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Three-dimensional visualization of connexin 43 on the human cardiomyocytes
Jacek Kołcz1, Bartłomiej Rajwa, Justyna Drukała
1Department of Pediatric Cardiac Surgery, Polish-American Children's Hospital, Collegium Medicum, Jagiellonian University, Cracow, Poland.
Insights
Connexin 43 distribution is abnormal on the surface of cardiomyocytes in tetralogy of Fallot, unlike healthy hearts where it
Area of Science:
- Cardiovascular Biology
- Cellular Biology
- Developmental Biology
Background:
- Gap junctions, formed by connexin proteins, are vital for human heart development.
- Altered connexin 43 expression is linked to right ventricular outflow tract abnormalities.
- Tetralogy of Fallot involves right ventricular outflow tract narrowing and hypertrophy.
Purpose of the Study:
- To investigate connexin 43 distribution on human cardiomyocytes from tetralogy of Fallot patients.
- To compare connexin 43 distribution in tetralogy of Fallot with normal hearts.
Main Methods:
- Isolation and culture of cardiomyocytes from surgical biopsies.
- Immunofluorescence staining for connexin 43.
- Confocal microscopy and 3D volume rendering for distribution analysis.
Main Results:
- Connexin 43 exhibited irregular surface distribution on tetralogy of Fallot cardiomyocytes.
- In control hearts, connexin 43 was localized to intercalated disks only.
- Significant differences in connexin 43 organization were observed between the two groups.
Conclusions:
- Disturbed connexin 43 distribution in tetralogy of Fallot may impair heart development.
- Abnormal connexin 43 may contribute to right ventricular hypertrophy and arrhythmias.
- This finding highlights connexin 43's role in congenital heart disease pathogenesis.
Abstract:
Gap junctions created by a family of connexin proteins play an important role in the development of human heart. It has been previously shown that the abnormalities of right ventricular outflow tract are related to an altered level of expression of connexin 43. The right ventricular outflow tract narrowing, stenosis, or atresia of the main pulmonary artery and hypertrophy of the right ventricle are observed in tetralogy of Fallot. The aim of the current study was to determine the distribution of connexin 43 on the surface of human cardiomyocytes obtained during reparative surgery for tetralogy of Fallot. Connexin 43 distribution in these cells was compared with distribution of connexin 43 in cardiomyocytes obtained from patients without right ventricular outflow tract pathology. Cardiomyocytes isolated from tissue biopsy were cultured on collagen substratum, fixed with paraformaldehyde, and incubated with goat antihuman connexin 43 antibodies and secondary donkey antigoat antibodies conjugated with fluorescent indocarbocyanine. Z-series of optical sections were recorded using a laser scanning confocal microscope. Three-dimensional data stacks were visualized using volume-rendering techniques. Images of connexin 43 fluorescence revealed a pattern of three-dimensional distribution of connexin on the surface of an individual cardiomyocyte. Cardiomyocytes from tetralogy of Fallot and hearts with normal right ventricular outflow tract differ in the organization of connexin 43. Cardiomyocytes from tetralogy of Fallot hearts revealed disturbed distribution of connexin 43. The protein is located irregularly on the entire surface of the cell. In the controls, connexin 43 can be visualized within the intercalated disks only. These disturbances may influence heart maturation, cause hypertrophy of the right ventricle, and induce severe arrhythmias in children with tetralogy of Fallot.