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Knockout of VDAC1 in H9c2 Cells Promotes Oxidative Stress-Induced Cell Apoptosis through Decreased Mitochondrial Hexokinase II Binding and Enhanced Glycolytic Stress

BACKGROUND/AIMS: The role of VDAC1, the most abundant mitochondrial outer membrane protein, in cell death depends on cell types and stimuli. Both silencing and upregulation of VDAC1 in various type of cancer cell lines can stimulate apoptosis. In contrast, in mouse embryonic stem (MES) cells and mou...

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Autores principales: Yang, Meiying, Sun, Jie, Stowe, David F., Tajkhorshid, Emad, Kwok, Wai-Meng, Camara, Amadou K. S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7898235/
https://www.ncbi.nlm.nih.gov/pubmed/32901466
http://dx.doi.org/10.33594/000000274
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author Yang, Meiying
Sun, Jie
Stowe, David F.
Tajkhorshid, Emad
Kwok, Wai-Meng
Camara, Amadou K. S.
author_facet Yang, Meiying
Sun, Jie
Stowe, David F.
Tajkhorshid, Emad
Kwok, Wai-Meng
Camara, Amadou K. S.
author_sort Yang, Meiying
collection PubMed
description BACKGROUND/AIMS: The role of VDAC1, the most abundant mitochondrial outer membrane protein, in cell death depends on cell types and stimuli. Both silencing and upregulation of VDAC1 in various type of cancer cell lines can stimulate apoptosis. In contrast, in mouse embryonic stem (MES) cells and mouse embryonic fibroblasts (MEFs), the roles of VDAC1 knockout (VDAC1(−/−)) in apoptotic cell death are contradictory. The contribution and underlying mechanism of VDAC1(−/−) in oxidative stress-induced cell death in cardiac cells has not been established. We hypothesized that VDAC1 is an essential regulator of oxidative stress-induced cell death in H9c2 cells. METHODS: We knocked out VDAC1 in this rat cardiomyoblast cell line with CRISPR-Cas9 genome editing technique to produce VDAC1(−/−) H9c2 cells, and determined if VDAC1 is critical in promoting cell death via oxidative stress induced by tert-butylhydroper-oxide (tBHP), an organic peroxide, or rotenone (ROT), an inhibitor of mitochondrial complex I by measuring cell viability with MTT assay, cell death with TUNEL stain and LDH release. The mitochondrial and glycolytic stress were examined by measuring O(2) consumption rate (OCR) and extracellular acidification rate (ECAR) with a Seahorse XFp analyzer. RESULTS: We found that under control conditions, VDAC1(−/−) did not affect H9c2 cell proliferation or mitochondrial respiration. However, compared to the wildtype (WT) cells, exposure to either tBHP or ROT enhanced the production of ROS, ECAR, and the proton (H(+)) production rate (PPR) from glycolysis, as well as promoted apoptotic cell death in VDAC1(−/−) H9c2 cells. VDAC1(−/−) H9c2 cells also exhibited markedly reduced mitochondria-bound hexokinase II (HKII) and Bax. Restoration of VDAC1 in VDAC1(−/−) H9c2 cells reinstated mitochondria-bound HKII and concomitantly decreased tBHP and ROT-induced ROS production and cell death. Interestingly, mitochondrial respiration remained the same after tBHP treatment in VDAC1(−/−) and WT H9c2 cells. CONCLUSION: Our results suggest that VDAC1(−/−) in H9c2 cells enhances oxidative stress-mediated cell apoptosis that is directly linked to the reduction of mitochondria-bound HKII and concomitantly associated with enhanced ROS production, ECAR, and PPR.
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spelling pubmed-78982352021-09-09 Knockout of VDAC1 in H9c2 Cells Promotes Oxidative Stress-Induced Cell Apoptosis through Decreased Mitochondrial Hexokinase II Binding and Enhanced Glycolytic Stress Yang, Meiying Sun, Jie Stowe, David F. Tajkhorshid, Emad Kwok, Wai-Meng Camara, Amadou K. S. Cell Physiol Biochem Article BACKGROUND/AIMS: The role of VDAC1, the most abundant mitochondrial outer membrane protein, in cell death depends on cell types and stimuli. Both silencing and upregulation of VDAC1 in various type of cancer cell lines can stimulate apoptosis. In contrast, in mouse embryonic stem (MES) cells and mouse embryonic fibroblasts (MEFs), the roles of VDAC1 knockout (VDAC1(−/−)) in apoptotic cell death are contradictory. The contribution and underlying mechanism of VDAC1(−/−) in oxidative stress-induced cell death in cardiac cells has not been established. We hypothesized that VDAC1 is an essential regulator of oxidative stress-induced cell death in H9c2 cells. METHODS: We knocked out VDAC1 in this rat cardiomyoblast cell line with CRISPR-Cas9 genome editing technique to produce VDAC1(−/−) H9c2 cells, and determined if VDAC1 is critical in promoting cell death via oxidative stress induced by tert-butylhydroper-oxide (tBHP), an organic peroxide, or rotenone (ROT), an inhibitor of mitochondrial complex I by measuring cell viability with MTT assay, cell death with TUNEL stain and LDH release. The mitochondrial and glycolytic stress were examined by measuring O(2) consumption rate (OCR) and extracellular acidification rate (ECAR) with a Seahorse XFp analyzer. RESULTS: We found that under control conditions, VDAC1(−/−) did not affect H9c2 cell proliferation or mitochondrial respiration. However, compared to the wildtype (WT) cells, exposure to either tBHP or ROT enhanced the production of ROS, ECAR, and the proton (H(+)) production rate (PPR) from glycolysis, as well as promoted apoptotic cell death in VDAC1(−/−) H9c2 cells. VDAC1(−/−) H9c2 cells also exhibited markedly reduced mitochondria-bound hexokinase II (HKII) and Bax. Restoration of VDAC1 in VDAC1(−/−) H9c2 cells reinstated mitochondria-bound HKII and concomitantly decreased tBHP and ROT-induced ROS production and cell death. Interestingly, mitochondrial respiration remained the same after tBHP treatment in VDAC1(−/−) and WT H9c2 cells. CONCLUSION: Our results suggest that VDAC1(−/−) in H9c2 cells enhances oxidative stress-mediated cell apoptosis that is directly linked to the reduction of mitochondria-bound HKII and concomitantly associated with enhanced ROS production, ECAR, and PPR. 2020-09-09 /pmc/articles/PMC7898235/ /pubmed/32901466 http://dx.doi.org/10.33594/000000274 Text en http://creativecommons.org/licenses/by/4.0/ This article is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND). Usage and distribution for commercial purposes as well as any distribution of modified material requires written permission.
spellingShingle Article
Yang, Meiying
Sun, Jie
Stowe, David F.
Tajkhorshid, Emad
Kwok, Wai-Meng
Camara, Amadou K. S.
Knockout of VDAC1 in H9c2 Cells Promotes Oxidative Stress-Induced Cell Apoptosis through Decreased Mitochondrial Hexokinase II Binding and Enhanced Glycolytic Stress
title Knockout of VDAC1 in H9c2 Cells Promotes Oxidative Stress-Induced Cell Apoptosis through Decreased Mitochondrial Hexokinase II Binding and Enhanced Glycolytic Stress
title_full Knockout of VDAC1 in H9c2 Cells Promotes Oxidative Stress-Induced Cell Apoptosis through Decreased Mitochondrial Hexokinase II Binding and Enhanced Glycolytic Stress
title_fullStr Knockout of VDAC1 in H9c2 Cells Promotes Oxidative Stress-Induced Cell Apoptosis through Decreased Mitochondrial Hexokinase II Binding and Enhanced Glycolytic Stress
title_full_unstemmed Knockout of VDAC1 in H9c2 Cells Promotes Oxidative Stress-Induced Cell Apoptosis through Decreased Mitochondrial Hexokinase II Binding and Enhanced Glycolytic Stress
title_short Knockout of VDAC1 in H9c2 Cells Promotes Oxidative Stress-Induced Cell Apoptosis through Decreased Mitochondrial Hexokinase II Binding and Enhanced Glycolytic Stress
title_sort knockout of vdac1 in h9c2 cells promotes oxidative stress-induced cell apoptosis through decreased mitochondrial hexokinase ii binding and enhanced glycolytic stress
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7898235/
https://www.ncbi.nlm.nih.gov/pubmed/32901466
http://dx.doi.org/10.33594/000000274
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