Mechanical regulation of cardiac fibroblast profibrotic phenotypes

Abstract

Cardiac fibrosis is a serious condition currently lacking effective treatments. It occurs as a result of cardiac fibroblast (CFB) activation and differentiation into myofibroblasts, characterized by proliferation, extracellular matrix (ECM) production and stiffening, and contraction due to the expression of smooth muscle α-actin. The mechanical properties of myocardium change regionally and over time after myocardial infarction (MI). Although mechanical cues are known to activate CFBs, it is unclear which specific mechanical stimuli regulate which specific phenotypic trait; thus we investigated these relationships using three in vitro models of CFB mechanical activation and found that 1) paracrine signaling from stretched cardiomyocytes induces CFB proliferation under mechanical conditions similar to those of the infarct border region; 2) direct stretch of CFBs mimicking the mechanical environment of the infarct region induces a synthetic phenotype with elevated ECM production; and 3) progressive matrix stiffening, modeling the mechanical effects of infarct scar maturation, causes smooth muscle α-actin fiber formation, up-regulation of collagen I, and down-regulation of collagen III. These findings suggest that myocyte stretch, fibroblast stretch, and matrix stiffening following MI may separately regulate different profibrotic traits of activated CFBs.

FIGURE 1:

Maintaining a quiescent CFB phenotype by controlling substrate stiffness. Immunofluorescence staining for (A) SMA (green) and (B) vinculin. F-actin and nuclei were stained with phalloidin (red) and DAPI (blue), respectively. (C) Cell area and (D) proliferation rate (% of proliferating cells per hour) of CFBs plated on gels of 3, 8, 10, 20, and 50 kPa HA gels. One-way ANOVA with Tukey’s post hoc test was used in C and D. N = 6 and 4, respectively. (E) Heat map showing relative mRNA expression levels of collagen (col) 1a1, 1a2, and 3a1, fibronectin (FN1), TNC, SPARC, OPN, POSTN, THBS1, LOX, BMP1, matrix metalloproteinase (MMP) 2, TIMP1, and TIMP2 normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in CFBs plated on HA gels with 3, 6, 8, 10, 20, and 50 kPa stiffness. Expression across genes was lowest at 8 kPa stiffness, as indicated by the black frame. Values in each box represent raw 2^(-delta Ct) values for comparison of expression levels among genes. *Denotes significant effect of stiffness as tested by one-way ANOVA and Kruskal-Wallis tests for parametric and nonparametric data, respectively. *P < 0.05, ***P < 0.001, N = 8–12.

FIGURE 2:

Paracrine signaling from stretched CMs induces CFB proliferation. (A) Schematic illustration of the principles of the paracrine signaling model. (B) Proliferation rate of CFB cultures alone or in coculture with nonstretched CMs or stretched CMs. CFBs were plated on PA gels with 8 kPa stiffness. Two-way ANOVA showed significant effect of culture type*** and Tukey’s post hoc test as indicated in figures. N = 3–6. (C) Proliferation rate of CFBs in CM coculture or treated with conditioned media from cocultures. Two-way ANOVA showed an effect of culture type***, conditioned media***, and a significant interaction**. Tukey’s post hoc test results are indicated in figures. N = 3. (D) Relative expression of chemokine (C-X-C motif) ligand 1 (CXCL1), CSF-1, interleukin-1 receptor antagonist (IL-1ra), cluster of differentiation 54 (CD54), PDGF-B, and FGF2 mRNA normalized to GAPDH mRNA in CMs stretched for 4 and 24 h. *Denotes significant difference from 0 h control as determined by Student’s t test. N = 9 (control) and 4 (stretch). (E) Proliferation rate following 24 h stimulation with recombinant CSF-1 and/or PDGF-B. One-way ANOVA showed significant effect of stimulation*** and Tukey’s post hoc test results as indicated. N = 3. (F) Relative proliferation rate of CFBs in cocultures in the presence of PDGF-B and CSF-1 receptor antagonists (AG and GW, respectively). Two-way ANOVA showed an effect of culture type*** and blockers*** as well as significant interaction* between the two. Tukey’s post hoc test results are indicated in the figure. N = 3. Cell area (G) and morphology (H) of CFBs on 8 kPa alone or in coculture with nonstretched and stretched CMs. One-way ANOVA showed significant effect of culture type* on cell area and Tukey’s post hoc test results as indicated in figure. *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 3:

Stretch promotes ECM remodeling by CFBs. (A) Schematic illustration of the principles of the stretch model. (B) Collagen (col) 1a1, 1a2, and 3a1 and fibronectin (FN1) mRNA normalized to GAPDH mRNA in CFBs on 3 and 8 kPa gels subjected to 0%, 3%, and 6% stretch (x-axis). One-way ANOVA was used to test significant effect of stretch with Tukey’s post hoc tests for specific comparisons. Statistical results are indicated in the figure. N = 4 (control) and 12 (stretch). (C) Quantification and immunoblot of FN protein normalized to total protein determined by Ponceau staining. N = 4 (3 kPa) and 8 for (8 kPa). (D) Fold difference in LOX, BMP-1, OPN, POSTN, TNC, SPARC, THBS1, matrix metalloproteinase (MMP) 2, and TIMP 1 and 2 mRNA expression normalized to GAPDH mRNA. Significance was determined by multiple Student’s t tests corrected for multiple comparisons using Holm-Sidak’s post hoc test. p Values displayed in D are the uncorrected p values from the t test. N = 4 (control) and 12 (stretch). (E) Relative proliferation of CFB controls (C) or with 3% stretch (S). N = 3. (F) SMA mRNA normalized to GAPDH mRNA. N = 4 (control) and 12 (stretch). (G) Immunofluorescence staining for SMA (green) and DAPI staining for nuclei (blue). (H) Immunoblot for total and phophorylated focal adhesion kinase (FAK and pFAK, respectively) normalized to GAPDH. N = 4. Student’s t test was used to determine significant changes for C, E, F, and H. *P < 0.05, **P < 0.01, ***P < 0.001; n.s., nonsignificant.

FIGURE 4:

Matrix stiffening causes cell spreading, SMA fiber formation, up-regulation of collagen I, and down-regulation of collagen III. (A) Schematic illustration of the principles of the stiffening model. (B) immunofluorescence staining for SMA (green). F-actin and nuclei were stained with phalloidin (red) and DAPI (blue), respectively. (C) Collagen (col) 1a1, 1a2, and 3a1, fibronectin (FN1), LOX, and OPN mRNA normalized to GAPDH mRNA in CFBs on 8 kPa gels stiffened to 30 kPa. N = 4 (control) and 12 (stiffened). (D) Immunofluorescence staining for FN (green) and quantification of staining intensity 48 h after gels stiffening. N = 8. F-actin was stained with phalloidin (red). Student’s t tests were applied to determine significant changes. *P < 0.05; n.s., nonsignificant.

FIGURE 5:

Proposed model of mechanical regulation of CFBs following MI. 1) Paracrine signaling involving PDGF-B and CSF-1 from stretched CMs in the border region leads to proliferation of CFBs. 2a) Stretch of the infarct region during the acute phase after MI (ECM stiffness ∼3 kPa) promotes collagen I, LOX, BMP-1, POSTN, SPARC, THBS1, TIMP1 and 2, while inhibiting TNC expression by CFBs. Proliferation is increased during this phase. 2b) Stretch of the infarct region during the remodeling phase (ECM stiffness ∼8 kPa) also up-regulates collagen I in addition to collagen III, FN, and TNC, while it down-regulates the matricellular protein OPN. Proliferation is decreased during this phase. Stretch causes up-regulation of SMA mRNA during both the acute and remodeling phases; however, incorporation into stress fibers requires an accompanying increase in matrix stiffness. 3) At late stages of infarct healing, ECM stiffening to pathological stiffnesses (∼30 kPa) dominates the mechanical environment. This promotes a contractile CFB phenotype due to SMA fiber formation that has elevated, albeit dampened, collagen I production compared with that of stretched CFBs. Collagen III and OPN expression is reduced, supporting scar maturation and healing. However, continuous stretch of CFBs at this stage will cause persistent ECM production and thus development of pathological fibrosis.