In vivo expression of myosin essential light chain using plasmid expression vectors in regenerating frog skeletal muscle.

It is well established that mutations in specific structural elements of the motor protein myosin are directly linked to debilitating diseases involving malfunctioning striated muscle cells. A potential way to study the relationship between myosin structure and function is to express exogenous myosin in vivo and determine contractile properties of the transgenic muscle cells. However, in vivo expression of functional levels of contractile proteins using transient transgenesis in skeletal muscle has not been demonstrated. Presently, we used in vivo gene transfer to express high levels of full-length myosin light chain (MLC) in skeletal muscle fibers of Rana pipiens. Anterior tibialis (AT) muscles were injected with cardiotoxin to cause degeneration and then injected at various stages of regeneration with plasmid expression vectors encoding full-length MLC1(f). In fibers from the most robustly transfected muscles 3 weeks after plasmid injections, trans-MLC1(f) expression averaged 22-43% of the endogenous MLC1(f). Trans-MLC1(f) expression was the same whether a small epitope tag was placed on the C- or N-terminus and was highly variable along individual fibers. Confocal microscopy of skinned fibers showed correct sarcomeric incorporation of trans-MLC1(f). The expression profile of myosin heavy chain isoforms 21 days after transfection was similar to normal AT muscle. These data demonstrate the feasibility of using in vivo gene transfer to probe the structural basis of contractile protein function in skeletal muscle. Based on these promising results, we discuss how further improvements in the level and consistency of myosin transgene expression may be achieved in future studies, and the therapeutic potential of plasmid gene transfer in regenerating muscle.

Identification of myosin light chains in Rana pipiens skeletal muscle and their expression patterns along single fibres.

Isoforms of myosin heavy chain (MHC) and myosin light chain (MLC) influence contractile kinetics of skeletal muscle. We previously showed that the four major skeletal muscle fibre types in Rana pipiens (type 1, type 2, type 3 and tonic; amphibian nomenclature) contain four unique MHC isoforms. In the present study we defined the MLCs expressed in each of these R. pipiens fibre types. The MLC composition of single MHC-typed fibres was determined from western blots using a panel of monoclonal MLC antibodies. A total of seven MLCs were identified, including four types of MLC1, two of MLC2 and a single MLC3. Twitch fibre types (types 1, 2 and 3) expressed MLC1(f) and MLC2(f), while tonic fibres contained a unique set of isoforms, MLC1(Ta), MLC1(Tb) and MLC2(T). MLC3 was expressed primarily in type 1, type 1-2 and type 2 fibres. Surprisingly, some frogs displayed a striking pattern of MLC expression where a unique isoform of MLC1 (MLC1(x)) was coexpressed along with the normal MLC1 isoform(s) in all fibre types. MLC1(x) was either expressed in all fibres of a given frog or was completely absent. The intraspecific polymorphism in MLC1 expression is likely to have a genetic basis, but is unlikely to be caused by allelic variation. The ratio of MLC3/MLC1 increased in direct proportion to the percentage of type 1 MHC, but was only weakly correlated. The variability in MLC3/MLC1 within a fibre type was extremely large. Both the MHC isoform and MLC3/MLC1 ratio varied significantly between 1 mm segments along the length of fibres. For all segments combined, MLC3/MLC1 increased with the percentage of type 1 MHC, but the correlation between segments was weaker than between fibres.

Myosin isoforms in anuran skeletal muscle: their influence on contractile properties and in vivo muscle function.

Functional studies on isolated single anuran skeletal muscle cells represent classic experiments from which much of our understanding of muscle contraction mechanisms have been derived. Because of their superb mechanical stability when isolated, single anuran fibers provide a uniquely powerful model system that can be exploited to understand the relationship between myosin heavy chain (MHC) and myosin light chain (MLC) composition and muscle fiber function. In this review, we summarize historic and recent studies of MHC and MLC expression patterns in the fiber types of anuran species. We extend the traditional classification scheme, using data from recent reports in which frog MHCs have been cloned, to reveal the molecular basis of frog muscle fiber types. The influence of MHC and MLC isoforms on contractile kinetics of single intact fibers is reviewed. In addition, we discuss more subtle questions such as variability of myosin coexpression along a single cell, and its potential influence on contractile function. The frog jump is used as a model system to elucidate principles of muscular system design, including the role of MHC isoforms on in vivo muscle function. Sequence information is used from cloned frog MHCs to understand the role of specific regions of the myosin motor domain in regulating contractile function and the evolutionary origins of fast and slow amphibian MHCs. Finally, we offer promising future possibilities that combine molecular methods (such as recombinant gene transfer) with single cell contractile measurements to address questions regarding myosin structure/function and gene regulation.

Cloning and characterization of the S1 domain of four myosin isoforms from functionally divergent fiber types in adult Rana pipiens skeletal muscle.

The motor properties of myosin reside in the globular S1 region of the myosin heavy chain (MHC) subunit. All vertebrates express a family of MHC isoforms in skeletal muscle that have a major influence on the mechanical properties of the various fiber types. Differences in molecular composition of S1 among MHC isoforms within a species have not been studied to any great detail. Presently, we have isolated, cloned and sequenced the S1 subunit of four MHC isoforms from skeletal muscle in Rana pipiens that are specifically expressed in four mechanically divergent fiber types. Paired analysis showed that the overall amino acid identity was higher between the three S1 isoforms expressed in twitch fibers than between the twitch and tonic isoforms. Relatedness in amino acid composition was evaluated in regions reported to govern cross-bridge kinetics. Surface loops 1 and 2, thought to influence motor velocity and ATPase, respectively, were both highly divergent between isoforms. However, the divergence in the loops was roughly equal to that of the amino-terminal region, a domain considered less important for motor function. We tested the hypothesis that the loops are more conserved in pairs of isoforms with more similar kinetics. Comparisons including other vertebrate species showed no tendency for loops from pairs with similar kinetics to be more conserved. These data suggest that the overall structure of loops 1 and 2 is not critical in regulating the kinetic properties of R. pipiens S1 isoforms. Cloning of this family of frog S1 isoforms will facilitate future structure/function studies of the molecular basis of variability in myosin cross-bridge kinetics.

Skeletal muscle myosin II structure and function.

Recent experimental advances in structural biology, biophysics, and molecular biology have dramatically increased our understanding of the molecular mechanism of muscle contraction, as well as the assembly of myosin filaments. Future studies are required to detail, for example, the molecular cause of the conformational change during the power stroke and ATP hydrolysis, as well as the nature of the communication between nucleotide and actin binding sites. Based on the structural and functional homology between myosin and other molecular motors, these findings have implications not only for understanding muscle contraction, but for understanding numerous aspects of motility in all cellular systems as well.

Quantitative analysis of muscle fibre type and myosin heavy chain distribution in the frog hindlimb: implications for locomotory design.

To investigate the design of the frog muscular system for jumping, fibre type distribution and myosin heavy chain (MHC) isoform composition were quantified in the hindlimb muscles of Rana pipiens. Muscles were divided into two groups: five large extensor muscles which were predicted to shorten and produce mechanical power during jumping (JP), and four much smaller muscles commonly used in muscle physiology studies, but that do not shorten or produce power during jumping (NJP). fibres were classified as one of four different types (type 1, 2, 3 or tonic) or an intermediate type (type 1-2) based on their relative myosin-ATPase reactivity and MHC immunoreactivity in muscle cross-sections according to previous nomenclature established for amphibian skeletal muscle. Type 1 fibres correspond to the fastest and most powerful of the twitch fibres, and type 3 fibres are the slowest and least powerful. Myosin-ATPase histochemistry revealed that the JP muscles were composed primarily of type 1 fibres (89%) with a small percentage of type 2 (7%) and intermediate type 1-2 fibres (4%). The fibre type composition of NJP muscles was more evenly distributed between type 1 (29%), type 2 (46%) and type 1-2 (24%) fibres. Tonic fibres comprised less than 2% of the muscle cross-section in both JP and NJP groups. Similarly, MHC composition determined by quantitative SDS-PAGE revealed that JP muscles were composed predominantly of type 1 MHC (86%), with a balance of type 2 MHC (14%). The opposite pattern was found for MHC composition in the NJP muscles: type 1 (28%), type 2 (66%) and type 3 (6%). These results demonstrate that the large extensor muscles that produce the power required for jumping have a fibre type distribution that enables them to generate high levels of mechanical power, with the type 1 isoform accounting for 85-90% of the total MHC content.

Four novel myosin heavy chain transcripts define a molecular basis for muscle fibre types in Rana pipiens.

1. Differential expression of myosin heavy chain (MHC) isoforms dramatically affects mechanical and energetic properties of skeletal muscle fibre types. As many as five different fibre types, each with different mechanical properties, have been reported in frog hindlimb muscles. However, only two frog MHC isoforms have previously been detected by SDS-PAGE and only one adult hindlimb MHC isoform has been cloned. 2. In the present study, four different fibre types (type 1, type 2, type 3 and tonic) were initially identified in adult Rana pipiens anterior tibialis muscle based on myosin ATPase histochemistry, size and location. Each fibre type exhibited unique reactivity to a panel of MHC monoclonal antibodies. Single fibre analysis using SDS-PAGE revealed that MHCs from immunohistochemically defined type 1, type 2 and type 3 fibres ran as three distinct isoform bands, while MHC of tonic fibres co-migrated with type 1 MHC. The combined data from immunohistochemistry and SDS-PAGE suggests that Rana fibre types are composed of four different MHCs. 3. Four novel MHC cDNAs were cloned and expression of the corresponding transcripts was measured in single immuno-identified fibres using specific polymerase chain reaction (PCR) primer pairs. Each of the four transcripts was found to be primarily expressed in a different one of the four fibre types. 4. Coexpression of MHC isoforms was observed only between types 1/2 and types 2/3 at both the protein and mRNA level. 5. These data provide a molecular basis for differentiation between frog fibre types and permit future molecular studies of MHC structure/function and gene regulation in this classic physiological system. 6. Comparison of sequence homology among amphibian, avian and mammalian MHC families supports the concept of independent evolution of fast MHC genes within vertebrate classes subsequent to the amphibian/avian/mammalian radiation.

Muscle function during jumping in frogs. I. Sarcomere length change, EMG pattern, and jumping performance.

We determined the influence of temperature on muscle function during jumping to better understand how the frog muscular system is designed to generate a high level of mechanical power. Maximal jumping performance and the in vivo operating conditions of the semimembranosus muscle (SM), a hip extensor, were measured and related to the mechanical properties of the isolated SM in the accompanying paper [Muscle function during jumping in frogs. II. Mechanical properties of muscle: implication for system design. Am. J. Physiol. 271 (Cell Physiol. 40): C571-C578, 1996]. Reducing temperature from 25 to 15 degrees C caused a 1.75-fold decline in peak mechanical power generation and a proportional decline in aerial jump distance. The hip and knee joint excursions were nearly the same at both temperatures. Accordingly, sarcomeres shortened over the same range (2.4 to 1.9 microns) at both temperatures, corresponding to myofilament overlap at least 90% of maximal. At the low temperature, however, movements were made more slowly. Angular velocities were 1.2- to 1.4-fold lower, and ground contact time was increased by 1.33-fold at 15 degrees C. Average shortening velocity of the SM was only 1.2-fold lower at 15 degrees C than at 25 degrees C. The low Q10 of velocity is in agreement with that predicted for muscles shortening against an inertial load.

Muscle function during jumping in frogs. II. Mechanical properties of muscle: implications for system design.

We characterized the design of the frog muscular system for jumping by comparing the properties of isolated muscle with the operating conditions of muscle measured during maximal jumps. During jumping, the semimembranosus muscle (SM) shortened with a V/Vmax (where V is shortening velocity and Vmax is maximal shortening velocity) where 90 and 100% of maximal power would be generated at 15 and 25 degrees C, respectively. To assess the level of activation during jumping, the SM was driven through the in vivo length change and stimulus conditions while the resulting force was measured. The force generated under the in vivo conditions at both temperatures was at least 90% of the force generated at that same V under maximally activated conditions. Thus the SM was nearly maximally activated, and shortening deactivation was minimal. The initial sarcomere length and duration of the stimulus before shortening were important factors that minimized shortening deactivation during jumping. Thus the frog muscular system appears to be designed to meet the three necessary conditions for maximal power generation during jumping: optimal myofilament overlap, optimal V/Vmax, and maximal activation.

Built for jumping: the design of the frog muscular system.

Frogs must generate a high level of mechanical power when they jump. The muscular system of frogs that jump is presumably designed to deliver these high powers. The length changes and activation pattern that muscles undergo during jumping were measured, and isolated muscle bundles were driven through this in vivo pattern. During jumping, muscles generated maximum power. Specifically, the muscle fibers (i) operated at optimal sarcomere lengths, (ii) operated at optimal shortening velocities, and (iii) were maximally activated during power generation. Thus, many different parameters must have evolved in concert to produce a system capable of this explosive jumping movement.

Histochemical and molecular determination of fiber types in chemically skinned single equine skeletal muscle fibers.

Until now, there has been no reliable method for histochemical determination of fiber types of single skinned muscle fibers. The major problem arises from the fact that most histochemical techniques use cross-sections of a large group of fibers and compare a given fiber with those surrounding it. This is not possible with a single skinned fiber which has been separated from a bundle to be used for mechanical analysis. A further problem is that the skinning procedure itself may alter the staining pattern. We have developed a procedure by which multiple cross-sections of single skinned fibers can be exposed to various histochemical reactions and the staining patterns compared on the same slide to those of frozen muscle and skinned bundles. By this procedure, three fiber types were distinguished by both Ca2+-ATPase and SDH reactions. The fiber typings determined from both enzyme systems correlated well with each other. Although we were able to differentiate only between slow and fast fibers by SDS-PAGE, these results corroborated the histochemical classification. This procedure will clearly be useful in skinned single muscle fiber mechanics experiments performed to determine functional differences among fiber types.

Determination of yttrium in rare earths by photon activation analysis.

The determination of yttrium in the presence of large amounts of the rare earths by the thermal neutron reaction (89)Y(n, gamma)(90)Y is complicated because of frequent problems of sample self-shielding from major constituents of the sample, and the difficulty of separating (90)Y, a pure beta-emitter, from other elements which are very similar chemically. A non-destructive photon activation analysis method has been developed for this determination. Bremsstrahlung from a 35-muA beam of 35-MeV electrons induces the photonuclear reaction (89)Y(gamma, n)(88)Y. Optimum sensitivity is obtained by coincidence counting of the 0.90 and 1.84 MeV gamma-rays associated with the decay of (88)Y. The detection limit is less than 1 mug of yttrium.