LCZ696 Ameliorates Oxidative Stress and Pressure Overload-Induced Pathological Cardiac Remodeling by Regulating the Sirt3/MnSOD Pathway

Figures

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  Figure 1. LCZ696 improved cardiac function in pressure overload-induced cardiac hypertrophy. (a) Representative images of echocardiography for sham or TAC mice treated with vehicle or LCZ696 for 4 weeks. (b) Cardiac function as determined by echocardiography (n = 6 mice per group). (c) Statistics analysis of HW/BW, HW/TL, and LW/TL for different groups (n = 6 mice per group). (d) Anatomical view of the whole heart in different groups under a dissecting microscope and representative images of cross-sectional HE staining. Data are presented as mean ± SEM. ∗∗∗p < 0.001 vs. sham+vehicle group, #p < 0.05 vs. TAC+vehicle group. BW: body weight; HW: heart weight; LW: lung weight; TL: tibia length; HE: hematoxylin-eosin; LVEF: left ventricular ejection fraction; LVFS: left ventricular fractional shortening; LVIDd: left ventricular end-diastolic diameter; LVIDs: left ventricular end-systolic diameter; TAC: transverse aortic constriction.

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  Figure 2. LCZ696 treatment mitigated TAC-induced pathological cardiac hypertrophy. (a) Representative cross-sectional HE staining images and WGA staining images for the sham or TAC mouse hearts treated with vehicle or LCZ696 for 4 weeks. (b) Statistics analysis of cross-sectional areas of cardiomyocytes based on HE staining (n > 100 cells per group). (c) mRNA levels of ANP and BNP in different treatment groups. mRNA levels are normalized to the GAPDH mRNA level and converted to fold change relative to the sham+vehicle group (n = 6 mice per group). Data are presented as mean ± SEM. ∗∗∗p < 0.001 vs. the sham+vehicle group, #p < 0.05 vs. the TAC+vehicle group. ANP: atrial natriuretic peptide; BNP: brain natriuretic peptide; WGA: wheat germ agglutinin; β-MHC: myosin heavy chain β.

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  Figure 3. LCZ696 treatment attenuated TAC-induced cardiac fibrosis. (a) Representative images of Masson trichrome staining in different treatment groups. (b) Representative images of Picric Sirius red (PSR) staining of cardiac interstitial and perivascular regions in different treatment groups. (c) Representative immunohistochemical staining of α-SMA for myofibroblasts. (d) Quantification of the percentage of left ventricular fibrosis area from (b) (n = 6 mice per group). (e) mRNA levels of collagen I, collagen III, TGF-β, and CTGF in the indicated groups (n = 6 mice per group). The results are normalized against GAPDH and converted to fold change relative to the sham+vehicle group (n = 6 mice per group). Data are presented as mean ± SEM. ∗∗∗p < 0.001 vs. the sham+vehicle group, #p < 0.05 vs. the TAC+vehicle group.

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  Figure 4. LCZ696 treatment mitigated TAC-induced oxidative stress. (a) Representative immunohistochemical staining images of nitrotyrosine (NT) performed to assess myocardial nitrotyrosine production in different groups. (b) Representative immunohistochemical staining images of 4 hydroxynonenal (4-HNE) performed to assess myocardial lipid peroxidation. (c) Representative immunofluorescence staining images of dihydroethidium (DHE) performed to assess myocardial ROS accumulation.

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  Figure 5. LCZ696 treatment alleviated TAC-induced myocardial apoptosis. (a) Representative TUNEL staining images performed to assess myocardial apoptosis in mouse hearts in different groups. (b, c) Western blot analysis and quantitative analysis of Bcl-2 and Bax protein levels in different cardiac homogenates. (d) The protein expression of Sirt3 and MnSOD levels as detected by Western blot. (e) Histograms showing the quantitative expression of Sirt3 and MnSOD, n = 3. (f) Expression levels of Sirt3 and MnSOD in hearts from different groups. (g) Histogram of the quantitative analysis of the data in (f), after normalization to the expression of GAPDH, n = 3. (h) Western blot analysis of p-AMPK and AMPK protein levels in different cardiac homogenates. (i) Quantitative analysis of the ratio of p-AMPK/AMPK, n = 3. ∗p < 0.05 vs. the sham+vehicle group, ∗∗∗p < 0.001 vs. the sham+vehicle group, #p < 0.05 vs. the TAC+vehicle group.

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  Figure 6. LCZ696 blocked the hypertrophic response in NRCMs. (a) Immunofluorescence staining of sarcomeric alpha-actinin (α-actinin) in primary neonatal rat cardiomyocytes (NRCMs) in the indicated treatment groups. (b) The cell surface area of the indicated groups (n > 50 cells per experimental group). (c) mRNA levels of ANP, BNP, and β-MHC in different treatment groups. The results were normalized against GAPDH and converted to fold change relative to the ctrl group. (d) Intracellular ROS levels in primary cardiomyocytes were quantified with DCFH-DA staining. (e) Mitochondrial ROS levels in primary cardiomyocytes were measured by MitoSOX staining. (f) Representative TUNEL staining of primary cardiomyocytes in different treatment groups. Data are presented as mean ± SEM. ∗∗∗p < 0.001 vs. the control group, #p < 0.05 vs. the PE treatment group.

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  Figure 7. LCZ696 upregulated Sirt3 expression. (a) NRCMs were treated with 50 μM PE for 24 h, and the expression of Sirt3 and MnSOD were detected by Western blotting. (b) Histograms showing the quantitative analysis of Sirt3 and MnSOD, n = 3. (c) NRCMS were transfected with small interfering RNA targeting Sirt3 (si-Sirt3, 50 nM) of scramble small interfering RNA (si-NC,50 nM) for 48 h and then treated as indicated. The expression of Sirt3 and MnSOD was determined with immunoblotting. (d) Histogram of the quantitative analysis of the data in (c), after normalization to the GAPDH levels, n = 3. (e) Representative images of immunofluorescence staining for Sirt3 and mitochondrial marker (COX IV) in NRCMs. ∗p < 0.05 vs. the control group, #p < 0.05 vs. the PE treatment group.

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  Figure 8. Sirt3 knockdown abolished the antihypertrophic effect of LCZ696 in PE-induced cardiomyocyte hypertrophy. (a) Immunofluorescence staining of α-actinin in primary neonatal rat cardiomyocytes (NRCMs) in the indicated groups. (b) The cell surface area in the indicated groups (n > 50 cells per experimental group), ∗∗∗p < 0.001 vs. the si-NC+vehicle group, #p < 0.05 vs. the si-NC+LCZ696 treatment group. (c, d) Western blot analysis and quantitative analysis of Bcl-2 and Bax protein levels from different treatment groups. (e) Representative images of DCFH-DA staining in NRCMs. (f) Representative images of MitoSOX staining in NRCMs. (g, h) Western blot analysis of p-AMPK and AMPK protein levels in different treatment NRCMs and quantitative analysis of the ratio of p-AMPK/AMPK, n = 3. ∗p < 0.05 vs. control group, #p < 0.05 vs. PE treatment group.