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Osteopontin-stimulated apoptosis in cardiac myocytes involves oxidative stress and mitochondrial death pathway: role of a pro-apoptotic protein BIK

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Abstract

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.

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Abbreviations

OPN:

Osteopontin

ARVMs:

Adult rat ventricular myocytes

ROS:

Reactive oxygen species

NADPH oxidase:

Nicotinamide adenine dinucleotide phosphate-oxidase

NOX:

NADPH oxidase

NAC:

N-acetyl-l-cysteine

SOD-2:

Mitochondrial superoxide dismutase-2

DMEM:

Dulbecco’s modified eagle medium

APO:

Apocynin

GLIB:

Glibenclamide

5-HD:

5-hydroxydecanoate

H2DCFDA:

2′,7′-dichlorohydrofluorescein diacetate

mitoKATP :

Mitochondrial KATP channels

MnTBAP:

Manganese (III) tetrakis (4-benzoic acid) porphyrin

CTL:

Control

MAM:

Mitochondrial-associated endoplasmic reticulum membrane

MHC-OPN:

OPN transgenic mice

Mut BIK:

Mutant BIK

ER:

Endoplasmic reticulum

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Acknowledgments

The authors appreciate the technical help received from Dr. Dennis Defoe, Dr. Robert V. Schoborg, Mr. Rolf Fritz, Ms. Jennifer Kintner, and Ms. Barbara A. Connelly.

Funding

This work was supported by Merit Review awards (BX002332 and BX000640) from the Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development, National Institutes of Health (R15HL129140), and funds from Institutional Research and Improvement account (to KS).

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Authors and Affiliations

  1. Department of Biomedical Sciences, James H Quillen College of Medicine, East Tennessee State University, PO Box 70582, Johnson City, TN, 37614, USA

    Suman Dalal, Qinqin Zha, Mahipal Singh & Krishna Singh

  2. Center for Inflammation, Infectious Disease and Immunity, East Tennessee State University, Johnson City, TN, 37614, USA

    Krishna Singh

  3. James H Quillen Veterans Affairs Medical Center, Johnson City, TN, 37614, USA

    Krishna Singh

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  1. Suman Dalal
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  2. Qinqin Zha
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Correspondence to Krishna Singh.

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Dalal, S., Zha, Q., Singh, M. et al. Osteopontin-stimulated apoptosis in cardiac myocytes involves oxidative stress and mitochondrial death pathway: role of a pro-apoptotic protein BIK. Mol Cell Biochem 418, 1–11 (2016). https://doi.org/10.1007/s11010-016-2725-y

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