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Umeå University Medical Dissertations
From the Department of Clinical Sciences, Ophthalmologyand 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
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Copyright by Daniel Kjellgren, 2004Illustrations by the author
ISBN 91-7305-638-3Printed 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
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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
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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
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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
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Abbreviations
ATPase Adenosine triphosphataseBM Basement membraneCMD Congenital muscular dystrophyEOM Extraocular muscleECM Extracellular matrixGL Global layerLn LamininLP Levator palpebraeLu Lutheran glycoproteinMAb Monoclonal antibodyMIF Multiply innervated fiberMTJ Myotendineous junctionMyBP-C Myosin binding protein CMyBP-Cfast Fast isoform of MyBP-CMyBP-Cslow Slow isoform of MyBP-CcMyBP-C Cardiac isoform of MyBP-CMyHC Myosin heavy chainMyHC -cardiac -cardiac isoform of MyHCMyHCemb Embryonic isoform of MyHCMyHCeom Extraocular isoform of MyHCMyHCI Slow isoform of MyHCMyHCIIa Fast isoform IIa of MyHCMyHCIIb Fast isoform IIb of MyHCMyHCIIx Fast isoform IIx of MyHCMyHCsto Slow tonic isoform of MyHCNMJ Neuromuscular junctionOL Orbital layerSDS-PAGE Sodium dodecyl sulfate polyacrylamide gel
electrophoresisSERCA Sarco(endo)plasmic reticulum Ca2+ATPaseSERCA1 SERCA fast isoformSERCA2 SERCA slow isoformSIF Singly innervated fiberSR Sarcoplasmic reticulumTn-C Tenascin-CT-tubules Transverse tubules
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Abstract
Introduction: The extraocular muscles (EOMs) are highly specialized and differso significantly from other muscles in the body that they are considered aseparate class of skeletal muscle, allotype. A thorough characterization of themolecular and cellular basis of the human EOM allotype has not been presentedalthough it is expected to contribute to a better understanding of the functionalproperties of these muscles and the reasons why they are selectively spared insome neuromuscular disorders.
Myosin is the major contractile protein in muscle and consists of two heavy andfour light chains. The myosin heavy chain (MyHC) isoforms contain both theATPase and the actin binding site and are therefore the best molecular markers offunctional 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 intothe sarcoplasmic reticulum (SR) mostly by SR Ca2+ATPase (SERCA). The majorSERCA isoforms in muscle are SERCA1 (fast) and SERCA2 (slow).
Myosin binding protein C (MyBP-C), the second most abundant thick filamentprotein in striated muscle, plays a physiological role in regulating contraction.There are 3 major isoforms of human MyBP-C: MyBP-Cfast, MyBP-Cslow andcardiac MyBP-C.
The laminins (Ln) are the major non-collagenous components of the basementmembrane (BM) surrounding muscle fibers and are important for muscle fiberintegrity. They are composed of an -, a - and a -chain, all of which exist inmultiple isoforms.
Methods: Adult human EOMs were studied with SDS-PAGE, immunoblots andimmunocytochemistry, 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 densitywere also determined.
Results: Important heterogeneity in fiber composition was the hallmark of thehuman EOMs. Three major groups of fibers could be distinguished. Fast fibersthat stained with anti-MyHCIIa, slow fibers that stained with anti-MyHCI andMyHCeompos/MyHCIIaneg-fibers that stained with neither of these antibodies butwith anti-MyHCI+IIa+eom and anti-MyHCeom. Approximately 62% of thesampled fibers in the global layer and 81% in the orbital layer were MyHCIIapositive fibers and 14% of the fibers in the global layer and 16% in the orbital
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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 orbitaland 7% in the global layer. MyHCeom was also present in some of the MyHCIIapositive fibers and slow fibers. MyHCemb was co-expressed in some of the fibersof all groups. The staining levels of each antibody varied among fibers.
Almost all MyHCIIa positive fibers contained SERCA1 and 86% of them alsocontained SERCA2, whereas <1% were unlabeled with both antibodies. Thirteenpercent of the slow fibers contained SERCA2 only, 86% contained both SERCA1and 2 and 1% were unstained. Biochemically SERCA2 was more abundant thanSERCA1.
MyBP-Cfast was not present in the EOMs and MyBP-Cslow was only detectedimmunocytochemically. Interestingly, two unrecognized bands were seen in theMyBP-C region in SDS-PAGE of whole muscle extracts of EOM but not of limbmuscle.
The extrasynaptical BM of the EOM muscle fibers contained Ln 2, 1, 2, 1,and to some extent also Ln 4 and 5 chains. The capillary density in the EOMswas very high (1050 +/-190 capillaries/mm2) and significantly (p<0.05) higher inthe orbital than in the global layer.
Conclusions: The human EOMs have a very complex fiber type compositionwith respect to the major proteins regulating fiber contraction velocity and force(MyHCs, MyBP-Cs) and fiber relaxation rate (SERCAs). There are alsoimportant differences in laminin isoform composition and capillary density whencompared to human limb muscle. The presence of additional laminin isoformsother than laminin-2 in the extrasynaptic BM, could partly explain the sparing ofthe EOMs in Ln 2 deficient congenital muscle dystrophy.
The co-existence of complex mixtures of several crucial protein isoforms providethe human EOMs with a unique molecular portfolio that a) allows a highlyspecific fine-tuning regime of contraction and relaxation, and b) impartsstructural properties that are likely to contribute to protection against certainneuromuscular diseases.
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Original papers
This thesis is based on the following original papers, which are referred to in thetext by their Roman numerals:
I. Kjellgren D, Thornell L-E, Andersen J, Pedrosa-Domellöf F. Myosin heavychain 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 humanextraocular muscles. Invest Ophthalmol Vis Sci. 2003;44:5057-62.
III. Kjellgren D, Stål P, Larsson L, Fürst D, Pedrosa-Domellöf F Uncoordinatedexpression of myosin heavy chains and myosin binding protein C isoforms inhuman extraocular muscles, Manuscript.
IV. Kjellgren D, Thornell L-E, Virtanen I, Pedrosa-Domellöf F. Laminin isoformsin human extraocular muscles. Submitted.
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Figure 1: Extraocular muscles and
levator palpebrae of the left orbit
Introduction
Vision is by far the most important of our senses for communication with theworld around us. The process of imaging is complex and includes the optics of theeye, the photoreceptors, the optical tract and the occipital cortex. In order tooptimize perception and maintain stereo vision in every gaze position, it isnecessary to perfectly control and co-ordinate the delicate movements of theeyeballs. The eyes are capable of highly specialized movements such as slowpursuit movements as well as rapid, instant change from one point of fixation toanother (saccades). Eye movements are the result of the action of the extraocularmuscles (EOMs).
Anatomy
There are six EOMs (fig 1): themedial, lateral, superior and inferiorrecti, the superior and inferioroblique. The four recti originatefrom the tendinous ring around theoptic canal, run forward in the orbitand insert into the eyeball at themedial, lateral, superior and inferiorside respectively. The obliquussuperior arises from the sphenoidbone just superior to the tendinousring, runs forward to the trochlea, afibrous pulley located at thesuperior and medial angle of theorbital anterior margin. Here itbecomes tendinous and runs backwardsand laterally, passing below the rectussuperior to be inserted into the superiorsurface of the eyeball. The obliquusinferior originates from the orbital floor, just lateral to the lacrimal fossa, runslaterally and posteriorly, between the rectus inferior and the orbit floor and insertsinto the inferior side of the eyeball (fig 1).
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Histology
The fibers of the EOMs are organized into two separate layers. The thin orbitallayer faces the orbital wall. The global layer consists of the central part of themuscle.1 This organization into layers is most clearly seen in the midbelly part ofthe muscles. In the oblique muscles the orbital layer may totally surround theglobal layer.2 A third layer – the marginal zone – covering parts of the outersurface has been described in human EOMs.3
Fiber types
Human skeletal muscle fibers are usually divided into three major types dependingon their physiological, biochemical and histochemical characteristics: 1) slowtwitch, fatigue resistant (type I fibers), 2) fast-twitch, fatigue resistant (type IIAfibers) and 3) fast-twitch fatigable (type IIB fibers). However, this generalcategorization into fiber types does not apply to more specialized muscles4 andespecially not to the EOMs.5 Six fiber types have been described in the EOMs ofseveral species on the basis of fiber location, innervation and color: 1) orbitalmultiply innervated, 2) orbital singly innervated, 3) global multiply innervated, 4)global red singly innervated, 5) global intermediate singly innervated and 6) globalpale singly innervated fibers.6 However, when studying the original papers2, 5, 7 it isclear that the urge to categorize has led to an oversimplification. In particular, theoriginal data show that the human EOMs are far more complex and that theproperties 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 theinnervation of the fibers in the orbital and global layers. The singly innervatedfibers (SIF) have a single synaptic site, or neuromuscular junction (NMJ), in themiddle region of the muscle fiber and have a twitch mode of contraction.9 TheSIFs contain a great amount of mitochondria throughout their length. In the globallayer the red SIFs have more mitochondria and the pale SIFs have fewer.10 Themultiply innervated fibers (MIF) of the global layer have a tonic mode ofcontraction, multiple innervation points along the fiber length and contain onlyfew mitochondria. In contrast, the multiple innervated fibers of the orbital layerhave a twitch mode of contraction and an endplate-like neuromuscular junction inthe midbelly part,1, 6 but multiple innervation points and tonic mode of contractionin the peripheral parts of the fiber. Accordingly, the midbelly part of the orbitalMIFs is also rich in mitochondria, as the SIFs.10 The peripheral parts of the MIFsof the orbital layer and all of the global MIFs contain fewer mitochondria. At thedistal end of the muscle, at the myotendinous junctions (MTJs), the global layer is
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Figure 3: Organisation of the SR and the T-tubules
Figure 2: Role of Ca2+
in contraction and relaxation of
muscle fibers
enclosed by a tangle of nerve endings called palisade endings,11 supposed to have aproprioceptive function.
Physiology
The extraocular muscles(EOMs) are unique inmany ways, as shown bydata collected fromseveral species. TheEOMs are among thefastest muscles in thebody1 and yet they areextremely fatigueresistant,12 as reviewed byPorter.9 The nontwitch
motor units, consisting ofMIFs, respond to stimulationwith slow tonic contractionand are highly fatigueresistant.13 In monkey EOMs, the twitch motor units, consisting of SIFs have beendivided into fast and slow according to their fusion frequency, although thedistribution of this parameter is unimodal without an apparent dividingfrequency.14 When tested for fatigue resistance, the twitch motor units have abimodal distribution of fatigable and fatigue resistant units.14 So depending ontheir physiological properties a system of five different types of EOM motor unitshave been proposed: 1) nontwitch (NT), 2) fast fatigable (FF), 3) fast fatigueresistant (FR), 4) slow fatigable (SF) and 5) slow fatigue resistant (S).15 Acorrelation between this division and the six histochemical fiber types described inthe EOMs (see previous section on fiber types) has not been made.
Muscle contraction
When a muscle fiber isactivated, the action potentialstimulus spreads along thesarcolemma and reaches thedepth of the muscle fiberthrough the transversetubules (t-tubules) (fig 2, 3).The T-tubules form junctionswith the terminal cisterna ofthe sarcoplasmatic reticulum
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Figure 4: Schematic illustration of myosin II
sarcomeric
(SR) and depolarization triggers the release of Ca2+ from the SR into thesarcoplasm. The raised Ca2+ concentration alters the structure of the actin boundproteins troponin and tropomyosin, moving them to the side making it possible forthe head of the myosin heavy chain (MyHC) to bind to actin. Then the myosinfilaments slide along the actin filaments and the muscle fiber contracts (fig 2).
During relaxation, Ca2+ ions are actively pumped from the myofibrilar space intothe lumen of the SR and the Ca2+ gradient between the SR and the cytosol isrestored. This is accomplished mostly by sarco(endo)plasmatic reticulumCa2+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). Bothheavy and light chains exist in multiple isoforms.18, 19 The sarcomeric myosinheavy 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 mannerand their expression isregulated by not fullyunderstood physiological,hormonal and tissuedependent factors. Since theMyHC contains both theATPase and the actin-binding site, it therebydetermines the speed ofcontraction and thecontraction force.18, 21, 22
Hence, the MyHCcomposition is regarded as thebest marker of the functionalheterogeneity among musclefibers.23
The MyHC isoforms can be classified into fast (MyHCIIa, IIx, IIb, eom), slow(MyHCI, -cardiac, slow tonic) and developmental (MyHCembryonic, fetal).24 Inhuman 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 andMyHCI, 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
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fiber types, in the rat, and the predominant expression of a given MyHC hasrecently been suggested, although not really investigated.28 In addition, the MyHCisoform 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 physiologicalproperty of muscle fibers is their relaxation rate. The relaxation rate reflects therate at which Ca2+ is transported back from the myofibrilar space into the lumen ofthe sarcoplasmic reticulum (SR). The major determinant of relaxation rate is theSERCA 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 resultsin further variations and at present at least seven SERCA isoforms have beendescribed in human.38 SERCA1a is present in fast-twitch skeletal muscle fibersand SERCA2a is found in cardiac and slow-twitch muscle fibers. SERCA1b ispresent in neonatal muscles, SERCA2b in almost all nucleate cells andSERCA3a/b/c in several types of non-muscle cells. Mutations of the three SERCAgenes have been associated with disease. For example, mutation of the SERCA1gene is associated with Brody's disease, a condition characterized by impairmentof skeletal muscle relaxation after exercise, stiffness and cramps.39 The distributionof SERCA1 has been reported as rather complex in rat and rabbit EOMs40 but dataon human EOMs are lacking.
Myosin binding protein C
Myosin binding protein C (MyBP-C) is, after myosin, the second most abundantthick filament protein in striated muscle41 and it has a regulatory role in sarcomereassembly42, 43 MyBP-C is composed of a single subunit of 130 kDa and both its C-and the N-terminals bind to myosin.44, 45 MyBP-C has an essential physiologicalrole in regulating contraction by modulating maximal shortening velocity.46
There are 3 major isoforms of MyBP-C in human muscle: fast skeletal (MyBP-Cfast), slow skeletal (MyBP-Cslow) and cardiac (cMyBP-C).47 MyBP-Cfast,detected both with in situ hybridization and immunocytochemistry, is present intype 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 coordinatedexpression of MyBP-Cslow in fibers containing MyHCI (slow), MyBP-Cfast infibers with MyHCIIx and an additional isoform, intermediate MyBP-C in fiberscontaining MyHCIIa in human limb muscle.49 Coordinated isoform changesindicating that MyBP-C expression is linked to MyHC expression have been
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Figure: 5 Laminin
reported during skeletal muscle hypertrophy in the rat.50 However, in the humanmasseter, a masticatory muscle with very special properties,4, 51, 52 the very complexMyHC composition of its fibers was not paralleled by an intricate MyBP-Cpattern.49
Basement membrane
The muscle fibers are surrounded and supported by a layer of specializedextracellular matrix (ECM) called the basement membrane (BM). BMs are presentunder all epithelial cells, in blood vessels and surrounding many cells includingmuscle cells and Schwann cells.53 The BM provides strength and elasticity, servesas a selective filter and is necessary for muscle fiber maintenance andregeneration.At the neuromuscular junction (NMJ), where the peripheral nerve connects withthe muscle fiber, a specialized BM continues between nerve and muscle,delineating the postsynaptic membrane.53 In the same way, the BM extends intothe deep junctional folds of the myotendineous junction (MTJ), where force istransmitted from muscle to tendon.54
Laminin
The major non-collagenous protein of thebasement membrane is laminin.55 Laminins(Ln) adhere to collagen and are essential forthe assembly of the BMs and appear veryearly in development.56 Laminins strengthenthe BM, connect the BM to the cells andfurthermore, interact with cells via integrinreceptors57 and -dystroglycan.58 Lamininsare glycoproteins composed of three subunits( , and chains). Presently there are 5different -chains ( 1-5), 3 -chains ( 1-3)and 3 -chains ( 1-3) known. Differentcombinations of these chains assembly intoat least 14 laminin isoforms.55, 59 Thepredominant laminin in the BM of muscleand peripheral nerve is Ln-2 ( 2 1 1).60 Atthe NMJ the composition of the BM changesfrom Ln-2 to Ln-4 ( 2 2 1), Ln-9 ( 4 2 1)and Ln-11 ( 5 2 1) and the juxtapositionedSchwann cells are covered with a BMcontaining Ln-2 and Ln-8 ( 4 1 1).61
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Figure 6: Dystrophin associated protein
complex
The BM at the MTJ is also specialized and contains Ln-4 ( 2 2 1),62, 63 but thereis also expression of Ln 5 and possibly Ln 1.62
Clinical implications
The EOMs differ from otherskeletal muscles in that they areoften spared in systemicneuromuscular diseases such asDuchenne muscle dystrophy(DMD), merosin deficientcongenital muscular dystrophy(CMD) and other musculardystrophies.64
DMD, a recessive x-linkeddisease, is caused by a defect inthe DMD gene at Xp21.2coding for dystrophin.Dystrophin is a cytoskeletalprotein located in closeassociation with thesarcolemma. The lack ofdystrophin leads to progressivemuscle fiber destruction, withdevastating consequences andtypical 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 CMDcases and is caused by a gene defect in LAMA2 affecting the Ln 2 chain.64, 66, 67
The clinical picture includes postnatal hypotonia with contractions, weakness anddelayed motor development. Death in the first year as a result of respiratory failureis not uncommon. Variants of CMD with partial laminin-2 deficiency often have aless severe phenotype.
Both laminin-2 and dystrophin are needed for an intact link between the ECM andthe cytoskeleton of muscle fibers, through the dystrophin associated proteincomplex (DAPC).64 DAPC includes many proteins e.g. dystroglycans,sarcoglycans and syntropin. It links to the BM and the ECM by laminin-2 and tothe intracellular actin cytoskeleton by dystrophin (fig 6). Defects involving the linkbetween ECM and the cytoskeleton (e.g. collagen, laminin, dystrophin, desmin)cause muscle dystrophy.64, 68
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The EOMs are remarkably unaffected in both DMD64 and merosin deficientCMD69 in spite of severe general muscle fiber degeneration in other muscles of thebody. The reason for this is still essentially unknown, and the thought that thisshould be simply due to the small size of the EOM fibers and lack of mechanicalstress64 or as a result of a disturbed calcium homeostasis, has been surpassed by amore wide approach where the unique properties and molecular diversity of theEOMs must be taken in consideration.70, 71
The EOMs are preferentially involved in Graves’ disease, myasthenia gravis andMiller-Fisher. Graves’ disease is an autoimmune disorder followinghyperthyreosis, with myosit, swelling of the EOMs and the optic nerve.72
Myasthenia gravis is caused by an autoimmune attack against theacetylcholinesterase receptors in all neuromuscular junctions. It results intiredness, ptosis and dysfunction of the EOMs. The ocular symptoms oftenprecede the general symptoms, but not always. Miller-Fisher syndrome is a rarevariant of Guillian-Barré syndrom, characterized by ophthalmoplegia, ataxia andareflexia. 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 basisof the unique phenotype of the EOMs is expected to provide further understandingof their properties and behavior in disease and thereby open the opportunity fornew therapeutical strategies.
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Aims of the study
The aims of the present investigation were to characterize the human EOMs withrespect to fundamental contractile and structural proteins, in particular:
• to investigate the MyHC composition of the fibers and to determinewhether the previous fiber type classifications correlate to the MyHCcomposition
• to investigate the SERCA composition of the fibers and to determinewhether there is a correlation between the MyHC and SERCA isoformcontent
• to investigate the MyBP-C composition of the fibers and to determinewhether there is a coordinated expression of MyHC and MyBP-C
• to investigate the composition of the basement membranes with respect tolaminin chains and to compare them with those of human limb muscle
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Materials and methods
Human EOMs (paper I-IV) and also the levator palpebrae (paper I and III) werecollected at autopsy or following enucleation. Samples were also taken frombiceps brachii, first lumbrical, quadriceps femoris, psoas and heart muscle forcomparison. All samples were collected from subjects with no previously knownneuromuscular disease. The study followed the Swedish Transplantation Law andhad the approval of the Medical Ethical Committee, Umeå University. In somecases the anterior part of the EOMs was not available since it had been removedtogether with the eye for donation purposes. The samples were mounted oncardboard, 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 ina cryostat (Reichart-Jung, Leica, Heidelberg, Germany).
Histochemistry
Ten µm-thick sections were treated to reveal ATPase activity after pre-incubationat 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 histochemicallyusing the acetylcholinesterase reaction75 (paper IV).
Immunocytochemistry
Five to 7 µm-thick sections were processed for immunocytochemistry,29 with abattery of well-characterized MAbs, each recognizing distinct MyHC isoforms(paper I, II and III), SERCA1 and SERCA2 (paper II), MyBP-C isoforms (paperIII) and laminin chain isoforms (paper IV). A MAb against tenascin-C (Tn-C) wasused to detect the MTJs (paper IV). For details see table 1.
Control sections were processed as above, except that the primary antibody wasomitted. 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 aSpot camera (RT color; Diagnostic Instruments, USA) connected to a Nikonmicroscope (Eclipse, E800, Tokyo, Japan).
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Fiber typing (paper I, II, III)
Two to eight areas that were recognizable in all serial sections processed with thedifferent MAbs used were studied in detail to determine the fiber composition. Thechoice was not completely random because the EOM fibers are so looselyarranged that they are difficult to follow even in consecutive series of sections.Therefore the areas chosen were limited to those where fiber identification waspossible in a long series of sections. In spite of this, care was taken to pick areasrepresentative of the orbital and global layer. The MyHC composition of over4000 fibers (paper I) and the SERCA composition of 1571 fibers (paper II) wereanalyzed in detail.
Fiber area (paper I)
The fiber area was measured on sections from rectus superior, obliquus superiorand levator palpebrae, stained with MAb 4C7 against laminin 5 chain,76 whichdelineated the contours of the muscle fibers. The area of a total of 885 fibers wasmeasured 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 adultEOMs, cardiac and limb muscle, as well as fetal limb muscle. SDS-PAGE wasperformed77 in a Mini Protean II unit (Bio-Rad, Glattbrug, Switzerland) at 75 V for22 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 identityof the MyHC bands separated by SDS-PAGE. After SDS-PAGE, proteins weretransferred to 0.45µm nitrocellulose membrane (Bio-Rad Laboratories) for 17hours at 30 V with the unit surrounded by a 15°C water bath. Monoclonalantibodies A4.840, 4A6, A4.74 and 2B6 were used to identify the bandscontaining MyHCI, MyHCeom, MyHCIIa and MyHCemb respectively.
Paper II: A crude microsomal fraction isolated from 11 individual EOM samplesthat were combined was used for immunoblot analysis.79, 80 Protein concentrationwas determined using bovine serum albumin as standard.81 A gel electrophoreticseparation of EOM proteins was performed and the proteins were transferred tonitrocellulose sheets.82 The sheets were processed83 with primary antibodies IID884
with affinity to the slow SERCA2 isoform and IIH1184 with affinity to the fastSERCA1 isoform.Immunodecoration was evaluated by the enhanced chemiluminescencetechnique.85 Densitometric scanning of enhanced chemiluminescence blots was
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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 adultEOMs, levator palpebrae and limb muscle.87 The acrylamide concentration in thestacking and running gels were 4 and 8% (w/v), respectively, and the gel matrixincluded 10% glycerol.50 The separating gels (160x180x0.75 mm) weresilverstained and scanned with a soft laser.
Capillaries (paper IV)
The number of capillaries was determined in representative areas of the orbital andglobal layer in sections from 5 EOM samples, stained with the antibodyrecognizing laminin 5 chain. All vessels with an outer diameter less than 15 µmwere assumed to be capillaries according to the definition put forward byJerusalem.88 The capillary density was calculated for both the global and theorbital layer in all 5 muscles. A total of 4861 capillaries were counted.
Statistics
Statistical analyses were conducted on both the fiber size and capillary densitydata. The fiber size was compared between the EOM and LP samples and betweenthe orbital and global layer samples. The capillary density was compared betweenthe orbital and the global layer samples. Unpaired t-test and one-way analysis ofvariance (ANOVA) were performed using the StatView 5.0.1 software (AbacusConcepts, Berceley, California, USA). A probability of rejecting the nullhypothesis of less than 0.05 was considered a statistically significant difference.Data is presented as means +/- standard deviations.
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Results
Biochemistry
Electrophoresis of whole muscle extracts of the sampled EOMs and LP revealedfour 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 humanEOMs (paper II, fig 4) revealed that the slow isoform SERCA2 was significantlymore 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 inlimb muscle (paper III, fig 1). These bands were seen above MyBP-Cslow and in aposition 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 infascicles. Two different layers of fibers could be identified. The orbital layer was 5to 30 fibers deep. The orbital layer covered at least one side of the four recti andthe superior oblique muscle, sometimes it completely surrounded the global layer.No marginal zone could be identified. In the LP no orbital/global layers werediscerned.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 fibersof both layers as the fibers with strong mATPase activity following pre-incubationat pH 4.3 and moderate mATPase activity at pH 10.4. Similarly, the remainingfibers with high ATPase activity at pH 10.4 and low activity at pH 4.3, wereassumed to be singly innervated.3 The NADH activity of the fibers in the EOMswas generally high and it was more homogeneous in the orbital than in the globallayer. The multiply innervated fibers tended to have lower NADH activity in bothlayers. Classification of the fibers into further, clearly defined subgroups based on
24
the ATPase or NADH activity was impossible due to variation of the level ofstaining in a continuum (paper I, fig 3).
Immunocytochemistry
The EOM fibers displayed important heterogeneity in immunostaining intensitywith the different antibodies used.
MyHC: Three fiber groups could be distinguished on basis of their pattern ofimmunoreactivity (paper I, fig 4-6). The majority of the fibers stained stronglywith anti-MyHCIIa, a smaller group of fibers were labeled with anti-MyHCI andthe remaining fibers were not stained with either of these two antibodies, but werereactive to anti-MyHCeom and anti-MyHCI+IIa+eom. These fibers were namedMyHCeompos/MyHCIIaneg-fibers. The MyHCeompos/MyHCIIaneg-fibers weregenerally larger than the otherfibers. The MyHCIIa positivefibers were evenly distributed inthe orbital layer, but in the globallayer there were large areasdevoid of this fiber type. Theseareas were instead filled withMyHCeompos/MyHCIIaneg-fibers.The MyHCI positive fibers wereevenly distributed in both layersand corresponded to the fiberswith strong mATPase activityfollowing pre-incubation at pH4.3 and moderate mATPaseactivity at pH 10.4 (presumablymultiply 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 anti-
SERCA1 (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)
25
Practically all fibers contained more than one MyHC isoform. A summary of thestaining patterns, layer distribution, presumed innervation type and size of theEOM fibers is given in table 2.
No fibers in the LP stained with anti-MyHCsto and less than 1% of them stainedwith anti-MyHCemb.
SERCA: In the orbital layer, anti-SERCA1 and anti-SERCA2 stained almost allfibers (paper II fig 1 and 2) and in the global layer only 37% of the fibers did notcontain both SERCA1 and SERCA2. A summary of the staining patterns observedis presented in table 3.
The distribution of MyHCIIa varied along the length of the EOMs whereas nodifference 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 EOMsamples immunostained with this MAb (paper III, fig 3, 4, 5). However, in twosamples from the proximalpart of one rectus superiorand one rectus inferiormuscle taken from the oldestsubjects (82 and 86 yearsrespectively), there were afew weakly to moderatelystained fibers, mostly locatedin the periphery of themuscles (paper III, fig 6).These fibers were MyHCIIapositive fibers orMyHCeompos/MyHCIIaneg-fibers.
Anti-MyBP-Cslow stained allfibers: the slow fibers strongly,the fast fibers moderately tostrongly and theMyHCeompos/MyHCIIaneg-fibers lightly to strongly (fig8 and paper III, fig 4, 5). Nocorrelation was foundbetween the staining patternof these two MAb and thepresence or absence ofMyHCemb in the fibers.
Figure 8: Photomicrograph of sections from a rectus inferior
muscle immunostained with A) anti-MyBP-Cslow, B) anti-
MyBP-Cfast, C) anti-MyHCI, D) anti-MyHCI+IIa+eom, E)
anti-MyHCsto, F) anti-MyHCIIa and G) anti-MyHC -cardiac.
(from paper III)
26
In the levator palpebrae anti-MyBP-Cfast stained some of the MyHCIIapos fibersand some of the MyHCeompos/MyHCIIaneg
-fibers heterogeneously. The fiberscontaining MyHCI were unstained with anti-MyBP-Cfast. Anti-MyBP-Cslowstained all fibers in the levator palpebrae strongly (paper III, fig 7).
Laminin chains: Anti-Ln 1 did not show immunoreactivity to any tissuestructure in the sampled sections (paper IV, fig 1). Anti-Ln 3 immunostained theblood vessels only (paper IV, fig 1). The extrasynaptical BM of adult human EOMmuscle fibers contained Ln 2, 1, 2, 1, and to some extent also Ln 4 and 5(fig 7 and paper IV, fig 1, 2, 5). Ln 2 was also detected in the BM of adult limbmuscle (paper IV, fig 5). At the NMJs there was increased expression of Ln 4,and also maintained expression of Ln 2, 5, 1, 2 and 1 (paper IV, fig 2,3).The MTJs contained Ln 2, 5, 1, 2 and 1 as described for skeletal muscle(paper IV, fig 4). MAb against Ln 2, 4, 5, 1, 2 and 1 stained theperineurium. The endoneurium stained strongly with all these antibodies exceptanti-Ln 5 that only immunostained the endoneurium moderately (paper IV, fig 1).The MAb against Lutheran glycoprotein immunostained blood vessels andperineurium in both EOMs and limb muscle, but the contours of the fibers werelabeled only in the EOMs (paper III, fig 7).The capillary density in the EOMs, determined in sections processed with anti-Ln 5, was 1050 +/-190 capillaries/mm2 (paper IV, figure 5). The capillary densitywas significantly (p<0.05) higher in the orbital layer (1170 +/-180capillaries/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-Ln 4. The first
lumbrical muscle (L), the global layer (GL) and the orbital layer (OL) of the EOM are indicated.
(from paper IV)
27
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 MyHCIMyHCIIaMyHCeom
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-Ln 1 62
5H2 Laminin 2-chain Anti-Ln 2 100
BM-2 Laminin 3-chain Anti-Ln 3 76
FC10 Laminin 4 chain Anti-Ln 4 101
4C7 Laminin 5-chain Anti-Ln 5 102
DG10 Laminin 1-chain Anti-Ln 1 103
C4 Laminin 2-chain Anti-Ln 2 104
113BC7 Laminin 1chain Anti-Ln 1 105
4A10 Tenascin-C Anti-tenascin 106
MCA-1982 Lutheran glycoprotein Anti-Lu 107
28
Table 2. Staining pattern, distribution, innervation and size of fibers in human EOMs
Fiber type: MyHCIIapos
MyHCIpos
MyHCeompos
/MyHCIIaneg
Staining:
Anti-MyHCI+IIa+eom + + +
Anti-MyHCIIa + - -
Anti-MyHCI - + -
Anti-MyHCeom +/- +/- +
Anti-MyHCsto - +* -
Anti-MyHC -cardiac - +/- -
Anti-MyHCemb +/- +/- +/-
Distribution:
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**
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
29
Table 3 Patterns of staining with MAbs against SERCA1 and 2 observed in thethree major groups of fibers
GLOBAL LAYER
Staining intensity with MAbagainst
Number of sampled fibers in each group accordingto MyHC content
SERCA1 SERCA2 IIa I IIaneg
/eompos
- - 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 MAbagainst
Number of sampled fibers in each group accordingto MyHC content
SERCA1 SERCA2 IIa I IIaneg
/eompos
- - 0 0 0
++ - 6 0 0
- ++ 0 0 0
+ + 0 1 0
++ + 559 1 0
+ ++ 0 134 0
++ ++ 137 42 4
702 178 4
30
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 MyHCisoforms in a single fiber
up to 5 up to 4 1 to 2
MyHC isoform variationalong the fiber length
yes - no
Number of SERCAisoforms in a single fiber
0 - 2 - 1
Presence of MyBP-Cfast trace amounts inpart of the muscle
in some fast fibers in all fastfibers
Ln chains inextrasynaptic 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 ofmuscle fiber
yes - no
- not examined
31
Discussion
The present study showed that the human EOMs have a very complex fiber typecomposition with respect to the major proteins regulating fiber contraction velocityand force (MyHCs, MyBP-Cs) and fiber relaxation rate (SERCAs). There werealso important differences in laminin isoform composition and capillary densitywhen compared to human limb muscle.
Validity
MyHCI, MyHCeom, MyHCIIa and MyHCemb as well as SERCA1 and 2 weredetected in the EOMs with immunocytochemistry, SDS-PAGE and immunoblots.All MyHC and SERCA antibodies used are well characterized and have beenextensively used.29, 86, 90, 94, 108-110 The specificity of the MyHC MAbs for humanmuscle has been well verified with immunoblots90 and withimmunocytochemistry.29, 90, 94 Therefore we can be sure that the complex patternsof reactivity obtained with immunocytochemistry reflect differences in theSERCA and MyHC composition of each myofiber.The MAbs against MyBP-C were raised against purified human C-protein and weestablished that they do not cross-react with cMyBP-C (paper III). Furtherconfirmation of their specificity with immunoblots is planned.The MAbs against laminin chain isoforms,62, 76, 100-105 Lutheran glycoprotein107 andtenascin-C106 are also well characterized and have been widely used both inhumans and other species.
EOM fiber type composition
ATPase activity: The mATPase activity of the fibers varied in a continuum fromlow to high. This is consistent with the fact that most of the human EOM fibersexamined contained more than one MyHC isoform. The mATPase reaction doesnot allow the discrimination of mixtures of MyHCs, particularly when both fastand developmental isoforms are present.24 The mATPase activity resides in thehead region of the MyHC molecule, and therefore data on the MyHC compositionhave more relevance and provide more information on the expected contractileproperties of the fibers.
MyHC: The human EOMs could be divided into three major fiber groups takinginto account their MyHC content: 1) slow fibers containing MyHCI, 2) fast fiberscontaining MyHCIIa, 3) fast fibers containing MyHCeom, but lacking bothMyHCIIa and MyHCI (fig 10). Most slow fibers contained both MyHCI and
32
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 slowisoform.
MyHCeom was additionally present in subgroups of the fibers defined by theircontent of MyHCI or IIa and, finally, MyHCemb was also found in subgroups ofall three major fiber types. Altogether, there were 22 distinct MyHC combinationsobserved in the human EOMs. Furthermore, differences in immunostainingintensity clearly showed that fibers with a given combination of MyHCs differedfrom each other in their relative amounts of the MyHC isoforms. In summary, theMyHC composition of the human EOM fibers varies in a continuum and is verycomplex.
Previous data on human EOMs have shown that fibers containing MyHCstocorrespond to the orbital and global multiply innervated fiber groups.3 The fiberscontaining MyHCI and MyHCsto represented approximately 16% of the total inthe orbital layer and 13% in the global layer, consistent with values reported forthe rat multiply innervated fibers.2 Another important finding regarding theheterogeneity of the fibers containing slow MyHC isoforms was the identification
33
of fibers containing MyHCI but not MyHCsto. We speculate that these fibers arelikely to be singly innervated.The genes for human MyHCIIb and MyHCeom have been sequenced20 and RNAtranscripts of MyHCIIb have been detected111 in human EOM samples. Thecorresponding MyHCs are therefore expected to be present in the EOMs. Inaddition, immunocytochemical data indicate the presence of MyHCsto in thesemuscles. To date there are no antibodies specific to human MyHCIIb available andit has not been established yet whether the SDS-PAGE band called MyHCeomreally corresponds to the product of the MYH13 (MyHCeom) gene or whether itcorresponds to the MYH4 (MyHCIIb) gene or both. We follow the previousnomenclature tentatively identifying the unique band in SDS-PAGE asMyHCeom.112, 113
MyHCIIx can be identified in human muscle fibers by immunocytochemistry,using a MAb that labels all MyHCs except MyHCIIx. Because practically allfibers in the EOMs co-expressed more than one MyHC isoform, this antibody is ofno 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 relativeamounts of SERCA1 and 2 among the fibers, indicating that also the amounts ofthe major Ca2+-pump protein isoforms in the fibers of the EOMs vary in acontinuum.
A small number of fibers were devoid of reactivity to the MAb against SERCA1and 2. These fibers were very few and may contain yet another isoform ofSERCA, perhaps specific for the EOMs. There are at least seven known SERCAisoforms in human, generated by alternative splicing38 and the existence ofadditional isoforms remains to be explored.
MyBP-C: The MyBP-C isoform composition of the EOM fibers, investigated withMAbs against MyBP-Cfast and slow, was not coordinated with the MyHCcomposition. No immunoreaction with anti-MyBP-Cfast was seen in the vastmajority of the samples and both the slow and fast MyBP-C bands were absent inwhole muscle extract electrophoresis.
The lack of immunoreaction to anti-MyBP-Cfast in most of the EOMs, and thefinding of unidentified bands in EOM whole muscle extracts investigated withSDS-PAGE, suggest the presence of new isoforms of MyBP-C in the EOMs.Although only MyBP-Cfast, slow, cardiac and intermediate have been identified inhuman skeletal muscle, multiple isoforms of MyBP-C are present in chickenmuscle.114
Longitudinal variation: The MyHC composition was not constant along thelength of the EOMs. Our material did not allow us to follow the same fibers alongtheir entire length but it clearly showed that the distribution of MyHCIIa varied
34
along the muscle length in both the global and the orbital layer, as previouslydescribed in rabbit.35 Furthermore, MyBP-Cfast was only found in a few fibers inthe proximal part of two muscles. In contrast, the distribution of SERCA isoformswas 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 isvery rich in blood vessels and nerves. Our study showed that the capillary densityof the EOMs was higher than that of any previously examined human muscle,52
which is in agreement with their outstanding physiological demands, highoxidative enzyme activity3 and higher overall vascular density of the orbitallayer.115
The distinct character of the human EOMs was also apparent in the laminincomposition of the extrasynaptic BM of the fibers. The presence of Ln 4, Ln 5and Ln 2 in extrasynaptic BM suggests that the BM of the EOMs contain more Lnisoforms than the BM of limb muscle. This is interesting since laminin is not onlyan important structural protein, but it is also highly involved in the signalingsystem between the ECM, cell surface receptors and the cytoskeleton of themuscle fibers.116, 117 The presence of several laminin isoforms based on differentalpha chains may in part explain the fact that the EOMs are spared in musculardystrophies such as merosin deficient CMD and DMD, perhaps by making the BMstructurally more stable, but also by providing alternative signal paths between theECM and the cells. The fact that Lutheran glycoprotein, a Ln 5 receptor, wasfound 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. looselyarranged fibers, presence of MyHC -cardiac, MyHCemb and MyHCeom,absence of MyBP-Cfast and slow bands and presence of two novel bands inwhole muscle extracts studied with SDS-PAGE. On the other hand severalfindings distinguished the LP from the EOMs, e.g. larger fiber size, lack oforganization into layers, lack of MyHCsto, presence of MyBP-Cfast in allsamples examined (table 4). These findings place the phenotype of the LP asintermediate between that of the EOMs and the limb muscles. The functionaldemands on the levator palpebrae muscle are high and its importance forsurvival is as obvious as for the EOMs. However, the nature and range ofmovements of the levator palpebrae are far less complex than those of theEOMs.
35
Human EOM versus EOM of other species
Clear distinctions in MyHC isoform content between the orbital and global layerswere 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 inthe rat EOMs whereas we found MyHCeom widely distributed in both layers.MyHCemb and -cardiac were more abundant in, though not restricted to, theorbital layer of human EOMs. MyHCemb is also found in both layers of rabbitEOMs.35
A correspondence between the MyHC content and six putative fiber types basedon color, location and innervation, was not present in human EOMs. In ourmaterial the vast majority of the fibers contained two or more MyHC isoforms anda distinction into six fiber groups based on the histochemical andimmunohistochemical staining was impossible. In the rat EOMs,28 the globalmultiply innervated fibers are proposed to contain mainly MyHCI whereas thesingly innervated fibers would presumably contain either predominantly MyHCIIa(red fibers), IIx (intermediate fibers) or IIb (white fibers). The orbital fibers areproposed to contain predominantly embryonic MyHC and either MyHCeom(singly innervated fibers) or MyHCI (multiply innervated fibers) in the middleregion.28
Rubinstein and Hoh28 reported that the rat orbital singly innervated fibers onlycontained MyHCemb in the distal and proximal portions of the EOM. Jacoby,40 onthe other hand, reported that both multiply and singly innervated fibers in the ratorbital 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 thirdsof the orbital singly innervated fibers, and all of these fibers were also stained byanti-MyHCIIa and/or anti-MyHCeom.
Our results show that, in human EOMs, most fibers contain both SERCAisoforms, clearly distinguishing them from the EOMs of rat and rabbit, whereSERCA1 is co-expressed with fast MyHC.40 Data on the distribution of SERCA2are missing in other species. SERCA2 is typically found in slow-twitch skeletalmuscle fibers and cardiac muscle fibers.118
These results indicate that caution is needed when extrapolating data from animalmodels to the human EOMs.
The present work on MyBP-C and laminins are, to the best of our knowledge, thefirst to address the EOMs, with the exception that Ln 2 has been detected in NMJsof mouse EOM.119 Therefore there are no data for comparison in other species.
36
Human EOM versus human limb muscle
In the EOMs we found mixtures of up to five different MyHC isoforms in a singlefiber according to their immunocytochemical reaction. A single fiber could e.g.stain with anti-MyHCI, sto, -cardiac, eom and emb. Human limb muscle fiberstypically contain one or at most two MyHC isoforms: MyHCI, I+IIa, IIa, IIa+IIxor 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 inchronically stimulated limb fast twitch fibers, in a dog model.120 The presence ofmore than one SERCA isoform in the same cell has been speculated to reflect theexistence of segregated calcium stores, each with its own particular role andloaded by a particular calcium pump.38 Further investigations will be needed to testthis hypothesis in muscle cells.Some EOM fibers were unstained by both SERCA markers, hitherto not reportedin human limb muscle.
The absence of MyBP-Cfast and the possibility for additional isoforms other thanMyBP-Cfast and slow clearly further separate the EOMs from limb muscle wherethe correlation between MyBP-Cfast and MyHCIIx, MyBP-Cintermediate andMyHCIIa, MyBP-Cslow and MyHCI is established.49
We found Ln 4 and Ln 5 at the NMJs and extrasynaptically in the EOMs. Ln 4has not been detected in adult human limb muscle BM, although it is presentduring development.101 The detection of Lutheran glycoprotein, a Ln 5 receptor,on the fiber surface in EOMs, a feature not seen in limb muscle, confirms thepresence of Ln 5 in extrasynaptical BM of the EOMs. These findings imply thatthe extrasynaptical BM of the EOMs might also contain laminin-8 ( 4 1 1), -9( 4 2 1), -10 ( 5 1 1) and/or -11 ( 5 2 1) in addition to the laminins found inextrasynaptical BM of human limb muscle, laminin-2 ( 2 1 1) and 4 ( 2 2 1).
Altogether our data further establish the distinctness of the EOMs in comparison tolimb muscle, with respect to contractile, Ca2+ transport and structural proteins aswell 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 complexmuscles in the body, with more features in common with other muscles innervatedby cranial nerves (e.g. laryngeal and masticatory muscles) than limb muscle. Themuscles of the vocal folds contain MyHCeom,96 MyHCsto and multiply innervatedfibers.121 The single fibers of the masseter contain up to 5 different MyHCisoforms including fetal, embryonic and -cardiac.4, 49
37
The structural and molecular complexity of the EOMs shown here, could in part beexplained by the physiological demands put on these muscles. For example, theslow fibers containing MyHCsto are likely to enable slow and sustainedcontraction with modest consumption of energy, allowing the EOMs to performfixation and slow pursuit movements. Small differences in the MyHC (e.g. co-expression 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 contractionvelocity and force as well as of relaxation rates that can be speculated to allowaltogether more versatile muscle activity than it would be the case if all slow fibershad the same composition. The same holds for the fast fibers containing the veryfast MyHCeom and/or MyHCIIb, which are probably involved in the very fast andexact movements, e.g. saccades.
But what really makes the EOMs unique is the distinct molecular profile of almosteach individual muscle fiber. This heterogeneity probably reflects the very smallsize of the motor units in the eye muscles and a very intricate functionaladaptation. In fact it may even represent an ultimate filter for fine motor control ina system that performs so very diverse and coordinated types of movement.
The simultaneous presence of multiple isoforms of a given protein represents avery advantageous evolutionary adaptation which a) allows a wider range offunctional capacity, e.g. of contraction and relaxation parameters; b) makes theEOMs less vulnerable to defects/changes involving a particular isoform, as theadditional isoforms can compensate for the defective one.
Future directions
The unique character of the EOMs in rodents has recently been confirmed at thegene level with both micro-array and SAGE techniques,122-124 particularly withrespect to metabolic pathways, structural components and regeneration markers.However, these studies rely on whole muscle RNA extractions and therefore thepotential transcript sources include the muscle fibers, satellite cells, fibroblasts,different types of nerve and blood vessel cells along with circulating cells. In orderto further assess the molecular basis of the unique human EOM allotype, we willin the future need to combine differential gene expression data with solidmorphological and biochemical techniques to be able to assign the correctmolecular portfolio to the different cell types. Basic knowledge on the structureand composition of the EOMs provides the platform for understanding theirunique functional properties and may provide clues to protection and susceptibilitymechanisms that ultimately can be used for new therapeutic approaches.
38
Conclusions
The present thesis further advances our knowledge of the distinct character of thehuman EOMs and consolidates the concept that they are a separate skeletal muscleallotype.
The EOMs differ from the limb muscles in basic features involving musclemorphology, 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 ofconnective 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 thelength 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 Ln 5 receptor
The EOMs differed from the levator palpebrae, which showed a phenotypesomewhat intermediate between that of the EOMs and the limb muscles.
Previous classifications into six fiber types in mammals do not reflect theheterogeneity seen in human EOMs and are not supported by data on the MyHCcomposition of the fibers.
There are important differences between the human EOMs and those of otherspecies and therefore caution is needed when extrapolating data from animalmodels.
The hallmark of the human EOMs was a remarkable heterogeneity in the staininglevels 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 basementmembrane of the EOMs could partly explain the selective sparing of these musclesin Ln 2 deficient congenital muscle dystrophy.
The co-existence of complex mixtures of several crucial protein isoforms providesthe human EOMs with a unique molecular portfolio that a) allows a highly specificfine-tuning regime of contraction and relaxation, and b) imparts structuralproperties that are likely to contribute to protection against certain neuromusculardiseases.
39
Acknowledgments
After the service, one hot Sunday in June, the parish clerk, whose face was prettyred, after having blown the organ’s bellow for over an hour, turned to the organistand said: “That was a nice postlude we played today!” The organist snorted, closedhis briefcase with the sheets of music and replied dryly: “We played? May Iremind you that I did the actual playing. I am an artist and you are only theprovider of air.”Next Sunday, the organist had prepared a mighty prelude by J.S. Bach, since thebishop 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 nothinghappened. Soon all the parish members and the bishop were looking at thedesperate organist, struggling with his instrument. Suddenly the embarrassingsilence 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 allthe wonderful persons who have contributed to this thesis.
First of all I thank the Lord, for creating the extraocular muscles in such a fantasticway 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 Departmentof Integrative Medical Biology, section for Anatomy. The attitude of thedepartment is inspiring, creative and forgiving. All people there have been equallyhelpful, but some people have been more equal than others:
I would like to thank my fantastic supervisor docent Fatima Pedrosa-Domellöf, forinvaluable help and support, throughout this study, and for showing me her secretdepot of chocolates (bottom left drawer of her desk, but don’t tell anyone).
My co-supervisor, professor Lars-Eric Thornell, for encouragement andconstructive criticism, and for setting an example in advanced house improvement.
Margaretha Enerstedt and Anna-Karin Olofsson for excellent technical assistanceand patience.
Per Stål, for help and encouragement and especially for lending me his high techcomputer 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: EvaCarlsson, Ji-Gou Yu, Mona Lindström and Lena Carlsson. Tomas Carlsson, Göran
40
Dahlgren and Ulf Ranggård for invaluable computer support. Anna-LenaThallander, department secretary. My fellow research students: Jing-Xia, DinaRadovanovic, Anders Eriksson and last but not least Berit Byström (theJukkasjärvi Blizzard).
At the Department of Clinical Sciences, Ophthalmology I would especially thankmy co-supervisor, professor Lillemor Wachtmeister, for invaluable help inproviding grants for this work, Eva Mönestam for giving me time for research inmy busy clinical schedule and Birgit Johansson, the department secretary, forhelping 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. Iwill 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 churchchoir of Ålidhem and my former floor hockey team for making my sparetime arare but pleasant enjoyment.
I am also in gratitude to my mother and father, Catharina and Per Kjellgren, forlovingly anticipating this ever since I was born. My sister Hanna for setting anexample. My mother and father in law, Barbro and Lars Berglund for practicalhelp with cars, tractors, lawn movers, orbital sanders and other necessities of life.
Finally and foremost I thank my wonderful family: Åsa - wife extraordinaire, andmy daughter Rebecka - the eighth wonder of the world, for giving my lifehappiness and meaning. I am indeed a fortunate husband!
This study was supported by grants from Stiftelsen KMA, the Swedish Society forMedical Research, the Swedish Research Council and the Medical Faculty ofUmeå University.
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