The crucial relationship between miRNA-27 and CSE/H 2 S, and the mechanism of action of GLP-1 in myocardial hypertrophy

Figures

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  Figure 1. Changes of miR-27a/b and CSE levels in TAC mice and Ang II-induced NMVCs. (A). Modeling of myocardial hypertrophy in mice. (B) Echocardiograms of mice in the control group (Sham) and TAC group (n = 6-10 mice). (C) Macroscopic view of Masson and HE-stained transverse sections showing myocardial fibrosis and in control and TAC hearts. HE × 400, Masson × 400. Scale bar: 100 μm (n = 3 mice). (D) Changes in miR-27a and miR-27b levels in TAC mice compared to the sham group (n = 5-6 mice). (E) qRT-PCR detecting changes in miR-27a and miR-27b in neonatal mouse ventricular cardiomyocytes (NMVCs) after 48 hours of Ang II treatment (n = 5-6 group). (F-I) Changes in mRNA and protein levels of CSE in hypertrophy models in vivo and in vitro (n = 5-6 mice or group). Averaged data are presented as mean ± SD; *P < 0.05, **P < 0.01. Note: CN represents the control group, Ang II represents angiotensin II, and TAC represents transverse aortic constriction.

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  Figure 2. The regulatory effect of miR-27a on CSE. (A). The sequence of miR-27 between 480-490 is highly conserved among humans, rats, mice, rabbits, and other organisms. (B). Sequence alignment between members of the miR-27 family and 3′-UTRs of CSE in humans and mice. The complementary sequences in CSE genes to the miR-27a/b seed regions are highlighted in red and blue, respectively. (C). Constructs of wild-type (WT) and mutant (MT) 3′UTRs of CTH were generated. (D, E). Luciferase reporter gene assay measuring the interactions between miR-27a/b and CSE in HEK293T cells (n = 3 group). (F, G). Changes in miR-27a and CSE mRNA expression were determined by qRT-PCR after miR-27a inhibition (miR-27aI) or overexpression (miR-27a) in cells (n = 5 group). (H, I). Cell supernatant was collected to assess the alterations in H2S expression after silencing or overexpressing miR-27a using the Elisa Kit, compared to the control (CN) and negative control (NC) groups (n = 5 group). (J, K). Western blot analysis was carried out to investigate changes in CSE protein expression after miR-27a inhibition or overexpression (n = 5 group). The figure legend from (D) applies to (E). The data are presented as the…

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  Figure 3. Silencing miR-27a protects cardiomyocyte hypertrophy. Relative expression of β-MHC, ANP, BNP, CSE, and miR-27a was assessed in NMVCs using qRT-PCR and Western blot analysis in various experimental conditions: CN, Ang II + negative control (NC), Ang II + miR-27a inhibitor (miR-27aI), and Ang II + miR-27aI + CSE siRNA (CSEI). The mRNA levels (A-E) and protein levels (F-I) were analyzed (n = 5 group). (J). H2S expression was quantified using an Elisa Kit (n = 5-6 group). (K, L). Cell area changes were determined by fluorescence imaging and statistical analysis for the four groups: CN, Ang II + NC, Ang II + miR-27aI, and Ang II + miR-27aI + CSEI (n = 40-50 cells). The magnification factor used was × 400, with the scale bar representing 20 μm. The figure legend from (L) applies to all. The data are expressed as mean ± SD, with statistical significance denoted by *P < 0.05 and **P < 0.01. Notably, CN denotes the control condition, and Ang II refers to angiotensin II.

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  Figure 4. rAAV9-anti-miR-27a affects myocardial hypertrophy by regulating CSE/H2S. (A-E). Statistical results of interventricular septal thickness in diastole (IVSd, mm) and systole (IVSs, mm), left ventricular posterior wall at end-diastole (LVPWd, mm) and end-systole (LVPWs, mm), Heart/body weight (H/B weight, mg/g) (n = 5 mice). (F). Echocardiograms of mice in Sham, TAC + NC, TAC+ miR-27aI. (G). Masson and HE staining were used to detect transverse sections and myocardial fibrosis in C57BL/6 after injecting rAAV9-anti-miR-27a for three weeks before TAC modeling. HE ×400, Masson ×400. Scale bar represents 100 μm. (n = 3 mice). (H, I). Detect the mRNA level and protein level of miR-27a and CSE in the heart of C57BL/6 mice after tail vein injection of rAAV9-anti-miR-27a (miR-27aI) and rAAV9-miR-27a (miR-27a) for three weeks. (K-O). Four weeks after modeling, qRT-PCR and Western blot experiments were used to detect the mRNA and protein expression changes of hypertrophic factors, CSE and miR-27a compared with Sham and TAC NC group. β-tubulin served as an internal control. (n = 4-6 mice). (J). Changes in the expression of H2S in serum. Figure legend from (K) applies to (J), (L-O). Averaged…

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  Figure 5. The effect of miR-27a on myocardial hypertrophy depends on the regulation of CSE/H2S. (A-E). rAAV9-miR-27a overexpressing adenovirus was injected into C57BL/6 mice at three weeks before TAC, and SPRC was intraperitoneal injected daily for eight days before surgery. Statistical results of interventricular septal thickness in diastole (IVSd, mm) and systole (IVSs, mm), left ventricular posterior wall at end-diastole (LVPWd, mm) (C) and end-systole (LVPWs, mm), and Heart/body weight (mg/g) in Sham, TAC + NC, TAC+ rAAV9-miR-27a (miR-27a), TAC+ miR-27a + SPRC groups (n = 5 mice). (F). Mouse echocardiogram. (G). Masson and HE staining were used to detect transverse sections and myocardial fibrosis in four groups. HE ×400, Masson ×400. Scale bar represents 100 μm. (n = 3 mice). Relative mRNA and protein expression of CSE and miR-27a (H, K) and hypertrophic marker factors (I, L, M) in the C57BL/6 was measured from Sham, TAC + NC, TAC + miR-27a, and TAC + miR-27a + SPRC by qRT-PCR and Western blot (n = 5-7 mice). β-tubulin served as an internal control. (J). Changes in the expression of H2S in serum (n = 5 group). Figure legend from (M) applies to all other bar graphs. Averaged data are…

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  Figure 6. Liraglutide/Dulaglutide protect cardiac hypertrophy by regulating CSE and miR-27a. NMVCs were exposed to Ang II for 48 hours and Liraglutide or Dulaglutide for another 24 hours. qRT-PCR analysis of hypertrophic markers (A, C) and CSE/miR-27a (B, D) from the heart of the different groups (n = 5 group). Figure legend from (B) also applies to (A), figure legend from (D) also applies to (C). (E) Mechanism diagram of miR-27a-3p regulation on myocardial hypertrophy. Averaged data are presented as the mean ± SD; *P < 0.05, **P < 0.01. Of note, CN control, Ang II angiotensin II.