Angiotensin II receptor blocker LCZ696 attenuates cardiac remodeling through the inhibition of the ERK signaling pathway in mice with pregnancy-associated cardiomyopathy

Figures

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  Fig. 1. LCZ696 alleviates cardiac injury in PAH mice and represses the Ang II receptor pathway. PAH mice were either treated with or without LCZ696. a Effect of LCZ696 on survival rate of PAH mice detected by Kaplan–Meier method (N = 7). b Heart size (scale bar = 100 mm) and ratio of heart weight/body weight of mice. c HE staining analysis of cardiac tissues (upper panels: scale bar = 50 mm; lower panels: bar = 25 μm). d Cardiac fibrosis observed by Masson’s trichrome staining (scale bar = 25 μm). e Apoptosis of cardiomyocytes detected by TUNEL staining (scale bar = 25 μm). f Western blot analysis of ACE2 protein. N = 7. *p < 0.05 vs. normal mice; #p < 0.05 vs. PAH mice. Measurement data (mean ± S.D.) among multiple groups were analyzed by one-way ANOVA, followed by Tukey post hoc test. Survival rate was calculated by the Kaplan–Meier method, and compared by a log-rank test

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  Fig. 2. LCZ696 alleviates Ang II-induced cardiac hypertrophy and fibrosis. Extracted cardiomyocytes were treated with Ang II (100 nM) alone or with LCZ696 (1.0 μM), or without any treatment. a Cardiac hypertrophy condition detected by 3[H]-leucine incorporation methodology. b mRNA expression of ANP, βMHC and TIMP2 in cardiomyocytes detected by RT-qPCR. c Size of cardiomyocytes detected by a-actinin immunofluorescence staining and percentage of cardiomyocyte size (scale bar = 25 μm). d Cardiac fibrosis assessed by 3[H]-proline incorporation methodology. e mRNA expression of collagen I, collagen III and TGF-β detected by RT-qPCR. F, Western blot analysis of ACE2 protein in cardiomyocytes. *p < 0.05 vs. cardiomyocytes without treatment; #p < 0.05 vs. cardiomyocytes treated with Ang II. Measurement data (mean ± S.D.) among multiple groups was analyzed by one-way ANOVA, followed by Tukey post hoc test. The experiment was conducted 3 times independently

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  Fig. 3. LCZ696 impedes Ang II-induced apoptosis of cardiomyocytes. Extracted cardiomyocytes were treated with Ang II (100 nM) alone or with LCZ696 (1.0 μM), or without any treatment. a Apoptosis of cardiomyocytes treated with LCZ696 in vitro detected by TUNEL staining (scale bar = 25 μm). b Quantitative analysis for AI of cardiomyocytes. c Western blot analysis of caspase 3 and cleaved-caspase 3 proteins in cardiomyocytes treated with LCZ696 or Ang II. *p < 0.05 vs. cardiomyocytes without treatment; #p < 0.05 vs. cardiomyocytes treated with Ang II. Measurement data (mean ± S.D.) among multiple groups was analyzed by one-way ANOVA, followed by Tukey post hoc test. The experiment was conducted 3 times independently

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  Fig. 4. LCZ696 diminishes cardiac hypertrophy and fibrosis induced by Ang II via suppression of the ERK phosphorylation. Cardiomyocytes were initially treated with either sh-NC or sh-ERK, or without any treatment. a mRNA and protein expression of ERK detected by RT-qPCR and western blot analysis. b Western blot analysis of the extent of ERK phosphorylation in cardiomyocytes upon different treatment. c Cardiac hypertrophy condition assessed by 3[H]-leucine incorporation methodology. d mRNA expression of ANP, βMHC and TIMP2 in cardiomyocytes detected by RT-qPCR. E, Cardiomyocyte fibrosis assessed by 3[H]-proline incorporation methodology. F, mRNA expression of collagen I, collagen III and TGF-β probed by RT-qPCR. G, Apoptosis of cardiomyocytes treated with LCZ696 as probed by TUNEL staining (scale bar = 25 μm). *p < 0.05 vs. cardiomyocytes without treatment; #p < 0.05 vs. cardiomyocytes treated with control + Ang II; &p < 0.05 vs. cardiomyocytes treated with Ang II + sh-NC; @p < 0.05 vs. cardiomyocytes treated with Ang II + sh-ERK; $p < 0.05 vs. cardiomyocytes treated with Ang II + sh-NC + LCZ696. Measurement data (mean ± S.D.) among multiple groups was analyzed by one-way ANOVA, followed…

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  Fig. 5. LCZ696 reduces cardiac remodeling through inhibiting the ERK signaling pathway. PAH mice were treated with sh-ERK with sh-NC as the control, or sh-ERK in the presence of LCZ696 with sh-NC in the presence of LCZ696 or control. a Heart size (scale bar = 100 mm) and ratio of heart weight/body weight in PAH mice. b HE staining of ventricular transection of heart (upper panels: scale bar = 50 mm; lower panels: scale bar = 25 μm). c Cardiomyocyte fibrosis observed by Masson’s trichrome staining (scale bar = 25 μm). d mRNA expression of collagen I, collagen III and TGF-β detected by RT-qPCR. e Isolectin B4 immunofluorescence of the capillary in ventricular tissues (blood vessel: yellow; cell membrane: red; nucleus: blue). f mRNA expression of ANP, βMHC and TIMP2 in cardiomyocytes detected by RT-qPCR. g Apoptosis of cardiomyocytes treated with LCZ696 detected by TUNEL staining (scale bar = 25 μm). h Western blot analysis of ERK protein and extent of ERK phosphorylation. *p < 0.05 vs. PAH mice treated with sh-NC; #p < 0.05 vs. PAH mice treated with sh-ERK; &p < 0.05 vs. PAH mice treated with sh-NC + LCZ696. N = 7. Measurement data (mean ± S.D.) among multiple groups was analyzed by…

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  Fig. 6. Potential molecular mechanisms of Ang II receptor inhibitor LCZ696 in PAH by regulating the ERK signaling pathway. In vivo and in vitro, LCZ676 reduces the expression of ANP, βMHC, TIMP2, Collagen I, Collagen III and TGF-β in cardiomyocytes by inhibiting ERK phosphorylation, thus diminishing cardiac hypertrophy and fibrosis, along with cardiomyocyte apoptosis, which ultimately attenuates cardiac remodeling in mice with PAH