Our overall goal is to determine signaling pathways and function in normal and diseased hearts. We have recently focused on the m2-muscarinic acetylcholine receptor (m2-AchR) activation of the Gi family of heterotrimeric G-proteins which inhibit heart rate, contraction and relaxation. This proposal replaces our previous grant on these pathways. This proposal tests the importance of beta2-adrenergic receptor (beta2AR) coupling Gi family members leading to changes in contraction-relaxation and cell survival both in vitro and in vivo. Critical unknowns are the specific pathways used through these multiple gene products and the physiologic consequences. To determine the specific gene products transducing these signals, we have produced knockouts of each of the Gi family members expressed in heart (Galphai2, Galphai3, and Galphao). Inactivation of the alpha subunits effectively removes the heterotrimer. These knockouts will allow us to determine the mechanism of inhibition of contraction and relaxation, first by determining the specific Gi-proteins, which are mediating the responses, and second determining how each knockout affects other signaling intermediates (Specific Aim 1). The beta2AR-Gi coupling has also been implicated in antiapoptotic pathways (Specific Aim 2). We will extend our analysis to determine the importance of these specific pathways in vivo both with acute and chronic beta-adrenergic stimulation using both pharmacological and genetic probes (Specific Aim 3). These pathways may be critical to the pathogenesis and treatment of cardiac hypertrophy and failure. Equally important, this proposal uses the cardiac cell as a model system and tests a new paradigm for maintaining specificity in G-protein signaling (Specific Aim 4). As discussed in Background and Significance, we have now established that each of the Gi family members have specific functions. In addition, multiple Gi family members activated by a single receptor transduce signals not just by overlapping phenotypes (i.e. family members can substitute for each other) or by a single family member to a single response, but also through required combinations. That much of the specificity is maintained by specific combinations of Gi pathways is a new paradigm. While initially unexpected, we have found a growing number of examples. This new paradigm requires us to broaden and reevaluate our concepts about how G-proteins maintain specificity in signaling and sort multiple inputs and outputs. We will test this paradigm by comparing the Gi family members required for 2 very different receptors.