Role of miR-1 and miR-133a in myocardial ischemic postconditioning

Abstract

Background: Ischemic postconditioning (IPost) has aroused much attention since 2003 when it was firstly reported. The role of microRNAs (miRNAs or miRs) in IPost has rarely been reported. The present study was undertaken to investigate whether miRNAs were involved in the protective effect of IPost against myocardial ischemia-reperfusion (IR) injury and the probable mechanisms involved.

Methods: Thirty SD rats weighing 250-300 g were equally randomized to three groups: Control group, where the rats were treated with thoracotomy only; IR group, where the rats were treated with ischemia for 60 min and reperfusion for 180 min; and IPost group, where the rats were treated with 3 cycles of transient IR just before reperfusion. The extent of myocardial infarction, LDH and CK activities were measured immediately after treatment. Myocardial apoptosis was detected by TUNEL assay. The myocardial tissue was collected after IR or IPost stimulation to evaluate the miRNAs expression level by miRNA-microarray and quantitative real-time RT-PCR. Real-time PCR was conducted to identify changes in mRNA expression of apoptosis-related genes such as Bcl-2, Bax and Caspase-9 (CASP9), and Western blot was used to compare the protein expression level of CASP9 in the three groups. The miRNA mimics and anti-miRNA oligonucleotides (AMO) were transferred into the cultured neonatal cardiomyocytes and myocardium before they were treated with IR. The effect of miRNAs on apoptosis was determined by flow cytometry and TUNEL assay. CASP9, as one of the candidate target of miR-133a, was compared during IR after the miR-133a mimic or AMO-133a was transferred into the myocardium.

Results: IPost reduced the IR-induced infarct size of the left ventricle, and decreased CK and LDH levels. TUNEL assay showed that myocardial apoptosis was attenuated by IPost compared with IR. MiRNA-microarray and RT-PCR showed that myocardial-specific miR-1 and miR-133a were down-regulated by IR, and up-regulated by IPost compared with IR. Furthermore, IPost up-regulated the mRNA expression of Bcl-2, down-regulated that of Bax and CASP9. Western blot showed that IPost also down-regulated the CASP9 protein expression compared with IR. The results of flow cytometry and TUNEL assay showed that up-regulation of miR-1 and miR-133a decreased apoptosis of cardiomyocytes. MiR-133a mimic down-regulated CASP9 protein expression and attenuated IR-induced apoptosis.

Conclusion: MiRNAs are associated with the protective effect of IPost against myocardial IR injury. IPost can up-regulate miR-1 and miR-133a, and decrease apoptosis of cardiomyocyte. Myocardial-specific miR-1 and miR-133a may play an important role in IPost protection by regulating apoptosis-related genes. MiR-133a may attenuate apoptosis of myocardiocytes by targeting CASP9.

Figure 1

IPost reduced the IR-induced infarct size of LV. (A) Representative mid-myocardial crosssections of TTC-stained hearts for IR and IPost. Dark blue area, nonischemic zone; remaining area, AAR; white area, infracted tissue; red area, viable myocardium. (B) AAR/LV was similar between IR and IPost groups. IPost significantly attenuated myocardial INF/LV and INF/AAR compared with IR (n = 10, *P < 0.05, compared with IR group).

Figure 2

LDH and CK assay of blood serum. The activities of CK and LDH were increased by IR, and IPost decreased them compared with IR (n = 10, *P < 0.05, compared with Con group; ^▲P < 0.05, compared wit IR group).

Figure 3

TUNEL assay. (A) TUNEL staining pictures, in which brown staininged cells were TUNEL positive cells (magnification, × 400). (B) The percent of TUNEL positive cells in the heart. TUNEL positive cells were increased by IR, and decreased by IPost (n = 10, *P < 0.05, compared with Con group; ^▲P < 0.05, compared wit IR group).

Figure 4

MiRNA-microarray compaired between Control and IR groups. 16 miRNAs were dysregulated by IR, of which 10 miRNAs were up-regulated and the other 6 miRNAs were down-regulated significantly. The green signal is labeled with cy5 and the red signal was labeled by cy3 (green: cy3 >cy5; yellow: cy3 = cy5; red: cy3 <cy5).

Figure 5

Regulation of miR-1 and miR-133a by IPost. MiR-1 and miR-133a were down-regulated in IR group, while IPost up-regulated them as compared with IR group (n = 10, *P < 0.05, compared with Con group; ^▲P < 0.05, compared wit IR group).

Figure 6

Regulation of apoptosis-related gene mRNA by IPost. Compared with Control group, IR increased the mRNA expression of Bax and CASP9. While IPost increased Bcl-2 mRNA expression, and decreased Bax mRNA expression (n = 10, *P < 0.05, compared with Con group; ^▲P < 0.05, compared with IR group).

Figure 7

The protein expression of CASP9 was regulated by IPost. (A) Western blot of CASP9 in different groups. (B) The relative quantity of CASP9 protein in different groups. IR up-regulated CASP9 protein compared with Con group, and IPost down-regulated CASP9 protein compared with IR. (n = 10, *P < 0.05, compared with Con group; ^▲P < 0.05, compared with IR group)

Figure 8

The expression of miR-133a and CASP9 protein after transferring the mimic or AMO. (A) Relative expression of miR-133a in different groups. MiR-133a was down-regulated by AMO-133a, and up-regulated by miR-133a mimic (n = 10, *P < 0.05, compared with IR group; ^▲P < 0.05, compared with AMO-133+IR group); (B) The relative quantity of CASP9 protein in different groups. AMO-133a up-regulated CASP9 protein, and miR-133a mimic down-regulated it(n = 10, *P < 0.05, compared with IR group).

Figure 9

MiR-133a mimic attenuates myocardiocyte apoptosis in vivo. (A) TUNEL staining pictures, in which brown stained cells were TUNEL positive cells (magnification, × 400). (B) The percent of TUNEL positive cells in the heart. MiR-133a mimic decreased the apoptosis ratio induced by IR, while AMO-133a increased the apoptosis ratio (n = 10, *P < 0.05, compared with IR group).

Figure 10

Representative diagrams of the flow cytometric readings for myocardiocytes stained with annexin V and propidium iodide (PI). (A) IR. (B) miR-1 mimic+ IR. (C) AMO-1 +IR. (D) miR-133a mimic+ IR. (E) AMO-133a inhibitor +IR. (F) The percentage of apoptosis induced by IR in each group. MiR-1 promoted cell aopoptosis during IR, but miR-133a inhibited cell apoptosis during IR. (*P < 0.05, compared with IR group compared with IR group)