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.
Chen
Xiongwen
X
Cardiovascular Research Center, Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Zhang
Xiaoying
X
Kubo
Hajime
H
Harris
David M
DM
Mills
Geoffrey D
GD
Moyer
Jed
J
Berretta
Remus
R
Potts
Sabine Telemaque
ST
Marsh
James D
JD
Houser
Steven R
SR
D000818:Animals; D017209:Apoptosis; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000032:analysis / Q000502:physiology*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology; D053181:Caspase 8; D020169:Caspases / Q000502:physiology; D002415:Cats; D002478:Cells, Cultured; D006352:Heart Ventricles; D008929:Mitochondria, Heart / Q000502:physiology*; D009200:Myocardial Contraction; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000473:pathology*; D010766:Phosphorylation; D021122:Protein Subunits; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000502:physiology; D051028:bcl-2-Associated X Protein / Q000502:physiology
D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C); D021122:Protein Subunits; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C505528:Casp8 protein, rat; D053181:Caspase 8; D020169:Caspases; D002118:Calcium