Regulation of sarcoplasmic reticulum calcium release in heart failure

项目总结/摘要 Ryanodine受体（RyR 2）调节肌浆网胞内Ca 2+释放 (SR)是心肌收缩力的重要决定因素。我们最近展示了 Ca 2 +/钙调蛋白依赖性蛋白激酶（CaMKII）磷酸化丝氨酸2814 （S2814）对RyR 2的作用，RyR 2增加SR Ca 2+释放，并作为重要的调节因子。 在更快的心率下增强心脏收缩力的机制。另一方面， CaMKII活性已被确定为充血性心力衰竭的重要信号缺陷 （瑞士法郎）。RyR 2的慢性CaMKII磷酸化已被认为是导致糖尿病的主要原因。 增强SR Ca 2+渗漏，这是CHF中收缩功能障碍的重要决定因素。 该项目的长期目标是确定细胞/分子机制， CaMKII调节RyR 2介导的SR Ca 2+释放和正常和衰竭心肌的收缩力 心脏，通过研究敲入小鼠，其中CaMKII磷酸化位点丝氨酸2814（S2814） RyR 2上的蛋白质是失活的（S2814 A）或组成型激活的（S2814 D）。我们的假设是 在正常心脏中，CaMK II磷酸化RyR 2上的S2814以增加SR Ca 2+释放， 心肌收缩力，而在衰竭心脏中，RyR 2-S2814的慢性CaMKII磷酸化 增强SR的舒张期Ca 2+渗漏，这会干扰SR Ca 2+负荷并导致 收缩力降低 具体目的是：1）确定RyR 2的CaMK II磷酸化对心脏的影响， 2）确定RyR 2-S2814的慢性CaMK II磷酸化是否足以 诱导心力衰竭; 3）评价RyR 2的CaMK II磷酸化的抑制是否 心力衰竭的治疗方法我们建议进行多学科研究，从体内 在基因改变的小鼠中的研究，在分离的心肌细胞中的Ca 2+成像研究，以及 RyR 2 Ca 2+释放通道的生物化学和单通道测量。预计 这些研究将促进我们对CaMKII依赖性机制的理解， 调节正常和衰竭心脏的心脏收缩力。动物模型 为该项目开发的药物可能对开发新药特别有用， 治疗充血性心力衰竭，这是美国死亡的主要原因。

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.