Uncoordinated Expression of Myosin Heavy Chains and Myosin-Binding Protein C Isoforms in Human Extraocular Muscles

Abstract

purpose. To examine the distribution of myosin-binding protein C (MyBP-C) in human extraocular muscles (EOMs) and to correlate the myosin heavy chain (MyHC) and the MyBP-C composition of the fibers.

methods. Samples from 17 EOMs, 3 levator palpebrae (LP), and 6 limb muscles were analyzed with SDS-PAGE and immunoblot or processed for immunocytochemistry with monoclonal antibodies (mAbs) against MyBP-C-fast, MyBP-C-slow, MyHCIIa, MyHCI, MyHCsto, MyHCα-cardiac, and MyHCemb.

results. In the limb muscle samples, fast fibers were labeled with anti-MyBP-C-fast and anti-MyBP-C-slow, whereas the slow fibers were immunostained with anti-MyBP-C-slow only, in accordance with previous studies. In 11 EOM samples MyBP-C-fast was not detected, and weak staining with anti-MyBP-C-fast was seen only in a few fibers in the proximal part of 2 muscles. The mAb against MyBP-C-slow labeled all fibers, but fibers containing MyHCI were generally more strongly stained. In the levator palpebrae, immunostaining with anti-MyBP-C-fast was present in some fibers labeled with anti-MyHCIIa and/or anti-MyHCeom. MyBP-C-fast and -intermediate were not detected biochemically in the EOMs.

conclusions. The lack of MyBP-C-fast and intermediate is an additional feature of the human EOM allotype. The true EOMs have a unique myofibrillar protein isoform composition reflecting their special structural and functional properties. The levator palpebrae muscle phenotype is intermediate between that of the EOMs and the limb muscles.

The functional properties of muscle fibers vary considerably between different muscles. ¹ The extraocular muscles (EOMs) are among the most complex muscles in the body and have been considered a separate muscle allotype, ² due to their special features. The EOMs contain fibers with a wide array of contractile properties, varying from extremely fast to fibers capable of sustained, tonic contractions. Previously, we sought to elucidate the molecular basis of this unique allotype of human EOMs at the fiber level, ^{3 4 5} by determining the patterns of expression of myosin heavy chain (MyHC; the major determinant of heterogeneity of contraction force and velocity ^{6 7} ) and of SERCA-1 and -2 (determinants of the relaxation rate ⁸ ). The fibers in the human EOMs have very complex MyHC composition patterns, with most fibers containing more than one MyHC isoform. Moreover, differences in the relative amounts of a given MyHC isoform are typically observed among fibers sharing a particular combination of isoforms. ³ Despite this heterogeneity, the fibers in the human EOMs can be divided grossly into three major fiber groups, based on their content of MyHCI, MyHCIIa, and MyHCeom. ³  

Myosin-binding protein C (MyBP-C) is, next to myosin, the second most abundant thick-filament protein in striated muscles. ⁹ It is located in the A band, in a restricted part of the cross-bridge–bearing region. ¹⁰ MyBP-C is a ≈130 kDa protein and both its C terminus and N terminus bind to myosin. ^{11 12} MyBP-C is presumed to have a regulatory, although not essential, role in sarcomere assembly ^{13 14} and to play a physiological role in regulating contraction by modulating unloaded shortening velocity. ¹⁵ The importance of MyBP-C for muscle function is indicated by the fact that mutations in the gene for cardiac MyBP-C (MyBP-C-card) cause familial hypertrophic cardiomyopathy. ^{16 17 18}  

There are three major isoforms of MyBP-C in human muscle: fast skeletal (MyBP-C-fast), slow skeletal (MyBP-C-slow), and MyBP-C-card. ¹⁹ MyBP-C-fast, detected both with in situ hybridization and immunocytochemistry, is present in fast fibers, whereas MyBP-C-slow is present in both slow and fast fibers, in human skeletal muscle. ²⁰ The cardiac isoform is restricted to the heart and has never been detected in conjunction with any other MyBP-C isoforms in cardiac or skeletal muscle. ²⁰ The three human MyBP-C genes have been mapped and sequenced. ^{21 22 23} Recent analysis of single fibers by SDS-PAGE, revealed the coordinated expression of MyBP-C-slow in fibers containing MyHCI (slow); MyBP-C-fast in fibers with MyHCIIx (fast); and an additional isoform, MyBP-C-intermediate, in fibers containing MyHCIIa (fast) in human limb muscle. ²⁴ Coordinated isoform changes, indicating that MyBP-C expression is linked to MyHC expression, have been reported during skeletal muscle hypertrophy in the rat. ²⁵ However, in the human masseter, a masticatory muscle with rather unique properties, ^{26 27 28} the very complex MyHC composition of its fibers was not paralleled by an intricate MyBP-C pattern. ²⁴  

Data on the MyBP-C composition of human EOMs and its correlation to the MyHC composition at the protein and cellular level are lacking. In the present study, we investigated the distribution of the fast and slow isoforms of MyBP-C in relation to the MyHC profile of the fibers and found further evidence of the uniqueness of the molecular portfolios of the fibers in the human EOMs. 

Material and Methods

The muscles were collected according to the ethical recommendations of the Swedish Transplantation Law, with the approval of the Medical Ethics Committee, Umeå University, and in compliance with the Declaration of Helsinki for research involving human tissue. Seventeen EOM samples were obtained at autopsy from six men and one woman (ages, 17, 26, 27, 34, 34, 81 and 86 years) who had had no known neuromuscular disease. The samples were mounted on cardboard, rapidly frozen in propane chilled with liquid nitrogen, and stored at −80°C until used. 

SDS-PAGE and Immunoblots

Whole-muscle extracts were prepared from one rectus superior, one rectus lateralis, two obliquus superior, one levator palpebrae (LP), and one brachioradialis muscle, as previously described. ²⁹ MyBP-C isoforms were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using 4% (wt/vol) stacking and 8% running gels. Gel buffer solutions included 10% glycerol. ²⁴ Proteins were identified on the basis of their molecular mass, immunoreactivity, and order of migration in comparison with purified rabbit MyBP-C-fast and reference samples prepared from one vastus lateralis muscle with mostly type 1 (slow-twitch) fibers and one vastus lateralis muscle with mostly type 2 (fast-twitch) fibers, as well as cultured human skeletal muscle cells rich in MyBP-C-fast. ^{30 31} The separating gels (160 × 180 × 0.75 mm) were silver stained and subsequently scanned. For immunoblot analysis, the separated proteins were transferred onto nitrocellulose sheets and incubated with antibodies against MyBP-C-slow and -fast (Table 1) . 

Immunocytochemistry

The samples for immunocytochemistry were taken from the rectus superior (n = 5), rectus inferior (n = 2), rectus medialis (n = 2), rectus lateralis (n = 2), and obliquus superior (n = 2) muscles. Eight samples were taken from the middle portion of the muscle, two from the distal part (close to the bulb), and three from the proximal part (close to anulus tendineus) of the muscle. For comparison, samples were also taken from human myocardium, LP (n = 2), biceps brachii, first dorsal interosseus, and vastus lateralis muscles. 

Serial cross sections, 5 μm thick, were processed for immunocytochemistry, with a panel of previously very well characterized monoclonal antibodies (mAb), recognizing MyBP-C and MyHC isoforms (Table 1) . The tissue sections were processed as previously described, ^{32 37} by using the indirect peroxidase complex (Dako, Copenhagen, Denmark) technique to visualize bound antibody. 

Processed sections were photographed under a microscope equipped with a charge-coupled device (CCD) camera (Nikon, Tokyo, Japan). The overall staining pattern of each section was examined, and representative areas of each muscle sample, including the orbital and global layers, were studied in detail. 

Results

SDS-PAGE and Immunoblots

Both MyBP-C-slow and -fast were identified in the limb muscle samples by SDS-PAGE (Fig. 1A)and in immunoblots (Fig. 1B) . In the EOMs, only MyBP-C-slow could be detected in the gels and immunoblots, whereas MyBP-C-fast and -intermediate were absent (Fig. 1) . 

Immunocytochemistry

Validation of Antibody Specificity.

The mAbs against MyBP-C-fast and slow did not label sections of human myocardium, indicating that they do not cross-react with the MyBP-C-card isoform. 

Limb Muscles.

Anti-MyBP-C-fast immunostained all fast fibers (containing MyHCIIa and/or MyHCIIx) strongly, whereas it did not label any fibers containing solely MyHCI (Fig. 2) . Anti-MyBP-C-slow stained all fibers strongly (Fig. 2) , in accordance with previous data. ²⁰  

Extraocular Muscles.

The antibodies against the different MyHC isoforms stained the EOMs heterogeneously (Fig. 3 4 5) . The MyHC composition of the individual fibers in the orbital and global layers was complex, because of the presence of multiple isoforms in each fiber and differences in the relative amounts of any given MyHC among the fibers. Three major groups of fibers were distinguished in both the orbital and global layers of the EOMs, according to their immunohistochemical staining patterns, as previously described ³ : (1) fast fibers that contain MyHCIIa and in addition may contain MyHCemb and/or MyHCeom; (2) slow fibers that contain MyHCI and may also contain MyHC-slow tonic, MyHCα-cardiac, MyHCemb, and/or MyHCeom; and (3) MyHCeom^pos/MyHCIIa^neg-fibers that lack MyHCI and MyHCIIa, but contain MyHCeom and may in addition contain MyHCemb. 

Anti-MyBP-C-fast did not label any of the fibers in 11 of the EOM samples examined (Figs. 3B 4B 5B) . However, in the proximal part of a rectus superior and of a rectus inferior muscle taken from the oldest subjects (81 and 86 years, respectively), this mAb labeled a few fibers weakly (Fig. 6) . These stained fibers were mostly, but not exclusively, located in the periphery of the muscles. Many of them were also reactive to anti-MyHCIIa, but there was no clear correlation between the staining pattern of anti-MyHCIIa and anti-MyBP-C-fast. 

Anti-MyBP-C-slow labeled all fibers (Figs. 3A 4A 5A) . The slow fibers were strongly stained, irrespective of whether they contained only MyHCI, MyHCI+MyHCsto, or MyHCI+MyHCsto+MyHCα-cardiac (Figs. 4 5) . The fast fibers (MyHCIIa) were labeled moderately to strongly with anti-MyBP-C-slow, whereas the MyHCeom^pos/MyHCIIa^neg-fibers were labeled lightly, moderately, or strongly by this mAb. At low magnification, the orbital layer appeared more heavily stained than the global layer, and the proximal and distal portions of the muscles appeared more stained than the middle portions, because the MyHCeom^pos/MyHCIIa^neg-fibers were more abundant in the global layer and in the midbelly region. No correlation was found between the staining pattern of anti-MyBP-C-slow and the presence or absence of MyHCemb, MyHCsto, or MyHCα-cardiac in the fibers (Fig. 5) . 

Levator Palpebrae.

Anti-MyBP-C-fast labeled some of the fast fibers and the MyHCeom^pos/MyHCIIa^neg fibers in the LP heterogeneously. The fibers containing MyHCI were not stained with anti-MyBP-C-fast. Anti-MyBP-C-slow labeled all fibers strongly, with less heterogeneity than in the EOMs (Fig. 7) . 

Discussion

The most important result of the present study is the striking difference in the MyBP-C composition between human EOM fibers and limb muscle, a finding that lends further support to the uniqueness of the EOMs as a separate muscle allotype. In addition, the present results further confirm the phenotype of the LP muscle as intermediate between that of the true EOMs and the limb muscles, as previously indicated on the basis of the MyHC composition. ³  

MyHC versus MyBP-C Composition

Human limb muscle fibers contain MyHCI, MyHCIIa, or MyHCIIx and occasional hybrid fibers contain two of these isoforms (I +IIa or IIa+IIx). ⁴⁰ Coordinated expression patterns for MyHC and MyBP-C isoforms have been reported for rat limb muscle in hypertrophy ²⁵ and for single human limb muscle fibers studied with SDS-PAGE, ²⁴ meaning that MyBP-C-fast dominates in fibers expressing MyHCIIx, MyBP-C-intermediate dominates in fibers containing MyHCIIa, and MyPB-C-slow correlates with a content of MyHC I. ²⁴ Our results confirm previous data in human limb muscle. ^{20 24} Moreover, we showed in the present study that there is no such coordination between MyHC and MyBP-C isoforms in the human EOMs. We were unable to detect MyBP-C-fast or intermediate in most of the fibers of the EOMs, although these muscles have a predominance of fast fibers when examined on the basis of their myosin heavy chain reactivity (71% of the fibers contain MyHCIIa and 13% are MyHCeom^pos/MyHCIIa^neg fibers ³ ) and they are among the fastest muscles in the body. This is an additional feature that further strengthens the identity of the EOMs as a separate allotype, distinct from limb muscle. In the masseter muscle, a member of the masticatory allotype, only one MyBP-C isoform, with a molecular mass similar to slow MyBP-C, was identified in single fibers with SDS-PAGE, irrespective of their MyHC composition. ²⁴ Whether this MyBP-C isoform detected in the masseter is identical with MyBP-C-slow or an additional isoform remains to be investigated. ²⁴ The MyHC composition of the fibers in the human EOMs and in the masseter is very complex. Most, if not all the fibers in the EOMs contain more than one MyHC isoform, and in both EOMs and masseter fibers, up to five different MyHC isoforms have been identified, ^{3 24 26} including developmental and α-cardiac MyHC isoforms. ^{24 26 41} In addition to the MyHC isoforms detected in our study, transcripts of MyHCIIb are also present in human EOMs (Andersen J, Pedrosa-Domellöf F, personal communication, 2000) and the presence of MyHCIIx cannot be excluded. ³  

MyBP-C Composition

In the present study, SDS-PAGE, immunoblots, and immunocytochemistry all revealed that the human EOMs lacked MyBP-C-fast. Furthermore, microarray data confirm at the RNA level that the human EOMs differ significantly from limb muscles in the level of expression of MyBP-C-fast, which is downregulated more than 100-fold in EOMs. ⁴² All these findings indicate that MyBP-C-fast is absent or present in only trace amounts in the human EOMs. MyBP-C intermediate was not detected biochemically in the EOMs but the lack of a specific antibody does not allow us to explore whether it may be present in amounts below the level of detection with SDS-PAGE. 

In contrast, at the RNA level, MyBP-C-slow appears to be essentially identical in human EOMs and limb muscle. ⁴² The MyBP-C-cardiac isoform has been detected only in heart muscle, ^{20 43} and expression profiling of the human EOMs versus limb did not reveal the cardiac isoform to be part of the EOM allotype. ⁴² Multiple isoforms of MyBP-C have been identified in chicken, ⁴⁴ but until now there is no evidence of the existence of isoforms other than MyBP-C-slow, -fast, -intermediate, and -cardiac in human muscle. Further studies with single-fiber SDS-PAGE in conjunction with genetic tools are needed to address the question of whether novel MyBP-C isoforms may exist in the human EOMs. 

Our understanding of the function of MyBP-C is still rather limited, despite the fact that mutations on the cardiac isoform are responsible for familial hypertrophic cardiomyopathies. ^{16 17 18} Recently, it has been shown that the S2 binding domain of MyBP-C is a modulator of contractility and that it works in a fashion that is at least partly independent of a “tether” with the MyHC molecule. ⁴⁵  

The lack of MyBP-C theoretically may result in higher contraction rates, since experimental extraction of MyBP-C leads to an increase in contraction rate and increases the Ca²⁺ sensitivity of the force–velocity curve. ⁴⁶ These changes were shown to be reversible by the readdition of MyBP-C. 

The lack of MyBP-C at the single muscle fiber level may have an impact on regulation of muscle contraction at low levels of activation in vivo. This notion is supported by in vitro experiments demonstrating an increase in the low-velocity phase of shortening at submaximal Ca²⁺ activation levels ¹⁵ and increase the Ca²⁺ sensitivity of force ⁴⁶ after chemical extraction of endogenous MyBP-C from skinned skeletal muscle fibers. The effects of removal of MyBP-C on regulation of contraction have been interpreted to be secondary to removal of a structural constraint of MyBP-C on the myosin S-1 domain leading to increased cross-bridge binding. Thus, lack of MyBP-C is expected to reduce an internal load on the myosin head, resulting in increased shortening velocity. Single-fiber experiments are needed to elucidate the role of MyBP-C on the contractile properties of the different fiber types in the human EOMs. However, the very small size of the EOM fibers poses a technical challenge in performing such experiments. 

Levator Palpebrae

We have previously shown that the human LP muscle shares common features with the true EOMs (e.g., loosely arranged fibers and the presence of MyHCα-cardiac, MyHCemb, and MyHCeom), but has a phenotype that is intermediate between the EOMs and the limb muscles (e.g., intermediate fiber size, lack of organization into layers, and lack of MyHCsto). ³ In the present study, immunocytochemistry indicates the presence of MyBP-C-slow in all fibers and MyBP-C-fast in some fast fibers. Taken together, these findings further confirm the phenotype of the LP as intermediate between that of the EOMs and the limb muscles. 

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

 Submitted for publication November 23, 2005; revised May 31, 2006; accepted August 15, 2006.

 Disclosure: D. Kjellgren,None; P. Stål, None; L. Larsson, None; D. Fürst, 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.

 

Antibodies Used for Immunocytochemistry

Antibody	Specificity	Short Name	Gene*	Reference
BB146	MyBP-C slow	Anti-MyBP-C slow	MYBPC1 alt MYBPCS	20
BB88	MyBP-C fast	Anti-MyBP-C fast	MYBPC2 alt MYBPCF	20
A4.74, †	MyHCIIa	Anti-MyHCIIa	MYH2	32 33
A4.951, †	MyHCI	Anti-MyHCI	MYH7	32 34
F88, ‡	MyHCα-cardiac	Anti-MyHCα-cardiac	MYH6	36
N2.261, †	MyHCI	Anti-MyHCI + IIa + eom	MYH7	32 34
	MyHCIIa		MYH2
	MyHCeom		MYH13
	MyHCα-cardiac		MYH6
ALD19, §	MyHC slow tonic	Anti-MyHCsto	?	37, 38
2B6, ∥	MyHCembryonic	Anti-MyHCemb	MYH3	32 37 39

*  Official gene nomenclature, according to OMIM (http://www.ncbi.nlm.nih.gov/omim/).

†  Obtained from The Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by The University of Iowa, Dept of Biological Sciences, Iowa City, IA.

‡  Gift from Jean J. Léger, Institut National de la Santé et de la Recherche Médicale, Unité 249, Montpellier, France.

§  Gift from Donald A. Fischman, Cornell University, Ithaca, New York.

∥  Gift from Alan Kelly, University of Chicago, Chicago, IL.

Figure 1.

 

(A) SDS-PAGE of whole muscle extracts from a limb muscle, the brachioradialis (BR), and a rectus medialis (RM). Note that the MyBP-C-slow band is present in both samples whereas the MyBP-C-fast band is present in the limb muscle sample only. M, molecular mass standard, showing 170 and 130 kDa. (B) Immunoblots treated with the antibodies against MyBP-C-slow (lanes 1–4) and -fast (lanes 5–8). No immunoreactivity was detected with anti-MyBP-C in the EOM samples (lanes 5 and 7). M, molecular mass standard; RM, rectus medialis; BR, brachioradialis; OS, obliquus superior; C, cultured human muscle cells rich in MyBP-C-fast.

Figure 2.

 

Photomicrographs of sections from a biceps brachii muscle immunostained with (A) anti-MyBP-C-fast, (B) anti-MyBP-C-slow, (C) anti-MyHCIIa, and (D) anti-MyHCI. Fibers containing MyHCIIa (arrowhead), MyHCIIx (open arrows), and MyHCI (solid arrows) are indicated. Note that anti-MyBP-C-fast immunolabeled the fibers containing MyHCIIa and MyHCIIx, whereas all fibers were labeled with anti-MyBP-C-slow.

Figure 3.

 

Photomicrographs of sections from a rectus inferior muscle immunostained with (A) anti-MyBP-C-slow, (B) anti-MyBP-C-fast, (C) anti-MyHCI, (D) anti-MyHCI+IIa+eom, (E) anti-MyHCsto, (F) anti-MyHCIIa, and (G) anti-MyHCα-cardiac. Note that all fibers were immunolabeled by anti-MyBP-C-slow, whereas no part of the section showed immunoreactivity with anti-MyBP-C-fast. Also note that there is an area in the global layer showing a somewhat lighter immunoreaction with anti-MyBP-C-slow and that this area is also weakly immunostained with anti-MyHCIIa.

Figure 4.

 

Photomicrographs of sections from the orbital layer of a rectus medialis muscle immunostained with (A) anti-MyBP-C-slow, (B) anti-MyBP-C-fast, (C) anti-MyHCI, (D) anti-MyHCI+IIa+eom, (E) anti-MyHCsto, (F) anti-MyHCIIa, (G) anti-MyHCα-cardiac, and (H) anti-MyHCemb. Arrows: fibers containing MyHCI; arrowheads: fibers containing MyHCIIa. Note that anti-MyBP-C-fast did not label any fibers in this muscle and that anti-MyBP-C-slow immunostained all fibers strongly. There was no correlation between the labeling with anti-MyHCemb and any of the other mAbs.

Figure 5.

 

Photomicrographs of sections from the global layer of a rectus medialis muscle. Immunostaining as in Figure 4 . Fibers containing MyHCIIa (arrowhead), MyHCI (solid arrow) or lacking both of these MyHC isoforms (open arrow) are indicated. Note that the fibers containing MyHCI have a stronger immunoreaction with anti-MyBP-C-slow than the other fibers. Also note that anti-MyHCα-cardiac and anti-MyHCemb stained only a few fibers, without correlation to the staining patterns of the other mAbs.

Figure 6.

 

Photomicrograph of a section from the proximal part of a rectus inferior immunostained with anti-MyBP-C-fast The orbital layer (OL) and the global layer (GL) are indicated. Scattered immunoreactivity, preferably in some fibers of the orbital layer is evident.

Figure 7.

 

Photomicrographs of sections from an LP muscle immunostained with (A) anti-MyBP-C-slow, (B) anti-MyBP-C-fast, (C) anti-MyHCI, and (D) anti-MyHCIIa, labeled as in Figure 5 . Note that some of the fibers containing MyHCIIa are immunostained to a variable degree with anti-MyBP-C-fast.

The authors thank Margaretha Enerstedt for excellent technical assistance.