VDAC1 in the diseased myocardium and the effect of VDAC1-interacting compound on atrial fibrosis induced by hyperaldosteronism

Figures

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  Figure 1. Overexpression of VDAC1 in human cardiac pathologies. (a,b) VDAC1 IHC staining in the left ventricle (LV) of patients post myocardial infraction (MI) relative to hearts without known cardiac disease. IHC staining presented as fold change relative to the mean intensity of the control samples. “MI short-term” represents samples defined as acute MI or granulation tissue. “MI long-term” represents the samples in which clinical data indicated the presence of scar tissue. Only non-infarcted zones with viable myocardium were analyzed. (b) Representative photomicrographs for each condition. Scale bars, 50 µm and enlargement of 20 µm. (b) Summarizing scatter plot of quantitative image processing analysis. Note gradual increase in VDAC1 expression levels between MI long-term and MI short-term. Also note, punctuated staining suggesting mitochondrial localization of VDAC1. (c,d) VDAC1 IHC staining in the LV of patients with cardiac diseases involving dilatation\hypertrophy. HF—Heart failure, TR—tricuspid regurgitation, RV—Right ventricle, CIHD—Chronic ischemic heart disease, MR—Mitral regurgitation. (c) Representative photomicrographs for each condition. Scale bars, 20 µm. (d) Summarizing…

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  Figure 2. The overexpression of VDAC1 in human cardiac pathologies does not signify changes in mitochondrial mass. Co-immunofluorescent staining of the human cardiac tissue arrays with a VDAC1 specific antibody (ab186321, Table S4) as well as an antibody for citrate synthase (CS) to quantify mitochondrial mass/amount. (a,b) analysis of the same MI samples as in Fig. 1a,b. (a) Representative photomicrographs for each condition demonstrating staining for VDAC1 (green), CS (red) and both (Merged) . Scale bars, 50 µm. (b) Summarizing scatter plot of quantitative image processing analysis. (c,d) analysis of the diseased LV samples as in Fig. 1c,d. (c) Representative photomicrographs for each condition demonstrating staining for VDAC1 (green), CS (red) and both (Merged) . Scale bars, 50 µm. (d) Summarizing scatter plot of quantitative image processing analysis.

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  Figure 3. Overexpression of VDAC1 in the LV of rats post MI. (a,b) Quantitative analysis of myocardial fibrosis in the LV of sham and MI rats using Masson’s trichrome (MT) staining. (a) Representative photomicrographs for each condition. Scale bars, 50 µm and enlargement of 20 µm. (b) Summarizing scatter plot of quantitative image processing analysis. Note, increased LV fibrosis in MI rats relative to sham, as expected. (c,d) VDAC1 IHC staining in the LV of MI and sham rats. IHC staining presented as fold change (%) relative to the mean intensity of the sham preparations. In the MI rats, only non-infarcted zones of the LV were analyzed. (c) Representative photomicrographs for each condition. Scale bars, 50 µm and enlargement of 20 µm. (d) Summarizing scatter plot of quantitative image processing analysis. Note increased expression of VDAC1 in LV of MI rats. The number of rats in each group is indicated (n = 6–10).

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  Figure 4. Overexpression of VDAC1 in the LA of rats post MI. (a,b) Analysis of LA fibrosis (similar representation as in Fig. 2a,b). Note prominent increase of LA fibrosis in MI rats. (c,d) Analysis of VDAC-1 in the LA (similar representation as in (c,d). Note increased expression of VDAC1 in LA of MI rats. The number of rats in each group is indicated (n = 6–11).

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  Figure 5. Atrial structural remodeling in rats exposed to excessive Aldo levels. (a,b) MT staining and its quantitative analysis of myocardial fibrosis in the LA of sham and Aldo rats. (a) Representative photomicrographs for each condition. Scale bars, 50 µm and enlargement of 20 µm. (b) Summarizing scatter plot of quantitative image processing analysis. Note increased LA fibrosis in Aldo rats. (c,d) IF staining of the expression of α-SMA in the atrial tissues of the sham and Aldo rats using IF staining. Two fields were analyzed from each rat. n represents the total number fields. (c): Representative photomicrographs for each condition. Scale bars, 50 µm (d) Summarizing scatter plot of quantitative image processing analysis. As expected, increased expression of α-SMA was noted in the Aldo treated atria in accordance with the elevated fibrotic load. (e,f) TUNEL analysis, performed by counting the total number of TUNEL-positive cells in each LA section normalized to its surface area (e) Representative photomicrographs for each condition. Scale bars, 50 µm. (f): Summarizing dot plot of quantitative image processing analysis. Note increased apoptosis in the LA of Aldo rats. The number of rats…

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  Figure 6. Increased atrial expression of VDAC1 in rats exposed to excessive Aldo levels. (a,b) VDAC1 expression in the LA evaluated using IHC staining. (a) Representative photomicrographs for each condition. (b) Summarizing scatter plot of quantitative image processing analysis. Note increased expression of VDAC1 in LA of Aldo rats relative to sham. (c,d) VDAC1 and Cyto c expression in the LA analyzed using IF staining (c). (d) Quantitative analysis of the IF data. The number of rats in each group is indicated (n = 6).

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  Figure 7. Attenuating effect of VBIT-4 on atrial fibrosis induced by hyperaldosteronism. (a) Schematic outline of the interventional study of VBIT-4 (see “Methods” section for technical details of the experimental protocol). (b,c) MT staining and quantitative analysis of myocardial fibrosis in the LA of Aldo and Aldo + VBIT-4-treated rats. (b) Representative photomicrographs for each condition. Scale bars, 50 µm and enlargement 20 µm. (c) Summarizing scatter plot of quantitative image processing analysis. Note a decrease in LA fibrotic area following VBIT-4 treatment. (d,e) IF staining of α-SMA expression in the atrial tissues of the sham and Aldo rats. (d) Representative photomicrographs for each condition. Scale bars, 5 µm. (e): Quantitative analysis of image expressing α-SMA. (f,g) TUNEL staining analysis (as in Fig. 4) in LA tissues of Aldo and Aldo + VBIT-4 treated rats. (f) Representative photomicrographs for each condition. Scale bars, 50 µm. (g) Summarizing scatter plot of quantitative image processing analysis. (h,i) VDAC1 IHC staining intensity in the LA. (h) Representative photomicrographs for each condition. Scale bars, 50 µm and enlargement 20 µm. (i) Summarizing scatter plot…