Cardiomyocytes (CMs) are building blocks and function units of the heart, and their dysfunction or loss is the root of heart failure. Similar with other specialized innate immune cells, CMs have their own innate immune molecular machinery. Ischemic or non-ischemic pathological stress activates CM innate immune signaling pathways, which stimulate pro-inflammatory cytokine release and reactive oxygen species (ROS) production. These innate immune responses are beneficial for defending CMs against pathogen invasion and for tissue repair, but can also cause myocardial damage. The ultimate goal of this project is to define new molecular mechanisms that regulate CM innate immune responses, which will shed light on the pathogenesis of pathogen or non-pathogen related cardiomyopathy. Damage/danger-associated molecular patterns (DAMPs) initiate innate immune responses by binding to pattern recognition receptors, and one of the best-characterized signaling axis is TLR4/NF-kB pathway. The Hippo-YAP pathway is crucial for heart development and cardiac regeneration, and disturbances in Hippo-YAP have been implicated in a range of heart diseases. IRF2BP2 (interferon regulatory factor 2 binding protein 2) is a transcription co-factor little studied in the heart. We recently found that YAP regulates CM innate immune responses by blunting TLR4/NF-kB signaling, and that IRF2BP2 is crucial for restraining TLR4 expression in the heart. In this project, we will test the hypothesis that YAP and IRF2BP2 are suppressors of CM innate immune responses. We propose the following two aims. Aim 1. Define the role of YAP in CM innate immune responses. In this aim, we will determine whether YAP regulation of CM innate immune responses depends on its transcriptional activity. Additionally, we will develop a novel YAP modRNA delivery system. We previously showed that transiently activating YAP with intra-myocardial delivered aYAP modRNA reduced heart injury. However, the in situ delivery method limits aYAP modRNA's clinical application. Here, we will test the efficacy of CM targeted and nano-particle packaged aYAP modRNA in a murine cardiac ischemia/reperfusion model. Aim 2. Define the role of IRF2BP2 in CM innate immune responses. In this aim, we will perform IRF2BP2 gain- and loss-of-function studies to define its role in CM innate immune responses. Additionally, we will investigate the molecular mechanism of how IRF2BP2 regulates CM innate immune responses. Using the new Irf2bp2 knock-in mouse line we have recently generated, we will perform IRF2BP2 ChIP-Seq to identify its direct targets. This proposal is innovative and significant, as it focuses on dissecting the basic molecular mechanisms that underlie CMs innate immune responses, and it aims to develop new therapeutic strategies for reducing myocardial infarction injury.