Myosin Effects On Thin Filament Activation In Slow-Twitch Human Soleus Muscle Fibers

It is well known that striated muscle thin filaments are activated by calcium but the level of activity at sub-saturating calcium is increased by myosin binding to actin. Observations on the effects of myosin binding on activation were made primarily withcardiac or fast-twitch skeletal muscle. Here we asked whether similar contributions of myosin to thin filament activation occur in slow-twitch skeletal muscle fibers that contain the same myosin heavy chain isoform as in the human heart. We exchanged endogenous troponin of slow-twitch single human soleus fibers for a well characterized, fast skeletal troponin complex labeled with IANBD (fsTnIANBD). This was used as a fluorescent sensor to report the effects of calcium and myosin on thin filament activation. Inducing maximal force development by increasing the calcium concentration from pCa 7.5 to pCa 4.5 generated a change in the emission intensity of fsTnIANBD of on average 32% (control). Increasing the calcium concentration in the same conditions but in presence of 50 µM of the myosin inhibitor para-amino-blebbistatin reduced force to 7% of maximum force in control conditons and reduced the change in the emission intensity of fsTnIANBD to 18% (or to 55% of the change observed in control conditions). Similar effects were detected at submaximal calcium concentrations. Additionally, when switching from isometric contraction to slightly-loaded shortening, changes in fluorescence indicating conformational changes of fsTnIANBD had rate constants at least 10 times higher than those of force redevelopment. Thus, our data with fsTnIANBD as thin filament activation sensor suggest that force-generating myosin heads contribute to thin filament activation at all activation levels in human slow-twitch skeletal muscle fibers. Importantly, the rate constant of force redevelopment is not limited by the much faster equilibration among different states of the thin filament.