During normal cardiac excitation-contraction coupling (ECC), the Ca2+ that drives contraction is released from the sarcoplasmic reticulum (SR) when the ryanodine receptor (RyR) channel opens. Abnormal RyR- mediated Ca2+ release is thought to be responsible for the diastolic SR Ca2+ leak flux (Jleak) which is associated with heart failure (HR) as well as certain dangerous cardiac arrhythmias. The fundamental issues to be resolved are delineation of 1) mechanisms that govern the amplitude and dynamics of diastolic SR Ca release (i.e. Jleak) and 2) potential roles of these mechanisms during cardiac pathophysiology. The overall hypothesis is that Jleak is increased by protein kinase activity, primarily due to RyR phosphorylation by Ca-calmodulin-dependent protein kinase (CaMKII), and that abnormally high Jleak levels increase the probability of cardiac arrhythmias. This hypothesis is tested using a unique multidisciplinary experimental approach which includes Ca2+ sparks &transients measurements, intra-SR a2+ detection, whole heart Ca2+ wave measurements and single RyR channel recording.
The specific aims are:
Specific Aim 1 : To define the role of both protein kinase A (PKA) and CaMKII in changing RyR activity and Jleak- The hypothesis is that CaMKII will increase RyR activity and Jleak and that this will be partially dependent upon a shift in Jleak as a function of total SR [Ca] ([Ca]SRT, aims a-d) and free SR [Ca] ([Ca]SR, aims e &f). We will: a) determine the effect of each kinase on Jleak as a function of [Ca]SRT and [Ca]SR in isolated, intact cardiac myocytes. b) define the mechanism by which ISO increases CaMKII activity, c) define the mechanism by which both CaMKII/PKA and [Ca]SR increase Jleak by measuring Ca spark- mediated flux (Jspark) in intact cardiac myocytes. d) define in artificial lipid bilayers the mechanism by which CaMKII/PKA increase single RyR activity.
Specific Aim 2 : To define the (patho)physiological significance of the Jleak. The hypothesis is that higher Jleak-mediated sub-sarcolemmal [Ca] raises the probability of generating arrhythmias. We will determine the effect of enhancing Jleak upon the generation of delayed after depolarizations (DADs) in intact cardiac myocytes.
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