Understanding Cardiac C-Looping Using Microscale In Vitro Models

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Defects in laterality are observed in more than 1 in 8000 live births and have significant clinical implications. The embryonic heart starts as a straight cardiac tube along the midline of the embryo, which is subsequently transformed into a c-shaped heart loop reliably toward the right side of the body. This cardiac c-looping is considered as the earliest evident event of left-right (LR) asymmetry breaking (also called chirality or handedness) of a human organ. The inversed lateralization of cardiac looping often leads to severe clinical outcomes, including dextrocardia, septum defects, double outlet right ventricle, and even death of fetuses and infants. Accumulating evidence suggests that asymmetric cardiac looping derives from an unknown tissue- intrinsic mechanism. Recently, we have recapitulated chiral morphogenesis on micropatterned surfaces and in 3D hydrogels and demonstrated that cardiac cells have a definite chirality before asymmetric looping. Protein kinase C (PKC) activators can reverse both cell chirality and cardiac c looping. Our rationale is that novel cell chirality based high-throughput platforms, together with a better understanding of molecular mechanisms of cell chirality, can facilitate the LR symmetry research. We propose to use a combination of micro-fabrication, hydrogel technology, live-cell imaging, molecular assays, traction force microscopy, high-throughput screening, ex vivo culture, and genetic mouse models as tools to elucidate the biophysical and biochemical mechanisms. Our objectives are to determine biomolecular and biomechanical mechanisms of PKC regulated cell chirality and asymmetric looping and to identify cytoskeletal mechanisms of cell chirality during cardiac c-looping. SPECIFIC AIM 1: Identify components and signaling pathways that regulate cardiac chirality with high- throughput screening and validate with ex ovo embryo culture. We will screen inhibitors/activators of PKC isoforms, their downstream effectors, possible substrates, and a small-molecule kinase library, determine mechanisms of action, and validate the findings with the whole-embryo ex ovo culture. SPECIFIC AIM 2: Determine the biomechanical role of cell chirality in multicellular morphogenesis. We will examine whether chiral mechanical forces are sufficient to induce cardiac c-looping using traction force microscopy and whether the cells on ventral myocardium exhibit intrinsic chiral biases. SPECIFIC AIM 3: Determine cytoskeletal mechanisms in cardiac cell chirality during c-looping. We will analyze the chirality of actin dynamics of cardiac cells, observe its change under drugs of interest, and confirm the findings with ex ovo whole-embryo culture and genetic mouse models. If the project is successful, we will be able to establish a set of novel high-throughput platforms for studying the biophysics of asymmetric cardiac looping by measuring cell chirality, and further our understanding of congenital heart disease. Also, this proposed research is transformative, and potentially open a new field of research: cell chirality, a fundamental cellular property defining symmetry breaking in tissue development.