studies of myosin isoforms in muscle cells: single cell mechanics...

CLINICAL ORTHOPAEDICS AND RELATED RESEARCHNumber 403S, pp. S51–S58© 2002 Lippincott Williams & Wilkins, Inc.

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Myosin, the motor protein in skeletal muscle, iscomposed of two subunits, myosin heavy chainand myosin light chain. All vertebrates expressa family of myosin heavy chain and myosin lightchain isoforms that together are primary deter-minants of force, velocity, and power in musclefibers. Therefore, appropriate expression ofmyosin isoforms in skeletal muscle is critical toproper motor function. Myosin isoform expres-sion is highly plastic and undergoes significantchanges in response to muscular injury, muscledisuse, and disease. Therefore, myosin isoformfunction and plasticity are highly relevant toclinical orthopaedic research, musculoskeletalsurgery, and sports medicine. Muscle from frogsoffers a special opportunity to study the struc-tural basis of contractile protein function becausesingle intact fibers can be isolated that maintainexcellent mechanical stability, allowing for high-resolution studies of contractile performance inintact cells. The current authors summarize re-

cent studies defining the myosin isoforms inmuscle from frogs and the relationship betweenmyosin isoforms and mechanical performanceof intact single muscle cells. Preliminary studiesalso are described that show the potential forsimple plasmid-based in vivo gene transfer ap-proaches as a model system to elucidate thestructural basis of muscle protein function in in-tact cells.

Introduction to Myosin IsoformsThe maximal force, velocity, and power pro-duced by muscle fibers are determined to alarge extent by the properties of myosin iso-forms. The myosin molecule in skeletal mus-cle is composed of two subunits, MHC andMLC. All vertebrates, including humans, ex-press a family of MHC and MLC isoforms.The compliment of myosin isoforms in a mus-cle is matched exquisitely to the properties of

Studies of Myosin Isoforms in MuscleCells: Single Cell Mechanics and

Gene Transfer

Gordon J. Lutz, PhD; and Richard L. Lieber, PhD

From the Departments of Orthopaedics and Bioengineer-ing, Biomedical Sciences Graduate Group, University ofCalifornia, Veterans Affairs Medical Center and Veter-ans Medical Research Foundation, San Diego, CA.Supported by National Institutes of Health grantsAR40050, AR45631 and AR46469 and a grant from theDepartment of Veterans Affairs.Reprint requests to G. J. Lutz, PhD, Drexel UniversityCollege of Medicine, Department of Pharmacology andPhysiology, Mail Stop 488, 8th Floor NCB, 245 N. 15thStreet, Philadelphia, PA 19102.DOI: 10.1097/01.blo.0000031308.06353.00

List of Abbreviations Used

CMV cytomegalovirusDNA deoxyribonucleic acidGFP green fluorescent proteinMHC myosin heavy chainMLC myosin light chainSDS-PAGE sodium dodecyl sulphate

polyacrylamide gelelectrophoresis

other structural and dynamic components, thattogether meet the functional requirements ofthe organism. Therefore, maintenance of thecorrect compliment of myosin isoforms is im-portant to maintaining normal motor function.

During the past 15 years, a series of detailedsingle muscle fiber studies of human and othermammals have characterized the relationshipbetween myosin isoforms and mechanical func-tion.2,15,17 These studies showed large differ-ences among fiber types in force-velocity prop-erties that correlated well with differences inMHC isoform composition. There also is sub-stantial evidence, especially in rodent fast twitchmuscle fibers, that maximal shortening veloc-ity (Vmax) increased significantly with the ratioof the light chains MLC3/MLC1.1 Althoughthese data have unquestionably establishedthat myosin isoforms are key regulators offorce-velocity properties, a major limitation isthat most studies have been done on chemi-cally skinned (membrane permeabilized) fibers.Skinning partially destabilizes mechanical func-tion and induces fiber swelling, making inter-pretation of mechanical data difficult, espe-cially at relatively high forces. Also, skinnedfiber experiments must be done well belowbody temperature, and extrapolation to 37� Cis problematic. In contrast, there have been rel-atively few data showing the direct relation-ship between myosin isoforms and mechani-cal function in intact cells.5,6

Frog Muscle Provides an Ideal System forStudying the Structural Basis of MyosinFunction in Intact CellsFrog muscle provides an ideal preparation tostudy the structural basis of contractile proteinfunction because intact single fibers can beisolated that maintain superb mechanical sta-bility. However, with a few notable exceptions(see below), frog muscle has not been ex-ploited to investigate myosin isoform kinetics,mainly because of incomplete definition of thefrog myosin isoform family. The MHC andMLC isoforms in skeletal muscle of Rana pip-iens recently were defined at the protein andtranscript levels7,9,11 (Table 1). This work ex-

tended previous detailed definitions of myosin-based fiber types of amphibian skeletal mus-cle.5,10,16,19,20 Four MHC isoforms and sevenMLC isoforms were characterized in Rana pip-iens (Table 1). Based on amphibian nomen-clature of fiber types, which incidentally is op-posite that used in mammals, the four MHCisoforms simply were defined as Type 1, Type2, Type 3, and tonic.

Relationship Between MechanicalPerformance and Myosin Isoforms inIntact Frog Muscle FibersA detailed analysis of the relationship betweenmyosin isoforms and mechanical function ofintact single fibers from Rana pipiens wasdone recently.13 The preparation for isolatinga single fiber is shown in Figure 1A. Contrac-tile data were obtained using a classic single fiberrecording system, including a spot-follower tomeasure precisely the length transients of a de-fined region of the fiber during contractions(Fig 1B).

To measure force-velocity properties, mus-cle fibers were driven through a series of isove-locity ramps while recording resultant forceproduction (Fig 2A, B). Examples of force-velocity and power-velocity curves for two rep-resentative fibers are shown in Figures 2C and2D, respectively. After the mechanics experi-ments, MHC and MLC isoform content weremeasured in each of the fibers by quantitativeSDS-PAGE. Analysis was restricted to fibersthat contained Type 1 or Type 2 MHCs, orfibers that coexpressed Type 1 and Type 2

Clinical OrthopaedicsS52 Lutz and Lieber and Related Research

TABLE 1. The MHC and MLC IsoformFamilies in Rana pipiens SkeletalMuscle

MHC isoform MLC isoforms

MHC1 MLC1f, ML2f, MLC3MHC2 MLC1f, MLC2f, MLC3MHC3 MLC1f, MLC2fMHCT MLC1Ta, MLC1Tb, MLC2T

This is a simplified version of the expression patterns previ-ously described.7 The MLC1x isoform is ignored for clarity.

MHCs. It previously was shown that these arethe predominant fiber types in Rana pipienshindlimb muscles, comprising greater than95% of the fibers.8 For MLCs, the ratio of

MLC3/MLC1 was quantified because this hasbeen shown to influence Vmax in rodent muscle.

A summary of the relationship between MHCisoforms and mechanical function is shown inFigure 3. Maximal shortening velocity (Vmax),velocity at 50% maximal tension (VP50), maxi-mal specific tension (Po/CSA; where Po is max-imal tension and CSA is fiber cross-sectionalarea) and maximal power (Wmax) all increasedsignificantly with the percentage of Type 1MHC (%MHC1; Fig 3). From the regressionanalysis in Figure 3, Vmax, VP50, Po/CSA, andWmax increased by 21.4%, 34.4%, 22.3%, and61.3%, respectively, as %MHC1 increased from0% to 100%. There was no significant correla-tion between MLC3/MLC1 and any of thesefour mechanical parameters (data not shown13).These data show that MHC isoforms have a po-tent influence over the full force-velocity rangeand maximal power production of intact fibersfrom Rana pipiens.

The influence of MHC isoforms on me-chanical function at velocities below Vmax es-pecially is important. Fibers are not used dur-ing normal motor function at Vmax, where theygenerate no power. In contrast, the influenceof MHC isoforms on Po/CSA and Wmax has amore obvious impact on normal motor func-tion. Wmax is the product of maximal specificisometric tension (Po/CSA), relative tension(P/Po; where P is tension) and velocity at thepoint of peak power. Multiple regression analy-sis showed that Po/CSA accounted for 70.4%of the variability in Wmax, whereas velocityand P/Po explained an additional 17.1% and11.6% of the variability, respectively. A moregeneral multiple regression analysis of 15 dif-ferent structural and mechanical parametersshowed that %MHC1 had the highest correla-tion with Wmax (r2 � 0.846).13

In an extensive series of studies, Lannergren5

and Lannergren and Hoh6 measured the contrac-tile properties of single intact fibers representingthe full range of fiber types from Xenopus laevis.Type 1 and Type 2 fibers (subclassified as Type1n and Type 2n) in Xenopus laevis containedunique MHC isoforms.5 The differences in con-tractile properties between Type 1 and Type 2

Number 403SOctober, 2002 Myosin Isoforms in Skeletal Muscle S53

Fig 1A–B. (A) Isolation of single intact musclefibers is shown. The anterior tibialis muscle fromRana pipiens is shown thinned to a monolayer ofcells, running from tendon to tendon. The fibersin this image are approximately 8 mm in length.For mechanical analysis, a single intact cell isdelicately isolated, with a small piece of tendon ateach end. (B) A single fiber contractile recordingapparatus is shown. Muscle fiber is secured in aRinger’s-filled chamber between a force trans-ducer and servomotor. Stimulation is deliveredthrough platinum plate electrodes. A spot-followersystem is used to measure the change in dis-tance within a defined region of the cell delin-eated by surface markers. The details of this me-chanics system were described previously.13

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fibers in Rana pipiens, reported here, are similarto the differences between Type 1n and Type 2nfibers in Xenopus laevis. Both studies showedthat MHC isoforms had a significant influenceon the full force-velocity relationship and max-imal mechanical power production in intactfibers. The importance of MHC isoforms onVmax also was implicated in work on Rana tem-poraria by Edman and colleagues.3

Importance of Myosin IsoformDistribution in Frog HoppingThe large differences in power production be-tween fibers with Type 1 and Type 2 MHCs

clearly is reflected in the overall design of thefrog muscular system. Large extensor mus-cles, which are responsible for producing themajority of power during jumping in frogs, arecomposed almost entirely of Type 1 MHC,whereas Type 2 MHC is predominant insmaller muscles that do not contribute as muchto jumping. This indicates the importance ofmyosin isoform expression patterns in thefunctional design of the muscular system. Inthe case of Rana pipiens frogs, a design that al-lows near maximal power to be delivered bythe major extensor muscles of the hindlimbduring jumping.12


Fig 2A–D. These graphs show single fiber mechanical recordings. (A) Sarcomere length transientsand (B) force production of an intact single frog muscle fiber are shown during a series of isovelocitycontractions over a range of shortening velocities. Fiber shortening velocities were 3.0, 6.0 and 12.5lengths per second (L/s) for traces a, b, and c, respectively. The period during which length and forcewere measured for force-velocity curves is indicated with thickened lines in the respective traces. Inpractice, approximately 13 to 15 such contractions were used to construct the force-velocity relation-ship. 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 mus-cle fiber are shown. (C, Inset) High velocity region of the force-velocity data is shown. Force is shownin relative units (P/Po). Data are based on experiments published previously.13 Temperature � 25� C

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Future Directions: In Vivo Gene Transferas a Model to Study the Structural Basis ofContractile Protein Function in IntactMuscle CellsIn vivo gene transfer is a rapidly expandingfield with enormous potential for understandingthe structural basis of contractile protein func-tion, gene regulation, and providing therapeu-tic treatment of musculoskeletal disorders (genetherapy). In this section preliminary experi-ments are described that show the potential forusing in vivo gene transfer of simple plasmidvectors to study the structural basis of myosinfunction in intact frog muscle cells. The ulti-

mate goal of these experiments is to expressfull-length recombinant MHC and MLC con-structs in frog muscle cells. These myosin con-structs will be designed to contain modificationsat strategic locations thought to be important inregulating cross-bridge kinetics. We will thenuse the mechanical recording system describedabove to measure the contractile properties ofthe transgenic cells. This system offers the ad-vantage that the functional outcome of the ma-nipulations of myosin structure, are determinedin the intact cells.

As a starting point, it was required to deter-mine whether high levels of in vivo plasmid


Fig 3A–D. The relationship between mechanical properties and MHC isoforms in intact frog singlemuscle fibers is shown. Data points are individual fibers (n � 12). Myosin heavy chain isoform contentis expressed as percentage of Type 1 MHC (%MHC1). Therefore, data points to the far right indicate100% Type 1 MHC, and those to the far left indicate 100% Type 2 MHC. Mechanical parameters mea-sured were (A) maximal shortening velocity (Vmax), (B) velocity at 50% of maximum isometric tension(VP50), (C) maximal specific isometric tension (Po/CSA) and (D) maximal mechanical power (Wmax). Lin-ear 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.13Temperature � 25� C

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transfection and expression could be achievedin frog muscle. For this purpose, a plasmidvector containing a CMV promoter (constitu-tive cellular promoter) driving the expressionof GFP was used. As expected, when theCMV-GFP plasmid was injected into normalfrog muscle the transfection efficiency essen-tially was 0. Two methods of boosting trans-fection efficiency, electroporation, and regen-eration will be documented.

Enhanced in vivo gene transfer by electro-poration has received attention.18 The CMV-GFP plasmid was injected directly into the an-terior tibialis muscle of an anesthetized frogand a series of electrical pulses were deliveredimmediately across the site of injection (Fig4A). Fifteen days after electroporation, nu-merous fluorescent cells were detected at thesite of injection (Fig 4B, C).

Enhanced plasmid transfection efficiencyin vivo also has been reported by direct injec-tion of naked DNA into regenerating muscle,without the need for electroporation.4,14 To in-duce degeneration, anterior tibialis muscleswere injected with cardiotoxin (cobra venom).Nine days after cardiotoxin, the muscle had un-dergone massive degeneration, but containeda bed of small regenerating muscle fibers (Fig 5).Muscles were injected 9 days after cardiotoxinwith the CMV-GFP plasmid. Twenty-one daysafter plasmid injection, the muscles containednumerous fluorescent fibers, marking the pres-ence of the transgene (Fig 5C, D). The hightransfection efficiency especially is appreciatedin the transverse section (Fig 5D).

The regeneration model has shown excel-lent promise for contractile protein structureand function studies. In pilot experiments with


Fig 4A–C. In vivo electroporation increased gene transfer efficiency and expression of CMV-GFP plas-mid DNA in frog muscle. (A) The anterior tibialis muscle was injected with an expression plasmid thatencoded GFP driven by the constitutive CMV cellular promoter, and the injection was followed imme-diately by three bipolar pulses (30 V, 30 ms each). The low voltage, long duration pulses increase plas-mid transfection efficiency through the process of electroporation. (B) GFP expression in muscle 15days after electroporation is shown. (C) A higher magnification view of the transfected region in Figure4B is shown.

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this system, expression and myofilament in-corporation of transgenic epitope-tagged MLC1freached levels nearly equal to the endogenousMLC1f.21 This encouraging result may be ac-counted for by the fact that with this system,transgene expression occurs coincident withthe growth of newly-forming sarcomeres, myo-filaments, and myofibers. Therefore, the levelof transMLC incorporated may not be limitedby protein turnover rates, as it would be whentransfecting mature muscle fibers.

Relevance to Clinical OrthopaedicsThe current authors have described the strongrelationship between MHC isoforms and me-chanical function, over the full force-velocityrange, in intact muscle cells. This further val-idates that expression of the correct compli-ment of myosin isoforms in muscle has a crit-ical impact on motor function and locomotion.Myosin isoform expression is extremely plas-tic and can be altered in response to a host ofconditions, including muscle disuse, and vari-


Fig 5A–D. Plasmid injection into regenerating muscle increased transfection efficiency and expressionof CMV-GFP plasmid DNA in frog muscle. (A) Normal muscle reacted with antiMHC (F8), shows clas-sic mosaic appearance (F8-positive fibers are Type 1). (B) Regenerating muscle 9 days after toxinshows a clear bed of small regenerating myofibers. (C) Longitudinal and (D) transverse views of mus-cle injected with CMV-GFP plasmid during regeneration (9 days after cardiotoxin) and harvested at 21days after plasmid injection are shown. Green fluorescent protein expression shows the high level oftransfection efficiency and expression in fibers that have regenerated to near adult size.

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ous types of muscle injury. Therefore, myosinisoform expression is highly relevant to clini-cal orthopaedics, musculoskeletal surgery, andsports medicine. In addition, pilot experimentsindicated a strong potential for plasmid-basedin vivo gene transfer into regenerating muscleas a model system to study contractile proteinstructure and function. Plasmid expression inregenerating muscle may have an equally brightfuture in studies of gene regulation during re-generation and muscle growth. These issuesare clinically relevant, because many clinicalorthopaedic applications involve, directly orindirectly, the degeneration and regeneration ofskeletal muscles (surgical removal of musclesto treat skeletal disorders).

AcknowledgmentsThe authors thank Shannon Bremner, Dustin Robin-son, Karen Sethi, Shashank Sirsi, Sarah Shapard-Palmer, Haiyan Yu, and Michael Bade for technicalassistance.

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