MicroRNA-410-5p exacerbates high-fat diet-induced cardiac remodeling in mice in an endocrine fashion

Figure 1. Cardiac fibrosis and expression of fibrotic markers in the heart of rats fed HFD. (a) Cardiac matrix deposition in Masson-stained sections of rats fed HFD and control rats. Heart tissues from the HFD group show focal regions of fibrosis in the interstitium (Left panels). Quantitative analysis of fibrotic area (Right panel). These results are means ± SD of four experiments (n = 10 in each experiment). ***P < 0.001 relative to control (normal group). Scale bar, 20 μm. (b) The mRNA levels of fibrotic genes in the heart of rats were detected by qPCR. All results are standardized to the levels of GADPH and the means ± SD of three experiments (n = 10 in each experiment). *P < 0.05; **P < 0.01 relative to control (normal group). (c) The levels of fibrotic proteins were measured using Western blotting (n = 6 in each experiment). Representative Western blots are shown. (d) Quantification of the ratio of proteins to β-actin. These results are the means ± SD of three experiments. *P < 0.05; **P < 0.01 relative to control (normal group).

Figure 2. HFD impairs LV function. (a) M-mode echocardiogram. Comparison of general appearance of rats fed HFD (lower panel) and control rats (upper panel). The measurement of interventricular septum (LVS) and LV posterior wall (LVPW), LV end-systolic diameter (LVESD), and LV end-diastolic diameter (LVEDD) is shown. (b) LVS, LVPW, LVESD, and LVEDD. (c) LV ejection fraction (EF). (d) LV fractional shortening23. These results are means ± SD of three experiments (n = 10 in each experiment). *P < 0.05; **P < 0.01 relative to control (normal group).

Figure 3. The TGFβ signaling is activated in the heart of rats fed HFD. (a) The levels of phosphorylated Smad 2 were increased in the heart of rats fed HFD (n = 6 in each experiment). Representative Western blots are shown. (b) Quantification of the ratio of proteins to β-actin. These results are the means ± SD of three experiments. **P < 0.01 relative to control (normal group). (c) The protein levels of Smad 7 were reduced in the heart of rats fed HFD (n = 6 in each experiment). (d) Quantification of the ratio of proteins to β-actin. These results are the means ± SD of three experiments. *P < 0.05 relative to control (normal group).

Figure 4. The miRNA expression profiles in the heart of rats. (a) Heat maps show significantly up- and down-regulated mRNAs in the heart of rats fed HFD and control rats for 24 weeks (n = 7 in each experiment). miRNA expression profiles were clustered hierarchically. (b) Volcano Plot for differential miRNA expression in the heart of rats. The green dost represent the most significantly upregulated miRNAs (more than five-fold, P < 0.05), including miR-3583-3p, -410-5p, -146b-3p, -764-5p, -330-5p, and -324-3p, in the heart of rats fed HFD for 24 weeks. The arrow indicates miR-322-5p. (c) qPCR analysis of the six most-up-regulated miRNAs in the heart of rats. All results are standardized to the levels of 18S rRNA and the means ± SD of three experiments (n = 6 in each experiment). *P < 0.05; **P < 0.01 relative to control (normal group).

Figure 5. Smad 7 is a target gene of miR-410-5p. (a) Complementarity between the 3′UTR of Smad 7 gene and miR-410-5p. For constructing of Smad 7-3′UTR (Mut) reporter, the putative miR-410-5p binding site was replaced with the sequence as shown below (Mut). Mutated seeds were marked in red, Watson-Crick base pairing with a straight line and U-G wobbles with a dotted line. (b) The putative target sites of Smad 7 3′-UTR, which are marked in red, are conserved in mammals. (c) Luciferase analysis of a reporter vector harboring the 3′-UTR of Smad 7 (WT and Mut) in H9c2 cells transfected with negative control (NC) or miR-410-5p for 36 h. These results are means ± SD of three experiments. **P < 0.01 relative to NC. (d) Western blot analysis in H9c2 cells transfected with NC or miR-410-5p for 36 h. Representative Western blots are shown. (e) Quantification of the ratio of proteins to β-actin. These results are the means ± SD of four experiments. *P < 0.05; **P < 0.01 relative to NC.

Figure 6. Overexpression of miR-410-5p promotes cardiac fibrosis. The rats were infected with lentivirus particles containing PGLV3-miR-410 or PGLV3-NC. After 21 days injection, animals were euthanized for further analysis. (a) The protein levels of genes in the TGFβ pathway were measured using Western blotting (n = 6 in each experiment). Representative Western blots are shown. (b) Quantification of the ratio of proteins to β-actin. These results are the means ± SD of three experiments. **P < 0.01; ***P < 0.001 relative to negative control (NC). (c) Masson’s staining of heart in rats. Heart tissues from miR-410-5p overexpression group showed focal regions of fibrosis in the interstitium (Left panels). Quantitative analysis of fibrotic area (Right panel). These results are means ± SD of three experiments (n = 10 in each experiment). ***P < 0.001 relative to NC. Scale bar, 20 μm. (d) The mRNA levels of fibrotic genes in the heart of rats were detected by qPCR. All results are standardized to the levels of GAPDH and the means ± SD of three experiments (n = 6 in each experiment). *P < 0.05 relative to NC. (e) The protein levels of fibrotic genes were measured using Western blotting (n = 6 in…

Figure 7. miR-410-5p from the kidney and adipose tissues promotes cardiac fibrosis. (a) The levels of pre-miR-410 (a) and miR-410-5p (b) were determined by qPCR in different tissues of rats fed normal diet and HFD, respectively. All results are standardized to the levels of 18S rRNA and the means ± SD of three experiments (n = 10 in each experiment). *P < 0.05; **P < 0.01 relative to control (normal group). (c,d) The levels of miR-410-5p in serum (c) and exosomes (d) of rats fed normal diet and HFD. These results are the means ± SD of three experiments (n = 10 in each experiment). *P < 0.05 relative to control. (e) The levels of miR-410-5p in the heart of rats fed normal diet with kidney- or adipocyte-specific overexpression of miR-410. NPHS2-miR-410, kidney-specific overexpression; aP2-miR-410, adipocyte-specific overexpression. *P < 0.05 relative to negative control (NC). All results are standardized to the levels of 18S rRNA and the means ± SD of three experiments (n = 10 in each experiment). (f) The levels of miR-410-5p in exosomes of rats fed normal diet with kidney- or adipocyte-specific overexpression of miR-410. These results are the means ± SD of three experiments (n = 10 in each…

Figure 8. A proposed mechanism of miR-410-5p-induced cardiac fibrosis in rats with obesity.