Targeting mir128-3p alleviates myocardial insulin resistance and prevents ischemia-induced heart failure

Figures

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  Figure 1.. IRS1 is downregulated in failing hearts and prolonged hypoxia-stressed cardiomyocytes. (A) Experimental design of male C57BL/6N mice subject to 4w-MI. (B) M-mode images captured by echocardiography determined cardiac dysfunction after 4w-MI, measured by fractional shortening (FS%) and end-diastolic and systolic left ventricular internal diameter (dLVID and sLVID) (N = 8 mice). (C) Infarct area (from one heart) detected by triphenyltetrazolium chloride (TTC) staining (N = 5 mice). Scale bar: 1 mm. (D) Interstitial fibrosis in the non-infarct region determined by Masson’s Trichrome staining (N = 8 mice). Scale bar: 100 μm. (E) RNA-sequencing (RNA-Seq) heat map depicts expression levels of the selected genes in the category of glycose pathways. * indicates genes identified as significantly different between sham and MI for four weeks (Log2 fold change according to adjusted P value < 0.05) (N = 2 mice). (F–G) qPCR validation and immunoblot analyses of IRS1, IRS2, INSR, SLC2A1 (also known as GLUT1), and SLC2A4 (also known as GLUT4) in the myocardium (N = 6–8 mice). (H) Insulin-dependent pathway evaluated by immunoblots after insulin injection (1 U/kg for 30 mins). (I) IRS1 and IRS2…

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  Figure 2.. Hypoxia-upregulates mir128-3p and degrades Irs1. (A) Mature mir128-3p was increased in 4w-MI myocardium (N = 6–8 mice). Data were normalized against mature mir191-5p expression. (B) In situ hybridization with digoxigenin-labeled mir128-3p probes (right) was accompanied with consecutive section stained by H and E (left) (N = 3–5 mice). Scale bar: 20 µm. (C) Upregulation of mir128-3p in ARCMs after 6 hr hypoxia (N = 4 experiments). (D) Location of Pri-mir128-1 hosted by human R3HDM1 gene and stem-loop structure of Pre-mir128-1. (E) Pri-mir128-1 level was determined by qPCR (N = 5 experiments). (F) Alignment of conserved mir128-3p sequence among mammals. (G) Sequences of mir128-3p conserved complementarity in human, mouse, and rat at Irs1 3’UTR. (H) Irs1 3’UTR luciferase reporter assay was performed in rat H9C2 cardiomyocytes transfected with pmiRGlo, pmiRGlo-Irs1 3’UTR, or mutant form (pmiRGlo-mut-Irs1 3’UTR). Luciferase activity was suppressed by mir128-3p mimic (N = 4 experiments). (I–J) qPCR and immunoblots illustrated miR-128–3 p mimic repression of IRS1 in ARCMs (N = 4–6 experiments). (K) Irs1 3’UTR luciferase reporter activity recovered by mir128-3p antagomiR (N = 4…

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  Figure 2—figure supplement 1.. MiRNA analysis in 4w-MI myocardium. (A) Mature mir144-3p and mir145-5p in 4w-MI myocardium were measured using TaqMan Advanced miRNA qPCR (N = 6–8 mice). Data were normalized against mature mir191-5p expression. (B) In situ hybridization with digoxigenin-labeled mir128-3p probes was quantified using ImageJ software (N = 3–5 mice). Groups were compared using the Mann-Whitney test. (C) Pri-mir128-1 and Pri-mir128-2 measurement in 4w-MI myocardium using TaqMan Gene expression (N = 6–8 mice). Data were normalised against 18S expression. Data presented as mean ± SEM. Groups were compared using two-tailed Student’s t-test. Figure 2—figure supplement 1—source data 1.MiRNA analysis in 4w-MI myocardium.

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  Figure 2—figure supplement 2.. Mir128-3p level in rat cardiomyocytes after hypoxia. Mature mir128-3p was measured using TaqMan Advanced miRNA qPCR (N = 6 experiments). Data were normalized against mature mir191-5p expression. Data presented as mean ± SEM. Groups were compared using one-way ANOVA followed by Bonferroni post hoc tests. Figure 2—figure supplement 2—source data 1.Mir128-3p level in rat cardiomyocytes after hypoxia.

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  Figure 2—figure supplement 3.. Pri-mir128-2 location and structure. Location of Pri-miR-128-2 hosted by human ARPP21 gene (GRCh38:3:35638945:35794496:1, Ensembl ENSG00000172995) and the stem-loop structure of Pre-miR-128-2.

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  Figure 2—figure supplement 4.. Pri-mir128-1 level in rat cardiomyocytes after hypoxia. Pri-mir128-1 in cardiomyocytes was performed using TaqMan Gene expression (N = 5 experiments). Data were normalised against 18S expression. Data presented as mean ± SEM. Groups were compared using one-way ANOVA followed by Bonferroni post hoc tests. Figure 2—figure supplement 4—source data 1.Pri-mir128-1 level in rat cardiomyocytes after hypoxia.

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  Figure 2—figure supplement 5.. Mir128-3p overexpression and inhibition optimization. Validation of mir128-3p mimic and antagomir. (A) mir128-3p mimic and (B) antagomir validation was carried out on H9C2 cells at different concentrations. Mature miRNA was measured by TaqMan Advanced miRNA qPCR. Data were normalized against mature mir191-5p expression (N = 3 experiments). Data presented as mean ± SEM. Groups were compared using one-way ANOVA followed by Bonferroni post hoc tests. Figure 2—figure supplement 5—source data 1.Mir128-3p overexpression and inhibition optimization.

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  Figure 3.. MAPK7 regulation of CEBPβ transcriptionally represses mir128-3p. (A) Pri-mir128-1 promotor luciferase activity in H9C2 was increased by long-term hypoxia (N = 4 experiments). (B) Schematic figure showing CEBP consensus binding sites in the intronic promoter region between exon1 and exon2 of R3hdm1 gene conserved in human, rat, and mouse. (C–D) Cebpβ was downregulated in myocardium after 4w-MI (N = 6–8 mice) and ARCMs (N = 4 experiments) stressed by short and long-lasting hypoxia. (E–F) Measurements of Pri-mir128-1, mir128-3p and Irs1 were performed in ARCMs with CEBPβ overexpression (Cebpb o/e) under long-term hypoxia or Cebpb knockdown in response to short-term hypoxia (N = 4–6 experiments). (G) Pri-mir128-1 promotor luciferase activity was augmented by CEBPβ deficiency (N = 3 experiments). (H) MAPK7 (also known as ERK5) phosphorylation and expression were decreased in the myocardium four weeks after MI (N = 7 mice). (I) MAPK7 was activated by acute hypoxic stress, while it was declined by prolonged stimulation in ARCMs (N = 3 experiments). (J–K) qPCR analyses of Cebpb and mir128-3p in Mapk7-deficient or Mapk7-overexpressing (Mapk7 o/e) ARCMs (N = 6 experiments). (L) Chromatin…

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  Figure 3—figure supplement 1.. Hypoxia effect on R3hdm1 expression. Luciferase reporter assay performed on H9C2 cells transfected with pGL3 or pGL3-R3hdm1 promoter (−1.5 kbp of transcription start site TSS downstream of R3hdm1 exon1) demonstrates that R3hdm1 promoter luciferase activity was not affected during different periods of hypoxia. Data were normalized to renilla expression and control (N = 3 experiments). Data presented as mean ± SEM. Groups were compared using one-way ANOVA followed by Bonferroni post hoc tests. Figure 3—figure supplement 1—source data 1.Hypoxia effect on R3hdm1 expression.

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  Figure 3—figure supplement 2.. Mapk7 and Cebpa expression in 4w-MI myocardium. (A) Cebpa transcript expression was not changed in 4w-MI myocardium, while (B) Mapk7 was decreased measured by qPCR (N = 6–8 mice). Data were normalized against 18S expression. Data presented as mean ± SEM. Groups were compared using two-tailed Student’s t-test. Figure 3—figure supplement 2—source data 1.Mapk7 and Cebpa expression in 4w-MI myocardium.

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  Figure 3—figure supplement 3.. Role of CEBPβ in miR128-3p and Irs1 regulation in H9C2 cells. CEBPβ repression of mir128-3p retains Irs1 in H9C2 cardiomyocytes. (A) Cebpb was knocked down more than 70% by siRNA transfection with Lipofectamine LTX (N = 6 experiments). (B) Human Cebpb overexpression (Cebpb o/e) in H9C2 cells showing LAP as the dominant isoform. qPCR detection of mir128-3p and Irs1 in H9C2 cardiomyocytes with (C) Cebpb overexpression under long-term hypoxia or (D) siRNA-mediated Cebpb knockdown under short-term stress (N = 5–6 experiments). (E) Pri-mir128-2 was not affected by Cebpb overexpression or knockdown (N = 4–6 experiments). Data were normalized against 18S expression. Data presented as mean ± SEM. Comparisons between two groups were performed using two-tailed Student’s t-test. Two-way ANOVA followed by Bonferroni post hoc tests was employed as appropriate. Figure 3—figure supplement 3—source data 1.Role of CEBPβ in miR128-3p and Irs1 regulation in H9C2 cells.

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  Figure 3—figure supplement 4.. MAPK7 activation and expression in short and long-term hypoxia. MAPK7 activation was observed in neonatal rat cardiomyocytes (NRCMs) in response to short-term hypoxia, whereas MAPK7 was decreased by prolonged stress. Quantification is provided (N = 3–6 experiments). Data presented as mean ± SEM. One-way ANOVA followed by Bonferroni post hoc tests was employed to compare groups. Figure 3—figure supplement 4—source data 1.MAPK7 activation and expression in short and long-term hypoxia.