Culturing of Cardiac Fibroblasts in Engineered Heart Matrix Reduces Myofibroblast Differentiation but Maintains Their Response to Cyclic Stretch and Transforming Growth Factor β1

Abstract

Isolation and culturing of cardiac fibroblasts (CF) induces rapid differentiation toward a myofibroblast phenotype, which is partly mediated by the high substrate stiffness of the culture plates. In the present study, a 3D model of Engineered Heart Matrix (EHM) of physiological stiffness (Youngs modulus ~15 kPa) was developed using primary adult rat CF and a natural hydrogel collagen type 1 matrix. CF were equally distributed, viable and quiescent for at least 13 days in EHM and the baseline gene expression of myofibroblast-markers alfa-smooth muscle actin (Acta2), and connective tissue growth factor (Ctgf) was significantly lower, compared to CF cultured in 2D monolayers. CF baseline gene expression of transforming growth factor-beta1 (Tgfβ1) and brain natriuretic peptide (Nppb) was higher in EHM-fibers compared to the monolayers. EHM stimulation by 10% cyclic stretch (1 Hz) increased the gene expression of Nppb (3.0-fold), Ctgf (2.1-fold) and Tgfβ1 (2.3-fold) after 24 h. Stimulation of EHM with TGFβ1 (1 ng/mL, 24 h) induced Tgfβ1 (1.6-fold) and Ctgf (1.6-fold). In conclusion, culturing CF in EHM of physiological stiffness reduced myofibroblast marker gene expression, while the CF response to stretch or TGFβ1 was maintained, indicating that our novel EHM structure provides a good physiological model to study CF function and myofibroblast differentiation.