Ischemic Postconditioning Protects against Aged Myocardial Ischemia/Reperfusion Injury by Transcriptional and Epigenetic Regulation of miR-181a-2-3p

Figure 2

IPostC inhibits aged myocardial I/R injury through miR-181a-2-3p. (a) The cluster heat map showed the differentially expressed miRNAs in aged cardiomyocytes after HPostC (fold , ). Red and blue indicate the miRNA expression level. The original expression values of miRNAs were normalized using -score normalization. (b) The detailed schematic of miR-181a-2-3p from miRNA sequencing analysis. (c, d) The expression of miR-181a-2-3p was validated by qRT-PCR both in aged cardiomyocytes () after HPostC and in aged myocardium () after IPostC. (e) Western blot analysis of LC3B-II and p62 expression in aged cardiomyocytes transfected with miR-181a-2-3p mimic or inhibitor after HPostC in the presence of CQ (). (f) Predicted binding of miR-181a-2-3p to AMBRA1 3-UTR by TargetScan (http://www.targetscan.org/vert_72/). (g) The reporter constructs containing the wild type (WT) and mutant type (Mut) 3-UTR regions of AMBRA1 were transfected into HEK293 cells with miR-181a-2-3p mimic or inhibitor. Relative luciferase activities were normalized by Renilla luciferase activities (). (h) AMBRA1 expression was detected by Western blot both in aged myocardium after IPostC (left, ) and aged cardiomyocytes after HPostC (right, ). (i) Western blot analysis of AMBRA1 expression in aged cardiomyocytes transfected with miR-181a-2-3p mimic or inhibitor after HPostC (). (j) The expression of LC3B-II and p62 was detected by Western blot in aged cardiomyocytes transfected with sh-AMBRA1 in the presence of CQ (). (k) Fluorescent GFP-LC3 and RFP-LC3 puncta in aged cardiomyocytes transfected with sh-AMBRA1 after HPostC. Numbers of autophagosomes and autolysosomes in each cell were quantified (scale  μm) (). Data were presented as the .

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