Osteopontin-stimulated apoptosis in cardiac myocytes involves oxidative stress and mitochondrial death pathway: role of a pro-apoptotic protein BIK.
Increased osteopontin (OPN) expression in the heart, specifically in myocytes, associates with increased myocyte apoptosis and myocardial dysfunction. Recently, we provided evidence that OPN interacts with CD44 receptor, and induces myocyte apoptosis via the involvement of endoplasmic reticulum stress and mitochondrial death pathways. Here we tested the hypothesis that OPN induces oxidative stress in myocytes and the heart via the involvement of mitochondria and NADPH oxidase-4 (NOX-4). Treatment of adult rat ventricular myocytes (ARVMs) with OPN (20 nM) increased oxidative stress as analyzed by protein carbonylation, and intracellular reactive oxygen species (ROS) levels as analyzed by ROS detection kit and dichlorohydrofluorescein diacetate staining. Pretreatment with NAC (antioxidant), apocynin (NOX inhibitor), MnTBAP (superoxide dismutase mimetic), and mitochondrial KATP channel blockers (glibenclamide and 5-hydroxydecanoate) decreased OPN-stimulated ROS production, cytosolic cytochrome c levels, and apoptosis. OPN increased NOX-4 expression, while decreasing SOD-2 expression. OPN decreased mitochondrial membrane potential as measured by JC-1 staining, and induced mitochondrial abnormalities including swelling and reorganization of cristae as observed using transmission electron microscopy. OPN increased expression of BIK, a pro-apoptotic protein involved in reorganization of mitochondrial cristae. Expression of dominant-negative BIK decreased OPN-stimulated apoptosis. In vivo, OPN expression in cardiac myocyte-specific manner associated with increased protein carbonylation, and expression of NOX-4 and BIK. Thus, OPN induces oxidative stress via the involvement of mitochondria and NOX-4. It may affect mitochondrial morphology and integrity, at least in part, via the involvement of BIK.
Department of Biomedical Sciences, James H Quillen College of Medicine, East Tennessee State University, PO Box 70582, Johnson City, TN, 37614, USA.
Suman
S
Dalal
Department of Biomedical Sciences, James H Quillen College of Medicine, East Tennessee State University, PO Box 70582, Johnson City, TN, 37614, USA.
Qinqin
Q
Zha
Department of Biomedical Sciences, James H Quillen College of Medicine, East Tennessee State University, PO Box 70582, Johnson City, TN, 37614, USA.
Mahipal
M
Singh
Department of Biomedical Sciences, James H Quillen College of Medicine, East Tennessee State University, PO Box 70582, Johnson City, TN, 37614, USA. singhk@etsu.edu.|Center for Inflammation, Infectious Disease and Immunity, East Tennessee State University, Johnson City, TN, 37614, USA. singhk@etsu.edu.|James H Quillen Veterans Affairs Medical Center, Johnson City, TN, 37614, USA. singhk@etsu.edu.
Krishna
K
Singh
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D005786:Gene Expression Regulation; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008929:Mitochondria, Heart / Q000235:genetics / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000473:pathology; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D053495:Osteopontin / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress; D051381:Rats; D017207:Rats, Sprague-Dawley
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C111741:Bik protein, mouse; D024101:Mitochondrial Proteins; C506896:Spp1 protein, mouse; C506897:Spp1 protein, rat; D053495:Osteopontin; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C422943:Nox4 protein, mouse
Apoptosis; BIK; Myocyte; Osteopontin; Reactive oxygen species