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）患者的主要死亡原因。越来越多的证据表明，细胞内Ca 2+的失调是这些心律失常的一个关键因素。重要的是，肌细胞Ca 2+释放的改变也被广泛认为是心力衰竭病理生理学的中心参与者。具体而言，HF心肌细胞显示肌浆网（SR）Ca 2+储存减少和SR Ca 2+瞬变减少。然而，Ca 2+依赖性心律失常（例如，延迟后去极化，DAD）通常由SR Ca 2+过载诱导。关于心力衰竭的一个关键但尚未解决的问题是：在SR Ca 2+含量全面降低的情况下，HF期间如何发生Ca 2+依赖性心律失常？人们对这一悖论理解甚少。我们的长期目标是获得一个机制的理解，钙离子依赖性心律失常在HF。最近，我们观察到显着重塑的t-小管（TT）系统和孤儿兰尼碱受体（RyRs）的心肌细胞分离自自发性高血压大鼠（SHR）与明显的HF。根据我们发表的初步结果，我们假设在HF期间，TT结构重塑在不稳定的Ca 2+稳态中起重要的机制作用，因此Ca 2+依赖性血管生成。此外，我们预测信号通路（例如，PKA过度磷酸化和CaMKII上调）用于补偿对原发性损伤的反应，可调节TT重塑。为了验证这些假设，我们将提出三个具体的目标：目标1-评估TT系统的改变和衰竭心肌细胞中EC偶联功能障碍之间的关系;目标2-确定TT重构在HF中Ca 2+依赖性血管生成中的作用;目的3-确定CaMK II和PKA信号在TT超微结构重塑、异常Ca 2+信号传导、和Ca 2+依赖性血管生成。为了实现这些目标，我们将结合联合收割机最先进的技术，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;和数字图像处理。我们将使用这些技术来检查来自小鼠实验HF模型的分离的肌细胞和完整的心脏，包括对照和遗传小鼠，其中磷酸化被“全面”抑制（AC 3-I）或其中特定的CaMKII和PKA磷酸化位点被禁用（RyR-S2814 A，RyR-S2808 A）。我们预计，完成拟议的项目将提高我们的钙依赖性心律失常和心脏性猝死在人类心肌病的机制的理解，并提供重要的见解，为预防和治疗人类HF和致命的心律失常的有效治疗策略的发展。

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.