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Previous work has shown that ablation or mutation of nonmuscle myosin heavy chain II-B (NMHC II-B) in mice results in defects in the heart and brain with death occurring between embryonic day 14.5 (E14.5) and birth (Tullio, A. N., Accili, D., Ferrans, V. J., Yu, Z. X., Takeda, K., Grinberg, A., Westphal, H., Preston, Y. A., and Adelstein, R. S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 12407-12412). Here we show that mice ablated for NMHC II-A fail to develop a normal patterned embryo with a polarized visceral endoderm by E6.5 and die by E7.5. Moreover, A(-)/A(-) embryoid bodies grown in suspension culture constantly shed cells. These defects in cell adhesion and tissue organization are explained by loss of E-cadherin and beta-catenin localization to cell adhesion sites in both cell culture and in the intact embryos. The defects can be reproduced by introducing siRNA directed against NMHC II-A into wild-type embryonic stem cells. Our results suggest an essential role for a single, specific nonmuscle myosin isoform in maintaining cell-cell adhesions in the early mammalian embryo.

Thrombin receptor (ThR) plays a significant role in myocyte contractility and hypertrophy. Heart myocyte ischemic damage, caused by insufficient blood supply, is the leading cause of heart infarction. Here we demonstrate that when primary myocyte cultures are subjected to hypoxic stress, ThR mRNA levels are reduced markedly. This takes place also in vivo in a model of ischemic pig heart, exhibiting reduced levels of ThR compared with normal heart sections. Prior activation of ThR however, by either thrombin receptor-activating peptide (TRAP) or by alpha-thrombin resulted in full protection of ThR mRNA levels under hypoxia. The effect appeared specific to ThR because the addition of TRAP did not affect the hypoxic damage as shown by the levels of lactic dehydrogenase release and up-regulated GLUT-1, a glucose transporter gene. This protection effect took place not only in primary myocytes but also in NIH3T3 fibroblasts. ThR protection occurs via specific cell signaling events because activation of the receptor by TRAP, following interruption of the signaling cascade by calphostin C, a protein kinase C inhibitor, resulted in loss of ThR mRNA protection. Because Ras and Src are part of the ThR signaling cascade, the introduction of either dominant ras or src oncogenes to NIH3T3 murine fibroblasts gave rise to similar protection of ThR mRNA levels under hypoxic conditions without the exogenous addition of TRAP. Likewise, ThR mRNA protection was obtained after transfection with proto-oncogene vav. The 95-kDa protein Vav undergoes tyrosine phosphorylation after ThR activation, serving thus as part of the receptor machinery cascade. We therefore conclude that the initiation of the signaling cascades either exogenously by TRAP or within the cell via src or ras, as well as via vav oncogene interconnecting G-binding protein to the tyrosine kinase pathway, ultimately results in ThR protection under hypoxia. We present hereby, a novel concept of activated receptors, which under minimal oxygen tension protect their otherwise decaying mRNA. Maintaining the level of ThR that plays an active role in normal myocyte function may provide a significant repair mechanism in ischemic tissue, assisting in the regaining of normal myocyte functions.