Abstract

The ryanodine receptor Ca²⁺ channel (RyRC) constitutes the Ca²⁺-release pathway in sarcoplasmic reticulum (SR) of cardiac muscle. A direct mechanical and a Ca²⁺-triggered mechanism (Ca²⁺-induced Ca²⁺ release) have been proposed to explain the in situ activation of Ca²⁺ release in cardiac muscle. A variety of chemical oxidants have been shown to activate RyRC; however, the role of modification induced by oxygen-derived free radicals in pathological states of the muscle remains to be elucidated. It has been hypothesized that oxygen-derived free radicals initiate Ca²⁺-mediated functional changes in or damage to cardiac muscle by acting on the SR and promoting an increase in Ca²⁺ release. We confirmed that superoxide anion radical (O₂⨪) generated from hypoxanthine-xanthine oxidase reaction decreases calmodulin content and increases ⁴⁵Ca²⁺ efflux from the heavy fraction of canine cardiac SR vesicles; hypoxanthine-xanthine oxidase also decreases Ca²⁺ free within the intravesicular space of the SR with no effect on Ca²⁺-ATPase activity. Current fluctuations through single Ca²⁺-release channels have been monitored after incorporation into planar phospholipid bilayers. We demonstrate that activation of the channel by O₂⨪ is dependent of the presence of calmodulin and identified calmodulin as a functional mediator of O₂⨪-triggered Ca²⁺ release through the RyRC. For the first time, we show that O₂⨪ stimulates Ca²⁺ release from heavy SR vesicles and suggest the importance of accessory proteins such as calmodulin in modulating the effect of O₂⨪. The decreased calmodulin content induced by oxygen-derived free radicals, especially O₂⨪, is a likely mechanism of accumulation of cytosolic Ca²⁺ (due to increased Ca²⁺ release from SR) after reperfusion of the ischemic heart.