Introduction
The extraocular muscles (EOM) are unique in their structure and function. The extraordinary functional demands including globe rotation, imposed upon these muscles made them the fastest and the most fatigue resistant skeletal muscles [
10]. Compared to other skeletal muscles they are not respecting the traditional fiber type classification schemes. EOM differ in their histochemical profile, type of innervation and fiber type distribution [
48,
51].The sequential development of EOM fiber types is believed to be conserved across the mammalian species, but may follow a different sequence in frontal and lateral-eyed species [
36]. In rodents, the composition of EOM is in principle similar to that of other mammals, including humans, however species differences were reported and mostly concern muscle fiber type characteristics [
16].
Several attempts at classification of fiber types in EOM of different mammals, i.e. rodents, monkeys, cats, sheep, rabbits and humans have been made by studying the morphology, histochemical characteristics and /or ultrastructure [
1,
2,
7,
9,
19,
25,
27,
30,
31,
39,
48,
55]. However, the most pertinent classification system used at present is still descriptive and incorporates different classification schemes [
48]. It distinguishes among six fiber types in EOM according to (i) their location in the global (GL) or orbital layer (OL), (ii) type of innervation (i.e. singly (SIF) and multiply (MIF) innervated fibers) [
35], (iii) the staining for the myofibrillar adenosintriphosphatase (mATPase) reaction after preincubation in acid and alkaline medium (slow or type I and fast or type II fibers), and (iv) metabolic profile (oxidative, oxidative-glycolytic, glycolytic). Still, this scheme remains limited in recognizing the full extent of the muscle fiber heterogeneity in EOM, not considering the myosin heavy chain (MyHC) isoform composition, as suggested by McLoon and co-workers [
28].
In fact, MyHC isoforms are the ones that determine the activity of mATPase and the shortening velocity of myofibers. In large human and rat skeletal muscles four fiber types (I, IIc, IIa, IIb in rat and I, IIc, IIa, IIx in humans) can be distinguished with the histochemical reaction for mATPase after preincubation in alkaline and acid media. Each fiber type expresses corresponding MyHC isoform, except type IIc fibers, which co-express MyHC-1 and -2a [
12,
35]. However, in rat, an additional fast MyHC, i.e., IIx or IId (henceforth -2x) can be distinguished immunohistochemically [
4,
42].
In spite of all the complex research, the classification of muscle fiber types in EOM is still not fully clarified, and neither is the correlation between its structure and function. The studies that considered the MyHC composition of the rat or human EOM [
4,
6,
8,
13,
19,
20,
23,
34,
40,
41,
51,
55,
56] applied different methods and therefore the results are mostly not comparable. The reported expression of MyHC isoforms in EOM is not consistent although it is generally agreed that EOM express all the MyHC isoforms present in other striated muscles. Besides these common MyHC isoforms, the EOM-specific extraocular (MyHC-eom), α-cardiac (MyHC-α) and developmental MyHC isoforms, i.e., MyHC-embryonic (-emb) and -neonatal (-neo) are expressed in EOM as well.
The parallel data on serial histo- and immunohistochemical EOM profiles of MyHC isoforms in rat and human EOM have been scarce so far. In this study, we applied the above-mentioned methods and in case of human EOM in situ hybridization as well to obtain an additional insight into the fiber type characteristics and MyHC isoform expression in normal rat and human ocular medial rectus muscles (MR). The purpose of this study was also to determine whether the rat EOM could serve as a model for human EOM structure in experiment and disease, despite rat’s less complex visuomotor repertoire.
Discussion
In this study we have proved that almost identical muscle fiber types exist in rat and human extraocular muscles. The majority of fibers are hybrid fibers, co-expressing two or more MyHC isoforms. To our knowledge in this study the expression of MyHC-2b isoform and MyHC gene transcripts in human EOM muscle has been demonstrated for the first time. Furthermore, to our knowledge this is the first study in which the distribution of MyHC isoforms in the extraocular muscle fibers of different species was compared.
We confirmed that muscle fibers of human and rat MR muscle are organized into a thicker GL and a thinner OL [
33]. Previously described marginal zone (MZ) adjacent to the OL was more evident in human than in rat ocular MR muscles, however it was thin and was to our observation similar to the fibers positioned between the OL and the GL [
55].
As previously found in rat [
48,
49], the muscle fibers of larger diameter (from 20 to 40 μm) with various metabolic activity were present in the GL, while in the OL smaller, (less than 20 μm), mostly highly oxidative muscle fibers were found in both species. The human MR muscles were larger due to the higher absolute number of muscle fibers, while the muscle fiber diameters were within the range of rat muscle fiber diameters. Interestingly, the muscle fibers of monkey are larger, more variable in size, and fewer than in human EOM (e.g., 8,000–11,800 fibers in monkey vs. 17,700 to 24,500 fibers in humans) [
20,
21].
In large skeletal muscles, three to four major fiber types can be revealed by mATPase histochemistry [
12]. However, in ocular MR muscles the mATPase histochemistry alone was insufficient to completely distinguish fiber subtypes although a variable incubation time in alkaline and acid media had been used within a wide span of pH values [
39,
50,
51].
Nevertheless, following the already described simplified classification system applied so far for EOM fiber types according to which six fiber types can be distinguished regarding to their location, type of innervation [
36,
37,
48,
55], the mATPase activity and metabolic profile [
37]. Slow fibers determined in this study obviously corresponded to multiply innervated fibers (MIF), while fast fibers corresponded to singly innervated fibers (SIF) described in other studies [
37,
48]. In the GL we could identify at least three fast fiber types (labeled as 3, 4, and 5), besides one slow or one hybrid slow/fast fiber type (labeled as 6). Fiber type 3 was, oxidative and thus obviously fatigue resistant, fiber type 5 was glycolytic and must be fatigable, and fiber type 4 was of intermediate oxidative-glycolytic type (Table
1 and Fig.
2). In the OL, additionally, one fast oxidative fiber type (number 1, Table
1, Fig.
2) in both, human and rat MR muscles was identified.
In both species the single slow type fibers in the GL (number 6 in Table
1 and Fig.
2) and OL (number 2, Table
1, Fig.
2) exhibited low oxidative and low glycolytic activity. These were fatigable pure slow or hybrid fibers coexpressing slow MyHC with other fast MyHC isoforms. The functional role of these fibers is not well understood. Since these global slow type fibers, (called also MIF fibers), develop first they probably allow proprioceptive information to be used in visual system development (36). Orbital fast fibers (called also SIF fibers) mature the last and may directly impact the range and precision of eye movements. They contain the largest bunches of mitochondria, which is consistent with the elevated fatigue resistance and a sustained level of eye position maintenance.
To better define the fiber types according to their oxidative capacity, the reaction for SDH was used in the present study, as the activity of the other marker for oxidative metabolism, the nicotineamide adenine dinucleotide dehydrogenase (NADH) is generally very high in EOM and it hardly distinguishes among fiber types, especially in human EOM [
19,
50]. However, the reported proportions of GL slow fibers in human EOM vary, ranging from 10 to 30% [
55]. Such variability in results is most probably due to the variable length of muscle fibers, extending from the proximal to the distal part of human extraocular muscle [
2,
27,
39,
40,
48,
55].
The unique extraocular MyHC isoform (MyHC-eom), considered as a fast one and not detected in limb muscles, is obviously abundantly expressed in EOM [
3,
56]. Until recently, its presence could only be proved with SDS polyacrylamide gel electrophoresis (SDS-PAGE). The share of MyHC-eom confirmed in rat EOM homogenates was 25 % of the total muscle myosin [
3,
51]. In this study, many OL muscle fibers of both species stained positively with the commercially available monoclonal antibody 4A6 against MyHC-eom. On the contrary, the fibers of the GL were labeled less intensively with this antibody (Fig.
5). Therefore we assume that MyHC-eom is co-expressed with other isoforms in many fibers, especially in the OL. Similarly, Rubinstein and Hoh [
40] found MyHC-eom in orbital fast fibers in rat EOM as well Kjellgren and co-workers reported that approximately 25% fibers in GL of other three human extraocular rectus muscles (the MR was not analyzed), co-expressed MyHC -eom and -2a, but contrary to our findings they found very few MyHC-eom expressing fibers (3%) in the OL [
19]. Further, in the rabbit EOM the MyHC-eom expression was determined in both, the GL and OL [
5], while in dog its presence was confirmed only in the OL [
5].
The presence of another fast MyHC isoform, i.e., -2x, expressed in large skeletal muscles as well, was not clearly confirmed in EOM due to lack of an antibody specific to MyHC-2x and due to similar migration of MyHC-2a and -2x isoforms in gels after SDS-PAGE in previous studies [
3,
19]. However, applying an antibody specific to MyHC-2x (6H1), in this study we undoubtedly demonstrated that MyHC-2x isoform is abundantly expressed in human EOM fibers and in many rat muscle fibers of both layers.
Furthermore, applying monoclonal antibodies BF-F3 in rat and 10F5 in humans we confirmed the presence of MyHC-2b isoform, not only in rat but in human EOM as well. To our knowledge this is the first study in which MyHC-2b isoform has been revealed in any human skeletal muscle, though 2b MyHC transcripts were demonstrated in human external abdominal oblique and masseter muscles [
17]. It should be stressed that the 10F5 antibody did not label any fiber in large limb muscles (biceps femoris, vastus intermedius, vastus lateralis), but it did in human EOM, indicating that MyHC-2b isoform is expressed in these “specific, very fast contracting” muscles.
Furthermore, the expression of MyHC-2b isoform in human MR was additionally confirmed with undoubtedly revealed expression of MyHC-2b transcripts by in situ hybridization technique (Fig.
4). Though the expression of MyHC gene transcripts in human EOM did not correlate well with MyHC isoform expression, it was not completely uninformative as the probe specific for MyHC-2b transcripts hybridized in MR, but gave absolutely negative results in the large limb muscles (not shown), placed on the same slide as MR muscles and processed simultaneously for in situ hybridization technique.
An explanation for such discrepancy in MyHC gene and isoform expression could be a possible posttranscriptional MyHC gene regulation not only for MyHC-2b but also for other isoforms [
15]. Another possibility for so ambiguous results of in situ hybridization technique could also be that the expression of MyHC transcripts in EOM is less abundant than in large skeletal muscles and that the method is not sensitive enough to offer better results, though good results were obtained in large skeletal muscles [
47].
Similarly, as found in human EOM, MyHC-2b expression has been detected in bovine EOM but not in trunk and limb muscles [
26,
54]. According to previous studies [
7,
40,
51,
52], MyHC-2b is the predominant isoform in the rat EOM global region (50%). In dog, the predominance of MyHC-2b isoform (50%) in the GL of the muscle belly was also found, though like in humans it was not found in large skeletal muscles [
46]. In the OL of the belly region MyHC-eom predominated (65–75% of total MyHC) [
5]. Evaluating mRNA levels by competitive polymerase chain reaction high expression of MyHC transcripts coding for fast MyHC isoforms (MyHC-2x 29.9%, MHC-2a 29.3%, MyHC-2b 24.5%) was also found in the adult rat EOM [
23].
Another specificity of EOM muscles is the expression of developmental MyHC isoforms even in adult stage. The presence of MyHC-emb and -neo isoforms predominantly in OL within the midbelly region of adult EOM, reported previously [
34,
40,
51,
53,
55] and confirmed in this study, seems to be a unique characteristic of the muscles innervated by cranial nerves. However, it was found that the pattern of MyHC isoform expression may change along the length of rabbit and rat EOM fibers, whereby the majority of fast and developmental MyHC isoforms expressing fibers were present in the middle muscle region of the OL and less in the GL [
18,
25,
28,
41].
The characteristic co-expression of two to three MyHC isoforms for EOM has been described in skeletal muscles as well [
43,
45,
47,
49,
51], however the co-expression in such a great extent (over 60% of muscle fibers) is a unique feature of EOM. Moreover, the hybrid fibers are even more numerous in human than in rat MR muscles as found in this study (Table
1, Figs.
3 and
5). However, the large share of hybrid fibers in human EOM could also be ascribed to the non-specificity of antibodies as they were not human specific, while in rat their specificity has been proved [
42]. Nevertheless, the EOM fibers obviously exhibit multiple patterns of gene regulation [
56], and due to continuous myogenesis triggered by the unique EOM stem cells [
29], the individual EOM fibers exhibited co-expression of multiple MyHC isoforms in both muscle layers of both species. The epigenetic influences, i.e., a visual maldevelopment, metabolic and neuromuscular diseases, probably essentially contribute to the understanding how the EOM phenotype is established [
8,
11,
19,
20,
28].
The comparison of EOM ontogeny in different species confirms that the sequential development of fiber types is conserved in frontal eyed animals but may follow a different sequence in lateral-eyed species. Although different cranial nerves innervate different extraocular muscles the EOM are similar in their function and structure. Actually, no difference in the fiber type composition of all six EOM among cat, rabbit, guinea pig and rat was found [
2]. It is also assumed that the EOM fiber types are largely conserved across mammalian species [
37].
In conclusion, the neuromuscular junction formation appears to follow similar pathways in rat, monkey, and humans [
36]. In the human and rat MR six muscle fibers types can be distinguished that are arranged within two muscle layers and are of similar size, in spite of large difference in body size of the two species [
50,
51]. However, the muscle bundles in the human MR muscles are larger and more numerous. Due to the more extensive connective tissue that surrounds individual fibers and bundles in human MR muscle, the fiber distribution does not appear as regular as in rat MR muscles. What is the functional consequence of this kind of the muscle arrangement, as well as greater extent of MyHC isoform co-expression in human than in rat MR, remains unclear and so does the overall understanding of the complex MR muscle physiology and pathophysiology.
Nevertheless, similar structure of human and rat MR muscle and similar characteristics of fiber types justify rat MR muscles to be a suitable model for experimental studies which would lead to better understanding of human MR muscles in experiment or disease.