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Sarco(endo)plasmic Reticulum Ca2ⴙ ATPases (SERCA1 and -2) in Human Extraocular Muscles Daniel Kjellgren,1 Michelle Ryan,2 Kay Ohlendieck,2 Lars-Eric Thornell,3,4 and Fatima Pedrosa-Domello ¨ f 3,4 PURPOSE. To investigate the composition of the fibers in human extraocular muscles (EOMs) with respect to the sarco(endo) plasmic reticulum Ca2⫹ATPases (SERCA)-1 and -2 and to investigate possible correlations between SERCA and myosin heavy chain (MyHC) composition. METHODS. EOM samples were processed for immunocytochemistry with monoclonal antibodies specific against SERCA1 (fast isoform), SERCA2 (slow isoform), or different MyHCs. A total of 1571 fibers were analyzed. Microsomal EOM fractions were analyzed with SDS-PAGE and immunoblots. RESULTS. The fast fibers, containing MyHCIIa, accounted for 79% of the fibers in the orbital layer (OL) and 74% in the global layer (GL). More than 99% of these fibers contained SERCA1, and 86% of them coexpressed SERCA1 and -2. Almost all slow fibers stained with SERCA2; 54% of those in the GL and all in the OL coexpressed SERCA1 and -2. Fifteen percent of the fibers in the GL and less than 1% in the OL were MyHCeompos/ MyHCIIaneg fibers. All these contained SERCA1 and in the OL also stained strongly with anti-SERCA2. Biochemically SERCA2 was more abundant than SERCA1. CONCLUSIONS. The human EOMs had a very complex pattern of expression of the major protein regulating fiber relaxation rate. The coexistence of SERCA1 and -2, together with complex mixtures of MyHCs in most of the fibers provide the human EOMs with a unique molecular portfolio that allows a highly specific fine-tuning regimen of contraction and relaxation. (Invest Ophthalmol Vis Sci. 2003;44:5057–5062) DOI:10.1167/ iovs.03-0218
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he extraocular muscles (EOMs) are among the fastest and yet most fatigue-resistant skeletal muscles in the body. They perform very complex movements ranging from rapid saccades to slow vergence and pursuit movements, and their phenotype is so unique that they are considered a separate class of skeletal muscle (allotype).1,2 The unique character of the EOMs in small rodents has recently been confirmed at the gene level with both microarray and SAGE techniques,3–5 but
From the Departments of 1Clinical Science, Section of Ophthalmology, and 3Integrative Medical Biology, Section of Anatomy, University of Umeå, Umeå, Sweden; the 2Department of Biology, National University of Ireland, Maynooth, Ireland; and the 4Department of Musculoskeletal Research, University of Ga¨vle, Ga¨vle, Sweden. Submitted for publication March 4, 2003; revised June 10, 2003; accepted June 12, 2003. Disclosure: D. Kjellgren, None; M. Ryan, None; K. Ohlendieck, None; L.-E. Thornell, None; F. Pedrosa-Domello ¨ f, 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. Corresponding author: Fatima Pedrosa-Domello ¨ f, Department of Integrative Medical Biology, Section of Anatomy, Umeå University, S-901 85 Umeå, Sweden;
[email protected]. Investigative Ophthalmology & Visual Science, December 2003, Vol. 44, No. 12 Copyright © Association for Research in Vision and Ophthalmology
extensive work is necessary before these data can be interpreted at the cell and organelle level. Corresponding genotype data are not available for human EOMs, and we have recently shown that they differ significantly from those of other species with respect to fiber composition.6 The major determinants of the functional properties of a muscle are the force of contraction and rate of both contraction and relaxation. The contraction force and contraction velocity are dictated by the myosin heavy chain (MyHC) composition of the muscle fibers.7 We have described the unique MyHC composition of the fibers in human EOMs.6 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 tubuli. The T-tubuli form junctions with the terminal cisterna of the sarcoplasmic reticulum (SR), depolarization triggers the release of Ca2⫹ from the SR into the sarcoplasm, and the muscle fiber contracts. The relaxation rate of a muscle fiber reflects the rate at which Ca2⫹ is transported back from the myofibrillar space into the lumen of the SR. The Ca2⫹ ions are actively pumped from the myofibrillar 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 (SERCAs) using adenosine triphosphate (ATP) hydrolysis as the source of energy.8 SERCAs are the major protein component of the SR. Three differentially expressed genes encode SERCA proteins in human: SERCA1, -2, and -3.9,10 Differential splicing of the primary transcripts results in further variations, and at present at least seven SERCA isoforms have been described in humans.11 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 nonmuscle cells. Mutations of the three genes have been associated with disease. For example, mutation of the SERCA1 gene is associated with Brody disease, a condition characterized by impairment of skeletal muscle relaxation after exercise, stiffness, and cramps.12 Data on SERCA expression in human EOMs are not available. The distribution of SERCA1 has been reported to be rather complex in rat and rabbit EOMs.13 To elucidate further the cellular and molecular basis of the extraordinary features of the human EOMs we determined biochemically and immunohistochemically the relative abundance and distribution of the fast (SERCA1) and slow (SERCA2) SERCA isoforms that are muscle-specific markers of the sarcoplasmic reticulum. We have also investigated the patterns of distribution of SERCA1 and -2 at the fiber level and their relation to the MyHC content of the fibers.
MATERIAL
AND
METHODS
Muscle Samples Seven EOM samples obtained at autopsy from five individuals (four men and one woman), ages 17, 26, midthirties, 34, and 81 were used
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TABLE 1. Data on the Antibodies Used for Immunocytochemistry Antibody
Specificity
Short Name
Gene*
NCL-SERCA1† NCL-SERCA2† A4.74‡ A4.840‡ F88§ 4A6㛳 N2.261‡
SERCA1 SERCA2 MyHClla MyHCl MyHC␣-cardiac MyHCextraocular MyHCl MyHClla MyHCeom MyHC␣-cardiac MyHCsto MyHCembryonic
Anti-SERCA1 Anti-SERCA2 Anti-MyHClla Anti-MyHCl Anti-MyHC␣-cardiac Anti-MyHCeom Anti-MyHCl⫹lla⫹eom
ATP2A1 ATP2A2 MYH1 MYH7 MYH6 MYH13 MYH7 MYH1 MYH13 MYH6 ? MYH3
ALD19¶ 2B6#
Anti-MyHCsto Anti-MyHCemb
Reference 29 29,30 31,32 32,33 34 35–37 32,33
15,38 15,32,39
* Official gene nomenclature according to OMIM (http://www.ncbi.nlm.nih.gov/omim/). † Purchased from Novocastra Laboratories Ltd, Newcastle-upon-Tyne, UK. Anti-SERCA1 is specific against the human SERCA1 isoform expressed in type II (fast) skeletal muscle fibers and anti-SERCA2 recognizes the human SERCA2a isoform present in type I (slow) skeletal muscle fibers as well as the SERCA2b isoform present in all cell types. However, the staining observed with the SERCA antibodies was exclusively present in the muscle fibers, and no other cells were labeled, indicating that the affinity of the SERCA2 antibody for the 2b isoform is apparently very low and below the level of detection of our study. ‡ Obtained from The Developmental Studies Hybridoma Bank, developed under the auspices of the National Institute of Child Health and Human Development and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA. § Gift from Jean J. Leger, National Institute of Health and Medical Research, Unit 249, Montpellier, France. 㛳 Gift from Joseph F. Y. Hoh, University of Sydney, NSW, Australia. ¶ Gift from Donald A. Fischman, Cornell University, Ithaca, NY. # Gift from Alan Kelly, University of Chicago, Chicago, IL.
for immunocytochemistry. Three samples were taken from the rectus superior, two from the rectus medialis, one from the obliquus superior and one from the rectus lateralis. The samples available were taken from the middle portion of each muscle, and one sample was also taken from the anterior part of one of the rectus medialis. In addition, 11 other EOM samples were pooled together for biochemical analysis. The samples were obtained in accordance with the ethical recommendations of the Swedish Transplantation Law, with the approval of the Medical Ethics Committee, Umeå University, and adhered to the tenets of the Declaration of Helsinki for research involving human tissue.
Immunocytochemistry The samples were mounted on cardboard and rapidly frozen in propane chilled with liquid nitrogen and stored at ⫺80°C until used. Series of 80 cross sections, 5 m thick, were cut from each muscle sample in a cryostat (Reichert-Jung, Vienna, Austria). The sections were processed for immunocytochemistry, with previously characterized monoclonal antibodies (mAbs), recognizing two SERCA and six MyHC isoforms (Table 1). Some of the sections were double stained with the mAb NCL-merosin against the laminin ␣2 chain,14 to outline the fibers and make their identification easier. The tissue sections were processed as previously described.15 Control sections were processed as just described, except that the primary antibody was omitted. No staining was observed in the control sections. The sections were photographed under a microscope equipped with a charge-coupled device (CCD) camera (Carl Zeiss Meditec, Oberkochen, Germany). The overall staining pattern of each section was examined, and one area of the orbital layer and one of the global layer of each muscle sample were studied in detail. The staining pattern in 1571 fibers was analyzed.
Biochemistry mAbs (mAb IID8 to the slow SERCA2 isoform of the Ca 2⫹ATPase and mAb IIH11 to the fast SERCA1 isoform of the Ca2⫹ATPase) were purchased from Affinity Bioreagents (Golden, CO). Peroxidase-conjugated secondary antibodies, acrylamide stock solutions, and protease
inhibitors were from Roche Molecular Biochemicals (Lews, UK) and immunoblotting chemiluminescence substrates were obtained from Pierce & Warriner (Chester, UK). Immobilon-P nitrocellulose was purchased from Millipore Corp. (Bedford, MA). All other chemicals used in the isolation of membrane vesicles and the electrophoretic separation of proteins were of analytical grade and obtained from Sigma-Aldrich Co. (Poole, UK). For membrane isolation, eleven individual EOM samples were combined and yielded approximately 1 g of wet tissue weight. Muscles were finely minced and homogenized at 0°C to 4°C in 10 volumes of 50 mM HEPES (pH 7.4), 10% (wt/vol) sucrose, 0.02% (wt/vol) sodium azide, and 3 mM MgCL2. Buffers were supplemented with a protease inhibitor cocktail consisting of 0.2 mM serine protease inhibitor (Pefabloc; Roche), 1.4 M pepstatin A, 0.3 M E-64, 1 M leupeptin, 1 mM EDTA, and 0.5 M soybean trypsin inhibitor to avoid proteolytic degradation.16 A crude microsomal fraction was isolated by a standard subcellular fractionation procedure, as has been described in detail.17 Microsomal pellets were resuspended at the protein concentration of 10 mg/mL and used immediately for immunoblot analysis. Protein concentration was determined by the method of Bradford,18 using bovine serum albumin as standard. The gel electrophoretic separation of EOM proteins was performed with 7% (wt/vol) resolving gels using a 5% (wt/vol) stacking gel in the presence of sodium dodecyl sulfate and dithiothreitol. Due to the limited amount of material available, a electrophoresis system (MiniMP3; Bio-Rad Laboratories, Herts, UK) was used, whereby 30 g protein was loaded per well and electrophoresed at 280 V/h.19 The electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets was performed by the method of Towbin et al.20 Blocking, incubation with primary and secondary antibodies, and washing of nitrocellulose sheets was performed as previously described.21 Immunolabeling was evaluated by the enhanced chemiluminescence technique.22 Densitometric scanning of enhanced chemiluminescence blots was performed on a computing densitometer (model 300S; Molecular Dynamics Sunnyvale, CA; ImageQuant, ver. 3.0 software).23
SERCA1 and -2 in Human Eye Muscles
IOVS, December 2003, Vol. 44, No. 12 TABLE 2. SERCA Staining Pattern (Fiber Count) SERCA Staining SERCA1 Orbital layer ⫺ ⫹⫹ ⫺ ⫹ ⫹⫹ ⫹ ⫹⫹ Total fiber count Global layer ⫺ ⫹⫹ ⫺ ⫹ ⫹⫹ ⫹ ⫹⫹ Total fiber count
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Orbital Layer
MyHC Group
SERCA2
lla
l
llaneg/eompos
⫺ ⫺ ⫹⫹ ⫹ ⫹ ⫹⫹ ⫹⫹
0 6 0 0 559 0 137 702
0 0 0 1 1 134 42 178
0 0 0 0 0 0 4 4
⫺ ⫺ ⫹⫹ ⫹ ⫹ ⫹⫹ ⫹⫹
1 161 0 35 267 1 40 505
2 0 35 1 2 42 0 82
0 54 0 22 24 0 0 100
⫺, no staining; ⫹, light staining; ⫹⫹, strong staining.
RESULTS Immunocytochemistry Three major groups of fibers were distinguished according to their immunohistochemical staining patterns, as previously described6: (1) fast fibers that stained with anti-MyHCIIa, (2) slow fibers that stained with anti-MyHCI, and (3) MyHCeompos/ MyHCIIaneg fibers that stained with neither anti-MyHCIIa nor anti-MyHCI but were labeled with anti-MyHCI⫹IIa⫹eom and anti-MyHCeom. Practically all slow fibers also stained with anti-MyHCsto and some of them were also stained with antiMyHC␣-cardiac. Staining with the SERCA1 and -2 antibodies was restricted to the muscle fibers in all samples studied. The staining patterns of the two SERCA antibodies were different in the orbital and the global layer (Table 2).
Anti-SERCA1 and anti-SERCA2 stained almost all fibers in the orbital layer (Fig. 1). Anti-SERCA1 stained 85% of the fibers strongly and 15% of the fibers moderately to lightly. AntiSERCA2 stained 36% of the fibers strongly and 63% moderately to lightly. One percent of the fibers were unstained by antiSERCA2. The fibers were smaller and more tightly arranged than in the global layer. The fast fibers accounted for 79% of the sampled fibers in the orbital layer. All fast fibers were strongly stained with anti-SERCA1. Anti-SERCA2 stained approximately 20% of the fast fibers strongly and 80% lightly to moderately. Less than 1% of them were unstained by anti-SERCA2. The slow fibers accounted for 20% of the sampled fibers in the orbital layer. All of them stained with anti-SERCA2, 99% strongly and 1% lightly. Anti-SERCA1 labeled all these slow fibers, 25% strongly and 75% moderately to lightly. The remaining fibers (MyHCeompos/MyHCIIaneg fibers) accounted for less than 1% (four fibers out of 884 sampled) of the fibers in the orbital layer. These four fibers stained heavily with both anti-SERCA1 and anti-SERCA2.
Global Layer In general, the global layer seemed more lightly and heterogeneously stained than the orbital layer (Fig. 2). Anti-SERCA1 stained 80% of the fibers strongly and 15% moderately to lightly. Anti-SERCA2 in contrast stained 17% of the fibers strongly and 51% moderately to lightly. Only 3 of 687 sampled global fibers were unstained by both anti-SERCA1 and antiSERCA2. The fast fibers accounted for 74% of the fibers in the global layer. One of the 505 fast fibers sampled was unstained by both anti-SERCA1 and anti-SERCA2. Seven percent of the fast fibers stained lightly with both anti-SERCA1 and anti-SERCA2. The remaining fast fibers stained strongly with anti-SERCA1 and were unstained (32%), lightly stained (53%), or strongly stained (8%) with anti-SERCA2. The slow fibers accounted for 12% of the sampled fibers in the global layer. Fifty-one percent of the slow fibers sampled
FIGURE 1. Photomicrographs of six serial sections from the orbital layer of a rectus medialis muscle immunostained with (A) anti-SERCA1, (B) anti-MyHCIIa, (C) anti-MyHCeom, (D) anti-SERCA2, (E) antiMyHCI, and (F) anti-MyHCsto. The sections shown in (C) and (F) were also stained with the mAb NCL-merosin against the laminin ␣2 chain, which stained the fibers’ contours. Black arrows: fibers stained with anti-MyHCI; arrowheads: fibers stained with anti-MyHCIIa; white arrows: MyHCeompos/MyHCIIaneg fibers. Note that anti-SERCA1 stained all fibers, but that many slow fibers were generally more weakly stained. Anti-SERCA2 stained almost all fibers, but there was a tendency for the slow fibers to be more strongly stained. The MyHCeompos/MyHCIIaneg fibers are strongly stained by both SERCA markers.
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FIGURE 2. Photomicrographs of six serial sections from the global layer of a rectus medialis muscle. Immunostaining and arrow labeling as in Figure 1. Note that the slow fibers are strongly stained by anti-SERCA2, but practically unstained by anti-SERCA1 and that both the fast fibers and the MyHCeompos/MyHCIIaneg-fibers are strongly stained by anti-SERCA1, but unstained or weakly stained by antiSERCA2.
stained strongly with anti-SERCA2 and were lightly stained with anti-SERCA1. Forty-three percent of the slow fibers were also strongly stained with anti-SERCA2 but were unstained with anti-SERCA1. Approximately 2% of the slow fibers were unstained by both anti-SERCA1 and anti-SERCA2. One percent were lightly stained by both SERCA antibodies and approximately 2% showed stronger staining with anti-SERCA1 (fast isoform) than with anti-SERCA2. The MyHCeompos/MyHCIIaneg fibers accounted for approximately 15% of the fibers in the global layer. Fifty-four percent of them were strongly stained with anti-SERCA1 only, 22% were lightly stained by both SERCA markers, and 24% were strongly stained with the fast isoform (SERCA1) and lightly labeled with the mAb against the slow isoform (SERCA2).
the areas sampled from the global and orbital layer, respectively) in the human EOMs. The present data showed that most (99% in the orbital and 63% in the global layer) of the fibers
Longitudinal Variation Samples from both the middle and the anterior parts of one rectus medialis muscle were available (Fig. 3). Although there was a clear difference between the anterior and midbelly parts in the staining pattern of anti-MyHCIIa in the global layer, no major difference was evident in the staining patterns of antiSERCA1 and anti-SERCA2 (Fig. 3). There was no apparent correlation between the staining patterns observed with the SERCA markers and with anti-MyHC-embryonic (not shown).
Biochemistry The relative abundance of fast and slow markers of the SERCA isoforms was determined biochemically in a microsomal fraction isolated from human EOM fibers. The Coomassie-stained gel (Fig. 4A) exhibited two major high molecular mass band regions of apparent 100 and 200 kDa, mostly probably representing the sarcoplasmic reticulum Ca2⫹ pump and MyHCs, respectively. The relative density, measured as the percentage of intensity of enhanced chemiluminescence of a control band set at 100%, of the slow isoform (SERCA2 ⫽ 95% ⫾ 2.3% SEM) was significantly higher (P ⬍ 0.00181, unpaired t-test, twotailed) than that of its fast counterpart (SERCA1 ⫽ 88% ⫾ 0.7% SEM) in immunoblots (Figs. 4B, 4C).
DISCUSSION The present study of the distribution of the major Ca2⫹ pump protein of the longitudinal tubules and the terminal cisternae, emphasizes the highly complex and unique structure of the human EOMs. Biochemically, the most abundant SERCA isoform in the EOMs was the slow isoform SERCA2, although the fibers containing slow MyHC were a minority (12% and 20% in
FIGURE 3. Two series of photomicrographs of serial sections, from the anterior (left) and midbelly part (right) of the same rectus medialis muscle immunostained with the mAbs shown on the left. Note the large areas in the global layer of the midbelly with an abundance of MyHCeompos/MyHCIIaneg fibers, unstained by anti-MyHCIIa and antiMyHCI, but stained by anti-MyHCI⫹IIa⫹eom, and that these fibers were strongly stained by anti-SERCA1 and more lightly stained or unstained by anti-SERCA2. OL, orbital layer; GL, global layer.
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FIGURE 4. Immunoblot analysis of EOM microsomes. Shown is a Coomassie-stained gel (A) and immunoblots (B, C) labeled with antibodies to (B) the slow isoforms of the Ca2⫹ATPase (SERCA2) and (C) the fast isoforms of the Ca2⫹ATPase (SERCA1). Left: sizes of molecular mass standards (in kilodaltons), as deduced from rat myofibrillar proteins.
contained both SERCA1 and -2, and a wide spectrum of staining intensities was observed. This is in contrast to the reports on limb muscle, where SERCA1 is the only isoform present in fibers containing fast MyHC and SERCA2 is the isoform seen in fibers containing slow MyHC.8 In general, the fast fibers and the MyHCeompos/MyHCIIaneg fibers were more strongly stained by the antibody against the fast isoform SERCA1, and the slow (tonic) fibers were more strongly stained by anti-SERCA2 (the slow isoform). However, there was considerable variation in the levels of staining—in particular, staining with anti-SERCA2 in the fast fibers of the global layer. Immunocytochemistry is not a quantitative method, but differences in the staining intensity of the fibers with a single antibody indicate heterogeneity in the amount of epitope detected. In the case of the SERCA isoforms, the wide spectrum of staining intensities observed can be interpreted as variation in the relative densities of the SERCA1 or -2 present. Furthermore, most possible staining combinations (both MyHC and SERCA isoforms) were found among the fibers of the global layer. In other words, not only do SERCA1 and -2 coexist in the same fiber but there seems also to be wide variation in the relative proportions of each isoform present in a given fiber type. Similarly, we have also shown that practically all EOM fibers have complex mixtures of two or more MyHC isoforms.6 Taken together, these data indicate that the fibers of the human EOM have a unique molecular portfolio that allows extremely fine tuning of their contraction and relaxation properties. The functional demands on the EOMs are very high. The EOMs are constantly active and, even at rest with the eye in primary position, two thirds of the motor neurons are at threshold.24 The coexpression of SERCA isoforms in most of the fibers probably reflects the extraordinary functional features of the EOMs. The EOM fibers must both contract and relax extremely fast in saccades and finely adjust tension in slow pursuit movements and fixation. Experimentally, it has
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been observed that chronically stimulated fast-twitch muscle fibers can express SERCA1 and -2a simultaneously25 and even switch from SERCA1 to SERCA2a completely, if stimulated for sufficient time. A small number of fibers in the human EOMs were apparently devoid of SERCA1 and -2. These fibers were very few and may be in a transitional state. For instance, SERCAs can be significantly modified by chronic stimulation and ageing processes.11 Another possible explanation would be the presence of yet unidentified SERCA isoforms, possibly specific for the EOMs. The MyHC composition of the fibers varies along the length of the EOMS in human6 and other species.26,27 In the rat EOMs SERCA1 is expressed in the midbelly of the muscle, but it disappears at the ends.13 We found no evidence of variation in the distribution of the SERCA isoforms when the middle and anterior portion of a single rectus medialis were compared. Further studies are needed to elucidate the possible heterogeneity in SERCA content along the length of the human EOM fibers. The human EOMs differ from those of other species with respect to the SERCA composition. In rat and rabbit EOMs all fibers of the orbital layer and the singly innervated fibers of the global layer contain SERCA1, but the multiply innervated fibers of the global layer do not.13 We found that more than half of multiply innervated fibers of the global layer, identified by their MyHC content,6,28 in addition to SERCA2 also contain the fast isoform SERCA1 in the human EOMs. This is in agreement with data showing that there are important differences between the human EOMs and those of other species in fiber type and MyHC composition.6
Acknowledgments The authors thank Margaretha Enerstedt and Anna-Karin Olofsson for excellent technical assistance.
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