Abstract 4369026: Proteasomal Degradation of MyD88 Directs TGFβ Induced Fibroblast Transformation in Heart Failure

Background: Cardiac fibrosis is a pathological hallmark of heart failure (HF) leading to cardiac dysfunction. Cardiac fibroblasts (CFB) are central mediators of both cardiac fibrosis and cardiac inflammation through their ability to recognize extrinsic cues such as profibrotic cytokines, and damage associated molecular patterns (DAMPs) which signal through MyD88. As inflammatory therapeutic targets governing pro-fibrotic CFB transformation are highly desirable, we hypothesized that MyD88 regulates CFB activity and fibrosis in HF. Methods: We subjected WT and MyD88-specific CFB -/- mice to PE/ANGII infusion and transverse aortic constriction (TAC) for 4 weeks, and characterized fibrosis and myofibroblasts by histologic and molecular biologic analyses. We leveraged publicly available single-cell RNA-sequencing data from preclinical and human hearts to measure Myd88 expression. We used primary mouse and human fibroblasts for in vitro studies of MyD88 in pro-fibrotic activity. Results: We found downregulation of MyD88 expression in mouse and human CFB in the failing human heart. TGFβ treatment of mouse and human CFB ex vivo also downregulated MyD88 gene and protein expression. Deletion of MyD88 in vitro enhanced myofibroblast transformation while overexpression of MyD88 prevented transformation. CFB-MyD88 -/- mice exhibited enhanced fibrosis, increased myofibroblast number, and worse systolic function after PE/ANGII or TAC compared to WT littermates. Mechanistically, we found that TGFβ acted both transcriptionally and through the Ubiquitin-Proteasome-System (UPS) to downregulate MyD88, which enhanced downstream TGFβ signaling by alleviating an inhibitory interaction between MyD88 and SMAD4. Specifically, TGFβ induces SMURF2 expression to degrade MyD88, and proteasomal inhibition or SMURF2 knockdown prevented MyD88 degradation and CFB transformation in mouse and human cells. Conclusion: Our data characterize a novel regulatory mechanism in myofibroblast transformation by which MyD88 is degraded in response to TGFβ through the UPS. This study highlights the diversity of functions for MyD88 in non-immune cells, adds to our understanding of TGFβ signaling, and highlights SMURF2 as a novel anti-fibrotic therapeutic target in HF.