The Expression Of Connexin 43 In Children With Tetralogy Of Fallot

Objective:Tetralogy of Fallot(TOF) is the most frequent, cyanotic congenital heart defect in which conotruncal anomalies, right ventricle dysfunction and life-threatening arrhythmias occur.Abnormalities in the expression and distribution of Connexin 43(Cx43) in cardiomyocytes may lead to anomalous conotruncal embryogenesis and disturbances in the maturation and function of the heart. In this study,age-related changes in the expression and spatial distribution of Cx43 in cardiomyocytes from TOF children compared to patients without right ventricular outflow tract pathology were determined Immunohistochemistry, Confocal microscopy and flow cytometry were used to assess the changes.Method:Myocardial samples from the right ventricular outflow tract were collected from 44 patients undergoing reparative cardiac surgery. In patients with TOF, samples were taken from the site of the ventriculotomy. The TOF patients were grouped by age. Group Ia consisted of 18 younger patients (3 months to 3 years). Group Ib consisted of 12 older children (3years -6years). The control group (group II) consisted of 14 patients with ventricular septal defect and without the RVOT pathology. Ages ranged from 3months to 6years. The tissue collected within the lumen of the needle was carefully removed. We measured expression and spatial distribution of Cx43 in cardiomyocytes from TOF children compared to patients without right ventricular outflow tract pathology by Immunohistochemistry, Confocal microscopy and flow cytometry. Result:1.with immunohistochemical S-P method,we found that The cell size was unequal and cell density became lower than that of the control group. Immunolabeling of connexin 43 was confined to the intercalated disks in the normal ventricularmy ocytes of control group. But The expression of Cx43 had a punclate distribution in cytoplasm and over the entire surface of the cardiocyte,and a few located at intercalated disk of ventricular myocardium in the TOF group. The results of image analyzer showed that the Cx43 area was 21.45±7.75, the Cx43 percentage was 31.74±10.44,the Average gray degree was 72.36±0.43 in the TOF group; the Cx43 area was 33.82±8.23, the Cx43 percentage was 40.22±10.14,the Average gray degree was 99.80±0.53.in the control group the amount of connexin43 expression was lower in TOF group than that of control group. The defference was significant(P<0.05).2. The expression of Cx43 was determined with flow cytometry. The mean fluorescence intensity (MFI) of positive cells were analysed within the gated cell population. The intensity of Cx43 fluorescence in the TOF group Ia was 87.33±8.4,in the TOF group Ib was101.42±7.9. In the control group, the intensity of fluorescence of Cx43 was 133.37±23.9. The defference was significant(P<0.05) .3. In the control group, the distribution of Cx43 was as expected. The fluorescencelabel formed clusters of immunofluorescence that were confined to the cell terminals oriented transversely to the long axes of the myocytes. Typical longitudinal slices of the Cx43-labeled control cardiomyocytes are shown . All staining was present within intercalated discs at the ends of the cells. The distribution of Cx43 in patients with TOF differed significantly from controls. Typical confocal slices through longitudinally sectioned Cx43 immunolabeled right ventricular cardiomyocytes from the TOF group are presented .The immunolabeling conjugated with Cx43 is distributed over the circumferences of the cardiomyocytes.the protein was irregularly distributed on the surface of the cell.CONCLUSIONS:1.In children with tetralogy of Fallot, the age-related process of redistribution ofCx43 connected with the morphological and functional maturation of the heartdoes not occur properly, and detected levels of Cx43 are found outside the intercalated discs. The anomalous distribution pattern of Cx43 on the surface of cardiomyocytes from TOF hearts does not depend on the age of the patient and is identical in both infants and patients several years of age.2.There are essential differences in the distribution of cardiomyocyte Cx43 between TOF patients and children without RVOT pathology. Cardiomyocytes from TOF hearts revealed a disturbed, immature-like distribution of Cx43, whereas hearts without RVOT pathology revealed a normal, mature-like arrangement of this protein. These disturbances may create the basis for ventricular arrhytmia in children with TOF.3.The Cx43 redistribution and morphological maturation of the normal cardiac tissue take place in early infancy. This fact can support the strategy of early total correction of the defect.