We completed our original Specific Aims directed at studying the effects of Al adenosine receptor (A1AR) activation on the developing heart. We found that adenosine is the dominant humoral regulator of embryonic cardiac function, that early A1AR expression results from NKX2.5 and GATA4 activation of the A1AR promoter, and that A1AR activation inhibits cardiac cell division leading to cardiac hypoplasia. We also expanded our studies to examine cardiac effector systems during early heart formation. In the course of these experiments we found that Rho GTPase signaling plays a very important role in heart formation. In this competitive renewal, we will focus on the influence of Rho GTPase activity on mammalian cardiac development and the regulation of Rho activity by receptors for adenosine and other neuromodulators. GTPases are small intracellular signaling molecules that transduce the effects of extracellular signals and include Rho, which activates Rho-associated kinase (ROCK) to influence important cellular events. Recent evidence suggests that these factors play essential roles in the mature myocardium. Yet, their role in heart development is not known. Suggesting that GTPases are important in heart formation, we found that blockade of the Rho/ROCK effector system leads to major cardiac malformations. Thus, we hypothesize that Rho GTPases play a critical role in heart formation. We also hypothesize that cardiac Rho/ROCK activity is regulated by G protein-coupled receptors (GPCRs) during early developmental stages. To test these hypotheses and provide insights into the role of GTPases in the developing heart, the following Specific Aims are proposed. (1) We will characterize the temporal and spatial patterns of Rho/ROCK expression during embryogenesis. (2) We will examine the effects of Rho/ROCK signaling on heart development. (3) We will examine mechanisms by which Rho/ROCK action effects heart development. (4) We will examine GPCR regulation of Rho/ROCK activity in the embryonic heart. These experiments will build on the experience we acquired in studying the effects of adenosine on the developing heart. We anticipate that these studies will identify Rho and ROCK as critical signaling molecules in heart development and reveal that cardiac GTPase activity transduces the effects of adenosine.
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