Functional Coupling between Ito and ICa in Cardiomyocytes

描述（由申请人提供）：在美国，心脏肥大和随后的心力衰竭（HF）每年导致超过260，000人死亡。近年来的研究表明，心肌细胞内钙离子（[Ca 2 +]i）的病理性升高是心肌肥厚的主要触发因素。有趣的是，瞬时外向钾通道（Ito）的抑制也会导致细胞生长和心肌肥大。因此，Ito可能参与了心肌细胞[Ca ~（2+）]I的功能调节。我们最近报道，阻断Ito显著促进心室肌细胞L型钙电流（伊卡），我们认为Ito和伊卡之间存在功能关联，Ca 2 +/钙调蛋白依赖性激酶II（CaMKII）是潜在的调节信号。该建议的一般假设是，大量的CaMKII与Ito通道以其非活性形式形成分子复合物。然而，当Ito通道阻断剂与通道结合或Ito通道被敲低时，偶联的CaMKII将从复合物中解离并随后被激活，从而导致CaMKII引起的伊卡通道磷酸化增加。在这里，我们扩展我们的工作来测试这些假设。具体目标1中的实验将测试Ito通道亚基的分子偶联是否通过Kv4.3在肌细胞中的过表达阻止CaMKII活化（即CaMKII-Ito复合物充当CaMKII储库）。将确定CaMK II分子中的Kv4.3结合位点，以测试Kv4.3是否与CaMK II的功能位点偶联，从而阻止CaMK II活化。在具体目标2中，我们将测试Ito通道表达的减少或4-AP与Ito通道的结合是否引起CaMK II解离和活化。具体目标3将证明CaMKII从复合物中的解离是心肌细胞中CaMKII和伊卡功能活化的重要机制。这项工作将Ito改变与钙通道功能和随后的细胞内Ca 2+稳态和Ca 2+相关信号转导联系起来。这项研究的预期结果可能会破译一个重要的机制，牵连Ito下调激活的CaMKII和伊卡，重要的贡献者的触发和发展的心脏肥大，HF，和致命的室性心律失常。公共卫生相关性：目前，500万美国人患有心脏肥大和心力衰竭，并且这种病态疾病的发病率正在迅速增加。由于对这些机制的了解有限，治疗这些疾病的治疗手段令人遗憾地无效。该研究提示细胞膜钾通道（Ito）参与调节细胞钙内流通道（伊卡）的功能，这可能为该疾病的发生提供新的机制。

DESCRIPTION (provided by applicant): Cardiac hypertrophy and the subsequent heart failure (HF) kill more than 260,000 lives a year in The United States. Recent studies demonstrated that the pathological increase of intracellular calcium [Ca2+]i is a primary trigger for cardiac hypertrophy. Interestingly, inhibition of transient outward K+ channel (Ito) also causes cell growth and cardiac hypertrophy. Thus, Ito may participate in the functional regulation of [Ca2+]I in myocytes. We recently reported that blocking of Ito significantly facilitates L-type calcium current (ICa) in ventricular myocytes and we suggested that there is a functional association between Ito and ICa and that Ca2+/Calmodulin- dependent kinase II (CaMKII) is the underlying regulatory signal. The general hypothesis for this proposal is that a significant amount of CaMKII forms a molecular complex with Ito channel in its inactive form. The coupled CaMKII, however, will be dissociated from the complex and subsequently activated when Ito channel blockers binds to the channel or when Ito channel is knocked down, causing an increased ICa channel phosphorylation by CaMKII. Here, we extend our work to test these hypotheses. Experiments in specific aim 1 will test whether molecular coupling of Ito channel subunits prevents CaMKII from activation (i.e. that the CaMKII-Ito complex serves as a CaMKII reservoir) by over-expression of Kv4.3 in myocytes. The Kv4.3 binding sites in CaMKII molecule will be determined to test whether Kv4.3 couples to the functional sites of CaMKII, preventing CaMKII from activation. In specific aim 2, we will test whether reduction of Ito channel expression or binding of 4-AP to Ito channel causes CaMKII dissociation and activation. Specific aims 3 will demonstrate that the dissociation of CaMKII from the complex is an important mechanism for the functional activation of CaMKII and ICa in cardiomyocytes. This work will link Ito alteration to the calcium channel function and subsequent intracellular Ca2+ homeostasis and Ca2+-related signaling. The anticipated results from this study may decipher an important mechanism that implicates Ito down-regulation in the activation of CaMKII and ICa, the important contributors to the triggering and development of cardiac hypertrophy, HF, and lethal ventricular arrhythmias. PUBLIC HEALTH RELEVANCE: Currently, five million Americans are suffering from cardiac hypertrophy and heart failure, and the incidence of this morbid disease is increasing rapidly. Owing to the limited understanding of the mechanisms, the therapeutic means for treating these diseases are disappointingly ineffective. The proposed study implicates a cell membrane potassium channel (Ito) in regulating the cellular calcium influx channel (ICa) function, which may decipher a new mechanism for this morbid disease.