Myosin Light Chain 1 Isoforms in Slow Fibers from Global and Orbital Layers of Canine Rectus Muscles

Abstract

purpose. Initial results of an examination of the low molecular mass (≤45 kDa) protein composition of canine rectus muscle homogenates, based on gel electrophoresis, revealed a distinct difference between the global and orbital layers in the myosin light chain (MLC)-1 region. The objectives of the present study were, therefore, to identify isoforms of MLC1 in homogenates of the global and orbital layers of adult canine rectus muscles and to determine the MLC1 isoform expression pattern among single muscle fibers isolated from both layers.

methods. Muscle homogenates and single fibers from the global and orbital layers of canine rectus muscles were analyzed, using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) was used to identify a protein band in the orbital layer that comigrated with MLC1 in the adult canine atrium.

results. Adult canine extraocular rectus muscles expressed embryonic skeletal/atrial MLC1 (MLC1_E/A), in addition to the fast-type MLC1 (MLC1_F) and slow-type MLC1 (MLC1_S) isoforms expressed in limb skeletal muscles. MLC1_E/A was detected in slow fibers of the orbital but not the global layer, and MLC1_S was detected in slow fibers in only the global but not the orbital layer. Densitometric analysis of gel bands from homogenates supported these results, with significantly greater amounts of MLC1_S in the global layer and of MLC1_E/A in the orbital layer.

conclusions. MLC1_E/A is expressed in rectus muscles of adult dogs. Furthermore, two types of slow fibers, distinguished on the basis of MLC1 isoform expression, exist in separate layers of canine rectus muscles.

Vertebrate skeletal muscle myosin is a hexameric protein, composed of two heavy chains (MHC; ∼223 kDa each) and four light chains (MLC; ∼15–25 kDa). ¹ Each myosin molecule contains two essential MLCs (referred to as MC1 and MLC3) and two regulatory MLCs (MLC2). The amino terminus, or head region, of MHC binds adenosine triphosphate (ATP) and is the site of ATP hydrolysis. Myosin and actin are the predominant constituents of thick filaments and thin filaments, respectively, in striated muscle. The MHC head also contains an actin-binding site. On activation, myosin molecules bind to actin molecules, thus forming crossbridges. Cycles of crossbridge formation and dissociation (i.e., myosin attaching to actin and detaching), which also involve hydrolysis of ATP, are the basis of muscle contractions. The liberated energy is used to generate force and drive muscle shortening to perform work. 

It is well accepted that the MHC isoform composition of individual muscle fibers is a major determinant of shortening velocity and, therefore, of power output. ² There is also evidence that the essential (or alkali) MLCs (MLC1 and -3) modulate the rate of contraction ^{3 4} by increasing or decreasing the attachment and detachment rates of myosin-actin crossbridges. MLC1 has a longer N terminus than MLC3, and the N terminus of MLC1 binds to the C terminus of actin and therefore could play an important modulatory role in actin-activated myosin adenosine triphosphatase (ATPase) activity, shortening velocity and power output. ^{5 6}  

The expressed isoforms of MHC vary among different muscles and among individual fibers within a muscle. There are marked differences in MHC isoform expression between limb muscles and craniofacial muscles, ⁷ with the latter expressing a greater number of isoforms, which is believed to contribute to the more complex and precise motor functions served by craniofacial muscles. Contractile properties of extraocular muscles from several species have been studied ^{8 9 10 11 12} and span a much broader range than those of limb muscles, to drive eye rotations that vary from extremely slow to very fast. The greater range of extraocular muscle contractions is largely believed to be due to the MHC isoform composition, ^{13 14 15 16 17 18 19} the mechanisms associated with Ca²⁺ release/uptake, ^{20 21 22} and the concentration of the Ca²⁺-binding protein, parvalbumin ²³ (higher in fast-type fibers ^{24 25} ) in these muscles. Given that MLCs can modulate crossbridge kinetics and, therefore, contractile properties, it is important to determine whether the MLC isoform composition of extraocular muscles differs from that of limb muscles and provides yet another potential mechanism for modulating contraction kinetics. Typical slow and fast skeletal muscle fibers express slow MLC1_S and MLC2_S and fast MLC1_F, MLC2_F, and MLC3, respectively. These isoforms can be readily separated electrophoretically and identified on the basis of known molecular weights and myofibrillar protein stoichiometry. A preliminary examination of myofibrillar protein isoform composition of canine rectus muscles, using protein gel electrophoresis, suggested the presence of an additional band migrating in the vicinity of MLC1 that was not observed in samples of limb muscles. The objective of this study was therefore to examine thoroughly the MLC isoform composition of canine extraocular muscles and of single fibers from the same muscles to test whether variations in MLC isoforms exist in these muscles and among individual fibers. The results demonstrate that there are at least two types of slow fibers in canine extraocular muscles, based on electrophoretic analysis: slow fibers expressing typical slow isoforms of MLC1 and -2 and an additional type expressing MLC1_E/A and slow MLC2. The results also suggest that these two slow fiber types are localized in separate rectus muscle layers. 

Methods

Lateral, medial, dorsal (superior), and ventral (inferior) rectus muscles, tibialis cranialis (TC), deep portion of the medial or lateral gastrocnemius muscle (DG), right atrium, and left ventricle of adult beagle hounds (either gender and weighing approximately 10–15 kg) were harvested after death by intravenous injection of pentobarbital). The care and use of the animals in this study were in accordance with an institutionally approved protocol and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Canine rectus muscles were used for this study, because the global and orbital layers can be easily distinguished from each other in this species, using a dissecting microscope, even at low magnification (e.g., 6×). The rectus muscles were isolated from the globe and pinned to a Petri dish lined with silicone rubber (Sylgard; Dow Corning Corp., Midland, MI) and containing cold relaxing solution (2.0 mM EGTA, 4.0 mM MgATP, 1.0 mM free Mg²⁺, 10.0 mM imidazole, and sufficient KCl to achieve an ionic strength of 180 mM [pH 7.00]). ²⁶ The two layers are readily distinguished from each other by bright illumination, when the muscle is placed with the orbital side down. The orbital layer is C-shaped in cross-section, and the global layer is centrally positioned when the muscle is placed in this orientation. Care was taken to avoid using samples that included fibers from both layers. Homogenates of portions from both layers of each rectus muscle and of the atrium, ventricle, TC, and DG were prepared as previously described. ²⁷ The portions of the global and orbital layers that were adjacent to each other were not included in the samples, to ensure analysis of pure global and pure orbital layers. Portions of single fibers from TC and DG, known to be either fast-type or slow-type fibers from an analysis of their MHC isoform composition, were also included in the electrophoretic analyses to assist in identifying the fast and slow MHC and MLC isoforms of extraocular fibers. Canine atrial and ventricular samples were used as standards to compare cardiac and extraocular MHC and MLC isoforms. 

Bundles of fibers were prepared from the remaining portions of the global and orbital layers of each rectus muscle and of the TC and DG. The bundles were tied to glass capillary tubes and stored for a minimum of 24 hours in glycerinating solution (same ionic composition as the relaxing solution with 50% of the water substituted with glycerol) at −20°C. Segments of single fibers were isolated from bundles in cold relaxing solution, using a dissecting microscope. A total of 120 fibers were isolated from bundles prepared from medial and lateral rectus muscles and the low molecular mass protein isoform composition of each fiber was analyzed, using SDS-PAGE. Fifteen fibers were isolated from both layers of four rectus muscles (three lateral recti and one medial rectus from three dogs; i.e., 60 global fibers and 60 orbital fibers). The length of each fiber segment was measured with a ruler submersed in the relaxing solution. The width of each segment was estimated by comparison with submersed pieces of black surgical nylon monofilament of known diameter. The volume of each fiber segment was calculated from the length and width, assuming a circular circumference. Each fiber segment was soaked in relaxing solution containing 1% Triton X-100 (Sigma-Aldrich) for 5 minutes, rinsed in relaxing solution without Triton and transferred to a 0.5 mL microcentrifuge tube. Two microliters of gel sample buffer per nanoliter of fiber volume were added to each tube, and the samples were allowed to sit for 30 minutes at room temperature before being heated at 65°C for 2 minutes. ²⁷ After heating, samples were placed on ice for 5 minutes. The samples were stored at −40°C until loading onto gels. 

All the gels in this study were run in commercial gel units (SE600; 16 × 18-cm plates; Hoefer Scientific Instruments, San Francisco, CA). Gels used to examine MHC isoform composition were prepared as previously described, ²⁸ with modifications. The stacking gel (pH 6.8) consisted of 4% total acrylamide and 5% glycerol. The separating gel (pH 8.8) consisted of 7% total acrylamide and 30% glycerol. An acrylamide-to-N,N′-methylene-bis-acrylamide ratio of 50:1 was used for these stacking and separating gels, which were run at constant 300 V for 25 hours at 8°C. Stacking and separating gels, which were used to examine low molecular mass (≤45 kDa) proteins and to generate bands for matrix-assisted desorption ionization time-of-flight (MALDI-TOF) analysis, consisted of 4% and 12% total acrylamide, respectively. Glycerol was not included in these gels. The acrylamide-to-N,N′-methylene-bis-acrylamide ratio was 50:1 and 200:1 in the stacking gels (pH 6.8) and separating gels (pH 9.3), respectively. The gels were run at constant 24 mA/gel at 18°C until the dye front migrated to the bottom edge (∼3.5 hours). All the gels in this study, except the gels used to isolate bands for MALDI-TOF analysis (below), were fixed and silver stained, as described. ²⁷ Pictures of the gels were taken with a video camera (model XC-ST70, Nikon, Tokyo, Japan) and stored using on computer (Simple PCI, ver. 3.6.0.1.8 software; Compix Imaging Systems, Cranberry Township, PA). The gels were densitometrically scanned (model GS 300; Hoefer Scientific Instruments) to measure the relative amounts of the different isoforms of MLC1 in a homogenate. 

MALDI-TOF-mass spectrometry (MS) was conducted in the Campus Chemical Instrument Center at The Ohio State University to identify the protein postulated to be MLC1_E/A, based on its observed comigration with MLC1 in canine atrium. The gel used to generate the bands to be analyzed with MALDI-TOF was fixed with 50% ethanol and 10% acetic acid for 1 hour and then stained with Coomassie blue (0.025% Coomassie brilliant blue R-250 in 40% methanol and 7% acetic acid) for 1 hour. The gel was then destained with 50% methanol and 5% acetic acid until the background became clear. The band of interest was cut out and stored in 5% acetic acid solution at 4°C until MALDI-TOF-MS analysis. The gel slice was trimmed to minimize background polyacrylamide material and washed in 50% methanol and 5% acetic acid for several hours, dried with acetonitrile (ACN), and reconstituted with dithiothreitol (DTT) solution to reduce the cysteines. Iodoacetamide was added to alkylate the cysteines, and the gel was washed again in cycles of ACN and ammonium bicarbonate. The gel slice was digested at room temperature overnight with sequencing grade trypsin from Promega (Madison WI) with a kit (Montage In-Gel Digestion Kit; Millipore, Bedford, MA), according to the manufacturer’s recommended protocols. The peptides were extracted from the polyacrylamide with 50% ACN and 5% formic acid several times and pooled together. The extracted pools were concentrated in a speed vac (ThermoSavant, Holbrook, NY) to approximately 25 μL. Salt buffers from the protein sample were cleaned (ZipTips; Millipore) according to the manufacturer’s directions. α-Cyano-4-hydroxy cinnamic acid was used as the matrix and prepared as a saturated solution in 50% ACN/0.1% trifluoroacetic acid (in water). Allotments of 1 μL of matrix and 1 μL of sample were thoroughly mixed together; 0.5 μL of this was spotted on the target plate and allowed to dry. MALDI- TOF was performed on a mass spectrometer (Reflex III; Bruker, Breman, Germany) operated in linear, positive ion mode with an N₂ laser. Laser power was used at the threshold level necessary to generate a signal. Accelerating voltage was set to 28 kV. 

Other MLC gel bands were identified on the basis of migration relative to (1) molecular mass markers (not shown) and to MLC bands in canine atrium, ventricle, and fast and slow limb fibers and (2) the known stoichiometry of MLC proteins relative to other myofibrillar proteins. ²⁹ The predominant isoform of MLC2 that is expressed in slow skeletal muscle is identical with ventricular MLC2. ³⁰  

The statistical significance of differences between mean values obtained from scanning densitometry was tested with ANOVA followed by the Student t-test. P < 0.05 was considered to be significant. 

Results

MLC1_E/A Levels in Homogenates of Global and Orbital Muscle Layers

The protein band in homogenates of rectus muscles that was observed on 12% acrylamide gels and that was postulated to be the atrial isoform of MLC1, based on its electrophoretic comigration with MLC1 in atrial tissue, was positively identified by MALDI-TOF-MS. The z-score obtained from MALDI-TOF MS with 52% sequence coverage was 1.16, corresponding to a probability between 0.85 and 0.90. Therefore, nano-liquid chromatography-tandem mass spectrometry (LC/MS/MS) was conducted, and the results strongly suggested the presence of one protein in the gel band. The combined MALDI and nano-LC/MS/MS results yielded identification of the band as MLC1_E/A, with a probability close to 1.0. 

Atrial MLC1 is identical with the MLC1 isoform normally expressed in embryonic skeletal muscle ³¹ and is thus referred to in this report as MLC1_E/A. Gel analysis of homogenates of global and orbital layers from each rectus muscle of six dogs (Fig. 1) , including quantitation of the relative amounts of the three isoforms of MLC1—the slow isoform of MLC1 (MLC1_S), the fast isoform of MLC1 (MLC1_F), and MLC1_E/A—revealed significant differences between the layers (Fig. 2) . The relative level (i.e., percent of total MLC1) of MLC1_S was significantly higher in the global layer, whereas the relative level of MLC1_E/A was significantly higher in the orbital layer. MLC1_F did not differ in relative amount between the global and orbital layers. The differences in the relative levels of MLC1_S and MLC1_E/A were consistent among all rectus muscles examined. 

MLC Isoforms in Global and Orbital Single Fibers

Single fibers (SFs) were isolated from the global and orbital layers of rectus muscles and analyzed electrophoretically for MLC isoform composition. Three types of fibers, distinguished on the basis of electrophoretic migration of MLC isoforms, were detected (Fig. 3) . Fibers with exclusively MLC1_F and MLC2_F, were isolated from both layers of the rectus muscles and were designated as SF-EO_F. Fast-type vertebrate fibers also express MLC3, ¹ another fast-type MLC isoform, but this protein could not be identified on gels with certainty, possibly because of low expression levels. Both of the other types of canine extraocular muscle fibers appeared to be slow types, based on the presence of MLC2_S in these fibers and on the single MHC isoform band in these fibers that comigrated with the single MHC band in a sample of canine left ventricle and a prominent band in the deep portion of the gastrocnemius muscle (Fig. 4) . It should be noted that the predominant MHC isoform in canine ventricle is MHC-β ³² which is identical with the slow-type MHC isoform (MHC-I) in slow-twitch fibers in limb skeletal muscle ^{33 34} and is distinct from the slower-migrating MHC-α isoform that is expressed, along with MHC-β, in canine atria. One type of slow fiber in canine rectus muscles expresses MLC1 and -2 isoforms that comigrate with MLC1_S and -2_S in slow-type limb fibers and this slow fiber type is designated SF-EO_S. All the SF-EO_S fibers were isolated from the global layer of rectus muscles. The other slow fiber type expressed MLC2_S, but did not express normal levels of either MLC1_S or MLC1_F. Instead, this slow fiber type expressed a prominent protein that migrated slightly faster than MLC1_F and was not expressed in SF-EO_F or in SF-EO_S fibers. This MLC1 isoform comigrated with the MLC1 isoform in canine atrium (Fig. 3) and, therefore, with the protein in samples of rectus muscle homogenates that was identified by MALDI-TOF-MS as MLC1_E/A. All the slow-type fibers expressing MLC1_E/A, designated SF-EO_E/A, were isolated from the orbital layer of rectus muscles (Fig. 5) . Small amounts of proteins comigrating with MLC1_S and MLC1_F were detected in several SF-EO_E/A fibers, but these minor bands were not identified. 

Additional Features of Global and Orbital Fibers

There was sufficient sample from 35 of the single fibers to analyze both MHC and MLC isoform compositions in each fiber, using the two gel formats. All 5 of the fast fibers and all 10 of the slow fibers from the global layer that were dually analyzed had exclusively fast and slow, respectively, isoforms of MHC and MLC. All 10 fast fibers from the orbital layer had fast isoforms of MHC and MLC and all 10 of the slow fibers (SF-EO_E/A) from the orbital layer had an MHC isoform that comigrated with slow-type MHC and had MLC1_E/A and MLC2_S. No hybrid fibers (that is, fibers expressing mixtures of fast and slow isoforms of MHC or MLC) were detected. The SF-EO_E/A fibers may be considered hybrid fibers, because an MLC1 isoform, MLC1_E/A, that is normally expressed in atria, was expressed in these fibers with MLC2_S, which is normally found in ventricle and slow-twitch skeletal muscle. 

SF-EO_E/A fibers appeared, in general, to have smaller diameters and had a clearer appearance when transilluminated and viewed with a dissecting microscope at 30× to 40× magnification, compared with the other fibers from the orbital layer, which expressed fast isoforms of MHC and MLC and which had a grainy and cloudy appearance. 

Discussion

Our results demonstrate that there are two distinct slow fiber types in canine rectus muscles, based on MLC1 isoform expression, and suggest that the two types are segregated into separate layers. All the SF-EO_S fibers, which express the MLC1_S isoform found in typical slow type muscle fibers, were isolated from the global layer, and all the SF-EO_E/A fibers, which express MLC1_E/A, were isolated from the orbital layer of rectus muscles. However, the presence of some SF-EO_S fibers in the orbital layer and some SF-EO_E/A fibers in the global layer of canine rectus muscles cannot be ruled out, because the inner most portions of these layers (i.e., adjacent to each other) were not included in the analyses to avoid contamination of one layer with the other. A major question that should be addressed is the functional significance of the expression of MLC1_E/A in any type of skeletal muscle fiber. The segregated localization of two distinct slow fiber types in the two layers suggests that each type might contribute to oculomotor function in a unique manner. Overexpression of MLC1_E/A in transgenic mouse hearts resulted in increased rates of isolated muscle shortening, myosin-driven thin filament sliding in an in vitro motility assay, and whole-heart rate of pressure development and relaxation. ³⁵ Upregulation of MLC1_E/A in ventricles of cardiomyopathic human hearts is associated with increased rates of muscle shortening and force generation, presumably as a compensatory mechanism to offset partially the decreased contractility of the failing heart. ^{36 37} The essential myosin light chains have the ability to bind to actin. ^{6 38} Morano and Haase ³⁹ have reported that MLC1_E/A has a lower affinity for actin, compared with that of ventricular MLC1 (same as MLC1_S in global slow fibers), which could allow faster crossbridge cycling and thereby provide a mechanism for faster contractile properties in fibers expressing MLC1_E/A. Whether canine SF-EO_E/A have faster rates of shortening and/or isometric force generation than do SF-EO_S fibers remains to be determined. Measurements of contractile properties of identified SF-EO_S fibers and SF-EO_E/A should provide valuable information on the roles of these two fiber types in ocular rotation. 

MLC1_E/A is normally expressed in embryonic skeletal muscle at embryonic ages and in cardiac atria at all ages. However, MLC1_E/A is also expressed in adult human masseter muscle. ^{40 41} The expression of MLC1_E/A in adult skeletal muscle appears to be limited to some craniofacial muscles, suggesting that innervation of muscle by cranial nerves and/or developmental origin, both of which differ between these two muscle groups (reviewed by Spencer and Porter ⁴² ), may underlie the persistent expression of this protein. 

Extensive heterogeneity exists in myofibrillar protein expression in extraocular muscles. For example, MHC isoforms expressed in adult limb muscles, as well as MHC isoforms in limb muscles at embryonic and neonatal stages and in atrial myocardium, are expressed in adult extraocular muscles. ⁷ Our results show that the heterogeneity in the light chain subunit of myosin in extraocular muscle is greater than previously reported. Presumably, the large heterogeneity in myosin expression in extraocular muscles is related to the broad range of oculomotor contractions. Another distinction from limb muscle is the variation in MHC isoform expression along the length of extraocular muscles. ⁴³ However, we did not examine whether differences exist in MLC1_E/A expression along the length of individual fibers in this study. Furthermore, fast and slow troponin T isoform expression in extraocular muscles differs from that in limb muscles. ^{44 45} More recently, results from microarray studies reveal a broad range of differences in protein expression between extraocular and limb muscles, including the expression of several myofibrillar proteins. ^{46 47} Fischer at al. ⁴⁶ reported the expression of MLC1_E/A RNA in rat extraocular muscles, at a level that is significantly greater than in fast limb muscle. Our results show that MLC1_E/A is, in fact, expressed at the protein level in canine rectus muscles. 

Several schemes for classifying extraocular muscle fiber types have been proposed (also reviewed by Spencer and Porter ⁴² ). The most popular scheme to date describes six fiber types in rectus muscles of several species, including two slow types segregated in global and orbital layers. Fibers of these two types are multiply innervated (MIF), whereas all the other types are singly innervated (SIF). The MIFs in the global layers appear to be slow, based on histochemical staining for myosin ATPase activity. The SIFs in both layers appear to be fast, based on the same criterion. Orbital MIFs are unusual in that they do not exhibit the pH-sensitivity that normally distinguishes fast from slow fibers. Unfortunately, canine extraocular fiber types have not been described. It is possible, though, that the two slow fiber types identified in this study, on the basis of MLC1 isoform expression, correspond to the two MIF types in the global and orbital layers of other species. 

The two slow fiber types described in this report could be partially responsible for different roles of the global and orbital layers in oculomotor mechanics. Demer et al. ⁴⁸ proposed the active pulley hypothesis to explain the roles of the two layers in eye rotations. Determination of the contractile properties of SF-EO_S fibers and SF-EO_E/A fibers, as well as the other fiber types in both layers of the same muscles, should provide valuable information for better understanding of the cellular basis of rectus muscles in driving eye rotations. 

 Supported by National Science Foundation Grant 0133613.

 Submitted for publication July 10, 2003; revised August 27 and September 22, 2003; accepted September 28, 2003.

 Disclosure: S. Bicer, None; P.J. Reiser, 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.

 

Figure 1.

 

(A) The low molecular mass region of a silver-stained gel onto which homogenates of the global (G) and orbital (O) layers of lateral (L), medial (M), dorsal (D), and ventral (V) rectus muscles were loaded. (B) The low molecular mass region of another silver-stained gel onto which homogenates of the global and orbital layers of the ventral rectus muscle, a single glycerinated (skinned) slow fiber from the deep portion of the DG_SF, and a single glycerinated fast fiber from the tibialis cranialis (TC_SF) were loaded. The single fibers in (B) were identified as being slow and fast, respectively, through analysis of their MHC isoform composition on another gel (not shown). Note the greater amount of MLC1_E/A in the orbital layer of each rectus muscle. Slow and fast isoforms of MLC1 and -2 (MLC1_S, -1_F, -2_S, and -2_F).

Figure 2.

 

Mean ± SEM levels of the slow, fast, and embryonic/atrial isoforms of MLC1 (MLC1_S, -1_F, and -1_E/A), relative to total MLC1 content, of the global and orbital layers of all four rectus muscles from six dogs. *Significant differences (P < 0.05) between the global and orbital layers of each muscle.

Figure 3.

 

Low molecular mass region of a silver-stained gel. RA, right atrium; LV, left ventricle; EO_E/A, slow extraocular fiber with embryonic/atrial MLC1; EO_S, slow extraocular fiber; EO_F, fast extraocular fiber; TC_F, fast fiber from the tibialis cranialis; DG_S, slow fiber from the deep portion of the gastrocnemius muscle. Other abbreviations are the same as in Figure 1 . Lane M: biotinylated molecular mass markers. Dotted line: the MLC1_E/A band in the EO_E/A fiber. See Figure 4 for the MHC isoform composition of the same fibers.

Figure 4.

 

The MHC region of a silver-stained gel onto which homogenates of canine left ventricle (LV), right atrium (RA), and single fibers (portions of the same fibers run on the gel shown in Fig. 3 ) from a lateral rectus muscle and limb muscles were loaded. EO_E/A, slow extraocular fiber with embryonic/atrial MLC1; EO_S, slow extraocular fiber; EO_F, fast extraocular fiber; TC_F, fast fiber from the tibialis cranialis; DG_S, slow fiber from the deep portion of the gastrocnemius muscle; MHC-α and MHC-β, isoforms of cardiac MHC; MHC-I, slow (type I) isoform of skeletal muscle MHC (same protein as MHC-β); MHC-fast, fast isoforms of skeletal muscle MHC.

Figure 5.

 

Two identical silver-stained gels onto which single muscle fibers, isolated from the global and orbital layers of a lateral rectus muscle, were loaded. Abbreviations are the same as in Figure 1 . Note that all the slow-type fibers (EO_E/A) in the orbital layer expressed MLC1_E/A, whereas none of the slow type fibers (EO_S) in the global layer or the fast-type fibers (EO_F) in both layers, expressed this protein.

The authors thank Christine A. Lucas and Joseph F. Y. Hoh for providing a prepublication copy of their manuscript.