Characterization of cardiac fibroblast-extracellular matrix crosstalk across developmental ages provides insight into age-related changes in cardiac repair

Abstract

Heart failure afflicts an estimated 6.5 million people in the United States, driven largely by incidents of coronary heart disease (CHD). CHD leads to heart failure due to the inability of adult myocardial tissue to regenerate after myocardial infarction (MI). Instead, immune cells and resident cardiac fibroblasts (CFs), the cells responsible for the maintenance of the cardiac extracellular matrix (cECM), drive an inflammatory wound healing response, which leads to fibrotic scar tissue. However, fibrosis is reduced in fetal and early (<1-week-old) neonatal mammals, which exhibit a transient capability for regenerative tissue remodeling. Recent work by our laboratory and others suggests this is in part due to compositional differences in the cECM and functional differences in CFs with respect to developmental age. Specifically, fetal cECM and CFs appear to mitigate functional loss in MI models and engineered cardiac tissues, compared to adult CFs and cECM. We conducted 2D studies of CFs on solubilized fetal and adult cECM to investigate whether these age-specific functional differences are synergistic with respect to their impact on CF phenotype and, therefore, cardiac wound healing. We found that the CF migration rate and stiffness vary with respect to cell and cECM developmental age and that CF transition to a fibrotic phenotype can be partially attenuated in the fetal cECM. However, this effect was not observed when cells were treated with cytokine TGF-β1, suggesting that inflammatory signaling factors are the dominant driver of the fibroblast phenotype. This information may be valuable for targeted therapies aimed at modifying the CF wound healing response and is broadly applicable to age-related studies of cardiac remodeling.

Keywords: cardiac development; cardiac fibroblast; extracellular matrix; fibrosis; wound healing.

FIGURE 1

AFM data of fetal, neonatal, and adult cardiac fibroblasts (N ≥ 3 for all groups). GF, gelatin–fibronectin; fECM, fetal cardiac ECM; aECM, adult cardiac ECM. *, p < 0.05; ** p < 0.01; ***, p < 0.001; ****, p < 0.0001.

FIGURE 2

(A–C) Representative images of scratch assay used for analysis at 0–12 h. (D) Scratch assay data of fetal, neonatal, and adult CFs on gelatin–fibronectin (GF), fetal cardiac ECM (fECM), and adult cardiac ECM (aECM) coatings. N = 3 for all groups. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

FIGURE 3

Immunofluorescence imaging of fetal, neonatal, and adult cardiac fibroblasts treated with TGF-β1 and left untreated. Cells were cultured on TCP, gelatin–fibronectin, fetal ECM, or adult ECM. Red, SMA; green, Ki67; blue, DAPI. N > 5 for all groups. Scale bars = 100 um.

FIGURE 4

Image quantification of D3 fetal, neonatal, and adult cardiac fibroblasts when cultured on gelatin–fibronectin, fetal ECM, adult ECM, and TCP. (A) Fibroblast (untreated) percentage of α-SMA positive cells. (B) TGF-β1-treated cardiac fibroblast percentage of α-SMA-positive cells. (C) Fold change vs. Day 0 of Ki67 in untreated fibroblasts. (D) Fold change vs Day 0 of Ki67 in TGF-β1-treated cardiac fibroblasts. N > 5 for all groups. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. ECM-to-ECM comparisons are also evaluated and reported with significance in Supplementary Table S1.

FIGURE 5

Western blot analysis of fetal, neonatal, and adult cardiac fibroblasts. (A) Western blot bands for all tested fibroblast ages cultured on fetal cECM, adult cECM, and gelatin–fibronectin treated with TGF-β1 and those left untreated. (B) Relative abundance of α-SMA in both TGF-β1-treated and untreated cardiac fibroblasts, normalized to GAPDH; groups normalized to the gelatin–fibronectin group. (C) Relative abundance of total SMAD2/3 in both TGF-β1-treated and untreated cardiac fibroblasts, normalized to GAPDH; groups normalized to the gelatin–fibronectin group. N = 3 for all groups. *, p < 0.05, differences in the cECM type are significant for the tested CF age. Bars (p < 0.05) indicate significance between cell ages cultured on adult cECM.