Altered Myocardial Structure in Post-natal Tetralogy of Fallot: A Substrate for Interpretation of Ventricular Function and Dysfunction?

BACKGROUNDIn tetralogy of Fallot (ToF), changes in right ventricular function (as assessed by strain or TAPSE) reflect altered myocardial structure. Direct three-dimensional anatomical evidence supporting these changes remains limited. The objective of this study was to non-destructively characterize myocardial architecture in pediatric ToF hearts using Hierarchical Phase-Contrast Tomography (HiP-CT) and structure tensor analysis. METHODSTwenty ToF and control pediatric hearts were imaged at the European Synchrotron, ESRF. Myocyte orientation was assessed through structure tensor analysis and distributed high-performance computing. A region-specific framework was developed for analysis of the right ventricle. The predominant direction of myocardial aggregates (their helical angle) was compared across ventricular regions. RESULTSSignificant differences in orientation were found in all ToF segments vs controls (left ventricle, right ventricular inlet, right ventricular outflow tract, septum; p < 0.001). Myocytes in the ToF right ventricular inlet were more circumferential overall, with regional heterogeneity. Contrary to traditional models, no discrete middle layer was found in the ToF right ventricle; instead, a shift towards more circumferentially orientated myocytes and disrupted septal and outflow components was observed. Right ventricular contribution to the septum was greater in ToF (47.3% vs 34.0%; p = 0.0026), with extension of ventricular insertion points disrupting septal architecture. There were more longitudinally oriented myocytes in the ToF right ventricular outflow tract, consistent with hypertrophied septoparietal trabeculations. Left ventricular structure in ToF demonstrated a greater proportion of circumferentially oriented myocytes compared to controls. CONCLUSIONSWe reveal profound alterations in ToF myocardial organization which broadly align with clinical observations and provide the first open-access HiP-CT congenital heart disease data as a basis for future computational modelling. Clinical PerspectiveWhole-heart HiP-CT demonstrates a loss of normal LV-RV distinction in the ToF myocardium, alongside extensive septal disarray. These findings provide a structural substrate for RV dysfunction, ventricular-ventricular interaction, and arrhythmogenesis in ToF, challenging traditional layer-based models of ventricular myocardium. Understanding myocardial organization as a continuous, developmentally patterned three-dimensional structure is essential for accurate interpretation of ventricular mechanics and disease progression. Although HiP-CT imaging is not applicable in vivo, the structural phenotypes identified in this study generate testable hypotheses for clinical imaging. Future work should focus on correlating ex-vivo measures with in-vivo imaging markers derived from cardiac magnetic resonance, including strain, TAPSE, and assessment of ventricular interactions. Investigating myocardial phenotype across the life-course, from fetal life to adulthood, paired with multi-omics mapped to these three-dimensional datasets, may help elucidate mechanisms underlying myocardial remodeling in ToF and support the development of novel therapeutic approaches.