Regulation of sarcoplasmic reticulum calcium release in heart failure

项目摘要/摘要 兰尼定受体(RyR2)调节肌浆网细胞内钙释放 (Sr)，是心脏收缩能力的重要决定因素。我们最近展示了 钙/钙调蛋白依赖的蛋白激酶(CaMKII)使丝氨酸2814磷酸化 (S2814)在RyR2上，它增加了SR钙的释放，是一个重要的调节因素 以更快的心率增强心脏收缩能力的机制。另一方面，上调了 CaMKII活性已被认为是充血性心力衰竭的一个重要信号缺陷 (瑞士法郎)。RyR2的慢性CaMKII磷酸化被认为是导致 肌浆网钙离子漏出增加，这是CHF收缩功能障碍的重要决定因素。 这个项目的长期目标是定义细胞/分子机制，通过它 CaMKII调节RyR2介导的肌浆网钙离子释放和心力衰竭 心脏，通过研究CaMKII磷酸化位点丝氨酸2814(S2814)的敲打小鼠 在RyR2上被灭活(S2814A)或被结构性激活(S2814D)。我们的假设是 在正常心脏中，CaMKII磷酸化RyR2上的S2814以增加SR钙离子释放和 心肌收缩能力，而在衰竭的心脏中，RyR2-S2814的慢性CaMKII磷酸化 增加SR的舒张期钙泄漏，从而干扰SR的钙负荷并导致 收缩能力下降。 其具体目的是：1)明确RyR2的CaMKII磷酸化对心脏的影响 2)确定RyR2-S2814的慢性CaMKII磷酸化是否足以 3)评价抑制RyR2的CaMKII磷酸化是否 治疗心力衰竭。我们建议进行多学科的研究，范围从体内 对转基因小鼠的研究，对分离心肌细胞的钙成像研究，以及 RyR2钙释放通道的生化和单通道测量。这是意料之中的 这些研究将促进我们对CaMKII依赖机制的理解 心脏收缩功能正常和衰竭心脏的收缩能力的调节。动物模型 为这一项目开发的药物可能对开发新的 治疗充血性心力衰竭，这是美国的主要死亡原因。

PROJECT SUMMARY/ ABSTRACT Ryanodine receptors (RyR2) regulate intracellular Ca2+ release from the sarcoplasmic reticulum (SR), and are important determinants of cardiac contractility. We have recently demonstrated that the enzyme Ca2+/calmodulin-dependent protein kinase (CaMKII) phosphorylates serine 2814 (S2814) on RyR2, which increases SR Ca2+ release and serves as an important regulatory mechanism to enhance cardiac contractility at faster heart rates. On the other hand, upregulated CaMKII activity has been identified as an important signaling defect in congestive heart failure (CHF). Chronic CaMKII phosphorylation of RyR2 has been proposed as a principal cause of enhanced SR Ca2+ leak, which is an important determinant of contractile dysfunction in CHF. The long-term goal of this project is to define the cellular/molecular mechanisms by which CaMKII regulates RyR2-mediated SR Ca2+ release and cardiac contractility in normal and failing hearts, by studying knockin mice in which the CaMKII phosphorylation site serine 2814 (S2814) on RyR2 is either inactivated (S2814A) or constitutively activated (S2814D). Our hypothesis is that in normal hearts, CaMKII phosphorylates S2814 on RyR2 to increase SR Ca2+ release and cardiac contractility, whereas in failing hearts chronic CaMKII phosphorylation of RyR2-S2814 enhances diastolic leak of Ca2+ from the SR, which interferes with SR Ca2+ loading and causes depressed contractility. The specific aims are to: 1) Define the effects of CaMKII phosphorylation of RyR2 on cardiac contractility; 2) Determine whether chronic CaMKII phosphorylation of RyR2-S2814 is sufficient to induce heart failure; 3) Evaluate whether inhibition of CaMKII phosphorylation of RyR2 is therapeutic in heart failure. We propose to conduct multidisciplinary studies ranging from in vivo studies in genetically-altered mice, Ca2+ imaging studies in isolated cardiomyocytes, and biochemical and single channel measurements of RyR2 Ca2+ release channels. It is anticipated that these studies will advance our understanding of CaMKII-dependent mechanisms underlying the regulation of cardiac contractility in both normal and failing hearts. The animal models developed for this project may be particularly useful for the development of new drugs for the treatment of congestive heart failure, the leading cause of death in the United States.