Myosin Heavy Chain Isoforms in Human Extraocular Muscles

Abstract

purpose. To investigate the myosin heavy chain (MyHC) composition of human extraocular (EOM) and levator palpebrae (LP) muscle fibers.

methods. Adult human EOMs and LP were studied with SDS-PAGE, immunoblots, and immunocytochemistry, with antibodies against six MyHC isoforms. Myofibrillar adenosine triphosphatase (mATPase) and reduced nicotinamide adenine dinucleotide (NADH)-TR activity and fiber area were also determined.

results. Most of the fibers in both layers stained strongly with anti-MyHCIIa. Approximately 14% of the fibers in the global layer and 16% in the orbital layer were labeled with anti-MyHCI. The remaining 24% of the fibers in the global layer and 3% in the orbital layer were not stained with either of these two antibodies, but were reactive to anti-MyHCeom (MyHCeom^pos/MyHCIIa^neg fibers). The fibers stained with anti-MyHCI had acid-stable mATPase activity, and the remainder of the fibers had alkaline-stable mATPase activity. Almost all the slow fibers stained with both anti-MyHCI and anti-MyHCslow tonic in both layers. Anti-MyHCα-cardiac stained approximately 26% of these slow fibers in the orbital layer and 7% in the global layer. Some slow fibers in both layers lacked staining with anti-MyHCslow tonic or with anti-MyHCα-cardiac. MyHCemb and/or MyHCeom were also present in some of the fibers of all the groups. The LP did not stain with anti-MyHCslow tonic.

conclusions. The present study revealed that the human EOMs have a very complex fiber type and MyHC composition and differ significantly from the EOMs of other species. The features of the LP were distinct from those of the four recti, the obliquus superior, and the limb muscles.

The myosin isoforms are the major determinant of the functional heterogeneity of the key contractile and biochemical properties of skeletal muscle fibers, including velocity of shortening, adenosine triphosphate (ATP) consumption, and power. ¹ Myosin is the major contractile protein in muscle, and it is a multimeric complex of two heavy and four light chains. Both heavy and light chains exist in multiple isoforms. ^{2 3} The myosin heavy chain (MyHC) contains the adenosine triphosphatase (ATPase) and the actin binding site and thereby determines speed of contraction as well as contraction force. ^{2 4} Hence, MyHC composition is regarded as the best marker of functional heterogeneity among muscle fibers. Data on the myosin heavy chain composition of the fibers in human extraocular muscles are scarce. ^{5 6} The purpose of the present study was to determine the MyHC composition of the fibers in the human EOMs, to gain further insight into the functional properties of these unique muscles. 

The EOMs are among the most fascinating muscles in the human body. They are responsible for highly coordinated and complex movements that are as diverse as the fast precise saccades, the smooth slow pursuit and vergence movements, and fixation at a given position. The complexity of actions performed by the EOMs is reflected in their cytoarchitecture and fiber type composition, which differ from ordinary skeletal muscles in many respects. The fibers of the EOMs are organized into two layers: a thin orbital layer facing the orbital wall and a global layer facing the bulb. ⁷ Recently, a third layer—the marginal zone, covering parts of the outer surface—has been described in human EOMs. ⁶  

Considerable effort has been put into classifying the extraocular fibers using different histochemical stains. ^{8 9} Although the original paper by Mayr ⁸ describes the singly innervated fibers of the global layer of the rat as representing a “continuous spectrum of muscle fibers whose diameters and staining intensities vary in inverse proportions,” and clearly states that “The dark, intermediate, and pale fibers—together 90% of the fibers in the global layer—are difficult to be counted separately, as they constitute a continuous fiber spectrum with all kinds of transitional forms between two extremes,” a classification into six fiber types is presented for the rat EOMs. Similarly, in a study of the human EOMs, it is stated that the “NADH-TR and SDH activity was classified into three grades from low (+) to intense (+++) although they showed a continuous transition” ⁹ and 29 groups of fibers with different histochemical profiles were merged into a classification of 6 fiber types, which excluded approximately 10% of the fibers in the orbital layer. The need to simplify and organize has clearly been very strong, and it has become generally accepted that there are six fiber types in the EOMs of all mammalian species studied to date. ^{10 11} These six fiber types are described on the basis of fiber location, innervation, and color as follows: orbital multiply innervated, orbital singly innervated, global multiply innervated, global red singly innervated, global intermediate singly innervated, and global pale singly innervated fibers. ^{9 12} However, Wasicky et al. ⁶ found only five fiber types in human EOMs on the basis of location and oxidative activity: global granular singly innervated, global coarse singly innervated, global multiply innervated, orbital singly innervated, and orbital multiply innervated fibers. 

The adult EOMs have been reported to express a large number of MyHC isoforms, including a specific fast isoform, MyHCeom, ^{11 13 14} in addition to the typical slow and fast MyHC isoforms. ^{3 15} The MyHC repertoire of the EOMs also includes: embryonic and fetal MyHC, ^{5 13 16} slow tonic MyHC (MyHCsto), ^{17 18} and MyHCα-cardiac. ¹⁹ A correspondence between the six fiber types and MyHC content in the rat EOMs has been suggested, but not actually shown. ¹¹  

Most data on the EOMs have been collected in other species, ^{8 11 16 20 21 22 23 24 25 26 27 28} and, in particular, only a few studies consider the MyHC composition of the human EOMs. ^{5 6 19 29} In a recent study, ⁶ the distribution of fast unspecified, slow, embryonic, and fetal MyHCs was investigated at the fiber level in human EOMs, but data on the distribution of the remaining MyHCs are not available. The purpose of the present study was to characterize thoroughly the human EOMs and the levator palpebrae superior (LP), with respect to fiber type composition and their MyHC content, by using SDS-PAGE and immunocytochemistry with a large battery of monoclonal antibodies (mAbs). Our results show an impressive level of complexity in the MyHC composition of the fibers in the human EOMs, most likely reflecting a wide array of contractile properties. ²⁸  

Material and Methods

Fourteen EOM samples were obtained from five males (ages 17, 32, 34, mid-30s, and 81) and one female (age 26) at autopsy, according to the ethical recommendations of the Swedish Transplantation Law, with the approval of the Medical Ethics Committee, Umeå University, and adhering to the tenets of the Declaration of Helsinki. 

The samples were obtained from the posterior and middle parts of the rectus superior (five samples), rectus lateralis (three samples), obliquus superior (two samples), rectus inferior (one sample), and levator palpebrae (three samples). The anterior part of the muscles was not available because of donation procedures. The samples were rapidly frozen in propane chilled with liquid nitrogen and stored at −80°C until used. Series of 10 to 35 cross sections, 5 to 10 μm thick, were treated to display ATPase activity after preincubation at pH 4.3, 4.6, and 10.4 ³⁰ or processed for immunocytochemistry ⁵ with well-characterized mAbs, each recognizing distinct MyHC isoforms (Table 1) . The specificity of mAb 4A6 against MyHCeom has been assessed in human tissue with immunohistochemistry, ⁵ and it does not react with MyHCI, MyHCIIa, MyHCIIx, MyHCα-cardiac, MyHCemb, or MyHCfetal. In immunoblots mAb 4A6 labels the MyHCeom band weakly. Control sections were processed the same as experimental sections, except that the primary antibody was replaced by normal serum from the same species and at the same dilution. No staining was observed in the control sections. 

Two to eight areas from each of the muscles were studied in detail. The selection of these areas was not completely random, because it was necessary to choose areas recognizable in all sections processed with each of the mAbs to be able to establish the staining pattern of the individual fibers. The staining patterns of 3203 fibers were analyzed in detail. The fiber area was measured on sections from rectus superior, obliquus superior, and levator palpebrae, stained with mAb 4C7 against laminin α5 chain, ³⁷ which delineated the contours of the muscle fibers. The areas of 885 fibers were measured with an image analysis system (IBAS; Kontron Elektronik GmBH, Eching, Germany). 

Whole muscle extracts were prepared from frozen samples of adult EOMs. The samples were taken from two rectus superior, one rectus lateralis, one obliquus superior and one levator palpebrae. Samples were also taken from psoas muscle (MyHCI, MyHCIIa, MyHCIIx), heart (MyHCI, MyHCα-cardiac), and fetal limb muscles (MyHCI, MyHCfetal, MyHCemb), as previously described. ³⁸ SDS-PAGE was performed ³⁹ (Mini Protean II; Bio-Rad Laboratories, 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 then stained silver ⁴⁰ and photographed. 

Immunoblot analysis (WesternBreeze kit; Invitrogen, La Jolla, CA) was used to further establish the identity of the MyHC bands separated by SDS-PAGE. After SDS-PAGE, proteins were transferred to 0.45-μm nitrocellulose membrane (Bio-Rad Laboratories) for 17 hours at 30 V with the unit surrounded by a 15°C water bath. mAbs A4.840, 4A6, A4.74, and 2B6 were used to identify the bands containing MyHCI, MyHCeom, MyHCIIa, and MyHCemb, respectively (see Table 1 for references). 

Results

Biochemistry

Electrophoresis of whole-muscle extracts of the sampled EOMs and LP revealed four MyHC bands that were identified ⁴¹ as MyHCI (the fastest migrating band), MyHCeom, MyHCIIa, and MyHCemb (the slowest migrating band) both by comparison with the migration patterns of extracts with known MyHC composition (Fig. 1) and with immunoblots (Fig. 2) . The relative proportions of these MyHCs varied among the EOM samples tested. In some cases, MyHCI was present in trace amounts only. 

Morphology

The fibers in the EOMs were small, round, and loosely arranged in fascicles. The relative positions of the fibers varied through a series of sections, and adequate care was taken to identify correctly the same fiber in consecutive sections. 

Two different layers of fibers were identified in all four recti plus the obliquus superior. The orbital layer was 5 to 30 fibers deep, and in some cases it completely surrounded the global layer, whereas in other cases it covered only one side of the muscle. A marginal zone, as described by Wasicky et al., ⁶ with large fibers and much connective tissue, was not easily identified in our samples, because the outermost part of the orbital layer generally consisted of small and closely arranged fibers. In the LP no orbital or global layers were discerned. 

The size of the fibers varied greatly (Table 2) . Fibers in the orbital layer had significantly smaller mean areas (260 ± 160 μm²) than fibers in the global layer (440 ± 200 μm²; P < 0.0001), in agreement with earlier studies. ¹²  

Enzyme Histochemistry

A small subpopulation of fibers in both layers exhibited strong mATPase activity after preincubation at pH 4.3 and moderate mATPase activity at pH 10.4 (Fig. 3) . In a previous study, fibers with a similar pattern of mATPase activity were assumed to correspond to the global and orbital multiply innervated fibers. ⁶ The remaining fibers had more homogeneously high ATPase activity at pH 10.4 and displayed low or practically no activity at pH 4.3 (Fig. 3) . Such fibers have been assumed to correspond to the singly innervated fibers of both layers. ⁶ Although we did not investigate the innervation pattern of the fibers, we will use the terms “singly” and “multiply” innervated, as did Wasicky et al. ⁶ . The mATPase activity of the fibers at pH 4.6 was somewhat higher than at pH 4.3. The reduced nicotinamide adenine dinucleotide (NADH) activity in the fibers in the EOMs was generally high, and it was more homogeneous in the orbital than in the global layer (Fig. 3) . In the global layer, there was appreciable heterogeneity both in the level of NADH activity and in the appearance of NADH staining. The multiply innervated fibers tended to have lower NADH activity in both layers. The NADH activity of the singly innervated fibers in the global layer varied in a continuum, from very high to low, and distinction among them for classification into further fiber groups was not possible. 

Immunocytochemistry

The EOMs revealed distinct patterns of immunoreactivity to each antibody used. A striking feature of the EOMs was the heterogeneity in fiber composition among neighboring fascicles (Fig. 4) . 

Anti-MyHCI+IIa+eom stained all the fibers in the EOMs (Fig. 5A) . Anti-MyHCIIa stained most of the fibers in the EOMs strongly to moderately, but there were areas in the global layer that were practically devoid of stained fibers (Figs. 4 5B) . Anti-MyHCI and anti-MyHCsto had an almost identical staining pattern consisting of few heavily stained and in general evenly distributed fibers in both layers (Figs. 5C 5D) . However, a small amount of fibers stained with anti-MyHCI but not with anti-MyHCsto. Staining with anti-MyHCα-cardiac (Fig. 5E) and anti-MyHCemb (Fig. 4) was preferentially found in the fibers of the orbital layer, but their staining patterns were not similar at the fiber level. Anti-MyHCeom stained fibers in both layers and the staining intensity was often higher in the areas unstained by anti-MyHCIIa (Fig. 5F) . 

The EOMs

In the global layer, three fiber types were distinguished on the basis of their patterns of immunoreactivity. The majority of the fibers (approximately 60% in the areas sampled) stained strongly with anti-MyHCIIa, approximately 15% of the fibers were labeled with anti-MyHCI, and the remaining 25% of the fibers were not stained with either of these two antibodies, but were reactive to anti-MyHCeom and anti-MyHCI+IIa+eom. These will hereafter be called MyHCeom^pos/MyHCIIa^neg fibers (Fig. 6) . The MyHCeom^pos/MyHCIIa^neg fibers were generally larger (590 ± 210 μm²) than the fibers stained with anti-MyHCIIa (320 ± 190 μm²) or with anti-MyHCI (280 ± 140 μm²) in both layers (P < 0.0001). 

The slow fibers, in the sense that they were stained with anti-MyHCI, had acid-stable ATPase activity (Fig. 7) , whereas the fibers stained with anti-MyHCIIa and the MyHCeom^pos/MyHCIIa^neg fibers had alkaline-stable ATPase activity (not shown). In the orbital layer, approximately 80% of the fibers were stained with anti-MyHCIIa, 17% were stained by anti-MyHCI, and only a few fibers (3%) were MyHCeom^pos/MyHCIIa^neg fibers. Almost all the slow fibers stained with both anti-MyHCI and anti-MyHCsto in both layers. Furthermore, anti-MyHCα-cardiac stained approximately 30% of the slow fibers in the orbital and 7% in the global layer. Eight percent of the slow fibers stained with anti-MyHCI but not with anti-MyHCsto or with anti-MyHCα-cardiac in both layers. 

MyHCemb was coexpressed in some of the fibers of all the groups, and MyHCeom, detected by mAb 4A6, was also present to some extent in the groups containing MyHCIIa and MyHCI. Sporadic fibers (<1%) that did not fit into any of these staining patterns were also observed. In the orbital layer of one sample, there were a few fibers stained with anti-MyHCsto but not with anti-MyHCI or anti-MyHCemb. 

Levator Palpebrae Superioris

The fibers in the LP were significantly larger (470 ± 320 μm²) than those in the EOMs (340 ± 200 μm²; P < 0.0001). Of the fibers sampled in the LP, 77% stained with anti-MyHCIIa and 20% with anti-MyHCI, and 3% were MyHCeom^pos/MyHCIIa^neg fibers. No fibers stained with anti-MyHCsto. Nevertheless, there were two fibers in the sampled areas that stained with anti-MyHCI and anti-MyHCα-cardiac in addition to anti-MyHCeom. There was no capsule surrounding these two fibers, and they did not seem to be muscle spindle fibers. Half of the fibers in the LP stained with anti-MyHCeom and less than 1% of them stained with anti-MyHCemb. 

Discussion

The MyHC composition of the human EOMs at the fiber level proved to be much more complex than anticipated. 

MyHC Content and Antibody Specificity

Four MyHCs (MyHCI, MyHCeom, MyHCIIa, and MyHCemb) were present in the EOMs and LP muscles in sufficient amounts to be detected by SDS-PAGE. In addition, the antibodies used are well characterized, and their specificity has been further verified in immunoblots. ³¹ Therefore, we can be sure that the complex patterns of reactivity obtained with immunocytochemistry reflect differences in the MyHC composition of each myofiber and each EOM. 

ATPase Activity and MyHC Composition

Most of the human EOM fibers examined contained more than one MyHC isoform. This fits with the observed continuum in mATPase activity. The mATPase reaction does not allow the discrimination of mixtures of MyHCs, particularly when both fast and developmental isoforms are present. ⁴² The fibers containing MyHCI and MyHCsto exhibited strong mATPase activity at pH 4.3 and moderate at pH 10.4. This pattern of activity is similar to that reported for bag2 fibers in human muscle spindles, which also contain both MyHCI and MyHCsto. ³¹ 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. 

Fiber Type Composition

The MyHC isoforms can be classified as fast (MyHCIIa, IIx, IIb, eom), slow (MyHCI, α-cardiac, slow tonic), or developmental (embryonic, fetal). ⁴² The human EOMs contained two groups of fibers with adult fast MyHC isoforms: fibers containing MyHCIIa and fibers without MyHCIIa or MyHCI, but containing MyHCeom. Some of these fibers containing fast IIa MyHC were also stained with the mAb against MyHCeom. Furthermore, all these fibers containing adult fast MyHCs, showed moderate to strong staining with the mAb against MyHCemb. 

There were also fibers containing the adult slow isoform MyHCI. Fibers containing only MyHCI were rare, and most slow fibers coexpressed MyHCI and MyHCsto. Published data have shown that fibers with the acid stable ATPase activity reported in this study ⁶ and containing MyHCsto ¹⁸ correspond to the previously described orbital and global multiply innervated fiber groups. In the areas sampled, the fibers containing MyHCI and MyHCsto represented approximately 16% of the total in the orbital layer and 13% in the global layer, which roughly fits the values reported for the rat multiply innervated fibers. ⁸ However, our data showed that a subpopulation of fibers expressing both MyHCI and MyHCsto also expressed MyHCα-cardiac, which is also a slow isoform. Additional staining with anti-MyHCemb and anti-MyHCeom was unexpectedly present in many fibers containing slow isoforms. In summary, our data show important heterogeneity among the slow fibers, and we speculate that these fibers containing MyHCI but not MyHCsto are likely to be singly innervated. 

In a recent immunohistochemical study of the rat EOM, a possible correspondence between the previously described six fiber types ¹⁰ and the predominant expression of a given MyHC has been suggested, although not really investigated ¹¹ : Global multiply innervated fibers were 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 were proposed to contain predominantly embryonic MyHC and either MyHCeom (singly innervated fibers) or MyHCI (multiply innervated fibers) in the middle region. ¹¹ An earlier study, ⁴³ in contrast, reported that both multiply and singly innervated fibers in the rat orbital layer contain MyHCII near the endplate band and MyHCemb at the ends. Apart from that, the staining pattern of rat EOM fibers is obviously not as complex as that of human EOM fibers. In particular, the clear distinctions in immunoreactivity between the orbital and global layers reported in the rat ¹¹ were not present in the human muscles. Notably, MyHCeom ^{11 16} and MyHCα-cardiac ¹¹ are restricted to the orbital layer in the rat, whereas we found these isoforms in fibers of both layers, although anti-MyHCeom was often more common in the orbital layer, especially in the MyHCeom^pos/MyHCIIa^neg fibers. Rubinstein and Hoh ¹¹ also found that the rat orbital singly innervated fibers contain only MyHCemb in the distal and proximal portion of the EOM. We studied sections from all parts of the EOMs, except the most anterior part, but found no section where anti-MyHCemb stained more than two thirds of the orbital singly innervated fibers, and all these fibers were also stained by anti-MyHCIIa and/or anti-MyHCeom. 

The simpler features of adult rat EOMs in comparison with those of humans are generally in agreement with the fundamental differences reported in the developing patterns of these muscles in the two species. ^{5 16}  

Variation and Complexity

Marked variation in fiber composition was noted among the fascicles of any given EOM. The differences observed were quantitative rather than qualitative and were related to the relative abundance of each fiber type. They further illustrate the complexity of these muscles and suggest the presence of distinct contractile properties, even within parts of each layer. Altogether, our data indicate that the cytoarchitecture of the human EOMs is far more complex than could be attributed to the presence of two separate layers, given that all fiber types were present in both layers but with a very heterogeneous distribution within the layers. Some fascicles appear clearly more suited for very fast contractions, as they are almost purely composed of MyHCeom^pos/MyHCIIa^neg fibers whereas others may be more apt for intermediately fast contractions, where MyHCIIa predominates. Still other regions have a more balanced fiber type composition involving both slow and fast fiber types. Such a heterogeneous fiber type distribution among adjacent regions of the same layer probably allows and/or reflects a wide range of contractile behaviors and the capacity to switch among very distinct motor tasks. Furthermore, each of the fiber types exhibited further variation in the level of staining with the mAb against MyHCemb, which can be interpreted as an additional level of complexity probably allowing further fine-tuning of their range of contractile properties. 

Finally, the MyHC composition was not constant along the length of the EOMs. Our specimens did not allow us to follow the same fibers along their entire length, but it clearly showed that the distribution of MyHCIIa varied along the muscle length in both the global and orbital layers (Fig. 4) . In the rat, heterogeneity in MyHCemb composition has been reported along fibers of the orbital layer. ^{11 43} However, no such heterogeneity has been noted for the fibers of the global layer, showing again that the human EOMs differ significantly from those of other species. The increased complexity of the human EOMs in comparison to other species may reflect the human’s much-expanded oculomotor range and increased reliance on binocular vision and vergence. Further studies are needed to elucidate the variation in MyHC composition along the length of the human EOMs. 

Additional MyHCs

The genes for human MyHC2B and MyHCeom have been sequenced from human extraocular muscle samples ⁴⁴ and 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 EOM gene or whether it corresponds to the MyHC2B gene or both. Studies are under way at our laboratory to clarify this question. In the meantime, we use the previous nomenclature that tentatively identifies the unique band in SDS-PAGE as MyHCeom. ^{20 41}  

MyHCIIx can be identified in human muscle fibers by immunocytochemistry, using an mAb that labels all MyHCs except MyHCIIx. Because practically all fibers in the EOMs coexpressed more than one MyHC isoform, this antibody is of no use. 

Further Studies

The question regarding the organization of these different mixtures of MyHCs into myofibrils along the EOM fibers remains to be investigated, and it is likely to provide further information on the functional properties of the fibers comprising each of the individual EOMs. 

 Supported by grants from Stiftelsen KMA, the Swedish Society for Medical Research, the Swedish Research Council and the Medical Faculty of Umeå University.

 Submitted for publication June 28, 2002; revised September 13, 2002; accepted October 8, 2002.

 Disclosure: D. Kjellgren, None; L.-E. Thornell, None; J. Andersen, None; F. Pedrosa-Domellöf, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Data on the Antibodies Used

Antibody	Specificity	Short Name	Reference
A4.74*	MyHCIIa	Anti-MyHCIIa	31 32
A4.840*	MyHCI	Anti-MyHCI	23 31
N2.261*	MyHCI	Anti-MyHCI+IIa+eom	23 31
	MyHCIIa
	MyHCextraocular
	MyHCα-cardiac
ALD19, †	MyHCslow tonic	Anti-MyHCsto	33 34
F88, ‡	MyHCα-cardiac	Anti-MyHCα-cardiac	35
4A6, §	MyHCextraocular	Anti-MyHCeom	5 26 27
2B6, ∥	MyHCembryonic	Anti-MyHCemb	31 34 36

Figure 1.

 

SDS-PAGE of human obliquus superior (MyHCI, MyHCeom, MyHCIIa, MyHCemb), psoas muscle (MyHCI, MyHCIIa, MyHCIIx), fetal limb muscle (MyHCI, MyHCfetal, MyHCemb), and atrium (MyHCI, MyHCα-cardiac). MyHCemb and MyHCIIx migrated together, but the uppermost band of the obliquus superior extract was labeled by mAb 2B6 recognizing MyHCemb (not shown). Note that MyHCeom migrated slightly slower than MyHCα-cardiac.

Figure 2.

 

Immunoblot of human obliquus superior, rectus superior, and psoas muscle, labeled with mAb N2.261. mAb N2.261 labeled MyHCeom in addition to MyHCI and MyHCIIa. The amount of MyHCI transferred to the nitrocellulose membrane was below detection level in the obliquus superior sample.

Table 2.

 

Differences in Fiber Size between Muscles, Muscle Layers, and Fiber Types

	Fiber Count	Mean Area (μm2)
Muscle
EOMs	756	340 ± 200
Levator	123	470 ± 320
Layer
Orbital	415	260 ± 160
Global	341	440 ± 200
Fiber type (EOM), containing
MyHCIIa	541	320 ± 190
MyHCI	135	280 ± 140
MyHCeompos/MyHCIIaneg	80	590 ± 210

Figure 3.

 

Photomicrograph of the orbital layer (left column) and global layer (right column) from three sections of a rectus medialis, treated to show NADH activity (top row), mATPase activity at pH 10.4 (middle row) and mATPase activity at pH 4.3 (bottom row). Arrows: examples of slow, presumably multi-innervated fibers with high mATPase activity at pH 4.3.

Figure 4.

 

Photomicrograph of four sections, from the posterior (left column) and midbelly (right column) portions of the same rectus superior immunostained with anti-MyHCIIa (top row) and antiMyHCemb (bottom row). Notice that large areas in the global layer of the midbelly section are practically unstained by anti-MyHCIIa and that many fibers in the same areas are stained with anti-MyHCemb. OL, orbital layer; GL, global layer.

Figure 5.

 

Photomicrographs of six sections from rectus medialis, global layer, immunostained with (A) anti-MyHCI+IIa, (B) anti-MyHCIIa, (C) anti-MyHCI, (D) anti-MyHCsto, (E) anti-MyHCα-cardiac, and (F) anti-MyHCeom. Sections in (E) and (F) were also stained with anti-laminin, to facilitate identification of the fibers. Anti-MyHCemb did not stain any fibers in this area (not shown). Note the global layer (GL) to the left and the orbital layer (OL) to the right. (✶) An area of the global layer with many MyHCeom^pos/MyHCIIa^neg fibers; arrows: fibers stained with anti-MyHCI but not with anti-MyHCsto.

Figure 6.

 

Photomicrographs of five sections from obliquus superior, global layer, immunostained with (A) anti-MyHCI+IIa+eom, (B) anti-MyHCIIa, (C) anti-MyHCI, (D) anti-MyHCsto, and (E) anti-MyHCeom. (○) Two fibers stained with anti-MyHCIIa; arrow: a fiber stained with anti-MyHCI; (⋆) two MyHCeom^pos/MyHCIIa^neg-fibers.

Figure 7.

 

Photomicrographs of two sections from rectus superior, global layer, treated to show mATPase activity at pH 4.3 (A) and immunostained with anti-MyHCI (B). Note the similarity in staining pattern. Arrows: examples of fibers.

The authors thank Mona Lindström, Margaretha Enerstedt, and Anna-Karin Olofsson for excellent technical assistance.