Probing Estradiol Effects on Super-Relaxed Myosin in Muscle Fibers with Fluorescence

Abstract

Muscle myosins use the energy from ATP hydrolysis to perform mechanical work during muscle contraction. Recent work suggests that the interactions between the catalytic and light-chain-binding domains of myosin, the S2 coiled-coil fragment, and the thick-filament backbone, stabilize an auto-inhibited structural state of myosin, termed the super-relaxed state (SRX), with slowed ATP turnover kinetics. The super-relaxed state is hypothesized to play an important role in regulating contractility and thermogenesis in skeletal, cardiac muscle, and smooth muscle. We are using quantitative confocal microscopy of fluorescent ATP turnover in skinned skeletal and cardiac muscle fibers to measure the mole fraction of myosin molecules in the super-relaxed state, over a range of perturbations including animal age, sex, and hormonal state, post-translational modification of sarcomeric regulatory proteins, and small molecule modulators. From this work, we are mapping the determinants and regulatory correlates that control partitioning of myosin molecules into this important, poorly understood biophysical state. This work is supported by grants NIH R01AR032961-33 and T32AR007612-15 (to DDT).