Effect Of Hypothermic Hypoxic Reoxygenation Cardiac Fibroblasts On Viability And Expression Of Connexin 43 In Neonatal Rat Cardiomyocytes In Vitro In Transwell System

Objective:The study is aimed to observe the effect of hypothermic hypoxic reoxygenation cardiac fibroblasts on beating frequency,apoptosis rate,Cx43-m RNA,Cx43 protein and phosphorylated Cx43 of cardiomyocytes after Transwell cultured with neonatal rat cardiomyocytes in vitro,which will lay a foundation for the study of antiarrhythmic effect of cardiac fibroblasts.Methods:10 healthy adult SD rats(5 males and 5 females),weighing 280 ± 20 g,were mated to breed neonatal rats.The ventricular tissues of 1-3 days old SD neonatal rats were cut and collected,and the cardiac fibroblasts and cardiomyocytes from neonatal SD rats were isolated and purified by differential adherent method for primary culture.Immunofluorescence was used to identify cardiomyocytes specific actin and cardiac fibroblasts specific vimentin,and the purity of cardiomyocytes and cardiac fibroblasts was determined.Part of fibroblasts were treated with hypothermia hypoxia(1 hour)and reoxygenation(4 hours),then cardiomyocytes and cardiac fibroblasts were divided into two groups for experiment: In control group(Group C),cardiomyocytes and normal cardiac fibroblasts were Transwell cultured for 20 hours;In hypoxia-reoxygenation group(Group HR),cardiomyocytes and hypothermic hypoxic and reoxygenated cardiac fibroblasts were Transwell cultured for 20 hours.The microporous membrane in Transwell chamber can physically isolate cardiac fibroblasts from cardiomyocytes and avoid the contact between the two kinds of cells,but the culture medium and cell secretions can pass through the microporous membrane freely.After Transwell culture,the beating frequency of cardiomyocytes in the two groups was observed under inverted microscope,and the apoptosis rate of cardiomyocytes in Group C and Group HR were detected by flow cytometry.The survival rate of cardiomyocytes were detected by MTT method.The expression level of Cx43-m RNA was analyzed by real-time PCR,and the relative expression levels of connexin 43 protein and phosphorylated connexin 43 protein were analyzed by Western blotting.Results:The purity of cardiomyocytes and cardiac fibroblasts was 94% and 92%respectively,which were identified by actin and vimentin immunofluorescence.The beating frequency of cardiomyocytes in Group HR was slower than that in Group C(P < 0.05).The results of flow cytometry showed that compared with Group C,the apoptosis rate of cardiomyocytes in Group HR increased(P < 0.05).The survival rate of cardiomyocytes in Group HR decreased via MTT method(P < 0.05).PCR results showed that the expression of Cx43-m RNA in Group HR was lower than that in Group C(P < 0.05).Western blotting results showed that compared with Group C,the expression levels of connexin 43 total protein and phosphorylated connexin 43 were decreased in Group HR(P < 0.05).Conclusions:Without direct contact with cardiomyocytes,the cardiac fibroblasts of neonatal SD rats were treated with hypothermia hypoxia reoxygenation,and then Transwell cultured with cardiomyocytes,which could slow down the beating frequency of cardiomyocytes and reduce the survival rate of cardiomyocytes,increase the apoptosis of cardiomyocytes,and also decreased the expression of Cx43-m RNA,Cx43 total protein and phosphorylated Cx43.