Ca2+ influx-induced sarcoplasmic reticulum Ca2+ overload causes mitochondrial-dependent apoptosis in ventricular myocytes.

Increases in Ca2+ influx through the L-type Ca2+ channel (LTCC, Cav1.2) augment sarcoplasmic reticulum (SR) Ca2+ loading and the amplitude of the cytosolic Ca2+ transient to enhance cardiac myocyte contractility. Our hypothesis is that persistent increases in Ca2+ influx through the LTCC cause apoptosis if the excessive influx results in SR Ca2+ overload. Feline ventricular myocytes (VMs) in primary culture were infected with either an adenovirus (Ad) containing a rat Cav1.2 beta2a subunit-green fluorescent protein (GFP) fusion gene (Adbeta2a) to increase Ca2+ influx or with AdGFP as a control. Significantly fewer beta2a-VMs (21.4+/-5.6%) than GFP-VMs (99.6+/-1.7%) were viable at 96 hours. A fraction of beta2a-VMs (20.8+/-1.8%) contracted spontaneously (SC-beta2a-VMs), and viability was significantly correlated with the percentage of SC-beta2a-VMs. Higher percentages of apoptotic nuclei, DNA laddering, and cytochrome C release were detected in beta2a-VMs. This apoptosis was prevented with pancaspase or caspase-3 or caspase-9 inhibitors. L-type calcium current (I(Ca-L)) density was greater in beta2a-VMs (23.4+/-2.8 pA/pF) than in GFP-VMs (7.6+/-1.6 pA/pF). SC-beta2a-VMs had higher diastolic intracellular Ca2+ (Indo-1 ratio: 1.1+/-0.1 versus 0.7+/-0.03, P<0.05) and systolic Ca2+ transients (1.89+/-0.27 versus 0.80+/-0.08) than GFP-VMs. Inhibitors of Ca2+ influx, SR Ca2+ uptake and release, mitochondrial Ca2+ uptake, mitochondrial permeation transition pore, calpain, and Bcl-2-associated X protein protected beta2a-VMs from apoptosis. These results show that persistent increases in Ca2+ influx through the I(Ca-L) enhance contractility but lead to apoptosis through a mitochondrial death pathway if SR Ca2+ overload is induced.