Ca and Na Transport in Heart Failure

描述(由申请者提供)：心力衰竭(HF)是美国人的主要死亡原因。一个中心方面是心肌收缩能力的降低，许多证据表明，心肌细胞钙处理的改变，特别是肌浆网钙含量的减少是主要原因。心肌细胞钙和钠的调节通过钠/钙交换紧密相连(我们已经对此进行了深入研究)。我们的总体目标是了解心衰时钙钠调节的改变。我们着重于深入分析肌浆网钙调节(目标1和2)和心肌细胞钠转运(目标3和4)的关键方面，包括正常心肌细胞的基本机制和定量问题，以及这些问题在HF中是如何变化的。我们将使用我们的特征良好的非缺血型兔心衰模型(&人心肌细胞)，利用转基因和基因敲除小鼠进行关键的机制研究。使用交指荧光、共聚焦、电生理和生化方法，我们将解决4个问题： 1.肌浆内游离[Ca]([Ca]sr)。使用我们的新方法直接测量(Ca)sr，我们将测试a)是否存在重要的空间(Ca)sr梯度，b)为什么在HF(低(Ca)sr，SR体积或Ca缓冲)中SR Ca负荷低，c)磷蛋白(PLB)是否降低SR Ca-ATPase效率，以及d)(Ca)sa如何在E-C耦合过程中动态地终止SR Ca释放。 2.氢氟酸钙泄漏及PKA和CaMKII效应。舒张期心衰时SR-Ca渗漏和蛋白激酶的作用存在争议。我们将使用我们的新方法&钙火花来测量泄漏。我们将阐明作为SR Ca负荷的函数在HF中如何改变SR Ca泄漏，以及PKA和CaMKII如何在对照和HF心肌细胞中调节SR Ca泄漏(包括在HF中SR蛋白的表达和磷酸化状态的变化)。 3.心衰时的NA内流。(Na)+在HF中升高，我们发现TTX敏感的静息Na内流增加是主要原因(例如，vs.Na/H或Na/Ca交换)。我们将测试在HF刺激过程中是否也是如此，以及HF中对TTX敏感的Na内流是否可归因于缓慢失活或窗口Na电流。 4.磷脂酶和Na/K-ATPase的调节。PLM是Na/K-ATPase的内源性调节因子，也是心脏内PKA的主要靶点。我们将检验假设：a)内源性PLM抑制Na/K-ATPase，并且这种抑制可以通过依赖PKA的磷酸化来解除，b)PLM表达水平调节Na/K-ATPase表达，以及c)在HF中，PLM低表达和/或高磷酸化解释了为什么在HF低表达Na/K-ATPase不会抑制钠泵功能。 这些研究将交织在心肌细胞E-C偶联、对照和HF中钙和钠调节的定量基础机制研究中(关于肌浆网钙转运、在E-C偶联过程中SRCa释放如何停止、PKA和CaMKII效应、Na内流、NCX和Na/K-ATPase)。我们将测试明确的机制假说，这将丰富我们对心肌细胞中钙和钠调节的基本理解，也为这些在心力衰竭中如何改变提供了新的见解。这将有助于开发治疗人类心力衰竭的新的治疗靶点和策略。

DESCRIPTION (provided by applicant): Heart failure (HF) is a major cause of death in the US. A central aspect is reduced cardiac contractility, and much evidence indicates that altered myocyte Ca handling, particularly reduced SR Ca content is centrally responsible. Myocyte Ca & Na regulation are tightly linked by Na/Ca exchange (which we have studied in depth). Our overall goal is to understand altered Ca & Na regulation in HF. We focus on in-depth analysis of key aspects of SR Ca handling (Aim 1 & 2) and myocyte Na transport (Aim 3 & 4), including basic mechanistic & quantitative issues in normal myocytes, and also how these change in HF. We will use our well characterized nonischemic rabbit HF model (& human myocytes) with key mechanistic studies using transgenic and knockout mice. Using interdigitated fluorescence, confocal, electrophysiological and biochemical approaches, we will address 4 issues: 1. Intra-SR free [Ca] ([Ca]sR). Using our new method to directly measure (Ca)sR,we will test a) if important spatial (Ca)sR gradients exist, b) why SR Ca load is low in HF (low (Ca)sR, SR volume or Ca buffering), c) if phospholamban (PLB) reduces SR Ca-ATPase efficiency, and d) how (Ca)sa may dynamically function in terminating SR Ca release during E-C coupling. 2. SR Ca leak in HF and PKA & CaMKII effects. Diastolic SR Ca leak in HF and protein kinase effects are controversial. We will use Ca sparks & our novel method to measure leak. We will clarify how leak is altered in HF as a function of SR Ca load, and how PKA and CaMKII modulate SR Ca leak in control & HF myocytes (including changes in expression & phosphorylation state of SR proteins in HF. 3. Na influx in HF. (Na)+ is elevated in HF and we showed that elevated TTX-sensitive resting Na influx is largely responsible (e.g. vs. Na/H or Na/Ca exchange). We will test whether this is also true during stimulation in HF, and whether the TTX-sensitive Na influx in HF is attributable to slowly inactivating or window Na current. 4. Phospholemman (PLM) and Na/K-ATPase modulation. PLM is an endogenous regulator of Na/K-ATPase, and a major PKA target in heart. We will test hypotheses that a) endogenous PLM inhibits Na/K-ATPase, and that inhibition is relieved by PKA-dependent phosphorylation, b) PLM expression level modulate Na/K-ATPase expression, and c) in HF, lower PLM expression and/or higher phosphorylation explain why lower Na/K-ATPase expression in HF does not depress Na-pump function. These studies will interweave both quantitative fundamental mechanistic studies of cardiac myocyte E-C coupling, Ca and Na regulation in control and HF (regarding SR Ca transport, how SRCa release shuts off during E-C coupling, PKA & CaMKII effects, Na influx, NCX and Na/K-ATPase). We will test explicit mechanistic hypotheses which will enrich our fundamental understanding of Ca and Na regulation in heart cells, but also provide new insight into how these are altered in HF. This should help in developing new therapeutic targets and strategies for the treatment of human HF.