Oxidative stress and ca(2+) release events in mouse cardiomyocytes

Abstract

Cellular oxidative stress, associated with a variety of common cardiac diseases, is well recognized to affect the function of several key proteins involved in Ca(2+) signaling and excitation-contraction coupling, which are known to be exquisitely sensitive to reactive oxygen species. These include the Ca(2+) release channels of the sarcoplasmic reticulum (ryanodine receptors or RyR2s) and the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Oxidation of RyR2s was found to increase the open probability of the channel, whereas CaMKII can be activated independent of Ca(2+) through oxidation. Here, we investigated how oxidative stress affects RyR2 function and SR Ca(2+) signaling in situ, by analyzing Ca(2+) sparks in permeabilized mouse cardiomyocytes under a broad range of oxidative conditions. The results show that with increasing oxidative stress Ca(2+) spark duration is prolonged. In addition, long and very long-lasting (up to hundreds of milliseconds) localized Ca(2+) release events started to appear, eventually leading to sarcoplasmic reticulum (SR) Ca(2+) depletion. These changes of release duration could be prevented by the CaMKII inhibitor KN93 and did not occur in mice lacking the CaMKII-specific S2814 phosphorylation site on RyR2. The appearance of long-lasting Ca(2+) release events was paralleled by an increase of RyR2 oxidation, but also by RyR-S2814 phosphorylation, and by CaMKII oxidation. Our results suggest that in a strongly oxidative environment oxidation-dependent activation of CaMKII leads to RyR2 phosphorylation and thereby contributes to the massive prolongation of SR Ca(2+) release events.