Myosin Heavy Chain Isoforms Influence Stretch Activation and Cross Bridge Kinetics of Drosophila Muscles

Myosin is the molecular motor that powers muscle contraction. Variation between myosin isoforms is a major determinant of muscle mechanical properties including shortening velocity and perhaps stretch activation (SA). We investigated the effect of Ca2+ concentration and myosin heavy chain (MHC) isoforms on SA, and attempted to determine the rate-limiting step of the myosin cross-bridge cycle for maximum shortening velocity. First, to understand the relationship between Ca2+ concentration, stretch-activated tension (FSA) and Ca2+-activated tension (F O), we measured FSA and power generation in skinned Drosophila indirect flight muscles (IFMs). We found that a large SA response was induced in Ca2+ activated IFMs upon a 1% muscle length increase. At pCa 4.5, FSA and FO made up ~70% and 30% of total active tension (FO+FSA), respectively. We found that IFM power output increased with increasing [Ca2+], indicating that Drosophila may regulate power output by varying Ca2+ levels instead of adjusting the number of motor units recruited. The contribution to power enhancement over the physiological Ca 2+ range of pCa 5.7 to pCa 5.4 from FSA was 4-fold greater than from FO, suggesting that FSA plays a major role in regulating IFM power output during insect flight. Second, we observed the effect of MHC isoforms on SA and power generation of Drosophila jump muscles (TDT) by replacing the native TDT MHC with the embryonic MHC isoform (EMB) and the indirect flight muscle MHC isoform (IFI). We found that the TDT muscle displayed only minimal SA and could not produce positive power under oscillatory conditions at pCa 5.0. However, it was transformed to be moderately stretch-activatable and could produce positive power when the EMB MHC isoform was expressed. We found that Pi increased EMB, but not wild type, SA force and power. Based on this observation, we propose a mechanism by which myosin isoforms can endow a muscle with a moderate amount of SA that includes myosin strain sensitivity and Pi affinity. In contrast, we found that expressing IFI from the highly SA IFM muscle in TDT muscles neither affected SA properties nor allowed positive power generation under oscillatory conditions. This suggests the IFI MHC isoform in Drosophila TDT is insufficient to enable SA, and that the IFM uses another mechanism to attain its very high FSA. Finally, to evaluate if shortening velocity of fast myosins is limited by Pi release rather than ADP release, we observed the effect of Pi concentration on unloaded shortening velocity (Vslack) of Drosophila TDT muscles expressing the IFI, the native TDT and EMB-3b MHC isoforms. We found that Vslack of fibers expressing the IFI isoform was greatest (∼ 145% of control TDT), followed by TDT and EMB-3b (∼ 65% of control TDT). Increasing [Pi] decreased the isometric tension of all three transgenic fibers, whereas Vslack was not significantly altered. This result suggests that Pi release rate does not limit the maximum shortening velocity of fast muscle types.