Introduction: The role of RhoA in cardiac fibroblasts (CF) is still poorly understood. During cardiac remodeling CF undergo a transition towards a myofibroblast phenotype thereby showing an increased proliferation and migration rate. Both processes involve the remodeling of the cytoskeleton. Since RhoA is known to be a major regulator of the cytoskeleton, we analyzed its role in CF and its effect on myofibroblast characteristics in 2D and 3D models. Results: Downregulation of RhoA in neonatal rat CF by about 80% was accompanied by a disorganization of higher order actin structures including stress fibers, geodesic domes and focal adhesion sites. Functionally, the knockdown of RhoA increased the
adhesion velocity on plastic and collagen surfaces. On a molecular level, the expression of actin cytoskeleton-associated proteins investigated were found unchanged besides
a decrease in the myofibroblast marker α-sm-actin. Interestingly, in RhoA-depleted neonatal rat CF the fraction of acetylated tubulin, which is involved in intracellular vesicle- dependent transport processes, was found elevated. This data led to the investigation of serum response factor (SRF) activity and secretory processes with focus on profibrotic factors like CTGF, TGFβ and collagens. It was found that SRF activity as well as the expression of the investigated
profibrotic factors is regulated in a complex manner by RhoA signaling in CF. In assays accessing three different types of migration, we demonstrate that RhoA/ROCK/Dia1 are important for 2D migration and the repression of RhoA and Dia1 signaling accelerates 3D migration. Additionally it was found that proliferation of myofibroblasts rely on RhoA and tubulin acetylation. Finally, we show that a downregulation of RhoA in NRCF impacts the viscoelastic and contractile properties of engineered tissues. While engineered connective tissues containing RhoA knockdown cells show a delayed failure point, engineered heart muscle complemented with the same cells display a reduction in contractile force by 50%. Conclusion: Dependend on the environmental conditions RhoA positively or negatively influences myofibroblast characteristics by differential signaling cascades. Reduction of RhoA leads to an increase in viscoelasticity and a decrease in contractile force in engineered cardiac tissue.

Title

Acta Physiologica

Publisher

Wiley

Publisher Country

Germany

Indexing

Thomson Reuters

Impact Factor

5.97

Publication Type

Both (Printed and Online)

Volume

216

Year

2016

Pages

55-56