Role of Tissue Biomechanics in the Formation and Function of Myocardial Trabeculae in Zebrafish Embryos

Introduction:: Cardiac trabeculae are uneven ventricular muscular structures on the endocardial surface at the outer curvature zone of the embryonic ventricle. They are important as trabecular malformations are associated with embryonic death. However, whether and how trabeculation confer biomechanical advantages to the heart’s function is not understood, and is investigated here. The formation of trabeculation is a mechanosensitive process, as it can be prevented by stopping the heart and blood flow. However, we previously showed [1] that there is no distinctive difference in fluid wall shear stress characteristics between regions that trabeculate and those that do not. We thus hypothesize that cardiac tissue stresses play a role in trabeculation formation, and investigate this here.


Materials and Methods:: High-resolution, 4D imaging of zebrafish embryonic hearts at 2-5 days post fertilization (dpf) is performed with light-sheet microscopy in a line with myocardium fluorescence, Tg(cmlc2:EGFP). Ventricular reconstruction and motion tracking are performed to obtain 3D myocardial strains, to evaluate how trabeculation affects strains. Finite Element (FE) modelling of zebrafish embryonic heart is performed with and without trabeculae artificially smoothed out to understand how trabeculation affects tissue stresses. Passive pressurization of pharmacologically stopped heart is performed, paired with FE modelling of the same process, to obtain spatial variability of tissue stiffness, to assess how trabeculation affect stiffness homogeneity. We further tested if increasing ventricular pressure and myocardial stresses with vasopressin can rescue the defective trabeculation formation in gata1 morphant zebrafish embryos.


Results, Conclusions, and Discussions:: In the trabeculated hearts (3-5 dpf), the trabeculated outer curvature region shows higher strains compared to the non-trabeculated inner curvature region, but in pre-trabeculated hearts (2 dpf), the opposite occurs. Motion tracking further shows trabeculations exhibit larger contractile strains than surrounding compact tissues. These suggest that trabeculation improves deformability of cardiac walls.

Geometric analysis shows that between 2 and 3dpf, when trabeculations formation, there are thickness and geodesic curvature changes at regions of trabeculation, which FE modelling showed will lead to stress concentration in those region. Comparisons between FE models with and without trabeculations show that the existence of cardiac trabeculae formation reduces such high stresses without sacrificing stroke volume and pressure generation. These results suggests that trabeculations has a structural support and stress reduction function.

Passive pressurization of embryonic hearts paired with FE modelling of the same process shows the pre-trabeculation normal ventricle, and gata1 morphant ventricle with disrupted trabeculation, there is substantial spatial heterogeneity of tissue stiffness. However, in normal ventricles after trabeculation, stiffness becomes very homogeneous. This suggests that trabeculation also has the function reduce spatial variability of tissue stiffness.

In gata1 morphants, the absence of blood cells reduced blood viscosity and thus cardiac pressures, and trabeculation development is disrupted. When gata1 morphants are treated with vasopressin, a potent vasoconstrictor, between 1dpf and 4dpf, ventricular pressures and thus wall tissue stresses increase. Interestingly, this results in the restoration of trabeculation formation to be similar to normal embryos. This thus demonstrates that tissue stresses are important for trabeculation formation.

In conclusion, our results show that trabeculations may have the function of improving wall deformability, reducing wall stresses, and maintaining homogeneity of wall stiffness, and that tissue stresses are important stimuli for formation of trabeculations during cardiac development.


Acknowledgements (Optional): : Imperial College PhD scholarship, BHF Centre of Research Excellence (RE/18/4/34215), and NSF Grant (NSF 1936519 (J.L.)) for funding.

References (Optional): : [1] Cairelli AG, Chow RW, Vermot J, Yap CH. Fluid mechanics of the zebrafish embryonic heart trabeculation. PLOS Computational Biology. 2022 Jun 6;18(6):e1010142.