miR-24 inhibits apoptosis and represses Bim in mouse cardiomyocytes

Abstract

Acute myocardial infarction (MI) involves necrotic and apoptotic loss of cardiomyocytes. One strategy to salvage ischemic cardiomyocytes is to modulate gene expression to promote cell survival without disturbing normal cardiac function. MicroRNAs (miRNAs) have emerged as powerful regulators of multiple cellular processes, including apoptosis, suggesting that regulation of miRNA function could serve a cardioprotective function. In this study, we report that miR-24 (miRNA-24) expression is down-regulated in the ischemic border zone of the murine left ventricle after MI. miR-24 suppresses cardiomyocyte apoptosis, in part by direct repression of the BH3-only domain-containing protein Bim, which positively regulates apoptosis. In vivo expression of miR-24 in a mouse MI model inhibited cardiomyocyte apoptosis, attenuated infarct size, and reduced cardiac dysfunction. This antiapoptotic effect on cardiomyocytes in vivo was partially mediated by Bim. Our results suggest that manipulating miRNA levels during stress-induced apoptosis may be a novel therapeutic strategy for cardiac disease.

Figure 1.

miR-24 is down-regulated early after MI and inhibits apoptosis. (A and B) qPCR of miR-24 was performed on RNA extracted from the BZ and DZ of hearts 24 h, 3 d, and 1 and 4 wk after coronary artery ligation (MI) or sham surgery. (C) In situ hybridization using miR-24 locked nucleic acid (LNA) probe on heart sections from MI and sham operated mice 24 h after surgery. (D) TUNEL staining of infarcted hearts 24 h after coronary artery ligation. Cardiomyocytes were costained with antibody to α-Actinin. DAPI was used for nuclear staining. (E) Percentage of TUNEL-positive cardiomyocytes (CM) at BZ of hearts over time after MI. n = 3 for each time point. (F and G) TUNEL and α-Actinin staining on primary cardiomyocytes transfected with mimic control, miR-24 mimic, or miR-24^mut mimic (F) or transfected with inhibitor control, miR-24 inhibitor, or miR-24^mut inhibitor (G). (H and I) Quantification of percentage of TUNEL-positive cardiomyocytes in F and G. (J and K) Quantification of percentage of Caspase 3–positive cardiomyocytes. All experiments were repeated three times (technical triplicates) with biological triplicates (n = 3). Error bars indicate SEM (*, P < 0.05). Bars, 50 µm.

Figure 2.

Bim is a direct target of miR-24. (A) qPCR of Bim mRNA from cardiomyocytes transfected with control (mock transfection with Lipofectamine 2000 only), miR-24 mimic, or miR-24 inhibitor. (B) Western blot comparing protein levels of Bim in control (mock transfection) and miR-24 mimic– or miR-24 inhibitor–expressing cells. Quantification compared with control (set as 1) is shown above the panel. (C) Conserved miR-24 binding sites in the Bim 3′ UTR and relative luciferase activity (RLA) in HeLa cells expressing miR-24 mimic, miR-24 precursor (in expression vector, pEF–miR-24), or inhibitor compared with control. Base pairs highlighted in color (miR-24 in red and Bim 3′ UTR in green) are seed sequences that are complimentary between miRNA and target. Mutations in miR-24 binding sites for luciferase assay in 3′ UTR are indicated. Controls are set up as 1, indicated by the dashed line. (D) Primary cardiomyocytes (CM) expressing control siRNA or Bim siRNA were stained with AnnexinV, PI, TUNEL, or activated Caspase 3. Dot plots show representative staining by flow cytometry, and arrows indicate the quadrant of AnnexinV⁺PI⁻ apoptotic cells. (E) Primary cardiomyocytes cotransfected with Bim siRNA and miR-24 inhibitor or control inhibitor were stained with AnnexinV, PI, TUNEL, or activated Caspase 3. Dot plots show representative staining by flow cytometry, and arrows indicate the quadrant of AnnexinV⁺PI⁻ apoptotic cells. All experiments were repeated three times (technical triplicates) with biological triplicates (n = 3). Bar graphs show mean ± SEM (*, P < 0.05).

Figure 3.

In vivo delivery of miR-24 inhibits apoptosis. (A and B) Immunohistochemistry of control mimic– or miR-24 mimic–treated heart sections marking TUNEL-positive cardiomyocytes costained with α-Actinin antibody within the BZ of infarcted hearts. Quantification is shown in B. (C) Quantification for activated Caspase 3⁺ cardiomyocytes (CM) at BZ. (D) Representative pictures of Evans blue/TTC staining on four continuous slices of LV from representative hearts of control mimic– or miR-24 mimic–injected mouse. (E) Blinded quantification of size of the AAR and infarct size as described in Materials and methods. All staining in this figure was performed on hearts 24 h after MI. Measurements were repeated three times (technical triplicates), with biological sample size indicated in each panel. The mean number from technical triplicates was used for statistical calculation. Error bars indicate SEM (*, P < 0.05). Bars: (A) 50 µm; (D) 500 µm.

Figure 4.

In vivo delivery of miR-24 blunts effects of MI. After MI, miR-24 or control mimic was injected along the BZ of infarct. (A) EF, SV, and cardiac output (CO) of the LV were measured by MRI 12 wk after MI. (B) M-mode echocardiography of representative hearts 3 d after MI. (C) Fractional shortening (FS), EF, and cardiac output at varying time points were measured using high-resolution echocardiography. Data were collected 1 d before and 3 d, 4 wk, and 12 wk after MI in a blinded fashion. (D) qPCR of ANF and BNP on RNA extracted from BZ of MI hearts 24 h after injection of control or miR-24 mimic. (E) Stress-responsive miRNAs were measured by qPCR 24 h after MI. Data in D and E are shown relative to control, indicated by dashed lines. (F and G) Azan staining was performed on heart sections 4 wk after MI with miR-24 or control mimic injected. RV, right ventricle. Bars, 500 µm. (H and I) Quantification of scar size was calculated by measurement of both scar area (H) and scar circumference (I). (J) Western blots for activated Caspase 3, Caspase 12, activated Caspase 12, Caspase 9, Bim, and GAPDH in protein extracts from control mimic– or miR-24 mimic–injected hearts 24 h after MI, as well as hearts without MI (sham). Relative quantification of Western blots is shown to the right of each panel; comparison was made between control mimic (set as 1) and miR-24 mimic after MI. The vertical black line indicates that intervening lanes have been spliced out. For all histograms, sample size (n) is indicated for each group. Each experiment was repeated three times (technical triplicates), and the mean number was used for statistical analyses. Error bars indicate SEM (*, P < 0.05; **, P < 0.01).

Figure 5.

In vivo inhibition of Bim reduces miR-24 inhibitor–induced apoptosis. (A and B) After MI, control or Bim siRNA was injected along the BZ of infarct. Immunohistochemistry of heart sections labeling TUNEL-positive cardiomyocytes, marked by α-Actinin antibody within the BZ. DAPI indicates nuclei. Quantification of TUNEL⁺ or activated Caspase 3⁺ cells is shown in B. (C and D) Cardiomyocyte (CM) apoptosis was determined by TUNEL and activated Caspase 3 stainings on heart sections at the BZ of infarcted hearts with miR-24 or control inhibitor injected. (E) Western blots for activated Caspase 3, Caspase 12, activated Caspase 12, Caspase 9, Bim, and GAPDH in protein extracts from control inhibitor– or miR-24 inhibitor–injected hearts 24 h after MI, as well as hearts without MI (sham). Relative quantification of Western blots is shown to the right of each panel; comparison was made between control inhibitor (set as 1) and miR-24 inhibitor after MI. The vertical black line indicates that intervening lanes have been spliced out. (F) TUNEL stainings on MI heart sections coinjected with Bim siRNA or control siRNA and miR-24 inhibitor. (G) Quantification of TUNEL⁺ or activated Caspase 3⁺ cardiomyocytes. All experiments were repeated three times (technical triplicates), with biological duplicates indicated in each panel. Error bars indicate SEM (*, P < 0.05; **, P < 0.01). All data in this figure were collected 24 h after MI. Bars, 50 µm.