Functional Coupling between Ito and ICa in Cardiomyocytes

描述（由申请人提供）：心脏肥厚和随后的心力衰竭（HF）在美国每年夺去超过26万人的生命。最近的研究表明，细胞内钙[Ca2+]i的病理性增加是心肌肥厚的主要触发因素。有趣的是，瞬时外向K+通道（Ito）的抑制也会导致细胞生长和心脏肥大。因此，Ito可能参与了肌细胞中[Ca2+]I的功能调节。我们最近报道了Ito阻滞显著促进心室肌细胞的l型钙电流（ICa），我们认为Ito和ICa之间存在功能关联，Ca2+/钙调素依赖性激酶II （CaMKII）是潜在的调节信号。该建议的一般假设是，大量CaMKII与Ito通道形成非活性形式的分子复合物。然而，偶联的CaMKII将从复合物中解离，并随后在Ito通道阻滞剂与通道结合或Ito通道被敲除时被激活，导致CaMKII增加ICa通道磷酸化。在这里，我们扩展了我们的工作来检验这些假设。特异性目的1的实验将测试Ito通道亚基的分子偶联是否通过在肌细胞中过表达Kv4.3来阻止CaMKII的激活（即CaMKII-Ito复合体作为CaMKII储存库）。确定CaMKII分子中Kv4.3的结合位点，检测Kv4.3是否与CaMKII的功能位点偶联，阻止CaMKII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通道的结合是否会导致CaMKII解离和激活。Specific aims 3将证明CaMKII与复合物的分离是CaMKII和ICa在心肌细胞中功能激活的重要机制。这项工作将把Ito改变与钙通道功能和随后的细胞内Ca2+稳态和Ca2+相关信号传导联系起来。本研究的预期结果可能揭示了Ito下调CaMKII和ICa激活的重要机制，CaMKII和ICa是心肌肥厚、HF和致死性室性心律失常的触发和发展的重要因素。公共卫生相关性：目前，500万美国人患有心脏肥厚和心力衰竭，这种病态疾病的发病率正在迅速增加。由于对机制的了解有限，治疗这些疾病的治疗手段令人失望地无效。本研究提示细胞膜钾通道（Ito）参与调控细胞钙内流通道（ICa）的功能，这可能为这一病态疾病的发病机制提供新的线索。

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.