Project Summary The extracellular matrix (ECM) is essential for maintaining cardiac structure and function. Aside from providing structural support, the ECM relays molecular signals to cells via cellular attachment complexes and ECM- sequestered growth factors to maintain cellular homeostasis. In response to acute injury or pathological stress, excess ECM deposition occurs that can contribute to cardiac dysfunction. Transforming growth factor β (TGFβ) is an ECM-sequestered growth factor that is released and activated by cardiac stress. Once released from the ECM, TGFβ induces differentiation of quiescent fibroblasts into myofibroblasts which secrete ECM components. However, the requirement for baseline TGFβ signaling in the heart remains unknown. Three TGFβ isoforms exist encoded by three separate genes (Tgfb1, Tgfb2, and Tgfb3) although the specific roles of each TGFβ isoform are not understood. To investigate the role of TGFβ ligands in regulating cardiac homeostasis, we generated mice with cardiomyocyte (CM)-specific deletion of TGFβ ligands (Tgfb1/2/3fl/fl-αMHC-Cre). These mice developed heart failure by 10 weeks of age accompanied by cardiac dilation without signs of fibrosis suggesting defective ECM deposition. In contrast to previous studies demonstrating that TGFβ receptor deletion had minimal effect on cardiac development, microarray analysis of Tgfb1/2/3fl/fl-αMHC-Cre hearts demonstrated a defect in CM differentiation indicating a key role for the ECM in driving CM maturation. To circumvent a developmental role of TGFβ, we employed a Tamoxifen-inducible CM-specific mouse model (Tgfb1/2/3fl/fl-αMHC-MCM) to delete TGFβ ligands from the adult heart. These mice developed heart failure 12-16 weeks post-tamoxifen treatment suggesting that TGFβ is required to maintain cardiac function in the adult heart. Surprisingly, fibroblast-specific deletion of Tgfb1/2/3 did not develop cardiac dysfunction indicating that TGFβ is primarily generated by CMs. Altogether, we hypothesize that TGFβ acts as a critical regulator of ECM-cell cross talk in the heart by signaling to fibroblasts to promote ECM production, which ultimately provides signals back to CMs to maintain their maturation. To address this hypothesis, we will use Tgfb1/2/3fl/fl-αMHC-MCM mice to determine the role of TGFβ in maintaining CM differentiation. Specifically, we will assess these mice for cardiac function, CM maturation and ECM composition and organization. Furthermore, we will perform RNA analysis at multiple timepoints both before and after the onset of cardiac dysfunction to determine the transcriptional role of TGFβ in maintaining CM maturation. Additionally, we aim to decipher the differential roles of each specific ligand (TGFβ 1, 2, or 3) in regulating cardiac function. The proposed studies will further assess the role of TGFβ in mediating CM-cardiac fibroblast crosstalk by studying fibroblast activity using in vitro models. Lastly, we will examine the composition, organizatio...