Chronic activation of human cardiac fibroblasts in vitro attenuates the reversibility of the myofibroblast phenotype

Activation of cardiac fibroblasts and differentiation to myofibroblasts underlies development of pathological cardiac fibrosis, leading to arrhythmias and heart failure. Myofibroblasts are characterised by increased α-smooth muscle actin (α-SMA) fibre expression, secretion of collagens and changes in proliferation. Transforming growth factor-beta (TGF-β) and increased mechanical stress can initiate myofibroblast activation. Reversibility of the myofibroblast phenotype has been observed in murine cells but has not been explored in human cardiac fibroblasts. In this study, chronically activated adult primary human ventricular cardiac fibroblasts and human induced pluripotent stem cell derived cFbs (hiPSC-cFbs) were used to investigate the potential for reversal of the myofibroblast phenotype using either subculture on soft substrates or TGF-β receptor inhibition. Culture on softer plates (25 or 2 kPa Young’s modulus) did not alter proliferation or reduce expression of α-SMA and collagen 1. Similarly, culture of myofibroblasts in the presence of TGF-β inhibitor did not reverse myofibroblasts back to a quiescent phenotype. Chronically activated hiPSC-cFbs also showed attenuated response to TGF-β receptor inhibition and inability to reverse to quiescent fibroblast phenotype. Our data demonstrate substantial loss of TGF-β signalling plasticity as well as a loss of feedback from the surrounding mechanical environment in chronically activated human myofibroblasts.


Materials and methods
Human cardiac fibroblast culture. Human cardiac fibroblasts (HCF) obtained from the ventricles of explanted hearts taken at the time of cardiac transplantation were purchased from PromoCell GmbH (C-12375) and received cryopreserved at passage 2. The donors were aged 60 and 63. These cells were used as a model of chronically-activated myofibroblasts as found in chronic heart failure. These HCFs were thawed and cultured using Fibroblast Growth Medium 3 (PromoCell®, C-23025) for up to 10 passages (split ratio 1:10) in T75 tissue culture flasks and kept in a humidified incubator at 37 °C and 5% CO 2 . These cells were used for experimentation between P5 and P10 and kept in culture for roughly 4 weeks. HCFs were cultured on stiff polystyrene culture plates (Young's modulus [E] = 3 GPa) from Corning before subculture on soft (E = 2 kPa and 25 kPa) culture plates (Matrigen Softwell, SW6-EC-2/SW6-EC-25). Matrigen Softwell culture plates achieve lower Young's modulus than standard polystyrene or glass cultureware through a transparent, two-dimensional layer of bisacrylamide crosslinked polyacrylamide hydrogel along the entire cell culture surface. The product is supplied sterile and tissue-culture treated. Standard cell culture procedures such as cell culture media, dissociation with TryPLE Express (Thermo Fisher Scientific, 12,604,013) and cell culture dish area (9.6 cm 2 per well) were kept constant across all tissue cultureware during experiments. In order to promote efficient adhesion of HCFs, all cultureware were coated with 10 μg/mL fibronectin (Sigma-Aldrich, F1141). Fibronectin is produced by cardiac fibroblasts and is a principal component of the cardiac ECM 32,33 .
In vitro treatments. The following drugs were added to the respective culture media as summarised in Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed using TaqMan™ gene expression chemistry (Table 2). Samples were run in triplicate. GAPDH was used as an endogenous housekeeping gene and inter-plate calibrator. Samples with GAPDH C T values greater than two standard deviations from the mean C T of the dataset were excluded from analysis. Relative gene expression values were determined by 2 -ΔΔCT method using vehicle-treated cells for comparison.
Western blotting. Cell pellets were re-suspended in 50 µL RIPA buffer (Sigma-Aldrich, R0278) with 1% Halt™ Protease and Phosphatase Inhibitor Cocktail 100X (Thermo Scientific, 78441) and incubated on ice for 30 min. Protein concentration was determined by DC™ protein assay (Bio-Rad, 5000116) prior to dilution in 2X sample loading buffer and incubation at 95 °C for 5 min.
Samples (up to 20 ug per lane) were subjected to SDS-PAGE on 4-15% precast gels (Bio-Rad, 4561086). Proteins were transferred onto PVDF membranes using Trans-Blot Turbo™ Mini Transfer Packs and Transfer System  www.nature.com/scientificreports/ (Bio-Rad, 1704156/1704150). Membranes were blocked in 5% milk containing tris-buffered saline with 0.1% Tween 20 (Bio-Rad, 1610734/1710781) before probing with primary and secondary antibodies as listed in Table 3. Visualisation and quantification of protein band density was performed using the Odyssey Fc imaging System and Image Studio Lite v5.2 (Li-Cor) without any post-capture image manipulation. GAPDH was used as loading control; the band intensity of the protein of interest (POI) was normalised to the GAPDH loading control and presented as a ratio -POI/GAPDH -to account for protein loading inconsistencies.
Statistical methods. Data are presented as mean ± standard error of the mean. Differences between group means were examined using nested one-way analysis of variance (ANOVA) or Student's t test and were considered statistically significant when P < 0.05. Subgroups for the nested analysis were replicates done on different days with different passage numbers. Statistical analysis was conducted using GraphPad Prism 9.

Results
Long-term culture of human cardiac fibroblasts leads to myofibroblast transition. Myocardium stiffness has been associated with activation of cardiac fibroblasts to myofibroblasts 24 . Culture of primary healthy murine cardiac fibroblasts on stiff plastic plates is shown to induce myofibroblast activation 30 . HCFs were obtained from the explanted hearts of patients undergoing cardiac transplantation and cultured in a 2D monolayer on "stiff " polystyrene plates (E = 3 GPa) for up to 10 passages in order to chronically activate HCFs. These cells were then exposed to 10 ng/mL TGF-β, a well-established inducer of the myofibroblast phenotype, for 2-4 days to assess their ability to further activate-2 days for immunofluorescence and proliferation experiments (to avoid high confluency) and 4 days for qPCR and western blotting. α-SMA and collagen 1 were utilised as markers of fibroblast activation and transition to myofibroblasts. Vimentin was used as a marker for cardiac fibroblasts.
Following long term culture on stiff substrates, α-SMA was detected by western blot and immunofluorescence staining in HCF cultured on 3 GPa polystyrene plates indicating baseline activation ( TGF-β treatment did not significantly affect α-SMA expression, as assessed by immunofluorescence (Figs. 2d and f, Fig. S2). Secondary antibody only controls show that this was not due to non-specific binding or background fluorescence staining (Fig. S3). This lack of TGF-β-induced response was maintained at each passage tested post thaw (p6-p9), when assessed by immunofluorescence ( Fig. 2f and Fig. S4a). Total α-SMA expression as measured by relative integrated density did not significantly change from passage 6 to 9 (Fig. S4a). Similarly, percentage of cells positive for α-SMA fibres did not significantly change during long term culture from p6-p9 (Fig. 2f). Interestingly, long term culture indicated a non-significant decrease % of α-SMA positive cells (p = > 0.05) with long term culture indicating possible de-differentiation of HCF.
These results clearly demonstrate that standard 2D culture on stiff plastic of human cardiac fibroblasts, isolated from diseased hearts, leads to persistent myofibroblast transition and renders them unresponsive to TGF-β stimulation.
Chronically activated human fibroblasts cannot transition back to quiescent phenotype. We then investigated whether the established myofibroblast phenotype can be reversed. Two approaches for myofibroblast de-activation were employed, via reduction in mechanical stress (using sub-culture on softer substrates, 25 and 2 kPa) or via TGF-β receptor inhibition (using small molecule inhibitor SD208). Culture on 25 kPa or 2 kPa soft substrates did not result in significant reduction in α-SMA expression in myofibroblasts, as measured by immunofluorescence (Fig. 3a). These findings were supported by RT-PCR analysis of ACTA 2 and COL1A1 mRNA transcripts, which showed comparable levels of mRNA expression between myofibroblasts cultured on the stiff and soft substrates (Fig. 3b). When proliferation was assessed over a 48-h period, no significant difference was seen between myofibroblasts grown on 3 GPa and 25 kPa (Fig. 3c). A trend towards non-significant reduction in proliferation of myofibroblasts grown on 2 kPa when compared with 3 GPa was observed (Fig. 3c). Cell size was unchanged on 25 kPa ( Fig. S1) but significantly decreased for myofibroblasts grown on 2 kPa substrates (Fig. S1) compared with 3 GPa. Table 3. Western blotting antibodies. *ab34710 anti-collagen 1 antibody has affinity to both α1 and 2 subunits. Two bands are seen at ~ 130 and 170 kDa. Both subunits were quantified and expressed as a total expression relative to GAPDH loading control.  www.nature.com/scientificreports/ TGF-β receptor inhibitor SD208 (30 nM) had no discernible effect on proliferation of myofibroblasts grown on 3 GPa (Fig. S5). There was also no significant effect on α-SMA expression, as measured by immunofluorescence, following treatment with SD208 (Fig. S5b). Similarly, percentage of cells positive for α-SMA was also unchanged in the presence of SD208 (Fig. S2).
We next assessed whether a dual approach of myofibroblast deactivation, via both subculture on soft substrates and in the presence of TGF-β inhibitors, can reverse myofibroblasts to their quiescent phenotype. Culture of myofibroblasts on 25 kPa substrate, in the presence of SD208, demonstrated no significant effect on proliferation (Fig. S6c), nor change in expression of α-SMA, as assessed by immunofluorescence ( Fig. S6d and e). As demonstrated in Fig. S6, mRNA and protein expression of α-SMA and Collagen 1 were also unaffected by addition of TGF-β, when cultured on 25 kPa ( Fig. S6a and b, summarised in Table 4).
Culture on 2 kPa in the presence of SD208 similarly yielded no significant differences in proliferation (Fig. S7c), nor expression of α-SMA ( Fig. S7d and e). Expression of α-SMA and Collagen 1 were also unaffected at the mRNA transcript or protein level ( Fig. S7a and b, summarised in Table 4). www.nature.com/scientificreports/ To examine whether longer-term TGF-β receptor inhibition is required to reverse chronically activated HCF, myofibroblasts were cultured from p5 to p9 in the presence or absence of TGF-β receptor inhibitor SB431542 (SB). At each passage, α-SMA expression was assessed by immunofluorescence. Total α-SMA expression as measured by relative integrated density did not significantly change from passage 6 to 9 (Fig. S4a). Similarly, % cells positive for α-SMA fibres did not significantly change compared to myofibroblasts cultured in the presence of SB (Fig. S4b). Our data also demonstrate that HCF are unresponsive to TGF-β at each passage tested (p6-p9) regardless of whether they are cultured in the presence or absence of SB (Fig. S4c).
These data show that myofibroblasts cannot be de-activated when subsequently cultured on softer substrates (25 kPa or 2 kPa) that more closely reflect that of the human myocardium. These data further demonstrate that human myofibroblasts do not respond to TGF-β treatment and cannot transition back to quiescent state when cultured on soft substrates, in the presence or absence of a TGF-β inhibitor.
Chronically activated hiPSC derived cardiac fibroblasts cannot be reversed to a quiescent phenotype. To further investigate whether chronic activation of human cardiac fibroblasts is reversible, separate experiments using human iPSC-cFbs were performed (see Fig. S8 for protocol schematic). hiPSC-cFbs were cultured on 3 GPa substrates in the presence of TGF-β receptor inhibitor SB431542 to generate a quiescent cardiac fibroblast or in the absence of SB431542 to generate an activated fibroblast (myofibroblast) phenotype. Quiescent cells showed a significant increase in α-SMA expression when exposed to TGF-β (Fig. 4a). Of note is that after 2 days of culture on stiff surfaces in the absence of TGF-β a two-fold increase in the number of quiescent cells positively expressing α-SMA was observed (Fig. 4a), indicating that culture on stiff substrates alone induces α-SMA expression.
As expected, exposure to TGF-β, or subsequently to inhibitors SD208 and SB431542, did not significantly modify α-SMA expression in activated hiPSC-cFbs, when compared to the control (Fig. 4b). This mirrors our observation with the activated HCFs; culture on stiff substrates (3 GPa) activates cardiac fibroblasts and makes them unresponsive to TGF-β stimulation and inhibition. In line with our previous observations (see Sect. "Longterm culture of human cardiac fibroblasts leads to myofibroblast transition"), culture of hiPSC-cFbs over 2 weeks in the absence of SB431542 reduced the number of α-SMA positive cells that display fibre-like staining pattern (see Fig. 4b).
An additional qualitative assessment of α-SMA expression was conducted over the period of 1 week using immunofluorescence. hiPSC-cFbs were cultured in the absence of a TGF-β receptor inhibitor. This data confirms that iPSC-cFbs are activated by culture on 3 GPa substrates alone, independent of TGF-β, albeit to a lesser extent (Figs. S9 and S10). This data also shows that activation by hard substrates begins within 24 h, with a peak at around 96 h of culture (Figs. S9 and S10).
Proliferation of hiPSC-cFbs was also assessed. Activated hiPSC-cFbs show a significantly reduced proliferation rate when compared to their quiescent counterparts (Fig. 5g). These data suggest that in hiPSC-cFbs, activation leads to a reduction in proliferation, rather than the expected increase in proliferation.
Quiescent hiPSC-cFbs demonstrated higher proliferation rates compared to the activated hiPSC-cFbs (Fig. 5g). A decrease in proliferative capacity was observed in response to TGF-β in quiescent hiPSC-cFbs (Fig. 5h). The proliferative capacity of quiescent hiPSC-cFbs showed a small but non-significant decrease when SB431542 was removed from the culture media, (Fig. 5h). Activated hiPSC-cFbs did not respond to either recombinant TGF-β or inhibition of the TGF-β signalling pathway by SB431542 or SD208 (Fig. 5i).
These results mirror that seen in the primary HCFs, demonstrating that chronically activated myofibroblasts cannot be reversed to a quiescent phenotype using TGF-β receptor inhibition. Table 4. Myofibroblast markers on soft culture plates by RT-PCR and western blot. Data presented as mean ± S.E.M. t test of TGF-β treated cells versus vehicle control. Western blot data presented as a ratio of protein of interest (POI) and GAPDH as a loading control.

RT-PCR
Gene of interest TGF-β mean fold change versus control P

Discussion
There have been significant efforts to identify effective therapeutic approaches against long-established cardiac fibrosis targets. However, clinical trials of drugs which target the mineralocorticoid receptor, renin-angiotensin system, natriuretic peptide degradation, vascular tone, and others have yielded mixed results in reducing fibrosisrelated outcomes in patients with heart failure with preserved and reduced ejection fraction and ischaemic heart disease despite encouraging preclinical data (Reviewed in 38 ). Given the central role of the myofibroblast www.nature.com/scientificreports/ in cardiac fibrosis, our study intended to investigate the potential for the reversal of activated human cardiac myofibroblasts to a more quiescent phenotype.
The principal findings of this study (Table 5) are that chronically activated primary human cardiac ventricular fibroblasts show a diminished response to activation by TGF-β and that neither culture on softer substrates, nor TGF-β receptor inhibition are able to reverse the observed myofibroblast phenotype. Our findings are confirmed in hiPSC-cFbs, indicating that chronic activation of human cardiac fibroblasts leads to a terminal differentiation into myofibroblasts. The lack of response to TGF-β was not associated with a change in expression of the TGF-β receptor itself (Fig. S11). We further demonstrate that culture of hiPSC-cFbs on stiff surfaces results in activation within 24 h, and intriguingly longer-term culture (> 96 h) results in the loss of fibrous α-SMA expression.
Proliferative capacity of human cardiac fibroblasts. Conflicting data exists concerning the correlation between cardiac fibroblast activation and proliferation. Intriguingly, our data highlights significantly higher proliferation rates in quiescent hiPSC-cFbs compared to the activated hiPSC-cFbs. Accordingly, TGF-β treatment reduced the proliferative capacity in quiescent hiPSC-cFbs but did not alter proliferation of activated hiPSC-cFbs or fibroblasts isolated from a patient. Similarly, Hecker et al. also showed that human lung myofibroblasts, activated using TGF-β, exhibited less proliferation than quiescent fibroblasts as well as an attenuated proliferative response to serum 19 . When using freshly isolated mouse cardiac fibroblasts, Herum et al. observed no changes in proliferation rates when cultured on multiple different substrates (3,8,10, and 50 kPa) as well as no changes when substrates were stiffened from 3 or 8 kPa to 30 kPa after 5 days of culture 29 . A further study using human cardiac fibroblasts found that although there was a higher density of vimentin positive cells in failing hearts, there was a significant decrease in the proliferative marker Ki-67 in the failing hearts when compared to the non-failing hearts. Myofibroblasts (α-SMA positive cells) from both groups showed similarly low levels of Ki-67 staining 22 . Other studies have implicated the renin-angiotensin-aldosterone system in the proliferation of cardiac fibroblasts 39,40 . Using freshly isolated rat cardiac fibroblasts, Olson et al. were able to attenuate myofibroblast differentiation and cardiac fibroblast proliferation by inhibiting mitogen-activated protein kinase mediated signalling with polyphenolresveratrol 41 . Whether the same affects would be observed in vivo remains to be studied.
Cardiac fibroblasts derive from mesenchymal cells with a variety of origins. These include, but are not limited to, the pro-epicardium, epicardium and cardiac endothelium through epithelial-or endothelial-to mesenchymal transformation (EMT and EndMT, respectively). Although cardiac fibroblasts show a decreased proliferative capacity once having undergone myofibroblast differentiation, cellular cues may instigate enhanced EMT/EndMT to maintain or increase cardiac fibroblast numbers within the heart 42,43 . The exact processes underpinning this require further investigation, where targeted attenuation of these mechanisms could present an alternative avenue to decreasing the fibrotic burden of the heart.

Reversal of myofibroblast phenotype through TGF-β inhibition.
Cardiac fibrosis, whilst generally thought of as a maladaptive and pathophysiological process, is essential to ensuring tissue integrity following acute injury. For example, fibrotic tissue replaces necrotic myocardium following myocardial infarction thus maintaining cardiac mechanical function and preventing myocardial rupture 17 . This replacement fibrosis leads to impaired systolic and diastolic function, and to arrhythmias, likely around its margins. In contrast, diffuse interstitial fibrosis appears to be a purely maladaptive response with adverse effects on both ventricular function and susceptibility to arrhythmia 44 . The key role of TGF-β in cardiac fibroblast activation and proliferation is well-studied. Clinical studies have consistently reported upregulated TGF-β expression in the myocardium of patients with heart failure [45][46][47] . The induction of fibrosis because of activation of myofibroblasts via small mothers against decapentaplegic (SMAD) and non-SMAD-mediated signalling pathways such as ERK, JNK and p38 has also been previously described 24,48,49 .
A recent study by Nagaraju et al. similarly investigated the potential for reversal of cardiac fibroblast activation using TGF-β receptor inhibition. Using freshly isolated human cardiac fibroblasts from explanted hearts of transplant recipients, the authors found that incubation with the TGF-β receptor inhibitor SD208 resulted in loss of α-SMA and reduced expression of profibrotic genes 22 . To achieve this, a high concentration of SD208 (3 µM) was utilised. In contrast, we found no effect on the activation status (proliferation rate and α-SMA expression)  36 . Inhibition of TGF-β or its signalling components has been shown to be an effective anti-fibrotic treatment in preclinical studies. Transgenic rodent studies have demonstrated that disruption of fibroblast-specific TGF-β receptor 1 produced cardiac-protective effects against fibrosis and diastolic dysfunction 35 . Further in vivo studies utilising neutralising monoclonal antibodies and small molecular inhibitors directed against TGF-β and TGF-β receptor 1, respectively, reported significant reductions in cardiac fibrosis in murine tissue 23,51,52 . However, anti-TGF-β therapies are associated with significant dose-limiting drug toxicity given the ubiquitous expression of TGF-β and its receptors in healthy tissue throughout the human body, which restricts the effectiveness of such treatments. Mitra et al. reported widespread dose-dependent complications associated with pan-TGF-β inhibition in both mouse and monkey models. These included vascular and cardiac valvular inflammation, systemic bleeding, bone dysplasia and increased mortality 53 . Studies employing targeted inhibition via small molecular inhibitors against TGF-β receptor 1 also reported similar cardiac and haemorrhagic complications 54,55 . Due to these challenges, there has yet to be an anti-TGF-β therapy that has successfully found the balance between achieving an adequate anti-fibrotic response whilst avoiding serious cardiovascular side effects, despite candidate drugs such as galunisertib showing initial promise in cancer trials 56,57 . Terminally differentiated cardiac myofibroblasts cannot be de-activated. The discrepancy between our reported findings of irreversibly activated myofibroblasts and that of Nagaraju et al. 22 may also be explained by differing cell culture approaches, specifically, the duration in culture and passage number prior to application of a de-activating agent or treatment. In their experiments, the authors utilised the isolated cardiac fibroblasts within 4 days of isolation without passaging, in contrast to our HCF and hiPSC-cFb cell lines, which were intentionally cultured long-term for up to 10 passages/2-4 weeks before usage to produce a chronically activated myofibroblast phenotype. Taken together, our results suggest the presence of a 'threshold' after which fibroblasts become unresponsive to a de-activating treatment, which warrants further discussion.
Studies have supported the concept that the plasticity of myofibroblast phenotype is only conserved and demonstrable when using freshly isolated cells 25 , or cells which have been cultured in 'activating' conditions for a short period of time 58 . Tomasek and Gabbiani 59 challenged the idea of the fibroblast and myofibroblast being discrete, categorical phenotypes. Instead, they proposed the presence of an intermediate 'protomyofibroblast' stage, encompassing a continuum of cellular processes and fibroblast phenotypes, which bridge the differentiation of fibroblasts into myofibroblasts 60 . In parallel, the potential for myofibroblast de-differentiation would also follow a similar principle and depend on where they lie on the continuum of phenotypes. Indeed, data from single cell RNA sequencing already provides evidence that there are several distinct subpopulations of cardiac fibroblasts in healthy hearts 9,10 , and at different stages of ischaemic injury in murine studies 61,62 .
Our immunofluorescence data show that chronically activated HCFs and hiPSC-cFbs exhibited an unusual α-SMA staining pattern. Though these cells are unresponsive to TGF-β, and therefore assumed to be activated, they exhibit less of the "classic" actin fibres as found in our quiescent hiPSC-cFbs following TGF-β treatment. Instead, the activated cardiac fibroblasts displayed dispersed α-SMA staining within the cytoplasm (Fig. 2e,  Fig. 3a and Fig. 4b). Use of secondary antibody-only controls shows that this effect is not due to non-specific staining (Fig. S3). The exact explanation for this is unclear, however we hypothesise that chronic culture in activating conditions may result in disassembly of actin fibres. This hypothesis may be supported by the decrease in α-SMA fibrous staining seen in Figs. S9 and S10 from 96 h to 1 week of activation by both hard substrates alone and in conjunction with TGF-β, as well as in Fig. S4 with a decrease from passage 6 to passage 9. The localisation of α-SMA within the cardiac fibroblast requires further investigation as a potential marker for late stage and/or irreversible activation status.
It is therefore conceivable that our cell culture approach, which bears resemblance to the persistent, longer duration of cardiac fibroblast activation at the latter stages of cardiac disease, resulted in irreversible terminal differentiation of myofibroblasts, a phenomenon that has been reported in other organ systems such as the lung 58,63 . The role of mechanical stiffness and myofibroblast activation. Our data show that human cardiac fibroblasts are activated by culture on stiff ~ 3 GPa substrates 58,63 . Chronically activated cardiac fibroblasts failed to transition back to a quiescent phenotype by subsequent culture on softer culture substrates (2 and 25 kPa), suggesting a loss of feedback from the surrounding mechanical environment. Healthy myocardium has a typical elastic modulus of between 10 and 30 kPa 25,64,65 , rising to over 100 kPa in diseased tissues 26,66 . Several other studies have indicated that prolonged culture on a hard plastic surface is sufficient to cause increased α-SMA expression in freshly isolated murine cardiac fibroblasts 40,65,[67][68][69] . However, the cellular mechanisms that regulate the cardiac fibroblast phenotype and function in response to mechanical stimuli are complex. Studies have reported some success in de-activating human and porcine myofibroblasts through the use of soft hydrogels 25,70 and three-dimensional microfabricated tissues associated with reduced tissue tension 71 . Still, the reversal tended to be incomplete, and experiments utilised freshly isolated cells cultured for shorter periods 40,69 , further supporting the concept of there being a range of protomyofibroblast states.
We also note that recommended culture of hiPSC-cFbs requires use of TGF-β receptor antagonist SB431542 at high concentrations (10 µM; IC 50 94 nM) to maintain quiescence 34,36 . It may be that use of soft substrates would allow the use of lower concentrations of TGF-β antagonism. In particular, the consequences of longerterm cardiac fibroblast culture in the presence of an inhibitor, and whether the effects of mechanical stress of the substrate can be fully suppressed over prolonged periods remain unclear.

Conclusion and future direction
The lack of efficacious treatments for the reversal of cardiac fibrosis is a product of the complex cellular pathways, which are common to extracellular matrix generation and turnover in both health and disease. The cardiac fibroblast is the principal cell type in the fibrotic process and we show, using diseased patient-derived cardiac fibroblasts, that their activation into myofibroblasts under chronic disease conditions represents a challenging phenotype to reverse in vitro. Our experiments using hiPSC-cFbs largely recapitulated these findings. Whether cardiac fibroblasts isolated from healthy human myocardium would yield similar findings presents an intriguing and important avenue for future investigation.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.