The non-hypertrophic role of calcineurin in regulating cardiac structure-function

描述（由申请人提供）：每年有超过500万美国人被诊断患有心力衰竭。心力衰竭通常伴有心脏的病理性生长。钙调磷酸酶信号是肥厚反应的重要调节因子。钙调磷酸酶的一种新的非肥厚作用正在出现，其中钙调磷酸酶可能直接维持活力和心脏功能，因为心脏特异性钙调磷酸酶缺失的小鼠心脏形态正常，但左心室功能差和过早死亡。迄今为止，钙调磷酸酶缺失对心功能的直接作用和代偿作用仍未得到解决，报告显示，钙调磷酸酶缺失的心肌功能结果既有积极的，也有消极的。在钙调磷酸酶缺乏症的心脏中发现了Ca2+处理基因表达的实质性改变。这表明钙调磷酸酶可能调节Ca2+循环蛋白，这是关键的功能成分，作为波动的细胞质Ca2+启动收缩和松弛。因此，本提案旨在验证钙调磷酸酶通过翻译后和nfat依赖的Ca2+处理调节直接调节肥厚独立的心脏活力和心室功能的假设。这项资助的特点是比较分析了钙调磷酸酶靶向siRNA到心肌细胞的急性基因转移和条件Lox-P靶向钙调磷酸酶小鼠模型，以阐明钙调磷酸酶损失对体外心肌细胞功能的直接和间接影响（目的），并确定Ca2+处理蛋白的磷酸化状态（目的2）。心肌细胞收缩性将在分离的心肌细胞中进行检查，这两种遗传模型使用高保真细胞缩短和Ca2+测量，而候选蛋白和蛋白质组学分析将确定Ca2+处理蛋白的磷酸化状态。基因拯救策略也将用于研究nfat依赖性转录是否导致钙调神经磷酸酶缺乏小鼠的生存能力低下（目的3）。这一建议将对阐明钙调磷酸酶在心脏中的额外调节作用作出重大贡献，从而为开发新的心力衰竭治疗策略奠定基础。外行语言：钙调磷酸酶依赖的心脏病理性生长与终末期心力衰竭有关。钙调磷酸酶在心功能中的一个新的生长无关的调节作用现在正在出现，表明钙调磷酸酶的缺失可以直接改变心肌功能。本研究旨在阐明钙调磷酸酶在细胞和组织水平上对心脏功能的直接和间接影响，而不依赖于心脏的生长。这项工作的结果应该在设计具有转化潜力的分子心力衰竭疗法方面取得重大进展。

DESCRIPTION (provided by applicant): Over 5 million Americans are diagnosed with heart failure yearly. Heart failure is generally accompanied by pathologic growth of the heart. Calcineurin signaling is an essential regulator of the hypertrophic response. A new non-hypertrophic role for calcineurin is emerging, in which calcineurin might directly maintain viability and cardiac performance, as mice with cardiac-specific calcineurin deletion have normal cardiac morphology but poor left ventricular function and premature death. To date the direct versus compensatory effects of calcineurin deletion on cardiac function remain unresolved, and reports show both positive and negative functional outcomes in calcineurin-deleted cardiac muscle. Substantial alterations in Ca2+ handling gene expression were identified in a screen of calcineurin deficient hearts. This suggests that calcineurin may regulate Ca2+ cycling proteins, which are critical functional components, as fluctuations in cytosolic Ca2+ initiate contraction and relaxation. Thus, this proposal seeks to test the hypothesis that calcineurin is directly regulating hypertrophy-independent cardiac viability and ventricular performance through post-translational and NFAT-dependent regulation of Ca2+ handling. This grant features a comparative analysis of acute gene transfer of calcineurin-targeted siRNA to cardiac myocytes and a conditional Lox-P targeted calcineurin mouse model to elucidate the direct versus indirect effects of calcineurin loss on cardiac myocyte function in vitro (aiml), and to determine the phosphorylation status of Ca2+ handling proteins (aim 2). Myocyte contractility will be examined in isolated cardiac myocytes from both genetic models using high fidelity cell shortening and Ca2+ measurements, while candidate protein and proteomic analysis will determine the phosphorylation status of Ca2+ handling proteins. A genetic rescue strategy will also be used to investigate if NFAT-dependent transcription underlies the poor survivability in calcineurin deficient mice (aim 3). This proposal should make substantial contributions to elucidating calcineurin's additional regulatory roles in the heart thereby laying the ground work for developing new therapeutic strategies for heart failure. Lay Language: Calcineurin-dependent pathologic growth of the heart is associated with end-stage heart failure. A new growth-independent regulatory role for calcineurin in cardiac function is now emerging that suggests calcineurin deletion can directly alter cardiac muscle function. This proposal is designed to elucidate the direct versus indirect effects of calcineurin on cardiac function independent of cardiac growth at the cellular and organismal level. The outcome of this work should make significant inroads into designing molecular heart failure therapies with translational potential.