UC Berkeley

Biological systems are frequently exposed to reactive oxygen species (ROS), which can be damaging to cellular function. Recent research suggests that ROS can also be used as signaling agents, modifying protein functions post-translationally, by reversibly modifying cysteines. The contractile functions of both skeletal and cardiac muscle are responsive to reactive oxygen species, but the mechanisms are unknown. We therefore developed methods to quantify, which cysteines are modified by exposure to oxidative reagents, and used these methods as a gateway for testing different mechanisms for how ROS affect function. From these techniques, we found that nearly every myofilament protein in both cardiac and skeletal muscle has one or more cysteines that can be modified by ROS in the environment of the myofilament lattice. Interestingly, we found that both the cysteines modified and the effects from ROS exposure depended on the contractile state of the muscle. Exposure in a relaxing buffer (ATP present) modified a large number of myosin thiols, and decreased the maximum ATPase and calcium sensitivity when compared to untreated myofibrils. In contrast, ROS exposure in a rigor buffer (no ATP) decreased the number of modified myosin thiols and increased the modification of actin when compared to treatment in relaxing buffer. Exposure in rigor buffer did not change the maximum myofibril ATPase, but did increase the basal ATPase and calcium sensitivity. Complementary studies in fast skeletal muscle tested the ability of different cysteine reagents to change calcium sensitivity. We found that different reagents affected calcium sensitivity in opposite directions, and found evidence that the difference in direction was due to differences in the type of modification, rather than a difference between cysteine sites. We narrowed down the proteins responsible for these effects to actin, essential light chain, tropomyosin and the myosin heavy chain. Further eliminating proteins with cysteines not unique to fast muscle, we found two cysteines in the myosin heavy chain, both in the neck region, that are the most likely to be responsible for the changes in calcium sensitivity. In summary these experiments shed significant light on a variety of mechanisms by which ROS can modify the contractile function of cardiac and skeletal muscle. These results may be important for understanding and eventually treatment of diseases that have been found to produce oxidative modifications and decreased function of muscle, including heart failure, muscular dystrophy, arthritis and cancer.