K8 - Soft matrix permits agonist-mediated cardiac myofibroblast activation

Introduction: : Cardiovascular disease continues to be the leading cause of death globally, and adverse extracellular matrix (ECM) remodeling often plays a critical role in reduced heart function during disease. Persistent activation of cardiac fibroblasts (CFs) can cause them to increase ECM deposition, leading to myocardial fibrosis. While inflammatory cytokines or increased ECM stiffnesses are necessary to activate CFs, the extent to which intrinsic matrix properties activate CFs is less clear. Genomic sequencing of murine myocardial infarction models show different subtypes of CF populations with a time dependence following injury, corresponding to cardiac remodeling. Further, in vitro models of CF activation show different cytokine profiles and RNA transcription levels depending on the stimuli used to activate the CFs. Understanding the contribution of the microenvironment to human CF response is crucial to designing models and therefore therapies for cardiac fibrosis; here, we tested the response of stem-cell derived CFs to prolonged culture in a soft niche and then how CFs respond to stiffened matrix or contractile agonists to mimic the onset of fibrosis.

Materials and

Methods: : Pluripotent stem cell-derived cardiac fibroblasts were cultured in fibroblast maintenance media then selectively replated on ECM of different stiffness with or without myofibroblast agonists to assess the persistence of ECM-induced activation. A combination of known myofibroblast agonists was used, composed of TGFβ-1, Angiotensin II, and low molecular weight Hyaluronic Acid for 48 hours. Immunofluorescence staining for proteins of interest were used to quantify activation levels. Bulk ATAC-sequencing was used to quantify the epigenomic accessibility of CFs, and differentially compared between samples to quantify the relative accessibility of individual peaks.

Results, Conclusions, and Discussions:: As classically seen with primary cells, stem-cell derived CFs cultured on tissue culture plastic for multiple passages and in the presence of myofibroblast agonists become activated myofibroblasts. These CFs activate quickly and show alpha-Smooth Muscle Actin (αSMA) in both the conditions treated with the agonists and the control condition; intensity and fiber formation increased with agonist treatment. Surprisingly, CFs on softened matrix became the most activated when exposed to myofibroblast agonists or a histone deacetylase inhibitor (Figure 1, A-B). These data suggested that CFs retain a degree of mechanical “memory”. ATAC-sequencing show that softened matrix facilitates the CF response to agonists via enhanced accessibility of myofibroblast transcription sites, allowing for a more robust response to agonists (Figure 1, C-E). Current work includes investigating these trends in prolonged culture on softer environments relative to stiffer environments. These results highlight the need for physically relevant in vitro models that account for the epigenetic and mechanical history of CFs. Further, these results could be used to study CF response at specific timepoints corresponding with changes to the mechanical environmental after injuries such as MI, or in diseases with prolonged cardiac remodeling.

Acknowledgements and/or References (Optional)::