Abstract P1007: Cardiac Fibroblast BAG3 Controls TGFBR2 Signaling And Contributes To Engineered Cardiac Tissue Function.

Introduction: Mutations in BAG3 result in dilated cardiomyopathy with cardiac fibrosis. Currently, the role of BAG3 in non-myocyte cell types is not known. We tested the hypothesis that BAG3 controls cardiac fibroblast (CF) function and that the loss of fibroblast-specific BAG3 is deleterious, using cells and engineered cardiac tissues (ECT) derived from pluripotent stem cells (iPSC). Methods: Isogenic iPSCs containing homozygous knockout (KO) of BAG3 were differentiated into CFs. Mass spectrometry measured the effect of BAG3 loss on CF proteome. EdU staining and flow cytometry were used to assess CF proliferation. Transforming growth factor beta (TGF) signaling pathway activity was measured using a luciferase reporter and Western blotting. Affinity purification mass spectrometry (AP-MS) identified high-confidence interactors of BAG3 in CFs. To assess the role of fibroblast-specific BAG3 in cardiac function, conditional KO ECTs were generated using wild-type cardiomyocytes mixed with either wild-type CFs or KO CFs. ECTs were functionally assessed using video microscopy and immunofluorescence. The effects of CF BAG3 loss on tissue gene expression are under study by single nuclear RNA sequencing. Results: Analysis of proteomics data revealed enrichment of TGF and proliferation pathways in KO cells. KO-CFs had increased proliferation measured by EdU staining and increased SMAD luciferase reporter activity. Western blotting showed that KO CFs have higher expression of TGF beta receptor 2 (TGFBR2) and activation of canonical TGF signaling. AP-MS identified TGFBR2 as an interacting partner of BAG3, suggesting BAG3 mediated TGFBR2 turnover. In ECT, the loss of CF BAG3 caused a reduction in cardiac tissue contractile force (50% reduction, n = 10-12 tissues per group). Immunofluorescence revealed significantly increased tissue fibrosis in CF-KO cardiac tissues quantified by vimentin staining. Conclusions: BAG3 loss results in increased TGFBR2 signaling and proliferation in cardiac fibroblasts. Conditional KO ECTs demonstrate that CF BAG3 affects tissue function. These data suggest that fibrosis related to BAG3 pathology may be a result of a primary deficiency in CFs rather than a secondary response to cardiomyocyte death.