The bone morphogenetic proteins BMP-2 and BMP-4 and the homeobox gene MSX-2 are required for normal development of many embryonic tissues. To elucidate their possible roles during the remodeling of the tubular heart into a fully septated four-chambered heart, we have localized the mRNA of Bmp-2, Bmp-4, Msx-2 and apoptotic cells in the developing mouse heart from embryonic day (E)11 to E17. mRNA was localized by in situ hybridization, and apoptotic cells by TUNEL (TDT-mediated dUTP-biotin nick end-labeling) as well as by transmission electron microscopy. By analyzing adjacent serial sections, we demonstrated that the expression of Msx-2 and Bmp-2 strikingly overlapped in the atrioventricular canal myocardium, in the atrioventricular junctional myocardium, and in the maturing myocardium of the atrioventricular valves. Bmp-4 was expressed in the outflow tract myocardium and in the endocardial cushion of the outflow tract ridges from E12 to E14. Msx-2 appeared in the mesenchyme of the atrioventricular endocardial cushion from E11 to E14, while Bmp-2 and Bmp-4 were detected between E11 and E14. Apoptotic cells were also detected in the mesenchyme of the endocardial cushion between E12 and E14. Our results suggest that BMP-2 and MSX-2 are tightly linked to the formation of the atrioventricular junction and valves and that BMP-4 is involved in the development of the outflow tract myocardium and of the endocardial cushion. In addition, BMP-2, BMP-4 and MSX-2 and apoptosis seem to be associated with differentiation of the endocardial cushion.

Tissue inhibitor of metalloproteinases 4 (TIMP4) is expressed highly in heart and found dysregulated in human cardiovascular diseases. It controls extracellular matrix remodeling by inhibiting matrix metalloproteinases (MMPs) and is implicated in processes including cell proliferation, apoptosis, and angiogenesis. Timp4-deficient mice (Timp4 ؊/؊ ) were generated to assess TIMP4 function in normal development and in models of heart disease. We deleted exons 1-3 of the Timp4 gene by homologous recombination. Timp4 ؊/؊ mice are born healthy, develop normally, and produce litters of normal size and gender distribution. These mice show no compensation by overexpression of Timp1, Timp2, or Timp3 in the heart. Following cardiac pressure overload by aortic banding, Timp4 ؊/؊ mice have comparable survival rate, cardiac histology, and cardiac function to controls. In this case, Timp4 deficiency is compensated by increased cardiac Timp2 expression. Strikingly, the induction of myocardial infarction (MI) leads to significantly increased mortality in Timp4 ؊/؊ mice primarily due to left ventricular rupture. The post-MI mortality of Timp4 ؊/؊ mice is reduced by administration of a synthetic MMP inhibitor. Furthermore, combining the genetic deletion of Mmp2 also rescues the higher post-MI mortality of Timp4 ؊/؊ mice. Finally, Timp4 ؊/؊ mice suffer reduced cardiac function at 20 months of age. Timp4 is not essential for murine development, although its loss moderately compromises cardiac function with aging. Timp4 ؊/؊ mice are more susceptible to MI but not to pressure overload, and TIMP4 functions in its capacity as a metalloproteinase inhibitor after myocardial infarction.Tissue inhibitors of metalloproteinases (TIMPs) 5 comprise a family of four endogenous inhibitors. Classically, matrix metalloproteinases (MMPs) and TIMPs are known as important regulators of extracellular matrix turnover during physiologic and numerous pathologic processes. Several other functions also have been ascribed to MMPs, many of which extend to their inhibitors (1). TIMPs also exhibit functions that appear to be independent of their metalloproteinase inhibitory capacity (2).TIMP4 is the most recently discovered member of the TIMP family. It inhibits several soluble MMPs (types 1, 2, 3, 7, 8, 9, 12, 13, 19, and 26) and membrane-type MMPs (MT1, MT2, and MT3) (3-7). TIMP4 also inhibits a disintegrin and metalloproteinase (ADAM) 28 and ADAM33 (7) but not ADAM10 (which is inhibited by TIMP1 and TIMP3) or ADAM17 (which is inhibited only by TIMP3) (2,7,8). Inhibition of the ADAMTS (ADAM with thrombospondin motifs) family by TIMP4 has not yet been reported. Although Timp genes 1, 2, and 3 are widely expressed, the Timp4 gene exhibits a restricted tissue expression pattern, with the highest expression in the heart, followed by brain, ovary, and skeletal muscle, and TIMP4 protein is detectable in the serum (9 -11). Several lines of evidence suggest a specific role in cardiovascular pathology: Timp4 is induced following endothelial injury of rat carotid art...

The transformation of the endocardial cushion into valves and septa is a critical step in cardiac morphogenesis as it initiates the development of the four-chambered heart. This transformation results from a region-specific balance between cellular proliferation, apoptosis, and differentiation. The development of the form and structure of the endocardial cushion is accompanied by precise patterns of abundant cell death having the morphological features of programmed cell death (apoptosis), which plays an important role in the elimination of redundant cells and in changes of phenotypic composition during histogenesis. Apoptosis is an essential process in morphogenesis as it balances mitosis in renewing tissues. It is controlled by one or more genetic programs that kill the targeted cell. However, the causes, role, and regulation of apoptosis in the developing endocardial cushion still remain to be determined. The clarification of the role of the apoptosis regulatory genes constitutes a major task in future studies of cell death in the developing heart. This new molecular histology of heart development awaits further experiments to clarify the interactive mechanisms that act to ensure the sculpting of the endocardial cushion into valves and septa by determining the size of the cushion cell populations. The relation between the expression of different factors and the modifications of the cushion region during cardiac development are reviewed. In addition, we review and summarize information on molecules identified in our experiments that imply the activity of a number of essential genes coinciding with the key steps in generating the overall architecture of the heart. We correlate their temporal and spatial expression with their proposed roles.

Apoptosis is an important mechanism in organogenesis, but its role in heart development has been poorly characterized. We have here studied apoptosis in the developing ventricular wall of mouse embryonic heart. Developing mice hearts on days 11 to 16 of gestation were studied using in situ end-labeling of degraded DNA (TUNEL), immunocytochemistry of regulatory genes Bcl-2 and Bax, and light and electron microscopy. TUNEL end-labeled apoptotic cells were found in the ventricular wall on days 11 to 16 of gestation. The proportions of apoptotic cells of all cells in the ventricular wall differed between the trabecular and compact regions (P ϭ 0.003) and between the days of gestation (P ϭ 0.0001), the calculated apoptotic index was greater in the compact region at all ages except day 14.Ultrastructural analysis showed typical apoptotic shrinkage, chromatin degradation, and apoptotic bodies in several myoblastic and myocardial endothelial cells which were also positive by DNA end-labeling. Immunocytochemical reaction for the apoptosis checkpoint proteins in the ventricular wall showed clearly more Bcl-2 positive cells than Bax positive cells. The numerical densities of all cells in the compact and trabecular regions remained always higher in the compact region (P ϭ 0.04) despite the fact that apoptosis was present in both areas at the same time.In conclusion, apoptosis takes place in the developing myocardial muscle as well as the myocardial endothelium during ventricular morphogenesis on days 11 through 16 and decreases clearly on day 16. We suggest that apoptosis and its regulatory factors are closely involved in the morphogenesis of the ventricular wall of the mammalian heart.

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