Control of KATP channel expression by CaMKII: role in heart failure resistance

描述(由申请人提供)：心力衰竭在美国是一个巨大的公共卫生问题。在过去的二十年里，慢性心力衰竭的治疗已经取得了长足的进步，即使是最好的现代治疗方法，心力衰竭仍然与50%的5年死亡率有关。因此，寻找治疗和预防心力衰竭的新方法是医学上的主要挑战之一。ATP敏感性钾(KATP)通道是最丰富的心肌膜蛋白复合体之一，具有独特的调节膜兴奋性的能力，以响应细胞能量状态的变化。当被增加的细胞代谢需求激活时，KATP通道依赖的钾外流缩短心脏动作电位时程(Apd)。这种钾外流限制了钠和钙进入细胞，从而减少了离子动态平衡和收缩所需的能量，并延长了支持心肌松弛和ATP补充的舒张期。我们最近的工作发现，心脏优化时程和能量利用的能力取决于KATP通道的膜表达水平，KATP通道的膜表达水平影响KATP电流适应工作负荷变化的速度和效率。对细胞膜KATP通道表达调控机制的全面了解，可能为提高心脏能量效率和抗心力衰竭提供新的途径。根据我们的初步数据，我们假设膜上KATP通道的表达与整体心脏功能是通过钙/钙调蛋白依赖性蛋白激酶II(CaMKII)耦合的。这种高表达的多功能激酶以参与兴奋收缩偶联和兴奋性的多种蛋白为靶点，以支持增强的心脏性能，而它在病理生理条件下的持续激活促进了心肌细胞的死亡和功能障碍。我们提出了一条先前未知的下游信号通路，通过磷酸化Kir6.2形成孔的KATP通道亚基并随后内吞KATP通道来激活CaMKII。在CaMKII持续激活的情况下，KATP通道表达的下降将加剧心脏能量资源的耗竭，从而导致心肌损伤、细胞死亡和心力衰竭。我们预测，抑制CaMKII对心脏应激抵抗的已知有益影响将在很大程度上依赖于KATP通道的膜保持。在AIM1中，我们将通过使用标记的重组KATP通道亚单位、共聚焦免疫荧光成像以及心肌细胞和HEK293T细胞的分子生物学和膜片钳技术来确定依赖CaMKII的KATP通道内吞的机制。在目标2中，我们将研究KATP通道表达缺陷和心脏选择性CaMKII抑制的遗传性小鼠模型诱导的心力衰竭，以了解依赖CaMKII的KATP通道表达调控在定义心力衰竭的能量和功能缺陷的发生中所起的作用。

DESCRIPTION (provided by applicant): Heart failure is an enormous public health problem in the United States. Over the past two decades, there has been considerable progress in the treatment of chronic heart failure yet, even with the best of modern therapy, heart failure is stil associated with a 5-year mortality rate of 50%. Therefore, the search for new approaches to treatment and prevention of heart failure is one of the major challenges in medicine. The ATP-sensitive potassium (KATP) channel, one of the most abundant cardiac membrane protein complexes, has the unique ability to adjust membrane excitability in response to changes in the energetic status of the cell. When activated by increased cellular metabolic demand, KATP channel-dependent potassium efflux shortens cardiac action potential duration (APD). This potassium efflux limits sodium and calcium entry into the cell and thus reduces energy requirements for ion homeostasis and contraction, as well as prolongs the diastolic interval that supports myocardial relaxation and replenishment of ATP. Our recent work uncovered that that the ability of the heart to optimize APD and energy utilization depends on the membrane expression level of KATP channels which affects how quickly and efficiently KATP current can adapt to changes in workload. A complete understanding of mechanisms that control membrane KATP channel expression may reveal new avenues to promote cardiac energy efficiency and resistance to heart failure. Based on our preliminary data, we hypothesize that membrane KATP channel expression is coupled with overall cardiac function by calcium/calmodulin dependent protein kinase II (CaMKII). This densely expressed multifunctional kinase targets numerous proteins involved in excitation contraction coupling and excitability to support enhanced cardiac performance, while its persistent activation under pathophysiological conditions promotes cardiomyocyte death and dysfunction. We propose a previously unrecognized downstream signaling pathway of CaMKII activation through phosphorylation of the Kir6.2 pore-forming KATP channel subunit and consequent endocytosis of KATP channels. Under persistent CaMKII activation, the consequent reduction in KATP channel expression would aggravate depletion of cardiac energy resources thus contributing to myocardial injury, cell death and heart failure. We predict that the known beneficial effects on cardiac stress resistance that occur with CaMKII inhibition will depend significantly on membrane retention of KATP channels. In Aim1 we will define the mechanism for CaMKII-dependent endocytosis of KATP channels by use of tagged recombinant KATP channel subunits, confocal immunofluorescence imaging, and molecular biology and patch clamp techniques in cardiomyocytes and HEK293T cells. In Aim 2 we will study heart failure, induced in genetic mouse models with KATP channel expression deficits and cardioselective CaMKII inhibition, to understand the role of CaMKII-dependent KATP channel expression regulation in the generation of the energetic and functional deficits defining heart failure.