Molecular effects of lithium exposure during mouse and chick gastrulation and subsequent valve dysmorphogenesis.

BACKGROUND: Lithium (Li) has been associated with cardiac teratogenicity in the developing fetus. We took advantage of the association of therapeutic administration of Li with an increase in heart defects to gain insight into both normal and pathological heart and valve development with GSK-3 inhibition. The objective of this study was to define whether Li mimicry of canonical Wnt/beta-catenin signaling induces cardiac valve defects. METHODS: Li was administered by a single intraperitoneal injection to the pregnant mouse on embryonic day E6.75, much earlier than heretofore analyzed. On E15.5 developing heart defects were defined by Doppler ultrasound. The embryonic hearts were analyzed for changes in patterning of active canonical Wnt expression and nuclear factor of the activated T cells-c1 (NFATc1), both key regulators of valve development. Li-exposed chick embryos were used to define the early cell populations during gastrulation that are susceptible to GSK-3 inhibition and may relate to valve formation. RESULTS: Li exposure during gastrulation decreased the number of prechordal plate (PP) cells that reached the anterior intestinal portal, a region associated with valve development. Li decreased expression of Hex, an endoderm cardiac inducing molecule, normally also expressed by the PP cells, and of Sox 4 at the anterior intestinal portal and NFAT, critical factors in valvulogenesis. CONCLUSIONS: Cells existing already during gastrulation are associated with valve formation days later. The Wnt/beta-catenin signaling in PP cells is normally repressed by Wnt antagonists and Hex is up-regulated. The antagonism occurring at the receptor level is bypassed by Li exposure by its intracellular inactivation of GSK-3 directly to augment Wnt signaling.

MCT-4, A511/Basigin and EF5 expression patterns during early chick cardiomyogenesis indicate cardiac cell differentiation occurs in a hypoxic environment.

We have identified the presence of the hypoxia marker EF5 in the stage 4/5 chick heart fields. This suggests that cardiac cell differentiation occurs in a relatively anaerobic environment. Monocarboxylate transporter (MCT) studies in adult cardiac myocytes have demonstrated that MCTs catalyze proton-linked pyruvate and lactate transport activity. 5A11/Basigin is an ancillary protein that targets MCTs to the plasma membrane for their function. MCT-4 expression is most evident in cells with a high glycolytic rate associated with hypoxic energy production. Subsequent to the immunohistochemical localization of EF5 in the early heart field, we continued in our analysis during stages 5 to 12 for the expression of indicators of cellular glycolytic metabolism in the developing heart, such as MCT-4, MCT-1, and 5A11 (Basigin/CD147). Our observations indicate that MCT-4 and 5A11/Basigin are expressed early, in a differential left-right pattern, in the bi-lateral plate mesoderm, as the cardiac compartment is forming. At stage 11, MCT-4/5A11 continues to be highly expressed in the myocardial wall of the looping heart, but not in the dorsal mesocardium. RT-PCR analyses for MCT-1, -4, and 5A11 indicate that MCT-4 and 5A11 are expressed throughout precardiac, embryonic, and fetal stages in the heart. MCT-1 is first detected in the heart on embryonic day 3 and then remains expressed throughout development to hatching. These results indicate that cardiac precursor cells are equipped for differentiating in a hypoxic environment using anaerobic metabolism for energy production.