B Embryogenesis of Ventricular Myocardial Trabeculae – Novel Insights from Episcopic 3D Imaging and Fractal Analysis of Wild-type and Notch MIB1 Noncompaction Mouse Models

Introduction Left ventricular noncompaction (LVNC) is characterised by prominent ventricular myocardial trabeculations, composed of sheets of cardiomyocytes. They form early during cardiogenesis but their development is complex. Measuring trabecular complexity in animal models of cardiac disease is important as abnormal trabecular patterns are increasingly been recognised to coexist with several other cardiac conditions, not just LVNC. We describe an innovative approach that utilises fractal algorithms and high-resolution episcopic microscopy (HREM) to study the developmental timing of myocardial trabeculation in mouse and we validate it using a recently described LVNC mouse model (NOTCH pathway regulator Mib1 mutant). Methods HREM (2–3 μm resolution) analysis was performed prospectively on 123 embryonic mouse hearts consisting of wild-type (WT) NIMR:Parkes, WT C57BL/6 and Mib1 flox ; cTnT-cre mutant and WT littermates. HREM permits the 2D/3D imaging of tissue samples as they are physically sectioned. Datasets underwent fractal analysis using a box-counting approach (Figure 1). Results LV trabecular complexity showed a significant drop between E14.5 and E18.5 (Figure 2). Across all embryonic stages, the apical half of the LV retained the highest fractal dimensions (FD) when compared to the base. By E18.5 the myocardium was almost fully compacted registering the lowest FD. For the first time, we demonstrate that strain-specific differences in LV trabecular patterning exist in mouse because NIMR:Parkes compacts earlier than C57BL/6 (Figure 3). Reslicing experiments (Figure 4) and separate validation tests on Mib1 mutants and WT littermates (Fig.5) confirmed how the proposed methodology is a reliable and effective tool for the detection of mutagenesis-related differences in trabeculation. Conclusion Reported here is a method in which sequential, 2D sections of mouse embryo hearts may be analysed using a fractal algorithm to calculate ventricular trabecular complexity – a technique so sensitive, that small inter-strain differences in somitogenesis are detectable in mouse pups. Precise knowledge of the trabecular architecture as it presents itself in WT, is a prerequisite for the correct identification of pathological trabecular phenotypes in mouse models of cardiac disease, explaining the need for a quantitative fractal atlas of trabecular development. Fractal mathematics in combination with HREM has the potential to answer to many developmental biology questions in the heart, with future applicability to other organ systems and to other species.