Five Alternative Myosin Converter Domains Influence Muscle Power, Stretch Activation, and Kinetics.

Muscles have evolved to power a wide variety of movements. A protein component critical to varying power generation is the myosin isoform present in the muscle. However, how functional variation in muscle arises from myosin structure is not well understood. We studied the influence of the converter, a myosin structural region at the junction of the lever arm and catalytic domain, using Drosophila because its single myosin heavy chain gene expresses five alternative converter versions (11a-e). We created five transgenic fly lines, each forced to express one of the converter versions in their indirect flight muscle (IFM) fibers. Electron microscopy showed that the converter exchanges did not alter muscle ultrastructure. The four lines expressing converter versions (11b-e) other than the native IFM 11a converter displayed decreased flight ability. IFM fibers expressing converters normally found in the adult stage muscles generated up to 2.8-fold more power and displayed up to 2.2-fold faster muscle kinetics than fibers with converters found in the embryonic and larval stage muscles. Small changes to stretch-activated force generation only played a minor role in altering power output of IFM. Muscle apparent rate constants, derived from sinusoidal analysis of the chimeric converter fibers, showed a strong positive correlation between optimal muscle oscillation frequency and myosin attachment kinetics to actin, and an inverse correlation with detachment related cross-bridge kinetics. This suggests the myosin converter alters at least two rate constants of the cross-bridge cycle with changes to attachment and power stroke related kinetics having the most influence on setting muscle oscillatory power kinetics.

An embryonic myosin converter domain influences Drosophila indirect flight muscle stretch activation, power generation and flight.

Stretch activation (SA) is critical to the flight ability of insects powered by asynchronous, indirect flight muscles (IFMs). An essential muscle protein component for SA and power generation is myosin. Which structural domains of myosin are significant for setting SA properties and power generation levels is poorly understood. We made use of the transgenic techniques and unique single muscle myosin heavy chain gene of Drosophila to test the influence of the myosin converter domain on IFM SA and power generation. Replacing the endogenous converter with an embryonic version decreased SA tension and the rate of SA tension generation. The alterations in SA properties and myosin kinetics from the converter exchange caused power generation to drop to 10% of control fiber power when the optimal conditions for control fibers - 1% muscle length (ML) amplitude and 150 Hz oscillation frequency - were applied to fibers expressing the embryonic converter (IFI-EC). Optimizing conditions for IFI-EC fiber power production, by doubling ML amplitude and decreasing oscillation frequency by 60%, improved power output to 60% of optimized control fiber power. IFI-EC flies altered their aerodynamic flight characteristics to better match optimal fiber power generation conditions as wing beat frequency decreased and wing stroke amplitude increased. This enabled flight in spite of the drastic changes to fiber mechanical performance.

Disrupting the myosin converter-relay interface impairs Drosophila indirect flight muscle performance.

Structural interactions between the myosin converter and relay domains have been proposed to be critical for the myosin power stroke and muscle power generation. We tested this hypothesis by mutating converter residue 759, which interacts with relay residues I508, N509, and D511, to glutamate (R759E) and determined the effect on Drosophila indirect flight muscle mechanical performance. Work loop analysis of mutant R759E indirect flight muscle fibers revealed a 58% and 31% reduction in maximum power generation (P(WL)) and the frequency at which maximum power (f(WL)) is generated, respectively, compared to control fibers at 15 °C. Small amplitude sinusoidal analysis revealed a 30%, 36%, and 32% reduction in mutant elastic modulus, viscous modulus, and mechanical rate constant 2πb, respectively. From these results, we infer that the mutation reduces rates of transitions through work-producing cross-bridge states and/or force generation during strongly bound states. The reductions in muscle power output, stiffness, and kinetics were physiologically relevant, as mutant wing beat frequency and flight index decreased about 10% and 45% compared to control flies at both 15 °C and 25 °C. Thus, interactions between the relay loop and converter domain are critical for lever-arm and catalytic domain coordination, high muscle power generation, and optimal Drosophila flight performance.

Alternative versions of the myosin relay domain differentially respond to load to influence Drosophila muscle kinetics.

We measured the influence of alternative versions of the Drosophila melanogaster myosin heavy chain relay domain on muscle mechanical properties. We exchanged relay domain regions (encoded by alternative versions of exon 9) between an embryonic (EMB) isoform and the indirect flight muscle isoform (IFI) of myosin. Previously, we observed no effect of exchanging the EMB relay domain region into the flight muscle isoform (IFI-9b) on in vitro actin motility velocity or solution ATPase measurements compared to IFI. However, in indirect flight muscle fibers, IFI-9b exhibited decreased maximum power generation (P(max)) and optimal frequency of power generation (f(max)) to 70% and 83% of IFI fiber values. The decrease in muscle performance reduced the flight ability and wing-beat frequency of IFI-9b Drosophila compared to IFI Drosophila. Previously, we found that exchanging the flight muscle specific relay domain into the EMB isoform (EMB-9a) prevented actin movement in the in vitro motility assay compared to EMB, which does support actin movement. However, in indirect flight muscle fibers EMB-9a was a highly effective motor, increasing P(max) and f(max) 2.5-fold and 1.4-fold, respectively, compared to fibers expressing EMB. We propose that the oscillatory load EMB-9a experiences in the muscle fiber reduces a high activation energy barrier between two strongly bound states of the cross-bridge cycle, thereby promoting cross-bridge cycling. The IFI relay domain's enhanced sensitivity to load increases cross-bridge kinetics, whereas the EMB version is less load-sensitive.