Calcium signaling in cardiac myocytes: Contributions from mitochondria, sodium-calcium exchanger, and ryanodine receptor.

摘要

This dissertation explores three critical players in the cardiac Ca2+ signaling pathway. First, I examined the role of mitochondria in Ca2+ signaling by generating a Ca 2+ probe, mitycamE31Q, with 1.5μM affinity for Ca2+ directed to mitochondria. Using mitycamE31Q and Fura-2AM, in neonatal rat cardiomyocytes, I show that release of Ca2+ from the SR (caffeine-application) caused cytosolic Ca2+ rise, followed by slower mitochondrial uptake of calcium. Similarly, Ca2+ influx upon Na+ withdrawal increased both cytosolic and more slowly the mitochondrial Ca 2+. I also found two populations of mitochondria (central, peripheral) that on such interventions had differing patterns of Ca2+ release and uptake. My findings suggest that mitochondrial Ca2+ changes with changes in cytosolic Ca2+, though with slower kinetics, and reveal Ca2+-load dependent subpopulations of mitochondria.;The second Ca2+ signaling issue addressed was the shark-heart splice-variant of NCX, reported to have bimodal adrenergic regulation (protective mechanism against Ca2+-overload induced arrhythmia). To test the anti-arrhythmic role of shark NCX, two approaches were employed: 1) creation of transgenic mice that express shark NCX-myc-tag; 2) construction of an adenovirus containing shark NCX-YFP. Although the transgenic mice failed to show biochemical and functional evidence for transgene overexpression, the adenoviral model showed 3-4 times greater expression of shark NCX current with shark-like bimodal regulation by 8-Br-cAMP in neonatal rat cardiomyocytes. I conclude that shark NCX can be functionally expressed in the mammalian cardiomyocytes, but whether it can confer anti-arrhythmic properties to the mammalian heart remains undetermined.;The role of the 3rd major player (RyR2) in the Ca 2+ signaling pathway in iPS cell-derived cardiomyocyte model expressing the wild and mutant RyR2-F2483I causing catecholaminergic polymorphic ventricular tachycardia, CPVT1, were evaluated. I found that while ICa and ICa-induced Ca2+-transients were similar, caffeine-induced Ca2+-release and I NCX were smaller, confirming smaller Ca2+-stores in CPVT. Adrenergic agonists enhanced ICa, but differentially altered the CICR gain, diastolic Ca2+, and Ca 2+-sparks in mutant cells. We conclude that both iPS-CM lines express the adult cardiomyocyte Ca2+-signaling phenotype. RyR2 F2483I-mutant myocytes have aberrant unitary Ca2+-signaling, smaller Ca 2+-stores, higher CICR gains, and sensitized adrenergic regulation, consistent with functionally altered Ca2+-release profile of CPVT.