Mitochondria-resident SBK3 confers protection against pressure overload-induced heart failure in mice

Figures

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  Figure 1 The mitochondrial protein SBK3 is downregulated following Ang II administration (A) The expression of SBK3 dynamically fluctuated following Ang II treatment and was downregulated after 24 h in adult rat cardiomyocytes (ARCMs), as detected by western blot analysis (n = 3). (B) SBK3 was successfully detected by western blot analysis in the mitochondria of rat cardiomyocytes but not in the cytoplasm (n = 3). (C,D) AD-SBK3 was successfully transfected into ARCMs, as evidenced by (C) fluorescence microscopy (scale bar = 100 μm) and (D) western blot analysis. (E) SBK3 was localized in the mitochondria of ARCMs, as detected by immunofluorescence staining; scale bar = 25 μm.

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  Figure 2 Overexpression of SBK3 suppresses Ang II-induced hypertrophy in rat cardiomyocytes (A) Representative images and (B) quantitative analysis of ANP and BNP protein expressions detected by western blot analysis in adult rat cardiomyocytes after treatment with 5 μM Ang II for 24 h (n = 4). Ca2+ transients were visualized via confocal microscopy. (C) Representative images of peak values, valley values, and line scans of Ca2+ fluorescence intensity are shown from left to right; scale bar = 50 μm. (D) Effects of SBK3 overexpression on the peak Ca2+ fluorescence intensity (△Peak/F0) and decay time of ARCMs induced by Ang II. (E) Schematic representation of the fluorescence intensity (F/F0) vs time (s) in each experimental group. Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant; one-way ANOVA test, followed by the Student-Newman-Keuls test.

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  Figure 3 SBK3 knockdown aggravates Ang II-evoked hypertrophy in rat cardiomyocytes (A) Fluorescence microscopy was used to detect the effect of Lenti-SBK3-RNAi in neonatal rat cardiomyocytes (scale bar = 100 μm). (B) The content of SBK3 in rat cardiomyocytes transfected with Lenti-SBK3-RNAi was detected by western blot analysis, n = 3. (C,D) Effects of SBK3 knockdown on the mRNA levels of ANP and BNP in angiotensin II-evoked cardiomyocytes were detected by RT-PCR, n = 4. (E) Hypertrophic areas of cardiomyocytes in NRCMs were detected by immunofluorescence staining (phalloidin staining, scale bar = 50 μm). (F) Quantitative analysis of the cardiomyocyte surface area. The average value of 10 cardiomyocytes in each visual field was taken as independent data. In total, 9 fields of view were taken from each group, and three parallel experiments were performed, n = 27. Rat cardiomyocytes were treated with 1 μM Ang II for 24 h. Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant; one-way ANOVA test, followed by the Student-Newman-Keuls test.

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  Figure 4 Overexpression of SBK3 in the heart via the AAV9 vector reduces mortality in mice subjected to TAC (A–C) AAV9-SBK3 was successfully expressed in mouse hearts, as determined by (A) immunofluorescence staining of tissues, (B) qPCR analysis (n = 3) and (C) western blot analysis. (D) A schematic diagram of the experimental program. (E) Kaplan-Meier survival analysis was conducted to evaluate the impact of SBK3 overexpression on the survival rate of TAC mice. (F) The actual number of living mice, n = 9–11; the AAV9-Flag + TAC group was supplemented with three additional mice due to high mortality. However, these additional deaths were not included in the overall death statistics. Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ns: not significant; one-way ANOVA test or Kaplan-Meier survival analysis.

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  Figure 5 Overexpression of SBK3 in the heart restores cardiac function in mice subjected to TAC (A) Representative images of M-mode echocardiography in mice. (B–G) Mice underwent M-mode echocardiography every two weeks to evaluate (B,C) left ventricular posterior wall (LVPW) thickness, (D,E) interventricular septal (IVS) thickening at the diastole and systole, (F) ejection fraction (EF), and (G) fractional shortening (FS). Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant; two-way ANOVA.

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  Figure 6 Overexpression of SBK3 in the heart suppresses cardiac hypertrophy in mice subjected to TAC (A–E) Overexpression of SBK3 in the heart inhibited TAC-induced hypertrophy, as evidenced by (A) representative photographs of mouse hearts (scale bar = 1 mm), (B) representative images of the cross-sectional area of cardiomyocytes (scale bar = 50 μm) and (C) quantitative analysis, (D) HW/BW ratio, LVW/BW, and mRNA expression of (E) ANP and BNP in mice challenged with Ang II (1000 ng/kg/min). n = 8–10 mice per group. Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant; one-way ANOVA test, followed by the Newman-Keuls test.

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  Figure 7 Overexpression of SBK3 in the heart suppresses myocardial fibrosis in mice subjected to TAC (A–F) Overexpression of SBK3 in the heart inhibited TAC-induced myocardial fibrosis, as evidenced by (A,B) Masson’s trichrome staining (scale bar = 50 μm) and quantitative analysis of the mRNA expressions of (C) α-SMA and (D) Colla-1 and the levels of (E) CK and (F) LDH; n = 8–10 mice per group. Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant; one-way ANOVA test, followed by the Newman-Keuls test.

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  Figure 8 AAV9-SBK3 is beneficial for maintaining the morphology and function of mitochondria in mice subjected to TAC (A) Representative mitochondrial transmission electron microscopy images of cardiac tissue from each mouse group. (B–G) Mitochondrial respiratory chain complexes I–V (B: representative images of western blot analysis, C: quantitative analysis), OPA1, Mfn1, Mfn2 (D: representative images, E: quantitative analysis), and Drp1, p-Drp1 (F: representative images, G: quantitative analysis) were rebalanced following SBK3 overexpression in mice subjected to TAC. NADH-Q oxidoreductase (complex I), succinate-Q oxidoreductase (complex II), UQ-cytochrome C oxidoreductase (complex III), ATP synthetase (complex V). n = 8–10 mice per group. Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant; one-way ANOVA test followed by the Newman-Keuls test.