Ca regulation in cardiac myocytes is central to excitation-contraction coupling (ECC) and is also involved in hypertrophic nuclear signaling, mitochondrial energy regulation and cell death. Two important and Ca regulatory systems, Ca-calmodulin (CaM) dependent protein kinase II (CaMKll) and inositol (1,4,5)P3 receptors (InsPsR) are present in myocytes, and have been increasingly implicated in regulating ECC, arrhythmogenesis and nuclear signaling (due in part to studies in this PPG). Indeed, CaMKll is emerging as a nodal point in ECC, arrhythmogenesis and signaling in hypertrophy (Hyp) and heart failure (HF). Overall goals here are to understand better how Ca and CaMKll function in cardiac myocytes with respect to acute Ca signaling (ECC & arrhythmogenesis), mitochondrial function and how both InsPaR and CaMKll may be involved in nuclear signaling (via class II histone deacetylases, HDACs) in hypertrophy & HF. Main experimental methods include confocal fluorescence imaging (of Ca indicators & other fluorescent probes) and voltage clamp in isolated adult cardiac myocytes and hearts (including from transgenic and knockout mice, and HF rabbits). This project has 4 aims. Aim 1 will measure acute CaMKIIS effects on ECC, and also dynamics of CaM & CaMKll signaling in myocytes, including effects on Ca current facilitation, Na current, SR Ca release (diastolic & systolic) and frequency-dependent acceleration of relaxation. Aim 2 will test novel hypotheses about Ca/CaM-dependent signaling bv CaMKll & calcineurin to the nucleus via HDAC & NFAT to better understand neurohumoral signaling in Hyo & HF. Aim 3 will assess how mitochondrial [Cal is regulated in cardiac myocytes, quantitatively and locally, with an eye toward mitochondrial and cell death mechanisms. Aim 4 will assess altered CaMKll signaling in Hyp & HF regarding ECC, arrhythmias & nuclear signaling. The proposed work will be highly interdigitated with all three other projects in the PPG, taking full advantage of complementary perspectives and expertise of the PPG team. The results will provide comprehensive new information regarding the roles of CaM, CaMKll, calcineurin, mitochondrial Ca and InsPa signaling in cardiac myocytes during ECC, arrhythmogenesis and nuclear signaling in normal ventricular myocytes and during hypertrophy and heart failure.

RELEVANCE (See instructions); Cardiac myocyte calcium signaling is critical to the electrical activity which synchronizes the heartbeat, the contraction that pumps blood, energetic balance at the mitochondrial level, and the control of gene transcription. In heart failure or arrhythmias things go wrong in these pathways, contributing to heart malfunction. Here we will provide novel experimental results in careful quantitative studies, which will help to understand the fundamental workings of these systems critical to health and cardiac disease