Inhibitory effect of (pro)renin receptor decoy inhibitor PRO20 on endoplasmic reticulum stress during cardiac remodeling

Figures

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  FIGURE 1. PRR expression is positively associated with cardiac remodeling. (A) The plasma concentration of soluble PRR in mice after 1, 4 and 8 weeks of transversus aortic constriction (TAC). (B) Immunoblot analysis of PRR in mouse failing hearts and quantitative analysis of the PRR protein level normalized to GAPDH. (C) Immunoblot analysis of PRR in Ang II-induced cardiomyocytes (CMs) and quantitative analysis of the PRR protein level normalized to GAPDH. (D) Immunoblot analysis of PRR in Ang II-induced cardiac fibroblasts (CFs) and quantitative analysis of the PRR protein level normalized to GAPDH. ∗ p-value < 0.05. n = 5 per group. Data are expressed as mean ± SD.

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  FIGURE 2. PRR decoy inhibitor PRO20 ameliorates HF in mice after 8 weeks of TAC surgery. (A) The ratios of heart weight and body weight (HW/BW) in TAC-induced mice with and without PRO20 treatment for 8 weeks (250 μg/kg/d and 500 μg/kg/d). (B) Representative images of Masson staining of heart sections from TAC-induced mice and quantitative analysis of cardiac fibrosis. (C) Representative images of wheat germ-agglutinin (WGA) staining of heart sections from TAC-induced mice and quantitative analysis of cardiac hypertrophy. (D) Relative mRNA expression of cardiac hypertrophy markers (ANP and BNP) and Collagen Iα in TAC-induced hearts with and without PRO20 treatment. (E–H) Echocardiographic analyses of left ventricular ejection fraction (LVEF), fractional shorting (FS), left ventricular mass (LV mass) and left ventricular internal diameter at end-systole (LVIDs) after 8 weeks of TAC operation. (I) Representative images of CD31 staining of heart sections from TAC-induced mice and quantitative analysis of angiogenesis. ∗ p-value < 0.05. n = 5 per group. Data are expressed as mean ± SD.

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  FIGURE 3. PRR decoy inhibitor PRO20 suppresses oxidative stress in TAC-induced mice. (A) Representative immunofluorescent images of the production of reactive oxygen species assessed by dihydroethidium (DHE, stained in red) in hearts from TAC-induced mice with and without PRO20 treatment for 8 weeks. Nuclei were counterstained with DAPI (blue). (B–D) The content of SOD, MDA and NADPH oxidase in hearts from TAC-induced mice with and without PRO20 treatment for 8 weeks ∗ p-value < 0.05. n = 5 per group. Data are expressed as mean ± SD.

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  FIGURE 4. Inhibition of PRR by PRO20 alleviates CM hypertrophy and CF activation. (A) Immunoblot analysis of ANP and BNP in Ang II-induced CMs and quantitative analysis of the ANP and BNP protein level normalized to GAPDH. (B) Immunoblot analysis of α-SMA and Collagen Iα in Ang II-induced CFs and quantitative analysis of the α-SMA and Collagen Iα protein level normalized to GAPDH. (C) Effect of PRO20 on CF proliferation assessed by Edu assay. (D) Boyden chamber assay was performed to evaluate the migration of CFs with and without PRO20 treatment (10 nM) in the presence of Ang II. ∗ p-value < 0.05. n = 5 per group. Data are expressed as mean ± SD.

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  FIGURE 5. PRR decoy inhibitor PRO20 promotes intracellular cAMP content and suppresses endoplasmic reticulum stress. (A–B) The intracellular content of cAMP and cGMP in Ang II-induced CFs with and without PRO20 treatment (10 nM). #p < 0.05 vs. PBS group; ∗ p-value < 0.05 vs. Ang II group at indicated time point. (C–D) The intracellular content of cAMP and cGMP in Ang II-induced CMs with and without PRO20 treatment (10 nM). #p < 0.05 vs. PBS group; ∗ p-value < 0.05 vs. Ang II group at indicated time point. (E) Immunoblot analysis of IRE1, PERK and cleaved N-terminal cytoplasmic domain of ATF-6 (ATF-6 [N]) in Ang II-induced CFs and quantitative analysis of the IRE1 and PERK phosphorylation and the ATF-6 [N] activation. ∗ p-value < 0.05. (F) Immunoblot analysis of IRE1, PERK and ATF-6 [N] in Ang II-induced CMs and quantitative analysis of the IRE1 and PERK phosphorylation and the ATF-6 [N] activation. ∗ p-value < 0.05. n = 5 per group. Data are expressed as mean ± SD.

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  FIGURE 6. Effects of PRO20 on CM hypertrophy and CF activation induced by ER stressor TG. (A) Immunoblot analysis of ANP and BNP in TG-induced (5 μM) CMs with and without PRO20 treatment and quantitative analysis of the ANP and BNP protein level normalized to GAPDH. (B) Immunoblot analysis of α-SMA and Collagen Iα in CFs with and without TG treatment and quantitative analysis of the α-SMA and Collagen Iα protein level normalized to GAPDH. (C) Effect of PRO20 on TG-induced CF proliferation assessed by Edu assay in indicated groups. (D) Boyden chamber assay was performed to evaluate the migration of CFs in indicated groups. ∗ p-value < 0.05. n = 5 per group. Data are expressed as mean ± SD.

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  FIGURE 7. Schematic diagram of the mechanism. We systematically analyzed the mechanism of the PRO20 effect on cardiac remodeling, and found that PRO20 inhibited both oxidative stress and ER stress signaling pathways.