Transforming Growth Factor β1 Modulates Sex Differences in Cardiac Myofibroblast Activation on Hydrogel Biomaterials

Background and Purpose Cardiac fibrosis is a pathological process in which the myocardium stiffens due to the overproduction of extracellular matrix (ECM) proteins. Cardiac fibroblasts activate to myofibroblasts in response to the inflammatory cytokine transforming growth factor beta1 (TGF-β1) to promote fibrotic scarring. Biological sex also influences cardiac fibrosis progression and patient outcomes, where males exhibit increased fibrotic scarring after acute inflammation relative to females. At the cellular level, sex differences in TGF-β1-mediated cardiac myofibroblast activation processes have not been clearly defined. We hypothesized that TGF-β1 would cause sex-specific cardiac myofibroblast activation levels and alter the secretion of bioactive molecules to modulate sex differences in cardiac fibrosis. Methods Primary left ventricle cardiac fibroblasts were isolated from male and female C57BL/6 J mice and cultured on hydrogel biomaterials mimicking native myocardial ECM stiffness and treated with TGF-β1 and/or the TGF-β1 receptor inhibitor SD208. We assessed myofibroblast activation via immunofluorescence for α-smooth muscle actin (α-SMA) and Mothers against decapentaplegic homolog 2/3 (SMAD2/3) localization, quantified myofibroblast gene expression, and performed cytokine arrays to analyze secreted factors. Results Male myofibroblasts exhibited increased α-SMA stress fiber formation, increased SMAD2/3 localization, and greater resistance to SD208 inhibition compared to female myofibroblasts on hydrogels at various time points tested. Sex differences in relative secreted cytokine abundance were also determined, with male CFs secreting increased vascular endothelial growth factor (VEGF) and female CFs producing increased periostin and fibroblast growth factor 21 in response to TGF-β1. Conclusion Our findings establish that TGF-β1 mediates sex differences in cardiac myofibroblast activation on hydrogels and secreted factors that may modulate the myocardial microenvironment. Our work underscores the importance of using hydrogels as cell culture platforms to recapitulate sex-specific cardiac fibrosis phenotypes as a steppingstone towards identifying sex-dependent therapeutic interventions for cardiac fibrosis.