microRNA regulatory mechanisms of mitoKATP in myocardial ischaemia–reperfusion injury in rats

We aimed to set up a rat isolated heart ischaemia–reperfusion model to screen miRNA characteristics and a role for mitochondrial (mitoKATP). Male Sprague–Dawley (SD) rats (250–300 g) were anaesthetised using i.p. sodium pentobarbital and heparin, and hearts were quickly removed. Isolated hearts were set into a Langendorff system with Krebs–Hensleit (K-H) to facilitate ischaemia–reperfusion. The protocol used four groups (n=12): groups N, I/R, DZ, and 5-HD. In group N, hearts were perfused with K-H solution for 120 min continuously. In group I/R, hearts were perfused with K-H solution for 20 min, after which perfusion was stopped for 30 min followed by reperfusion for 70 min. In group DPO, after 20 min perfusion, we stopped the perfusion for 30 min, and then perfused hearts for MitoKATP specific opener DZ for 5 min. In group 5-HD, after 20 min perfusion, we stopped the perfusion for 25 min, and then perfused hearts for mitoKATP specific blocker 5-HD for 5 min. The perfusion flow rate was adjusted during the 20 min stabilisation period to maintain an average perfusion pressure between 60 and 70 mm Hg. Except for group N, the rest of the groups were maintained at room temperature or 32°C during the irrigation period, and the mean perfusion pressure was maintained between 60 and 70 mm Hg during perfusion. The following measurements were made. (1) Cardiac haemodynamic indexes were recorded at 20 min perfusion and the end of reperfusion. (2) At the end of reperfusion, myocardial infarct size was detected by TTC. (3) At the end of reperfusion, deenzyme cryopreservation tube was used to store myocardial tissue, after mRNA and miRNA fragmentation, and sequencing libraries were constructed and sequenced using the Illumina sequencing platform. (4) miRNAs expression by quantitative reverse transcription PCR (RT–qPCR). (5) Analysis of the biological regulatory pathways and networks was performed. At the end of the stabilisation period, with the exception of the HR group, there was a statistical difference in cardiac haemodynamic parameters (P<0.05). When compared with group N, infarct size measured by TTC increased in group I/R (P<0.05); compared with group I/R, myocardial infarct size of group DZ decreased (P<0.05); Compared with group DZ, myocardial infarct size of group 5-HD increased (P<0.05). In the miRNA sequencing results, compared with group N, the number of mir ‘up-regulations’ in group I/R was 192 and the number of mir ‘down-regulations’ was 77; compared with group I/R, the number of mir ‘up-regulations’ in group DZ was 118 and the number of mir ‘down-regulations’ was 48; compared with group DZ, the number of mir up-regulations in group 5-HD was 37 and the number of mir ‘down-regulations’ was 123. (4) Eight mi-RNAs with high expression and distinct differences were selected using RT–qPCR: rno-miR-30c-5p,rno-miR-30d-5p, rno-miR-128-3p, rno-miR-148a-3p, rno-miR-328a-3p, rno-miR-676, rno-miR-novel-chr1-36162, and rno-miR-novel-chr5_59812; their involvement in possible regulatory pathways and networks were further analysed using Gene Ontology and KEGG Pathway databases. We suggest that DZ may reduce myocardial IRI via opening of mitoKATP.