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

Abstract

Rationale: Cardiac fibroblasts are key effector cells in the pathogenesis of cardiac fibrosis. Transforming growth factor (TGF)-beta/Smad3 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: The present 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 wild-type 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-beta1 inhibited murine cardiac fibroblast proliferation; these antiproliferative effects were mediated via Smad3. Smad3-/- fibroblasts were functionally defective, exhibiting impaired collagen lattice contraction when compared with wild-type cells. Decreased contractile function was associated with attenuated TGF-beta-induced expression of alpha-smooth muscle actin. In addition, Smad3-/- fibroblasts had decreased migratory activity on stimulation with serum, and exhibited attenuated TGF-beta1-induced upregulation of extracellular matrix protein synthesis. Upregulation of connective tissue growth factor, an essential downstream mediator in TGF-beta-induced fibrosis, was in part dependent on Smad3. Connective tissue growth factor 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.

Figure 1

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).

Figure 2

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).

Figure 3

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).

Figure 4

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).

Figure 5

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).

Figure 6

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).

Figure 7

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).

Figure 8

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.