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. Author manuscript; available in PMC: 2014 Jun 4.
Published in final edited form as: Microsc Microanal. 2011 Jul 11;17(4):555–562. doi: 10.1017/S1431927611000390

Age-Dependent Expression of Collagen Receptors and Deformation of Type I Collagen Substrates by Rat Cardiac Fibroblasts

Christopher G Wilson 1,†, John W Stone 2,‡, Vennece Fowlkes 1, Mary O Morales 1, Catherine J Murphy 3, Sarah C Baxter 4, Edie C Goldsmith 1,★
PMCID: PMC4045481  NIHMSID: NIHMS587673  PMID: 21740617

Abstract

Little is known about how age influences the ways in which cardiac fibroblasts interact with the extracellular matrix. We investigated the deformation of collagen substrates by neonatal and adult rat cardiac fibroblasts in monolayer and three-dimensional (3D) cultures, and quantified the expression of three collagen receptors [discoidin domain receptor (DDR) 1, DDR2, and β1 integrin] and the contractile protein alpha smooth muscle actin (α-SMA) in these cells. We report that adult fibroblasts contracted 3D collagen substrates significantly less than their neonate counterparts, whereas no differences were observed in monolayer cultures. Adult cells had lower expression of β1 integrin and α-SMA than neonate cultures, and we detected significant correlations between the expression of α-SMA and each of the collagen receptors in neonate cells but not in adult cells. Consistent with recent work demonstrating age-dependent interactions with myocytes, our results indicate that interactions between cardiac fibroblasts and the extracellular matrix change with age.

Keywords: cardiac fibroblast, extracellular matrix, collagen, integrin, discoidin domain receptor

Introduction

Cardiac fibroblasts comprise a substantial fraction of the cells in the adult heart (~30% in mice, ~60% in rats) (Grove et al., 1969; Banerjee et al., 2007) and serve important roles in developmental and pathologic remodeling of the myocardial extracellular matrix (ECM) (Baudino et al., 2006). During early postnatal development, cardiac fibroblasts synthesize a highly organized ECM composed primarily of type I collagen. In pathological situations, such as following a myocardial infarction, cardiac fibroblasts generate a collagen-dense scar that mechanically stabilizes the injured tissue. Typically the fibrotic response persists and ultimately compromises cardiac function (Weber & Brilla, 1993; Weber et al., 1994). Both adult and neonate cardiac fibroblasts express receptors for soluble factors that promote ECM biosynthesis (Sadoshima & Izumo, 1993; Villarreal et al., 1993), but it is unknown how the cells’ capacity to remodel the ECM changes with age.

Cardiac fibroblasts can be distinguished from other cells in the heart by expression of discoidin domain receptor 2 (DDR2) (Goldsmith et al., 2004; Morales et al., 2005). DDR2 and its related family member DDR1 are receptor tyrosine kinases that bind fibrillar collagens and appear to operate independently of integrins (Vogel et al., 2000). The DDRs mediate renal and pulmonary fibrosis (Matsuyama et al., 2005; Flamant et al., 2006) and type I collagen colocalizes with DDR2+ cells in the developing heart (Goldsmith et al., 2010), suggesting that this class of receptors is important for collagen remodeling in the myocardium. Cardiac fibroblasts also express integrin receptors for type I collagen including the α1, α2, and β1 subunits, and these integrins are essential for contraction of three-dimensional (3D) collagen substrates (Burgess et al., 1994; Carver et al., 1995). Expression of the cytoskeletal protein alpha smooth muscle actin (α-SMA) is positively correlated with fibroblast-mediated contraction of 3D collagen lattices (Arora & McCulloch, 1994; Hinz et al., 2001; Lijnen et al., 2003). Expression of collagen-binding integrins and α-SMA thus appears to be predictive for collagen remodeling behavior, whereas much less is known about the relationship between DDRs and contraction of collagen substrates.

The objectives of this study were to (1) compare the collagen contraction behavior of neonate and adult cardiac fibroblasts in monolayer and 3D culture, and (2) investigate the quantitative relationships between collagen receptors (DDR1, DDR2, and β1 integrin) and the contractile protein α-SMA in these populations. We found that adult cardiac fibroblasts contracted a 3D collagen scaffold substantially less than neonate cardiac fibroblasts, a difference not observed in monolayer cultures. Quantitative age-dependent differences in the expression of collagen receptors and α-SMA at the mRNA and protein levels were observed in monolayer and 3D cultures. Regression analyses indicated that in adult cells expression of collagen receptors (DDR1, DDR2, and β1 integrin) were decoupled from expression of the contractile protein α-SMA.

Materials and Methods

Isolation of Cardiac Fibroblasts

Cells were isolated from neonate (4 day old) and adult (~8–12 week old) Sprague Dawley rats as previously described (Banerjee et al., 2007) using a protocol approved by the University of South Carolina Institutional Animal Care and Use Committee. For each independent experiment, neonatal hearts (approximately 30) were pooled for the isolation of cells, whereas each independent adult experiment was conducted using cells isolated from an individual animal. Whole hearts were aseptically dissected from the animals, minced in Krebs-Ringer bicarbonate buffer, digested with 100 U/mL collagenase, and mechanically dissociated by passing through a 14 gauge cannula. Digests were moved to T75 flasks, and fibroblasts were isolated by attachment to the flasks over 2 h. Cells were expanded through passage 3 or 4 in growth medium composed of Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% newborn bovine serum, 5% fetal bovine serum, 100 U/mL penicillin G, 100 μg/mL streptomycin, and 10 μg/mL gentamicin.

Synthesis of Gold Nanorods

Gold nanorods were synthesized via a seed-mediated surfactant-driven approach (Jana et al., 2001a, 2001b). Briefly, nanoparticulate gold seeds (~4 nm diameter) were prepared by reducing 250 μM chloroauric acid with 0.01 M sodium borohydride in the presence of 0.1 M hexadecyltrimethylammonium bromide (CTAB) in 18 Mω cm water. After 15 min, the seed solution was serially diluted three times, 10-fold each, in solutions of 250 μM chloroauric acid/0.1 M CTAB/500 μM ascorbic acid. The reaction proceeded overnight at room temperature, and the rod-shaped products (mean length: 376 ±105 nm, mean width: 26 ± 5 nm) were purified and concentrated by centrifugation.

Monolayer Culture and Two-Dimensional Strain Measurements Using Nanoparticles

For experiments with monolayer cultures, two-chamber chamber slides were coated with type I collagen and gold nanorods as previously described (Stone et al., 2007). Briefly, pepsin-extracted acid-solubilized bovine type I atelocollagen (Advanced BioMatrix, Inc., San Diego, CA, USA) was neutralized by the addition of 0.2 N 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 9.0 and 10× alpha minimal essential medium (αMEM) on ice at volumetric ratios of 8:1:1, respectively. The neutralized collagen (final concentration ~2.4 mg/mL) was added to each well of the chamber slide, excess collagen was removed from the wells, and the adsorbed collagen was allowed to polymerize for 2–24 h in a standard tissue culture incubator (37°C, 95% relative humidity, 5% CO2). A second adsorbed layer of collagen, doped with as-prepared gold nanorods (10% v/v) was applied to each well and allowed to polymerize for 3 h in a culture incubator. The final thickness of the collagen coating was ~200 μm. The addition of as-prepared gold nanorods does not appear to influence the polymerization of type I collagen (Wilson et al., 2009) and has been used previously by our group for generating strain maps of fibroblasts on collagen films (Stone et al., 2007). These particles efficiently scatter visible light and thus permit high contrast dark-field imaging and subsequent computation of substrate deformation by digital image correlation algorithms (Orendorff et al., 2005).

Neonate or adult cardiac fibroblasts were plated on the collagen-coated chamber slides at 105 cells per well (~2.4 × 104 cells/cm2) in the presence of 10 μM CellTracker Orange (Invitrogen, Carlsbad, CA, USA) and allowed to adhere in a culture incubator. In pilot experiments we determined that 5 h was the minimum time for >90% of the cells to adhere and spread on these substrates. After 5 h the cultures were rinsed with fresh growth medium to remove nonadhered cells, the upper chambers were removed from the slides, and the cultures were coverslipped. A single, randomly chosen field from each culture was imaged at 10 min intervals for 1 h by dark-field and epifluoresence microscopy (excitation filter 510–560 nm/emission filter 590 nm) on a Nikon E600 microscope with a 20× objective and SPOT Insight camera (Diagnostic Instruments, Sterling Heights, MI, USA) at room temperature. Two-dimensional (2D) strain maps were computed using particle tracking algorithms applied to the time series of dark-field images with Vic2D software (Correlated Solutions, Columbia, SC, USA). The reference image was captured at 5 h post-plating. In parallel with the imaging, identically prepared cultures (not imaged) were used for isolation of protein or RNA at 5, 5.5, and 6 h post-plating.

Three-Dimensional Collagen Substrate Contraction Assays

For 3D cultures, neonate or adult cardiac fibroblasts were suspended in neutralized type I collagen (prepared as described above) at 105 cells/mL. Cell-collagen suspensions were moved to 24-well tissue culture plates preadsorbed with 5% bovine serum albumin in DMEM and allowed to polymerize in a cell culture incubator. After 2 h, gels were detached from the wells in a 1 mL growth medium. Images of contracting gels were captured for up to 24 h with a Canon EOS Rebel digital camera, and gel areas were measured using ImageJ. Identically prepared cultures were collected at 0, 5, 6, 12, and 24 h for isolation of protein or RNA.

Western Blotting and Quantitative Polymerase Chain Reaction

Cell lysates for blotting were prepared by harvesting monolayer and 3D cultures in ice cold RIPA buffer (65 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, pH 7.4) with protease inhibitors (10 μM PMSF, 10 μg/mL leupeptin, 0.2 U/mL aprotinin, 50 μM iodoacetamide, 5 μM sodium orthovanadate, and 100 μM sodium fluoride). Samples for RNA isolation were prepared by harvesting cultures in 0.5–1 mL Trizol. The lysates and RNA samples were snap frozen in liquid nitrogen and stored at −20°C prior to analysis.

For western blotting, lysates were thawed on ice and mechanically homogenized by brief ultrasonication. Lysates were then centrifuged at 10k × g for 15 min, and the supernatants assayed for protein content by a detergent compatible colorimetric method (Bio-Rad Laboratories, Hercules, CA, USA). Equal quantities of protein were fractionated by SDS-PAGE, transferred to nitrocellulose, and blotted for DDR1 (Santa Cruz Biotechology, Inc., Santa Cruz, CA, USA), DDR2 (Santa Cruz), β1 integrin subunit (Santa Cruz), or α-SMA (Abcam, Cambridge, MA, USA). Each blot was stripped using Reblot Plus (Chemicon, now Millipore, Billerica, MA, USA) according to the manufacturer’s instructions and probed for GAPDH (Chemicon) as a loading control. Blots were developed with a chemiluminescent substrate and subsaturation exposure to radiographic film. Films were scanned as digital images, and ImageJ software was used to quantify the integrated pixel density of bands migrating at the expected molecular weight of each target protein. For each blot and each sample, the pixel density for the target was normalized by the pixel density of GAPDH.

For analysis of RNA, samples were thawed on ice and homogenized by ultrasonication. After centrifugation, RNA was extracted with chloroform, loaded onto RNeasy mini columns (Qiagen, Valencia, CA, USA) and processed according to the manufacturer’s directions with the optional on-column DNase 1 treatment. The RNA was then analyzed for quantity and quality by spectrophotometry at λ = 260 and 280 nm. 200 ng of total RNA was converted to cDNA using the iScript cDNA synthesis kit (Bio-Rad Laboratories) and then subjected to real-time polymerase chain reaction (PCR) using IQ SybrGreen Supermix (Bio-Rad Laboratories) and primers specific for DDR1 (forward: 5′-AGCCGGAATCTCTACGC-3′; reverse: 5′-CACAGGGTCACTCCGAA-3′), DDR2 (forward: 5′-ATCCA-ACTCCATCTTCTG-3′; reverse: 5′-ATTCTGACCACTGAC-TTG-3′), β1 integrin subunit (forward: 5′-GCGATCAGG-AGAACCACAGAAG-3′, reverse: 5′-ACAAATGAGCCAA-AGCCAATGC-3′), α-SMA (forward: 5′-GGAGTGATGGTT-GGAATGG-3′; reverse: 5′-ATGATGCCGTGTTCTATCG-3′), and the reference gene ARBP (forward: 5′-TAGAGGGT-GTCCGCAATG-3′; reverse: 5′-GAAGGTGTAGTCAGTC-TCC-3′). Data were analyzed by the ΔCt method of Pfaffl (2001), in which the threshold cycle number for detection of a target gene is determined relative to the threshold cycle number for a reference gene.

Statistics

Data were analyzed by the general linear model (GLM) using MiniTab version 11. Linear regressions of ln-transformed data were performed with SPSS version 16. Differences and regressions were considered statistically significant for p < 0.05.

Results

Our initial approach to examining the ECM remodeling behavior of neonate and adult cardiac fibroblasts was to quantify the deformation of a thin collagen substrate in monolayer cultures. Representative reference images of Cell-Tracker Orange-labeled cells and scattering from gold nanorods embedded in the collagen substrate are given in Figure 1A. The morphology of neonate and adult cells was similarly spread and stellate. Over 1 h of particle tracking, the peak principal strains (postive, tensile, ε1 and negative, compressive, ε2) reached a maximum at ε1 ~ 6.3% and ε2 ~ −5.5% for adults and at ε1 ~ 3.7% and ε2 ~ −3.3% for neonates (Fig. 1B); these differences between adult and neonate fibroblasts were not statistically significant. Similarly, the mean principal strains (averaged over the entire field of view, Fig. 1C) and the distributions of principal strain magnitudes (Fig. 1D) were similar for adult and neonate cultures. Collectively, these data indicate that deformation of collagen substrates by adult and neonate cardiac fibroblasts is indistinguishable in monolayer culture.

Figure 1.

Figure 1

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Deformation of collagen substrates in monolayer cultures of adult and neonate cardiac fibroblasts. A: i: Fluorescence image of CMRA-labeled adult rat cardiac fibroblasts cultured on a collagen-coated coverslip. ii: Dark-field image of the same field of view shown in panel i. Gold nanoparticles efficiently scatter light producing a yellow-green color, thus permitting high-resolution particle tracking for calculating strain fields. The diffuse gray-blue background is light scattered by the collagen film. iii: Overlay of fluorescence and dark-field images. B: Time courses of maximum principal strains (ε1 and ε2) generated by adult and neonate cardiac fibroblasts. C: Principal strains averaged over the field of view after 6 h monolayer culture (1 h of particle tracking). D: Histograms of principal strain magnitudes over each field of view. Data are mean ±SD (adult, n = 6; neonate, n = 5).

We examined culture time- and age-dependent changes in the adhesion/contraction apparatus during monolayer substrate deformation by quantifying the expression of mRNAs encoding collagen receptors DDR1, DDR2, and β1 integrin subunit, and the contractile protein α-SMA. Expression of DDR1, DDR2, and β1 were found to be significantly different for adult and neonate cardiac fibroblasts (Fig. 2A–C), with neonate cells exhibiting higher levels of expression (lower ΔCt) for all three receptors. Expression of DDR2 also significantly increased with culture time in both adult and neonate cultures. Nenoate cardiac fibroblasts had significantly higher expression of α-SMA than adult cells (Fig. 2D), and for both ages expression of α-SMA decreased with time in culture as substrate deformation plateaued. These results indicate that while neonates generally have higher expression of these mRNAs than the adult cells, the culture time-dependent shifts in transcription are similar between adult and neonate cardiac fibroblasts.

Figure 2.

Figure 2

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mRNA expression of collagen receptors (A) DDR1, (B) DDR2, (C) β1 integrin, and (D) α-SMA vary with age and time in monolayer culture. Gene expression was quantified by real-time PCR; ΔCt indicates the difference between threshold cycle numbers for the target gene (e.g., DDR1) and the internal standard (ARBP, acidic ribosomal phosphoprotein). Data are mean ±SD from three independent experiments. Inset tables list p values for each term in the general linear model ANOVA. NS = p >0.1. ★ denotes a significant difference (p <0.05) between adult and neonate.

Analysis of expression of DDR1, DDR2, β1 integrin, and α-SMA at the protein level also revealed similarities and differences between adult and neonate cardiac fibroblasts in monolayer cultures. Culture time was found to have significant effects on DDR1, DDR2, and β1 integrin protein levels (Fig. 3A–C), whereas significant age-dependent differences were detected only for DDR2 and β1 integrin. The GLM statistical analysis revealed a significant effect of an interaction (between culture time and age) on β1 integrin, suggesting that regulation of this receptor is distinct between adult and neonate cells. Consistent with the patterns in mRNA expression, α-SMA protein levels decreased with culture time in both adult and neonate cardiac fibroblast cultures and were not significantly different from one another (Fig. 3D), likely contributing to the lack of observed differences in collagen deformation. These data describe additional differences between adult and neonate cells in monolayer culture and suggest that mRNA and protein expression are not tightly coupled in these cells, particularly for the receptor proteins.

Figure 3.

Figure 3

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Protein expression of collagen receptors (A) DDR1, (B) DDR2, and (C)β1 integrin and (D)α-SMA vary with age and time in monolayer culture. Protein expression was quantified by densitometric analysis of western blots and are reported in arbitrary units of integrated pixel density. The density of each protein band was normalized by the density of the GAPDH band on the reprobed membrane. Data are mean ±SD from three independent experiments. Inset tables list p values for each term in the general linear model ANOVA. NS = p > 0.1.

We next investigated age-dependent differences in the deformation of 3D collagen hydrogels by cardiac fibroblasts (Fig. 4A). Culture time and cell age had significant effects on the extent of hydrogel compaction (p < 0.05) over 24 h (Fig. 4B). Neonate cardiac fibroblast populations consistently contracted the collagen hydrogels more than the adult populations (58% versus 35%). In contrast to the results of the monolayer deformation studies, these data clearly indicate that neonatal cells have a greater capacity to deform and/or remodel their ECM than adult cells.

Figure 4.

Figure 4

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Neonate cardiac fibroblasts contracted a 3D collagen scaffold significantly more than adult cardiac fibroblasts. A: Representative images of cell-seeded collagen hydrogels. B: Contraction was quantified by measuring the area (A) over 24 h and normalizing each measurement by the initital area (A0). Data are mean ±SD from four independent experiments. ★ denotes a significant difference (p < 0.05) between adult and neonate.

Analysis of mRNAs encoding collagen receptors and α-SMA in the 3D cultures demonstrated significant differences in expression of DDR2 with culture time, in expression of β1 integrin with culture time and cell age, and in expression of α-SMA with culture time and cell age (Fig. 5). Although the changes in DDR2 expression were not substantial, a pattern of expression in both adult and neonate cells marked by a peak ΔCt at ~5 h and subsequent decay (increase in expression) through 24 h is consistent with a minor role for this receptor in compaction and/or remodeling of the collagen scaffold. As in the monolayer cultures, α-SMA mRNA expression decreased with time, suggesting that as the cell populations approach an equilibrium state of substrate deformation, transcription of α-SMA decreases (independent of the age of the cells). Neonate cells exhibited lower ΔCts (higher expression) than the adult cells for both β1 integrin and α-SMA throughout the culture period, suggesting that these two proteins contributed significantly to the age-dependent difference in hydrogel compaction.

Figure 5.

Figure 5

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mRNA expression of collagen receptors (A) DDR1, (B) DDR2, and (C) β1 integrin, and (D) α-SMA vary with age and time in 3D culture. Gene expression was quantified by real-time PCR; ΔCt indicates the difference between threshold cycle numbers for the target gene (e.g., DDR1) and the internal standard (ARBP, acidic ribosomal phosphoprotein). Data are mean ±SD from three independent experiments. Inset tables list p values for each term in the general linear model ANOVA. NS = p > 0.1. ★ denotes a significant difference (p < 0.05) between adult and neonate.

Expression of collagen receptor and α-SMA proteins generally corroborated the patterns of mRNA expression in the 3D cultures (Fig. 6). We detected significant effects of culture time on the expression of DDR1 and DDR2, independent of the age of the cells. Expression of these proteins fluctuated substantially but essentially in synchrony, with peaks in expression at ~5 and ~12 h, and minima at ~6 and ~24 h. Both culture time and age of the cell were found to be statistically significant factors in the expression of β1 integrin protein, and as in the monolayer cultures the interaction term was also a significant factor. In contrast to the monolayer cultures, however, neonate cardiac fibroblasts generally had higher expression of β1 integrin than the adult populations. Expression of α-SMA protein was also higher in neonate than adult cardiac fibroblasts in the 3D cultures, further implicating these components of the contractile apparatus in the observed age-dependent differences in substrate deformation.

Figure 6.

Figure 6

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Protein expression of collagen receptors (A) DDR1, (B) DDR2, and (C) β1 integrin and (D) α-SMA vary with age and time in 3D culture. Protein expression was quantified by densitometric analysis of western blots and are reported in arbitrary units of integrated pixel density. The density of each protein band was normalized by the density of the GAPDH band on the reprobed membrane. Data are mean ±SD from three independent experiments. Inset tables list p values for each term in the general linear model ANOVA. NS = p > 0.1. ★ denotes a significant difference (p < 0.05) between adult and neonate.

To investigate the basis for age-dependent differences in 3D substrate deformation, we explored the relationships between expression of DDRs, β1 integrin, and α-SMA proteins via regression analyses. For both adult and neonate populations, we observed statistically significant (p < 0.05) power law relationships between DDR1 and DDR2 proteins independent of age and culture condition (Fig. 7A), suggesting a tight coupling in the regulation of these receptors. In contrast, significant regressions were detected between β1 integrin and DDR2 for cells cultured in 3D collagen hydrogels, independent of age, but not for cells cultured in monolayer (Fig. 7B). We detected a significant regression between α-SMA and β1 integrin in neonate 3D cultures, but not in adult 3D cultures or either adult or neonate monolayer cultures (Fig. 7C). Similarly, there was a significant correlation between α-SMA and DDR2 expression in neonate 3D cultures, but not in adult 3D cultures (Fig. 7D); significant regressions were also detected for both adult and neonate monolayer cultures. An identical pattern of correlation was observed for α-SMA and DDR1 expression (not shown). Collectively, these results underscore the distinct behavior of cells cultured in monolayer compared to 3D culture and suggest that the reduced contraction of the 3D collagen substrate by adult cardiac fibroblasts is due in part to a disruption of the correlated β1 integrin/α-SMA and DDR/α-SMA expression patterns observed in neonate cells.

Figure 7.

Figure 7

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Regressions of receptor and α-SMA protein expression reveal age- and culture condition-dependent correlations. Data were ln-transformed and then analyzed by linear regression. Regression curves for monolayer cultures are given by dashed lines, and regressions for 3D cultures are given by solid lines. ★ denotes a statistically significant correlation (p < 0.05).

Discussion

A primary function of cardiac fibroblasts is to maintain the myocardial ECM, and throughout life these cells participate in the deposition, turnover, and organization of structural proteins including type I collagen. Cardiac fibroblasts engage fibrillar collagens through DDR- and integrin-class receptors, and exert stresses on the ECM through intracellular contractile machinery composed of proteins such as α-SMA. The results of this study demonstrate that adult cardiac fibroblasts contract 3D collagen substrates significantly less than neonate populations and implicate a decoupling of DDR/α-SMA expression in the basis for this age-dependent difference.

An age-dependent difference in contraction of collagen substrates by cardiac fibroblasts was detected for 3D substrates but not for thin collagen films in monolayer cultures. This may be explained in part by the differences in cell-ECM interactions reported to exist between 2D and 3D cell cultures (Cukierman et al., 2001); monolayer culture conditions do not appear to support the assembly of in vivo–like cell-ECM adhesions and may thus obscure differences in collagen contraction that are readily observed in 3D cultures. We have previously reported that peak strains within cardiac fibroblast-contracted 3D collagen gels approach −0.40 (i.e., 40% compression) (Baxter et al., 2008), whereas the peak strains in our monolayer cultures plateaued at ±~0.10. Age-related differences in contractility then may only emerge in the context of substrates mechanically permissive for greater deformation. Due to their adhesion to the underlying glass, the collagen films have ~10-fold higher stiffness than the free-floating collagen gels, and this difference in compliance limits cell-induced deformation of the substrate. Substrate compliance, however, has also been shown to influence expression of α-SMA and α2 and β1 integrin subunits in fibroblasts (Arora et al., 1999) and, more generally, the morphology and phenotype of cells (Engler et al., 2004, 2006, 2008). Interestingly, in contrast to our results, Knezevic et al. (2002) reported no differences in the capacity of neonate and adult rat cardiac fibroblasts to contract type I collagen gels. Those experiments were performed with a higher cell density, a lower collagen concentration, and a different gel geometry than those in the current study, and a recent report indicates that contractile cooperativity between fibroblasts is dependent on the cell density and mechanical boundary conditions of 3D collagen gels (Fernandez & Bausch, 2009). Finally, the relatively short timecourse describing the behavior of the monolayer cultures, limited by our experimental setup, may be insufficient to detect age-dependent differences in contraction; our data suggest, however, that an equilibrium contraction of the collagen films was reached by <1 h.

Age-dependent differences in the expression of mRNAs encoding collagen receptors and α-SMA expression were detected in both monolayer and 3D cultures. Over the duration of the monolayer cultures, DDR1, DDR2, β1 integrin, and α-SMA were expressed at higher levels in neonate fibroblasts than in adult-derived cells. Concomitant with collagen film contraction, transcription of mRNAs for α-SMA decreased and for DDR2 increased with time (p < 0.05) in both neonate and adult cultures. Although we did not detect a significant effect of age on DDR1 or DDR2 expression in the 3D cultures, β1 integrin and α-SMA were again expressed higher in neonate compared with adult cells and the temporal patterns of DDR2 and α-SMA expression were similar between monolayer and 3D cultures for both neonate and adult cardiac fibroblasts. Collectively these results indicate that there are intrinsic age-dependent differences in the level of expression of these mRNAs and that the transcriptional regulation of DDR1, DDR2, β1 integrin, and α-SMA was age independent in our experiments. Our observations of age-dependent expression patterns are consistent with those in a recent report demonstrating that cardiac fibroblasts from embryonic hearts had higher expression of mRNAs for ECM proteins and growth factors, such as fibronectin and pleiotrophin, respectively, than fibroblasts from adult hearts (Ieda et al., 2009).

Regression analyses revealed age-dependent correlations between the expression of DDR1, DDR2, β1 integrin, and α-SMA proteins. Most strikingly, the significant correlations between β1 integrin and α-SMA expression, between DDR2 and α-SMA expression, and between DDR1 and α-SMA expression observed in 3D neonate cultures were absent in adult 3D cultures. This decoupling of collagen receptor and α-SMA expression in the adult cells may underlie the age-dependent differences in contraction of 3D collagen substrates if coordinated expression of the collagen receptors with α-SMA is required for contraction. Transcription of α-SMA is regulated in part by integrin-associated focal adhesion kinase signaling (Chan et al., 2009), which suggests that β1 integrin is an upstream regulator of α-SMA in fibroblasts. A regulatory connection between either DDR1 or DDR2 and α-SMA expression has yet to be described; however, the expression of α-SMA was not affected by disruption of DDR2 in skin fibroblasts (Olaso et al., 2002). We have previously found that a DDR2-function blocking antibody significantly inhibited contraction of collagen gels by neonate cardiac fibrobasts by ~15% (M.O. Morales and E.C. Goldsmith, unpubished observations), indicating that DDR2 serves a quantitative role in substrate deformation, although smaller in nature than that previously reported for β1 integrin (Carver et al., 1995). In addition, the DDRs are potent transcriptional regulators of matrix metalloproteinases (MMPs), including MMP2 (Olaso et al., 2001), MMP9 (Hou et al., 2002; Matsuyama et al., 2005), and MMP13 (Xu et al., 2005), which participate in the collagen degradation and cell migration required for contraction of 3D collagen gels. We did not observe significant correlations between DDR expression and collagen gel contraction (not shown), but our results support the hypothesis that coordinated expression of DDRs with α-SMA favors increased contraction of collagen substrates by cardiac fibroblasts.

Summary

This study provides insight into the dynamic nature of cardiac fibroblasts by demonstrating significant effects of age on the contraction of collagen substrates and on the expression of α-SMA and DDR- and integrin-class receptors. Adult cells have a diminished capacity to deform 3D collagen substrates compared to neonatal cells. This difference appears to be due to lower expression levels of β1 integrin and α-SMA, but disruption of the coordinated expression of DDRs with α-SMA may also contribute. These results are significant in the context of understanding mechanisms by which cardiac fibroblasts interact with and remodel the ECM throughout postnatal life.

Acknowledgments

The authors would like to thank Cheryl Cook, Wayne Carver, and Charity Fix for assisting with the isolation of cardiac fibroblasts. These studies were supported by funds from the National Institutes of Health (NIH) grant number HL73937 and Grant-in-Aid number 0855249E from the American Heart Association. C.G.W. was supported by a fellowship administered through NIH grant P20 RR-016461 from the National Center for Research Resources.

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