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. Author manuscript; available in PMC: 2011 Aug 6.
Published in final edited form as: Circ Res. 2010 Jun 3;107(3):418–428. doi: 10.1161/CIRCRESAHA.109.216101

Smad3 signaling critically regulates fibroblast phenotype and function in healing myocardial infarction

Marcin Dobaczewski *, Marcin Bujak *, Na Li *, Carlos Gonzalez-Quesada *, Leonardo H Mendoza *, Xiao-Fan Wang †, Nikolaos G Frangogiannis *
PMCID: PMC2917472  NIHMSID: NIHMS221616  PMID: 20522804

Abstract

Rationale

Cardiac fibroblasts are key effector cells in the pathogenesis of cardiac fibrosis. Transforming Growth Factor (TGF)-βSmad3 signaling is activated in the border zone of healing infarcts and induces fibrotic remodeling of the infarcted ventricle contributing to the development of diastolic dysfunction.

Objective

Our study explores the mechanisms responsible for the fibrogenic effects of Smad3 by dissecting its role in modulating cardiac fibroblast phenotype and function.

Methods and results

Smad3 null mice and corresponding wildtype (WT) controls underwent reperfused myocardial infarction protocols. Surprisingly, reduced collagen deposition in Smad3 −/− infarcts was associated with increased infiltration with myofibroblasts. In vitro studies demonstrated that TGF-β1 inhibited murine cardiac fibroblast proliferation; these anti-proliferative effects were mediated via Smad3. Smad3 −/− fibroblasts were functionally defective, exhibiting impaired collagen lattice contraction when compared to wildtype cells. Decreased contractile function was associated with attenuated TGF-β-induced expression of α-smooth muscle actin. In addition, Smad3 −/− fibroblasts had decreased migratory activity upon stimulation with serum, and exhibited attenuated TGF-β-induced upregulation of extracellular matrix protein synthesis. Upregulation of connective tissue growth factor (CTGF), an essential downstream mediator in TGF-β-induced fibrosis, was in part dependent on Smad3. CTGF stimulation enhanced extracellular matrix protein expression by cardiac fibroblasts in a Smad3-independent manner.

Conclusions

Disruption of Smad3 results in infiltration of the infarct with abundant, hypofunctional fibroblasts that exhibit impaired myofibroblast transdifferentiation, reduced migratory potential, and suppressed expression of fibrosis-associated genes.

Keywords: fibroblasts, myocardial infarction, transforming growth factor-β, growth factors, fibrosis

Introduction

Activated fibroblasts are critically involved in both reparative and fibrotic processes. In normal tissues, resident fibroblasts are quiescent, producing limited amounts of extracellular matrix proteins and exhibiting few actin-associated cell-matrix and cell-cell contacts. 1 Tissue injury triggers alterations in the mechanical microenvironment and activates cytokine and growth factor-mediated pathways resulting in differentiation of fibroblasts into myofibroblasts, 2,3,4 phenotypically modulated cells characterized by the presence of a microfilamentous contractile apparatus enriched with α-smooth muscle actin (α-SMA). In the healing wound, activated myofibroblasts are the main source of extracellular matrix proteins. 5,6

Release of biologically active transforming growth factor (TGF)-β1 plays an important role in activation of fibroblasts in wound repair, both by promoting myofibroblast transdifferentiation and by enhancing synthesis of extracellular matrix proteins. 7,8 TGF-β1 exerts many of its effects through a cascade of intracellular effectors, the Smads. 9 Extensive evidence suggests that activation of the Smad2/3 cascade plays an essential role in extracellular matrix protein gene expression 10 and regulates fibrous tissue deposition in a variety of experimental models. 11,12,13 Smad3 deficiency attenuates collagen deposition in a model of bleomycin-induced pulmonary fibrosis 14 and protects from the development of renal fibrosis after ureteral obstruction. 15 Experiments from our laboratory have demonstrated a critical role for Smad3 signaling in fibrotic remodeling of the infarcted heart. 16 Smad3 null mice had significantly reduced collagen deposition in the scar and in the remodeling non-infarcted ventricle, and exhibited reduced dilative remodeling and attenuated diastolic dysfunction following myocardial infarction. 16 Although Smad3 is essential for the development of cardiac fibrosis the mechanisms responsible for its profibrotic actions have not been systematically investigated.

Our current study examines the effects of Smad3 gene disruption on phenotype and function of cardiac fibroblasts. We found that Smad3 absence results in infiltration of the infarcted heart with a large number of cells that exhibit impaired functional properties and defective matrix synthetic capability. Smad3 null fibroblasts showed impaired myofibroblast transdifferentiation, reduced migratory potential and reduced capacity to contract collagen pads upon TGF-β1 stimulation. Upregulation of the essential TGF-β-induced fibrogenic mediator connective tissue growth factor (CTGF) in the infarcted myocardium was in part dependent on Smad3. Our findings suggest that Smad3 gene disruption abrogates the anti-proliferative effects of TGF-β1 in the infarcted heart and results in accumulation of abundant but functionally defective fibroblasts attenuating adverse fibrotic remodeling of the infarcted ventricle.

Methods

All animal studies were approved by the animal protocol review committee at Baylor College of Medicine. Smad3 −/− mice 17,16 and wildtype (WT) C57/BL/6 controls underwent reperfused myocardial infarction experiments using a closed-chest model of coronary occlusion/reperfusion. 18

Sections from paraffin-embedded hearts were immunolabeled with anti-Ki-67 and anti-α-SMA antibodies to allow identification of proliferating myofibroblasts. Identification of apoptotic cells was performed using fluorescent In situ Cell Death Detection Kit (Roche). 19 The collagen content in infarcted hearts network was assessed using picrosirius red staining and a hydroxyproline assay. Single cell suspensions were prepared from infarcted hearts after 72h of reperfusion and underwent flow cytometric analysis of α-SMA and collagen I expression.

In order to investigate the role of Smad3 signaling in fibroblast phenotype and function, mouse fibroblasts from WT and Smad3 −/− hearts were isolated by enzymatic digestion. Growth factor-stimulated cells were used for collagen lattice contraction assay, transwell migration assay, 19 cell proliferation assay, 19 immunofluorescence, flow cytometry, RNA and protein extraction. Real time PCR was used to assess expression of extracellular matrix proteins. α-SMA expression in infarcted hearts and isolated cells was assessed using established western blotting protocols. 16

Results

Smad3 null infarcts exhibit increased myofibroblast infiltration, but attenuated collagen deposition

α-SMA immunofluorescence was used to identify infarct myofibroblasts as spindle-shaped immunoreactive cells located outside the vascular media (Figure 1A-B). Smad3 absence resulted in formation of a highly cellular wound with reduced deposition of collagen. Quantitative analysis showed that Smad3 null mice had significantly increased cellular content and higher myofibroblast density in the infarcted heart (Figure 1C-E). Histochemical sirius red staining (Figure 1G-H) and a biochemical hydroxyproline assay (Figure 1I) demonstrated that, despite the enhanced accumulation of myofibroblasts, collagen deposition was significantly reduced in Smad3 null infarcts. Flow cytometry using single cell suspensions harvested from infarcted hearts after 72h of reperfusion confirmed the histological findings demonstrating that Smad3 null infarcts contained more cells, and had a higher number of α-SMA+/collagen I+ myofibroblasts (Figure 1F). Assessment of mean fluorescent intensity for collagen I in infarct myofibroblasts demonstrated that cells isolated from Smad3 null infarcts had lower collagen content than WT cells (Figure 1J-K).

Figure 1.

Figure 1

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Smad3 null mice have reduced collagen deposition in the infarcted heart when compared with WT mice despite the presence of a higher number of myofibroblasts. A-B: Immunofluorescent staining for α-SMA in sections from WT (A) and Smad3 null (B) infarcts after 72h of reperfusion identified infarct myofibroblasts as spindle-shaped cells located outside the vascular media (red - arrows). Sections were counterstained with DAPI to label nuclei. C: Smad3 −/− infarcts had significantly higher cellular content than WT infarcts after 72h- 7days of reperfusion. D: Myofibroblast density was also significantly higher in Smad3 null infarcts after 72h-7days of reperfusion. E: Myofibroblasts were a significantly higher percentage of nucleated cells in Smad3 infarcts after 72h of reperfusion. F: Flow cytometric analysis using isolated single cell suspensions harvested from infarcted hearts after 72 h of reperfusion confirmed that Smad3 null mice had significantly more cells and a higher number of a-SMA+/collagen I + myofibroblasts per mg of infarcted heart than WT animals. G - I: In contrast, collagen deposition in the infarcted and remodeling heart was attenuated in the absence of Smad3. Sirius red staining identified the collagen network in WT (G) and Smad3 null (H) infarcts. Quantitative analysis of collagen content using a hydroxyproline assay demonstrated markedly reduced collagen deposition in infarcted Smad3 null hearts after 72h and 7 days of reperfusion (I). J-K: Flow cytometry was used to quantitatively assess collagen content in infarct myofibroblasts after 72h of reperfusion. J: Histograms of collagen I fluorescent intensity for representative Smad3 null (black) and WT animals (white) are shown. K: Infarcted Smad3 null animals had significantly lower mean fluorescent intensity (MFI) for collagen type I in α-SMA+/collagen I + myofibroblasts than WT animals (**p<0.01, *p<0.05 vs. corresponding WT).

The role of Smad3 in cardiac fibroblast proliferation

In order to examine whether the increased myofibroblast density in Smad3 null infarcts is due to enhanced proliferative activity, we identified proliferating myofibroblasts using dual immunohistochemical staining for α-SMA and ki-67 (Figure 2A-F). Quantitative analysis showed that the density of proliferating myofibroblasts in the infarcted heart peaked after 72h of reperfusion (Figure 2G). Smad3 null mice had a significantly higher number of proliferating myofibroblasts per area of infarcted heart when compared with WT animals. However, when normalized to the total number of myofibroblasts present in the infarcted heart, the percentage of proliferating myofibroblasts was comparable between Smad3 null and WT mice (Figure 2H).

Figure 2.

Figure 2

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The role of Smad3 in myofibroblast proliferation in vitro and in healing myocardial infarction. A-F: Dual immunohistochemical staining for α-SMA in order to identify myofibroblasts (red -arrow) and ki-67 to label proliferating cells (black – arrowhead) was used to examine the effects of Smad3 gene disruption on myofibroblast proliferation in the infarcted heart. Representative sections from WT (A, control; B, 1h ischemia/72h reperfusion; C, 1h ischemia/7 days reperfusion) and Smad3 −/− (D, control; E, 1h ischemia/72h reperfusion; F, 1h ischemia/7 days reperfusion) animals are shown. G: The peak density of proliferating ki-67+ myofibroblasts (cells/mm2) was significantly higher in Smad3 null infarcts after 72h of reperfusion (**p<0.01 vs. corresponding 24h experiment, #p<0.05 vs. corresponding WT). H: The percentage of infarct myofibroblasts exhibiting ki-67 expression was increased after 72h of reperfusion (**p<0.01 vs. corresponding 24h experiment), but was comparable between WT and Smad3 null animals. I: In vitro TGF-β1 inhibited WT cardiac fibroblast proliferation in a dose-dependent manner (*p<0.05, **p<0.01 vs. WT control). In contrast, TGF-β1 did not significantly affect proliferation of Smad3 null fibroblasts (pNS). In comparison to WT cells, Smad3 null fibroblasts exhibited significantly higher proliferative activity for all experimental conditions (#p<0.05 vs. corresponding WT cells).

The antiproliferative actions of TGF-β1 on isolated cardiac fibroblasts are dependent on Smad3

Serum-deprived Smad3 −/− cardiac fibroblasts exhibited higher basal proliferative activity than corresponding WT cells (Figure 2I). TGF-β1 stimulation reduced proliferation of serum-deprived WT murine cardiac fibroblasts in a dose-dependent manner (Figure 2I). In contrast, TGF-β1 did not significantly affect proliferation of Smad3 null fibroblasts (pNS), indicating that the anti-proliferative effects of TGF-β1 are reduced in the absence of Smad3.

WT and Smad3 null infarcts exhibit comparable rates of apoptosis

Termination of the fibrogenic response in tissue repair is dependent on apoptosis of wound fibroblasts. In order to examine whether increased myofibroblast density in Smad3 null infarcts is due to their reduced apoptosis during infarct maturation we identified apoptotic cells in the healing infarct using TUNEL staining (Figure 3A-G). The density of apoptotic cells per area of infarcted heart was comparable between WT and Smad3 null animals after 24h-7days of reperfusion (Figure 3H). However, the percentage of nuclei exhibiting apoptosis was significantly lower in Smad3 null infarcts after 72 h of reperfusion (Figure 3I). Dual fluorescence for TUNEL and α-SMA showed rare apoptotic myofibroblasts in the infarcted heart (Figure 3G). The relative absence of apoptotic myofibroblasts in the infarct may reflect loss of contractile protein content as these cells become apoptotic or may be due to their rapid clearance from the healing infarct.

Figure 3.

Figure 3

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Effects of Smad3 deficiency on cellular apoptosis in the infarcted heart. A-I: Dual fluorescence was performed to identify apoptotic nuclei with TUNEL staining (green – arrows) and α-SMA immunofluorescence to label myofibroblasts (red – arrowheads). Sections were counterstained with DAPI (blue). Representative sections from WT (A, 1h ischemia/24h reperfusion; B, 1h ischemia/72h reperfusion; C, 1h ischemia/7 days reperfusion) and Smad3 null infarcts (D, 1h ischemia/24h reperfusion; E, 1h ischemia/72h reperfusion; F, 1h ischemia/7 days reperfusion) are shown. G: Apoptotic α-SMA+ myofibroblasts were very rarely found in the infarcted myocardium in both WT and Smad3 null animals (arrow) reflecting their rapid clearance from the infarcted heart or disruption of loss of the myofibroblast phenotype as the cells undergo apoptosis. H: Quantitative analysis showed that Smad3 null and WT infarcts had comparable number of apoptotic cells per surface area (pNS). I: The percentage of cells exhibiting apoptosis was significantly lower in Smad3 null infarcts after 72h of reperfusion (*p<0.05 vs. WT).

TGF-β1-mediated contraction of fibroblast-populated collagen lattices is partially dependent on Smad3

In order to examine the effects of Smad3 absence on fibroblast function, we assessed the ability of Smad3 null cells to elicit wound contraction using an assay of fibroblast-populated collagen lattices (Figure 4). In the absence of serum, WT fibroblasts induced more extensive lattice contraction than Smad3 null fibroblasts (Figure 4A, D, G). Stimulation with 5% serum induced marked lattice contraction in gels populated with WT or Smad3 null fibroblasts resulting in comparable changes in size between groups (Figure 4B, E, G). Although TGF-β1 stimulation enhanced collagen contraction in both WT and Smad3 null fibroblasts; contraction was markedly attenuated in lattices populated with Smad3 null cells (Figure 4C, F, G).

Figure 4.

Figure 4

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Smad3 mediates TGF-β-induced contraction of fibroblast-populated collagen lattices. The role of Smad3 in cardiac fibroblast contractile function was investigated using fibroblast-populated collagen pads. Representative experiments using WT (A-C) and Smad3 null (D-F) cardiac fibroblasts are shown (A and D, serum-deprived cells; B and E, 5% serum; C and F, 25ng/ml TGF-β1). Quantitative analysis demonstrated that lattice area was significantly higher in lattices populated with serum-deprived Smad3 null cells in comparison to corresponding WT cells. TGF-β1 markedly reduced collagen lattice area in pads populated with WT cells; this effect was attenuated in lattices populated with Smad3 null fibroblasts (*p<0.05, **p<0.01 vs. corresponding WT).

Smad3 null cardiac fibroblasts exhibit reduced α-SMA expression upon stimulation with TGF-β1

In order to examine the role of Smad3 on phenotypic modulation of cardiac fibroblasts into myofibroblasts we compared α-SMA expression in WT and Smad3 null cardiac fibroblasts upon stimulation with TGF-β1. WT cardiac fibroblasts showed robust upregulation of α-SMA mRNA after 24h of stimulation (Figure 5A); α-SMA mRNA synthesis was significantly attenuated in Smad3 null cells. Flow cytometric analysis (Figure 5B-C) and western blotting (Figure 5D-E) confirmed the essential role of Smad3 in TGF-β1-mediated β-SMA upregulation. Baseline mean fluorescent intensity was comparable between WT and Smad3 null cardiac fibroblasts (Figure 5B). TGF-β1 stimulation for 24h significantly increased the mean intensity of α-SMA expression in WT, but not in Smad3 null cardiac fibroblasts (Figure 5C). Western blotting demonstrated that TGF-β1-induced α-SMA protein upregulation was abrogated in Smad3 null cells (Figure 5D-E). Moreover, dual fluorescent staining for α-SMA and Alexa Fluor 694-labeled phalloidin showed that Smad3 absence results in impaired organization of the polymerized actin cytoskeleton and reduced incorporation of α-SMA-positive filaments (Figure 5F-K) in TGF-β1-stimulated fibroblasts.

Figure 5.

Figure 5

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TGF-β1-induced α-SMA upregulation in cardiac fibroblasts is dependent on Smad3. A: qPCR demonstrated that TGF-β1 stimulation for 24h induced marked upregulation of α-SMA mRNA in WT cardiac fibroblasts. α-SMA induction was abrogated in Smad3 null cells (**p<0.01 vs. corresponding WT. ##p<0.01 vs. WT control). B-C: Flow cytometry confirmed the essential role of Smad3 in TGF-β1-mediated α-SMA upregulation. Baseline mean fluorescent intensity was comparable between WT (white area) and Smad3 null (black area) cardiac fibroblasts (B). TGF-β1 stimulation for 72h resulted in significantly higher α-SMA expression in WT cells (white) in comparison to Smad3 null (black) fibroblasts. D-E: Western blotting demonstrated that TGF-β1-mediated α-SMA upregulation was abrogated in Smad3 null cardiac fibroblasts (*p<0.05 vs. corresponding WT cells). In order to visualize α-SMA incorporation into the cytoskeleton WT (F-H) and Smad3 −/− (I-K) fibroblasts were stimulated with 25 ng/ml TGF-β1 for 3 days and stained with Alexa Fluor 694 labeled phalloidin (which labels the actin filaments) (F, I) and FITC-conjugated anti-α-SMA antibody (G, J). Note the impaired formation of cytoskeletal fibers and reduced incorporation of α-SMA in Smad3 null myofibroblasts (I- K).

In vivo studies using flow cytometric analysis demonstrated that Smad3 null infarct myofibroblasts had significantly lower α-SMA expression than WT cells (Online Supplement).

Smad3 signaling mediates cardiac fibroblast migration

Incubation with 1% serum significantly increased cardiac fibroblast migration (Figure 6A-B, E). Upon stimulation with serum, Smad3 null cardiac fibroblasts had significantly diminished migratory activity when compared with WT cells (Figure 6). TGF-β1 exerted a modest effect on migration of WT fibroblasts that did not reach statistical significance, and had no effect on Smad3 null cells (Figure 6E).

Figure 6.

Figure 6

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Cardiac fibroblast migration induced by serum is in part dependent on Smad3. A transwell migration assay was used to study the role of Smad3 in fibroblast migration. A-D: Representative images of WT (A-B) and Smad3 null (C-D) fibroblasts that migrated toward 0% serum (A,C) and 1% serum (B,D) E: Quantitative analysis demonstrated that serum (1%) stimulation induced cardiac fibroblast migration. Smad3 null cardiac fibroblasts showed reduced migratory capacity in comparison to WT cells. (**p<0.01, *p<0.05 vs. corresponding WT. ##p<0.01 vs. WT control. ^^p<0.01 vs. KO control).

TGF-β1-mediated induction of extracellular matrix proteins synthesis in cardiac fibroblasts is dependent on Smad3

TGF-β1 induced marked upregulation of type I collagen (Figure 7A), type III collagen (Figure 7B) and fibronectin (Figure 7C) mRNA in WT cardiac fibroblasts that peaked after 4h of stimulation. TGF-β1-induced extracellular matrix protein synthesis was significantly impaired in Smad3 null cardiac fibroblasts (Figure 7A-C).

Figure 7.

Figure 7

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Smad3 null cardiac fibroblasts exhibit impaired matrix-synthetic capacity in response to TGF-β1 stimulation. qPCR analysis demonstrated that TGF-β1 stimulation markedly upregulated type I collagen (A), type III collagen (B), and fibronectin (C) mRNA expression in WT cardiac fibroblasts. TGF-β1-induced extracellular matrix protein transcription was markedly attenuated in Smad3 null fibroblasts. (**p<0.01, *p<0.05 vs. corresponding WT. ##p<0.01, #p<0.05 vs. WT control. &p<0.05 vs. KO control). D. Late, but not early, CTGF upregulation in the infarcted is dependent on Smad3. qPCR demonstrated that CTGF mRNA levels are markedly upregulated in the infarcted WT myocardium after 6-72h of reperfusion. Smad3 null mice had reduced CTGF expression after 24-72h of reperfusion; however, peak mRNA levels after 6h of reperfusion were comparable between WT and Smad3 −/− infarcts. (**p<0.01, *p<0.05 vs. WT sham. ##p<0.01 vs. KO sham. ^^p<0.01, &p<0.05 vs. corresponding WT). E. TGF-β1-mediated CTGF upregulation was dependent, in part, on Smad3. TGF-β1 stimulation upregulated CTGF synthesis by WT cardiac fibroblasts. Smad3 absence was associated with attenuated TGF-β1-induced CTGF upregulation (**p<0.01 vs. WT control. ##p<0.01 vs. KO control. ^p<0.05 vs. corresponding WT). F-H: CTGF markedly upregulated fibroblast-derived extracellular matrix protein mRNA synthesis in a Smad3-independent manner. CTGF induced comparable type I collagen (F), type III collagen (G) and fibronectin (H) mRNA upregulation in WT and Smad3 null cardiac fibroblasts. Co-stimulation of WT cardiac fibroblasts with TGF-β1 and CTGF had an additive effect on extracellular matrix protein mRNA expression. In contrast, collagen and fibronectin mRNA synthesis in Smad3 null cells upon co-stimulation with CTGF and TGF-β1, was comparable with the levels of expression observed in CTGF-stimulated cells (**p<0.01, *p<0.05 vs. WT control. ##p<0.01 vs. KO control. ^p<0.05 vs. corresponding WT).

Late, but not early CTGF upregulation in the infarcted heart is Smad3-dependent

Because CTGF is an important downstream effector of the profibrotic actions of TGF-β7, we examined whether Smad3 loss results in reduced CTGF upregulation in healing myocardial infarcts. In WT animals reperfused infarction resulted in early upregulation of CTGF mRNA after 6h of reperfusion (Figure 7D). CTGF mRNA levels remained elevated after 24-72h of reperfusion. Although early CTGF upregulation was not affected by the absence of Smad3, CTGF expression after 24-72h of reperfusion was significantly reduced in Smad3 null infarcts (Figure 7D). Thus, sustained induction of CTGF expression in the infarcted heart was Smad3-dependent.

TGF-β-induced CTGF upregulation in cardiac fibroblasts is in part dependent on Smad3

TGF-β1 upregulated CTGF synthesis by WT fibroblasts, peaking after 4h of stimulation (Figure 7E). Smad3 null fibroblasts had significantly attenuated CTGF induction upon stimulation with TGF-β1 (Figure 7E) indicating that TGF-β1-mediated CTGF upregulation is, at least in part, Smad3-dependent.

CTGF stimulates synthesis of fibrosis-associated genes by cardiac fibroblasts in a Smad3-independent manner

We next examined the effects of CTGF on cardiac fibroblast gene expression. Incubation with CTGF significantly enhanced type I collagen (Figure 7F), type III collagen (Figure 7G) and fibronectin (Figure 7H) mRNA synthesis by fibroblasts peaking after 4h of stimulation. Co-stimulation of fibroblasts with both CTGF and TGF-β1 induced significantly higher extracellular matrix protein expression than each one of the mediators alone. CTGF induced comparable upregulation of collagen and fibronectin mRNA in WT and Smad3 −/− cells suggesting that its effects on cardiac fibroblasts are independent of Smad3 (Figure 7F-H). In contrast, the stimulatory effects of CTGF and TGF-β1 co-incubation were significantly attenuated in Smad3 null cells.

DISCUSSION

Following myocardial infarction, cardiac fibroblasts migrate into the infarct border zone, where they proliferate and undergo myofibroblast transdifferentiation 20,21,22,23 promoting contraction of the scar. Activated myofibroblasts are the main source of extracellular matrix proteins in the infarcted and remodeling heart. 6 We have previously demonstrated that selective activation of the TGF-β/Smad3 pathway in the infarct border zone is critically involved in the pathogenesis of fibrotic cardiac remodeling, and contributes to the development of diastolic dysfunction following reperfused infarction 16 by enhancing collagen deposition in the remodeling myocardium. We now report that several essential phenotypic and functional alterations of cardiac fibroblasts with a key role in development of fibrotic remodeling are dependent on Smad3 (Figure 8). In the absence of Smad3, the healing infarct is infiltrated by abundant, but defective, fibroblasts that exhibit disrupted myofibroblast transdifferentiation, diminished migratory capacity, and impaired contractile and matrix-synthetic function. The functional impairment of Smad3 null fibroblasts results in attenuated interstitial fibrosis of the remodeling ventricle.

Figure 8.

Figure 8

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Schematic figure illustrating the role of TGF-β/Smad3 signaling in cardiac fibroblast phenotype and function. Smad3 signaling is essential for myofibroblast transdifferentiation and critically regulates extracellular matrix protein synthesis. In addition, Smad3 activation plays an important role in regulation of cardiac myofibroblast migration and proliferative activity, and may modulate apoptosis. Upregulation of CTGF, a downstream mediator of TGF-β-induced fibrosis, is in part dependent on Smad3. These actions are important in cardiac repair, fibrosis and remodeling.

Surprisingly, reduced collagen deposition in remodeling infarcted Smad3 null hearts was associated with significantly increased peak myofibroblast density (Figure 1). Expansion of the myofibroblast population in Smad3 null infarcts may be due to loss of the antiproliferative effects of TGF-β. Although extensive evidence suggests that the TGF-β/Smad3 pathway exerts growth-inhibitory effects on a variety of cell types, 24 its role in fibroblast proliferation appears to be dependent on the context and on the unique characteristics of fibroblasts populating various sites of injury. Thus, TGF-β induced proliferation in dermal fibroblasts, but attenuated the proliferative response in oral fibroblasts; both responses were dependent on Smad3 25,26. In the mouse heart transgenic overexpression of a mutant human TGF-β1 that prevents tethering of the latent complex to the extracellular matrix leading to enhanced TGF-β activity resulted in reduced fibroblast proliferation in the absence of injury, but markedly enhanced proliferative activity in the injured heart. 27 Our experiments demonstrated anti-proliferative effects of TGF-β on cardiac fibroblasts in vitro; these actions were abrogated in the absence of Smad3 (Figure 3). However, in vivo the proliferation index was comparable between Smad3 null and WT infarcts, suggesting that in the complex and dynamic environment of the healing infarct contextual factors may modulate the effects of TGF-β/Smad3 signaling.

Because, as the wound matures, mesenchymal cell populations in the healing infarct are cleared through apoptosis, we examined the effects of Smad3 deficiency on apoptosis of granulation tissue cells. TUNEL staining demonstrated that the density of apoptotic cells was comparable between Smad3 null and WT animals; whereas the percentage of infarct cells exhibiting apoptosis was somewhat lower in Smad3 null infarcts after 72h of reperfusion (Figure 4). Apoptotic myofibroblasts were rarely found in the infarcted heart reflecting their rapid clearance by professional phagocytes, or indicating loss of myofibroblast phenotype as the cells undergo apoptosis (Figure 3).

Beyond its involvement in regulating the proliferative activity of fibroblasts in the healing infarct, Smad3 signaling also played an important role in their functional activation. The abundant myofibroblasts accumulating in Smad3 null infarcts showed marked impairment of key functional responses. In a wound contraction assay, collagen lattices populated with Smad3 null cardiac fibroblasts exhibited markedly attenuated gel contraction when compared to pads populated with WT cells (Figure 4). Defective contractile function of Smad3 −/− cardiac fibroblasts was associated with reduced expression of α-SMA mRNA and protein, and with disturbed formation of contractile microfilaments upon stimulation with TGF-β, indicating impaired myofibroblast transdifferentiation (Figure 5). The role of the Smad3 pathway in α-SMA transcription has been previously demonstrated in fibroblast populations from non-cardiac tissues. 28 Experiments using embryonal mouse fibroblasts showed that receptor-regulated Smads, and in particular Smad3, are rate limiting for α-SMA enhancer activation. 29 In addition, transfection with a Smad3-expressing plasmid markedly increased α-SMA expression, whereas transfection with an antisense Smad3 plasmid attenuated α-SMA synthesis in rat pulmonary fibroblasts. 30 Thus, the Smad3 pathway appears to govern acquisition of the myofibroblast phenotype by fibroblasts in both reparative and fibrotic responses, and mediates TGF-β-induced contraction of collagen gels. 31

Smad3 signaling is also involved in fibroblast migration. Using a transwell assay we found that serum stimulation induced a robust migratory response in isolated cardiac fibroblasts, whereas the effects of TGF-β1 were modest and statistically insignificant (Figure 6). Smad3 absence attenuated serum-mediated fibroblast migration. The findings indicate that the highly proliferative Smad3 null cardiac fibroblasts infiltrating the infarcted myocardium exhibit impaired migratory capacity. Previous studies have suggested that signaling flux through Smad3 is critically involved in TGF-β-mediated chemotaxis in cutaneous wound healing and in fibrotic processes. 32,33

Smad3 absence also markedly reduced the matrix-synthetic ability of cardiac fibroblasts. Fibronectin, type I and type III collagen transcription was upregulated in cardiac fibroblasts upon stimulation with TGF-β1; this effect was markedly attenuated in the absence of Smad3 (Figure 7A-C). These findings may explain the marked reduction in collagen deposition observed in infarcted Smad3 null hearts, despite the presence of abundant myofibroblasts (Figure 1). Previous studies have identified several extracellular matrix proteins as Smad-dependent TGF-β targets in human dermal and in mouse embryonal fibroblasts. 10

Beyond the attenuation of direct TGF-β-mediated fibroblast responses, Smad3 gene disruption may reduce fibrosis by decreasing expression of CTGF, a TGF-β-inducible fibrogenic mediator with a central role in the pathogenesis of fibrosis. 7 TGF-β-mediated induction of CTGF is dependent on Smad3 in cutaneous fibroblasts, 34 and lesional skin from Smad3 −/− mice showed reduced CTGF expression. 28 Our experiments showed that CTGF mRNA levels were markedly increased in the infarcted murine myocardium after 6-72h of reperfusion. Although the early peak of CTGF expression was not dependent on Smad3, CTGF levels during the proliferative phase of healing (after 24-72h of reperfusion) were markedly reduced in Smad3 null infarcts (Figure 7). This observation may reflect different stimulatory signals responsible for CTGF induction, or may indicate distinct cellular sources of CTGF at various stages of healing. The effects of late attenuation of CTGF induction in Smad3 null infarcts may be particularly important for the fibrotic response, diminishing the activity of infarct myofibroblasts, that accumulate in the murine myocardium after 3-5 days of reperfusion. 18 Our in vitro studies demonstrated that CTGF stimulation induced intense upregulation of collagen and fibronectin synthesis by cardiac fibroblasts in a Smad3-independent manner (Figure 7). Co-stimulation of WT cardiac fibroblasts with TGF-β and CTGF had additive effects on extracellular matrix protein expression. In contrast, in Smad3 null cells, CTGF stimulation, and co-stimulation with TGF-β and CTGF, induced comparable collagen and fibronectin mRNA expression levels. Although a specific CTGF receptor has not been identified, CTGF functions are independent of the TGF-β/Smad3 pathway and appear to be mediated through integrins, proteoglycans and the low density lipoprotein receptor-related protein. 7,35 While TGF-β causes the induction of skin fibrosis, CTGF is required for persistent fibrous tissue formation. 36 Moreover, CTGF is a necessary cofactor for activation of TGF-β-mediated adhesive cascades in embryonic fibroblasts 37 and activates TGF-β signals by direct binding in the extracellular space. 38

Our findings contribute new information essential for understanding the role of the TGF-β/Smad3 pathway in fibrotic cardiac remodeling. Despite its anti-proliferative effects, Smad3 signaling promotes fibrogenic actions, essential in the pathogenesis of cardiac fibrosis mediating myofibroblast transdifferentiation and wound contraction, inducing fibroblast migration and upregulating synthesis of extracellular matrix proteins. In addition, activation of the Smad3 pathway results in upregulation of CTGF that further enhances the fibrogenic properties of infarct fibroblasts. Because Smad3 activation is primarily localized in the infarct border zone and in the adjacent remodeling myocardium, 16,39 Smad3-mediated actions result in expansion of the fibrotic process into non-infarcted areas and accentuate diastolic dysfunction. Thus, Smad3 inhibition may be a promising therapeutic approach in conditions associated with fibrotic cardiac remodeling.

Novelty and Significance.

What Is Known?

  • Transforming Growth Factor (TGF)-β1 participates in the pathogenesis of cardiac remodeling by activating cardiac fibroblasts; promoting myofibroblast transdifferentiation, and by enhancing extracellular matrix protein synthesis.

  • The profibrotic actions of TGF-β may be mediated through a cascade of intracellular effectors, the Smads, or through Smad-independent pathways.

  • TGF-β/Smad3 signaling is involved in tissue fibrosis; however, the mechanisms responsible for the profibrotic actions of the Smad3 pathway in the infarcted myocardium remain unknown.

What New Information Does this Article Contribute?

  • Despite a reduction in the amount of collagen in the infarcted heart, Smad3-null mice exhibit increased infiltration of the infarct with fibroblasts, possibly due to loss of the Smad3-dependent anti-proliferative effects of TGF-β on cardiac fibroblasts.

  • Smad3-null fibroblasts are functionally impaired, exhibiting reduced α-smooth muscle actin (α-SMA) expression and defective myofibroblast transdifferentiation, reduced migratory potential, diminished capacity to contract collagen pads upon TGF-β1 stimulation, and attenuated matrix-synthetic capacity.

  • Connective Tissue Growth Factor (CTGF) induction in the infarcted heart and in stimulated cardiac fibroblasts is in part dependent on Smad3, and exerts pro-fibrotic actions through Smad3-independent pathways.

Activation of TGF-β/Smad3 signaling in the infarct border zone is involved in the pathogenesis of post-infarction cardiac remodeling. Because the Smad3 pathway may promote fibrous tissue deposition in the remodeling ventricle, we studied the effects of Smad3 loss on murine cardiac fibroblast phenotype and function in vivo and in vitro. Our study demonstrates that Smad3 disruption results in infiltration of the infarct with abundant, but dysfunctional fibroblasts that exhibit impaired myofibroblast transdifferentiation, decreased capacity to contract collagen pads, reduced migratory potential, and suppressed expression of fibrosis-associated genes. The increased myofibroblast density in Smad3-null infarcts may be due to loss of the anti-proliferative actions of TGF-β. Moreover, upregulation of CTGF, an essential downstream mediator in TGF-β-induced fibrosis, was in part dependent on Smad3; however, CTGF-mediated fibrogenic actions were independent of Smad3 signaling. Thus, activation of the Smad3 pathway in the injured myocardium induces fibrosis by promoting myofibroblast transdifferentiation, recruitment and activation, and by enhancing synthesis of other fibrogenic signals, such as CTGF, while preventing uncontrolled cellular proliferation. Smad3 inhibition may be a promising therapeutic strategy in conditions associated with cardiac fibrosis.

Supplementary Material

Supp1

Acknowledgments

SOURCES OF FUNDING: This work was supported by R01 HL-76246 and R01 HL-85440, the Alkek endowment and the Medallion Foundation.

Non-standard Abbreviations and Acronyms

α-SMA

α-smooth muscle actin

TGF-β

transforming growth factor-β

CTGF

connective tissue growth factor

WT

wildtype

Footnotes

Dobaczewski: Smad3 in cardiac fibrosis

Subject codes: [151] Ischemic biology - basic studies; [147] Growth factors/cytokines; [137] Cell biology/structural biology; [115] Remodeling.

DISCLOSURES: None

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