Myofibroblasts (MFs) differentiate from fibroblasts and function to facilitate wound closure. Under pathological conditions, MFs fail to undergo apoptosis and continue to remodel the extracellular matrix, leading to increased contracture of connective tissue. MFs are characterized as having "super‐mature" focal adhesions and expression of smooth muscle α‐actin (SMAA). Myocardin‐related transcription factors A and B (MRTF‐A/MAL/MKL‐1 and MRTF‐B/MKL‐2) are putative mechanical stress‐induced co‐activators that activate expression of contractile proteins such as SMAA. Small interfering RNA (siRNA) has been used to study the effects of MRTF‐A and MRTF‐B reduction on the MF contractile phenotype in rat embryonic fibroblasts. Decreased SMAA levels, as determined by immunoblot analyses, and reduced contractile force generation, as determined by a wrinkling assay, were observed in cells transfected with MRTF‐A and ‐B‐targeted siRNA. Immunocytochemistry has shown a decrease in the size and number of focal adhesions in these cells when compared to control cells. Finally, transiently transfected myocardin, which acts similarly to constitutively‐active MRTF‐A, increased SMAA promoter activity and increased the contractile ability of cells plated on a deformable wrinkling substrate. These results suggest MRTFs play a critical role in regulating MF formation and function. (Funded by NIH 2R01GM60651)Grant Funding SourceNIH

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Myofibroblasts are specialized fibroblasts that facilitate wound closure through expression of smooth muscle (SM) contractile proteins and generation of contractile force on the surrounding matrix. We hypothesize mechanoregulation of SM α‐actin (SMAA) expression in myofibroblasts is mediated by actin dynamics regulating localization of myocardin related transcription factor‐A (MRTF‐A); on compliant substrata, high levels of G‐actin will sequester MRTF‐A, while on stiff substrata decreased G‐actin levels will release MRTF‐A to interact with the SMAA promoter. Rat embryonic fibroblasts cultured on novel deformable substrata of varying stiffness were analyzed. Increasing substratum stiffness resulted in a graded increase in SMAA promoter activity and endogenous mRNA expression, focal adhesion and stress fiber assembly, and MRTF‐A nuclear localization, suggesting that the lack of a critical stiffness acts as a switch. The actin polymerizing agents, jasplakinolide and cytochalasin D, promoted nuclear localization of MRTF‐A and increased SMAA expression, independent of substratum stiffness. Similarly, a constitutively nuclear MRTF‐A promoted SMAA expression independent of substratum stiffness. This work demonstrates that actin dynamics, in response to the mechanical environment, regulate nuclear localization of MRTF‐A and thereby expression of SMAA. Funded by NIH grant R01 GM60651.Grant Funding SourceNIH grant R01 GM60651