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Am J Physiol Heart Circ Physiol 293: H1053-H1064, 2007. First published May 4, 2007; doi:10.1152/ajpheart.00935.2006
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Differential and combined effects of cardiotrophin-1 and TGF-beta1 on cardiac myofibroblast proliferation and contraction

Vanja Drobic, Ryan H. Cunnington, Kristen M. Bedosky, Joshua E. Raizman, Vinit V. Elimban, Sunil G. Rattan, and Ian M. C. Dixon

Institute of Cardiovascular Science, St. Boniface General Hospital Research Centre, Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada

Submitted 29 August 2006 ; accepted in final form 30 April 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Myofibroblasts respond to an array of signals from mitogens and cytokines during the course of wound healing following a myocardial infarction (MI), and these signals may coordinate ventricular myofibroblast proliferation. Furthermore, myofibroblasts are contractile and contribute to wound contraction by imparting mechanical tension on surrounding extracellular matrix. Although TGF-beta1, CT-1, and PDGF-BB participate in various stages of post-MI wound healing, their combined net effect(s) on myofibroblast function is unknown. We investigated myofibroblast proliferation, expression of cell cycle proteins, and contractile function of cells treated with TGF-beta1 and/or CT-1. We confirmed that TGF-beta1 (10 ng/ml) suppresses proliferation of these cells, whereas CT-1 (10 ng/ml) and, for comparative purposes, PDGF-BB (1 ng/ml) treatments were associated with proliferation. Specific TGF-beta1 treatment ablated CT-1-induced myofibroblast proliferation. TGF-beta1 effects were specific, as they were suppressed by either TGF-beta-neutralizing antibody or viral Smad7 overexpression. TGF-beta1 treatment also increased expression of p27 and decreased expression of cyclin E and Cdk2 in primary cells. CT-1 (10 ng/ml) treatment of myofibroblasts had no effect on collagen gel deformation versus controls, whereas TGF-beta1 (10 ng/ml) and PDGF (10 ng/ml) treatments were associated with significant cell contraction; again, TGF-beta1-mediated contraction was unaffected by CT-1. Alone, CT-1 and TGF-beta1 treatments exert opposing effects on myofibroblast function, whereas in combination TGF-beta1-mediated effects supersede those of CT-1 (and PDGF-BB). Thus TGF-beta1 and CT-1 exert differential effects on myofibroblast proliferation and contraction in vitro, and we suggest that a balance of these effects may be important for the execution of normal cardiac wound healing.

transforming growth factor-beta1; Smad7

WOUND HEALING POSTMYOCARDIAL INFARCT (post-MI) heart is characterized by time-dependent changes in ventricular architecture manifested as remodeling of both the infarct site and the noninfarcted myocardium (55). Changes in ventricular ultrastructure are characterized by fibroblast and myofibroblast migration and proliferation as well as abnormally elevated collagen secretion. Differentiated myofibroblasts play a central role in cardiac matrix remodeling, as they contribute to abnormal expansion of the interstitium and ultimately in cardiac fibrosis. Fibroblasts migrate from the border zone between infarcted and noninfarcted myocardium and enter the infarcted region where they proliferate, thereby restoring cellularity of the affected zone (33). Within days after MI, cardiac fibroblasts in the infarct scar differentiate into cardiac myofibroblasts (20). Compared with relatively quiescent fibroblasts, cardiac myofibroblasts are hypersecretory for fibrillar collagens, and these collagen subtypes provide tensile strength to the healing wound and stabilize the scar, preventing rupture (9). Proliferation of cardiac myofibroblasts is associated with increased collagen deposition, stiffening of the myocardium, cardiac dysfunction, and heart failure.

The availability of specific cytokines contributes to coordination of cardiac wound healing. Specifically, transforming growth factor-beta1 (TGF-beta1) is a known pleiotropic growth factor that downregulates proliferation of neonatal cardiac myocytes (39) and cardiac fibroblasts (34). TGF-beta1 also acts as a stimulus for the differentiation of cardiac fibroblasts to myofibroblasts (11) as a profibrotic factor for increased collagen production and deposition (21). We and others (52) have previously observed that Smad7 functions as a repressor of canonical TGF-beta1 signaling (i.e., receptor-activated Smad) and that its effects include suppression of fibrillar collagen secretion. Cardiotrophin-1 (CT-1) is a novel cytokine implicated in regulating cell proliferation, cell migration, and production of collagen (17). Furthermore, platelet-derived growth factor-BB (PDGF-BB) is a profibrotic factor in cardiac fibroblasts (5), is a potent chemoattractant (38) for fibroblasts, smooth muscle cells, and endothelial cells, and induces cardiac myofibroblast proliferation (35, 41).

Both TGF-beta1 and CT-1 are expressed during the course of normal wound healing in post-MI heart (17). Although the individual specific effects of these cytokines on fibroblasts have been described, the combined effect of CT-1 and TGF-beta1 on contractile cardiac myofibroblasts is unknown. We have compared the effects of CT-1 and TGF-beta1 (and, for comparative purposes, PDGF-BB) on the induction of cardiac myofibroblast proliferation and collagen gel deformation, with the latter as an indicator of myofibroblast contractility. We hypothesize that TGF-beta1 will oppose the effects of CT-1 and PDGF-BB on myofibroblast proliferation by affecting the expression of the cell cycling proteins crucial to the progression of cell proliferation. We also suggest that CT-1 will have no effect on myofibroblast cell contraction, whereas both TGF-beta1 (suppressed by Smad7) and PDGF-BB (suppressed by AG1290) will induce significant myofibroblast contraction. We found that CT-1 and PDGF-BB are modest and strong stimuli for proliferation of cardiac myofibroblasts, respectively, whereas TGF-beta1 inhibits proliferation of these cells. Moreover, TGF-beta1 downregulates expression of cyclin E and cyclin-dependent kinase 2 (Cdk2) in cardiac myofibroblasts, proteins normally required for cell cycle progression. TGF-beta1 treatment also increased the expression of cell cycle inhibitor p27, which may contribute to inhibition of cellular proliferation. CT-1 treatment did not induce a contractile response in myofibroblasts that differed from baseline, whereas both TGF-beta1 and PDGF-BB induced significant contraction; TGF-beta1-dependent contraction of myofibroblasts was completely inhibited by overdriven expression of Smad7. Taken together, the data indicate that TGF-beta1 may serve as a potent stimulus for matrix reorganization via myofibroblast contraction, whereas CT-1 may be an important early signal for myofibroblast chemotaxis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Culture media [Dulbecco's modified Eagle's medium (DMEM-F-12) and minimum essential medium], fetal bovine serum (FBS), and antibiotics (penicillin, streptomycin) were purchased from GIBCO-BRL (Grand Island, NY). Bovine collagen solution was purchased from Stem Cell Technologies (Vancouver, BC, Canada). Primary antibodies for {alpha}-smooth muscle actin ({alpha}-SMA) and smooth muscle myosin (SMemb) were purchased from Sigma-Aldrich Canada (Oakville, ON, Canada) and Abcam (Cambridge, MA), respectively. Primary antibodies for cyclin E and Cdk2 were obtained from Biosource (Camarillo, CA). Primary antibody specific to p27 was ordered from Cell Signaling Technology (Beverly, MA). Alexa Fluor-conjugated secondary antibodies were purchased from Molecular Probes (Eugene, OR). Prestained low-molecular-weight marker secondary antibodies (anti-mouse, anti-rabbit) were obtained from Bio-Rad Laboratories (Hercules, CA). Polyvinylidene difluoride (PVDF) blotting membranes were obtained from Roche Diagnostics (Laval, QC, Canada). The enhanced chemiluminescence (ECL Plus) and the protein assay kit were purchased from Sigma-Aldrich. Recombinant human CT-1, TGF-beta1, TGF-beta-neutralizing antibody (AF-101-NA), and PDGF-BB were purchased from R&D Systems (Minneapolis, MN). AG1296, a PDGF-BB inhibitor, was obtained from Calbiochem (La Jolla, CA). A primary mouse monoclonal antibody specific for an amino acid motif on the carboxy terminus of human actin (C-2) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and used to correct for lane loading in all Western blot analyses (catalog no. sc-8432).

Isolation and culture of rat cardiac myofibroblasts. Male adult Sprague-Dawley rats in the weight range of 150–175 g were killed for preparation of cardiac fibroblasts according to the methods of Brilla et al. (4), with minor modifications. Rat hearts were subjected to Langendorff perfusion for 20 min at a flow rate of 5 ml/min at 37°C with recirculatory Joklik's medium containing 0.1% collagenase and 2% bovine serum albumin (BSA). Liberated cells were collected by centrifugation at 2,000 rpm for 5 min and resuspended in DMEM-F-12. Cells were seeded on 100-mm noncoated culture flasks at 37°C with 5% CO2 and grown in DMEM-F-12 supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1 µl/ml ascorbate. Cells that attached to the bottom of the flasks during 3 h of incubation were further maintained in serum-supplemented medium. Nonadherent cells, including myocytes, were removed by the medium change after 3 h of incubation. Cells used for this study were of passage one (P1).

Protein extraction and assay. Prior to stimulation, cells were starved for 24 h in 0% FBS-DMEM-F-12. Cells were stimulated for 24 h with CT-1, TGF-beta1, and PDGF, and stimulation was stopped by rinsing the cells twice with ice-cold phosphate-buffered saline (PBS). Lysis of the cells was accomplished by addition of 120 µl of RIPA lysing buffer (pH = 7.6) containing 150 mM NaCl, 1.0% nonidet P-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris, phosphatase inhibitors (10 mM NaF, 1 mM Na3VO4, and 1 mM EGTA), and protease inhibitors (4 µM leupeptin, 1 µM pepstatin A, and 0.3 µM aprotinin). Collected cells were allowed to lyse in RIPA buffer on ice for 1 h. Subsequently, cells were sonicated for 5 s. Insoluble portion (membrane fraction) was removed by centrifugation at 14,000 rpm for 15 min at 4°C. Supernatant was collected and stored at –20°C. The total protein concentration of all samples was measured using the bicinchoninic acid method, as previously described (44).

Western blot analysis of target proteins. The samples of cell lysates were mixed with Laemmli loading buffer [final concentration; 125 mM Tris·HCl (pH = 6.8), 5% glycerol, 2.5% SDS, 5% 2-mercaptoethanol, and 0.125% bromophenol blue] and boiled for 5 min. Equal amounts of protein samples (15 µg) were resolved by 10% SDS-polyacrylamide gel electrophoresis with the help of prestained low-molecular-weight marker (10 µl). Separated proteins were electrophoretically transferred onto 0.45-µM PVDF membranes. PVDFs were blocked overnight at 4°C in Tris-buffered saline with 0.2% Tween 20 (TBS-T) containing 3% BSA and probed with primary antibodies for 1 h at room temperature. Primary antibodies were diluted 1:500 in 3% BSA and incubated overnight at 4°C. The incubation period of secondary antibodies was 1 h at room temperature with the dilution 1:2,000 in 0.2% TBS-T containing 1% BSA. Secondary antibodies included horseradish peroxidase (HRP) anti-mouse for recognition of cyclin E and Cdk2 and HRP-labeled anti-rabbit for detection of p27. Protein bands on Western blots were visualized by ECL Plus according to the manufacturer's instructions and developed on X-ray film (Kodak). Equal protein loading was confirmed by immunoblotting analysis against actin.

Immunocytochemistry. Immunofluorescent staining with {alpha}-SMA and SMemb was performed to detect endogenous expression of these proteins in P1 cardiac myofibroblasts as well as expression in these cells treated in the presence of PDGF-BB (50 ng/ml) and TGF-beta1 (10 ng/ml). Cells were seeded onto glass coverslips in six-well dishes and allowed to grow for 24 h in DMEM-F-12 supplemented with 10% FBS until they were ~60% confluent. Cell growth was arrested by the addition of serum-free DMEM-F-12 for 24 h. After 24 h of serum starvation, cells were stimulated with either PDGF-BB (50 ng/ml) or TGF-beta1 (10 ng/ml) for 24 h.

Immunofluorescent staining was performed as previously described (23). Briefly, cells were washed three times with cold PBS, fixed in 4% paraformaldehyde for 15 min, rendered permeable with 0.1% Triton X-100 in PBS for 15 min, and then incubated with anti-mouse {alpha}-SMA or anti-mouse SMemb for 90 min. After cells were washed three times with PBS they were incubated for 1.5 h in the dark with anti-mouse- or anti-rabbit-conjugated Alexa Fluor secondary antibodies. All antibodies were diluted in 1x PBS containing 1% BSA, with ratios of 1:200 for {alpha}-SMA and SMemb and 1:700 for Alexa Fluor. Following another washing cycle with PBS, nuclei were stained with Hoechst 33342 (10 ng/ml) for 30 s. A further round of three washes with PBS followed. After coverslips were thoroughly dried, they were mounted to slides using Crystal Mount mounting medium and examined under a microscope (Nikon) equipped with epifluorescence optics. Digital pictures were photographed at x400 magnification using appropriate filters.

Tritiated thymidine incorporation to myofibroblasts. Approximately 2.5 x 104 cells/ml (counted with a hemacytometer) were resuspended in DMEM-F-12 containing 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1 µl/ml ascorbate. Cells attached to the bottom of each well of a 24-well culture plate. Cells were allowed to adhere for 24 h and then rendered quiescent by incubation in DMEM-F-12. Subsequently, cells were stimulated for 24 h by addition of TGF-beta1 (10 ng/ml), CT-1 (10 ng/ml), and PDGF-BB (1 ng/ml) in DMEM-F-12 containing 1% FBS. For the last 4 h of stimulation, cells were pulse labeled with tritiated thymidine ([3H]thymidine). Cold 20% trichloroacetic acid was used to precipitate DNA from cell lysates, which were filtered through GF/A filters (Fisher). Three milliliters of scintillation fluid (ICN Pharmaceuticals, Costa Mesa, CA) was added to each vial, where a dried filter was placed. beta-Emission was measured with a scintillation counter (LS6500; Beckman Coulter, Fullerton, CA).

Collagen gel deformation assay for myofibroblast contractility. Collagen type I gel matrices were prepared according to Takayama and Mizumachi (46). Briefly, collagen type I gel was prepared by gently mixing 7 ml of cold collagen I solution (3 mg/ml) and 2 ml of fivefold-concentrated cold DMEM-F-12. The pH of the solution was adjusted with 1 M NaOH to 7.4. The final volume was adjusted to 10 ml. Six-hundred-microliter aliquots were added to each well of the 24-well culture plate (Falcon) and set at 37°C overnight to solidify. Once the collagen gel solidified, it was measured that 600-µl gel aliquots produced 3-mm thick gels. Suspension of cardiac myofibroblasts (10 x 104 cells/ml DMEM-F-12 containing 10% FBS) was plated onto each well containing solidified collagen type I gel. Cells were allowed to adhere and grow at 37°C for 24 h, and we confirmed that cells grew on the surface of the matrices and did not infiltrate them beyond the matrix surface. Cells were rendered quiescent by serum starving for 24 h at 37°C. Prior to the cell stimulation, collagen type I gel was detached from each well with the use of a surgical blade (Fisher Scientific). Cells were then stimulated by addition of various cytokines to the medium. Wells were photographed at 0, 2, 4, 6, and 24 h after initiation of contraction. Gel area was determined for each well and each treatment using Measure Gel custom-made software. Area was plotted as means ± SE. One-way ANOVA was used to test groups for significance.

Viral transduction of P1 myofibroblasts with Smad7 adenovirus. Replication-deficient human adenovirus vector expression mouse Smad7 under the control of cytomegalovirus promoter was kindly provided by Dr. Anita Roberts (National Institutes of Health, Bethesda, MD). Briefly, P1 myofibroblasts were seeded onto preformed collagen type I gels in a 24-well culture dish in the presence of 10% FBS-DMEM-F-12 and were allowed to reach cell confluence of 60–70%. Once desired confluence was reached, medium was changed to low serum (2% FBS-DMEM-F-12), and P1 myofibroblasts were transduced with either Smad7 or LacZ adenovirus (at 100 multiplicities of infection). After a 24-h transduction period, cells were washed twice with 1x PBS and then starved for 24 h by changing medium to 0% FBS-DMEM-F-12. Cell starving was followed by stimulation with TGF-beta1 at 10 ng/ml.

Statistical analysis. All values are expressed as means ± SE. Unless specifically stated in the figure legends, Student's t-test was used to compare means between two groups and one-way ANOVA was used to compare differences among multiple groups. Significant differences among groups were defined as *P ≤ 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Phenotype of passaged fibroblastic cells. We characterized the P1 cells harvested from adult rat left ventricles, using standard markers to confirm that these cells are myofibroblasts. In previous studies from our laboratory (16, 17), we have shown that CT-1 treatment (10 ng/ml) was not associated with any alteration of the myofibroblast phenotype after passage and exposure to 10% serum. Figure 1 shows typical expression of {alpha}-SMA and embryonic SMemb in untreated cultured P1 cells as well as those exposed to PDGF-BB and TGF-beta1. Although the latter treatment was associated with an increase in expression in the intensity of myofibroblast markers, the different groups of cells show a retention of the myofibroblast phenotype.


Figure 1
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  		Fig. 1. {alpha}-Smooth muscle actin ({alpha}-SMA) and smooth muscle myosin (SMemb) expression in primary passaged fibroblastic cardiac cells: the effect of treatment of transforming growth factor-beta1 (TGF-beta1) and platelet-derived growth factor-BB (PDGF-BB). {alpha}-SMA and SMemb are specific phenotypic markers of hypersecretory and contractile myofibroblasts. This figure shows endogenous expression of these proteins in fibroblastic cells after the first passage and exposure to 10% FCS followed by 24-h serum starvation compared with expression after 24-h exposure to PDGF-BB (50 ng/ml) and TGF-beta1 (10 ng/ml). Cells retained the myofibroblastic phenotype across all groups compared.

 
Effects of CT-1 and TGF-beta1, alone and in combination, on P1 ventricular myofibroblast DNA synthesis. To assess cell proliferation in P1 cardiac myofibroblasts, we have measured the incorporation of [3H]thymidine into DNA. As shown in our results, CT-1 (Fig. 2A) and PDGF-BB (Fig. 2B) stimulated DNA synthesis compared with the control group (myofibroblasts cultured in the presence of 1% FBS). In contrast, TGF-beta1 inhibited DNA synthesis compared with the control group (Fig. 2B). The stimulatory effects of CT-1 and PDGF-BB were ablated and reduced significantly, respectively, by concomitant addition of TGF-beta1 (Fig. 2, A and B). Simultaneous addition of CT-1 and PDGF-BB stimulated DNA synthesis to a higher extent than either factor added alone (Fig. 2C), indicating a possible synergistic effect.


Figure 2
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  		Fig. 2. Incorporation of tritiated thymidine ([3H]thymidine) into DNA by cardiac myofibroblasts. Cells were grown for 24 h in 10% FBS DMEM and rendered quiescent by 24-h incubation in 0% FBS DMEM. A: cytokines [cardiotrophin-1 (CT-1), 10 ng/ml; TGF-beta1, 10 ng/ml] were added in the presence of 1% FBS-DMEM-F-12 for 24 h. B: cytokines (TGF-beta1, 10 ng/ml; PDGF-BB, 1 ng/ml) were added in the presence of 1% FBS-DMEM-F-12. C: CT-1 (10 ng/ml) and PDGF-BB (1 ng/ml) were added in the presence of 1% FBS-DMEM-F-12. Data are expressed as means ± SE, where n = 5 for each group. Student's t-test was used to check for statistical significance. *P ≤ 0.05 vs. control (1% FBS); #P ≤ 0.05 vs. CT-1; {dagger}P ≤ 0.05 vs. PDGF-BB.

 
Individual and combined effects of CT-1 and TGF-beta1 on the expression of cyclin E in myofibroblasts. Expression of cyclin E varies throughout the cell cycle, and the cyclin E/Cdk2 complex is necessary for the progression of the cell cycle into S phase, and therefore, increased expression of cyclin E is a marker of G1 to S phase transition (47). CT-1 and/or TGF-beta1 or PDGF-BB were added in the presence of 1% FBS-DMEM-F-12; low levels of serum stimulation provide a nominal baseline stimulus for myofibroblast proliferation, against which the specific effects of either CT-1 or TGF-beta1 were then measured according to previously published techniques (10). Western analysis (Fig. 3) revealed that TGF-beta1 induced a decrease in cyclin E protein expression versus 1% FBS control values. Conversely, treatment of the cells with CT-1 (and PDGF-BB) alone led to a slight increase (statistically insignificant when the densitometric ratios of cyclin E/actin band intensity were compared) in the expression of cyclin E compared with controls. In contrast, combination treatment of CT-1 and TGF-beta1 was associated with a significant dropout of cyclin E expression in treated myofibroblasts, and the same loss of expression was observed in PDGF-BB- plus TGF-beta1-treated cells. Serum-starved myofibroblasts served as the negative control in these experiments. These data indicate that TGF-beta1 effects supersede those of CT-1.


Figure 3
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  		Fig. 3. Cyclin E expression in primary cardiac myofibroblasts. A: Western blot analysis of cyclin E expression in cultured primary cardiac myofibroblasts (0% FBS starvation for 48 h, total cell lysates) treated with CT-1 (10 ng/ml), TGF-beta1 (10 ng/ml), or PDGF-BB (1 ng/ml) for 24 h in the presence of 1% FBS. Representative Western blots show expression of cyclin E (~49 kDa) and actin (~43 kDa) protein, indicating relatively even protein loading. B: histographic representation of quantified data of immunoreactive cyclin E from the groups of samples shown in A (quantified by densitometric scanning). *P ≤ 0.05 vs. control (1% FBS); data are expressed as means ± SE (n = 3).

 
Individual and combined effects of CT-1 and TGF-beta1 on the expression of Cdk2 in myofibroblasts. Increased expression of Cdk2 is observed during the cell's transition from G1 to S phase. Cdk2 is a component of cyclin E/Cdk2 complex needed for cell proliferation to occur (47). Western blot analyses of the Cdk2 protein expression (Fig. 4) revealed that CT-1 or PDGF-BB-treated myofibroblasts tended to exhibit increased expression of Cdk2 compared with the 1% FBS-treated control cells. Treatment of myofibroblasts with TGF-beta1 (in the presence of 1% FBS) caused a significant decrease in Cdk2 expression that was comparable to the levels in quiescent cells. Coincubation of TGF-beta1 in combination with CT-1 (or PDGF-BB) was associated with significant reductions of Cdk2 expression versus controls. Thus TGF-beta1 treatment dominated the apparent stimulatory effects of CT-1 expression on Cdk2.


Figure 4
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  		Fig. 4. Expression of cyclin-dependent kinase 2 (Cdk2) in primary cardiac myofibroblasts. A: Western blot analysis of Cdk2 expression in cultured primary cardiac myofibroblasts (0% FBS starvation for 48 h, total cell lysates) treated with CT-1 (10 ng/ml), TGF-beta1 (10 ng/ml), or PDGF-BB (1 ng/ml) for 24 h in the presence of 1% FBS. Representative Western blots show expression of Cdk2 (~33 kDa) and actin (~43 kDa) protein, indicating relatively even protein loading. B: histographic representation of quantified data of immunoreactive Cdk2 from the groups of samples shown in A (quantified by densitometric scanning). *P ≤ 0.05 vs. control (1% FBS); data are expressed as means ± SE (n = 3).

 
Individual and combined effects of CT-1 and TGF-beta1 on the expression of p27 in myofibroblasts. Accumulation of p27 prevents progression of the cell cycle into the S stage, thereby inhibiting cell proliferation. The function of p27 in the inhibition of the cell cycle is to inhibit cyclin E/Cdk2 complex formation. Thus relatively high cellular expression of p27 is a marker of nonproliferating cells (58). Western blot analysis of the protein levels of p27 (Fig. 5) revealed that TGF-beta1 induced a significant increase in the expression of p27 compared with control values (1% FBS-treated cells). Treatment with CT-1 (or PDGF-BB) alone did not alter the expression of p27 versus controls, whereas coincubation of TGF-beta1 with CT-1 (or PDGF-BB) was associated with significantly increased expression of p27, similar to that of TGF-beta1 induction alone. Thus TGF-beta1 treatment of myofibroblasts was the only cytokine that affected p27 expression either alone or in the presence of CT-1.


Figure 5
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  		Fig. 5. p27 Expression in primary cardiac myofibroblasts. A: Western blot analysis of p27 expression in cultured primary cardiac myofibroblasts (0% FBS starvation for 48 h, total cell lysates) treated with CT-1 (10 ng/ml), TGF-beta1 (10 ng/ml), or PDGF-BB (1 ng/ml) for 24 h in the presence of 1% FBS. Representative Western blots show expression of p27 (~27 kDa) and actin (~43 kDa) protein, indicating relatively even protein loading. B: histographic representation of quantified data of immunoreactive p27 from the groups of samples shown in A (quantified by densitometric scanning). *P ≤ 0.05 vs. control (1% FBS); {dagger}P ≤ 0.05 vs. CT-1; #P ≤ 0.05 vs. PDGF-BB; data are expressed as means ± SE (n = 4).

 
Individual and combined effects of CT-1 and TGF-beta1 on collagen type I gel deformation in myofibroblasts. Deformation, i.e., contraction of collagen type I gel matrices, is an in vitro model of wound contraction previously used as a model for assessment of cardiac fibroblast contractility (7). We examined the influence of CT-1 and TGF-beta1 singularly or in combination on the contraction of cardiac myofibroblasts. Upon analysis of the gel surface area, we determined that CT-1 (Fig. 6) had no effect on the deformation of the collagen type I gel compared with the matrix contraction imparted by untreated cells cultured on the surface of the matrix (control group). Conversely, TGF-beta1 treatment was associated with a potent stimulatory effect on gel matrix contraction versus control (Fig. 7). Notably, significant gel contraction was found as early as 4 h after TGF-beta1 treatment. To confirm that the effect of gel contraction is TGF-beta1 receptor dependent, a TGF-beta1-neutralizing antibody (TGF-beta1 NA; R&D Systems) was used to inhibit TGF-beta1 receptor's function. A 24-h treatment with TGF-beta1 NA (1 µg/ml) resulted in significant ablation of TGF-beta1-induced gel contraction (Fig. 8). Because we have previously shown that TGF-beta1 may signal via canonical receptor-activated Smads in cardiac fibroblasts (23, 25, 54), we sought to investigate a possible mechanism for TGF-beta1-mediated gel contraction and thus tested the effect of overdriven Smad7, a known inhibitor of the TbetaR1-mediated phosphorylation of R-Smads, on TGF-beta1-associated myofibroblast contractility. In myofibroblasts that were subjected to overdriven, i.e., exogenous virally driven, Smad7 expression and then treated with TGF-beta1, we observed a significant inhibition of TGF-beta1-mediated gel contraction (Fig. 9). In parallel experiments, we found that PDGF-BB induced gel contraction (Fig. 10) within 2 h after stimulation and that AG1296 (PDGF-BB-linked tyrosine kinase inhibitor, 0.005 ng/ml) significantly inhibited PDGF-BB-induced gel contraction (Fig. 8).


Figure 6
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  		Fig. 6. Collagen type I gel contraction assay: myofibroblast contraction. Myofibroblasts were seeded onto the surface of polymerized gels and allowed to grow to confluence in 10% FBS-DMEM-F-12. Cells were rendered quiescent in 0% FBS-DMEM-F-12 for 24 h and treated with CT-1 (10 ng/ml). Gels were then physically released from the plastic plate edges, and surface gel contraction is noted by the apparent diameter of the gel surface. A: representative photographs show control and CT-1-treated wells at 0 and 24 h after stimulation and the intermediate density lipoprotein (IDL)-based computer estimate of gel surface area. B: graphical representation of quantified data of control and CT-1 treatment ranging from time 0, 2, 4, 6, and 24 h after treatment with CT-1. Gel contraction was quantified using Measure Gel computer software. Surface area was measured and plotted as means ± SE, where n = 5. One-way ANOVA followed by Student-Newman-Keuls (SNK) post hoc analysis was used to test for statistical significance.

 

Figure 7
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  		Fig. 7. Collagen type I gel contraction assay: myofibroblast contraction. Passage one (P1) cardiac myofibroblasts were seeded onto the surface of polymerized gels and allowed to grow to confluency in 10% FBS-DMEM-F-12. Cells were rendered quiescent in 0% FBS DMEM-F-12 for 24 h and treated with TGF-beta1 (10 ng/ml). Gels were then physically released from the plastic plate edges, and surface gel contraction is noted by the apparent diameter of the gel surface. A: representative photographs show control and TGF-beta1-treated wells at 0 and 24 h after stimulation and the IDL-based computer estimate of gel surface area. B: graphical representation of quantified data of control and TGF-beta1 treatment ranging from time 0, 2, 4, 6, and 24 h after treatment with TGF-beta1. Gel contraction was quantified using Measure Gel computer software. Surface area was measured and plotted as means ± SE, where n = 5. One-way ANOVA followed by SNK post hoc analysis was used to test for statistical significance.

 

Figure 8
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  		Fig. 8. Histographic representation of myofibroblast contraction 24 h posttreatment with TGF-beta1: collagen type I gel contraction assay. P1 cardiac myofibroblasts were seeded on top of the preformed collagen type I gel and grown to confluency in 10% FBS-DMEM-F-12. Cells were rendered quiescent in 0% FBS DMEM-F-12 for 24 h and stimulated with TGF-beta1 (10 ng/ml), PDGF-BB (10 ng/ml), NA-TGF-beta1 (1 µg/ml), and AG1296 (0.005 ng/ml). Gels were then physically released from the plastic plate edges, and surface gel contraction was noted by the apparent diameter of the gel surface. Photographs were taken at 0 and 24 h following the treatments.

 

Figure 9
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  		Fig. 9. Histographic representation of myofibroblast contraction in Smad7 overexpressing cells 24 h after TGF-beta1 treatment: collagen type I gel contraction assay. P1 cardiac myofibroblasts were seeded on top of the polymerized collagen type I gel and grown to confluency in 10% FBS-DMEM-F-12. One group was infected with Smad7 adenovirus for 24 h at 100 multiplicities of infection in 2% FBS-DMEM-F-12. Cells were starved in 0% FBS-DMEM-F-12 for 24 h and treated with TGF-beta1 (10 ng/ml). Gels were then physically released from the plastic plate edges, and surface gel contraction was noted by the apparent diameter of the gel surface. Photographs were taken at 0 and 24 h following treatment with TGF-beta1, and gel surface was then measured with Measure Gel computer software and plotted as means ± SE, n = 5. Student's t-test was used to determine statistical significance. *P ≤ 0.05 vs. control (untreated cells); {dagger}P ≤ 0.05 vs. TGF-beta1.

 

Figure 10
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  		Fig. 10. Collagen type I gel contraction assay: myofibroblast contraction. P1 cardiac myofibroblasts were seeded onto the surface of polymerized gels and allowed to grow to confluency in 10% FBS-DMEM-F-12. Cells were rendered quiescent in 0% FBS-DMEM-F-12 for 24 h and treated with PDGF-BB (10 ng/ml). Gels were then physically released from the plastic plate edges, and surface gel contraction is noted by the apparent diameter of the gel surface. A: representative photographs show control and PDGF-BB-treated wells at 0 and 24 h after stimulation and the IDL-based computer estimate of gel surface area. B: graphical representation of quantified data of control and PDGF-BB treatment ranging from time 0, 2, 4, 6, and 24 h after treatment with PDGF-BB. Gel contraction was quantified using Measure Gel computer software. Surface area was measured and plotted as means ± SE, where n = 5. One-way ANOVA followed by SNK post hoc analysis was used to test for statistical significance.

 
When CT-1 was coincubated together with TGF-beta1, the contractile potency, observed in TGF-beta1 contraction of the gel, was not different from that of TGF-beta1 alone (Fig. 11, A and B); a similar result was observed when CT-1 and PDGF-BB were coincubated (Fig. 11, E and F). We suggest that TGF-beta1-treated cells are stimulated for near-maximal contraction with or without PDGF treatment. Thus CT-1 did not exert any effect on the ability of PDGF-BB to induce gel contraction. When TGF-beta1 and PDGF-BB treatments were combined, gel contraction was comparable, as seen in PDGF-BB-induced gel contraction alone (Fig. 11, C and D). Finally, in a separate TGF-beta1 concentration response study we found a progressive increase in gel contraction from 0 to 100 ng/ml, with a near-maximal contractile response at 10 ng/ml (Fig. 12).


Figure 11
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  		Fig. 11. Collagen type I gel contraction assay of myofibroblasts: the effect of combination treatment regimens of CT-1/TGF-beta1, PDGF-BB/TGF-beta1, and CT-1/PDGF-BB. P1 cardiac myofibroblasts were seeded onto the surface of polymerized gels and allowed to grow to confluency in 10% FBS-DMEM-F-12. Cells were rendered quiescent (serum starved) in 0% FBS-DMEM-F-12 for 24 h and then were treated with various cytokines. Gels were then physically released from the plastic plate edges, and surface gel contraction was noted by the apparent diameter of the gel surface. Surface area was measured and plotted as means ± SE; in all experiments, n = 5. One-way ANOVA followed by SNK post hoc analysis was used to test for statistical significance. A: representative photographs show control and combined CT-1- (10 ng/ml) plus TGF-beta1-treated (10 ng/ml) wells at 0 and 24 h after stimulation and the IDL-based computer estimate of gel surface area. B: graphical representation of quantified data of control and CT-1 plus TGF-beta1 treatment ranging from time 0, 2, 4, 6, and 24 h after treatment with CT-1 plus TGF-beta1. C: representative photographs show control and combined TGF-beta1- (10 ng/ml) plus PDGF-BB-treated (10 ng/ml) wells at 0 and 24 h after stimulation and the IDL-based computer estimate of gel surface area. D: graphical representation of quantified data of control and TGF-beta1 plus PDGF-BB treatment ranging from time 0, 2, 4, 6, and 24 h after treatment with TGF-beta1 plus PDGF-BB. E: representative photographs show control and combined CT-1 (10 ng/ml) plus PDGF-BB-treated (10 ng/ml) wells at 0 and 24 h after stimulation and the IDL-based computer estimate of gel surface area. F: graphical representation of quantified data of control and CT-1 plus PDGF-BB treatment ranging from time 0, 2, 4, 6, and 24 h after treatment with CT-1 plus PDGF-BB.

 

Figure 12
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  		Fig. 12. Collagen type I gel contraction assay of myofibroblasts: the effect of TGF-beta1 concentration-dependent treatment on P1 cardiac myofibroblast-mediated contraction. Cardiac myofibroblasts were seeded onto the surface of polymerized gels and allowed to grow to confluency in 10% FBS-DMEM-F-12. Cells were serum starved in 0% FBS-DMEM-F-12 for 24 h and then were treated with TGF-beta1. Gels were then physically released from the plastic plate edges, and surface gel contraction was noted by the apparent diameter of the gel surface. Surface area was measured and plotted as means ± SE; in all experiments, n = 3–15. One-way ANOVA followed by SNK post hoc analysis was used to test for statistical significance.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Improved treatment of acute MI has led to an increase in both post-MI survivorship and the incidence of congestive heart failure associated with MI (2). This syndrome remains a major cause of death in the general population (26). Post-MI induction of necrosis and inflammation is linked to the de novo synthesis and release of a host of cytokines that impact on cardiac fibrosis and dysfunction (13). Because post-MI wound healing and subsequent global cardiac fibrosis are known disease modifiers in the pathogenesis of post-MI heart failure, the characterization of the combined effect(s) of key cytokines is a warranted and novel approach. In particular, TGF-beta1 signaling is enhanced in post-MI heart with attendant elevation of expression of R-Smads (23, 24). Despite the importance of TGF-beta1 as a key profibrotic stimulus, recent work (17) has indicated that other specific cytokines may exert either synergistic or antagonistic (e.g., CT-1) effects to facilitate post-MI wound healing, and it has been suggested (37) that the timing of altered expression of a given cytokine is important in cardiac wound healing. Because elevated TGF-beta1 and CT-1 expression occurs rapidly following infarction and is maintained in vivo, the chronic masking of specific effects is likely. Thus functional opposition (or redundant support of function) among cytokines may confound the success of therapeutic attempts to inhibit the effects of any given factor in vivo. For example, attempts to target the inflammatory response to reduce infarct size were largely unsuccessful in clinical trials despite initial experimental results that supported such an attempt (15).

We and others (1, 17, 51) have shown that cardiac myofibroblasts are maintained in the infarct scar for extended periods after the initial infarct, and we have carried out a number of studies (17, 24, 52) that indicate elevated levels of TGF-beta1 and CT-1 in the infarct scar of an experimental model of post-MI heart failure. The main effect of PDGF-BB in cardiac tissue is stimulation of cell proliferation and migration (42). Because PDGF is, in our hands, is a potent positive stimulus for myofibroblast proliferation and a strong profibrotic stimulus (6), we felt it would serve as a positive control by which to compare CT-1. CT-1 is unique insofar as we found that it possesses a relatively weak proliferative effect in combination with an antifibrotic effect (17). As myofibroblast proliferation plays a central role in the development of myocardial fibrosis, including that of the infarct scar in post-MI heart (45), the study of PDGF isoforms is of considerable interest.

TGF-beta1 is a major contributor to initiation and development of cardiac fibrosis (28, 30, 34) and cardiac myofibroblast differentiation (11). On the other hand, CT-1 has been identified as a cytoprotective factor (3) and an antifibrotic factor (17), and, unlike other IL-6 superfamily members, its expression is rapidly elevated (and remains elevated) in the infarct scar weeks after MI (17). CT-1 effects on fibroblastic cells have only recently been addressed; it is known to induce migration as well as increase DNA and protein synthesis in cardiac myofibroblasts in vitro (18). PDGF-BB is characterized as a proliferative factor stimulating vascular smooth muscle cell growth (36), an important mediator of cardiac fibroblast/myofibroblast phenotypic switching (35) and stimulator of ECM reorganization and wound contraction (48).

The main findings of the present study address the combined effects of CT-1 and TGF-beta1 (and for comparative purposes, PDGF-BB) on 1) proliferation of primary myofibroblasts in serum-free culture conditions and possible mechanisms for these changes, specifically altered expression of cyclin E, Cdk2, and p27, and 2) contraction of cardiac myofibroblasts plated on the surface of prepared collagen matrices. Finally, we document 3) a novel post-TbetaR1 receptor Smad-based mechanism for the activation of myofibroblast contraction by TGF-beta1. Because the proliferative effects of 10 ng/ml CT-1 (and 1 ng/ml PDGF-BB) were completely ablated by coincubation with TGF-beta1 in P1 myofibroblasts, we confirm that TGF-beta1 has a negative proliferative effect on cardiac cells (34, 39). However, our work is the first to describe the effect of TGF-beta1 on the proliferation of fully differentiated adult rat primary cardiac myofibroblasts.

We addressed the individual and combined effects of CT-1 and TGF-beta1 on myofibroblast contraction and confirmed that TGF-beta1 (12, 19, 32) and PDGF (8, 32, 32) are potent stimuli for contraction. Cardiac myofibroblasts repopulate the infarct zone following necrosis of myocytes, express {alpha}-SMA and embryonic SMemb, and contribute to wound contraction (14, 49). That CT-1 treatment does not alter basal cellular contraction supports the suggestion that this cytokine may function in opposition to profibrotic TGF-beta1 and PDGF-BB signals in cardiac myofibroblasts. This possibility seems especially likely if one also considers its other effects, such as the reduction of collagen secretion (17), as well as its function as a mild proliferative and migratory stimulus for cardiac myofibroblasts. Freed et al. (16) have shown that normalized collagen secretion by cardiac myofibroblasts is significantly decreased in the presence of CT-1 versus unstimulated control cultures. Thus, despite its proliferative effect on these cells, net collagen secretion per cell is diminished in the presence of CT-1, and thus CT-1's effect may be to oppose the profibrotic signals of TGF-beta1 and PDGF-BB. Furthermore, both leukemia inhibitory factor (LIF) and CT-1 activate the LIF/gp 130 receptor dimer complex (57). Although the most notable function of LIF is the prevention of stem cell differentiation (43), recent evidence suggests that LIF inhibits phenotypic switching of cardiac fibroblasts to myofibroblasts. Furthermore, LIF is associated with decreased matrix metalloproteinase function, which highlights its putative role in modulation of excessive ECM remodeling (53).

The floating, anchored, or stress relaxed collagen matrix models (22) are used to examine ECM reorganization and myofibroblast contractility. We employed a modified anchored assay wherein the gel edge is released the gel from the plate at time zero for treatment. Myofibroblasts (as seen in Fig. 1) are distributed on the surface of the matrix, and the reduction of diameter of the matrices' surface is measured. Thus collagen gel deformation reflects tractional remodeling of the matrices and is an in vitro model of wound contraction.

We demonstrate, for the first time, that overdriven Smad7 (an inhibitory Smad or I-Smad) expression is associated with significant attenuation of TGF-beta1-mediated myofibroblast contraction. This finding may be distinguished from other findings indicating Smad7 inhibition of type I collagen lattice shrinkage in dermal fibroblasts (27) on the basis of differences in function. Unlike dermal wound healing, cardiac myofibroblasts remain in the infarct scar for months and years after the initial wound healing phase is completed (9). Cleutjens et al. suggest that continuous mechanical stimulation imparted by the beating heart on the infarct scar, e.g., mechanical stress and strain, may be linked to the chronic presence of cardiac myofibroblasts. Thus cardiac matrix turnover in the infarct scar may be different from healed dermal wounds. A comparison of R-Smad2 expression (24, 27) reveals that skin fibroblasts express relatively low levels, whereas cardiac myofibroblasts express high levels of this protein. Thus similar cells from different organ systems may carry out different functions that may require differential expression of R-Smads. Nevertheless, our result of Smad7-mediated ablation of gel contraction was directly comparable to the recent finding of Kopp et al. (27). In this respect, Smad7 is known to competitively inhibit TbetaRI-mediated phosphorylation of R-Smads or directly downregulate R-Smad target gene transcription, respectively (31, 56). As R-Smad activation is linked to activation of collagen genes (50), canonical Smad signaling is closely tied to collagen expression. The precise mode of Smad7-mediated inhibition of myofibroblast contraction is unknown but may operate via inhibition of R-Smad phosphorylation, the direct degradation of TGF-beta1 receptors, or both (40).

One of the main goals of this study was to assess the effect of coincubation of CT-1 and TGF-beta1 on myofibroblast proliferation, as both are elevated in the infarct scar in vivo (18, 24, 52). Alone, TGF-beta1 significantly reduces expression of cyclin E and Cdk2 in myofibroblasts, supporting the idea that TGF-beta1 decreases cyclin E/Cdk2 complexes and thus decreases proliferation in these cells. For the purpose of comparison, the effect of CT-1 and PDGF-BB on the expression of cyclin E and Cdk2 was investigated. Each cytokine was added in the presence of 1% FBS; since we postulated that TGF-beta1 will act to downregulate proliferation of cardiac myofibroblasts, we sought control conditions favorable for basal proliferation. Low serum-mediated induction of cyclin E is relatively strong, and we found that 1% FBS is a sufficient stimulus for myofibroblast proliferation. It is possible that low-serum incubation of myofibroblasts may alter CT-1 and PDGF-BB receptor expression and/or influence the function of CT-1 and PDGF-BB ligands. In this regard, we have observed that CT-1 alone in serum-free medium is a stimulus for cyclin E protein expression (unpublished observation). The combination of CT-1 and PDGF-BB coincubation elicited a significant increase in Cdk2 expression, whereas treatment of cells with either CT-1 or PDGF-BB alone was associated with only a mild increase in Cdk2 protein levels. Finally, we have shown that TGF-beta1 dramatically downregulates expression of Cdk2 under low-serum conditions and that the TGF-beta1-specific effect dominated other effects of either CT-1 or PDGF-BB.

The p27 protein is known to impair formation of the cyclin E/Cdk2 complex, resulting in the inhibition of cellular proliferation (47). Our data (29) confirmed that TGF-beta1 treatment of myofibroblasts stimulated an increase in the expression of p27 in association with ablation of cell proliferation. CT-1 treatment of myofibroblasts was not associated with significant alteration of p27 expression versus low-serum-treated control cultures, supporting CT-1's role as a proliferative factor and indicating that its effects are not additive to those of TGF-beta1 in this cell type. Furthermore, coincubation of TGF-beta1 with CT-1 significantly increased p27 expression to the same level of TGF-beta1 treatment alone, suggesting that CT-1 did not influence TGF-beta1-mediated cell proliferation.

In conclusion, our study addressed the combined effects of CT-1 and TGF-beta1 on myofibroblast proliferation and contraction. TGF-beta1 treatment ablated CT-1-induced myofibroblast proliferation, and TGF-beta1 treatment also increased expression of p27 and decreased expression of cyclin E and Cdk2 in primary cells. TGF-beta1 effects were ablated by ectopic Smad7 overexpression. CT-1 treatment of myofibroblasts had no effect on collagen gel deformation versus controls, whereas TGF-beta1 and PDGF-BB treatments caused myofibroblast contraction. TGF-beta1-mediated contraction was unaffected by CT-1 alone. In combination, CT-1 and TGF-beta1 treatments exert opposing effects on myofibroblast function. Finally, TGF-beta1-mediated effects supersede those of CT-1 (and PDGF-BB). Thus TGF-beta1 and CT-1 exert differential effects on myofibroblast proliferation and contraction in vitro, and we suggest that a balance of these effects may be important for the execution of normal cardiac wound healing.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study is supported by grants from the Canadian Institutes for Health Research (cardiotrophin-1-related work) and the Heart and Stroke Foundation of Manitoba (Smad7 experiments). V. Drobic, R. H. Cunnington, and K. M. Bedosky were the recipients of student traineeships (St. Boniface General Hospital and Research Foundation). I. M. C. Dixon is a scholar of the Myles Robinson Memorial Heart Fund and the recipient of the 2007 HSF R. E. Beamish Memorial award.


   ACKNOWLEDGMENTS
 
We acknowledge the assistance of Dr. Anders Nygren (University of Calgary), who provided the specific intermediate density lipoprotein-based computer software used to measure the surface area of collagen gels, and Dr. Lisa Chilton (University of Calgary) in assessing myofibroblast contractility. The assistance of Drs. Larry V. Hryshko and Mark Hnatowitch in the characterization of myofibroblast culture on the type I collagen matrix preparations is also greatly appreciated.


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. M. C. Dixon, Professor of Physiology, Institute of Cardiovascular Science, St. Boniface General Hospital Research Centre, 351 Tache Ave., Winnipeg, MB, Canada R2H 2A6 (e-mail: idixon{at}sbrc.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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K. B. Walsh and J. Zhang
Neonatal rat cardiac fibroblasts express three types of voltage-gated K+ channels: regulation of a transient outward current by protein kinase C
Am J Physiol Heart Circ Physiol, February 1, 2008; 294(2): H1010 - H1017.
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00935.2006v1
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