Ca and Na Transport in Heart Failure

描述（由申请人提供）：心力衰竭（HF）是美国的主要死因。 一个中心方面是降低的心肌收缩力，并且许多证据表明改变的肌细胞Ca处理，特别是减少的SR Ca含量是中枢负责的。 肌细胞Ca & Na调节与Na/Ca交换紧密相关（我们已经深入研究）。 我们的总体目标是了解HF中改变的Ca & Na调节。 我们专注于深入分析SR Ca处理（目标1和2）和肌细胞Na转运（目标3和4）的关键方面，包括正常肌细胞中的基本机制和定量问题，以及这些在HF中的变化。 我们将使用我们良好表征的非缺血性兔HF模型（和人心肌细胞），并使用转基因和基因敲除小鼠进行关键机制研究。 使用交叉荧光，共聚焦，电生理和生物化学方法，我们将解决4个问题： 1. SR内游离[Ca]（[Ca]sR）。 使用我们直接测量（Ca）sR的新方法，我们将测试a）是否存在重要的空间（Ca）sR梯度，B）为什么SR Ca负荷在HF中低（低（Ca）sR、SR体积或Ca缓冲），c）受磷蛋白（PL B）是否降低SR Ca-ATP酶效率，以及d）（Ca）sa如何在E-C偶联期间动态地起作用以终止SR Ca释放。 2. HF和PKA & CaMKII效应中的SR Ca泄漏。 HF中舒张期SR Ca泄漏和蛋白激酶效应存在争议。 我们将使用钙火花&我们的新方法来测量泄漏。 我们将阐明HF中作为SR Ca负荷的函数的渗漏如何改变，以及PKA和CaMKII如何调节对照和HF心肌细胞中的SR Ca渗漏（包括HF中SR蛋白的表达和磷酸化状态的变化）。 3. HF中的Na内流。 (Na)在HF中，Na+升高，并且我们表明，TTX敏感性静息Na内流升高是主要原因（例如，相对于Na/H或Na/Ca交换）。 我们将测试在HF中的刺激期间是否也是如此，以及HF中的TTX敏感性Na内流是否归因于缓慢失活或窗口Na电流。 4. 磷脂膜蛋白（PLM）和Na/K-ATP酶调节。 PLM是Na/K-ATP酶的内源性调节剂，也是心脏中PKA的主要靶点。 我们将检验以下假设：a）内源性PLM抑制Na/K-ATP酶，并且抑制被PKA依赖性磷酸化缓解，B）PLM表达水平调节Na/K-ATP酶表达，以及c）在HF中，较低的PLM表达和/或较高的磷酸化解释了为什么HF中较低的Na/K-ATP酶表达不抑制Na泵功能。 这些研究将交织心肌细胞E-C偶联的定量基础机制研究，对照和HF中的Ca和Na调节（关于SR Ca转运，SRCa释放如何在E-C偶联期间关闭，PKA & CaMKII效应，Na内流，NCX和Na/K-ATP酶）。 我们将测试明确的机制假说，这将丰富我们对心脏细胞中钙和钠调节的基本理解，但也提供了新的见解，这些是如何改变的HF。 这将有助于开发新的治疗靶点和治疗人类HF的策略。

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.