Cardiac Excitation-contraction coupling (ECC) is mediated by calcium-induced calcium release, where calcium influx through voltage gated calcium channels opens ryanodine receptors and triggers massive release of calcium from intracellular stores. Alterations in the cellular events of ECC predispose the heart to arrhythmia. IP3 receptor (IP3R) activity exerts positive inotropic and arrhythmogenic effects on ECC in atrial myocytes. During heart failure, the expression of IP3R is increased; whether this results in changes in ECC or arrhythmogenesis remains unclear. The goal of this proposal is to investigate the role of IP3R-dependent signaling on ECC (Specific Aim 1) and arrhythmogenesis (Specific Aim 2) during heart failure. Studies will utilize ventricular myocytes isolated from normal rabbits as well as from the well-characterized rabbit heart failure model. IP3R-dependent signaling will be induced using application of the I PS-liberating agonist Endothelin-1 or via direct application of IP3. Inhibition of IP3R-dependent signaling will be achieved using both pharmacological inhibition and the expression of an IP3 affinity trap which binds and buffers intracellular IP3. Fluorescence microscopy with calcium sensitive dyes will monitor intracellular calcium (epifluorescence microscopy, indo-1) and subcellular calcium release events (laser scanning confocal fluorescence microscopy, fluo-4). Further, patch clamp techniques will be used to record membrane potential and ion channel currents.
Specific Aim 1 investigates the hypothesis that IP3R-dependent signaling exerts positive inotropic effects on ECC in ventricular myocytes, particularly during heart failure. In this Aim the effects of IP3R-dependent signaling on basal intracellular calcium, action potential-induced calcium transients, and elementary calcium release events (calcium sparks and puffs) will be examined.
Specific Aim 2 tests the hypothesis that IP3R-dependent signaling contributes to arrhythmogenesis in heart failure ventricular myocytes. Here, the effects of IP3R-dependent signaling on the frequency of arrhythmogenic calcium signals (spontaneous calcium release, calcium waves, and calcium alternans) and changes in membrane potential (Early and Delayed Afterdepolarizations, spontaneous action potentials) will be investigated. Arrhythmia represents the major cause of sudden death during heart failure. It is expected that results from this proposal will give needed insight into the mechanisms of arrhythmia during heart failure, which may aid treatment of individuals with cardiac disease. ? ? ? ?
