Cellular and molecular mechanisms regulating cardiac chamber morphology

About This Grant

PROJECT SUMMARY The establishment of properly shaped cardiac chambers depends upon the patterned organization of cardiomyocytes of the appropriate shapes and sizes. For example, the distinct features of the cardiomyocytes that populate the outer curvature (OC) and inner curvature (IC) of the embryonic ventricle underlie the characteristic properties of each of these territories and create the precise dimensions of the developing chamber. The bulging, convex OC and the bent, concave IC are critical components of ventricular morphology, yet we lack a complete picture of the mechanisms that establish the divergent attributes of OC and IC cells. To elucidate the fundamental pathways that control chamber curvature formation, we take advantage of the opportunities available in the zebrafish embryo, where we can resolve distinct features of individual cells, follow these distinguishing traits over time, and identify the genes that regulate OC and IC divergence. Our studies in zebrafish show that ventricular cardiomyocytes acquire distinct squamous and cuboidal morphologies in the OC and IC regions, respectively. Importantly, these divergent cell shapes are preceded by distinct patterns of actomyosin cytoskeletal organization in the OC and IC cells. Moreover, we find that actomyosin dynamics, biomechanical forces, and tbx5a-regulated pathways all help to regulate these OC and IC attributes. Together, our data suggest a novel model in which extrinsic and intrinsic factors collaborate to create curvature-specific cytoskeletal features that, in turn, drive regional patterns of cell shape change in the OC and IC. To test this model, we will begin by identifying the cellular mechanisms that drive curvature-specific modes of cell shape change. In our first aim, we will determine the dynamics of F-actin colocalization with relevant partners in the OC and IC, and we will evaluate how components of focal adhesion complexes influence OC and IC morphogenesis. In addition, we will assess the impact of extension of cellular projections and differential composition of the extracellular matrix on the modes of OC and IC cell shape change. Then, in our second aim, we will identify the molecular mechanisms that create curvature-specific actomyosin dynamics. Specifically, we will test the roles of a roster of candidate effectors that may modulate the actomyosin cytoskeleton in OC or IC cardiomyocytes. These candidates include components of the Rho GTPase pathway and actin-modifying factors such as Adducin 3a. Additional candidates will emerge from single-cell transcriptomic analysis of differential gene expression in the OC and IC and from proximity-based labeling and proteomic identification of relevant F-actin-associated proteins. Ultimately, by revealing new connections between key regulatory pathways, downstream molecular effectors, their impacts on subcellular dynamics, and the resulting cell behaviors, our proposed studies will uncover essential mechanisms underlying chamber curvature formation. Over the long term, this work has the potential to shed light on the ways in which chamber morphogenesis can go awry in cases of congenital heart disease.