Fig 2: Altered mitochondrial structure and function in VDAC2-KO.a Representative transmission electron microscopy image at ×800, ×5000 and ×15,000 magnifications (scale bar, 10 μm, 1 μm, and 500 nm, respectively) (n = 3); b Number of mitochondria per unit area measured using Adobe Photoshop CC 2019 (p = 0.0365) (n = 18 images, N = 3); c Mitochondrial volume fraction measured using Adobe Photoshop CC 2019 using 22 × 14 grid (p = 0.0001) (n = 18 images, N = 3); d, e Western blot image of DRP1 and lane-loading control VINCULIN and quantification using Image Studio Lite (version 5.2.5) (p = ns) (n = 6); f Representative mitochondrial calcium uptake image produced using MATLAB (n = 3); g, h Mitochondrial calcium uptake in the first 30 s (p = ns) and 60 s of calcium pulse (p = 0.0300) (n = 3), respectively; i–k Western blot image of MCU (p = 0.0014) and NCLX (p = 0.0169) along with respective lane-loading control GAPDH and quantification using Image Studio Lite (version 5.2.5) (n = 6). p-value: unpaired two-tailed t-test performed in all comparisons. Data are represented as ±SEM.

Fig 3: Partial rescue of HF phenotype upon VDAC2 reintroduction.a Western blot image of VDAC2-GFP and lane-loading control VINCULIN and quantification using Image Studio Lite (version 5.2.5) (KO GFP vs. KO VDAC2-GFP p = 0.0568) (n = 3); b qRT-PCR of Vdac2 normalized to Vcl (Vinculin) (KO GFP vs. KO VDAC2-GFP p = 0.0044) (n = 3); c–g Serial echocardiography parameter including ejection fraction (EF) (week 6: WT GFP vs. KO GFP p = 0.0036, week 10: WT GFP vs. KO GFP p = 0.0008, WT VDAC2-GFP vs. KO VDAC2-GFP p = 0.0456, KO GFP vs. KO VDAC2-GFP p = 0.0359), fractional shortening (FS) (week 6: WT GFP vs. KO GFP p = 0.0091; week 10: WT GFP vs. KO GFP p = 0.0043, WT VDAC2-GFP vs. KO VDAC2-GFP p = 0.0652), left ventricular end diastolic diameter (LVEDD) (pre: WT GFP vs. KO GFP p = 0.0169, WT VDAC2-GFP vs. KO VDAC2-GFP p = 0.0407; week 10: WT GFP vs. KO GFP p = 0.0107, KO GFP vs. KO VDAC2-GFP p = 0.0444), end diastolic and end systolic volumes (EDV (pre: WT GFP vs. KO GFP p = 0.0032; week 10: WT GFP vs. KO GFP p = 0.0104, KO GFP vs. KO VDAC2-GFP p = 0.0378) and ESV (week6: WT GFP vs. KO GFP p = 0.03, week 10: WT GFP vs. KO GFP p = 0.0008, KO GFP vs. KO VDAC2-GFP p = 0.0066)) respectively (n = 3). p-value: unpaired t-test performed in all comparisons between KO GFP and KO VDAC2-GFP in panels a and b. Ordinary one-way ANOVA was performed for echocardiographic data and panel b. Data are represented as ±SEM.

Fig 4: 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 5: Altered action potential in VDAC2-KO cardiomyocytes.a Heart rate (HR), beats per minute (BPM) (p = 0.0005) (n = 6); b Representative ECG of 16-week-old mice (time frame, 100 ms); c Representative action potential image of 16-week-old mice cardiomyocyte (time frame, 100 ms); d, e Action potential repolarization duration at 50% and 90%, respectively (two-tailed Welch test: APD90 p = ns, APD50 p = 0.0148) (WT n = 10, KO n = 18, N = 3). p-value: unpaired two-tailed t-test and two-tailed Welch test performed. Data are represented as mean ± SEM.