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

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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.