Journal Article

Maintaining resting cardiac fibroblasts in vitro by disrupting mechanotransduction

Gilles, George, Herum, Kate M., McCulloch, Andrew D., Brakebusch, Cord H.

2020

Overview

Mechanical cues activate cardiac fibroblasts and induce differentiation into myofibroblasts, which are key steps for development of cardiac fibrosis. In vitro, the high stiffness of plastic culturing conditions will also induce these changes. It is therefore challenging to study resting cardiac fibroblasts and their activation in vitro. Here we investigate the extent to which disrupting mechanotransduction by culturing cardiac fibroblasts on soft hydrogels or in the presence of biochemical inhibitors can be used to maintain resting cardiac fibroblasts in vitro. Primary cardiac fibroblasts were isolated from adult mice and cultured on plastic or soft (4.5 kPa) polyacrylamide hydrogels. Myofibroblast marker gene expression and smooth muscle α-actin (SMA) fibers were quantified by real-time PCR and immunostaining, respectively. Myofibroblast differentiation was prevented on soft hydrogels for 9 days, but had occurred after 15 days on hydrogels. Transferring myofibroblasts to soft hydrogels reduced expression of myofibroblast-associated genes, albeit SMA fibers remained present. Inhibitors of transforming growth factor β receptor I (TGFβRI) and Rho-associated protein kinase (ROCK) were effective in preventing and reversing myofibroblast gene expression. SMA fibers were also reduced by blocker treatment although cell morphology did not change. Reversed cardiac fibroblasts maintained the ability to re-differentiate after the removal of blockers, suggesting that these are functionally similar to resting cardiac fibroblasts. However, actin alpha 2 smooth muscle (Acta2), lysyl oxidase (Lox) and periostin (Postn) were no longer sensitive to substrate stiffness, suggesting that transient treatment with mechanotransduction inhibitors changes the mechanosensitivity of some fibrosis-related genes. In summary, our results bring novel insight regarding the relative importance of specific mechanical signaling pathways in regulating different myofibroblast-associated genes. Furthermore, combining blocker treatment with the use of soft hydrogels has not been tested previously and revealed that only some genes remain mechano-sensitive after phenotypic reversion. This is important information for researchers using inhibitors to maintain a \"resting\" cardiac fibroblast phenotype in vitro as well as for our current understanding of mechanosensitive gene regulation.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Actin / Animals / Bioengineering / Biology and Life Sciences / Cell Differentiation - drug effects / Cell morphology / Collagen / Connective tissue / Cytology / Differentiation / Disruption / Experiments / Fibers / Fibroblasts / Fibroblasts - cytology / Fibroblasts - drug effects / Fibrosis / Gene expression / Gene regulation / Genes / Genotype & phenotype / Growth factors / Heart cells / Hydrogels / Inhibitors / Kinases / Liquid oxygen / Lysyl oxidase / Mechanical properties / Mechanotransduction / Mechanotransduction, Cellular - drug effects / Medicine and Health Sciences / Mice / Morphology / Muscles / Myocardium - cytology / Phenotype / Phenotypes / Phenotypic reversion / Physical Sciences / Physiological aspects / Polyacrylamide / Protein kinase / Receptor, Transforming Growth Factor-beta Type I - antagonists & inhibitors / Reversion / rho-Associated Kinases - antagonists & inhibitors / Smooth muscle / Social Sciences / Stiffness / Substrates / Transcription factors / Transforming growth factor-b