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(Circulation Research. 2005;97:1009.)
© 2005 American Heart Association, Inc.

Cellular Biology

Ca²⁺ Influx–Induced Sarcoplasmic Reticulum Ca²⁺ Overload Causes Mitochondrial-Dependent Apoptosis in Ventricular Myocytes

Xiongwen Chen, Xiaoying Zhang, Hajime Kubo, David M. Harris, Geoffrey D. Mills, Jed Moyer, Remus Berretta, Sabine Telemaque Potts, James D. Marsh, Steven R. Houser

From the Cardiovascular Research Center (X.C., H.K., D.M.H., G.D.M., J.M., R.B., S.R.H.) and Departments of Physiology (D.M.H., G.D.M., S.R.H.) and Microbiology and Immunology (X.Z.), Temple University School of Medicine, Philadelphia, Pa; and the Department of Internal Medicine (S.T.P., J.D.M.), University of Arkansas for Medical Sciences, Little Rock.

Correspondence to Dr Steven Houser, Department of Physiology, Temple University School of Medicine, 3400 N Broad St, Philadelphia, PA 19140. E-mail steven.houser{at}temple.edu

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 ß_2a subunit-green fluorescent protein (GFP) fusion gene (Adß_2a) to increase Ca²⁺ influx or with AdGFP as a control. Significantly fewer ß_2a-VMs (21.4±5.6%) than GFP-VMs (99.6±1.7%) were viable at 96 hours. A fraction of ß_2a-VMs (20.8±1.8%) contracted spontaneously (SC-ß_2a-VMs), and viability was significantly correlated with the percentage of SC-ß_2a-VMs. Higher percentages of apoptotic nuclei, DNA laddering, and cytochrome C release were detected in ß_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 ß_2a-VMs (23.4±2.8 pA/pF) than in GFP-VMs (7.6±1.6 pA/pF). SC-ß_2a-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 ß_2a-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.

Key Words: L-type calcium channel • ß_2a subunit • apoptosis • ventricular myocyte • primary culture

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