Skip to main content
PMC home page
PLOS ONE logoLink to PLOS ONE
. 2020 Oct 26;15(10):e0241390. doi: 10.1371/journal.pone.0241390

Maintaining resting cardiac fibroblasts in vitro by disrupting mechanotransduction

George Gilles 1, Andrew D McCulloch 1,2, Cord H Brakebusch 3, Kate M Herum 3,*
Editor: Philip C Trackman4
PMCID: PMC7588109  PMID: 33104742

Abstract

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.

Introduction

Activation of cardiac fibroblasts is a key step in development of cardiac fibrosis. In response to increased mechanical stress caused by increased left ventricular pressure or myocardial stiffening, cardiac fibroblasts differentiate into myofibroblasts characterized by de novo gene expression of actin alpha 2 smooth muscle (Acta2) and assembly of contractile smooth muscle α-actin (SMA) fibers, as well as enhanced production of typical cardiac extracellular matrix (ECM) genes including collagen (Col) 1a1, 1a2, 3a1 and the collagen cross-linking enzymes lysyl oxidase (Lox) [1] and Lox-like 2 [2]. In addition, expression of connective tissue growth factor (Ctgf) [3] and periostin (Postn) [4] is associated with the myofibroblasts phenotype. In vivo, the increases in myofibroblast markers are accompanied by decreased expression of transcription factor 21 (Tcf21), a marker of resting cardiac fibroblasts [4]. These changes in cardiac fibroblast phenotype alter ECM composition and structure leading to increased stiffness of the heart, which can compromise diastolic function. Importantly, studying cardiac fibroblast activation in vitro is hampered by rapid myofibroblast differentiation in response to the high stiffness of plastic culturing conditions [1]. We here investigate to what extent disrupting mechanotransduction can be used to maintain resting cardiac fibroblasts in vitro, thereby enabling the study of biomechanical activation of resting cardiac fibroblasts.

Soft hydrogels can be used as culturing substrate to eliminate the effect of high substrate stiffness on cardiac fibroblast activation [1, 5]. However, the use of soft hydrogels have some limitations including small sample size and the need for frequent, time-consuming isolation of primary cells for direct plating onto hydrogels. Furthermore, it is not known for how long cardiac fibroblasts remain inactivated when cultured on hydrogels. We have previously used soft hydrogels to maintain resting cardiac fibroblasts for up to 5 days [5], but some experiments, such as CRISPR/Cas9 gene editing requires longer cell culture durations. Here we examine the time frame for maintaining resting cardiac fibroblasts on soft hydrogels.

Instead of changing the mechanical environment, it may be possible to transiently inhibit the cardiac fibroblast’s ability to sense mechanical stress and thereby render cardiac fibroblasts unresponsive to the stiff in vitro culturing conditions. Although ECM gene expression was not examined, culturing cardiac fibroblasts in the presence of a transforming growth factor β receptor (TGFβR) inhibitor was previously shown to prevent stiffness-induced development of SMA fibers [6]. However, experiments required continuous culturing with TGFβR inhibitor which would interfere with subsequent experiments studying cardiac fibroblast activation.

Several other mechanotransduction signaling pathways are known to regulate cardiac fibroblast activation [7, 8]. We and others have previously shown that calcineurin-nuclear factor of activated T cells (NFAT) signaling is important for myofibroblast differentiation [9, 10], and that Rho-associated protein kinase (ROCK) regulates the myofibroblast-associated transcription factor myocardin-related transcription factor A (MRTF-A) by altering cytoskeletal dynamics [11]. Although it is known that these pathways are important for myofibroblast differentiation, their relative contributions and the effect of dual inhibition have not previously been tested. Here we examine whether single or combined treatment with TGFβRI, ROCK and calcineurin inhibitors can prevent and/or reverse myofibroblast marker expression. Furthermore, we test whether “reversed” cells have preserved mechanosensitivity, and whether culturing “reversed” cardiac fibroblast on hydrogels may serve as a method for studying resting cardiac fibroblasts in vitro, even after prolonged culturing time and multiple passages on plastic.

Methods

Cardiac fibroblast isolation and culturing

The project was approved by the veterinarian at Department of Experimental Medicine, University of Copenhagen (project license #P18-289). Mice were briefly anesthetized with isoflurane (open drop) and euthanized by cervical dislocation. Cardiac fibroblasts were isolated from adult mice (C56Bl6, Taconic, Denmark) as previously described [5]. Cells were expanded on plastic and treated with Y27632 (25μM), SB431542 (10μM) and cyclosporine A (CsA;1μM) for 3 days, adding fresh blocker solutions every 24h, or plated directly onto soft collagen I-coated polyacrylamide hydrogels with a stiffness of 4.5 kPa that were generated as previously described [12].

Immunostaining

Cardiac fibroblasts were fixed in 4% PFA, permeabilized with 0.1% triton, quenched with 25 mM glycine and blocked in 5% goat serum. Primary and secondary antibodies were diluted in 2% goat serum. Mouse anti-SMA antibody (1A4 clone, # 14-9760-82, Thermo Fisher, Denmark) was diluted 1:1000 and 488-Alexa secondary antibody (anti-mouse) was diluted 1:2000. SMA was quantified by taking the average GFP intensity for cells on 10 micrographs per cover slip, three cover slips (N = 3) per condition. The same exposure time and gain was used for all images and quantification was performed using Cell Profiler [13]. The “No 1AB negative control” was used to determine suitable exposure time (no signal in No 1AB control). Values were normalized to non-treated controls for each round of experiments. The experiments were done with cardiac fibroblasts from two separate isolations.

RNA isolation, cDNA synthesis and real-time PCR

RNA isolation was performed using GenElute Mammalian Total RNA Miniprep Kit (Sigma), and cDNA using the TaqMan Reverse Transcription Reagents kit (#N8080234, Applied Biosystems). Nanodrop was used to determine concentration and quality of RNA. A “no template negative control” and “no reverse transcriptase (RT) negative control” was included in the cDNA synthesis and subsequent RT-PCR runs. Real-time PCR was performed using SYBR Green and primers for collagen (Col) 1a1, Col3a1, Acta2, connective tissue growth factor (Ctgf), lysyl oxidase (Lox), periostin (Postn), transcription factor 21 (Tcf21). mRNA was normalized to housekeeping gene ribosomal 18S. Since one Ct value reflects a doubling of mRNA, Ct values were transformed to a linear scale by exponentiation. Ct levels are inversely proportional to the amount of target nucleic acid in the sample; therefore, ΔCt is a negative value. The data presented in Fig 2F were presented as 2-ΔCt. All other mRNA data were presented as relative to the control group.

Fig 2. Relieving cells of high stiffness delays, but does not prevent, myofibroblast differentiation.

Fig 2

Open in a new tab

A) Schematic illustration of protocol used for experiments in B and C. Cardiac fibroblasts were cultured on plastic or soft gels for 3, 9 or 15 days (d) and then analysed. B) Immunostaining for smooth muscle α-actin (SMA; green) in cardiac fibroblasts. Nuclei were stained with DAPI (blue). Scale bar 100μm. C) mRNA expression of myofibroblast markers and Tcf21 at 13 days after seeding. Y-axis value, 2-ΔCt, represents mRNA levels for each gene relative to 18S rRNA. ΔCt is the Ct value of the gene of interest minus the Ct value of 18S rRNA. D) Schematic illustration of protocol used for experiments in E and F. Cardiac fibroblasts were cultured on plastic for 9d where after they were either transferred to soft gels of kept on plastic for an additional 4d. E) Immunostaining for SMA (green) in cardiac fibroblasts. Nuclei were stained with DAPI (blue). Scale bar 100μm. F) mRNA expression of myofibroblast markers and Tcf21. Significant differences were determined by Student’s t-test corrected for multiple comparisons using Holm-Sidak test. N = 8 (C, plastic and 3d N = 4 (C, 9d), N = 3 (C, 15d) and N = 3 (F).

Statistics

Normal distribution was determined by D’Agostino & Pearson test. For comparisons of multiple groups, one-way ANOVA with Dunnett’s post-hoc test was used. For two-group comparisons Student’s t-test was used (two-tailed) andcorrected for multiple comparisons using Holm-Sidak test. *p<0.05, **p<0.01, ***p<0.001, not significant (n.s.).

Results

The myofibroblast phenotype peaks at day nine of culture on plastic

To determine the effect of a stiff culturing substrate on myofibroblast differentiation, we measured the expression of myofibroblast-associated genes at different time points after plating cells on plastic. As expected, we observed a rapid increase in the classical myofibroblast marker Acta2 mRNA (Fig 1A) and protein (Fig 1B) in response to plastic culture conditions, resulting in the formation of clear SMA fibers in the cytoplasm (Fig 1C). Acta2 mRNA and SMA staining intensity peaked at 9 days where after it declined. Expression of Col1a1 and Lox also increased during culture on plastic (Fig 1D and 1E), but started to decline after 12 days, indicating that myofibroblasts do not comprise a permanently differentiated state.

Fig 1. Cardiac fibroblast differentiation during culture on plastic.

Fig 1

Open in a new tab

Actin alpha 2 smooth muscle (Acta2) mRNA (A) and smooth muscle α-actin (SMA) staining intensity (B) in cardiac fibroblasts plated on plastic for different times. C) Representative immunostaining images for SMA. Scale bar 100μm. D) mRNA expression of collagen (Col) 1a1 and lysyl oxidase (Lox) in cardiac fibroblast cultured on plastic for different durations of time. One-way ANOVA with Dunnett’s multiple comparisons test as indicated for significant differences compared to the 1 day time point. N = 3 (2h, 1d, 2d, 3d, 9d, 12d and 15d), N = 5 (5d), N = 9 (7d). mRNA was normalized to ribosomal 18S.

Culturing cardiac fibroblasts on soft gels delays myofibroblast differentiation

We previously showed that culturing cardiac fibroblasts on soft hydrogels, mimicking the stiffness of the healthy heart, prevented myofibroblast differentiation over the course of several days (5). To examine the time frame of this effect, we examined marker gene expression after plating cardiac fibroblasts directly onto soft (4.5kPa) hydrogels and culturing them for 3, 9 and 15 days (Fig 2A).

SMA staining intensity was weak in cardiac fibroblasts cultured on soft gels for 3 and 9 days, but strong after 15 days on gels (Fig 2B). Cardiac fibroblasts cultured for 12 days on soft gels also showed development of SMA fibers in some cells, but not all (data not shown), suggesting that the specific time point for differentiation may vary among cells. As expected, low stiffness conditions were accompanied by reduced expression of myofibroblast markers Acta2, Col1a1 and Ctgf at 3 and 9 days, and Lox at 3 days when compared to cells cultured on plastic (Fig 2C). In contrast, Col3a1 mRNA was unaltered at these time points, suggesting that regulation of Col3a1 is not regulated by stiffness. Importantly, myofibroblast marker expression was no longer reduced in cardiac fibroblasts on soft gels compared to plastic after 15 days, and Col3a1 was even 8-fold increased (Fig 2C). These data show that myofibroblast differentiation can be delayed, but not prevented, by using soft hydrogels as the substrate.

Transferring myofibroblasts from plastic to soft gels (Fig 2D) did not reduce SMA fibers (Fig 2E), and surprisingly, Acta2 mRNA was significantly increased (Fig 2F), suggesting that there may be a temporal window during which Acta2 expression can be reversed. In contrast to Acta2, the remaining myofibroblast markers were markedly decreased after four days on soft gels, suggesting that once the myofibroblast phenotype is established, a stiff substrate is necessary for continued expression of many myofibroblast-associated genes.

Inhibiting ROCK and TGFβRI is most efficient for preventing and reversing the myofibroblast phenotype

We then tested the effect of blocking mechanotransduction signaling pathways (Fig 3A) by using single and combined treatments with the ROCK inhibitor Y27632 (Y27), the TGFβRI inhibitor SB431542 (SB), and the calcineurin inhibitor CsA (Fig 3B). To determine optimal blocker concentrations, we studied responses in SMA fiber formation. Concentrations >10 μmol/L for both Y27 and SB efficiently reduced SMA fiber formation, suggesting inhibition of mechanotransduction (Fig 3C and 3D). Since the half-life for Y27 has been reported to be 12–16 h [14], we used a concentration of 25 μmol/l for Y27. Interestingly, SB concentrations higher than 10μmol/l induced a different morphology compared to lower concentrations (Fig 3E) which did not resemble typical fibroblast morphology. Therefore, we used a concentration of 10 μmol/l for SB, and replaced blocker solutions every 24 h. CsA was applied at a final concentration of 1 μmol/L according to our previous publication [9].

Fig 3. Effect of different concentration of ROCK and TGFβRI inhibitors on smooth muscle α-actin fibers.

Fig 3

Open in a new tab

A) Schematic diagram (modified from Herum et al., JCM, 2017) of the three main mechanically activated signalling pathways that lead to myofibroblast differentiation and pro-fibrotic gene expression. B) Inhibition points using the inhibitors Y27632, SB431542 and cyclosporine A (CsA). Immunostaining of cardiac fibroblasts for smooth muscle α-actin (SMA) in cardiac fibroblasts treated with different concentrations of Y27632 (C) and SB431542 (D and E). The micrograph in E is the same as in D. Phalloidin was used to stain F-actin (C) and DAPI to stain nuclei (D and E). Scale bar 100μm (C) and 200μm (D and E).

The protocols used to test if blockers could prevent or reverse myofibroblast differentiation are illustrated in Fig 4A and 4B. Inhibiting ROCK or TGFβRI with Y27 and SB, respectively, prevented SMA expression and fiber formation, and altered cell morphology into smaller cells (Fig 4C and 4E). Y27 also induced a more dendritic appearance compared to the other groups, resembling the morphology of cardiac fibroblasts cultured on soft gels. Surprisingly, calcineurin inhibition with CsA increased staining intensity and had no effect on cell morphology. Combining Y27 and SB reduced SMA to the same extent as SB alone, however, cell morphology resembled cells treated with Y27 inhibitor alone. Combined treatment with all three inhibitors did not further reduce SMA compared with Y27 and SB co-treatment, and cells appeared smaller and more compact, possibly indicating reduced cell viability or proliferation in this group (Fig 4C).

Fig 4. Inhibiting ROCK and TGFβRI is most efficient for preventing and reversing the myofibroblast phenotype.

Fig 4

Open in a new tab

A and B) Schematic illustration of treatment regimen. Red arrows indicate the addition of fresh inhibitor solution every 24h. C and D) Immunostaining for smooth muscle α-actin (SMA) in cardiac fibroblasts treated with blockers of ROCK (Y27), TGFβRI (SB) and calcineurin (CsA). Scale bar 100μm. E and F) Quantification of SMA staining intensity. G and H) Expression of myofibroblast markers actin alpha 2 smooth muscle (Acta2), connective tissue growth factor (Ctgf), collagen (Col) 1a1, Col1a2, Col3a1, lysyl oxidase (Lox) and periostin (Postn), and the marker of resting cardiac fibroblasts, transcription factor 21 (Tcf21). One-way ANOVA as indicated beside gene name and Dunnett’s multiple comparisons test for significant differences compared to the “no treatment” control as indicated in the heat map. N = 3 (E and F), 4 (G) 4–7 (H).

For the reverse experiment, SB reduced SMA staining intensity, and this effect was augmented by adding Y27 or CsA (Fig 4D and 4F). No clear differences in cell morphology were observed for the reversed cardiac fibroblasts (Fig 4D), indicating that inhibiting mechanotransduction signaling at this time point is not sufficient to reverse cell morphological phenotype.

SB prevented Acta2 upregulation, confirming that TGFβRI signaling is essential for myofibroblast differentiation (Fig 4G). However, including Y27 additionally prevented induction of Lox and Postn, which are important regulators of ECM maturation and stiffening in the heart. Interestingly, only CsA prevented Col1a2 expression. In contrast to the other myofibroblast markers, Col3a1 was significantly increased by combined Y27 and SB treatment (Fig 4G).

For reversing myofibroblast gene expression, SB was the single blocker with largest inhibitory effect on myofibroblast markers (Fig 4H). In general, combinations of SB and the other blockers had a larger effect. Similar to the prevention experiment, blocker combinations with CsA were effective in reducing Col1a2 mRNA, while the combination of SB and Y27 increased Col3a1 expression (Fig 4H).

Taken together, the combination of inhibiting ROCK and TGFβRI had the strongest inhibitory effect on myofibroblast differentiation. Thus, we proceeded with this combination to study more closely the reversal of myofibroblast phenotype at different time points after isolation.

Reversibility of myofibroblasts depends on the duration of prior culturing time on plastic

To examine whether the reversibility of myofibroblasts is lost after longer culturing time on plastic, we examined the effect of combined treatment with SB and Y27 on myofibroblasts cultured on plastic for 9 (Fig 5A) and 16 days (Fig 5B). While myofibroblasts markers Col1a1, Lox and Postn were reduced by blocker treatment at both times, Acta2, Ctgf were only affected at the early time point, suggesting that expression of these genes can only be fully reversed within the first week of culturing on plastic (Fig 5C and 5D). Furthermore, the induction of Col3a1 expression in response to inhibitor treatment only occurred at the early time point, indicating that long culture times on plastic render cardiac fibroblasts more resistant to these pharmacological inhibitors.

Fig 5. Reversibility of myofibroblasts depends on the duration of prior culturing time on plastic.

Fig 5

Open in a new tab

A and B) Schematic illustrations of protocols used in C and D, respectively. Cardiac fibroblasts were cultured on plastic for 6 (A) or 13 (B) days (d) and then treated with blockers of ROCK (Y27) and TGFβRI (SB) for 3d. C and D) mRNA expression of myofibroblast markers actin alpha 2 smooth muscle (Acta2), connective tissue growth factor (Ctgf), collagen (Col) 1a1, Col3a1, lysyl oxidase (Lox) and periostin (Postn) in cardiac fibroblasts treated with Y27 and SB (reversed; grey bars) relative to untreated cardiac fibroblast (control; black bars). The myofibroblast marker gene measurements in D are also shown in the heat map of Fig 4H. Significant differences were determined by Student’s t-test. N = 6 (C) and N = 3 (D).

Reversed cardiac fibroblasts re-differentiate on plastic but not on soft gels

To determine whether reversed cardiac fibroblasts recapitulated the mechanosensitivity and plasticity of freshly isolated resting cardiac fibroblasts, we examined the ability of reversed cardiac fibroblasts to re-differentiate on plastic after removing inhibitors, and whether this could be prevented by culturing cells on soft gels (Fig 6A). SMA fibers were present in cardiac fibroblasts on plastic and gels (Fig 6B), suggesting the presence of a myofibroblast phenotype regardless of stiffness. Despite SMA fibers and increased Acta2 mRNA, myofibroblast marker genes Ctgf, Col1a1, Col1a2 and Col3a1 remained low when cells were cultured on soft hydrogels (Fig 6C white bars), whereas all myofibroblast markers, except Col3a1, increased during re-differentiation on plastic (Fig 6C, black bars). These results indicate that the expression of some genes, such as the collagen I genes, maintain mechanosensitivity after blocker treatment. Since Col3a1 was increased by treatment with Y27 and SB (Fig 4H), expression was not further increased during re-differentiation but rather inhibited by culturing on soft hydrogels (Fig 6C). In contrast, Acta2, Lox and Postn were increased in cardiac fibroblasts on plastic and soft gels after blocker removal, suggesting that re-activation of TGFβRI and/or ROCK signaling is occurring regardless of matrix stiffness and are strong inducers of these genes [15, 16]. Taken together, these results suggest that transient inhibition of mechanotransduction changes the mechanosensitivity of some genes.

Fig 6. Reversed cardiac fibroblasts re-differentiate on plastic.

Fig 6

Open in a new tab

A) Schematic illustration of re-differentiation protocol. Cardiac fibroblasts were cultured on plastic for 6 days (d) and then treated with blockers of ROCK (Y27) and TGFβRI (SB) for 3d, where after blockers were removed and cells transferred to soft gels for 4d. B) Immunostaining for smooth muscle α-actin (SMA; green). Nuclei are stained with DAPI (blue). Scale bar 100μm. C) mRNA expression of actin alpha 2 smooth muscle (Acta2), connective tissue growth factor (Ctgf), collagen (Col) 1a1, Col1a2, Col3a1, lysyl oxidase (Lox) and periostin (Postn) relative to reversed cells (grey bars), following re-differentiation on plastic (black bars) or soft gels (white bars). Significant differences were determined by Student’s t-test with Holm-Sidak correction for multiple comparisons. N = 3.

Discussion

In vitro studies of cardiac fibroblast activation are complicated by the rapid differentiation into myofibroblasts when primary cardiac fibroblasts are cultured on plastic [1]. Here we investigated whether soft hydrogels or biochemical inhibitors could maintain resting cardiac fibroblasts in vitro.

Although soft hydrogels initially prevented myofibroblast differentiation, SMA fibers and myofibroblast markers were eventually increased after 15 days. Myofibroblast differentiation might be induced by the high cell density at this time point. Indeed, high cell density has recently been shown to diminish the effect of hydrogel stiffness on cell phenotype. Atomic force measurements also suggested local strain stiffening of the hydrogel when cell density is high [17]. Thus, hydrogels are most suitable for short experiments over the course of days.

It is not clear whether cardiac fibrosis and the myofibroblast phenotype can be reversed [18, 19]. TGFβ signaling is crucial for myofibroblast differentiation [20–22] and is activated by tissue stiffness due to liberation from the latent protein binding [23, 24]. ROCK is also central for the mechanosensing and response of cells [7, 25]. As anticipated, the combined use of TGFβRI and ROCK inhibitors reversed the expression of myofibroblast markers. However, SMA fibers did not disappear completely after treatment with TGFβRI and ROCK inhibitors. Others have shown that the ability of TGFβRI inhibition to reverse myofibroblast differentiation is dependent on proliferative activity [6]. Interestingly, reduced proliferation has also been associated with high SMA expression, despite reduced contraction, suggesting that Acta2 expression may also modulate cellular functions other than contractility [26].

Since prolonged culture time is necessary for many experiments (e.g. CRISPR/Cas9 gene editing), it would be of great benefit to maintain a resting cardiac fibroblast phenotype in vitro. Reversed cardiac fibroblasts maintained the ability to become re-activated into myofibroblasts, and this was prevented by culturing on soft hydrogels. This could provide a potential method for studying activation of cardiac fibroblasts after initial expansion of primary cultures on plastic. One limitation to this approach is that, despite soft substrate culture conditions, SMA fibers re-appeared and Lox and Postn mRNA were increased suggesting partial myofibroblast differentiation after removing blockers.

The counter regulation of Col3a1 and Col1a1 by combined TGFβRI and ROCK inhibition was a novel finding of this study. In agreement with these results, we previously found differential effects on Col1a1 and Col3a1 expression in cardiac fibroblasts in response to substrate stiffening [5]. A shift in the ratio of collagen I/collagen III has been suggested be central for changes in myocardial stiffness [27, 28], due to the intrinsically higher stiffness of collagen I compared to collagen III fibers. Thus, Col3a1 expression might be considered beneficial in the context of fibrosis and diastolic function.

Controlling the in vitro environment is a challenge and because of the extensive plasticity of cardiac fibroblasts, variation in culturing conditions, apart from tissue stiffness, might affect cell phenotype. These include effects of serum composition, substrate protein coating, and number of cell passage. Based on our results, we recommend using blocker-induced reversal of myofibroblasts in combination with soft hydrogels to study the mechanical induction of collagen I and III expression, while other features of myofibroblasts such as SMA fibers, Lox and Postn should be studied in freshly isolated cardiac fibroblasts.

Acknowledgments

We are highly grateful to Volkan Turan and Jennifer Stowe for their excellent technical assistance.

Data Availability

All relevant data are within the manuscript.

Funding Statement

K.M.H. has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 795390. https://ec.europa.eu/programmes/horizon2020/en/ The project was supported in part by NIH grants 1 R01 HL137100-01, U01 H127654 and 1 U01 HL126273. https://www.nih.gov/grants-funding The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  • 1.van Putten S, Shafieyan Y, Hinz B. Mechanical control of cardiac myofibroblasts. J Mol Cell Cardiol. 2016;93:133–42. 10.1016/j.yjmcc.2015.11.025 [DOI] [PubMed] [Google Scholar]
  • 2.Yang J, Savvatis K, Kang JS, Fan P, Zhong H, Schwartz K, et al. Targeting LOXL2 for cardiac interstitial fibrosis and heart failure treatment. Nat Commun. 2016;7:13710 10.1038/ncomms13710 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Dorn LE, Petrosino JM, Wright P, Accornero F. CTGF/CCN2 is an autocrine regulator of cardiac fibrosis. J Mol Cell Cardiol. 2018;121:205–11. 10.1016/j.yjmcc.2018.07.130 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Kanisicak O, Khalil H, Ivey MJ, Karch J, Maliken BD, Correll RN, et al. Genetic lineage tracing defines myofibroblast origin and function in the injured heart. Nat Commun. 2016;7:12260 10.1038/ncomms12260 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Herum KM, Choppe J, Kumar A, Engler AJ, McCulloch AD. Mechanical regulation of cardiac fibroblast pro-fibrotic phenotypes. Mol Biol Cell. 2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Driesen RB, Nagaraju CK, Abi-Char J, Coenen T, Lijnen PJ, Fagard RH, et al. Reversible and irreversible differentiation of cardiac fibroblasts. Cardiovasc Res. 2014;101(3):411–22. 10.1093/cvr/cvt338 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Herum KM, Lunde IG, McCulloch AD, Christensen G. The Soft- and Hard-Heartedness of Cardiac Fibroblasts: Mechanotransduction Signaling Pathways in Fibrosis of the Heart. J Clin Med. 2017;6(5). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Stempien-Otero A, Kim DH, Davis J. Molecular networks underlying myofibroblast fate and fibrosis. J Mol Cell Cardiol. 2016;97:153–61. 10.1016/j.yjmcc.2016.05.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Herum KM, Lunde IG, Skrbic B, Florholmen G, Behmen D, Sjaastad I, et al. Syndecan-4 signaling via NFAT regulates extracellular matrix production and cardiac myofibroblast differentiation in response to mechanical stress. J Mol Cell Cardiol. 2013;54:73–81. 10.1016/j.yjmcc.2012.11.006 [DOI] [PubMed] [Google Scholar]
  • 10.Davis J, Burr AR, Davis GF, Birnbaumer L, Molkentin JD. A TRPC6-dependent pathway for myofibroblast transdifferentiation and wound healing in vivo. Dev Cell. 2012;23(4):705–15. 10.1016/j.devcel.2012.08.017 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Small EM. The actin-MRTF-SRF gene regulatory axis and myofibroblast differentiation. J Cardiovasc Transl Res. 2012;5(6):794–804. 10.1007/s12265-012-9397-0 [DOI] [PubMed] [Google Scholar]
  • 12.Tse JR, Engler AJ. Preparation of hydrogel substrates with tunable mechanical properties. Curr Protoc Cell Biol. 2010; Chapter 10:Unit 10 6. [DOI] [PubMed] [Google Scholar]
  • 13.McQuin C, Goodman A, Chernyshev V, Kamentsky L, Cimini BA, Karhohs KW, et al. CellProfiler 3.0: Next-generation image processing for biology. PLoS Biol. 2018;16(7):e2005970 10.1371/journal.pbio.2005970 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Ishizaki T, Uehata M, Tamechika I, Keel J, Nonomura K, Maekawa M, et al. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. Mol Pharmacol. 2000;57(5):976–83. [PubMed] [Google Scholar]
  • 15.Voloshenyuk TG, Landesman ES, Khoutorova E, Hart AD, Gardner JD. Induction of cardiac fibroblast lysyl oxidase by TGF-beta1 requires PI3K/Akt, Smad3, and MAPK signaling. Cytokine. 2011;55(1):90–7. 10.1016/j.cyto.2011.03.024 [DOI] [PubMed] [Google Scholar]
  • 16.Lu M, Qin Q, Yao J, Sun L, Qin X. Induction of LOX by TGF-beta1/Smad/AP-1 signaling aggravates rat myocardial fibrosis and heart failure. IUBMB Life. 2019;71(11):1729–39. 10.1002/iub.2112 [DOI] [PubMed] [Google Scholar]
  • 17.Venugopal B, Mogha P, Dhawan J, Majumder A. Cell density overrides the effect of substrate stiffness on human mesenchymal stem cells’ morphology and proliferation. Biomater Sci. 2018;6(5):1109–19. 10.1039/c7bm00853h [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Frangogiannis NG. Can Myocardial Fibrosis Be Reversed? J Am Coll Cardiol. 2019;73(18):2283–5. 10.1016/j.jacc.2018.10.094 [DOI] [PubMed] [Google Scholar]
  • 19.Nagaraju CK, Robinson EL, Abdesselem M, Trenson S, Dries E, Gilbert G, et al. Myofibroblast Phenotype and Reversibility of Fibrosis in Patients With End-Stage Heart Failure. J Am Coll Cardiol. 2019;73(18):2267–82. 10.1016/j.jacc.2019.02.049 [DOI] [PubMed] [Google Scholar]
  • 20.Biernacka A, Dobaczewski M, Frangogiannis NG. TGF-beta signaling in fibrosis. Growth Factors. 2011;29(5):196–202. 10.3109/08977194.2011.595714 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Bujak M, Frangogiannis NG. The role of TGF-beta signaling in myocardial infarction and cardiac remodeling. Cardiovasc Res. 2007;74(2):184–95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Hinz B. The extracellular matrix and transforming growth factor-beta1: Tale of a strained relationship. Matrix Biol. 2015;47:54–65. 10.1016/j.matbio.2015.05.006 [DOI] [PubMed] [Google Scholar]
  • 23.Wipff PJ, Hinz B. Integrins and the activation of latent transforming growth factor beta1—an intimate relationship. Eur J Cell Biol. 2008;87(8–9):601–15. 10.1016/j.ejcb.2008.01.012 [DOI] [PubMed] [Google Scholar]
  • 24.Sarrazy V, Koehler A, Chow ML, Zimina E, Li CX, Kato H, et al. Integrins alphavbeta5 and alphavbeta3 promote latent TGF-beta1 activation by human cardiac fibroblast contraction. Cardiovasc Res. 2014;102(3):407–17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Ohashi K, Fujiwara S, Mizuno K. Roles of the cytoskeleton, cell adhesion and rho signalling in mechanosensing and mechanotransduction. J Biochem. 2017;161(3):245–54. 10.1093/jb/mvw082 [DOI] [PubMed] [Google Scholar]
  • 26.Shinde AV, Humeres C, Frangogiannis NG. The role of alpha-smooth muscle actin in fibroblast-mediated matrix contraction and remodeling. Biochim Biophys Acta Mol Basis Dis. 2017;1863(1):298–309. 10.1016/j.bbadis.2016.11.006 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Sullivan KE, Quinn KP, Tang KM, Georgakoudi I, Black LD 3rd. Extracellular matrix remodeling following myocardial infarction influences the therapeutic potential of mesenchymal stem cells. Stem Cell Res Ther. 2014;5(1):14 10.1186/scrt403 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Mukherjee D, Sen S. Collagen phenotypes during development and regression of myocardial hypertrophy in spontaneously hypertensive rats. Circ Res. 1990;67(6):1474–80. 10.1161/01.res.67.6.1474 [DOI] [PubMed] [Google Scholar]

Decision Letter 0

Philip C Trackman

Transfer Alert

This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present.

24 Jun 2020

PONE-D-20-17115

Maintaining resting cardiac fibroblasts in vitro by disrupting mechanotransduction

PLOS ONE

Dear Dr. Herum,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The reviewer identified several technical and conceptual areas that require revision. In particular, the study is largely descriptive without new insights into mechanistic relationships between culture conditions and outcomes. Most of the documented relationships as a function of culture conditions have been described elsewhere. Identification of molecular interactions that drive the differences in cell behavior or gene regulation as a function of culture conditions would provide new information to advance the field. The reviewer has provided a thorough identification of additional changes that will be required before the manuscript can be accepted.

Please submit your revised manuscript by September 1, 2020. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Philip C. Trackman, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please upload a copy of Figure 3, to which you refer in your text on page 15, 16 and 17 (Figure 3 cited, but Supplementary Figure 3 uploaded).  If the figure is no longer to be included as part of the submission please remove all reference to it within the text.

3. Thank you for stating the following in the Competing Interests section:

[A.D.M. is a co-founder of and has an equity interest in Insilicomed Inc. and an equity interest in Vektor Medical, Inc.. He serves on the scientific advisory board of Insilicomed, and as scientific advisor to both companies. Some of his research grants have been identified for conflict of interest management based on the overall scope of the project and its potential benefit to these companies. The author is required to disclose this relationship in publications acknowledging the grant support; however, the research subject and findings reported in this study did not involve the companies in any way and have no relationship with the business activities or scientific interests of either company. The terms of this arrangement have been reviewed and approved by the University of California San Diego in accordance with its conflict of interest policies.]

Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests).  If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: To understand the effect of mechanotransduction on fibroblasts to myofibroblasts differentiation of cardiac fibroblasts, it is highly critical to maintain the resting fibroblasts that are freshly isolated from tissues. The authors screened in vitro culturing conditions to slow down activation/differentiation of cardiac fibroblasts to myofibroblasts and recommend adding inhibitors specific to TGFβR1 and ROCK as well as seeding fibroblasts on soft hydrogels.

It is an important study, however, the manuscript needs to be revised thoroughly to address the following concerns.

Major issues

1. It is well established that TGFβR1 and ROCK play critical roles in fibroblasts to myofibroblasts differentiation and inhibitors of these signaling pathways inhibit the differentiation. The results described in this manuscript do not add anything substantially new to our current understandings. It seems that once fibroblasts are activated through mechanotransduction by ECM stiffening to form SMA fibers, SMA fibers persist even after transferring cells onto a soft hydrogel or treatment with inhibitors of TGFβR1 and ROCK, therefore changes in cell morphology are negligible. LOX and LOX-family of proteins are upregualted via TGFβR1 signaling pathways, therefore upregulation of LOX gene was detected when TGFβR1 blocker was removed in Panel A and C (Figure 5). Authors need to cite literature concerning LOX gene and TGFβ.

2. It is better to include a schematic diagram for relevant signaling pathways for fibroblasts to myofibroblasts differentiation.

3. The authors need to provide rational/explanation for why they examine the effect of calcineurin inhibitor cyclosporine A in this study.

4. Images (Panel C and D) in Figure 3 (labeled as Supllemantary Figure 3 in the manuscript) should be replaced with images in better resolution.

5. It is intriguing that LOX is highly upregulated when cells are cultured on a plastic (Figure 1, Panel F and Figure5, Panel C).The authors need to add their explanation/interpretation for this.

6. Figure legends in this manuscript need to be revised thoroughly. At the current state, it is not self explanatory.

Figure 1.

- Which data of Panel C do have N= 3 and which data of Panel C have N= 8?

- Which data (day) do correspond to Panel F? What is Y-axis in Panel F?

- Panel A and C, it is better to include more data points between day 9 and day 15 so that the critical time point for differentiation can be defined.

- Panel D describes fibroblasts cultured 9 days on plastic and switched to soft hydrogel and cultured for additional 4 days, but this is not explained in the legend. Is it necessary to incubate 9 days to see full differentiation? Some explanation for this experimental set-up needs to be provided.

- The text of this manuscript mentions Figures 2E and 2F but are actually Figures 1E and 1F, respectively.

Figure 2.

- The label “anti-SMA” in Panel A, B, C should be SMA.

- What are the differences between Panel B 50 μM and Panel C 50 μM?

Supplementary Figure 3.

- This is called Supplementary Figure 2 within the text, but it should be Figure 3.

- Panel A. “wth” should be “with”

- Panel C and D. Description of upper and lower panels for effect of blockers need to be provided.

Figure 4.

- Panel A and B, provide detailed procedures, e.g. Fibroblasts were cultured on plastic for 6 days (Panel A) and 13 days (Panel B).

- Panel C and D should include relative mRNA levels of these markers before addition of SB+Y27.

Other issues

1. There are too many non-essential and introductory elements in a sentence and that makes this manuscript difficult to follow.

2. Abstract

The last sentence, starting with Importantly, needs to be revised to address the conclusion of this paper and the message should be clearer.

3. Introduction

Line 6 from the top, lysyl oxidase-like 2 (LOXL2) should be added as LOXL2 has also shown to play critical role in cardiac interstitial fibrosis by Yang, J. et al Nature Comm. 2016, 7, 13710.

4. Results

The following sentences need to be revised for better understanding.

under Relieving cells of high stiffness delays, but does not prevent myofibroblast differentiation.

Line 2 in the second paragraph, “SMA staining intensity was low for cardiac fibroblasts cultured on soft gels for 3 and 9 days, but increased after 15 days on gels (Figure 1B), at which time cells were highly confluent.” � SMA staining and confluency between day 9 and day 15 should be included (e.g. day 11, day 13).

Line 6 in the second paragraph,

“Marker expression was no longer suppressed by soft compared with stiff culture substrates.”

“for the reverse experiment, SB inhibition, as well as all combinations of blockers, reduced SMA staining intensity, albeit the effect of blocker combinations were more pronounced.”

Minor

1. Full name and abbreviations.

Abstract:

Line 8 from the top, (SMA) should be inserted after smooth muscle α-actin.

Line 10 from the top, transforming growth factor β receptor I should be inserted before (TGFβRI) as well as Rho-associated protein kinase before (ROCK). Actin alpha2 smooth muscle gene should be inserted before (Acta2).

Introduction:

Line 4 from the top, full name for Acta 2 should be given.

Line 9 from the top, full name for Tcf21 should be given.

2. Some errors listed below need to be revised.

Abstract:

Line 4 from the top, “It is therefore challenging to study resting cardiac fibroblasts and their activation, in vitro.” The comma should be removed.

Line 6 from the top, “maintain resting cardiac fibroblasts for up to 5 days but some experiments” Insert comma before but

Introduction:

line 5 from the bottom, “However, the use of soft hydrogels have limitations” � “The use of soft hydrogels has some limitations”

Results:

Under “Inhibiting ROCK and TGFβRI is most efficient for preventing and reversing the myofibroblast phenotype” on the second page,

In the paragraph starting with “For the reverse experiment, “

“blocker combinations where most efficient in preventing and reversing” should read as “blocker combinations were most efficient at preventing and reversing”

in the paragraph starting with “We next measured…”

“only SB prevented Acta2 upregulation confirming that” Insert a comma after upregulation.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Decision Letter 1

Philip C Trackman

23 Sep 2020

PONE-D-20-17115R1

Maintaining resting cardiac fibroblasts in vitro by disrupting mechanotransduction

PLOS ONE

Dear Dr. Herum,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

The expert reviewer clearly appreciated the improvement in the manuscript. A few adjustments in the writing and a couple of clarifications are still needed, which would further improve the manuscript. Once these have all been addressed, then the manuscript will likely be suitable for publication. I understand that the reviewer probably did not find the high resolution figures available by clicking the link located at the upper right corner of each figure on the PLoS website, so your figures are fine as is with respect to resolution. 

==============================

Please submit your revised manuscript by October 30, 2020. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Philip C. Trackman, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors made efforts to address my concerns and the manuscript reads significantly better.

However, there are some issues that need to be sorted out before considering this manuscript for publication on this journal.

Major problem:

I am not sure why images are still at low resolution in this revision, although authors stated that they replaced older images with images in higher resolution. Perhaps it is due to how this PDF version of article was generated. Figures and legends are fuzzy with some background. Immunostaining images are not in publishable quality.

1. The authors response to the first critique under Major issues, “It is well established that TGFβR1 and ROCK play critical roles in fibroblasts to myofibroblasts differentiation and inhibitors of these signaling pathways inhibit the differentiation. The results described in this manuscript do not add anything substantially new to our current understandings.” is reasonable.

“Although it is known that TGFβRI and ROCK are important for myofibroblast differentiation,

their relative contributions and the effect of dual inhibition have not previously been tested.”

- This provides the rational for the research conducted in this study and should be included in the introduction.

“Our results bring novel information regarding the relative importance of these specific signaling

pathways in regulating different myofibroblast-associated genes. E.g. the finding that col3a1 was upregulated by dual inhibition was a novel finding of this paper. 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.”

- This part should be included in abstract and discussion/conclusion.

2. Abstract Line 11-12 “Myofibroblast differentiation was prevented after 3 and 9 days on soft hydrogels. However, after 15 days, myofibroblasts appeared.”

“after 3 and 9 days on soft hydrogels” �”at 3 and 9 days after transferring to soft hydrogels”

3. Figure 2

Figure legend should read, “mRNA expression of myofibroblast markers and Tcf21 at 13 days after seeding. Y-axis value, 2-∆Ct, represents mRNA levels for each gene relative to 18S rRNA. ∆Ct is the Ct value of the gene of interest minus the Ct value of 18S rRNA.”

The following explanation should be included in text. “One Ct value reflects a doubling of mRNA and Ct values are transformed to a linear scale by exponentiation. Since Ct levels are inversely proportional to the amount of target nucleic acid in the sample, ∆Ct is a negative value. The data presented in Panel C where first transformed using 2-∆Ct and then presented as relative to the control group to clearly show the effect of soft gels for each gene.”

The difference between Figure 2C (little effect between soft gel and plastic) and Figure 2F (very high difference after transferring to soft gel) is striking. Could this be related to confluency of cells? It seems unlikely that cells transferred to soft gels kept the exact same confluency as those kept on plastic.

4. Figure 4

Panels A and B should have days on the arrows similar to Figure 2.

5. Figure 5

Figure legend for panel B is missing. The authors need to include some explanation/discussion about the difference in the level of Col3a1. Why it is 2X increased for reversed in panel C but no effect in D?

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Decision Letter 2

Philip C Trackman

14 Oct 2020

Maintaining resting cardiac fibroblasts in vitro by disrupting mechanotransduction

PONE-D-20-17115R2

Dear Dr.Herum,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Philip C. Trackman, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors adequately addressed all my concerns. This manuscript is ready for publication on PLOS ONE.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Acceptance letter

Philip C Trackman

16 Oct 2020

PONE-D-20-17115R2

Maintaining resting cardiac fibroblasts in vitro by disrupting mechanotransduction

Dear Dr. Herum:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Philip C. Trackman

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    Attachment

    Submitted filename: Response to Reviewer.docx

    Click here for additional data file. (217.8KB, docx)
    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (16.9KB, docx)

    Data Availability Statement

    All relevant data are within the manuscript.

    Articles from PLoS ONE are provided here courtesy of PLOS

    RESOURCES