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Cardiac fibroblast BAG3 regulates TGFBR2 signaling and fibrosis in dilated cardiomyopathy

ABSTRACT: Loss of Bcl2-associated anthanogene 3 (BAG3) is associated with dilated cardiomyopathy (DCM). Recent studies show that BAG3 regulates sarcomere protein turnover in cardiomyocytes (CMs). However, the function of BAG3 in other cell types of the heart is unknown. In this study, we used an isogenic pair of BAG3 knockout and wild-type human induced pluripotent stem cells (hiPSC) to interrogate the function of BAG3 in hiPSC-derived cardiac fibroblasts (CFs). Generating a series of conditional knockout (cKO) engineered heart tissues, we determined the contribution of CF BAG3 to engineered heart tissue function. Despite normal CM genotype, the loss of fibroblast-specific BAG3 (FBKO) caused a reduction in contractile force and an increase in cardiac fibrosis in the engineered heart tissues, recapitulating the phenotype of DCM. Using culture substrates with defined stiffness, we decoupled the mechanical activation of fibroblasts from their ligand response. In BAG3-/- CFs, we observed an increased sensitivity to transforming growth factor β (TGFβ) signaling and activation of a fibrogenic response when cultured at physiological stiffness (8kPa). Mechanistically, loss of BAG3 increased transforming growth factor receptor 2 (TGFBR2) levels. BAG3 binds TGFBR2 and mediates its ubiquitination and proteasomal degradation. To further validate these results, we performed single-nuclei sequencing of cardiac tissue from DCM patients carrying pathogenic BAG3 mutations. Mutations in BAG3 increased fibrotic gene expression in cardiac fibroblasts. Together, these results extend our understanding of the roles of BAG3 in heart disease beyond the cardiomyocyte-centric view. This study also highlights the ability of hiPSC-based models and tissue engineering to answer questions about the cell-type specific aspects of cardiac disease.

ORGANISM(S): Homo sapiens

PROVIDER: GSE261750 | GEO | 2024/09/16

REPOSITORIES: GEO

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Project description:In vitro cultures of primary cardiac fibroblasts (CFs), the major extracellular matrix (ECM)-producing cells of the heart, are used to determine molecular mechanisms of cardiac fibrosis. However, the supraphysiologic stiffness of tissue culture polystyrene (TCPS) automatically triggers the conversion of CFs into an activated myofibroblast-like state, and serial passage of the cells results in the induction of replicative senescence. These dramatic phenotypic switches confound interpretation of experimental data obtained with cultured CFs. In an attempt to circumvent TCPS-induced activation and senescence of CFs, we utilized poly (ethylene glycol) (PEG) hydrogels as cell culture platforms with low and high stiffness formulations to mimic healthy and fibrotic cardiac ECM, respectively. As hypothesized, low hydrogel stiffness converted activated CFs into a quiescent state with reduced abundance of a-smooth muscle actin (a-SMA)-containing stress fibers. Unexpectedly, lower substrate stiffness concomitantly augmented CF senescence, marked by elevated senescence-associated b-galactosidase (SA-b-Gal) activity and increased expression of p16 and p21, which are cyclin-dependent kinase (CDK) inhibitors and markers of senescence. Using dynamically stiffening hydrogels with phototunable crosslinking capabilities, we demonstrate that substrate-induced CF senescence is partially reversible. RNA-sequencing analysis revealed widespread transcriptional reprogramming of CFs cultured on low stiffness hydrogels, with a dramatic reduction in the expression of profibrotic genes encoding ECM proteins, and an attendant increase in expression of NF-kB-responsive inflammatory genes that typify the senescence-associated secretory phenotype (SASP). Our findings further demonstrate that alterations in matrix stiffness profoundly impact CF cell state transitions, and suggest mechanisms by which CFs change phenotype in vivo depending on the stiffness of the myocardial microenvironment to which they are exposed.

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Cardiac fibroblast BAG3 regulates TGFBR2 signaling and fibrosis in dilated cardiomyopathy

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