Shirokova Natalia, Kang Chifei, Fernandez-Tenorio Miguel, Wang Wei, Wang Qiongling, Wehrens Xander H T, Niggli Ernst
Department of Pharmacology and Physiology, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey.
Department of Pharmacology and Physiology, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey.
Biophys J. 2014 Dec 16;107(12):2815-2827. doi: 10.1016/j.bpj.2014.10.054.
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.
细胞氧化应激与多种常见心脏疾病相关，众所周知，它会影响参与钙（Ca2+）信号传导和兴奋-收缩偶联的几种关键蛋白的功能，而这些蛋白对活性氧非常敏感。其中包括肌浆网的钙（Ca2+）释放通道（兰尼碱受体或RyR2）和钙/钙调蛋白依赖性蛋白激酶II（CaMKII）。研究发现，RyR2的氧化会增加通道的开放概率，而CaMKII可通过氧化独立于钙（Ca2+）被激活。在此，我们通过分析在广泛氧化条件下通透的小鼠心肌细胞中的钙（Ca2+）火花，研究了氧化应激如何在原位影响RyR2功能和肌浆网（SR）钙（Ca2+）信号传导。结果表明，随着氧化应激增加，钙（Ca2+）火花持续时间延长。此外，长时和非常长时（长达数百毫秒）的局部钙（Ca2+）释放事件开始出现，最终导致肌浆网（SR）钙（Ca2+）耗竭。CaMKII抑制剂KN93可防止释放持续时间的这些变化，并且在缺乏RyR2上CaMKII特异性S2814磷酸化位点的小鼠中未发生这种情况。持久钙（Ca2+）释放事件的出现与RyR2氧化增加同时发生，也与RyR-S2814磷酸化以及CaMKII氧化同时发生。我们的结果表明，在强氧化环境中，CaMKII的氧化依赖性激活导致RyR2磷酸化，从而导致肌浆网（SR）钙（Ca2+）释放事件大量延长。
