Cardiac-specific deletion of voltage dependent anion channel 2 leads to dilated cardiomyopathy by altering calcium homeostasis

Figures

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  Fig. 1. Cardiac-specific deletion of VDAC2 causes dilated cardiomyopathy and leads to increased mortality. a Western blot image of VDAC2 and lane-loading control VINCULIN and quantification in WT (Wild-type) and KO (Knock-out) using Image Studio Lite (version5.2.5) (p = 0.02) (n = 6); b Kaplan–Meier survival curve (n = 10) (p < 0.0001); c–j Echocardiography analysis of left ventricular ejection fraction (EF) (p = 0.000143), fractional shortening (FS) (0.000376), left ventricular end-diastolic and end-systolic volumes (EDV and ESV) (p = 0.0132 and p = 0.0311), left-ventricular posterior wall thickness at diastole and systole (LVPW,d and LVPW,s) (p = ns and p = 0.0244), left ventricular end-diastolic diameter (LVEDD) (8-week p = 0.004824; 12-week p = 0.007670 and 16-week p = 0.005986) (n = 6) and j left ventricular mass to body weight ratio (LVM/BW), respectively (n = 6); k Representative heart image of 16-week-old mice (scale bar, 5 mm); l Representative m-mode image from echocardiography (n = 1); m Differential expression of NPPA gene (n = 4); n Representative ventricular cardiomyocyte image using confocal microscopy (scale bar, 100 μm ); o, p Cardiomyocyte cell length (p < 0.0001) and…

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  Fig. 2. Altered gene and protein expression profiles indicate altered calcium signaling and regulation in VDAC2-KO. a, b Differential gene expression data derived from 16-week old cardiac ventricular tissue (n = 4); c–f Western blot image of PLN (p = 0.0042), pPLN (p = 0.0045), and NCX (p = 0.0121) along with lane-loading control GAPDH and respective quantification using Image Studio Lite (version 5.2.5) (n = 6); g, h Western blot image of PKA and pPKA and quantification using Image Studio Lite (version 5.2.5) (p = ns) (n = 6); i, j Western blot image of CASQ2 and lane-loading control GAPDH and respective quantification using Image Studio Lite (version 5.2.5) (p = 0.0111) (n = 6); k, l Western blot image of SERCA2 and lane-loading control VINCULIN and quantification using Image Studio Lite (version 5.2.5) (Mann–Whitney: p = 0.0260) (n = 6); m, n Western blot image of RYR2 and lane-loading control VINCULIN and quantification using Image Studio Lite (version 5.2.5) (p = 0.0018) (n = 6). p-value: unpaired two-tailed t-test and two-tailed Mann–Whitney test performed. Data are represented as mean ± SEM.

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  Fig. 3. Impaired calcium cycling in VDAC2-KO cardiomyocytes. a Representative calcium transient image of 16-week-old mice (Time frame, 500 ms); b Amplitude (F/F0) data (p < 0.0001) (WT n = 54, KO n = 38, N = 3); c Rate of calcium uptake (tau) (ms) (p = 0.0492) (WT n = 54, KO n = 38, N = 3); d Representative image of rate of calcium release of 16-week-old mice (Time frame, 1 s); e Rate of calcium release (F/F0/s) (p < 0.0001) (WT n = 34, KO n = 23, N = 3). p-value: unpaired two-tailed t-test performed in all comparisons. Data are represented as mean ± SEM.

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  Fig. 4. Cross-talk between mitochondria and ER/SR facilitates mitochondrial calcium cycling. a Representative co-immunoprecipitation image from a cardiac ventricular sample of 8-week-old mice (n = 3) in WT and KO (Input—non-IP sample; IgG—control antibody; IP—VDAC2 protein pull-down); b Representative proximity ligation assay image for VDAC2–SERCA2 and VDAC2–NCX1 interaction in 8-week-old mice (scale bar, 10 μm) (n = 3); c Representative immunofluorescence image from 16-week-old mice heart (scale bar, 10 μm) (n = 3).

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  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.

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  Fig. 6. 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.

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  Fig. 7. Increased cardiac fibrosis observed in VDAC2-KO. a Differential gene expression data from 16-week-old mice ventricular sample (n = 4); b KEGG pathway analysis of differentially expressed genes in KO indicates ECM production pathway as highest statistical significance (n = 4); c Representative Masson’s trichrome stain image (×0.5 magnification; scale bar, 2000 μm) (n = 3); d Fibrosis quantification using Aperio software and colocalization v9 algorithm (p = 0.0043) (n = 3); e Representative transmission electron microscopy image (scale bar, 5 μm) (n = 3); f Collagen quantification using ImageJ software (p = 0.0033) (n = 9 images, N = 3). p-value: unpaired two-tailed t-test performed in all comparisons. One-tail Fisher Exact test performed for KEGG pathway analysis. Data are represented as ±SEM.

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  Fig. 8. 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 =…

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  Fig. 9. Enhanced contractile force and accelerated relaxation speed in myocardial tissue upon VDAC2 activation. a Measurement of relative contractile force in organotypic myocardial slices from both Sham- (left panel) and TAC-operated mice (right panel) after superfusion with either 30 or 50 µM of the VDAC2 agonist efsevin. Each dot represents the quantification of twitch amplitudes over 20 s at indicated time points. The gray arrow indicates the start of efsevin superfusion (left panel: DMSO vs. 30 μM Efsevin: 10 min p = 0.016, 32 min p = 0.010, 37 min p = 0.0004, 42 min p = 0.0012 and 47 min p = 0.0062; DMSO vs. 50 μM Efsevin: 22 min p = 0.0007, 27–47 min p < 0.0001, respectively) (right panel: DMSO vs. 30 μM Efsevin: 17 min p = 0.0038, 22 min p = 0.0009, 27 min p = 0.00031, 32 min p = 0.00029, 37 min p = 0.00015, 42 min p = 0.00073; DMSO vs. 50 μM Efsevin: 22 min p = 0.0218, 27 min p = 0.0044, 32 min p = 0.0037, 37 min p = 0.0031, 42 min p = 0.0034). b Relaxation speed of twitches of myocardial slices shown in a (N = 8, n = 22 slices for TAC, N = 6, n = 14 slices for Sham) (left panel: DMSO vs. 30 μM Efsevin: 22 min p = 0.0072, 27 min p = 0.00019, 32 min p = 0.044, 37 min p = 0.0125,…