Substrate Stiffness Regulates Cardiac Fibroblast Phenotype in Volume Overload Induced Heart Failure

Abstract

ObjectiveDetermine whether substrate stiffness contributes to the hypo‐fibrotic phenotype of cardiac fibroblasts (CFs) isolated from a volume overload heart failure model.MethodsPrimary (P0) CFs were isolated from left ventricle (LV) tissue of sham operated (control) rats and rats subjected to 4 weeks of volume overload surgically induced by Aortocaval Fistula (ACF). CFs were plated on polyacrylamide gels at a range of stiffness (0.2 kPa to 50 kPa) similar to LV stiffness in vivo. The alignment of f‐actin and αSMA was assessed by immunostaining and used as an indicator of the pro‐fibrotic myofibroblast phenotype. ImageJ was used to quantify fluorescent intensity and morphology. Changes in gene expression were assessed by qRT‐PCR.ResultsSubstrate stiffness differentially affected CF morphology and αSMA expression. CFs on soft gels had a smaller spread area; CFs on stiff gels displayed stellate morphologies and αSMA aligned stress fibers (Fig A). Compared to sham, there was ~50% reduction in ACF CFs differentiation towards a myofibroblast phenotype (Fig B) on stiff substrates. Substrate stiffness elicited significantly different patterns of αSMA (~5‐fold), BNP (~3‐fold) and PPAR‐γ (~12‐fold for sham; ~6‐fold for ACF) mRNA expression between sham and ACF (FigC‐E).ConclusionsThis study provides evidence that substrate stiffness plays an important role in phenotype regulation by affecting cytoskeletal organization and mRNA expression. The changes in αSMA, BNP, and PPAR‐γ could provide some explanation for the distinct phenotype in ACF.image