Abstract: BACKGROUND: Our previous studies found that decreased expression of connexin 43 and its Ser368 phosphorylation after myocardial hypothermic ischemia-reperfusion was closely associated with decreased cardiac conduction velocity and reperfusion arrhythmia. 
OBJECTIVE: To observe the effect of changes in membrane-type matrix metalloproteinase 2, matrix metalloproteinase 2 and collagen type IV on the expression of connexin 43 and its Ser368 phosphorylation and electrical conduction in the myocardial extracellular matrix after hypothermic ischemia-reperfusion.
METHODS: Sixteen Langendorff extracorporeal cardiac perfusion models were successfully established from SD rats and randomly divided into a control group (n=8) and a hypothermic ischemia-reperfusion group (n=8). The control group was balanced perfused with 37 °C Krebs-Henseleit solution for 15 minutes and then continued to be perfused with 37 °C Krebs-Henseleit solution for 90 minutes. The hypothermic ischemia-reperfusion group was balanced perfused with 37 °C Krebs-Henseleit solution for 15 minutes, and then the heart was arrested for 60 minutes by injection of 4 °C Thomas solution. During the cardiac arrest, the periphery was protected by 4 °C Krebs-Henseleit solution. Half-volume 4 °C Thomas solution was reperfused 30 minutes after the arrest. After stopping the arrest, the heart was reperfused with 37 °C Krebs-Henseleit solution for 30 minutes. The occurrence of arrhythmias, rebeating time, and the duration of arrhythmias were recorded from the immediate time point to the end of the reperfusion period. Conduction velocity, absolute inhomogeneity, and inhomogeneity index were measured using the Mapping Lab multi-channel electrophysiological mapping system at the time of balanced perfusion for 15 minutes (T1), reperfusion for 15 minutes/continuous perfusion for 90 minutes (T2), and reperfusion for 30 minutes/continuous perfusion for 105 minutes (T3). The relative expression levels of membrane-type matrix metalloproteinase 2, matrix metalloproteinase 2, collagen type IV, connexin 43, and its Ser368 phosphorylation in ventricular tissue were detected by western blot assay. 
RESULTS AND CONCLUSION: (1) No arrhythmia occurred in the control group. There were six cases of arrhythmia in the hypothermic ischemia-reperfusion group during reperfusion. Rebeating time and duration of arrhythmias were (25.38±12.02) and (158.67±67.68) seconds, respectively. (2) The conduction isochronal diagrams at T1, T2, and T3 in the control group were uniform and regular in direction, and the conduction velocity at T2 and T3 was not different from that at T1 (P > 0.05). The conduction isochronal diagrams at T2 and T3 in the hypothermic ischemia-reperfusion group were uneven and irregular in direction, and the conduction velocity was slower than that at T1 (P < 0.01). The conduction velocity at T2 and T3 in the hypothermic ischemia-reperfusion group was slower than that in the control group (P < 0.01).  Conduction dispersion was greater in the hypothermic ischemia-reperfusion group than that in the control group at T2 and T3 (P < 0.05). (3) Compared with the control group, the protein expressions of membrane-type matrix metalloproteinase 2 and matrix metalloproteinase 2 in the hypothermic ischemia-reperfusion group were increased (P < 0.05 or P < 0.01), and the protein expression levels of type IV collagen, connexin 43 and its Ser368 phosphorylation were decreased (P < 0.05 or P < 0.01). (4) The results indicate that after hypothermic ischemia-reperfusion, myocardial extracellular matrix remodeling may mediate the downregulation of myocardial connexin 43 and its Ser368 phosphorylation, slowed conduction velocity and increased conduction dispersion, thereby increasing the risk of arrhythmia.

Key words: connexin 43, phosphorylated connexin 43, reperfusion arrhythmia, extracellular matrix, membrane-type matrix metalloproteinase 2, matrix metalloproteinase 2, collagen type IV, engineered tissue construction