Regulation of sarcoplasmic reticulum calcium release in heart failure

描述（由申请人提供）：Ryanodine受体（RyR 2）调节肌浆网（SR）的细胞内Ca 2+释放，是心肌收缩力的重要决定因素。我们最近已经证明，酶Ca 2 +/钙调蛋白依赖性蛋白激酶（CaMKII）磷酸化RyR 2上的丝氨酸2814（S2814），增加SR Ca 2+释放，并作为一个重要的调节机制，以提高心脏收缩力在更快的心率。另一方面，上调的CaMKII活性已被确定为充血性心力衰竭（CHF）中的重要信号传导缺陷。RyR 2的慢性CaMKII磷酸化已被认为是增强SR Ca 2+渗漏的主要原因，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通过研究敲入小鼠来确定CaMKII调节RyR 2介导的SR Ca 2+释放和正常和衰竭心脏的心肌收缩力的细胞/分子机制，其中RyR 2上的CaMKII磷酸化位点丝氨酸2814（S2814）被失活（S2814 A）或组成性激活（S2814 D）。我们的假设是，在正常心脏中，CaMKII磷酸化RyR 2上的S2814以增加SR Ca 2+释放和心脏收缩力，而在衰竭心脏中，RyR 2-S2814的慢性CaMKII磷酸化增强了SR的Ca 2+舒张渗漏，这干扰了SR Ca 2+负荷并导致收缩力下降。具体目标是：1）确定RyR 2的CaMKII磷酸化对心肌收缩力的影响; 2）确定RyR 2-S2814的慢性CaMKII磷酸化是否足以诱导心力衰竭; 3）评价RyR 2的CaMKII磷酸化的抑制是否在心力衰竭中具有治疗性。我们建议进行多学科的研究，从在体内的遗传改变的小鼠，在离体心肌细胞中的钙离子成像研究，RyR 2钙释放通道的生化和单通道测量。预计这些研究将促进我们对正常和衰竭心脏中心肌收缩力调节的CaMK II依赖性机制的理解。为该项目开发的动物模型可能对开发治疗充血性心力衰竭的新药特别有用，充血性心力衰竭是美国的主要死因。公共卫生相关性：细胞内钙释放通道渗漏是心力衰竭收缩功能障碍的重要决定因素。我们将使用遗传小鼠模型来研究钙调蛋白依赖性激酶如何调节健康和衰竭心脏细胞内钙释放通道的活性。这些研究可能有助于开发治疗心力衰竭的新药理学方法，心力衰竭是美国的主要死亡原因。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Leaky intracellular calcium release channels are an important determinant of contractile dysfunction in heart failure. We will use genetic mouse models to study how the enzyme calmodulin-dependent kinase regulates the activity of intracellular calcium release channels in healthy and failing hearts. These studies may facilitate the development of new pharmacological approaches for the treatment of heart failure, the leading cause of death in the United States.