miR-21 enhances the protective effect of loperamide on rat cardiomyocytes against hypoxia/reoxygenation, reactive oxygen species production and apoptosis via regulating Akap8 and Bard1 expression

Abstract

Effective therapies to reduce ischemia/reperfusion and hypoxia/reoxygenation injury are currently lacking. Furthermore, the effects of loperamide and microRNA (miR)-21 on hypoxia/reoxygenation injury of cardiomyocytes have remained to be elucidated. Therefore, the present study aimed to investigate the effect of loperamide and miR-21 on cardiomyocytes during hypoxia/reoxygenation injury, and to explore the underlying molecular mechanisms. H9c2 rat cardiomyocytes were pre-treated with loperamide prior to hypoxia/reoxygenation. The viability of H9c2 cells was measured with a cell counting kit 8 and apoptosis was detected with an Annexin V-phycoerythrin/7-aminoactinomycin D apoptosis kit. Furthermore, reactive oxygen species were detected with a specific kit. Genes regulated by miR-21 were screened with an mRNA chip and confirmed using reverse-transcription quantitative polymerase chain reaction analysis. The direct targeting relationship of miR-21 with certain mRNAs was then confirmed using a Dual-Luciferase Reporter Assay system. The results indicated that the apoptotic rate and reactive oxygen species levels in rat cardiomyocytes were markedly increased after hypoxia/reoxygenation treatment. Pre-treatment with loperamide significantly protected H9c2 cells against apoptosis and reactive oxygen species production after hypoxia/reoxygenation. The protection was markedly decreased by miR-21 inhibitor and enhanced by miR-21 mimics. Screening for genes associated with cardiomyocyte apoptosis revealed that the relative expression of A-kinase anchoring protein 8 (Akap8) and BRCA1 associated RING domain 1 (Bard1) was consistent with the experimental results on apoptosis and reactive oxygen species. Compared with the group treated by hypoxia/reoxygenation alone, pre-treatment with loperamide markedly decreased the expression of BRCA1-interacting protein C-terminal helicase 1, Akap8 and Bard1 after hypoxia/reoxygenation. The decrease in the expression of Akap8 and Bard1 was markedly attenuated by miR-21 inhibitor and enhanced by miR-21 mimics. miR-21 mimics directly targeted the 3'-untranslated region (UTR) of Akap8 and Bard1 mRNA to thereby decrease their expression. In conclusion, the protection of rat cardiomyocytes against hypoxia/reoxygenation-induced apoptosis and reactive oxygen species production by loperamide was markedly enhanced by miR-21. miR-21 directly targets the 3'-UTR of Akap8 and Bard1 mRNA and enhances the inhibitory effects of loperamide on Akap8 and Bard1 expression in rat cardiomyocytes after hypoxia/reoxygenation.

Keywords: cardiomyocytes; hypoxia; loperamide; miR-21; reoxygenation.

Figure 1.

Pre-treatment with loperamide (50 and 100 nm) increases the viability of rat cardiomyocytes after H/R. H9c2 rat cardiomyocytes were pre-treated with 0, 10, 50, 100 or 200 mM loperamide for 24 h prior to hypoxia/reoxygenation treatment. The viability of H9c2 cells was measured with a cell counting kit-8 cell viability assay. After H/R treatment, the viability of H9c2 cells was significantly decreased, which was significantly inhibited pre-treatment with 50 or 100 nm loperamide. Cells treated with 50 nm loperamide had the highest viability. Therefore, 50 nm loperamide was used in subsequent experiments. Values are expressed as the mean ± standard error of the mean (n=3/group). ***P<0.001 vs. control group; ^##P<0.01, ^###P<0.001 vs. H/R group. H/R, hypoxia/reoxygenation.

Figure 2.

(A) Representative plot images of flow cytometry. (B) Statistical analysis of cell apoptotic rate. miR-21 inhibitor decreases and miR-21 mimics enhance the protective effect of loperamide against H/R-induced apoptosis of H9c2 rat cardiomyocytes. Cell apoptosis was detected with an Annexin V-PE/7-AAD apoptosis assay kit and flow cytometric analysis. The apoptotic rate was obtained by quantification of early (Q3) and late (Q2) apoptotic cells. The apoptotic rate of rat cardiomyocytes was significantly increased after hypoxia/reoxygenation treatment, as compared with that in the control group. Pre-treatment with loperamide significantly protected H9c2 cells against apoptosis after H/R. The protective effect was markedly decreased by miR-21 inhibitor and enhanced by miR-21 mimics. Values are expressed as the mean ± standard error of the mean (n=3/group). ***P<0.001 vs. control group; ^###P<0.001 vs. H/R group; ⁺⁺⁺P<0.001 vs. H/R + loperamide group. H/R, hypoxia/reoxygenation; miR, microRNA; PE, phycoerythrin; 7-AAD, 7-aminoactinomycin D; Q2, quadrant 2.

Figure 3.

miR-21 inhibitor decreases and miR-21 mimics enhance the protective effect of loperamide against H/R-induced reactive oxygen species production in H9c2 rat cardiomyocytes. (A) Representative images for the detection of reactive oxygen species (scale bar, 100 µm). (B) Quantified levels of reactive oxygen species in the different experimental groups. Reactive oxygen species were detected with a reactive oxygen species assay kit. The reactive oxygen species levels in rat cardiomyocytes were significantly increased after H/R treatment, as compared with those in the control group. Compared with the group treated by H/R alone, pre-treatment with loperamide markedly decreased reactive oxygen species in H9c2 cells after H/R. The decrease was markedly alleviated by miR-21 inhibitor and enhanced by miR-21 mimics. Values are expressed as the mean ± standard error of the mean (n=3/group). ***P<0.001 vs. control group; ^###P<0.001 vs. H/R group; ⁺⁺P<0.01, ⁺⁺⁺P<0.001 vs. H/R + loperamide group. H/R, hypoxia/reoxygenation; IOD, integrated optical density; miR, microRNA.

Figure 4.

Akap8 and Bard1 expression is regulated by miR-21, and the inhibitory effects of loperamide on Akap8 and Bard1 expression in H9c2 rat cardiomyocytes after H/R are enhanced by miR-21. Following loperamide pre-treatment and H/R, miR-21-regulated genes were screened by mRNA chip. A pathway enrichment analysis was applied to screen out genes associated with cardiomyocyte apoptosis: Brip1, Akap8, Rad51b, Hspa14, Bard1, MXD4 and MTHFD1. The results of the mRNA chip analysis were confirmed by reverse transcription quantitative polymerase chain reaction. The relative expression of Akap8 and Bard1 was consistent with the results on apoptosis and reactive oxygen species in the experimental groups. The expression of Brip1, Akap8, Rad51b, Hspa14 and Bard1 in rat cardiomyocytes was significantly increased after H/R treatment, as compared with that in the control group. Compared with the group treated with H/R alone, pre-treatment with loperamide markedly decreased the expression of Brip1, Akap8 and Bard1 in H9c2 cells after H/R. The decrease in expression of Akap8 and Bard1 was markedly alleviated by miR-21 inhibitor and enhanced by miR-21 mimics. Values are expressed as the mean ± standard error of the mean (n=3/group). *P<0.05, ***P<0.001 vs. control group; ^###P<0.001 vs. H/R group; ⁺⁺P<0.01, ⁺⁺⁺P<0.001 vs. H/R + loperamide group. H/R, hypoxia/reoxygenation; Akap8, A-kinase anchoring protein 8; Bard1, BRCA1-associated RING domain 1; Brip1, BRCA1 interacting protein C-terminal helicase 1; Hspa14, heat shock protein family A (Hsp70) member 14; Rad 51b, RAD51 paralog B; MXD4, MAX dimerization protein 4; MTHFD1, methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1; miR, microRNA.

Figure 5.

miR-21 directly targets the 3′-UTR of Akap8 and Bard1 mRNA. Luciferase reporter vectors containing the putative binding sequence in the 3′-UTR of Akap8 (CL853-Gluc-Cluc-AKAP8-3′UTR) or Bard1 (CL852-Gluc-Cluc-BARD1-3′UTR) were constructed. 293 cells were divided into 7 experimental groups. miR-21 mimics or miR-21 inhibitor or the respective controls were co-transfected with reporter vector containing the Akap8 or the Bard1 3′-UTR into 293 cells according to using Lipofectamine^® 2000 reagent. At 36 h after transfection, the luciferase activity in the culture supernatant was measured using the Dual-Luciferase Reporter Assay system. miR-21 mimics decreased and miR-21 inhibitor increased the luciferase activity of the reporter vectors driven by the Adap8 and Bard1 3′-UTRs. Values are expressed as the mean ± standard error of the mean (n=3/group). **P<0.01, ***P<0.001, mimics vs. NC and control group; ^##P<0.001, inhibitor vs. NC and control group. NC, negative control; RLU, relative light units; Akap8, A-kinase anchoring protein 8; Bard1, BRCA1-associated RING domain 1; UTR, untranslated region.

Figure 6.

Predicted consequential pairing of miR-21 and 3′-UTR sequences of Akap8 and Bard1 mRNA. (A) Predicted consequential pairing of miR-21 and a seed sequence from the 3′-UTR of Akap8 mRNA; (B) Predicted consequential pairing of miR-21 and a seed sequence from the 3′-UTR of Bard1 mRNA. UTR, untranslated region; Akap8, A-kinase anchoring protein 8; Bard1, BRCA1-associated RING domain 1; miR, microRNA; rno, rattus norvegicus.