Calmodulin and Regulation of Cardiac Ryanodine Receptor and CaMKII

University Of California At Davis, Davis CA

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Abstract

SUMMARY Our overall goal is to understand the structural mechanisms for functional regulation of the cardiac ryanodine receptor (RyR2) and Ca/calmodulin-dependent kinase (CaMKII), two central signaling nodes causally implicated in numerous cardiac pathologies, including heart failure (HF) and arrhythmias. Our focus is on pathological states and their mitigation by drug-like compounds. Our structural studies using fluorescence resonance energy transfer in physiological environments will evolve and animate the high resolution but static cryo-EM/crystallography to the level of the dynamic heartbeat. This information is urgently needed to drive novel therapeutic strategies for arrythmia and HF. Our team has complementary ideas, expertise (molecular biophysics to confocal myocyte imaging and animal models) and a novel fluorescence tool to tackle important issues concerning these two key targets (RyR2 and CaMKII) in two parallel aims. RyR2 is the primary Ca-release channel in the sarcoplasmic reticulum (SR), and contributes to and is a therapeutic target for treating HF and arrhythmias. Our work has developed a new integrated mechanistic paradigm explaining pathological RyR2 dysfunction that promotes arrhythmias, and systolic and diastolic dysfunction in HF and diabetes. Importantly, the common pathological RyR2 structural state reduces calmodulin (CaM) binding to RyR2, which results in elevated arrhythmogenic SR calcium leak. These conformational structure-function shifts will be revealed by our FRET-based molecular tools that will also fuel drug discovery assays to identify novel RyR therapeutics. Aim 1 here characterizes the dynamics of CaM regulation of RyR2 in healthy and pathological states, resolving the structural and temporal implications of pathologic CaM-RyR2 in isolated SR and cardiomyocytes. Further, we propose to test the potential therapeutic impact of new candidate compounds on correcting pathological RyR2. CaMKII has emerged as a multifunctional central node in cardiac pathology, including HF, diabetes and arrhythmias. CaMKII becomes chronically active in disease by multiple pathways of post-translational modification (PTMs) that we have helped to uncover (autophosphorylation, oxidation, O-glycosylation and S- nitrosylation - all in a key regulatory domain of CaMKII). CaMKII and RyR2 conspire at the core of a vicious positive feedback cycle where CaMKII promotes RyR2 leak, to further activate CaMKII that can also regulate many other myocyte targets. We will target CaM-RyR dysfunction in disease and use this approach for understanding CaMKII dynamics and drug discovery. Aim 2 focuses on dynamic CaMKII structural/functional states induced by pathological PTMs, using fluorescently tagged CaMKII and a new CaMKII-specific activity reporter. Additionally, we will adapt these assays for high-throughput screening to identify compound research tools selective for the pathological PTM induced state of CaMKII.