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

描述(申请人提供)：每年有500多万美国人被诊断为心力衰竭。心力衰竭通常伴随着心脏的病理性生长。钙调神经磷酸酶信号是肥大反应的重要调节因子。钙调神经磷酸酶的一种新的非肥大作用正在出现，其中钙调神经磷酸酶可能直接维持存活和心脏功能，因为心脏特异的钙调神经磷酸酶缺失的小鼠心脏形态正常，但左心功能较差，并过早死亡。到目前为止，钙调神经磷酸酶缺失对心脏功能的直接和代偿影响仍未得到解决，报道显示钙调神经磷酸酶缺失的心肌有阳性和阴性两种功能结果。在钙调神经磷酸酶缺乏的心脏筛查中，发现了钙处理基因表达的实质性变化。这表明，钙调神经磷酸酶可能调节钙循环蛋白，这是关键的功能成分，因为细胞内钙的波动启动收缩和松弛。因此，这项建议试图验证这样的假设，即钙调神经磷酸酶通过翻译后和NFAT依赖的钙处理调节，直接调节肥厚非依赖性的心脏活性和心室功能。这项资助的特点是比较分析了钙调神经磷酸酶靶向siRNA到心肌细胞的急性基因转移和条件性Lox-P靶向钙调神经磷酸酶小鼠模型，以阐明钙调神经磷酸酶缺失对体外培养心肌细胞功能(AIML)的直接或间接影响，并确定钙处理蛋白的磷酸化状态(目标2)。我们将使用高保真细胞缩短和钙测量来检测两种遗传模型中分离的心肌细胞的收缩能力，而候选蛋白质和蛋白质组学分析将确定钙处理蛋白的磷酸化状态。基因拯救策略也将被用来调查NFAT依赖的转录是否是钙调神经磷酸酶缺陷小鼠生存能力差的基础(目标3)。这项建议将对阐明钙调神经磷酸酶在心脏中的额外调节作用做出实质性贡献，从而为开发新的心力衰竭治疗策略奠定基础。外行语言：依赖钙调神经磷酸酶的心脏病理性生长与终末期心力衰竭有关。钙调神经磷酸酶在心脏功能中的一种新的生长非依赖性调节作用正在出现，这表明钙调神经磷酸酶缺失可以直接改变心肌功能。这项建议旨在阐明钙调神经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，而不是心脏的生长。这项工作的结果应该会在设计具有翻译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.