Effects Of Adiponectin On Hypoxia-reoxygenated Cardiomyocytes

Effects of adiponectin on hypoxia-reoxygenated cardiomyocytesObjective:The myocardial cells in neonatal SD rats were cultured to establish the hypoxia-reoxygenation model,the pathophysiologic changes of which are similar to those of the myocardial ischemia-reperfusion injury in vivo.The purpose of the study was to investigate the effect of adiponectin on hypoxia-reoxygenation injury in cultured cardiomyocytes and do further research on its protective mechanism.Methods:The myocardial cells were isolated from the neonatal Sprague-Dawley rats by trypsin,and purified by treatment with the differential adhesion technique and the chemical inhibition method, then cultured in the Dulbecco's modified eagle culture medium consisting of 20%fetal bovine serum.The condition of cell growth and spontaneous beating were observed under the inverted microscope.The myocardial sarcomeric actin and cardiac troponin T were identified by immunofluorescent technique.Primary cultured myocardial cells of rats were divided into 6 groups at random:control group,the cells of which were cultured in routine condition;adiponectin group(APN group),the cells of which were incubated with adiponectin for 24 hours;hypoxia/reoxygenation group(H/R group),the cells of which were treated as follows:the normal cultured medium was replaced by hypoxia solution.Then the cells were cultured hermetically for 3 hours.After the hypoxia solution was replaced by reoxygenation solution,the cells were placed in the oxygenated container for 1 hour to establish the hypoxia/reoxygenation model;hypoxia/reoxygenation+ adiponectin group(H/R+APN group),the cells of which were subjected to the hypoxia/reoxygenation injury after the pretreatment of adiponectin for 24 hours; hypoxia/reoxygenation+adiponectin+AraA group(H/R+APN+AraA group),the cells of which were pretreated with APN and AraA for 24 hours before the injury;hypoxia/reoxygenation+ AraA group(H/R+AraA group),the cells of which were preconditioned with AraA for 24 hours before the injury.The indexes for observation were as follows:the cardiocyte morphology and beating rate were observed by inverted microscope and transmission electron microscope;the DNA ladder was examined by agarose gel electrophoresis and the cell apoptosis was demonstrated with flow cytometry.Furthermore,the content of MDA in the myocardial cells and the activity of SOD in the supematant of the culture medium were detected.Results:1.Identification of myocardial cells:The microscope showed that the cells grew into clusters and beat regularly,the frequency of which were between 90 to 120 beats per minute.When the primary antibodies of myocardial sarcomeric actin and troponin T were used,the cells emitted green fluorescence under the fluorescence microscope.2.The morphology of cardiocyte in every group:In the H/R group,the cells grew poorly and the electron microscope showed significant margination of the chromatin and apoptotic bodies. The beating rate was significantly slow in the H/R group(19.50±3.39 beat/min) compared with that of control group(99.83±4.22 beat/min)(p＜0.05).However,most cells in the H/R+APN group grew well,and only a few of the cells appeared chromatin condensation and hyperchromatic nuclei.Compared with H/R,the beating rate was significantly increased(p＜0.05).The cells in the H/R+APN+AraA group beat obviously slower than the H/R+APN group (p＜0.05).The beating rate of cells in the H/R+AraA group was 7.36±2.79 beat/min and the difference was clear when compared with H/R group(p＜0.05).In addition,the cells in APN group beat significantly faster than those of control group,and the difference was obvious(p＜0.05).3.The DNA electrophoresis results:It appeared a large DNA fragment in the control group and APN group.But in the H/R group,it showed the typical DNA ladder,which is the characteristic of apoptosis.And in the H/R+APN group,the DNA ladder disappeared and was close to normal.The DNA electrophoresis of the H/R+APN+AraA group and H/R+AraA group also showed the typical ladder.4.The percentage of apoptosis cells in every group:In the H/R group,the DNA histogram showed a typical apoptosis peak as hyodiplod,and the apoptosis rate was significantly increased when compared with the control group(48.43±4.18%vs 2.30±0.89%,p＜0.05).The apoptosis rate of the H/R+APN group was obviously lower than that of the H/R group(10.93±2.47%vs 48.43±4.18%,p＜0.05).The percentage of apoptosis cells in the H/R+APN+AraA group was 53.32±1.56%,and the difference was significant compared with the H/R+APN group(p＜0.05). The apoptosis rate of the H/R+AraA group was also higher than that of the H/R group(p＜0.05). In addition,The apoptosis rate of the APN group was lower than control group(1.06±0.46%vs 2.30±0.89%,p＜0.05).5.The content of MDA and the activity of SOD:Compared with control group,the MDA content of the H/R group was significantly increased(p＜0.05),and the SOD activity was decreased(p＜0.05).The MDA content of the H/R+APN group was significantly lower than that of the H/R group(p＜0.05),and the SOD activity was higher(p＜0.05).There was no difference between the APN group and the control group in either the MDA content or the SOD activity(p＞0.05).Conclusions:1.Adiponectin can reduce the apoptosis of myocardial cells which is induced by hypoxia-reoxygenation.And this effect can be reversed by AraA,the specific inhibitor of AMPK (AMP-activated protein kinase).2.Adiponectin also has anti-apoptotic effect on normal myocardial cells.3.Adiponectin can reduce the lipid peroxidation induced by hypoxia-reoxygenation,and protect the SOD activity of myocardial cells.Thus,adiponectin has antioxidative effect on cardiomyocytes in the hypoxia-reoxygenation injury.