Combining Stiffness and Stretch to Study Cardiac Fibroblast Pro‐Fibrotic Activity
Authors: Gilles George K. , McCulloch Andrew D. , Herum Kate M. 
Published: June 2021

Cardiac fibrosis is prevalent in many forms of heart disease and results from the excessive accumulation of extracellular matrix (ECM). When the heart is subjected to mechanical stress, it induces a signal in cardiac fibroblasts that enhances collagen production and induces differentiation into myofibroblasts. Myofibroblasts are characterized by expression of smooth muscle α‐actin (SMA) enabling them to “pull” on the newly synthesized collagen and thereby remodel the heart. Although these properties initially provide structural support to the mechanically‐stressed heart, persistent collagen production and myofibroblast activity will lead to myocardial stiffening and thereby compromise cardiac diastolic function. To date, there is no effective treatment for cardiac fibrosis reflecting incomplete understanding of the underlying mechanisms that convert mechanical stress into pro‐fibrotic activity of cardiac fibroblasts, as well as differential effects of stretch and stiffness. Previous data showed stiffness‐dependent responses to stretch between cardiac fibroblasts plated on hydrogels with stiffness in the lower (3 kPa) and upper (8 kPa) physiological range. We here study the pro‐fibrotic responses to stretch in cardiac fibroblasts plated on substrates with stiffness corresponding to the healthy (4.5 kPa) and fibrotic (40 kPa) heart. Cardiac fibroblasts were isolated from mouse ventricles and plated on collagen‐coated polyacrylamide (PA) gels with stiffness of 4.5 kPa or 40 kPa. Immunocytochemistry for SMA fibers demonstrated lack of SMA fibers at 4.5 kPa while prominent SMA fiber formation at 40 kPa. Staining for the focal adhesion protein vinculin revealed smaller focal adhesions in cardiac fibroblasts on 4.5 kPa compared to 40 kPa indicating differences in these important mechanotransduction sites. To examine the effect of stretch on cardiac fibroblasts plated on 4.5 and 40 kPa stiffness, PA gels were attached to polydimethylsiloxane (PDMS) stretch membranes and cardiac fibroblasts stretched (10 % area increase) for 24 h. mRNA was extracted to measure collagen 1a1, collagen 1a2, collagen 3a1, fibronectin, SMA, lysyl oxidase and osteopontin expression. In response to stretch, cardiac fibroblasts plated on gels with fibrotic stiffness exhibited less increase in fibrotic gene expression compared to cardiac fibroblasts on gels with physiological stiffness. Preliminary results indicate rapid upregulation of collagen 1a1 30 min after stretching when cardiac fibroblasts are plated on 4.5 kPa. In contrast, collagen 1a1 was downregulated at this time point when cardiac fibroblasts were plated on 40 kPa, indicating that the response to stretch is highly dependent on substrate stiffness and the initial phenotype of the cardiac fibroblast. It will be interesting to more closely examine the underlying mechanotransduction pathways that are activated at different stiffness and thereby possibly enable selective targeting of myofibroblasts.Support or Funding InformationThis work was supported in part by the Research Council of Norway (K.M.H.), National Institutes of Health grants (GM094503, GM103426, HL105242, HL122199, and HL105242) and by a California Institute of Regenerative Medicine grant (CIRM RT3‐07899).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.