Investigation of stretch- and stiffness-induced pro-fibrotic mechanotransduction activity in cardiac fibroblasts

Cardiac fibrosis is the excessive accumulation of extra-cellular matrix that is mainly regulated by the activation of cardiac fibroblasts and their differentiation into myofibroblasts. Mechanical forces are important regulators of cardiac fibroblast activation. However, it is not clear which fibrotic signaling pathways are activated by the specific mechanical cues. Therefore, we used in vitro stretch models as well as stiff and soft hydrogels to examine how mechanical stretch and stiffness impacts the pathways involved in cardiac fibroblasts’ ability to generate fibrotic phenotypes. Treatment of cardiac fibroblasts on plastic with transforming growth factor β receptor I inhibitor resulted in lower mRNA expression levels for key myofibroblast gene markers. Transforming growth factor β receptor I inhibitor also eliminated the stretch induced upregulation of key fibrotic genes for fibroblasts on soft gels but only eliminated upregulation of the smooth muscle α-actin gene on stiff gels. Surprisingly, inhibition of Rho kinase did not impact expression levels of pro-fibrotic genes for cardiac fibroblasts on plastic and hydrogels. Due to complications with the hydrogel stiffness, atomic force microscopy showed that hydrogels can change their stiffness after fabrication depending on the environment the gel is in, explaining the unusual cellular response we were experiencing with the fibroblasts on hydrogels. Overall, transforming growth factor β signaling does significantly influence how cardiac fibroblasts generate pro-fibrotic phenotypes. However due to the revelation of the changing stiffness with the hydrogels, more work is needed to determine whether this pathway is more involved in stretch responses or stiffness responses. More research is needed to determine whether inhibiting transforming growth factor β signaling in cardiac fibroblasts can be used to maintain a freshly isolated phenotype while culturing large quantities of cardiac fibroblasts for longer periods on plastic tissue culture substrates