Investigating the potential for reversal of myofibroblast activation in human cardiac fibroblasts in 2D culture
Authors: Hall C , Law JP , Reyat JS , Fabritz L , Kirchhof P , Gehmlich K , Weston C , Townend JN , Ferro CJ , Denning C , Pavlovic D 
Published: May 2021

Abstract

Funding Acknowledgements
Type of funding sources: Public Institution(s). Main funding source(s): BHF MRC

Introduction
Cardiac fibroblasts (cFbs) are responsible for deposition of extracellular matrix in the heart, providing support to the contracting myocardium and contributing to a myriad of physiological signalling processes. Prolonged and excessive activation of cFbs, via stimulation by transforming growth factor β (TGF-β), causes conversion of cFbs into myofibroblasts. Myofibroblasts are believed to cause pathological cardiac remodelling and to contribute to heart failure and arrhythmias. Reversion of myofibroblasts into cFbs has been demonstrated in rodent cells; it has yet to be explored in human cells.

Purpose
To characterise the effects of long-term 2D standard culture on the activation status of human cFbs. To identify the potential for human myofibroblasts to dedifferentiate back to cFbs.

Methods
Primary human cFbs were cultured in Corning Costar flasks (Young’s modulus E = ∼3GPa) for up to 10 passages. Cells were subsequently plated onto dishes with a Young’s modulus of ∼3GPa, 25kPa and 2kPa in the presence or absence of TGF-β (10ng/ml) and/or TGF-β receptor I inhibitor SD208 (10nM) for up to 4 days. The proliferative capacity of the cells was assessed using the CyQUANT NF® assay. Cells were assessed for mRNA and protein expression of myofibroblast activation markers α-smooth muscle actin (α-SMA) and collagen-1 by qPCR and western blotting. The localised distribution of α-SMA was assessed by confocal microscopy.

Results
Human cardiac fibroblasts robustly expressed α-SMA. Proliferation was significantly decreased at 2kPa compared to higher Young’s moduli (mean percentage change over 2 days: 2kPa = 115.1, 25kPa = 191.4, 3GPa = 205.9, p &lt; 0.0001). qPCR analysis revealed no significant changes in expression of myofibroblast gene markers α-SMA and collagen 1 at either ∼3GPa, 25kPa or 2kPa Young’s Moduli in the presence or absence of TGF-β treatment (median fold change (interquartile range [IQR]) versus control: TGF-β(α-SMA, 3GPa) = 1.226 (0.820); TGF-β(Collagen 1, 3GPa) = 1.636 (1.403); TGF-β(α-SMA, 25kPa) = 1.069 (7.030); TGF-β(Collagen 1, 25kPa) = 1.103 (0.411); TGF-β(α-SMA, 2kPa) = 0.800 (5.021); TGF-β(Collagen 1, 2kPa) = 1.629 (7.092); n = 2-3). These data was confirmed by western blotting (median relative protein expression (IQR) versus control: TGF-β(α-SMA, 3GPa) = 1.012 (0.500); TGF-β(Collagen 1, 3GPa) = 1.008 (1.466); TGF-β(α-SMA, 25kPa) = 1.321 (2.282); TGF-β(Collagen 1, 25kPa) = 0.944 (1.125); TGF-β(α-SMA, 2kPa) = 1.142 (0.705); TGF-β(Collagen 1, 2kPa) = 0.283 (1.127), p &gt; 0.05; n = 2-3). TGF-β or SD208 treatment did not affect α-SMA expression when assessed by confocal microscopy.

Conclusions
Long-term culture of human cFbs in 2D format leads to a robust and persistent activation of myofibroblasts that is unresponsive to TGF-ß activation or inhibition. Ongoing work is focussed on investigating whether human myofibroblast de-differentiation is possible.