Background Reperfusion arrhythmia (RA) is a relatively common complication of myocardial ischaemia-reperfusion injury during extracorporeal circulation endocardial direct vision surgery, and it is of great significance to explore the pathogenesis of RA.

Objective To investigate the protective effect of hypoxic preconditioned H9C2 cell conditioned medium on hypothermic hypoxia-reoxygenation H9C2 cardiomyocytes, so as to provide experimental support for the reduction of RA.

Methods H9C2 cells were randomly divided into five groups, negative control group (equal volume of DMEM medium containing 10% fetal bovine serum cultured normal cardiomyocytes, Group C), hypoxia group (hypoxic conditioned medium cultured with hypothermic H/R cardiomyocytes, Group H), normoxia group (normoxic conditioned medium cultured with hypothermic H/R cardiomyocytes, Group N), recombinant protein MMP-2 group (200 ng/mL recombinant protein MMP-2 medium cultured with hypothermic H/R cardiomyocytes, MMP-2 group), and MMP-2 inhibitor group (0.03 mol/L ARP-100 medium cultured with hypothermic H/R cardiomyocytes, ARP-100 group). Cardiomyocyte viability was detected by CCK8 assay; cardiomyocyte apoptosis rate was detected by Hoechst33342 staining and Annexin V/PI double staining by flow cytometry; gap junction intercellular communication was detected by scrape-loading dye transfer technique; MMP-2 activity was detected by gelatin zymography in the cell culture medium; MMP-2, Cx43 and p-Cx43 protein expression were detected by Western blot; and the expression of Cx43 on the cardiomyocyte membranes was detected by immunofluorescence.

Results Compared with the group C, cardiomyocyte viability in the N group was decreased (P<0.01), apoptosis was increased (P<0.01), gap junction intercellular communication was attenuated, MMP-2 expression was upregulated (P<0.01), while both Cx43 and p-Cx43 expression were downregulated (P<0.01), and Cx43 fluorescence intensity was attenuated on cardiomyocyte membranes (P<0.05). Compared with the N group, cardiomyocyte viability in the H group was elevated (P<0.01), apoptosis was reduced (P<0.01), gap junction intercellular communicatio was enhanced, MMP-2 activity was decreased (P<0.05), MMP-2 expression was downregulated (P<0.01), both Cx43 and p-Cx43 expression were upregulated (P<0.01), and Cx43 fluorescence intensity was enhanced on cardiomyocyte membranes (P<0.05). Compared with the MMP-2 group, cardiomyocytes Cx43 and p-Cx43 expression in the H group and ARP-100 group were upregulated (P<0.01), Cx43 fluorescence intensity on the cardiomyocyte membranes was enhanced (P<0.01), with enhanced gap junction intercellular communication.

Conclusion Hypoxia preconditioned H9C2 cell conditioned medium improves gap junction intercellular communication between hypothermic H/R cardiomyocytes, reduces cardiomyocyte apoptosis, and enhances cardiomyocyte activity through inhibition of MMP-2 activity-mediated upregulation of Cx43 and p-Cx43 expression.