Myofibroblast differentiation in cardiomyocyte-cardiac fibroblast bilayer tissues on tunable synthetic fibrous matrices

Abstract

Cardiac pathologies are the leading cause of death in the United States with fibrosis implicated in many of these diseases. A hallmark of cardiac fibrosis is the differentiation of cardiac fibroblasts (CFs) into myofibroblasts (MFs) that synthesize and remodel the cardiac extracellular matrix (ECM). Previous studies have shown that modifications to the mechanics of the extracellular environment and cellular crosstalk between cardiomyocytes (CMs) and CFs are integral to this transformation. However, many of their in vitro settings fail to accurately recapitulate the mechanics and architecture of the native heart, let alone integrate different cell types. Here, we developed a novel bi-layer tissue platform (BLT) consisting of layers of CFs and CMs separated by a tunable synthetic fibrous matrix to mimic the ECM mechanics of healthy or diseased tissue states. Using this platform, we sought to understand how the synergy between mechanical cues and heterotypic cell-cell communication maintains tissue homeostasis vs. drives cardiac fibrosis. Dextran vinyl sulfone fibers were electrospun onto microfabricated PDMS substrates containing an array of square microwells (2x2 mm). Matrix alignment and stiffness were controlled by modulating the fiber collection mandrel speed and UV light exposure, respectively. CMs and CFs were seeded onto opposite sides of the matrix. Immunostaining for αSMA was conducted to assess MF differentiation. Our findings demonstrate that physical cues from the ECM play a critical role in MF differentiation, as supported by altered cellular mechanosensing as a function of matrix architecture and mechanics. Interestingly, co-culture with CMs hindered MF differentiation potentially through protective paracrine signaling. Future work will explore the influence of matrix mechanics on CM-CF communication and identify the mechanisms by which CMs inhibit MF differentiation by secretomics analyses and RNA sequencing.