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

描述(申请人提供)：心律失常是心力衰竭(HF)患者的主要死亡原因。越来越多的证据表明，细胞内钙离子调节失调是这些心律失常的关键因素。重要的是，心肌细胞钙释放的改变也被广泛认为是心力衰竭的病理生理过程中的中心角色。特别是，HF心肌细胞显示肌浆网(SR)钙存储减少，肌浆网钙瞬变减少。然而，钙依赖性心律失常(如延迟后除极，DADS)通常是由肌质网钙超载引起的。有关心力衰竭的一个关键但尚未解决的问题是：在肌浆网钙离子含量普遍下降的情况下，心衰期间如何发生钙离子依赖型心律失常？人们对这一悖论知之甚少。我们的长期目标是从机制上了解心衰时钙离子依赖的心律失常。我们最近在自发性高血压大鼠(SHR)显性心衰分离的心肌细胞中观察到T管(TT)系统和孤立的兰尼定受体(RyRs)的显着重构。根据我们已发表的和初步的结果，我们推测在心力衰竭过程中，TT结构重构在不稳定的钙稳态中起着重要的机制作用，从而抑制钙抑制的心律失常的发生。此外，我们预测心肌细胞用来补偿原发损伤的信号通路(如PKA过度磷酸化和CaMKII上调)可能调节TT重塑。为了验证这些假说，我们将针对三个特定的目标：目的1-评估衰竭心肌细胞中TT系统的改变与EC偶联功能障碍之间的关系；目的2-确定TT重塑在心力衰竭钙依赖型心律失常发生中的作用；目的3-明确CaMKII和PKA信号在心力衰竭模型中TT超微结构重塑、异常的钙信号转导和钙依赖的心律失常发生中的作用。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、松散密封的膜片钳)、高分辨率共聚焦成像、免疫荧光和数字图像处理。我们将使用这些技术来检测来自小鼠实验性心力衰竭模型的分离的心肌细胞和完整的心脏，包括对照组和遗传小鼠，在这些小鼠中，磷酸化被‘全局’抑制(AC3-I)或特定的CaMKII和PKA磷酸化位点被禁用(RyR-S2814A，RyR-S2808A)。我们预计，完成该项目将提高我们对人类心肌病钙依赖型心律失常和心源性猝死的机制的理解，并为开发有效的防治人类心力衰竭和致命性心律失常的治疗策略提供重要的见解。

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.