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

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Uncoordinated Expression of Myosin Heavy Chains and Myosin-Binding Protein C Isoforms in Human Extraocular Muscles ARTICLES Uncoordinated Expression of Myosin Heavy Chains and Myosin-Binding Protein C Isoforms in Human Extraocular Muscles Daniel Kjellgren,1 Per Stål,2 Lars Larsson,3 Dieter Fu¨rst,4 and Fatima Pedrosa-Domello¨f1,2 PURPOSE. To examine the distribution of myosin-binding pro- special features. The EOMs contain fibers with a wide array of tein C (MyBP-C) in human extraocular muscles (EOMs) and to contractile properties, varying from extremely fast to fibers correlate the myosin heavy chain (MyHC) and the MyBP-C capable of sustained, tonic contractions. Previously, we sought composition of the fibers. to elucidate the molecular basis of this unique allotype of 3–5 METHODS. Samples from 17 EOMs, 3 levator palpebrae (LP), and human EOMs at the fiber level, by determining the patterns 6 limb muscles were analyzed with SDS-PAGE and immunoblot of expression of myosin heavy chain (MyHC; the major deter- 6,7 or processed for immunocytochemistry with monoclonal anti- minant of heterogeneity of contraction force and velocity ) 8 bodies (mAbs) against MyBP-C-fast, MyBP-C-slow, MyHCIIa, My- and of SERCA-1 and -2 (determinants of the relaxation rate ). HCI, MyHCsto, MyHC␣-cardiac, and MyHCemb. The fibers in the human EOMs have very complex MyHC composition patterns, with most fibers containing more than RESULTS. In the limb muscle samples, fast fibers were labeled one MyHC isoform. Moreover, differences in the relative with anti-MyBP-C-fast and anti-MyBP-C-slow, whereas the slow amounts of a given MyHC isoform are typically observed fibers were immunostained with anti-MyBP-C-slow only, in 3 accordance with previous studies. In 11 EOM samples MyBP- among fibers sharing a particular combination of isoforms. C-fast was not detected, and weak staining with anti-MyBP-C- Despite this heterogeneity, the fibers in the human EOMs can be divided grossly into three major fiber groups, based on their fast was seen only in a few fibers in the proximal part of 2 3 muscles. The mAb against MyBP-C-slow labeled all fibers, but content of MyHCI, MyHCIIa, and MyHCeom. fibers containing MyHCI were generally more strongly stained. Myosin-binding protein C (MyBP-C) is, next to myosin, the second most abundant thick-filament protein in striated mus- In the levator palpebrae, immunostaining with anti-MyBP-C-fast 9 was present in some fibers labeled with anti-MyHCIIa and/or cles. It is located in the A band, in a restricted part of the cross-bridge–bearing region.10 MyBP-C is a Ϸ130 kDa protein anti-MyHCeom. MyBP-C-fast and -intermediate were not de- 11,12 tected biochemically in the EOMs. and both its C terminus and N terminus bind to myosin. MyBP-C is presumed to have a regulatory, although not essen- CONCLUSIONS. The lack of MyBP-C-fast and intermediate is an tial, role in sarcomere assembly13,14 and to play a physiological additional feature of the human EOM allotype. The true EOMs role in regulating contraction by modulating unloaded short- have a unique myofibrillar protein isoform composition reflect- ening velocity.15 The importance of MyBP-C for muscle func- ing their special structural and functional properties. The leva- tion is indicated by the fact that mutations in the gene for tor palpebrae muscle phenotype is intermediate between that cardiac MyBP-C (MyBP-C-card) cause familial hypertrophic car- of the EOMs and the limb muscles. (Invest Ophthalmol Vis Sci. diomyopathy.16–18 2006;47:4188–4193) DOI:10.1167/iovs.05-1496 There are three major isoforms of MyBP-C in human muscle: fast skeletal (MyBP-C-fast), slow skeletal (MyBP-C-slow), and he functional properties of muscle fibers vary considerably MyBP-C-card.19 MyBP-C-fast, detected both with in situ hybrid- between different muscles.1 The extraocular muscles T ization and immunocytochemistry, is present in fast fibers, (EOMs) are among the most complex muscles in the body and whereas MyBP-C-slow is present in both slow and fast fibers, in have been considered a separate muscle allotype,2 due to their 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 From the 1Department of Clinical Sciences, Ophthalmology, Umeå three human MyBP-C genes have been mapped and se- University, Umeå, Sweden; the 2Department of Integrative Medical quenced.21–23 Recent analysis of single fibers by SDS-PAGE, Biology, Section of Anatomy, Umeå University, Umeå, Sweden; the revealed the coordinated expression of MyBP-C-slow in fibers 3Department of Clinical Neurophysiology, Academic Hospital, Uppsala 4 containing MyHCI (slow); MyBP-C-fast in fibers with MyHCIIx University, Uppsala, Sweden; and the Institute for Biochemistry and (fast); and an additional isoform, MyBP-C-intermediate, in fibers Biology, Department of Molecular Cell Biology, University of Bonn, 24 Bonn, Germany. containing MyHCIIa (fast) in human limb muscle. Coordi- Supported by grants from Stiftelsen KMA, Synfra¨mjandet, the nated isoform changes, indicating that MyBP-C expression is Swedish Research Council and the Medical Faculty of Umeå University. linked to MyHC expression, have been reported during skeletal Submitted for publication November 23, 2005; revised May 31, muscle hypertrophy in the rat.25 However, in the human mas- 2006; accepted August 15, 2006. seter, a masticatory muscle with rather unique properties,26–28 Disclosure: D. Kjellgren,None; P. Stål, None; L. Larsson, None; the very complex MyHC composition of its fibers was not D. Fu¨rst, None; F. Pedrosa-Domello¨f, None paralleled by an intricate MyBP-C pattern.24 The publication costs of this article were defrayed in part by page Data on the MyBP-C composition of human EOMs and its charge payment. This article must therefore be marked “advertise- ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. correlation to the MyHC composition at the protein and cellu- Corresponding author: Fatima Pedrosa-Domello¨f, Department of lar level are lacking. In the present study, we investigated the Integrative Medical Biology, Section of Anatomy, Umeå University, distribution of the fast and slow isoforms of MyBP-C in relation S-901 87 Umeå, Sweden; [email protected]. to the MyHC profile of the fibers and found further evidence of Investigative Ophthalmology & Visual Science, October 2006, Vol. 47, No. 10 4188 Copyright © Association for Research in Vision and Ophthalmology Downloaded from iovs.arvojournals.org on 09/27/2021 IOVS, October 2006, Vol. 47, No. 10 MyBP-C in Extraocular Muscles 4189 the uniqueness of the molecular portfolios of the fibers in the ble 1). The tissue sections were processed as previously described,32,37 human EOMs. by using the indirect peroxidase complex (Dako, Copenhagen, Den- mark) technique to visualize bound antibody. Processed sections were photographed under a microscope MATERIAL AND METHODS equipped with a charge-coupled device (CCD) camera (Nikon, Tokyo, The muscles were collected according to the ethical recommendations Japan). The overall staining pattern of each section was examined, and of the Swedish Transplantation Law, with the approval of the Medical representative areas of each muscle sample, including the orbital and Ethics Committee, Umeå University, and in compliance with the Dec- global layers, were studied in detail. laration 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 RESULTS neuromuscular disease. The samples were mounted on cardboard, rapidly frozen in propane chilled with liquid nitrogen, and stored at SDS-PAGE and Immunoblots Ϫ80°C until used. Both MyBP-C-slow and -fast were identified in the limb muscle SDS-PAGE and Immunoblots samples by SDS-PAGE (Fig. 1A) and in immunoblots (Fig. 1B). In the EOMs, only MyBP-C-slow could be detected in the gels Whole-muscle extracts were prepared from one rectus superior, one and immunoblots, whereas MyBP-C-fast and -intermediate were rectus lateralis, two obliquus superior, one levator palpebrae (LP), and absent (Fig. 1). one brachioradialis muscle, as previously described.29 MyBP-C iso- forms were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using 4% (wt/vol) stacking and 8% running Immunocytochemistry 24 gels. Gel buffer solutions included 10% glycerol. Proteins were iden- Validation of Antibody Specificity. The mAbs against tified on the basis of their molecular mass, immunoreactivity, and order MyBP-C-fast and slow did not label sections of human myocar- of migration in comparison with purified rabbit MyBP-C-fast and refer- dium, indicating that they do not cross-react with the MyBP-C- ence samples prepared from one vastus lateralis muscle with mostly card isoform. type 1 (slow-twitch) fibers and one vastus lateralis muscle with mostly Limb Muscles. Anti-MyBP-C-fast immunostained all fast fi- type 2 (fast-twitch) fibers, as well as cultured human skeletal muscle bers (containing MyHCIIa and/or MyHCIIx) strongly, whereas 30,31 cells rich in MyBP-C-fast. The separating gels (160 ϫ 180 ϫ 0.75 it did not label any fibers containing solely MyHCI (Fig. 2). mm) were silver stained and subsequently scanned. For immunoblot Anti-MyBP-C-slow stained all fibers strongly (Fig. 2), in accor- analysis, the separated proteins were transferred onto nitrocellulose dance with previous data.20 sheets and incubated with antibodies against MyBP-C-slow and -fast Extraocular Muscles. The antibodies against the different (Table 1). MyHC isoforms stained the EOMs heterogeneously (Fig. 3, 4, 5). The MyHC composition of the individual fibers in the Immunocytochemistry orbital and global layers was complex, because of the presence The samples for immunocytochemistry were taken from the rectus of multiple isoforms in each fiber and differences in the relative superior (n ϭ 5), rectus inferior (n ϭ 2), rectus medialis (n ϭ 2), rectus amounts of any given MyHC among the fibers.
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