Faculty Collaboration Database

Knockout of VDAC1 in H9c2 Cells Promotes Oxidative Stress-Induced Cell Apoptosis through Decreased Mitochondrial Hexokinase II Binding and Enhanced Glycolytic Stress. Cell Physiol Biochem 2020 Sep 09;54(5):853-874

Date

09/10/2020

Pubmed ID

32901466

Pubmed Central ID

PMC7898235

DOI

10.33594/000000274

Scopus ID

2-s2.0-85090868984 (requires institutional sign-in at Scopus site)   10 Citations

Abstract

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-butylhydroperoxide (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 O₂ 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.

Author List

Yang M, Sun J, Stowe DF, Tajkhorshid E, Kwok WM, Camara AKS

Authors

Amadou K. Camara PhD Professor in the Anesthesiology department at Medical College of Wisconsin
David F. Stowe MD, PhD Professor in the Anesthesiology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Apoptosis
Cell Line
Cell Proliferation
Cell Survival
Gene Knockout Techniques
Glycolysis
Hexokinase
Mitochondria
Mitochondrial Membranes
Myocytes, Cardiac
Oxidative Stress
Rats
Signal Transduction
Voltage-Dependent Anion Channel 1
tert-Butylhydroperoxide