Analyses of form-function relationships during heart looping are directly related to technological advances. Recent advances in four-dimensional optical coherence tomography (OCT) permit observations of cardiac dynamics at high-speed acquisition rates and high resolution. Real-time observation of the avian stage 13 looping heart reveals that interactions between the endocardial and myocardial compartments are more complex than previously depicted. Here we applied four-dimensional OCT to elucidate the relationships of the endocardium, myocardium, and cardiac jelly compartments in a single cardiac cycle during looping. Six cardiac levels along the longitudinal heart tube were each analyzed at 15 time points from diastole to systole. Using image analyses, the organization of mechanotransducing molecules, fibronectin, tenascin C, α-tubulin, and nonmuscle myosin II was correlated with specific cardiac regions defined by OCT data. Optical coherence microscopy helped to visualize details of cardiac architectural development in the embryonic mouse heart. Throughout the cardiac cycle, the endocardium was consistently oriented between the midline of the ventral floor of the foregut and the outer curvature of the myocardial wall, with multiple endocardial folds allowing high-volume capacities during filling. The cardiac area fractional shortening is much higher than previously published. The in vivo profile captured by OCT revealed an interaction of the looping heart with the extra-embryonic splanchnopleural membrane providing outside-in information. In summary, the combined dynamic and imaging data show the developing structural capacity to accommodate increasing flow and the mechanotransducing networks that organize to effectively facilitate formation of the trabeculated four-chambered heart.

Flectin, a protein previously described to be expressed in a left-dominant manner in the embryonic chick heart during looping, is a member of the nonmuscle myosin II (NMHC-II) protein class. During looping, both NMHC-IIA and NMHC-IIB are expressed in the mouse heart on embryonic day 9.5. The patterns of localization of NMHC-IIB, rather than NMHC-IIA in the mouse looping heart and in neural crest cells, are equivalent to what we reported previously for flectin. Expression of full-length human NMHC-IIA and -IIB in 10 T1/2 cells demonstrated that flectin antibody recognizes both isoforms. Electron microscopy revealed that flectin antibody localizes in short cardiomyocyte cell processes extending from the basal layer of the cardiomyocytes into the cardiac jelly. Flectin antibody also recognizes stress fibrils in the cardiac jelly in the mouse and chick heart; while NMHC-IIB antibody does not. Abnormally looping hearts of the Nodal ⌬ 600 homozygous mouse embryos show decreased NMHC-IIB expression on both the mRNA and protein levels. These results document the characterization of flectin and extend the importance of NMHC-II and the cytoskeletal actomyosin complex to the mammalian heart and cardiac looping. Developmental Dynamics 237:3577-3590, 2008.

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Lithium (Li), a mood stabilizing drug, and elevated homocysteine (HCy), a metabolite in the folic acid (FA) cycle, are linked to induction of human congenital heart defects. We determined noninvasively by echocardiography, that exposure of the mouse embryo by an i.p. injection to the dam of Li or HCy during gastrulation on embryonic day E6.75 induces cardiac and valve defects. A single dose of 125 μl Li (25 mM) or HCy (75 μM) on E5.5 to 6.5 results in mouse embryonic lethality; on E6.75 to 7.0, both induce tricuspid and semilunar valve defects (Li: n=131 ; HCy: n=78). The tricuspid valve septal leaflet fails to delaminate, a characteristic of Ebstein’s Anomaly. Use of Li during human pregnancy has been associated with Ebstein’s Anomaly. Li mimics the Wnt/β-catenin (β-cat) signaling pathway by inactivating glycogen synthase kinase-3β. During chick cardiac specification, Li, Wnt 3A, or HCy exposure adversely affects chick cardiogenesis with severity of anomalies based on timing of early exposure. To initiate cardiogenesis, the secreted Wnt antagonist Dickkopf-1 acts extracellularly on the endoderm to upregulate Hex , an inducer of cardiomyogenesis. Exposure of stages 3+/4− chick embryos to Li/Wnt3A/ HCys inhibits Hex and Islet-1 gene expression in the cardiogenic crescent via an intracellular mechanism, thus augmenting inhibitory Wnt/β-cat signaling. FA deficiency leads to elevated HCys levels. We hypothesize that HCys/FA metabolism intersects with Wnt/β-cat signaling and that mechanistically FA supplementation acts by overriding Wnt/β-cat inhibition of gene expression in the embryonic heart fields that leads to cardiac defects. FA, known to protect against HCys-mediated neural tube defects, was tested for protective effects against Li/Wnt3A/HCy during cardiogenesis. With all three experimental exposures, FA addition results in reexpression of the cardiac inducers Hex and Islet-1 at high levels in the chick heart fields. In the mouse, no valve defects were detected in HCys/FA exposed embryos (n=27). Regimen and concentration of FA supplementation necessary to fully rescue Li effects are being tested. In conclusion, folate supplementation potentiates the repression of Wnt/β-cat signaling and protects formation of heart and valve defects. This research has received full or partial funding support from the American Heart Association, AHA Greater Southeast Affiliate (Alabama, Florida, Georgia, Louisiana, Mississippi, Puerto Rico & Tennessee).