Abstract Heart disease is the leading cause of death globally and poses an enormous public health and economic burden. In the United States, prevalence and mortality due to heart failure have even increased in recent years, underscoring the need for a greater understanding of pathophysiological signaling mechanisms that underlie cardiac maladaptation. The cardiac myocyte is a highly specialized cell-type with a complex and unique cytoarchitectural landscape. However, mechanisms that topologically orient intracellular signaling events in cardiomyocytes remain ill-defined. While the roles of phosphorylation in cardiac signal transduction have been studied for decades, very little is known regarding regulation of signaling by lipid modifications. Palmitoylation or S-acylation, the reversible attachment of fatty acids onto protein cysteines, is an optimal mechanism to regulate dynamic association of proteins with signaling complexes that nucleate at intracellular membranes. We found that protein palmitoylation modulates localized signaling in cardiomyocytes and participates in cardiac hypertrophy and failure. Specifically, our recent data indicate that palmitoylation of Ras-related C3 botulinum toxin substrate 1 (Rac1) exerts profound spatiotemporal control of cardiomyocyte Rac1 signaling in vivo. The small GTPase Rac1 is a critical regulator of pathogenic signaling and oxidative stress in cardiac hypertrophy, heart failure, and cardiac arrhythmia that functions in part through regulation of the NADPH oxidase 2 (Nox2) complex that regionally generates reactive oxygen species in response to hypertrophic stimulation. Here, we will exploit regulation of localized Rac1 signaling in cardiomyocytes by palmitoylation to reveal the microscale intracellular signal transduction landscape in cardiac hypertrophy. This proposal will test the central hypothesis that palmitoylation of Rac1 targets signaling activity to discrete cardiomyocyte membrane microdomains to elicit activation of unique effector pathways and pathophysiologic responses. In this application we will manipulate Rac1 palmitoylation status in vivo in the context of cardiac hypertrophy to achieve the following aims: (1) determine the role of Rac1 palmitoylation in cardiac hypertrophy, compartmentalization of Nox2 activity, and oxidative stress and (2) uncover effectors and signaling pathways regulated by palmitoylated versus depalmitoylated proteoforms of Rac1. We will achieve these aims in part using a novel mouse model with inducible knock-in mutation of the Rac1 palmitoylation site (Rac1 Cys178Ser). These studies will establish a paradigm for palmitoylation as a lipidation switch mechanism for spatially encoding cardiomyocyte signal transduction circuitry and will identify pathogenic microdomain signaling events that may provide more targeted vantage points for treatment of cardiac hypertrophy and heart failure.