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 Adenosine triphosphatase BM Basement membrane CMD Congenital muscular dystrophy EOM Extraocular muscle ECM Extracellular matrix GL Global layer Ln Laminin LP Levator palpebrae Lu Lutheran glycoprotein MAb Monoclonal antibody MIF Multiply innervated fiber MTJ Myotendineous junction MyBP-C Myosin binding protein C MyBP-Cfast Fast isoform of MyBP-C MyBP-Cslow Slow isoform of MyBP-C cMyBP-C Cardiac isoform of MyBP-C MyHC Myosin heavy chain MyHC
-cardiac
-cardiac isoform of MyHC MyHCemb Embryonic isoform of MyHC MyHCeom Extraocular isoform of MyHC MyHCI Slow isoform of MyHC MyHCIIa Fast isoform IIa of MyHC MyHCIIb Fast isoform IIb of MyHC MyHCIIx Fast isoform IIx of MyHC MyHCsto Slow tonic isoform of MyHC NMJ Neuromuscular junction OL Orbital layer SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SERCA Sarco(endo)plasmic reticulum Ca2+ATPase SERCA1 SERCA fast isoform SERCA2 SERCA slow isoform SIF Singly innervated fiber SR Sarcoplasmic reticulum Tn-C Tenascin-C T-tubules Transverse tubules

6 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 Ln
2, 1, 2, 1, and to some extent also Ln
4 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 Ln
2 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 and laterally, passing below the rectus Figure 1: Extraocular muscles and superior to be inserted into the superior levator palpebrae of the left orbit 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 with slow tonic contraction muscle fibers 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 Figure 3: Organisation of the SR and the T-tubules the sarcoplasmatic reticulum 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 actin- binding site, it thereby determines the speed of contraction and the contraction force.18, 21, 22 Hence, the MyHC composition is regarded as the Figure 4: Schematic illustration of myosin II 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 C- and 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 (MyBP- Cfast), 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 Figure: 5 Laminin Schwann cells are covered with a BM containing Ln-2 and Ln-8 (
411).61

16 The BM at the MTJ is also specialized and contains Ln-4 (
221),62, 63 but there is also expression of Ln
5 and possibly Ln
1.62

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 muscle fiber destruction, with Figure 6: Dystrophin associated protein 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 Ln
2 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 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)

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/MyHCIIaneg- fibers. Anti-MyBP-Cslow stained all fibers: the slow fibers strongly, the fast fibers moderately to strongly and the MyHCeompos/MyHCIIaneg- fibers lightly to strongly (fig 8 and paper III, fig 4, 5). No correlation was found between the staining pattern Figure 8: Photomicrograph of sections from a rectus inferior muscle immunostained with A) anti-MyBP-Cslow, B) anti- of these two MAb and the MyBP-Cfast, C) anti-MyHCI, D) anti-MyHCI+IIa+eom, E) presence or absence of anti-MyHCsto, F) anti-MyHCIIa and G) anti-MyHC
-cardiac. MyHCemb in the fibers. (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-Ln
1 did not show immunoreactivity to any tissue structure in the sampled sections (paper IV, fig 1). Anti-Ln
3 immunostained the blood vessels only (paper IV, fig 1). The extrasynaptical BM of adult human EOM muscle 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). Ln2 was also detected in the BM of adult limb muscle (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 the perineurium. The endoneurium stained strongly with all these antibodies except anti-Ln
5 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 anti- Ln
5, 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-Ln
4. 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 Anti-MyHCI+IIa+eom 90, 91 MyHCIIa MyHCeom MyHC
-cardiac 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-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: 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

28 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 Number of sampled fibers in each group according against to MyHC content SERCA1 SERCA2 IIa I IIaneg/eompos --120 ++ - 161 0 54 -++0350 ++35122 ++ + 267 2 24 +++1420 ++ ++ 40 0 0 505 82 100

ORBITAL LAYER Staining intensity with MAb Number of sampled fibers in each group according against to MyHC content SERCA1 SERCA2 IIa I IIaneg/eompos --000 ++ - 6 0 0 -++000 ++010 ++ + 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, I,
-cardiac, IIa, IIb, I, IIa, IIx IIb, eom, emb, fetal eom, emb, fetal Number of MyHC up to 5 up to 4 1 to 2 isoforms in a single fiber MyHC isoform variation yes - no along the fiber length Number of SERCA 0 - 2 - 1 isoforms in a single fiber Presence of MyBP-Cfast trace amounts in in some fast fibers in all fast part of the muscle fibers Ln chains in
2,
4,
5, 1, 2, -
2, 1, 2, 1 extrasynaptic BM 1 Ln chains at NMJs
2,
4,
5, 1, 2, -
2,
4,
5, 1, 1 2, 1 Ln chains at MTJs
2,
5, 1, 2, 1 -
2,
5, 1, 2, 1 Lu present in BM of yes - no muscle fiber - 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 Ln
4, Ln
5 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 Ln
5 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 Ln
4 and Ln
5 at the NMJs and extrasynaptically in the EOMs. Ln
4 has not been detected in adult human limb muscle BM, although it is present during 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 the presence of Ln
5 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. 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 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 Ln
5 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 Ln
2 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 References

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