Ca 2+ Influx–Induced Sarcoplasmic Reticulum Ca 2+ Overload Causes Mitochondrial-Dependent Apoptosis in Ventricular Myocytes

Abstract

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