Calcium regulation in cardiac myocytes is central to excitation-contraction coupling (ECC) and is also involved in hypertrophic nuclear signaling. Two important and ubiquitous Ca regulatory systems, Ca-calmodulin dependent protein kinase II (CaMKII) and inositol (1,4,5)P3 receptors (InsP3R) are present in myocytes, and have been implicated in altering ECC, arrhythmogenesis and nuclear signaling. However, surprisingly littlie is known about how these effects occur. Ca-dependent pathways implicated in regulating transcription in hypertrophy (Hyp) and heart failure (HF) include CaMKII &calcineurin (CaN) and these may function via nuclear translocation of key factors (NFAT &HDAC) which alter transcription. Overall goals here are to understand better how CaMKII and InsP3R function in cardiac myocytes with respect to acute Ca signaling (ECC &arrhythmogenesis) and in nuclear signaling (via NFAT &HDAC) in hypertrophy &HF. Four highly synergistic multidisciplinary projects are planned. Project I (Bers) focuses on cellular aspects of CaMKII in 3 aims concerning: 1) acute CaMKII effects on ECC, 2) Ca-dependent nuclear signaling via a proposed lnsP3R-CaMKII-HDAC pathway, &3) altered CaMKII signaling in Hyp &HF (regarding ECC, arrhythmias &HDAC activation). Project II (Blatter) focuses on cellular aspects of IP3Rs in 3 aims (all qHyp &HF) concerning: 1) acute IP3R-mediated effects on ECC, 2) the role of InsP3R in arrhythmogenesis and how CaMKII modulates lnsP3R function, and 3) Ca coding and IP3R involvement in NFAT signaling to the nucleus. Project III (Miqnery) focuses on molecular characterization of 1) the direction that nuclear InsP3Rs face and their physical interactions with CaMKII (&CaM &CaN), 2) CaMKII-dependent phosphorylation of InsP3R and modulation of function, 3) manipulation of InsP3R-CaMKII interaction, 4) InsP3R isoform expression &localization in atrial &ventricular myocytes (Hyp &HF), 5) generating novel fluorescent [InsP3] sensors (FIREs). Project IV (Brown) focuses on CaMKII and InsP3R regulation at in vivo and biochemical levels concerning: 1) development of Hyp &HF in knockout mice lacking cardiac lnsP3R2 or CaMKIIlambda (the dominant myocyte isoforms), 2) differential activation of cytosolic vs. nuclear CaMKII, plus development of a fluorescent CaMK activity sensor (CaMKAR), 3) differential target phosphorylation by CaMKII isoforms, &4) cardiac InsP3 formation and regulation by CaMKII. Three scientific cores will support these aims. Core B (Myocytes &HF Rabbits) will isolate myocytes from mice and rabbits (including Hyp mice and HF rabbits). Core C (Fluorescence Imaging) will provide instrumentation and expertise for fluorescent imaging. Core D (Genetic Mouse &Adenovirus) will develop unique mouse models (e.g. lnsPaR2- &CaMKIIlambda-KO) and adenoviral vectors for myocyte studies. The proposed work integrates experienced investigators with highly complementary expertise and perspective to tackle these questions in a highly interactive multidisciplinary approach. The results will greatly increase our understanding of the roles of CaMKII and InsP3R in cardiac myocytes during ECC, arrhythmogenesis and nuclear signaling in normal, Hyp and HF cardiac myocytes.
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