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

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.

Keywords: VDAC1 knockout; Oxidative stress; Mitochondria-bound hexokinase II; Extracellular acidification; Cell death/apoptosis; Bax.

Fig. 1.

Generation and identification of VDACl^−/− H9c2 cells. (A) Rat VDACl genomic graph and the sites and sequences of two single guide (sg) RNAs in the rat genome. (B) Diagram of complex formation of sgRNA, T7 promoter, CRISPR guide RNA and Cas9 protein. (C) The gene mutation sequences in selected VDAC1^−/− H9c2 cell clones from sgRNA1 and 2. (D) Alignment of DNA sequencing traces of VDAC1^−/−C3 to WT H9c2 cells. Arrow in the box indicates a single nucleotide T deletion in the VDAC1^−/−C3 H9c2 cell clone. (E) Western blot analyses of selected WT and VDAC1^−/− H9c2 cell clones with VDAC1 specific and Tom-20 (loading control) antibodies. (F) Western blot analyses of selected WT and VDAC1^−/− H9c2 cell clones with VDAC2, VDAC3 and β-tubulin (loading control) antibodies.

Fig. 2.

Characteristics of mitochondrial morphology and mass of WT and VDAC1^−/− H9c2 cells. (A) Immunostaining of WT and VDAC1^−/−C1 H9c2 cells with COX IV antibody. (B) Histogram shows average values of two mitochondrial shape metrics, aspect ratio (left panel) and form factor (right panel) from 20 cells/group. * #P<0.05 vs. WT. (C) Histogram shows relative ratio of mtDNA Ct/β-actin DNA Ct detected by real time PCR from WT and VDAC1^−/−C1, C2 and C3 H9c2 cells. * P<0.05 vs. WT.

Fig. 3.

Characteristics of mitochondrial bioenergetics of WT and VDAC1^−/− H9c2 cells. (A) Cell mitochondrial stress test profile plot. (B) Traces of O₂ consumption rate (OCR) in WT and VDAC1^−/−C1, C2 and C3 H9c2 cells under normal conditions measured using the Seahorse XFp Analyzer. (C) Individual parameters for basal respiration, maximal respiration, ATP production and spare respiratory capacity for WT and VDAC1^−/−C1, C2 and C3 H9c2 cells. Data are expressed as means ± SEM, n = 8 wells from 2 independent experiments.

Fig. 4.

Cell viability and cell death induced by tBHP or ROT in WT and VDAC1^−/− H9c2 cells. (A) MTT assay (cell viability) of WT and VDAC1^−/− C1, C2 and C3 H9c2 cells after 20 h exposure with various concentrations of tBHP. (B) TUNEL assay of WT and VDAC1^−/−C1 H9c2 cells after 20 h exposure to various concentrations of tBHP. (C) Representative TUNEL staining (cell death) of WT and VDAC1^−/−C1 H9c2 cells after 20 h exposure to 125 μM of tBHP. Apoptotic nuclei were TUNEL stained (red) and counterstained with DAPI (blue) to label nuclei. The red and blue merged cells were counted as TUNEL positive cells. (D) LDH release from WT, VDAC1^−/−C1, C2 and C3 and VDAC1 restored in VDAC1^−/−C1 H9c2 cells after 3 h exposure to 50, 100 μM tBHP. (E) MTT assay of WT and VDAC1^−/−C1, C2 and C3 H9c2 cells viability after 48 h exposure to various concentrations of ROT. *P<0.05 vs WT, ^# P<0.05 vs VDAC1^−/−. Each experiment was repeated three times.

Fig. 5.

Assay of mitochondrial respiration in WT and VDAC1^−/−C1 H9c2 cells exposed to tBHP. (A) Traces of O₂ consumption rate (OCR) in WT and VDAC1^−/−C1 H9c2 cells with or without exposure to tBHP. (B) Summary of OCR in WT and VDAC1^−/−C1 H9c2 cells with or without exposure to tBHP. (C) Proton leak and (D) coupling efficiency in WT and VDAC1^−/−C1 H9c2 cells with or without exposure to tBHP. NS: not significant.

Fig. 6.

ROS generation in WT and VDAC1^−/− and VDAC1 restored VDAC1^−/−C1 H9c2 cells exposed to tBHP or rotenone with H₂ DCFDA staining. (A) WT, VDAC1^−/− C1, C2 and VDAC1^−/− C1+ restored VDAC1 H9c2 cell exposed to 50 μM tBHP. (B) WT, VDAC1^−/−C1 and VDAC1^−/−C1+ restored VDAC1 H9c2 cell exposed to 50 μM rotenone. *P<0.05 VS VDAC1^−/−C1+TBHP. Each experiment was repeated three times.

Fig. 7.

Effect of mitochondria-bound HKII on tBHP-induced cell death in WT and VDAC1^−/− H9c2 cells. (A) Western blot analysis of HKII level in WT and VDAC1^−/− H9c2 cells from total cell lysate. β-tubulin level was used as protein loading control. (B) Western blot analysis of mitochondrial HKII levels in WT and VDAC1^−/−H9c2 cells. (C) Western blot analysis of mitochondrial HKII levels in WT and VDAC1^−/−C1 H9c2 cells exposed to tBHP. (D) Western blot analysis of HKII level in mitochondrial and cytosolic fractions in WT, VDAC1^−/− C1 and VDAC1^−/−C1+ restored VDAC1 H9c2 cells. *P<0.05 vs. WT. Each experiment was repeated three times.

Fig. 8.

Glycolytic stress in WT and VDAC1^−/− H9c2 cells exposed to tBHP. Change in traces of OCR (A) and ECAR (B) in WT and VDAC1^−/−C1, C2 and C3 H9c2 cells without or with exposure to tBHP for 20 h. Data were expressed as means ± SEM, n = 8 wells from 2 independent experiments. *P<0.05 vs. WT+tBHP. (C) Total proton production rate (PPR_tot) and the respective contributions of PPR_resp and PPR_gly before (top panel) and after adding glucose (middle panel) and oligomycin (bottom panel) in WT and VDAC1^−/−C1 H9c2 cells without or with exposure to tBHP. *P<0.05 WT+tBHP vs. VDAC1^−/− +tBHP.

Fig. 9.

Effect of VDAC l^−/− on Bax association with mitochondria. Western blot analyses of Bax levels in mitochondria (A) and cytosol (B) isolated from WT and VDAC1^−/−C1 H9c2 cells treated with or without 20 μM of ROT for 20 h. COX IV, GAPDH and (β-tubulin were used as protein loading control and as mitochondrial and cytosol markers. *P<0.05 vs. WT non-ROT. #P<0.05 vs. VDAC1^−/−C1 non-ROT. Each experiment was repeated three times.