeå University. In some
cases the anterior part of the EOMs was not available since it had been removed
together with the eye for donation purposes. The samples were mounted on
cardboard, rapidly frozen in propane chilled with liquid nitrogen and stored at 80 °C until used.
Serial cross or longitudinal sections, 5-10 µm thick, were cut from each sample in
a cryostat (Reichart-Jung, Leica, Heidelberg, Germany).
Histochemistry
Ten µm-thick sections were treated to reveal ATPase activity after pre-incubation
at pH 4.3, pH 4.6 and pH 10.474 or stained for nicotinamide tetrazolium reductase
(NADH-TR)7 (paper I). Neuromuscular junctions were detected histochemically
using the acetylcholinesterase reaction75 (paper IV).
Immunocytochemistry
Five to 7 µm-thick sections were processed for immunocytochemistry,29 with a
battery of well-characterized MAbs, each recognizing distinct MyHC isoforms
(paper I, II and III), SERCA1 and SERCA2 (paper II), MyBP-C isoforms (paper
III) and laminin chain isoforms (paper IV). A MAb against tenascin-C (Tn-C) was
used to detect the MTJs (paper IV). For details see table 1.
Control sections were processed as above, except that the primary antibody was
omitted. No staining was observed in the control sections.
The sections were photographed with a CCD camera (Dage-MTI, Michigan City,
Indiana, USA) connected to a Zeiss microscope (Oberkochen, Germany) or with a
Spot camera (RT color; Diagnostic Instruments, USA) connected to a Nikon
microscope (Eclipse, E800, Tokyo, Japan).
20
Fiber typing (paper I, II, III)
Two to eight areas that were recognizable in all serial sections processed with the
different MAbs used were studied in detail to determine the fiber composition. The
choice was not completely random because the EOM fibers are so loosely
arranged that they are difficult to follow even in consecutive series of sections.
Therefore the areas chosen were limited to those where fiber identification was
possible in a long series of sections. In spite of this, care was taken to pick areas
representative of the orbital and global layer. The MyHC composition of over
4000 fibers (paper I) and the SERCA composition of 1571 fibers (paper II) were
analyzed in detail.
Fiber area (paper I)
The fiber area was measured on sections from rectus superior, obliquus superior
and levator palpebrae, stained with MAb 4C7 against laminin 5 chain,76 which
delineated the contours of the muscle fibers. The area of a total of 885 fibers was
measured using an image analysis system (IBAS, Kontron elektronik GMBH,
Eching, Germany).
Biochemistry (paper I, II and III)
Paper I: Whole muscle extracts were prepared from frozen samples of adult
EOMs, cardiac and limb muscle, as well as fetal limb muscle. SDS-PAGE was
performed77 in a Mini Protean II unit (Bio-Rad, Glattbrug, Switzerland) at 75 V for
22 hours, with the lower two thirds of the gel unit surrounded by a 7°C water bath.
The gels were silver stained78 and photographed.
Immunoblotting (WesternBreezeTM Kit) was used to further establish the identity
of the MyHC bands separated by SDS-PAGE. After SDS-PAGE, proteins were
transferred to 0.45µm nitrocellulose membrane (Bio-Rad Laboratories) for 17
hours at 30 V with the unit surrounded by a 15°C water bath. Monoclonal
antibodies A4.840, 4A6, A4.74 and 2B6 were used to identify the bands
containing MyHCI, MyHCeom, MyHCIIa and MyHCemb respectively.
Paper II: A crude microsomal fraction isolated from 11 individual EOM samples
that were combined was used for immunoblot analysis.79, 80 Protein concentration
was determined using bovine serum albumin as standard.81 A gel electrophoretic
separation of EOM proteins was performed and the proteins were transferred to
nitrocellulose sheets.82 The sheets were processed83 with primary antibodies IID884
with affinity to the slow SERCA2 isoform and IIH1184 with affinity to the fast
SERCA1 isoform.
Immunodecoration was evaluated by the enhanced chemiluminescence
technique.85 Densitometric scanning of enhanced chemiluminescence blots was
21
performed on a Molecular Dynamics 300S computing densitometer (Sunnyvale,
CA) with ImageQuant V3.0 software.86
Paper III: Whole muscle extracts were prepared from frozen samples of adult
EOMs, levator palpebrae and limb muscle.87 The acrylamide concentration in the
stacking and running gels were 4 and 8% (w/v), respectively, and the gel matrix
included 10% glycerol.50 The separating gels (160x180x0.75 mm) were
silverstained and scanned with a soft laser.
Capillaries (paper IV)
The number of capillaries was determined in representative areas of the orbital and
global layer in sections from 5 EOM samples, stained with the antibody
recognizing laminin 5 chain. All vessels with an outer diameter less than 15 µm
were assumed to be capillaries according to the definition put forward by
Jerusalem.88 The capillary density was calculated for both the global and the
orbital layer in all 5 muscles. A total of 4861 capillaries were counted.
Statistics
Statistical analyses were conducted on both the fiber size and capillary density
data. The fiber size was compared between the EOM and LP samples and between
the orbital and global layer samples. The capillary density was compared between
the orbital and the global layer samples. Unpaired t-test and one-way analysis of
variance (ANOVA) were performed using the StatView 5.0.1 software (Abacus
Concepts, Berceley, California, USA). A probability of rejecting the null
hypothesis of less than 0.05 was considered a statistically significant difference.
Data is presented as means +/- standard deviations.
22
Results
Biochemistry
Electrophoresis of whole muscle extracts of the sampled EOMs and LP revealed
four MyHC bands identified as: 1) MyHCI, 2) MyHCeom, 3) MyHCIIa and 4)
MyHCembryonic (paper I, fig 1).
SDS-PAGE and immunoblots of a microsomal fraction isolated from human
EOMs (paper II, fig 4) revealed that the slow isoform SERCA2 was significantly
more abundant than the fast isoform SERCA1 (p< 0.00181).
SDS-PAGE failed to reveal either MyBP-Cfast or MyBP-Cslow in the EOMs.
However, faint protein bands were observed in the EOMs and the LP, but not in
limb muscle (paper III, fig 1). These bands were seen above MyBP-Cslow and in a
position similar to one of the degradation products of the purified MyBP-C.
Morphology
The fibers in the EOMs were small, round in shape and loosely arranged in
fascicles. Two different layers of fibers could be identified. The orbital layer was 5
to 30 fibers deep. The orbital layer covered at least one side of the four recti and
the superior oblique muscle, sometimes it completely surrounded the global layer.
No marginal zone could be identified. In the LP no orbital/global layers were
discerned.
The size of the fibers varied greatly (table 2). Fibers in the orbital layer (260+/160 µm2) were significantly (p<0.05) smaller than fibers in the global layer
(440+/-200 µm2). The fibers in the LP were significantly (p< 0.05) larger (470+/320 µm2) than those in the EOMs (340+/-200 µm2).
Enzyme histochemistry
In accordance with a previous study3 we identified the multiply innervated fibers
of both layers as the fibers with strong mATPase activity following pre-incubation
at pH 4.3 and moderate mATPase activity at pH 10.4. Similarly, the remaining
fibers with high ATPase activity at pH 10.4 and low activity at pH 4.3, were
assumed to be singly innervated.3 The NADH activity of the fibers in the EOMs
was generally high and it was more homogeneous in the orbital than in the global
layer. The multiply innervated fibers tended to have lower NADH activity in both
layers. Classification of the fibers into further, clearly defined subgroups based on
23
the ATPase or NADH activity was impossible due to variation of the level of
staining in a continuum (paper I, fig 3).
Immunocytochemistry
The EOM fibers displayed important heterogeneity in immunostaining intensity
with the different antibodies used.
MyHC: Three fiber groups could be distinguished on basis of their pattern of
immunoreactivity (paper I, fig 4-6). The majority of the fibers stained strongly
with anti-MyHCIIa, a smaller group of fibers were labeled with anti-MyHCI and
the remaining fibers were not stained with either of these two antibodies, but were
reactive to anti-MyHCeom and anti-MyHCI+IIa+eom. These fibers were named
MyHCeompos/MyHCIIaneg-fibers. The MyHCeompos/MyHCIIaneg-fibers were
generally larger than the other
fibers. The MyHCIIa positive
fibers were evenly distributed in
the orbital layer, but in the global
layer there were large areas
devoid of this fiber type. These
areas were instead filled with
MyHCeompos/MyHCIIaneg-fibers.
The MyHCI positive fibers were
evenly distributed in both layers
and corresponded to the fibers
with strong mATPase activity
following pre-incubation at pH
4.3 and moderate mATPase
activity at pH 10.4 (presumably
multiply innervated).
Figure 7: Two series of serial
sections, from the anterior (left
column) and midbelly part (right
column) of the same rectus medialis
muscle immunostained with antiSERCA1 (first row), anti-SERCA2
(second row), anti-MyHCIIa (third
row), anti-MyHCI (fourth row) and
anti-MyHCIIa+I+eom (fifth row). OL
indicates orbital layer and GL global
layer.
(from paper II)
24
Practically all fibers contained more than one MyHC isoform. A summary of the
staining patterns, layer distribution, presumed innervation type and size of the
EOM fibers is given in table 2.
No fibers in the LP stained with anti-MyHCsto and less than 1% of them stained
with anti-MyHCemb.
SERCA: In the orbital layer, anti-SERCA1 and anti-SERCA2 stained almost all
fibers (paper II fig 1 and 2) and in the global layer only 37% of the fibers did not
contain both SERCA1 and SERCA2. A summary of the staining patterns observed
is presented in table 3.
The distribution of MyHCIIa varied along the length of the EOMs whereas no
difference was evident in the staining patterns of anti-SERCA1 and anti-SERCA2
(fig 7 and paper I, fig 4).
MyBP-C: Anti-MyBP-Cfast did not label any of the fibers in 11 of 13 EOM
samples immunostained with this MAb (paper III, fig 3, 4, 5). However, in two
samples from the proximal
part of one rectus superior
and one rectus inferior
muscle taken from the oldest
subjects (82 and 86 years
respectively), there were a
few weakly to moderately
stained fibers, mostly located
in the periphery of the
muscles (paper III, fig 6).
These fibers were MyHCIIa
positive fibers or
MyHCeompos/MyHCIIanegfibers.
Anti-MyBP-Cslow stained all
fibers: the slow fibers strongly,
the fast fibers moderately to
strongly and the
MyHCeompos/MyHCIIanegfibers lightly to strongly (fig
8 and paper III, fig 4, 5). No
correlation was found
between the staining pattern
of these two MAb and the
presence or absence of
MyHCemb in the fibers.
Figure 8: Photomicrograph of sections from a rectus inferior
muscle immunostained with A) anti-MyBP-Cslow, B) antiMyBP-Cfast, C) anti-MyHCI, D) anti-MyHCI+IIa+eom, E)
anti-MyHCsto, F) anti-MyHCIIa and G) anti-MyHC-cardiac.
(from paper III)
25
In the levator palpebrae anti-MyBP-Cfast stained some of the MyHCIIapos fibers
and some of the MyHCeompos/MyHCIIaneg-fibers heterogeneously. The fibers
containing MyHCI were unstained with anti-MyBP-Cfast. Anti-MyBP-Cslow
stained all fibers in the levator palpebrae strongly (paper III, fig 7).
Laminin chains: Anti-Ln1 did not show immunoreactivity to any tissue
structure in the sampled sections (paper IV, fig 1). Anti-Ln3 immunostained the
blood vessels only (paper IV, fig 1). The extrasynaptical BM of adult human EOM
muscle fibers contained Ln2, 1, 2, 1, and to some extent also Ln4 and 5
(fig 7 and paper IV, fig 1, 2, 5). Ln2 was also detected in the BM of adult limb
muscle (paper IV, fig 5). At the NMJs there was increased expression of Ln4,
and also maintained expression of Ln2, 5, 1, 2 and 1 (paper IV, fig 2,3).
The MTJs contained Ln2, 5, 1, 2 and 1 as described for skeletal muscle
(paper IV, fig 4). MAb against Ln2, 4, 5, 1, 2 and 1 stained the
perineurium. The endoneurium stained strongly with all these antibodies except
anti-Ln5 that only immunostained the endoneurium moderately (paper IV, fig 1).
The MAb against Lutheran glycoprotein immunostained blood vessels and
perineurium in both EOMs and limb muscle, but the contours of the fibers were
labeled only in the EOMs (paper III, fig 7).
The capillary density in the EOMs, determined in sections processed with antiLn5, was 1050 +/-190 capillaries/mm2 (paper IV, figure 5). The capillary density
was significantly (p<0.05) higher in the orbital layer (1170 +/-180
capillaries/mm2) than in the global layer (930 +/-110 capillaries/mm2).
Figure 9: Photomicrograph of a rectus lateralis muscle (left) and a lumbricalis muscle (right)
from the same subject mounted and sectioned together, immunostained with anti-Ln4. The first
lumbrical muscle (L), the global layer (GL) and the orbital layer (OL) of the EOM are indicated.
(from paper IV)
26
Tables
Table 1
Data on the antibodies used for immunocytochemistry
Antibody
Specificity
Short name
Reference
A4.74
MyHCIIa
Anti-MyHCIIa
89, 90
A4.840
MyHCI
Anti- MyHCI
90, 91
A4.951
MyHCI
Anti- MyHCI
91, 92
ALD19
MyHCsto
Anti-MyHCsto
93, 94
F88
MyHC-cardiac
Anti-MyHC-cardiac
95
4A6
MyHCextraocular
Anti-MyHCeom
29, 96, 97
N2.261
MyHCI
MyHCIIa
MyHCeom
MyHC-cardiac
Anti-MyHCI+IIa+eom
90, 91
2B6
MyHCembryonic
Anti-MyHCemb
90, 94, 98
NCL-SERCA1 SERCA1
Anti-SERCA1
84
NCL-SERCA2 SERCA2
Anti-SERCA2
99
BB146
MyBP-Cslow
Anti-MyBP-Cslow
48
BB88
MyBP-Cfast
Anti-MyBP-Cfast
48
163DE4
Laminin 1-chain
Anti-Ln1
62
5H2
Laminin 2-chain
Anti-Ln2
100
BM-2
Laminin 3-chain
Anti-Ln3
76
FC10
Laminin 4 chain
Anti-Ln4
101
4C7
Laminin 5-chain
Anti-Ln5
102
DG10
Laminin 1-chain
Anti-Ln1
103
C4
Laminin 2-chain
Anti-Ln2
104
113BC7
Laminin 1chain
Anti-Ln1
105
4A10
Tenascin-C
Anti-tenascin
106
MCA-1982
Lutheran glycoprotein Anti-Lu
107
27
Table 2. Staining pattern, distribution, innervation and size of fibers in human EOMs
Fiber type:
pos
MyHCIIa
pos
MyHCI
MyHCeom
pos
/MyHCIIa
Staining:
Anti-MyHCI+IIa+eom
+
+
+
Anti-MyHCIIa
+
-
-
Anti-MyHCI
-
+
-
+/-
+/-
+
Anti-MyHCsto
-
+*
-
Anti-MyHC-cardiac
-
+/-
-
+/-
+/-
+/-
Orbital layer
80%
17%
3%
Global layer
60%
15%
25%
Presumed type of innervation:
Single
Multiple
Single
Predominant SERCA staining:
SERCA 1
SERCA 2
SERCA 1 and 2**
Anti-MyHCeom
Anti-MyHCemb
Distribution:
Size:
320±190µm2 280±140µm2
590±210µm2
* Very few (< 1%) slow fibers were unstained with anti-MyHCsto
** SERCA 1 in the global layer and SERCA 1 and 2 in the orbital layer
28
neg
Table 3
Patterns of staining with MAbs against SERCA1 and 2 observed in the
three major groups of fibers
GLOBAL LAYER
Staining intensity with MAb
against
Number of sampled fibers in each group according
to MyHC content
IIa
neg
pos
SERCA1
SERCA2
IIa
I
/eom
-
-
1
2
0
++
-
161
0
54
-
++
0
35
0
+
+
35
1
22
++
+
267
2
24
+
++
1
42
0
++
++
40
0
0
505
82
100
ORBITAL LAYER
Staining intensity with MAb
against
Number of sampled fibers in each group according
to MyHC content
IIa
neg
pos
SERCA1
SERCA2
IIa
I
/eom
-
-
0
0
0
++
-
6
0
0
-
++
0
0
0
+
+
0
1
0
++
+
559
1
0
+
++
0
134
0
++
++
137
42
4
702
178
4
29
Table 4. Feature comparison between human EOMs, LP and limb muscle
EOM
Levator
Limb
Layers
orbital, global
no
no
Innervation
single, multiple
single
single
Fiber size
small
intermediate
large
ECM
abundant
abundant
sparse
Capillary density
very high
-
“normal”
Fiber form
round
round
“polygonal”
Fiber types
multiple
multiple
I, IIA, IIB
MyHC isoforms present
I, sto, -cardiac, IIa,
IIb, eom, emb, fetal
I, -cardiac, IIa, IIb,
eom, emb, fetal
I, IIa, IIx
Number of MyHC
isoforms in a single fiber
up to 5
up to 4
1 to 2
MyHC isoform variation
along the fiber length
yes
-
no
Number of SERCA
isoforms in a single fiber
0-2
-
1
Presence of MyBP-Cfast
trace amounts in
part of the muscle
in some fast fibers
in all fast
fibers
Ln chains in
extrasynaptic BM
2, 4, 5, 1, 2,
1
-
2, 1, 2, 1
Ln chains at NMJs
2, 4, 5, 1, 2,
1
-
2, 4, 5, 1,
2, 1
Ln chains at MTJs
2, 5, 1, 2, 1
-
2, 5, 1, 2,
1
Lu present in BM of
muscle fiber
yes
-
no
- not examined
30
Discussion
The present study showed that the human EOMs have a very complex fiber type
composition with respect to the major proteins regulating fiber contraction velocity
and force (MyHCs, MyBP-Cs) and fiber relaxation rate (SERCAs). There were
also important differences in laminin isoform composition and capillary density
when compared to human limb muscle.
Validity
MyHCI, MyHCeom, MyHCIIa and MyHCemb as well as SERCA1 and 2 were
detected in the EOMs with immunocytochemistry, SDS-PAGE and immunoblots.
All MyHC and SERCA antibodies used are well characterized and have been
extensively used.29, 86, 90, 94, 108-110 The specificity of the MyHC MAbs for human
muscle has been well verified with immunoblots90 and with
immunocytochemistry.29, 90, 94 Therefore we can be sure that the complex patterns
of reactivity obtained with immunocytochemistry reflect differences in the
SERCA and MyHC composition of each myofiber.
The MAbs against MyBP-C were raised against purified human C-protein and we
established that they do not cross-react with cMyBP-C (paper III). Further
confirmation of their specificity with immunoblots is planned.
The MAbs against laminin chain isoforms,62, 76, 100-105 Lutheran glycoprotein107 and
tenascin-C106 are also well characterized and have been widely used both in
humans and other species.
EOM fiber type composition
ATPase activity: The mATPase activity of the fibers varied in a continuum from
low to high. This is consistent with the fact that most of the human EOM fibers
examined contained more than one MyHC isoform. The mATPase reaction does
not allow the discrimination of mixtures of MyHCs, particularly when both fast
and developmental isoforms are present.24 The mATPase activity resides in the
head region of the MyHC molecule, and therefore data on the MyHC composition
have more relevance and provide more information on the expected contractile
properties of the fibers.
MyHC: The human EOMs could be divided into three major fiber groups taking
into account their MyHC content: 1) slow fibers containing MyHCI, 2) fast fibers
containing MyHCIIa, 3) fast fibers containing MyHCeom, but lacking both
MyHCIIa and MyHCI (fig 10). Most slow fibers contained both MyHCI and
31
Figure 10: Three major fiber groups of human EOMs, containing 1. MyHCI (I), 2. MyHCIIa
(IIa) and 3. MyHCeom but neither MyHCI nor IIa (eom). Additional isoforms of MyHC and/or
SERCA add to the number of possible isoform combinations. All together 88 subtypes of fibers
are possible.
MyHCsto and some of them contained also MyHC-cardiac, another slow
isoform.
MyHCeom was additionally present in subgroups of the fibers defined by their
content of MyHCI or IIa and, finally, MyHCemb was also found in subgroups of
all three major fiber types. Altogether, there were 22 distinct MyHC combinations
observed in the human EOMs. Furthermore, differences in immunostaining
intensity clearly showed that fibers with a given combination of MyHCs differed
from each other in their relative amounts of the MyHC isoforms. In summary, the
MyHC composition of the human EOM fibers varies in a continuum and is very
complex.
Previous data on human EOMs have shown that fibers containing MyHCsto
correspond to the orbital and global multiply innervated fiber groups.3 The fibers
containing MyHCI and MyHCsto represented approximately 16% of the total in
the orbital layer and 13% in the global layer, consistent with values reported for
the rat multiply innervated fibers.2 Another important finding regarding the
heterogeneity of the fibers containing slow MyHC isoforms was the identification
32
of fibers containing MyHCI but not MyHCsto. We speculate that these fibers are
likely to be singly innervated.
The genes for human MyHCIIb and MyHCeom have been sequenced20 and RNA
transcripts of MyHCIIb have been detected111 in human EOM samples. The
corresponding MyHCs are therefore expected to be present in the EOMs. In
addition, immunocytochemical data indicate the presence of MyHCsto in these
muscles. To date there are no antibodies specific to human MyHCIIb available and
it has not been established yet whether the SDS-PAGE band called MyHCeom
really corresponds to the product of the MYH13 (MyHCeom) gene or whether it
corresponds to the MYH4 (MyHCIIb) gene or both. We follow the previous
nomenclature tentatively identifying the unique band in SDS-PAGE as
MyHCeom.112, 113
MyHCIIx can be identified in human muscle fibers by immunocytochemistry,
using a MAb that labels all MyHCs except MyHCIIx. Because practically all
fibers in the EOMs co-expressed more than one MyHC isoform, this antibody is of
no use.
SERCA: The majority, but not all of the fibers contained both SERCA isoforms,
which is a unique feature of the EOMs. There were clear differences in the relative
amounts of SERCA1 and 2 among the fibers, indicating that also the amounts of
the major Ca2+-pump protein isoforms in the fibers of the EOMs vary in a
continuum.
A small number of fibers were devoid of reactivity to the MAb against SERCA1
and 2. These fibers were very few and may contain yet another isoform of
SERCA, perhaps specific for the EOMs. There are at least seven known SERCA
isoforms in human, generated by alternative splicing38 and the existence of
additional isoforms remains to be explored.
MyBP-C: The MyBP-C isoform composition of the EOM fibers, investigated with
MAbs against MyBP-Cfast and slow, was not coordinated with the MyHC
composition. No immunoreaction with anti-MyBP-Cfast was seen in the vast
majority of the samples and both the slow and fast MyBP-C bands were absent in
whole muscle extract electrophoresis.
The lack of immunoreaction to anti-MyBP-Cfast in most of the EOMs, and the
finding of unidentified bands in EOM whole muscle extracts investigated with
SDS-PAGE, suggest the presence of new isoforms of MyBP-C in the EOMs.
Although only MyBP-Cfast, slow, cardiac and intermediate have been identified in
human skeletal muscle, multiple isoforms of MyBP-C are present in chicken
muscle.114
Longitudinal variation: The MyHC composition was not constant along the
length of the EOMs. Our material did not allow us to follow the same fibers along
their entire length but it clearly showed that the distribution of MyHCIIa varied
33
along the muscle length in both the global and the orbital layer, as previously
described in rabbit.35 Furthermore, MyBP-Cfast was only found in a few fibers in
the proximal part of two muscles. In contrast, the distribution of SERCA isoforms
was fairly constant throughout the length of the sampled muscles.
Extracellular matrix
The ECM of the EOMs accounts for a substantial part of the muscle area and is
very rich in blood vessels and nerves. Our study showed that the capillary density
of the EOMs was higher than that of any previously examined human muscle,52
which is in agreement with their outstanding physiological demands, high
oxidative enzyme activity3 and higher overall vascular density of the orbital
layer.115
The distinct character of the human EOMs was also apparent in the laminin
composition of the extrasynaptic BM of the fibers. The presence of Ln4, Ln5
and Ln2 in extrasynaptic BM suggests that the BM of the EOMs contain more Ln
isoforms than the BM of limb muscle. This is interesting since laminin is not only
an important structural protein, but it is also highly involved in the signaling
system between the ECM, cell surface receptors and the cytoskeleton of the
muscle fibers.116, 117 The presence of several laminin isoforms based on different
alpha chains may in part explain the fact that the EOMs are spared in muscular
dystrophies such as merosin deficient CMD and DMD, perhaps by making the BM
structurally more stable, but also by providing alternative signal paths between the
ECM and the cells. The fact that Lutheran glycoprotein, a Ln5 receptor, was
found on the cell surface of the EOMs sustains this latter possibility.
Levator palpebrae
The LP had many features in common with the true EOMs, e.g. loosely
arranged fibers, presence of MyHC-cardiac, MyHCemb and MyHCeom,
absence of MyBP-Cfast and slow bands and presence of two novel bands in
whole muscle extracts studied with SDS-PAGE. On the other hand several
findings distinguished the LP from the EOMs, e.g. larger fiber size, lack of
organization into layers, lack of MyHCsto, presence of MyBP-Cfast in all
samples examined (table 4). These findings place the phenotype of the LP as
intermediate between that of the EOMs and the limb muscles. The functional
demands on the levator palpebrae muscle are high and its importance for
survival is as obvious as for the EOMs. However, the nature and range of
movements of the levator palpebrae are far less complex than those of the
EOMs.
34
Human EOM versus EOM of other species
Clear distinctions in MyHC isoform content between the orbital and global layers
were not seen in our material, although they have been reported in the rat.28
Notably, MyHCeom28, 30 and MyHC-cardiac28 are restricted to the orbital layer in
the rat EOMs whereas we found MyHCeom widely distributed in both layers.
MyHCemb and -cardiac were more abundant in, though not restricted to, the
orbital layer of human EOMs. MyHCemb is also found in both layers of rabbit
EOMs.35
A correspondence between the MyHC content and six putative fiber types based
on color, location and innervation, was not present in human EOMs. In our
material the vast majority of the fibers contained two or more MyHC isoforms and
a distinction into six fiber groups based on the histochemical and
immunohistochemical staining was impossible. In the rat EOMs,28 the global
multiply innervated fibers are proposed to contain mainly MyHCI whereas the
singly innervated fibers would presumably contain either predominantly MyHCIIa
(red fibers), IIx (intermediate fibers) or IIb (white fibers). The orbital fibers are
proposed to contain predominantly embryonic MyHC and either MyHCeom
(singly innervated fibers) or MyHCI (multiply innervated fibers) in the middle
region.28
Rubinstein and Hoh28 reported that the rat orbital singly innervated fibers only
contained MyHCemb in the distal and proximal portions of the EOM. Jacoby,40 on
the other hand, reported that both multiply and singly innervated fibers in the rat
orbital layer contain fast MyHC near the endplate band and MyHCemb at the ends.
In the human EOMs sampled, anti-MyHCemb never stained more than two thirds
of the orbital singly innervated fibers, and all of these fibers were also stained by
anti-MyHCIIa and/or anti-MyHCeom.
Our results show that, in human EOMs, most fibers contain both SERCA
isoforms, clearly distinguishing them from the EOMs of rat and rabbit, where
SERCA1 is co-expressed with fast MyHC.40 Data on the distribution of SERCA2
are missing in other species. SERCA2 is typically found in slow-twitch skeletal
muscle fibers and cardiac muscle fibers.118
These results indicate that caution is needed when extrapolating data from animal
models to the human EOMs.
The present work on MyBP-C and laminins are, to the best of our knowledge, the
first to address the EOMs, with the exception that Ln2 has been detected in NMJs
of mouse EOM.119 Therefore there are no data for comparison in other species.
35
Human EOM versus human limb muscle
In the EOMs we found mixtures of up to five different MyHC isoforms in a single
fiber according to their immunocytochemical reaction. A single fiber could e.g.
stain with anti-MyHCI, sto, -cardiac, eom and emb. Human limb muscle fibers
typically contain one or at most two MyHC isoforms: MyHCI, I+IIa, IIa, IIa+IIx
or IIx.49 Normal extrafusal fibers in human limb muscle do not contain MyHCeom,
embryonic or -cardiac.
Most EOM fibers contained both SERCA1 and 2, a feature only observed in
chronically stimulated limb fast twitch fibers, in a dog model.120 The presence of
more than one SERCA isoform in the same cell has been speculated to reflect the
existence of segregated calcium stores, each with its own particular role and
loaded by a particular calcium pump.38 Further investigations will be needed to test
this hypothesis in muscle cells.
Some EOM fibers were unstained by both SERCA markers, hitherto not reported
in human limb muscle.
The absence of MyBP-Cfast and the possibility for additional isoforms other than
MyBP-Cfast and slow clearly further separate the EOMs from limb muscle where
the correlation between MyBP-Cfast and MyHCIIx, MyBP-Cintermediate and
MyHCIIa, MyBP-Cslow and MyHCI is established.49
We found Ln4 and Ln5 at the NMJs and extrasynaptically in the EOMs. Ln4
has not been detected in adult human limb muscle BM, although it is present
during development.101 The detection of Lutheran glycoprotein, a Ln5 receptor,
on the fiber surface in EOMs, a feature not seen in limb muscle, confirms the
presence of Ln5 in extrasynaptical BM of the EOMs. These findings imply that
the extrasynaptical BM of the EOMs might also contain laminin-8 (411), -9
(421), -10 (511) and/or -11 (521) in addition to the laminins found in
extrasynaptical BM of human limb muscle, laminin-2 (211) and 4 (221).
Altogether our data further establish the distinctness of the EOMs in comparison to
limb muscle, with respect to contractile, Ca2+ transport and structural proteins as
well as capillary supply, as summarized in table 4 on page 30.
Complexity and function
This study emphasizes the fact that the EOMs are indeed among the most complex
muscles in the body, with more features in common with other muscles innervated
by cranial nerves (e.g. laryngeal and masticatory muscles) than limb muscle. The
muscles of the vocal folds contain MyHCeom,96 MyHCsto and multiply innervated
fibers.121 The single fibers of the masseter contain up to 5 different MyHC
isoforms including fetal, embryonic and -cardiac.4, 49
36
The structural and molecular complexity of the EOMs shown here, could in part be
explained by the physiological demands put on these muscles. For example, the
slow fibers containing MyHCsto are likely to enable slow and sustained
contraction with modest consumption of energy, allowing the EOMs to perform
fixation and slow pursuit movements. Small differences in the MyHC (e.g. coexpression of MyHCI+sto or MyHCI+sto+-cardiac or MyHCI+sto+cardiac+emb in varying proportions) and SERCA (SERCA-2 only or 1+2)
composition among the slow fiber group provide a wider range of contraction
velocity and force as well as of relaxation rates that can be speculated to allow
altogether more versatile muscle activity than it would be the case if all slow fibers
had the same composition. The same holds for the fast fibers containing the very
fast MyHCeom and/or MyHCIIb, which are probably involved in the very fast and
exact movements, e.g. saccades.
But what really makes the EOMs unique is the distinct molecular profile of almost
each individual muscle fiber. This heterogeneity probably reflects the very small
size of the motor units in the eye muscles and a very intricate functional
adaptation. In fact it may even represent an ultimate filter for fine motor control in
a system that performs so very diverse and coordinated types of movement.
The simultaneous presence of multiple isoforms of a given protein represents a
very advantageous evolutionary adaptation which a) allows a wider range of
functional capacity, e.g. of contraction and relaxation parameters; b) makes the
EOMs less vulnerable to defects/changes involving a particular isoform, as the
additional isoforms can compensate for the defective one.
Future directions
The unique character of the EOMs in rodents has recently been confirmed at the
gene level with both micro-array and SAGE techniques,122-124 particularly with
respect to metabolic pathways, structural components and regeneration markers.
However, these studies rely on whole muscle RNA extractions and therefore the
potential transcript sources include the muscle fibers, satellite cells, fibroblasts,
different types of nerve and blood vessel cells along with circulating cells. In order
to further assess the molecular basis of the unique human EOM allotype, we will
in the future need to combine differential gene expression data with solid
morphological and biochemical techniques to be able to assign the correct
molecular portfolio to the different cell types. Basic knowledge on the structure
and composition of the EOMs provides the platform for understanding their
unique functional properties and may provide clues to protection and susceptibility
mechanisms that ultimately can be used for new therapeutic approaches.
37
Conclusions
The present thesis further advances our knowledge of the distinct character of the
human EOMs and consolidates the concept that they are a separate skeletal muscle
allotype.
The EOMs differ from the limb muscles in basic features involving muscle
morphology, cell types and composition of four fundamentally important proteins.
In particular, the EOMs differ from the limb muscles in:
•
•
•
•
•
•
organization into orbital and global layers, fiber form and size, amount of
connective tissue and capillary supply
fiber type composition
MyHC composition, regarding the number and type of isoforms present,
co-existence of multiple isoforms in a single fiber and variation along the
length of the muscle
SERCA composition, with co-existence of both isoforms in a single fiber
MyBP-C composition, with lack of MyBP-Cfast and possibly with
presence of novel isoforms
Extrasynaptic basement membrane composition, with respect to laminin
chains and Ln5 receptor
The EOMs differed from the levator palpebrae, which showed a phenotype
somewhat intermediate between that of the EOMs and the limb muscles.
Previous classifications into six fiber types in mammals do not reflect the
heterogeneity seen in human EOMs and are not supported by data on the MyHC
composition of the fibers.
There are important differences between the human EOMs and those of other
species and therefore caution is needed when extrapolating data from animal
models.
The hallmark of the human EOMs was a remarkable heterogeneity in the staining
levels seen with most antibodies against the contractile and Ca2+-pump proteins,
indicating the presence of a wide continuum in the amounts of a given isoform.
The presence of additional laminin isoforms in the extrasynaptic basement
membrane of the EOMs could partly explain the selective sparing of these muscles
in Ln2 deficient congenital muscle dystrophy.
The co-existence of complex mixtures of several crucial protein isoforms provides
the human EOMs with a unique molecular portfolio that a) allows a highly specific
fine-tuning regime of contraction and relaxation, and b) imparts structural
properties that are likely to contribute to protection against certain neuromuscular
diseases.
38
Acknowledgments
After the service, one hot Sunday in June, the parish clerk, whose face was pretty
red, after having blown the organ’s bellow for over an hour, turned to the organist
and said: “That was a nice postlude we played today!” The organist snorted, closed
his briefcase with the sheets of music and replied dryly: “We played? May I
remind you that I did the actual playing. I am an artist and you are only the
provider of air.”
Next Sunday, the organist had prepared a mighty prelude by J.S. Bach, since the
bishop was present to ordain a new priest. The organist chose the tunes carefully,
closed his eyes in contemplation and started to play, but the organ remained silent.
The organist tried to change the tunes and started all over again, but nothing
happened. Soon all the parish members and the bishop were looking at the
desperate organist, struggling with his instrument. Suddenly the embarrassing
silence was broken by the voice of the parish clerk from behind the organ:
“Who is playing now, you schmuck?!”
To avoid similar episodes in my future work I think it is fair to give credit to all
the wonderful persons who have contributed to this thesis.
First of all I thank the Lord, for creating the extraocular muscles in such a fantastic
way that it was possible to write a thesis about them.
I am also grateful for having had the opportunity to do my work at the Department
of Integrative Medical Biology, section for Anatomy. The attitude of the
department is inspiring, creative and forgiving. All people there have been equally
helpful, but some people have been more equal than others:
I would like to thank my fantastic supervisor docent Fatima Pedrosa-Domellöf, for
invaluable help and support, throughout this study, and for showing me her secret
depot of chocolates (bottom left drawer of her desk, but don’t tell anyone).
My co-supervisor, professor Lars-Eric Thornell, for encouragement and
constructive criticism, and for setting an example in advanced house improvement.
Margaretha Enerstedt and Anna-Karin Olofsson for excellent technical assistance
and patience.
Per Stål, for help and encouragement and especially for lending me his high tech
computer when mine broke down a month before deadline (I loved the tiger rug).
All people in the “muscle group” not mentioned above who have helped me: Eva
Carlsson, Ji-Gou Yu, Mona Lindström and Lena Carlsson. Tomas Carlsson, Göran
39
Dahlgren and Ulf Ranggård for invaluable computer support. Anna-Lena
Thallander, department secretary. My fellow research students: Jing-Xia, Dina
Radovanovic, Anders Eriksson and last but not least Berit Byström (the
Jukkasjärvi Blizzard).
At the Department of Clinical Sciences, Ophthalmology I would especially thank
my co-supervisor, professor Lillemor Wachtmeister, for invaluable help in
providing grants for this work, Eva Mönestam for giving me time for research in
my busy clinical schedule and Birgit Johansson, the department secretary, for
helping me filling in applications and other necessary (and not so necessary)
papers.
I thank my colleagues at the Eye clininic of the University Hospital of Umeå,
especially my fellow vitreoretinal surgeons, Siv Åström and Mikael Andersson,
who have had to endure a heavy workload, due to my partial absence this spring. I
will be back soon, so fill her up with gas and oil!
I also thank Ismo Virtanen, Dieter O Fürst, Lars Larsson, Jesper Andersen,
Michelle Ryan and Kay Ohlendieck for collaboration on manuscripts.
In the real world I thank the members of MESK, Grotteatern, SHT, the church
choir of Ålidhem and my former floor hockey team for making my sparetime a
rare but pleasant enjoyment.
I am also in gratitude to my mother and father, Catharina and Per Kjellgren, for
lovingly anticipating this ever since I was born. My sister Hanna for setting an
example. My mother and father in law, Barbro and Lars Berglund for practical
help with cars, tractors, lawn movers, orbital sanders and other necessities of life.
Finally and foremost I thank my wonderful family: Åsa - wife extraordinaire, and
my daughter Rebecka - the eighth wonder of the world, for giving my life
happiness and meaning. I am indeed a fortunate husband!
This study was supported by grants from Stiftelsen KMA, the Swedish Society for
Medical Research, the Swedish Research Council and the Medical Faculty of
Umeå University.
40
41
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