Sacubitril Ameliorates Cardiac Fibrosis Through Inhibiting TRPM7 Channel

Figures

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  FIGURE 1. Sacubitril/valsartan effectively protected cardiac function and ameliorated cardiac fibrosis induced by ISO in rats. (A) The flow chart of the cardiac fibrosis model and drug treatment in animal experiment. (B) Heart to body weight ratio of rats in the control group, the ISO group, the valsartan group and the sacubitril/valsartan group. (C) On the 15th day of the experiment, the heart function of rats was measured by echocardiography. (D) Cardiac output, ejection fraction, fractional shortening and stroke volume were analyzed and calculated according to the results of echocardiography. (E) Masson trichrome staining of heart tissue sections of rats. Scale bar = 200 μm. (F) mRNA expression levels of Mmp9 and α-Sma in the left ventricular apexes of rats. (G) Protein levels of MMP9 and α-SMA in the left ventricular apexes of rats. The data presented are mean ± SD. n = 5/group. 18S RNA was used as the internal reference gene and β-actin was used as the internal reference protein for normalization and statistical analysis. ∗∗∗P < 0.001, ∗∗P < 0.01, and ∗P < 0.05.

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  FIGURE 2. LBQ657 significantly extenuated TGF-β1 induced cardiac fibroblast activation in MEF. (A) mRNA expression of Mmp9 and α-Sma in control MEF, MEF treated with TGF-β1 and MEF treated with LBQ657 + TGF-β1. (B) Protein levels of MMP9 and α-SMA in the rats. (C) The efficiency of the siRNA used to reduce the mRNA expression of TRPM7 in MEF. (D) mRNA expression of Trpm7, Mmp9 and α-Sma in MEF treated with TGF-β1, MEF transfected with si-TRPM7 and treated with TGF-β1, MEF treated with LBQ657 + TGF-β1. (E) Protein levels of TRPM7, MMP9 and α-SMA in the rats. The data presented are mean ± SD. Each group contained the results of three independent repeated trials. 18S RNA was used as the internal reference gene and β-actin was used as the internal reference protein for normalization and statistical analysis. ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, and ∗∗P < 0.01.

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  FIGURE 3. LBQ657 protected against fibrosis by blocking TRPM7 channel. (A) The virtual binding mode of NEPi and natural cholesterol hemisuccinateon ligand (as control) on TRPM7. NEPi (red) and cholesterol hemisuccinate (gray) are shown as a stick model. (B) Current amplitude of TRPM7-like current recorded in neonatal rat cardiac fibroblasts in control cell (black), LBQ657-10 μM treated cell (blue) and LBQ657-50 μM treated cell (red). (C) mRNA expression of Mmp9 and α-Sma in MEF treated with TGF-β1, MEF treated with 2-APB + TGF-β1, and MEF treated with LBQ657 + TGF-β1. (D) Protein levels of MMP9 and α-SMA in the rats. The data presented are mean ± SD. Each group contained the results of three independent repeated trials. 18S RNA was used as the internal reference gene and β-actin was used as the internal reference protein for normalization and statistical analysis. ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, and ∗∗P < 0.01.

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  FIGURE 4. LBQ657 reduced hypoxia-induced necrosis by inhibiting TRPM7-mediated Ca2+ influx in cardiomyocytes. (A) Comparison of TRPM7 protein levels in MEF and H9C2 cells. (B) Fluorescence intensity of Fluo-4-AM fluorescent dye in 488 nm excitation light of normoxic H9C2, hypoxic H9C2 and hypoxic H9C2 pre-treated with LBQ657. (C) Protein levels of caspase3, cleaved caspase3, LC3B-I and LC3B-II in hypoxic H9C2 and hypoxic H9C2 pre-treated with LBQ657. (D) Protein levels of RIPK1/RIPK3/MLKL/p-MLKL in hypoxic H9C2 and hypoxic H9C2 pre-treated with LBQ657. (E) Protein levels of RIPK1/RIPK3/MLKL/p-MLKL in normoxic H9C2 and normoxic H9C2 treated with LBQ657. (F) Protein levels of RIPK1/RIPK3/MLKL/p-MLKL in hypoxic H9C2 and hypoxic H9C2 pre-treated with BAPTA-AM. The data presented are mean ± SD. Each group contained the results of three independent repeated trials. β-actin was used as the internal reference protein for normalization and statistical analysis. ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, ∗∗P < 0.01, and ∗P < 0.05.

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  FIGURE 5. Schematic showing that sacubitril ameliorates cardiac fibrosis through inhibiting TRPM7-mediated Ca2+ influx in cardiac fibroblasts and cardiomyocytes. This study demonstrated that sacubitril metabolite LBQ657 could relieve the fibrotic response of fibroblasts and reduce cardiomyocyte PCD by blocking TRPM7 channel, thereby ameliorating cardiac fibrosis.