Two Mechanisms For Increasing Muscle Calcium Sensitivity by ROS

Abstract

Cardiac muscle is sensitive to reactive oxygen species (ROS). Most measurements of calcium sensitivity following ROS exposure have shown a decrease, but others have measured no change or even an increase. One difference between studies was the activation state when ROS was applied. We therefore sought to characterize ROS-induced changes in myofilament proteins and their functional effects under different conditions of [Ca] and [ATP]. First, we identified all reactive cysteines in myofilament proteins by exposing myofibrils to a fluorescent maleimide probe under varying calcium concentrations, followed by SDS-PAGE. Only cysteines in troponin C had reactivity modulated by [Ca]. To find functional effects, we measured myofibril ATPase after exposure to 100uM DTDP in solutions at pCa 4.0 or 9.0. Ca sensitivity increased only when DTDP had been added at pCa 4.0. Next, we compared the reactivity of cysteines in myofibrils exposed to DTDP under rigor or relaxing conditions. Rigor conditions decreased reactivity of myosin cysteines but increased reactivity of actin cysteines, compared to relaxing conditions. We then measured ATPase rates in myofibrils exposed to DTDP under rigor or relaxing solutions. Exposure to DTDP in relaxing solution decreased both the maximum ATPase rate and the calcium sensitivity compared to myofibrils not exposed to DTDP (control). In contrast, when DTDP was exposed in rigor solution the minimum ATPase rate was increased from control, and there was an increase in calcium sensitivity. In conclusion, we found two activation dependent mechanisms to increase calcium sensitivity: the first calcium-dependent and specific to troponin C, the second dependent on whether myosin was bound to actin.