https://doi.org/10.1007/s00392-025-02625-4
1Universitäts-Herzzentrum Freiburg - Bad Krozingen Institut für Experimentelle Kardiovaskuläre Medizin Freiburg im Breisgau, Deutschland; 2Universitäts-Herzzentrum Freiburg - Bad Krozingen Klinik für Herz- und Gefäßchirurgie Freiburg, Deutschland; 3Universitäts-Herzzentrum Freiburg - Bad Krozingen Klinik für angeborene Herzfehler und Pädiatrische Kardiologie Freiburg im Breisgau, Deutschland
Background:
Improved clinical management of patients with Tetralogy of Fallot (ToF) has extended survival and enhanced quality of life over the past decades. However, a major persisting concern is an increased risk for ventricular arrhythmias in patients with repaired ToF. Here, we investigate potentially pro-arrhythmic structural alterations in ventricular myocardium of patients with unrepaired and with repaired ToF, including increased fibrosis, changes in cellular composition, and altered (hetero-)cellular coupling via the gap junction protein Connexin-43 (Cx43).
Methods:
This study was approved by the Ethics Committee of the University of Freiburg, Germany (approval number 589/17). We examined right ventricular outflow tract (RVOT) myectomies, excised in the context of surgical interventions, from eight infants with unrepaired ToF (mean age 5.0 ± 3.1 months; at repair operation) and eight adults with repaired ToF (mean age 32.8 ± 16.7 years; at operative pulmonary valve replacement), as well as right ventricular or RVOT myocardium from five patients without cardiac disease (mean age 18.0 ± 5.9 years; rejected organ transplant donor tissue from international collaborators). After chemical preservation with 4 % paraformaldehyde, 150 µm tissue slices were immuno-labelled to visualise cardiomyocyte outlines with wheat germ agglutinin, non-myocytes with an anti-vimentin antibody, nuclei with 4’,6-diamidino-2-phenylindole, and gap junctions with an anti-Cx43 antibody. To quantify populations of non-myocytes, endothelial cells were labelled with an anti-CD31 antibody, macrophages with an anti-CD68 antibody, T- and B-lymphocytes with an anti-CD4 and anti-CD20 antibody. Imaging was performed using high-resolution 3-dimensional confocal microscopy (voxel size: 180 nm x 180 nm x 360 nm in X-Y-Z). Obtained image volumes were analysed using a custom-developed, semi-automated image processing workflow, allowing us to quantify the relative proportion of cardiomyocytes, fibroblasts, endothelial cells, immune cells, as well as the distribution of Cx43.
Results and conclusion:
Our dataset of 220 image volumes, each covering a physical volume of 184x184x100 µm³, allows for extensive micro-structural exploration. Pilot analyses suggest Cx43 lateralisation in infant unrepaired patients, and show cardiomyocyte hypertrophy in adult patients with repaired ToF, which was not observed in the control group (Fig. 1). We are conducting further analyses to characterize age- and disease-associated remodelling, linking our findings to clinical parameters in order to identify candidate factors that may predispose ToF patients to arrhythmias.
Fig. 1: Representative confocal images for each patient group: A: Patient with unrepaired ToF at the age of 11 months, B: Patient with repaired ToF at the age of 17 years, C: Control donor tissue from a patient at the age of 18 years. Blue: 4’,6-diamidino-2-phenylindole, magenta: anti-vimentin, red: anti-Cx43, yellow: wheat germ agglutinin. Scale bar: 30 µm.