Published online before print October 6, 2005, doi: 10.1161/01.RES.0000189270.72915.D1
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© 2005 American Heart Association, Inc.
Cellular Biology |
Ca2+ Influx–Induced Sarcoplasmic Reticulum Ca2+ Overload Causes Mitochondrial-Dependent Apoptosis in Ventricular Myocytes
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 ß2a subunit-green fluorescent protein (GFP) fusion gene (Adß2a) to increase Ca2+ influx or with AdGFP as a control. Significantly fewer ß2a-VMs (21.4±5.6%) than GFP-VMs (99.6±1.7%) were viable at 96 hours. A fraction of ß2a-VMs (20.8±1.8%) contracted spontaneously (SC-ß2a-VMs), and viability was significantly correlated with the percentage of SC-ß2a-VMs. Higher percentages of apoptotic nuclei, DNA laddering, and cytochrome C release were detected in ß2a-VMs. This apoptosis was prevented with pancaspase or caspase-3 or caspase-9 inhibitors. L-type calcium current (ICa-L) density was greater in ß2a-VMs (23.4±2.8 pA/pF) than in GFP-VMs (7.6±1.6 pA/pF). SC-ß2a-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 ß2a-VMs from apoptosis. These results show that persistent increases in Ca2+ influx through the ICa-L enhance contractility but lead to apoptosis through a mitochondrial death pathway if SR Ca2+ overload is induced.
Key Words: L-type calcium channel • ß2a subunit • apoptosis • ventricular myocyte • primary culture
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