Project Summary/ Abstract Ca-Calmodulin dependent protein kinase (CaMKII) is an important regulator of cardiac function, and dysfunction in pathological states, regulating ion channels, Ca transporters, myofilaments and nuclear transcription. CaMKII may normally fine-tune these processes. But in pathological conditions chronic autonomous CaMKII over-activation can hyerphosphorylate targets, contributing to arrhythmogenesis due to acute effects on several ion channels and Ca-handling proteins. Chronic CaMKII activation is also a hallmark of several pathological states and acute or genetic CaMKII inhibition can reduce arrhythmias and the progression of HF. Thus understanding fundamental aspects of CaMKII regulation in cardiac myocytes is critical understanding dysfunction and potential therapeutics. We and others discovered several novel post-translational modifications (PTMs) that can trap CaMKII in an activated state, rather than turning on & off rapidly with local Ca transients. Autophosphorylation, oxidation, GlcNAcylation and S-nitrosylation within a regulatory hotspot on CaMKII creates memory and autonomous activity, even when Ca/CaM falls. The functional synergy among these PTMs is unknown, but will be directly measured in myocytes in Aim 1. Dogma has been that CaMKII-dodecamers neither exchange subunits nor move appreciably in myocytes, but our preliminary data upends both dogmas, and this will be elucidated in Aim 2. S-nitrosylation, the newest regulatory PTM, can either promote autonomous activation (C290) or inhibit Ca/CaM activation (C273). Aim 3 will test the functional impact of these sites in adult myocytes and in acute ischemia/reperfusion and long-term pressure overload in intact animals. We will use multiple innovative fluorescence tools and methods, and animals to test these 3 major CaMKII Aims. The proposed studies will have major impact on our understanding of how CaMKII activity is regulated in heart, in ways that promote pathology and might be targets for therapeutic intervention.

Narrative Heart failure and cardiac arrhythmias affect millions of Americans. Ca-Calmodulin dependent protein kinase II (CaMKII) has been shown to regulate the function of numerous ion channels and calcium transporters in cardiac myocytes that govern normal excitability and contraction. CaMKII is known to be upregulated and chronically activated in multiple chronic cardiovascular diseases including, and has been implicated in the development of heart failure and arrhythmias. We will elucidate the actual molecular mechanisms that individually cause chronic CaMKII activation, how these functionally synergize, and how a novel regulation by S-nitrosylation may put the brakes on runaway CaMKII activation and its pathological consequences.