T-tubule remodeling and Ca2+-dependent arrhythmogenesis in cardiomyopathies.

描述（由申请人提供）：心律失常是心力衰竭（HF）患者死亡的主要原因。越来越多的证据表明，细胞内Ca2+的失调是这些心律失常的关键因素。重要的是，心肌细胞Ca2+释放的改变也被广泛认为是心力衰竭病理生理学的核心参与者。具体来说，HF肌细胞显示肌浆网（SR） Ca2+储存减少和SR Ca2+瞬态减少。然而，Ca2+依赖性心律失常（例如，去极化后延迟，DADs）通常是由SR Ca2+过载引起的。关于心力衰竭的一个关键但尚未解决的问题是：在整体SR Ca2+含量降低的情况下，HF期间Ca2+依赖性心律失常是如何发生的？人们对这一悖论的理解甚少。我们的长期目标是获得心衰Ca2+依赖性心律失常的机制理解。我们最近观察到从明显HF的自发性高血压大鼠（SHR）分离的心肌细胞中t小管（TT）系统和孤立的ryanodine受体（RyRs）的显著重塑。根据我们发表的和初步的结果，我们假设在心衰期间，TT结构重塑在不稳定的Ca2+稳态中起重要的机制作用，因此Ca2+依赖性心律失常。此外，我们预测肌细胞用于补偿原发性损伤的信号通路（如PKA过度磷酸化和CaMKII上调）可能会调节TT重塑。为了验证这些假设，我们将解决三个具体目标：目的1-评估TT系统改变与衰竭心肌细胞功能失调的EC偶联之间的关系；目的2-确定TT重构在心衰Ca2+依赖性心律失常发生中的作用；目的3-确定CaMKII和PKA信号在HF模型中TT超微结构重塑、异常Ca2+信号和Ca2+依赖性心律失常中的作用。为了实现这些目标，我们将结合最先进的技术，包括膜片钳（全细胞，松散密封膜片钳）；高分辨率共聚焦成像；免疫荧光;以及数字图像处理。我们将使用这些技术检查小鼠实验性HF模型中分离的肌细胞和完整的心脏，包括磷酸化被“全局”抑制（AC3-I）或特定CaMKII和PKA磷酸化位点被禁用（RyR- S2814A, RyR- s2808a）的对照组和遗传小鼠。我们预计，完成拟议的项目将提高我们对人类心肌病中Ca2+依赖性心律失常和心源性猝死的机制的理解，并为预防和治疗人类HF和致命性心律失常的有效治疗策略的发展提供重要见解。

DESCRIPTION (provided by applicant): Cardiac arrhythmias are the leading cause of death among heart failure (HF) patients. A growing body of evidence indicates that dysregulation in intracellular Ca2+ is a critical factor in these arrhythmias. Importantly, alterations in myocyte Ca2+ release are also widely considered the central player in the pathophysiology of heart failure. Specifically, HF myocytes display reduced sarcoplasmic reticulum (SR) Ca2+ stores and a reduction in SR Ca2+ transients. However, Ca2+-dependent arrhythmias (e.g., delayed after-depolarizations, DADs) are normally induced by SR Ca2+ overload. A critical, but unresolved question about heart failure is: how can Ca2+-dependent arrhythmias occur during HF, in a setting of globally decreased SR Ca2+ content? This paradox is poorly understood. Our long-term objective is to gain a mechanistic understanding of the Ca2+-dependent arrhythmias in HF. We recently observed striking remodeling of the t-tubule (TT) system and orphaned ryanodine receptors (RyRs) in cardiomyocytes isolated from spontaneously hypertensive rats (SHR) with overt HF. Based on our published and preliminary results, we hypothesize that during HF, TT structural remodeling plays an important mechanistic role in unstable Ca2+ homeostasis and therefore Ca2+-depedent arrhythmogenesis. Moreover, we predict that signaling pathways (e.g., PKA hyperphosphorylation and CaMKII upregulation) utilized by myocytes to compensate in response to primary insults may modulate TT remodeling. To test these hypotheses, we will address three specific aims: Aim 1- Evaluate the relationship between alterations in TT system and dysfunctional EC coupling in failing cardiomyocytes; Aim 2- Determine the role of TT remodeling in Ca2+-dependent arrhythmogenesis in HF; Aim 3- Define the role of CaMKII and PKA signaling in TT ultrastructural remodeling, abnormal Ca2+ signaling, and Ca2+-dependent arrhythmogenesis in HF models. To achieve these aims, we will combine state-of-the-art techniques, including patch-clamp (whole-cell, loose-sealed patch clamp); high-resolution confocal imaging; immunofluorescence; and digital image processing. We will use these techniques to examine isolated myocytes and intact hearts from mouse experimental HF models, including control and genetic mice in which phosphorylation is `globally' inhibited (AC3-I) or where specific CaMKII and PKA phosphorylation sites are disabled (RyR- S2814A, RyR-S2808A). We anticipate that fulfilling the proposed project will improve our understanding of the mechanisms underlying Ca2+-dependent arrhythmias and sudden cardiac death in human cardiomyopathies, and provide important insights into the development of effective therapeutic strategies for preventing and treating human HF and fatal cardiac arrhythmias.