Purpose: RhoA has been shown to be beneficial in cardiac disease models when overexpressed in cardiomyocytes whereas its role in cardiac fibroblasts (CF) is still poorly understood. During cardiac remodeling CF undergo a transition towards a myofibroblastic phenotype showing an increased proliferation and migration rate. Both processes involve the remodeling of the cytoskeleton, a process known to be strongly regulated by RhoA.
Methods: To investigate the role of RhoA in neonatal rat cardiac fibroblasts we used a lentivirus-based knockdown (RhoA-KD-CF). In addition, wild type (wt) CF were treated with 10 μM Fasudil for ROCK inhibition or 5 μg/mL Tubastatin A (Tub A) for tubulin-specific deacetylase HDAC6 inhibition. Cytoskeletal proteins were analyzed by immunoblot and immunofluorescence. Adhesion velocity and migration was determined by microscopy and the serum-driven proliferation rate by nuclei counting.
Results: Compared to control cells (shControl) RhoA-KD-CF develop an epithelial-like morphology lacking stress fibers and higher order actin structures like geodesic domes. The orientation of focal adhesions sites along the cell stress axis was also impaired. This phenotype could be mimicked by the treatment of CF with Fasudil. Furthermore, in RhoA-KD-CF cytoskeletal proteins were found to be unchanged except for a decrease in the myofibroblast marker smooth muscle actin by 43% and an increase in acetylated tubulin by 57% without a change in the expression of HDAC6. In order to analyze the impact of both changes we investigated the migration and proliferation rate of CF. First, the reduction of RhoA accelerated the adhesion but decelerated migration (shControl 4043±316 nm/h versus shRhoA 3021±153 nm/h) and second, the serum-driven proliferation rate of RhoA-KD-CF was reduced by 50%. Interestingly, treatment of wt-CF with Fasudil significantly decreased migration velocity by 62% but had no effect on the proliferation rate, whereas Tub A only slightly decreased migration velocity but reduced proliferation rate by 60%.
Conclusion: RhoA influences the two main myofibroblast characteristics, migration and proliferation, by interfering with the actin and tubulin cytoskeleton via ROCK and HDAC6, respectively.