The Research Of Electrophysiological Properties During Islet-1 Induce C3H10T1/2 Differentiation Into Cardiomyocytes

ObjectiveC3H10T1/2 cells were infected with Islet-1 lentiviral vector, and made it overexpress Islet-1. Overexpressing Islet-1 could specifically induce C3H10T1/2 cells differentiation into cardiomyocyte-like cells. The expression of ion channel genes and ion channel currents in experimental group were detected respectively by RT-PCR and Whole-cell patch clamp. Analysis of Islet-1 can induce C3H10T1/2 to differentiate into cardiomyocytes with electrophysiological function.MethodsC3H10T1/2 cells were cultured and transplanted in to 24-well plate within 1×104 cells per well. Islet-1 lentiviral vector was added into each well when the cells were overspread 60%-70%. The expression of GFP and morphological changes were observed by fluorescence microscopy. Transfection efficiency was examined by flow cytometry. The expression of Islet-1 was detected by western blot. Neonatal mouse cardiomyocyte was isolated and cultured by differential adhesion method in vitro, and identified with immunofluorescence. The expressions of sodium, potassium and calcium ion channel encoding genes were detected by RT-PCR. Whole cell patch clamp was used to record sodium, potassium and calcium channel currents. All these data in experimental group (C3H10T1/2 cells with Islet-1 vector) were compared with those in blank group (untreated C3H10T1/2 cells), negative group (blank lentiviral vectors), and positive group (neonatal mouse cardiomyocytes).Results1. The expression of GFP was detected by fluorescence microscope after transfected 96h. Lentiviral transfection efficiency of negative group was detected by flow cytometry was 89%, while the experimental group was 74%. The cells of blank and negative control group arranged disorderly, no obvious morphological difference, while the cells of experimental group showed tightly packed, the direction of convergence and the shuttle-type morphology, which was similar to myocardial cells after 4 weeks of lentivirus infection. The expressions of Islet-1 in experimental group were stably detected by western blot during cultured 4 weeks.2. Neonatal mouse cardiomyocyte was isolated and cultured by differential adhesion method. Neonatal mouse cardiomyocyte were adherent growth and had visible single cell beating (43-46 beats/min) in 24h. After 72h the cells began to fuse and achieved self-synchronization beating (50-110 beats/min). Detected with immunofluorescence, there were about 90%cells expressing cTnT and CX43, which can be used as positive control group in this research.3. Expression of ion channel encoding genes was detected by RT-PCR. In experimental group, expression of Scn5a, Kcnj2, Kcnel, Kcnd3, Cacanlc and Cacanlh were significantly higher than those in blank group and negative group (P<0.05).4. Whole cell patch clamp detected INa, Ito, Ikl, Iks and IT-Ca in experimental group. Ito and Iks were also detected in blank group and negative group, and others were failed to detect in blank group and negative group. The peak current of each ion channel in experimental group showed heterogeneity, and lower than those in positive group.ConclusionDuring Islet-1 induced C3H10T1/2 to differentiate specificly into cardiomyocytes, the expression of cardiac-specific sodium, potassium, calcium channel encoding genes increased, and the expression of the sodium, potassium, calcium channel currents were detected by whole cell patch clamp, which were similar to cardiomyocytes. This confirmed Islet-1 could induce C3H10T1/2 cells to differentiate into cardiomyocytes with electrophysiological function.