Knockout of AMPKα2 Blocked the Protection of Sestrin2 Overexpression Against Cardiac Hypertrophy Induced by Pressure Overload

Abstract

Objectives: Sestrin2 (Sesn2) has been demonstrated to be a cysteine sulfinyl reductase and protects cells from multiple stress insults, including hypoxia, endoplasmic reticulum stress, and oxidative stress. However, the roles and mechanisms of Sesn2 in pressure overload-induced mouse cardiac hypertrophy have not been clearly clarified. This study intended to investigate whether sestrin2 (Sesn2) overexpression could prevent pressure overload-induced cardiac hypertrophy via an AMPKα2 dependent pathway through conditional knockout of AMPKα2. Methods and results: Sesn2 expression was significantly increased in mice hearts at 2 and 4 weeks after aortic banding (AB) surgery, but decreased to 60-70% of the baseline at 8 weeks. Sesn2 overexpression (at 3, 6, and 9 folds) showed little cardiac genetic toxicity in transgenic mice. Cardiac dysfunctions induced by pressure overload were attenuated by cardiomyocyte-specific Sesn2 overexpression when measured by echocardiography and hemodynamic analysis. Results of HE and PSR staining showed that Sesn2 overexpression significantly alleviated cardiac hypertrophy and fibrosis in mice hearts induced by pressure overload. Meanwhile, adenovirus-mediated-Sesn2 overexpression markedly suppressed angiotensin II-induced neonatal rat cardiomyocyte hypertrophy in vitro. Mechanistically, Sesn2 overexpression increased AMPKα2 phosphorylation but inhibited mTORC1 phosphorylation. The cardiac protections of Sesn2 overexpression were also via regulating oxidative stress by enhancing Nrf2/HO-1 signaling, restoring SOD activity, and suppressing NADPH activity. Particularly, we first proved the vital role of AMPKα2 in the regulation of Sesn2 with AMPKα2 knockout (AMPKα2-/-) mice and Sesn2 transgenic mice crossed with AMPKα2-/-, since Sesn2 overexpression failed to improve cardiac function, inhibit cardiac hypertrophy and fibrosis, and attenuate oxidative stress after AMPKα2 knockout. Conclusion: This study uniquely revealed that Sesn2 overexpression showed little genetic toxicity in mice hearts and inhibited mTORC1 activation and oxidative stress to protect against pressure overload-induced cardiac hypertrophy in an AMPKα2 dependent pathway. Thus, interventions through promoting Sesn2 expression might be a potential strategy for treating pathological cardiac hypertrophy and heart failure.

Keywords: AMPKα; Sestrin2; cardiac hypertrophy; fibrosis; oxidative stress.

FIGURE 1

The expression of Sesn2 in hypertrophic hearts and Ang II-induced cardiomyocytes. (A) Representative immunoblots and quantitative results of Sesn2 protein expression in mice hearts at 2, 4, and 8 weeks after AB surgery (n = 6). (B) qPCR analysis of the mRNA levels of Sesn2 at 2, 4, and 8 weeks after AB surgery (n = 6). (C) Representative Western blots and statistical analysis of Sesn2 expression in NRCMs at 12, 24, and 48 h after Ang II treatment (n = 6). (D) Sesn2 mRNA expression levels in NRCMs treated with or without Ang II stimulation (n = 6). (E) Representative Western blots and statistical analysis of Sesn2 expression in the hearts of WT and TG mice, respectively (n = 6). (F) Sesn2 mRNA expression in WT and TG mice hearts (n = 6). The data were expressed as the mean ± SD.*p < 0.05 vs. indicated group. One-way analysis of variance (ANOVA) followed by Tukey post hoc tests were used for significance analysis.

FIGURE 2

Cardiomyocyte-specific overexpression of Sesn2 attenuated pressure overload-induced cardiac dysfunction. (A) Representative echocardiographic images in each group (n = 6). Echocardiography and pressure-volume loop were performed to evaluate mouse cardiac function at 8 weeks after AB surgery (n = 6). (B) heart rate (HR), (C) left ventricular end diastolic diameter (LVEDd), (D) left ventricular end systolic diameter (LVEDs), (E) left ventricular ejection fraction (EF), (F) LV fraction shortening (FS), (G) end diastolic pressure (EDP), (H) end systolic pressure (ESP), (I) end diastolic volume (EDV), (J) end systolic volume (ESV), (K) left ventricular maximal rate of pressure rise (dp/dt max), (L) left ventricular maximal rate of pressure decay (dp/dt min). Data were presented as the mean ± SD. *p < 0.05 vs indicated group. One-way analysis of variance (ANOVA) followed by Tukey post hoc tests were used for significance analysis.

FIGURE 3

Sesn2 overexpression alleviated pressure overload-induced hypertrophy and fibrosis in mice. (A) Representative images of gross hearts and HE staining in the indicated groups (n = 6). (B) Quantification of the cross-sectional area (CSA) of cardiomyocytes in the indicated mice (n ≥ 100 left ventricular cells). Statistical results of the (C) HW/BW, (D) HW/TL (E) and LW/BW of mice at 8 weeks after AB surgery(n = 9). Cardiac mRNA levels of. (F) ANP, (G) BNP, (H) α-MHC, and (I) β-MHC. (J) Picrosirius red (PSR) staining of histological sections of left ventricles (n = 6), (K) quantitative results of the left ventricular collagen volume (n ≥ 25 fields). L-O The mRNA levels of (L) Collagen I, (M) CTGF, (N) α-SMA, and (O) FN. ANP: atrial natriuretic peptide; BNP, brain natriuretic peptide; α-MHC, α-myosin heavy chain; β-MHC, β-myosin heavy chain; HW, heart weight; BW, body weight; TL, tibia length; HE, hematoxylin and eosin; CTGF, connective tissue growth factor; FN, fibronectin; αSMA, alpha-smooth muscle actin (αSMA). The data were expressed as the mean ± SD. *p < 0.05 vs. indicated group. One-way analysis of variance (ANOVA) followed by Tukey post hoc tests were used for significance analysis.

FIGURE 4

Sesn2 overexpression activated the AMPKα2 pathway and suppressed oxidative stress. (A) Representative Western blots of the phosphorylated and total protein expression of AMPKα2, mTORC1, ACC, P70s6k, P67, NOX2, Nrf2, HO-1, SOD1, and SOD2 at 8 weeks after AB surgery (n = 6). (B,C) Quantification of the proteins. (D) Examination of SOD activity (n = 6), (E) Examination of NADPH activity (n = 6), (F) Examination of MDA accumulation (n = 6). (G). Immunohistochemical staining for 4-hydroxynonenal (4-HNE). *p < 0.05 vs. indicated group. One-way analysis of variance (ANOVA) followed by Tukey post hoc tests were used for significance analysis.

FIGURE 5

Sesn2 overexpression alleviates Ang II- induced cardiomyocyte hypertrophy in vitro. NRCMs were transfected with adenovirus (Ad)-Sesn2 or Ad-GFP for 24 h, and then treated with Ang II for another 48 h. (A) Examination of Sesn2 overexpression in cardiomyocytes (n = 6). (B,C) Representative images of α-actinin staining and statistical results of cell surface area (n > 50 cells per group). mRNA expression of (D) ANP, (E) α-MHC and (F) β-MHC. (G) Representative Western blots of the phosphorylated and total proteins of AMPKα2, mTORC1, ACC, P67, NOX2, Nrf2, HO-1, SOD1, and SOD2 in NRCMs transfected with Ad-Sesn2 or Ad-GFP for 24 h and treated with Ang II for 48 h (n = 6). (H) Quantitative results of the Western blotting analysis. Examination of (I) SOD and (J) NADPH activities transfected with Ad-Sesn2 or Ad-GFP for 24 h and treated with Ang II for 48 h (n = 6). (K,L) DCFH-DA staining and ROS calculation in NRCMs transfected with Ad-Sesn2 or Ad-GFP and treated with Ang II for 48 h (n = 6). NRCM: neonatal rat cardiomyocytes. DCFH-DA: DCFH-DA; 2′,7′-dichlorodihydrofluorescein diacetate. The data were expressed as the mean ± SD from 3 independent experiments. *p < 0.05 vs. indicated group. One-way analysis of variance (ANOVA) followed by Tukey post hoc tests were used for significance analysis.

FIGURE 6

Sesn2 overexpression could not protect against cardiac remodeling in AMPKα2 knockout mice. (A) Representative Western blots of Sesn2 and AMPKα2 (n = 6). Echocardiography and pressure-volume loop parameters detected mouse cardiac function at 8 weeks after sham or AB surgery (n = 6): (B) LVEDd, (C) LVEDs, (D) EF, (E) FS, (F) dp/dtmax and (G) dp/dtmin. The ratios of (H) HW/BW, (I) HW/TL and (J) LW/BW at 8 weeks after sham or AB surgery (n = 9). (K) Representative images of gross hearts and HE-stained heart sections (n = 6). (L) Quantitative measurements of cell surface area (CSA) of cardiomyocytes (n = 60) (n ≥ 100 left ventricular cells). (M) RT-PCR detected ANP, BNP and βMHC mRNA expression (n = 6). (N) Representative images of PSR staining (n = 6). (O) Quantitative results of the left ventricular collagen volume (n = 6). (P) RT-PCR detected FN, Collagen I, CTGF, and α-SMA mRNA expression. The data were expressed as the mean ± SD. *p < 0.05 vs. indicated group. One-way analysis of variance (ANOVA) followed by Tukey post hoc tests were used for significance analysis.