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(Investigative Ophthalmology and Visual Science. 2004;45:138-143.)
© 2004 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.03-0716
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Myosin Light Chain 1 Isoforms in Slow Fibers from Global and Orbital Layers of Canine Rectus Muscles

Sabahattin Bicer and Peter J. Reiser

From the Department of Oral Biology, College of Dentistry, The Ohio State University, Columbus, Ohio.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
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 (MLC1E/A), in addition to the fast-type MLC1 (MLC1F) and slow-type MLC1 (MLC1S) isoforms expressed in limb skeletal muscles. MLC1E/A was detected in slow fibers of the orbital but not the global layer, and MLC1S 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 MLC1S in the global layer and of MLC1E/A in the orbital layer.

CONCLUSIONS. MLC1E/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 contraction3 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 studied8 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, parvalbumin23 (higher in fast-type fibers24 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 MLC1S and MLC2S and fast MLC1F, MLC2F, 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 MLC1E/A and slow MLC2. The results also suggest that these two slow fiber types are localized in separate rectus muscle layers.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
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., 6x). 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 x 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 MLC1E/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. {alpha}-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
Top
 Abstract
 Methods
 Results
Discussion
 References
 
MLC1E/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 MLC1E/A, with a probability close to 1.0.

Atrial MLC1 is identical with the MLC1 isoform normally expressed in embryonic skeletal muscle31 and is thus referred to in this report as MLC1E/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 (MLC1S), the fast isoform of MLC1 (MLC1F), and MLC1E/A—revealed significant differences between the layers (Fig. 2) . The relative level (i.e., percent of total MLC1) of MLC1S was significantly higher in the global layer, whereas the relative level of MLC1E/A was significantly higher in the orbital layer. MLC1F did not differ in relative amount between the global and orbital layers. The differences in the relative levels of MLC1S and MLC1E/A were consistent among all rectus muscles examined.



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  		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 DGSF, and a single glycerinated fast fiber from the tibialis cranialis (TCSF) 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 MLC1E/A in the orbital layer of each rectus muscle. Slow and fast isoforms of MLC1 and -2 (MLC1S, -1F, -2S, and -2F).

 


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  		FIGURE 2. Mean ± SEM levels of the slow, fast, and embryonic/atrial isoforms of MLC1 (MLC1S, -1F, and -1E/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.

 
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 MLC1F and MLC2F, were isolated from both layers of the rectus muscles and were designated as SF-EOF. 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 MLC2S 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-ß32 which is identical with the slow-type MHC isoform (MHC-I) in slow-twitch fibers in limb skeletal muscle33 34 and is distinct from the slower-migrating MHC-{alpha} isoform that is expressed, along with MHC-ß, in canine atria. One type of slow fiber in canine rectus muscles expresses MLC1 and -2 isoforms that comigrate with MLC1S and -2S in slow-type limb fibers and this slow fiber type is designated SF-EOS. All the SF-EOS fibers were isolated from the global layer of rectus muscles. The other slow fiber type expressed MLC2S, but did not express normal levels of either MLC1S or MLC1F. Instead, this slow fiber type expressed a prominent protein that migrated slightly faster than MLC1F and was not expressed in SF-EOF or in SF-EOS 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 MLC1E/A. All the slow-type fibers expressing MLC1E/A, designated SF-EOE/A, were isolated from the orbital layer of rectus muscles (Fig. 5) . Small amounts of proteins comigrating with MLC1S and MLC1F were detected in several SF-EOE/A fibers, but these minor bands were not identified.



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  		FIGURE 3. Low molecular mass region of a silver-stained gel. RA, right atrium; LV, left ventricle; EOE/A, slow extraocular fiber with embryonic/atrial MLC1; EOS, slow extraocular fiber; EOF, fast extraocular fiber; TCF, fast fiber from the tibialis cranialis; DGS, 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 MLC1E/A band in the EOE/A fiber. See Figure 4 for the MHC isoform composition of the same fibers.

 


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  		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. EOE/A, slow extraocular fiber with embryonic/atrial MLC1; EOS, slow extraocular fiber; EOF, fast extraocular fiber; TCF, fast fiber from the tibialis cranialis; DGS, slow fiber from the deep portion of the gastrocnemius muscle; MHC-{alpha} and MHC-ß, isoforms of cardiac MHC; MHC-I, slow (type I) isoform of skeletal muscle MHC (same protein as MHC-ß); MHC-fast, fast isoforms of skeletal muscle MHC.

 


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  		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 (EOE/A) in the orbital layer expressed MLC1E/A, whereas none of the slow type fibers (EOS) in the global layer or the fast-type fibers (EOF) in both layers, expressed this protein.

 
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-EOE/A) from the orbital layer had an MHC isoform that comigrated with slow-type MHC and had MLC1E/A and MLC2S. No hybrid fibers (that is, fibers expressing mixtures of fast and slow isoforms of MHC or MLC) were detected. The SF-EOE/A fibers may be considered hybrid fibers, because an MLC1 isoform, MLC1E/A, that is normally expressed in atria, was expressed in these fibers with MLC2S, which is normally found in ventricle and slow-twitch skeletal muscle.

SF-EOE/A fibers appeared, in general, to have smaller diameters and had a clearer appearance when transilluminated and viewed with a dissecting microscope at 30x to 40x 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
Top
 Abstract
 Methods
 Results
 Discussion
References
 
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-EOS fibers, which express the MLC1S isoform found in typical slow type muscle fibers, were isolated from the global layer, and all the SF-EOE/A fibers, which express MLC1E/A, were isolated from the orbital layer of rectus muscles. However, the presence of some SF-EOS fibers in the orbital layer and some SF-EOE/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 MLC1E/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 MLC1E/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 MLC1E/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 Haase39 have reported that MLC1E/A has a lower affinity for actin, compared with that of ventricular MLC1 (same as MLC1S in global slow fibers), which could allow faster crossbridge cycling and thereby provide a mechanism for faster contractile properties in fibers expressing MLC1E/A. Whether canine SF-EOE/A have faster rates of shortening and/or isometric force generation than do SF-EOS fibers remains to be determined. Measurements of contractile properties of identified SF-EOS fibers and SF-EOE/A should provide valuable information on the roles of these two fiber types in ocular rotation.

MLC1E/A is normally expressed in embryonic skeletal muscle at embryonic ages and in cardiac atria at all ages. However, MLC1E/A is also expressed in adult human masseter muscle.40 41 The expression of MLC1E/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 Porter42 ), 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 MLC1E/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 MLC1E/A RNA in rat extraocular muscles, at a level that is significantly greater than in fast limb muscle. Our results show that MLC1E/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 Porter42 ). 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-EOS fibers and SF-EOE/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.


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


   Footnotes
 
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.

Corresponding author: Peter J. Reiser, Department of Oral Biology, 305 West 12th Avenue, The Ohio State University, Columbus, OH 43210; reiser.17{at}osu.edu


   References
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 Abstract
 Methods
 Results
 Discussion
 References
 

  1. Sellers J. Myosin II. Myosins. 1999;14–19. Oxford University Press London.
  2. Moss RL, Diffee GM, Greaser ML. Contractile properties of skeletal muscle fibers in relation to myofibrillar protein isoforms. Rev Physiol Biochem Pharmacol. 1995;126:1–63.[ISI][Medline][Order article via Infotrieve]
  3. Bottinelli R, Betto R, Schiaffino S, Reggiani C. Unloaded shortening velocity and myosin heavy chain and alkali light chain isoform composition in rat skeletal muscle fibres. J Physiol. 1994;478:341–349.[ISI][Medline][Order article via Infotrieve]
  4. Lowey S, Waller GS, Trybus KM. Skeletal muscle myosin light chains are essential for physiological speeds of shortening. Nature. 1993;365:454–456.[CrossRef][Medline][Order article via Infotrieve]
  5. Timson DJ, Trayer HR, Trayer IP. The N-terminus of A1-type myosin essential light chains binds actin and modulates myosin motor function. Eur J Biochem. 1998;255:654–662.[ISI][Medline][Order article via Infotrieve]
  6. Trayer IP, Trayer HR, Levine BA. Evidence that the N-terminal region of A1-light chain of myosin interacts directly with the C-terminal region of actin: a proton magnetic resonance study. Eur J Biochem. 1987;164:259–266.[ISI][Medline][Order article via Infotrieve]
  7. Schiaffino S, Reggiani C. Molecular diversity of myofibrillar proteins: gene regulation and functional significance. Physiol Rev. 1996.371–423.
  8. Asmussen G, Gaunitz U. Mechanical properties of the isolated inferior oblique muscle of the rabbit. Pflugers Arch. 1981;392:183–190.[CrossRef][ISI][Medline][Order article via Infotrieve]
  9. Bach-y-Rita P, Ito F. In vivo studies on fast and slow muscle fibres in cat extraocular muscles. J Gen Physiol. 1966;49:1177–1198.
  10. Frueh BR, Hayes A, Lynch GS, Williams DA. Contractile properties and temperature sensitivity of the extraocular muscles, the levator and the superior rectus, of the rabbit. J Physiol. 1994;475:327–336.[Abstract/Free Full Text]
  11. Fuchs AF, Luschei ES. Development of isometric tension in simian extraocular muscle. J Physiol. 1971;219:155–166.[Abstract/Free Full Text]
  12. Luff AR. Dynamic properties of the inferior rectus, extensor digitorum longus, diaphragm and soleus muscles of the mouse. J Physiol. 1981;313:161–171.[Abstract/Free Full Text]
  13. Asmussen G, Traub I, Pette D. Electrophoretic analysis of myosin heavy chain isoform patterns in extraocular muscles of the rat. FEBS Lett. 1993;335:243–245.[CrossRef][ISI][Medline][Order article via Infotrieve]
  14. Brueckner JK, Itkis O, Porter JD. Spatial and temporal patterns of myosin heavy chain expression in developing rat extraocular muscle. J Muscle Res Cell Motil. 1996;17:297–312.[ISI][Medline][Order article via Infotrieve]
  15. Lucas CA, Hoh JFY. Distribution of developmental myosin heavy chains in adult rabbit extraocular muscle: identification of a novel embryonic isoform absent in fetal limb. Invest Ophthalmol Vis Sci. 2003;44:2450–2456.[Abstract/Free Full Text]
  16. McLoon LK, Rios L, Wirtschafer JD. Complex three-dimensional patterns of myosin isoform expression: differences between and within specific extraocular muscles. J Muscle Res Cell Motil. 1999;20:771–783.[CrossRef][ISI][Medline][Order article via Infotrieve]
  17. Rubinstein NA, Hoh JFY. The distribution of myosin heavy chain isoforms among rat extraocular muscle fiber types. Invest Ophthalmol Vis Sci. 2000;41:3391–3398.[Abstract/Free Full Text]
  18. Wieczorek DF, Periasamy M, Butler-Browne GS, Whalen RG, Nadal-Ginard B. Co-expression of multiple myosin heavy chain genes, in addition to a tissue-specific one, in extraocular musculature. J Cell Biol. 1985;101:618–629.[Abstract/Free Full Text]
  19. Stirn Kranjc B, Sketelj J, D’Albis A, Ambroz M, Erzen I. Fibre types and myosin heavy chain expression in the ocular medial rectus muscle of the adult rat. J Muscle Res Cell Motil. 2000;21:753–761.[CrossRef][ISI][Medline][Order article via Infotrieve]
  20. Block BA, O’Brien J, Meissner G. Characterization of the sarcoplasmic reticulum proteins in the thermogenic muscles of fish. J Cell Biol. 1994;127:1275–1287.[Abstract/Free Full Text]
  21. Jacoby J, Ko K. Sarcoplasmic reticulum fast Ca2+-pump and myosin heavy chain expression in extraocular muscles. Invest Ophthalmol Vis Sci. 1993;34:2848–2858.[Abstract/Free Full Text]
  22. Porter JD, Karathanasis P. The development of extraocular muscle calcium homeostasis parallels visuomotor system maturation. Biochem Biophys Res Commun. 1999;257:678–683.[CrossRef][ISI][Medline][Order article via Infotrieve]
  23. Heizmann CW, Berchtold MW, Rowlerson AM. Correlation of parvalbumin concentration with relaxation speed in mammalian muscles. Proc Natl Acad Sci. 1982;79:7243–7247.[Abstract/Free Full Text]
  24. Celio MR, Heizmann CW. Calcium-binding protein parvalbumin is associated with fast contracting muscle fibres. Nature. 1982;297:504–506.[CrossRef][Medline][Order article via Infotrieve]
  25. Hou T-T, Johnson JD, Rall JA. Parvalbumin content and Ca2+ and Mg2+ dissociation rates correlated with changes in relaxation rate of frog muscle fibres. J Physiol. 1991;441:285–304.[Abstract/Free Full Text]
  26. Reiser PJ, Moss RL, Giulian GG, Greaser ML. Shortening velocity in single fibers from adult rabbit soleus muscles is correlated with myosin heavy chain composition. J Biol Chem. 1985;260:9077–9080.[Abstract/Free Full Text]
  27. Blough ER, Rennie ER, Zhang F, Reiser PJ. Enhanced electrophoretic separation and resolution of myosin heavy chains in mammalian and avian skeletal muscles. Anal Biochem. 1996;233:31–35.[CrossRef][ISI][Medline][Order article via Infotrieve]
  28. Reiser PJ, Kline WO. Electrophoretic separation and quantitation of cardiac myosin heavy chain isoforms in eight mammalian species. Am J Physiol. 1998;274:H1048–H1053.
  29. Yates LD, Greaser ML. Quantitative determination of myosin and actin in rabbit skeletal muscle. J Mol Biol. 1983;168:123–141.[CrossRef][ISI][Medline][Order article via Infotrieve]
  30. Barton PJ, Cohen A, Robert B, et al. The myosin alkali light chains of mouse ventricular and slow skeletal muscle are indistinguishable and are coded by the same gene. J Biol Chem. 1985;260:8578–8584.[Abstract/Free Full Text]
  31. Barton PJR, Robert B, Fiszman MY, Leader DP, Buckingham ME. The same myosin alkali light chain gene is expressed in adult cardiac atria and in fetal skeletal muscle. J Muscle Res Cell Motil. 1985;6:461–475.[CrossRef][ISI][Medline][Order article via Infotrieve]
  32. Hamilton N, Ianuzzo CD. Contractile and calcium regulating capacities of myocardia of different sized mammals scale with resting heart rate. Mol Cell Biochem. 1991;106:133–141.[ISI][Medline][Order article via Infotrieve]
  33. Cuda G, Fananapazir L, Zhu WS, Sellers JR, Epstein ND. Skeletal muscle expression and abnormal function of beta-myosin in hypertrophic cardiomyopathy. J Clin Invest. 1993;91:2861–2865.
  34. Lompre A-M, Nadal-Ginard B, Mahdavi V. Expression of the cardiac ventricular {alpha}- and ß-myosin heavy chain genes is developmentally and hormonally regulated. J Biol Chem. 1984;259:6437–6446.[Abstract/Free Full Text]
  35. Fewell JG, Hewett T, Sanbe A, et al. Functional significance of cardiac essential light chain isoform switching in transgenic mice. J Clin Invest. 1998;101:2630–2639.[ISI][Medline][Order article via Infotrieve]
  36. Morano I, Hädicke K, Haase H, Böhm M, Erdmann E, Schaub MC. Changes in essential myosin light chain isoform expression provide a molecular basis for isometric force regulation in the failing human heart. J Mol Cell Cardiol. 1997;29:1177–1187.[CrossRef][ISI][Medline][Order article via Infotrieve]
  37. Morano M, Zacharzowski U, Maier M, et al. Regulation of human heart contractility by essential myosin light chain isoforms. J Clin Invest. 1996;98:467–473.[ISI][Medline][Order article via Infotrieve]
  38. Sutoh K. Identification of myosin-binding sites on the actin sequence. Biochem. 1982;21:3654–3661.[CrossRef][Medline][Order article via Infotrieve]
  39. Morano I, Haase H. Different actin affinities of human cardiac essential myosin light chain isoforms. FEBS Lett. 1997;408:71–74.[CrossRef][ISI][Medline][Order article via Infotrieve]
  40. Butler-Browne GS, Eriksson PO, Laurent C, Thornell LE. Adult human masseter muscle fibers express myosin isozymes characteristic of development. Muscle Nerve. 1988;11:610–620.[CrossRef][ISI][Medline][Order article via Infotrieve]
  41. Soussi-Yanicostas N, Barbet JP, Laurent-Winter C, Barton P, Butler-Brown GS. Transition of myosin isozymes during development of human masseter muscle. Development. 1990;108:239–249.[Abstract]
  42. Spencer RF, Porter JD. Structural organization of the extraocular muscles. Büttner-Enneuer JA eds. Neuroanatomy of the Oculomotor System. 1988;33–79. Elsevier Science Amsterdam.
  43. Jacoby J, Ko K, Weiss C, Rushbrook JI. Systematic variation in myosin expression along extraocular muscle fibres of the adult rat. J Muscle Res Cell Motil. 1989;11:25–40.
  44. Briggs MM, Jacoby J, Davidowitz J, Schachat FH. Expression of a novel combination of fast and slow troponin T isoforms in rabbit extraocular muscles. J Muscle Res Cell Motil. 1988;9:241–247.[CrossRef][ISI][Medline][Order article via Infotrieve]
  45. Briggs MM, Lin JJC, Schachat FH. The extent of amino-terminal heterogeneity in rabbit fast skeletal muscle troponin T. J Muscle Res Cell Motil. 1987;8:1–12.[CrossRef][ISI][Medline][Order article via Infotrieve]
  46. Fischer MD, Gorospe JR, Felder E, et al. Expression profiling reveals metabolic and structural components of extraocular muscles. Physiol Genom. 2002;9:71–84.[Abstract/Free Full Text]
  47. Khanna S, Merriam AP, Gong B, Leahy P, Porter JD. Comprehensive expression profiling by muscle tissue class and identification of the molecular niche of extraocular muscle. FASEB J. .Express article 10.1096/fj. 02–1108fje. Published online May 8, 2003.
  48. Demer JJL, Oh SY, Poukens V. Evidence for active control of rectus extraocular muscle pulleys. Invest Ophthalmol Vis Sci. 2000;41:1280–1290.[Abstract/Free Full Text]



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