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

• Published in: Circulation research Vol. 97; no. 10; pp. 1009 - 1017
• Main Authors: Chen, Xiongwen, Zhang, Xiaoying, Kubo, Hajime, Harris, David M, Mills, Geoffrey D, Moyer, Jed, Berretta, Remus, Potts, Sabine Telemaque, Marsh, James D, Houser, Steven R
• Format: Journal Article
• Language: English
• Published: United States American Heart Association, Inc 11.11.2005
• Subjects: Animals; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein - physiology; bcl-2-Associated X Protein - physiology; Calcium - metabolism; Calcium Channels, L-Type - analysis; Calcium Channels, L-Type - physiology; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases - physiology; Caspase 8; Caspases - physiology; Cats; Cells, Cultured; Heart Ventricles; Mitochondria, Heart - physiology; Myocardial Contraction; Myocytes, Cardiac - metabolism; Myocytes, Cardiac - pathology; Phosphorylation; Protein Subunits; Rats; Sarcoplasmic Reticulum - metabolism
• ISSN: 0009-7330; 1524-4571
• DOI: 10.1161/01.RES.0000189270.72915.D1

Increases in Ca influx through the L-type Ca channel (LTCC, Cav1.2) augment sarcoplasmic reticulum (SR) Ca loading and the amplitude of the cytosolic Ca transient to enhance cardiac myocyte contractility. Our hypothesis is that persistent increases in Ca influx through the LTCC cause apoptosis if the excessive influx results in SR Ca overload. Feline ventricular myocytes (VMs) in primary culture were infected with either an adenovirus (Ad) containing a rat Cav1.2 β2a subunit-green fluorescent protein (GFP) fusion gene (Adβ2a) to increase Ca influx or with AdGFP as a control. Significantly fewer β2a-VMs (21.4±5.6%) than GFP-VMs (99.6±1.7%) were viable at 96 hours. A fraction of β2a-VMs (20.8±1.8%) contracted spontaneously (SC-β2a-VMs), and viability was significantly correlated with the percentage of SC-β2a-VMs. Higher percentages of apoptotic nuclei, DNA laddering, and cytochrome C release were detected in β2a-VMs. This apoptosis was prevented with pancaspase or caspase-3 or caspase-9 inhibitors. L-type calcium current (ICa-L) density was greater in β2a-VMs (23.4±2.8 pA/pF) than in GFP-VMs (7.6±1.6 pA/pF). SC-β2a-VMs had higher diastolic intracellular Ca (Indo-1 ratio1.1±0.1 versus 0.7±0.03, P<0.05) and systolic Ca transients (1.89±0.27 versus 0.80±0.08) than GFP-VMs. Inhibitors of Ca influx, SR Ca uptake and release, mitochondrial Ca uptake, mitochondrial permeation transition pore, calpain, and Bcl-2-associated X protein protected β2a-VMs from apoptosis. These results show that persistent increases in Ca influx through the ICa-L enhance contractility but lead to apoptosis through a mitochondrial death pathway if SR Ca overload is induced.