Studies of Myosin Isoforms in Muscle Cells: Single Cell Mechanics and Gene Transfer

Transcription

1 CLINICL ORTHOPEDICS ND RELTED RESERCH Number 403S, pp. S51 S Lippincott Williams & Wilkins, Inc. Studies of Myosin Isoforms in Muscle Cells: Single Cell Mechanics and Gene Transfer Gordon J. Lutz, PhD; and Richard L. Lieber, PhD Myosin, the motor protein in skeletal muscle, is composed of two subunits, myosin heavy chain and myosin light chain. ll vertebrates express a family of myosin heavy chain and myosin light chain isoforms that together are primary determinants of force, velocity, and power in muscle fibers. Therefore, appropriate expression of myosin isoforms in skeletal muscle is critical to proper motor function. Myosin isoform expression is highly plastic and undergoes significant changes in response to muscular injury, muscle disuse, and disease. Therefore, myosin isoform function and plasticity are highly relevant to clinical orthopaedic research, musculoskeletal surgery, and sports medicine. Muscle from frogs offers a special opportunity to study the structural basis of contractile protein function because single intact fibers can be isolated that maintain excellent mechanical stability, allowing for highresolution studies of contractile performance in intact cells. The current authors summarize recent studies defining the myosin isoforms in muscle from frogs and the relationship between myosin isoforms and mechanical performance of intact single muscle cells. Preliminary studies also are described that show the potential for simple plasmid-based in vivo gene transfer approaches as a model system to elucidate the structural basis of muscle protein function in intact cells. List of bbreviations Used CMV DN GFP MHC MLC SDS-PGE cytomegalovirus deoxyribonucleic acid green fluorescent protein myosin heavy chain myosin light chain sodium dodecyl sulphate polyacrylamide gel electrophoresis From the Departments of Orthopaedics and ioengineering, iomedical Sciences Graduate Group, University of California, Veterans ffairs Medical Center and Veterans Medical Research Foundation, San Diego, C. Supported by National Institutes of Health grants R40050, R45631 and R46469 and a grant from the Department of Veterans ffairs. Reprint requests to G. J. Lutz, PhD, Drexel University College of Medicine, Department of Pharmacology and Physiology, Mail Stop 488, 8th Floor NC, 245 N. 15th Street, Philadelphia, P DOI: /01.blo Introduction to Myosin Isoforms The maximal force, velocity, and power produced by muscle fibers are determined to a large extent by the properties of myosin isoforms. The myosin molecule in skeletal muscle is composed of two subunits, MHC and MLC. ll vertebrates, including humans, express a family of MHC and MLC isoforms. The compliment of myosin isoforms in a muscle is matched exquisitely to the properties of S51

2 Clinical Orthopaedics S52 Lutz and Lieber and Related Research other structural and dynamic components, that together meet the functional requirements of the organism. Therefore, maintenance of the correct compliment of myosin isoforms is important to maintaining normal motor function. During the past 15 years, a series of detailed single muscle fiber studies of human and other mammals have characterized the relationship between myosin isoforms and mechanical function. 2,15,17 These studies showed large differences among fiber types in force-velocity properties that correlated well with differences in MHC isoform composition. There also is substantial evidence, especially in rodent fast twitch muscle fibers, that maximal shortening velocity (V max ) increased significantly with the ratio of the light chains MLC3/MLC1. 1 lthough these data have unquestionably established that myosin isoforms are key regulators of force-velocity properties, a major limitation is that most studies have been done on chemically skinned (membrane permeabilized) fibers. Skinning partially destabilizes mechanical function and induces fiber swelling, making interpretation of mechanical data difficult, especially at relatively high forces. lso, skinned fiber experiments must be done well below body temperature, and extrapolation to 37 C is problematic. In contrast, there have been relatively few data showing the direct relationship between myosin isoforms and mechanical function in intact cells. 5,6 TLE 1. The MHC and MLC Isoform Families in Rana pipiens Skeletal Muscle MHC isoform MHC1 MHC2 MHC3 MHCT MLC isoforms MLC1 f, ML2 f, MLC3 MLC1 f, MLC2 f, MLC3 MLC1 f, MLC2 f MLC1 Ta, MLC1 Tb, MLC2 T This is a simplified version of the expression patterns previously described. 7 The MLC1 x isoform is ignored for clarity. Frog Muscle Provides an Ideal System for Studying the Structural asis of Myosin Function in Intact Cells Frog muscle provides an ideal preparation to study the structural basis of contractile protein function because intact single fibers can be isolated that maintain superb mechanical stability. However, with a few notable exceptions (see below), frog muscle has not been exploited to investigate myosin isoform kinetics, mainly because of incomplete definition of the frog myosin isoform family. The MHC and MLC isoforms in skeletal muscle of Rana pipiens recently were defined at the protein and transcript levels 7,9,11 (Table 1). This work extended previous detailed definitions of myosinbased fiber types of amphibian skeletal muscle. 5,10,16,19,20 Four MHC isoforms and seven MLC isoforms were characterized in Rana pipiens (Table 1). ased on amphibian nomenclature of fiber types, which incidentally is opposite that used in mammals, the four MHC isoforms simply were defined as Type 1, Type 2, Type 3, and tonic. Relationship etween Mechanical Performance and Myosin Isoforms in Intact Frog Muscle Fibers detailed analysis of the relationship between myosin isoforms and mechanical function of intact single fibers from Rana pipiens was done recently. 13 The preparation for isolating a single fiber is shown in Figure 1. Contractile data were obtained using a classic single fiber recording system, including a spot-follower to measure precisely the length transients of a defined region of the fiber during contractions (Fig 1). To measure force-velocity properties, muscle fibers were driven through a series of isovelocity ramps while recording resultant force production (Fig 2, ). Examples of forcevelocity and power-velocity curves for two representative fibers are shown in Figures 2C and 2D, respectively. fter the mechanics experiments, MHC and MLC isoform content were measured in each of the fibers by quantitative SDS-PGE. nalysis was restricted to fibers that contained Type 1 or Type 2 MHCs, or fibers that coexpressed Type 1 and Type 2

3 Number 403S October, 2002 Myosin Isoforms in Skeletal Muscle S53 Fig 1. () Isolation of single intact muscle fibers is shown. The anterior tibialis muscle from Rana pipiens is shown thinned to a monolayer of cells, running from tendon to tendon. The fibers in this image are approximately 8 mm in length. For mechanical analysis, a single intact cell is delicately isolated, with a small piece of tendon at each end. () single fiber contractile recording apparatus is shown. Muscle fiber is secured in a Ringer s-filled chamber between a force transducer and servomotor. Stimulation is delivered through platinum plate electrodes. spot-follower system is used to measure the change in distance within a defined region of the cell delineated by surface markers. The details of this mechanics system were described previously. 13 MHCs. It previously was shown that these are the predominant fiber types in Rana pipiens hindlimb muscles, comprising greater than 95% of the fibers. 8 For MLCs, the ratio of MLC3/MLC1 was quantified because this has been shown to influence V max in rodent muscle. summary of the relationship between MHC isoforms and mechanical function is shown in Figure 3. Maximal shortening velocity (V max ), velocity at 50% maximal tension (V P50 ), maximal specific tension (P o /CS; where P o is maximal tension and CS is fiber cross-sectional area) and maximal power (W max ) all increased significantly with the percentage of Type 1 MHC (%MHC1; Fig 3). From the regression analysis in Figure 3, V max, V P50, P o /CS, and W max increased by 21.4%, 34.4%, 22.3%, and 61.3%, respectively, as %MHC1 increased from 0% to 100%. There was no significant correlation between MLC3/MLC1 and any of these four mechanical parameters (data not shown 13 ). These data show that MHC isoforms have a potent influence over the full force-velocity range and maximal power production of intact fibers from Rana pipiens. The influence of MHC isoforms on mechanical function at velocities below V max especially is important. Fibers are not used during normal motor function at V max, where they generate no power. In contrast, the influence of MHC isoforms on P o /CS and W max has a more obvious impact on normal motor function. W max is the product of maximal specific isometric tension (P o /CS), relative tension (P/P o ; where P is tension) and velocity at the point of peak power. Multiple regression analysis showed that P o /CS accounted for 70.4% of the variability in W max, whereas velocity and P/P o explained an additional 17.1% and 11.6% of the variability, respectively. more general multiple regression analysis of 15 different structural and mechanical parameters showed that %MHC1 had the highest correlation with W max (r ). 13 In an extensive series of studies, Lannergren 5 and Lannergren and Hoh 6 measured the contractile properties of single intact fibers representing the full range of fiber types from Xenopus laevis. Type 1 and Type 2 fibers (subclassified as Type 1n and Type 2n) in Xenopus laevis contained unique MHC isoforms. 5 The differences in contractile properties between Type 1 and Type 2

4 Clinical Orthopaedics S54 Lutz and Lieber and Related Research C D Fig 2 D. These graphs show single fiber mechanical recordings. () Sarcomere length transients and () force production of an intact single frog muscle fiber are shown during a series of isovelocity contractions over a range of shortening velocities. Fiber shortening velocities were 3.0, 6.0 and 12.5 lengths per second (L/s) for traces a, b, and c, respectively. The period during which length and force were measured for force-velocity curves is indicated with thickened lines in the respective traces. In practice, approximately 13 to 15 such contractions were used to construct the force-velocity relationship. Stimulus began at Time 0 and continued throughout the period shown. (C) Force-velocity and (D) power-velocity relationships for a relatively fast (filled circles) and slow (open circles) intact frog muscle fiber are shown. (C, Inset) High velocity region of the force-velocity data is shown. Force is shown in relative units (P/P o ). Data are based on experiments published previously. 13 Temperature 25 C fibers in Rana pipiens, reported here, are similar to the differences between Type 1n and Type 2n fibers in Xenopus laevis. oth studies showed that MHC isoforms had a significant influence on the full force-velocity relationship and maximal mechanical power production in intact fibers. The importance of MHC isoforms on V max also was implicated in work on Rana temporaria by Edman and colleagues. 3 Importance of Myosin Isoform Distribution in Frog Hopping The large differences in power production between fibers with Type 1 and Type 2 MHCs clearly is reflected in the overall design of the frog muscular system. Large extensor muscles, which are responsible for producing the majority of power during jumping in frogs, are composed almost entirely of Type 1 MHC, whereas Type 2 MHC is predominant in smaller muscles that do not contribute as much to jumping. This indicates the importance of myosin isoform expression patterns in the functional design of the muscular system. In the case of Rana pipiens frogs, a design that allows near maximal power to be delivered by the major extensor muscles of the hindlimb during jumping. 12

5 Number 403S October, 2002 Myosin Isoforms in Skeletal Muscle S55 C D Fig 3 D. The relationship between mechanical properties and MHC isoforms in intact frog single muscle fibers is shown. Data points are individual fibers (n 12). Myosin heavy chain isoform content is expressed as percentage of Type 1 MHC (%MHC1). Therefore, data points to the far right indicate 100% Type 1 MHC, and those to the far left indicate 100% Type 2 MHC. Mechanical parameters measured were () maximal shortening velocity (V max), () velocity at 50% of maximum isometric tension (V P50 ), (C) maximal specific isometric tension (P o /CS) and (D) maximal mechanical power (W max ). Linear regression analysis showed that each of the four mechanical parameters increased with %MHC1, and all regressions were statistically significant. Data are based on experiments published previously. 13 Temperature 25 C Future Directions: In Vivo Gene Transfer as a Model to Study the Structural asis of Contractile Protein Function in Intact Muscle Cells In vivo gene transfer is a rapidly expanding field with enormous potential for understanding the structural basis of contractile protein function, gene regulation, and providing therapeutic treatment of musculoskeletal disorders (gene therapy). In this section preliminary experiments are described that show the potential for using in vivo gene transfer of simple plasmid vectors to study the structural basis of myosin function in intact frog muscle cells. The ultimate goal of these experiments is to express full-length recombinant MHC and MLC constructs in frog muscle cells. These myosin constructs will be designed to contain modifications at strategic locations thought to be important in regulating cross-bridge kinetics. We will then use the mechanical recording system described above to measure the contractile properties of the transgenic cells. This system offers the advantage that the functional outcome of the manipulations of myosin structure, are determined in the intact cells. s a starting point, it was required to determine whether high levels of in vivo plasmid

6 Clinical Orthopaedics S56 Lutz and Lieber and Related Research transfection and expression could be achieved in frog muscle. For this purpose, a plasmid vector containing a CMV promoter (constitutive cellular promoter) driving the expression of GFP was used. s expected, when the CMV-GFP plasmid was injected into normal frog muscle the transfection efficiency essentially was 0. Two methods of boosting transfection efficiency, electroporation, and regeneration will be documented. Enhanced in vivo gene transfer by electroporation has received attention. 18 The CMV- GFP plasmid was injected directly into the anterior tibialis muscle of an anesthetized frog and a series of electrical pulses were delivered immediately across the site of injection (Fig 4). Fifteen days after electroporation, numerous fluorescent cells were detected at the site of injection (Fig 4, C). Enhanced plasmid transfection efficiency in vivo also has been reported by direct injection of naked DN into regenerating muscle, without the need for electroporation. 4,14 To induce degeneration, anterior tibialis muscles were injected with cardiotoxin (cobra venom). Nine days after cardiotoxin, the muscle had undergone massive degeneration, but contained a bed of small regenerating muscle fibers (Fig 5). Muscles were injected 9 days after cardiotoxin with the CMV-GFP plasmid. Twenty-one days after plasmid injection, the muscles contained numerous fluorescent fibers, marking the presence of the transgene (Fig 5C, D). The high transfection efficiency especially is appreciated in the transverse section (Fig 5D). The regeneration model has shown excellent promise for contractile protein structure and function studies. In pilot experiments with C Fig 4 C. In vivo electroporation increased gene transfer efficiency and expression of CMV-GFP plasmid DN in frog muscle. () The anterior tibialis muscle was injected with an expression plasmid that encoded GFP driven by the constitutive CMV cellular promoter, and the injection was followed immediately by three bipolar pulses (30 V, 30 ms each). The low voltage, long duration pulses increase plasmid transfection efficiency through the process of electroporation. () GFP expression in muscle 15 days after electroporation is shown. (C) higher magnification view of the transfected region in Figure 4 is shown.

7 Number 403S October, 2002 Myosin Isoforms in Skeletal Muscle S57 C D Fig 5 D. Plasmid injection into regenerating muscle increased transfection efficiency and expression of CMV-GFP plasmid DN in frog muscle. () Normal muscle reacted with antimhc (F8), shows classic mosaic appearance (F8-positive fibers are Type 1). () Regenerating muscle 9 days after toxin shows a clear bed of small regenerating myofibers. (C) Longitudinal and (D) transverse views of muscle injected with CMV-GFP plasmid during regeneration (9 days after cardiotoxin) and harvested at 21 days after plasmid injection are shown. Green fluorescent protein expression shows the high level of transfection efficiency and expression in fibers that have regenerated to near adult size. this system, expression and myofilament incorporation of transgenic epitope-tagged MLC1 f reached levels nearly equal to the endogenous MLC1 f. 21 This encouraging result may be accounted for by the fact that with this system, transgene expression occurs coincident with the growth of newly-forming sarcomeres, myofilaments, and myofibers. Therefore, the level of transmlc incorporated may not be limited by protein turnover rates, as it would be when transfecting mature muscle fibers. Relevance to Clinical Orthopaedics The current authors have described the strong relationship between MHC isoforms and mechanical function, over the full force-velocity range, in intact muscle cells. This further validates that expression of the correct compliment of myosin isoforms in muscle has a critical impact on motor function and locomotion. Myosin isoform expression is extremely plastic and can be altered in response to a host of conditions, including muscle disuse, and vari-

8 Clinical Orthopaedics S58 Lutz and Lieber and Related Research ous types of muscle injury. Therefore, myosin isoform expression is highly relevant to clinical orthopaedics, musculoskeletal surgery, and sports medicine. In addition, pilot experiments indicated a strong potential for plasmid-based in vivo gene transfer into regenerating muscle as a model system to study contractile protein structure and function. Plasmid expression in regenerating muscle may have an equally bright future in studies of gene regulation during regeneration and muscle growth. These issues are clinically relevant, because many clinical orthopaedic applications involve, directly or indirectly, the degeneration and regeneration of skeletal muscles (surgical removal of muscles to treat skeletal disorders). cknowledgments The authors thank Shannon remner, Dustin Robinson, Karen Sethi, Shashank Sirsi, Sarah Shapard- Palmer, Haiyan Yu, and Michael ade for technical assistance. References 1. ottinelli R, etto 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 478: , ottinelli R, Reggiani C: Human skeletal muscle fibres: Molecular and functional diversity. Prog iophys Mol iol 73: , Edman KP, Reggiani C, Schiaffino S, tekronnie G: Maximum velocity of shortening related to myosin isoform composition in frog skeletal muscle fibres. J Physiol 395: , Hallauer PL, Karpati G, Hastings KE: Skeletal muscle gene transfer: Regeneration-associated deregulation of fast troponin I fiber type specificity. m J Physiol Cell Physiol 278: , Lannergren J: Contractile properties and myosin isoenzymes of various kinds of Xenopus twitch muscle fibres. J Muscle Res Cell Motil 8: , Lannergren J, Hoh JFY: Myosin isoenzymes in single muscle fibres of Xenopus laevis: nalysis of five different functional types. Proc R Soc Lond iol Sci 222: , Lutz GJ, remner SN, ade MJ, Lieber RL: Identification of myosin light chains in Rana pipiens skeletal muscle and their expression patterns along single fibres. J Exp iol 204: , Lutz GJ, remner SN, Lajevardi N, Lieber RL, Rome LC: Quantitative analysis of muscle fiber type and myosin heavy chain distribution in the frog hindlimb: Implications for locomotory design. J Muscle Res Cell Motil 19: , Lutz GJ, Cuizon D, Ryan F, Lieber RL: Four novel myosin heavy chain transcripts define a molecular basis for muscle fibre types in Rana pipiens. J Physiol 508: , Lutz GJ, Lieber RL: Myosin isoforms in anuran skeletal muscle: Their influence on contractile properties and in vivo muscle function. Microsc Res Tech 50: , Lutz GJ, Razzaghi S, Lieber RL: Cloning and characterization of the S1 domain of four myosin isoforms from functionally divergent fiber types in adult Rana pipiens skeletal muscle. Gene 250:97 107, Lutz GJ, Rome LC: uilt for jumping: The design of frog muscular system. Science 263: , Lutz GJ, Sirsi SR, Shapard-Palmer S, remner SN, Lieber RL: Influence of myosin isoforms on contractile properties of intact muscle fibers from Rana pipiens. m J Physiol Cell Physiol 282:C835 C844, Marsh DR, Carson J, Stewart LN, ooth FW: ctivation of skeletal alpha-actin promoter during muscle regeneration. J Muscle Res Cell Motil 19: , Moss RL, Diffee GM, Greaser ML: Contractile properties of skeletal muscle fibers in relation to myofibrillar protein isoforms. Rev Physiol iochem Pharmacol 126:1 63, Rowlerson M, Spurway NC: Histochemical and immunohistochemical properties of skeletal muscle fibres from Rana and Xenopus. Histochem J 20: , Schiaffino S, Reggiani C: Molecular diversity of myofibrillar proteins: Gene regulation and functional significance. Physiol Rev 76: , Smith LC, Nordstrom JL: dvances in plasmid gene delivery and expression in skeletal muscle. Curr Opin Mol Ther 2: , Smith RS, Lannergren J: Types of motor units in the skeletal muscle of Xenopus laevis. Nature 217: , Smith RS, Ovalle Jr WK: Varieties of fast and slow extrafusal muscle fibres in amphibian hind limb muscles. J nat 116:1 24, Zhang J, Robinson D, Lutz G: In vivo gene transfer as a model for myosin structure function analysis in intact muscle cells. iophys J 82:412, bstract.