Dysregulation of calcium in the cardiac ventricular myocyte is prevalent in many pathological models of heart disease such as heart failure and sudden cardiac death. Over the steady state, plasma membrane (PM) calcium entry must equal plasma membrane calcium extrusion to maintain calcium homeostasis. The major PM entry pathway is the L-type Ca channel, and the major PM extrusion pathway is the Na-Ca exchanger (NCX). Calcium may enter the cytosol during diastole via PM leak, and via unitary releases of calcium from intracellular stores - labeled as calcium sparks. Sodium-Calcium exchanger extrusion of calcium during diastole contributes a depolarization of membrane potential. Thus, diastolic calcium fluxes, via forward mode NCX can trigger ventricular cardiac arrhythmias, and can contribute to sudden cardiac death. Diastolic sparks are well-characterized in terms of dimension and location, yet the mechanism of how they are triggered remains a mystery. However, my pilot data suggests that calcium entry through the L-type calcium channel is a required input for a diastolic spark to occur in developing mouse ventricular myocyte. Therefore, the overall purpose of this proposal is to determine the trigger and the source of diastolic calcium sparks. My preliminary data leads me to the overall driving hypothesis: the L-type calcium channel entry triggers diastolic calcium sparks. This proposal will provide major inisght to calcium handling in adult and pathological conditions (i.e. heart failure). This overall goal will be accomplished by separating L-type channel dependent and independent diastolic sparks. Understanding spark inititiation could lead to discovering a therapuetic target for cardiac arrhythmias. I will use fast-scanning 2D confocal imaging to study the amplitude, rate of rise, and kinetics of diastolic calcium sparks in embryonic, adult, and thoracic aortic banded mice (i.e. heart failure model). If time allows or if needed, simultaneous whole patch electrophysiology and calcium imaging can be used to dissect the two populations of diastolic sparks in the adult and their voltage dependence.
