β3 adrenoceptor agonist mirabegron protects against right ventricular remodeling and drives Drp1 inhibition

Figures

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  Figure 1. The increased expression of B3AR in the RV in MCT rats was further elevated by mirabegron. (A) Detection of the RNA expression of β3AR using RT-qPCR. (B) Representative immunoblots of β3AR in the three groups. (C) Quantification of the density of the immunoblots in the three groups. MCT: MCT rats; MCT + Mira: MCT rats with mirabegron treatment. Sample size: N=6 for each group. Significance codes: *, P<0.05; **, P<0.01. β3AR, β3 adrenoceptor; HC, healthy control; MCT, monocrotaline; RV, right ventricular.

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  Figure 2. Mirabegron reduces RV pressure and prevents RV deterioration. (A) Representative pressure curves of RV and PA in groups of healthy controls, MCT rats, and MCT rats with mirabegron treatment. (B) Quantification of mPAP in the three groups. (C) Quantification of Fulton index (RV/LV + septum) in the three groups. (D) Quantification of systemic SBP in the three groups. (E) Representative four-chamber views of echocardiography in groups of healthy controls, MCT rats, and MCT rats with mirabegron treatment. (F) Quantitative analysis of RVEDA. (G) Quantitative analysis of RVFAC. (H) Quantitative analysis of cardiac index. (I) Quantitative analysis of PAAT. HC: healthy control; MCT: MCT rats; MCT + Mira: MCT rats with mirabegron treatment. Sample size: N=6–8 for each group in RHC analysis, N=6 for each group in data of echocardiography. *, P<0.05; **, P<0.01; ***, P<0.001. NS, non-significance; RV, right ventricular; LV, left ventricular; PA, pulmonary artery; MCT, monocrotaline; mPAP, mean pulmonary arterial pressure; SBP, systolic blood pressure; RVEDA, RV end-diastolic area; RVFAC, RV fractional area change; PAAT, pulmonary artery acceleration time; RHC, right heart catheterization.

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  Figure 3. Mirabegron attenuated RV hypertrophy and fibrosis, and reduced RV structural and functional deterioration. (A) Representative Sirius Red (upper row) and WGA staining (lower row) images of the RV free wall in groups of healthy controls, MCT rats, and MCT rats with mirabegron treatment. Scale bar = 100 µm in Sirius Red images; Scale bar =50 µm in WGA images. (B) Representative immunoblots of Collagen 1, TIMP1, and BNP in the RV in groups of healthy controls, MCT rats, and MCT rats with mirabegron treatment. (C) Quantification of Sirius Red percentage of RV free wall in the three groups. (D) Analysis of cardiomyocyte diameter from WGA staining in RV free wall in the three groups. (E) Quantitative analysis of immunoblots of Collagen 1. (F) Quantitative analysis of immunoblots of TIMP1. (G) Quantitative analysis of immunoblots of BNP. HC: healthy control; MCT: MCT rats; MCT + Mira: MCT rats with mirabegron treatment. Sample size: N=8 for each group in histology data. N=6 for each group in immunoblotting data. *, P<0.05; **, P<0.01; ***, P<0.001. WGA, wheat germ agglutinin; TIMP1, tissue inhibitor of metalloproteinases 1; MCT, monocrotaline; BNP, brain natriuretic peptide; RV, right…

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  Figure 4. Mirabegron suppresses Drp1 and enhances AMPK signaling in the overload RV. (A) Representative immunoblots of Drp1, AMPK, mTOR, and their phosphorylated forms (p-Drp1 at Ser616 and Ser637, p-AMPK at Thr172, and p-mTOR at Ser2448) in the RV in groups of healthy controls, MCT rats, and MCT rats with Mirabegron treatment. (B) Quantification of the immunoblots of phospho-Drp1 at Ser616 (p-Drp1S616). The phosphorylation is presented as the ratio of p-Drp1S616 to total Drp1 or GAPDH (p-Drp1S616/t-Drp1 or p-Drp1S616/GAPDH). (C) Quantification of the immunoblots of p-Drp1 at Ser637 (p-Drp1S637). The phosphorylation is presented as the ratio of p-Drp1S637 to total Drp1 or GAPDH (p-Drp1S637/t-Drp1 or p-Drp1S637/GAPDH) (D) Quantification of the immunoblots of total Drp1. (E) Quantitative analysis of the phosphorylation of AMPK and mTOR. HC: healthy control; MCT: MCT rats; MCT + Mira: MCT rats with mirabegron treatment. Sample size: N=4–5 for each group. Significance codes: *, P<0.05; **, P<0.01; ***, P<0.001. NS, non-significance; Drp1, dynamin-related protein 1; AMPK, AMP-activated protein kinase; RV, right ventricular.

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  Figure 5. Mirabegron suppresses Drp1 and enhances the AMPK signaling in hypertrophied cardiomyocytes. H9c2 cardiomyocyte cells were treated with Ang II (600 mM), as well as mirabegron (10 µM), AMPK inhibitor Compound C (10 µM), and Drp1 inhibitor Mdivi-1 (20 µM), as indicated with “−” or “+” in the figure. Five groups were included in the cell experiments. (A) Representative immunoblots of Drp1, AMPK, mTOR, and their phosphorylated forms (p-Drp1 at Ser616 and Ser637, p-AMPKα at Thr172, and p-mTOR at Ser2448) in the groups. (B) Quantification of the p-Drp1 at Ser616 (presented as p-Drp1S616/t-Drp1). (C) Quantification of the p-Drp1 at Ser637 (presented as p-Drp1S637/t-Drp1). (D) Quantification of the immunoblots of total Drp1. (E) Quantitative analysis of the p-AMPKα. (F) Quantitative analysis of the p-mTOR. Sample size: N=5 for each group. Significance codes: *, P<0.05; **, P<0.01; ***, P<0.001. NS, non-significance; Drp1, dynamin-related protein 1; AMPK, AMP-activated protein kinase.

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  Figure 6. Proposed mechanism of the treatment effect of mirabegron on the RV. Mirabegron reduces RV pathological changes and restores RV metabolic status. The beneficial effect could be attributed to (I) promotion of mitochondrial metabolism through AMPK activation; and (II) suppression of mitochondrial fission by inhibiting Drp1. Drp1, dynamin-related protein 1; AMPK, AMP-activated protein kinase; RV, right ventricular.