Mechanobiology of Cardiac Outflow Tract Morphogenesis

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PROJECT SUMMARY Proper growth, septation, and maturation of the cardiac outflow tract (OFT) into valved aortic and pulmonary outlets are essential for oxygenated circulation after birth. 1-2% of live births and up to 30% of pre-term fetal deaths have congenital heart defects, many of which affect the remodeling of the valvuloseptal primordial tissues, called the proximal and distal outflow cushions. Despite much effort uncovering the genetic basis of early OFT cushion formation, this understanding has not explained the clinically relevant phases of growth, condensation and elongation into valves and septa. Further, emerging evidence suggests that the formation, growth, and maturation of the valvuloseptal appratus is coupled with that of the ventricles. Gross congenital valve malformations induce hemodynamic changes within the developing ventricles (via stenosis and/or regurgitation), leading to structural differences in their myofiber architecture and trabecular patterning. While many of these malformations are gestationally survivable, structrural valvular defects like mitral valve prolapse, which have a developmental origin, also incur premature ventricular failure and risk of sudden death. It is currently unknown how hemodynamic perturbations drive shared fetal ventricular and valvular remodeling, in part because prevailing genetic tools lack the power to separate genetic from hemodynamic causality. The Butcher lab has pioneered innovative technology 1) to quantify local in vivo mechanical forces within cardiac inflow, ventricular, and OFT domains, and register them with local in situ gene/protein expression, 2) to non-invasively visualize and precisely ablate intracardiac tissues without collateral damage in vivo, and 3) to directly assess local spatial cellular transcriptomes across entire thin sections. This CAROL Act Supplement will expand the current funded project to interrogate how valvular and ventricular remodeling is coupled to their shared hemodynamic environment. First, emerging state of the art high-resolution spatial transcriptomics will be applied to achieve first ever true single-cell spatial resolution across full-size fetal heart domains (10x10 mm areas). This will be applied to uniquely identify inflow atrioventricular, ventricular, and outflow tract cellular transcriptional profiles in embryos treated with sham or hemodynamically perturbed conditions leading to established cardiac structural malformations. This will be further performed at early and late stages of malformation, enabled by an innovative device for precise planar application of cryosections. Next, we will apply novel cellular neighborhood analysis tools to determine unique and shared neighborhoods that associate with local structural changes in the atrioventricular valves, compact and trabecular ventricular domains, and outflow tracts. Cellular neighborhood candidates will then be verified by secondary immunofluorescence methods. These results will dramatically improve our understanding of how valve-related malformations induce undesirable ventricular remodeling towards impaired functional longevity, and identify multi-cellular fingerprint signatures that could be predictive of these risks.