MicroRNA let-7-TGFBR3 signalling regulates cardiomyocyte apoptosis after infarction

Figures

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  Fig. 1. Expression of micro RNA let-7a and let-7f is decreased after acute myocardial infarction in experimental pigs and human patients. (A) Experimental design. Pigs were subjected to LAD ligation and sacrificed after 1, 2 or 4 h. Next-generation sequencing was performed on RNA extracts from both infarct and remote tissues. Tissue was also collected from sham animals and a 24-h post-MI group. (B—F) Stem-loop qPCR validation of miRNA expression in sham and infarcted hearts at different time points. I/R ratio indicates the ratio of miRNA expression level in the infarct compared to the remote area, which was used as an internal control for each individual pig. (G) TaqMan qPCR validation of let-7a and let-7f expression in sham and infarcted hearts. (H—I) U6-normalized let-7a and let-7f expression levels in the infarct and remote area at 24 h post MI. (J) Abundance of let-7 family members in human hearts based on NGS data from Meunier, J. et al., 2013 [25]. (K) Abundance of let-7 family members in pig hearts based on next-generation sequencing data derived from the remote area of three individual pigs. TPM: transcripts per million. (L) Human plasma miR-1 expression levels in healthy…

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  Fig. 2. AAV9-mediated let-7 knockdown increases cardiomyocyte apoptosis and reduces cardiac function after experimental myocardial infarction in mice. (A-B) Myocardial expression levels of let-7a and let-7f 6 weeks after injection of AAV9 viruses carrying either GFP (AAV-GFP) or TuD (AAV-TuD) expressing plasmids. (C) Experimental schematic. Mice were injected with AAV-GFP or AAV-TuD 6 weeks prior to induction of MI. Serial echocardiography was performed to evaluate heart function before surgery, 1 day and 28 days after MI. (D) Ejection fraction (EF) in AAV-TuD and AAV-GFP infected mice before MI. (E) Ejection fraction in AAV-TuD and AAV-GFP infected mice one day after MI. (F) Ejection fraction, fraction shortening (FS), end diastolic volume (EDV) and end systolic volume (ESV) in AAV-TuD and AAV-GFP infected mice one month after MI. (G) Representative trichrome staining of LV sections 28 days post-MI. (H) Quantification of LV scar length 28 days post-MI. (I) TUNEL staining of mouse peri-infarct tissue 1 day post MI. (J) Quantification of TUNEL positive cells in AAV-TuD and AAV-GFP hearts 1 day post-MI. (K) WGA staining for cell size quantification. (L) Quantification of cell size. (M) ANF…

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  Fig. 3. AAV9-mediated let 7 overexpression reduces cardiomyocyte apoptosis and improves cardiac function after experimental myocardial infarction in mice. (A-B) Let-7a/f overexpression in mouse hearts after infection with either AAV-let-7a/f or AAV-GFP. (C) Experimental schematic. Mice were intravenously injected with AAV9 viruses carrying let-7 expressing plasmid for at least six weeks before LAD ligation. Heart function was evaluated by echocardiography before surgery, one day and four weeks after MI. (D) Ejection fraction in AAV-let-7 and AAV-GFP infected mice before MI. (E) Ejection fraction in AAV-let-7 and AAV-GFP infected mice one day after MI. (F) Ejection fraction, fraction shortening, end diastolic volume and end systolic volume in AAV-let-7 and AAV-GFP infected mice one month after MI. (G) Trichrome staining of LV sections 28 days post-MI. (H) Quantification of scar length 28 days post-MI. (I-J) Reduction of cardiomyocyte apoptosis in the border zone following let-7a/f overexpression based on TUNEL staining. (K-L) Attenuation of cardiomyocyte hypertrophy in the remote area 28 days post-MI following let-7a/f overexpression based on WGA staining. Data are presented as means ±…

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  Fig. 4. HMGA2 and TGFBR3 are let-7 targeted genes and are highly expressed in cardiomyocytes after acute infarction. (A) Overview of target gene identification. A heart RISC-seq database [34] and TargetScan 6.0 prediction [35] were used to identify candidate genes from a common pool of 188. (B-E) Gene expression of Hmga2, Tgfbr1, Tgfbr3 and Trappc1 after transfection of let-7a and let-7f inhibitors in rat neonatal cardiomyocytes. Histograms and error bars indicate mean and SEM, respectively, from four independent experiments. Differences between the control group and other groups were analysed by one-way ANOVA with Dunnett's test. (*, P < .05; **, P < .001; **, P < .0001). (F) Western blot of Hmga2 in rat neonatal cardiomyocytes transfected with let-7a and let-7f mimics and inhibitors. (G) Western blot of Tgfbr3 in rat neonatal cardiomyocytes transfected with let-7a and let-7f mimics and inhibitors. Quantification of western blot is shown in the histogram. Histograms and error bars indicate mean and SEM, respectively, from two independent experiments. (H) Characterization of Tgfbr3 expression in sham and 1, 2 and 6 day post-MI in mice. Quantification of western blot is shown in the…

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  Fig. 5. MicroRNA let-7-Tgfbr3-p38 MAPK signalling regulates cardiomyocyte apoptosis. (A) Western blots of cleaved LC3B (marker of autophagy) and caspase 3 (marker of apoptosis) in rat neonatal cardiomyocytes 1 day after adenovirus-mediated Hmga2 and Tgfbr3 expression. (B) Dose dependent activation of Smad2 and p38 MAPK signalling following Tgfbr3 expression. MOI: multiplicity of infection. The relative ratios of phospho-Smad2 to Smad2 and phospho-p38 to p38 are indicated by the numbers at the bottoms of the lanes. (C) Induction of p38 downstream target gene MMP9 signalling, instead of canonical TGFβ signalling, following overexpression of Tgfbr3 in rat neonatal cardiomyocytes. The histograms and error bars indicate mean and SEM, respectively, from six independent experiments. Differences between groups were analysed by unpaired Student's t-test (**, P < .01; ***, P < .001). (D) Partial rescue of Tgfbr3 induced apoptosis in rat neonatal cardiomyocytes upon treatment with p38 MAPK inhibitor, SB239063. (E) Quantification of rescue of apoptosis by flow cytometry. Percentage of subG1 cells from three independent experiments is shown. (F) Western blot of Tgfbr3, p38 activity and caspase 3…

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  Fig. 6. Plasma level of TGFBR3 is increased after acute myocardial infarction in pigs and in human patients. (A) Pig plasma TGFBR3 expression levels at baseline, 1 day and 7 days post-sham and MI (n = 16). One-way ANOVA was performed with Dunnett's test. (**, P < .01) (B) Correlation of plasma TGFBR3 expression and ejection fraction in pigs. (C) Human plasma TGFBR3 expression levels at several time points: before therapeutic intervention (Before), after therapeutic intervention (After), 1 day and 1 week post-MI (MI-1D and MI-1 W). Differences between the before group and the MI-1D group were analysed by paired Student's t-test (*, P < .05). (D) Graphical summary. MicroRNA let-7a and let-7f are abundant in healthy cardiomyocytes to suppress the expression of pro-apoptosis factor TGFBR3. Meanwhile, microRNA let-7a and let-7f are enriched in the plasma and the concentration of plasma TGFBR3 is low (left). Once infarction occurs, the expression of let-7a and let-7f are decreased and TGFBR3 is de-repressed. The expression of TGFBR3 initiates p38 MAPK signalling and promotes myocyte apoptosis. Meanwhile, plasma let-7a and let-7f respond to the injury by reducing the amount of their expression.…