Accessibility of myofilament cysteines and effects on ATPase depend on the activation state during exposure to oxidants.

Abstract

Signaling by reactive oxygen species has emerged as a major physiological process. Due to its high metabolic rate, striated muscle is especially subject to oxidative stress, and there are multiple examples in cardiac and skeletal muscle where oxidative stress modulates contractile function. Here we assessed the potential of cysteine oxidation as a mechanism for modulating contractile function in skeletal and cardiac muscle. Analyzing the cysteine content of the myofilament proteins in striated muscle, we found that cysteine residues are relatively rare, but are very similar between different muscle types and different vertebrate species. To refine this list of cysteines to those that may modulate function, we estimated the accessibility of oxidants to cysteine residues using protein crystal structures, and then sharpened these estimates using fluorescent labeling of cysteines in cardiac and skeletal myofibrils. We demonstrate that cysteine accessibility to oxidants and ATPase rates depend on the contractile state in which preparations are exposed. Oxidant exposure of skeletal and cardiac myofibrils in relaxing solution exposes myosin cysteines not accessible in rigor solution, and these modifications correspond to a decrease in maximum ATPase. Oxidant exposure under rigor conditions produces modifications that increase basal ATPase and calcium sensitivity in ventricular myofibrils, but these effects were muted in fast twitch muscle. These experiments reveal how structural and sequence variations can lead to divergent effects from oxidants in different muscle types.