Rho GTPase cross-talk with syndecan-4 regulates pro-fibrotic mechanotransduction in the heart

Project Information

ROCKSOFT

Grant agreement ID: 795390

DOI

10.3030/795390

Project terminated on 30 November 2020

EC signature date 27 February 2018

Start date 1 April 2019

End date 31 March 2021

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 212 194,80

EU contribution

€ 212 194,80

Coordinated by

KOBENHAVNS UNIVERSITET
Denmark

Periodic Reporting for period 1 - ROCKSOFT (Rho GTPase cross-talk with syndecan-4 regulates pro-fibrotic mechanotransduction in the heart)

Reporting period: 2019-04-01 to 2021-03-31

Summary of the context and overall objectives of the project

Cardiac fibrosis is a major cause of diastolic heart failure, and is induced by increased mechanical stress of the left ventricle. Treatment is currently lacking, reflecting a lack of understanding of cardiac fibroblast physiology, the main cell type responsible for extracellular matrix production and development of fibrosis. Cardiac fibroblasts respond to mechanical stress by differentiating into myofibroblasts that produce large amounts of extracellular matrix, thus, leading to development of cardiac fibrosis. However, in vitro studies of cardiac fibroblasts have been hampered by the rapid differentiation into myofibroblasts in vitro due to the high stiffness of culturing conditions.
We tested whether treating cardiac fibroblasts with blockers of mechano-signaling pathways could prevent and/or reverse the myofibroblast phenotype in vitro. Furthermore, we examined how phenotypic markers change over time in culture and whether culturing cardiac fibroblasts on soft substrates could prevent myofibroblast differentiation.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Our results showed that the combination of Rho-associated kinase (ROCK) inhibitor and transforming growth factor β receptor I (TGFβRI) inhibitor, prevented and reversed myofibroblast differentiation in vitro. Also, alleviating cells from the mechanical cues from stiff culturing conditions preserved the “resting” cardiac fibroblast phenotype. These results were recently published in Plos One (Gilles et al., 2020).

We further examined the expression pattern of fibrosis-related genes in cardiac fibroblasts over time in culture, and found that the expression of extracellular matrix genes was dynamic during time in culture and that myofibroblast marker genes decreased after 9-12 days in culture. These results suggest either further differentiation of myofibroblasts into a new phenotype, or the existence of several cardiac fibroblast sub-types in vitro. Indeed, recent in vivo results using single cell RNA sequencing reveal the presence of several cardiac fibroblast sub-types in the healthy and diseased heart.

The results were presented at the Cardiac Mechano-Electrical Coupling (MEC) meeting 2019 in Freiburg, Germany, the European Society of Cardiology virtual meeting 2020, the annual Danish Society for Matrix Biology virtual meeting and at internal seminars at the Biotech Research & Innovation Centre (BRIC).

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

"The results obtained in this project describe a method for maintaining a ""resting"" cardiac fibroblast phenotype in vitro by inhibiting mechanically activated signaling pathways. This is of great importance for experiments requiring longer culturing time, such as CRISPR/Cas9 gene editing. Thus, it will be possible to reverse the myofibroblast phenotype after gene editing thereby enabling examination of pro-fibrotic factors and signaling pathways that trigger activation and differentiation of cardiac fibroblasts into myofibroblasts.

Furthermore, our finding that cardiac fibroblast extracellular matrix gene expression is dynamic and shifts during in vitro culture indicates the presence of yet unidentified phenotypic sub-types. This is in line with recent in vivo findings showing the presence of several cardiac fibroblast sub-types during the course of cardiac disease. The composition and ratios of these cardiac fibroblast sub-types may correspond to the differences observed in patients with fibrotic disease. Thus, a better understanding of cardiac fibroblast phenotypic diversity may reveal new targets for anti-fibrotic therapy, and thereby new treatments for the millions of people worldwide that are suffering from heart failure caused by cardiac fibrosis."

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