MicroRNA-221 protects myocardial contractility in myocardial ischemia/reperfusion injury through phospholamban

Figures

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  Fig 1. The level of miRNA 221 in the myocardium of MIRI mice was decreased. The myocardial tissues used for following detections were respectively treated with MIRI, MIRI + miRNA 221 agomir, MIRI+ negative control sequence. RT-PCR showed miRNA 221 levels were significantly diminished in the MIRI group. The results were presented by mean ± standard deviation and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test (n = 6).

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  Fig 2. miRNA 221 alleviated myocardial injury and inhibited apoptosis and necrosis of cardiac cells in MIRI mice. (A) HE staining, Masson’s trichrome staining and TUNEL staining of mouse heart tissue sections. Scale bar: 250 μm. (inset: higher magnification; scale bar: 50 μm). (B) The myocardial fibrotic area of myocardial tissue of mice. The results were presented by mean ± standard deviation and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test (n = 6). (C) The TUNEL- positive cells of myocardial cells. The results were presented by mean ± standard deviation and analyzed by Brown-Forsythe and Welch ANOVA tests followed by Dunnett’s T3 multiple comparisons test. (n = 6).

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  Fig 3. miRNA 221 improved left ventricular contractile function in MIRI mice. (A) Representative M-mode left ventricular echocardiographic images of mice. (B-E) In vivo assessment of heart function by echocardiography. The left ventricular ejection fraction (LVEF; B), left ventricular shortening fraction (LVFS; C), diastolic left ventricular internal diameter (LVIDd; D), systolic left ventricular internal diameter (LVIDs; E), Left Ventricular End-Diastolic Volume (LEDV; F), Left Ventricular End-Systolic Volume (LESV; G) of mice. The results were presented by mean ± standard deviation and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test (n = 6).

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  Fig 4. Altered expression of genes related to Ca2+ cycling or myocardial contraction. (A) mRNA expression of miR221, PLB, SERCA2, RYR2 and NCX1 in myocardial tissue of mice by RT-qPCR (n = 6). (B-C) Protein expression of PLB, p-PLB (Ser16), p-PLB (Thr17), SERCA2, RYR2 and NCX1 in the myocardial tissue of mice by western blot analysis (n = 3). The results were presented by mean ± standard deviation and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test.

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  Fig 5. PLB is a target gene of miRNA 221. (A) The predicted binding sites of miRNA 221 on the 3′UTR sequence of PLB gene. (B) The luciferase activity of PGL3-control, PGL3-WT-PLB and PGL3-mut-PLB. Co-transfection of miRNA 221 mimics in HEK293 cells inhibited the activity of a luciferase reporter containing 478bp 3′UTR of mouse PLB gene. Mutations in the predicted miRNA 221 binding sites in the PLB 3’UTR eliminated the inhibitory effects of miRNA 221 mimics. The results of luciferase activity were presented by mean ± standard deviation and analyzed using the two-tailed unpaired Student’s t-test (n = 5).

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  Fig 6. miRNA 221 alleviated calcium overload in myocardium and inhibited the expression of apoptosis-related genes in MIRI mice. (A) The calcium content in the myocardium of mice (n = 6). The results were presented by mean ± standard deviation and analyzed by the Brown-Forsythe and Welch ANOVA tests followed by Dunnett’s T3 multiple comparisons test. (B) mRNA expression of caspase3 and Cyt C in myocardial tissue of mice by RT-qPCR (n = 6). (C-D) Protein expression of caspase3 and Cyt C in the myocardial tissue of mice by western blot analysis (n = 3). The results were presented by mean ± standard deviation and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test.