Investigation of calcium modulation in cardiomyocytes by novel methods

描述（申请人提供）：肌层l型Ca2+通道CaV1.2在心脏兴奋-收缩耦合中起关键作用。CaV1.2功能异常，包括长开模式门控增加和肾上腺素能反应减弱，与心力衰竭和肥厚有关。反过来，CaV1.2激活的增加触发Ca2+响应信号通路，包括那些影响基因表达的信号通路，这有助于心力衰竭和肥厚的发病机制。不足为奇的是，CaV1.2受到其锚定的细胞类型特异性大分子复合物组分的严格调控。然而，由于无法在异源表达系统中概括和解剖CaV1.2在肌细胞中功能的关键方面，对肌细胞中CaV1.2调控的详细分子理解受到了阻碍。我们的目标是更好地了解这些大分子复合物的成分如何调节CaV1.2影响心脏收缩力、肥厚和心力衰竭的发展以及相关的电生理并发症。我们已经开发出新的工具来克服限制该领域进展的主要障碍，并允许我们在心肌细胞的背景下，利用生物化学和电生理技术，利用异源表达系统的力量，探索CaV1.2调控的分子方面。利用转基因（TG）方法，可以选择性和可靠地表达flag表位标记，二氢吡啶抗性CaV1.2通道亚基，在关键调控位点或与调控成分共价连接的突变，在成人心肌细胞和发育的所有阶段，我们建议在心肌细胞中确定：(a) c端蛋白水解裂解的作用，负责肾上腺素能调节CaV1.2电流的分子机制，以及心肌内Ca2+内流的肾上腺素能调节是否需要蛋白水解裂解；(b) Ca2+/钙调素依赖性蛋白激酶（CaMKII）与Ca2+通道亚基的关联和磷酸化在调节CaV1.2电流中的作用；(c)钙调蛋白（CaM）是否以Ca2+依赖的方式调节心肌细胞通道生物合成。使用新的方法从TG通道中分离Ca2+电流，并将这些电流与同一心肌细胞中的内源性通道进行比较，我们将确定心肌细胞中肾上腺素能和CaMKII调节CaV1.2的分子机制，并定义在生理条件下和心力衰竭开始后，CaM的Ca2+敏感性如何影响心肌细胞中CaV1.2的运输。这三个目标，应该提供关于Ca2+内流在心肌细胞调节的关键新认识，对理解心脏病理和负责心脏收缩性调节的分子机制高度相关。

DESCRIPTION (provided by applicant): CaV1.2, the sarcolemmal L-type Ca2+ channel, plays a key role in cardiac excitation-contraction coupling. Abnormalities in CaV1.2 function, including increased long-opening-mode gating and blunted adrenergic responsiveness, are associated with heart failure and hypertrophy. The increased activation of CaV1.2, in turn, triggers Ca2+-responsive signaling pathways, including those affecting gene expressions, which contribute to the pathogenesis of heart failure and hypertrophy. Not surprisingly, CaV1.2 is tightly regulated by components of cell type-specific macromolecular complexes that it anchors. A detailed molecular understanding of CaV1.2 regulation in myocytes has been hampered, however, by the inability to recapitulate and then dissect in heterologous expression systems key aspects of CaV1.2 function in myocytes. Our goals are to gain a better understanding of how CaV1.2 modulation by components of these macromolecular complex impacts cardiac contractility, the development of hypertrophy and heart failure, and the associated electrophysiological complications. We have developed novel tools to surmount major obstacles that have limited progress in the field, and allow us to probe molecular aspects of CaV1.2 regulation, using biochemical and electrophysiological techniques, within the context of cardiomyocytes, but with the power of a heterologous expression system. Using a transgenic (TG) approach that enables selective and reliable expression of FLAG-epitope tagged, dihydropyridine-resistant CaV1.2 channel subunits, harboring mutations at key regulatory sites or covalently linked to regulatory components, in adult cardiomyocytes and at all stages of development, we propose to determine in cardiomyocytes: (a) the role for proteolytic cleavage of the ¿1C C-terminus, the molecular mechanisms responsible for adrenergic regulation of CaV1.2 current and whether proteolytic cleavage is required for adrenergic regulation of Ca2+ influx in the heart; (b) the rol of Ca2+/calmodulin-dependent protein kinase (CaMKII) association with, and phosphorylation of, Ca2+ channel subunits in the regulation of CaV1.2 current; and (c) whether calmodulin (CaM) associated with the C-terminus of ¿1C regulates channel biosynthesis in cardiomyocytes in a Ca2+-dependent manner. Using novel methodologies to isolate Ca2+ currents from the TG channels and compare these currents to endogenous channels in the same cardiomyocyte, we will determine the molecular mechanisms of adrenergic and CaMKII regulation of CaV1.2 in cardiomyocytes and define how the Ca2+-sensitivity of CaM affects CaV1.2 trafficking in cardiomyocytes, both under physiological conditions and after initiation of heart failure. The three Aims, which should provide key new understandings concerning the regulation of Ca2+ influx in cardiomyocytes, are highly relevant towards understanding cardiac pathologies and the molecular mechanisms responsible for the modulation of cardiac contractility.