Effect Of Hypoxic Preconditioning On Connexin43Expression And Function In Isolated Myocardial Cells

Objective:To investigate the effect of hypoxic preconditioning on connexin43(Cx43) expression and its function in isolated myocardial cells by establishing the model of hypoxic/reoxygenation and reperfusion.To clarify the molecular mechanism of ischemic preconditioning (IPC) on ischemia-reperfusion (I/R) injury. By doing this, we hope that it lay the foundation of clinical application of ischemic preconditioning and of research and development of new drugs for ischemia-reperfusion injury.Method:Ventricular myocytes from2-old-day SD neonatal rats were isolated and cultured in vitro by Cell Culture Technique. After96hours of culturing, the cardiocytes were randomly divided into three groups:control group, hypoxic/reoxygenation-reperfusion group and hypoxia preconditioning (HPC) group. Then we observed the growth of the ventricular myocytes, investigated the gap junction communication (GJC) activity with Scrape-loading and dye transfer technique (SLDT), and examined the expression level of total Cx43and phosphorylated Cx43in myocardial cells by semiquantitative Western blot assay.Results:1. From the growth of the ventricular myocytes, we found that the beating rate was significantly slow in hypoxic/reoxygenation and reperfusion group (50±6beats/min) compared with that of control group (76±7beats/min),(P<0.01), or compared with that of HPC goup(70±5beats/min)(P<0.01). we did not find significant difference in beating rate between HPC group and control group (P>0.05), The viability of ventricular myocytes in hypoxic/reoxygenation and reperfusion group was significantly decreased when compared with that of control group(P<0.01); the viability of ventricular myocytes in HPC group was far better than in hypoxic/reoxygenation and reperfusion group (P<0.01); and the cell viability among groups were significant difference when it comes to multiple comparison.(P<0.01).2. The GJC activity of Cx43was determined by SLDT technique. A reduced number of Lucifer Yellow coupled cell layers was observed on either side of the scrape in cells of hypoxic/reoxygenation and reperfusion group compared with cells of control group. In the hypoxic/reoxygenation and reperfusion group, the Lucifer Yellow coupled cell layers can transport to the first or second layers from either side of the scrape. While in HPC group or control group, it can transport to the fourth layers or beyond.3. Western blotting assay showed that the expression level of total connexin43and phosphorylated Cx43in HPC group was obviously higher than that of hypoxic/reoxygenation and reperfusion group (P <0.01). There were no significant difference between HPC group and control group (P>0.05).Conclusion:The Cx43protein and phosphorylated Cx43expression level of Ventricular myocytes was continued by hypoxia preconditioning, which means less hypoxic/reoxygenation and reperfusion to induce dephosphrylation of Cx43. Consequently, the structure and function of Cx43and Gap junction was conserved, these activities will help myocardial cells resist ischemia-reperfusion (I/R) injury.