Cardiac arrhythmias remain a leading cause of morbidity and mortality in the US. In multiple forms of cardiac disease, arrhythmias result from disturbances in intracellular calcium (Ca) cycling, the process that normally couples electrical excitation to mechanical function. Despite significant progress, the fundamental mechanisms underlying Ca-dependent arrhythmias remain elusive, owing mainly to the complex, nonlinear nature of cardiac Ca signaling, which hinders the development of effective antiarrhythmic therapies. Based on our work in the previous funding period, we have established that Ca signaling refractoriness provides a powerful concept for understanding cardiac intracellular Ca handling at multiple biological scales (from molecular and subcellular domains to myocardial tissue and intact heart) in normal and diseased hearts. Ca signaling refractoriness refers to a state of temporary deactivation of the sarcoplasmic reticulum (SR) Ca release channels (Ryanodine receptors, RyRs) in the wake of systolic release from the sarcoplasmic reticulum (SR). This Ca signaling refractoriness is critical in maintaining stable Ca-induced Ca release (CICR) by preventing aberrant diastolic Ca release (DCR) - a cause of arrhythmias. Using this new framework, our proposed research will establish the subcellular and molecular bases of aberrant Ca release synchronization, including the refractory properties of functionally distinct Ca release units and determine how these properties are influenced by genetic and acquired intrinsic RyR defects as well as by extrinsic local Na/Ca microdomain homeostasis and SR Ca load. Our quantitative studies of aberrant Ca-excitation coupling will provide a new understanding of characteristic differences in arrhythmogenic properties of ventricular and atrial tissue including ectopic firing propensity and self-sustaining Ca/Vm oscillations. Our multiscale studies of inhibition of arrhythmogenic propensity through the targeting of both intrinsic and extrinsic mechanisms using genetic mouse models of arrhythmia and pre- clinical models of cardiomyopathy as well as preparations from failing and rejected donor human hearts. Taken together these will provide a proof-of-principle for new therapeutic strategies based on desynchronization of aberrant SR Ca release.
Cardiac arrhythmias (abnormal heart rhythms) remain a leading cause of death in the United States. While much has been learned about the causes of these arrhythmias in single cells from the heart, there remain questions about how events in individual cells cause the heart to malfunction. We seek to better understand arrhythmias with the long-term goal of improving arrhythmia prevention and treatment. SPECIFIC AIMS: The excitation-contraction coupling (ECC) process that initiates a normal heart beat relies on electrically- induced calcium (Ca) influx in cardiac myocytes to trigger Ca-induced Ca release (CICR) from the sarcoplasmic reticulum (SR). In various disease settings that include Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) and acquired cardiomyopathy, an aberrant process of 'retrograde' Ca- excitation coupling results in unprovoked Ca release (i.e. diastolic Ca release, DCR) which can result in improperly timed electrical impulses. It is believed that abnormal impulses generated in this manner contribute to the over 300,000 cases of sudden cardiac death occurring in the US each year. Despite significant progress, however, the fundamental mechanisms underlying Ca-dependent arrhythmias remain elusive, owing mainly to the complex, nonlinear nature of cardiac Ca signaling, which hinders the development of effective antiarrhythmic therapies. Based on our work in the previous funding period, we have established that Ca signaling refractoriness provides a robust basis for understanding cardiac intracellular Ca handling at multiple biological scales (from molecular and subcellular domains to myocardial tissue to intact heart) in normal and diseased hearts. Ca signaling refractoriness refers to a phase of temporary deactivation of RyR channels in the wake of systolic release, a phase that is critical in maintaining stable CICR by preventing spontaneous oscillations of DCR. Studies from the previous funding period suggest that 1) Ca release refractoriness is governed by luminal Ca (at least in part via CASQ2) and is influenced by multiple factors affecting RyR functional state and SR Ca content; and 2) shortened RyR2 refractoriness provides a substrate for synchronization of aberrant DCR events, which is required for the genesis of triggered arrhythmias. However, essential factors driving shortened Ca signaling refractoriness and synchronization of aberrant Ca release at molecular, subcellular, and tissue levels remain to be determined. We propose to define for the first time the synchronization mechanisms of diastolic release leading to triggered activity, including refractory properties of Ca release sites and the role of SR Ca uptake, using state-of-the-art subcellular, cellular and multicellular Ca/membrane potential (Vm) imaging in ventricular and atrial preparations, utilizing clinically relevant modelsof genetic and acquired arrhythmias as well as human preparations. In terms of molecular antiarrhythmic targets our focus will be on distinct subdomains and their modulation by local Na/Ca homeostasis (including 'fuzzy space' neuronal Na channels), local CaMKII and ROS activities, and SERCA2a-mediated SR Ca re-uptake. The Specific Aims are: Aim 1. Elucidate the molecular and subcellular determinants of diastolic Ca release synchronization in the genesis of Ca-dependent arrhythmias. We will 1) test the hypothesis that shortened Ca signaling refractoriness contributes to synchronization of arrhythmogenic Ca release and 2) determine the role of spatially/functionally distinct populations of Ca release units (CRUs), subdomain-specific Na/Ca signaling and SR Ca uptake using high resolution imaging in genetic models of CPVT; approaches will include comparative studies in ventricular vs. atrial myocytes using the characteristic differences in subcellular organization to elucidate fundamental aspects of DCR synchronization. Aim 2. Define the cellular and supracellular mechanisms of aberrant Ca release synchronization and its role in the genesis of Ca-dependent arrhythmias. We will 1) test the hypothesis that shortened Ca signaling refractoriness provides a substrate for myocardial synchronization of aberrant diastolic Ca release and subsequent ectopic activity, and 2) define the functional determinants, atrio-ventricular variations, and strategies of pharmacological modulation of aberrant Ca-excitation coupling using dual Vm/Ca imaging with detailed cellular resolution in genetic mouse models of CPVT. Aim 3. Validate the applicability and antiarrhythmic therapeutic potential of mechanisms discovered in aims 1 and 2 to in vivo settings, and to acquired disease in preparations from clinically relevant animal models and humans. We will test the hypotheses that 1) desynchronization of DCR by neuronal Na channel blockade inhibits arrhythmias in CPVT mice, whereas DCR synchronization by SERCA2a overexpression will exacerbate arrhythmia burden in this model, and 2) these basic mechanisms and consequences of arrhythmic DCR synchronization are applicable to acquired cardiac disease in canine and human preparations.
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