Extracellular Matrix and Cell Developmental Age Influence Cardiac Fibroblast Phenotype in the Context of Cardiac Tissue Engineering

Introduction:: Cardiac injury due to disease or myocardial infarction (MI) typically leads to cardiomyocyte (CM) death and fibrosis. This pathology is mediated in part by cardiac fibroblasts (CFs), which maintain the cardiac extracellular matrix (cECM). Upon injury in adults, cytokines (e.g., TGF-β1) stimulate CFs to differentiate into myofibroblasts: active, contractile α-smooth muscle actin-positive cells responsible for the increased collagen deposition that characterizes fibrosis (1). However, during fetal and early neonatal development, fibrosis is diminished and CMs are transiently capable of myocardial restoration. Our lab has shown that this is due in part to age-dependent cECM compositional changes, as fetal cECM (fECM) promotes greater CM expansion versus adult cECM (aECM) (2). Further, the developmental age of CFs themselves motivates cECM remodeling, with adult CFs upregulating fibrotic protein expression compared to fetal CFs in engineered cardiac tissues (3). We hypothesized that age-dependent differences in CF-ECM interactions may induce pro-fibrotic or pro-regenerative phenotypes in CFs. Understanding how developmental age impacts interplay between CFs and cECM may assist in development of pro-regenerative biomaterials and cardiac therapeutics.


Materials and Methods:: CFs were isolated from fetal (fCF), neonatal (nCF), and adult (aCF) rats and cultured on decellularized cECM from fetal and adult porcine hearts was solubilized using published methods (2). These cells were cultured with and without the addition of TGF-β1 to induce a myofibroblast phenotype., to determine cECM impact on unactivated CFs and myofibroblasts. Scratch assays and atomic force microscopy (AFM) were used to probe migration and evaluate cell stiffness, respectively. Myofibroblast activation was evaluated via α-SMA immunofluorescence staining, and proliferation was assessed via Ki67 expression. We further examined age-specific gene expression differences independent of cECM via RNA-seq. Statistics: ANOVA with Tukey post-hoc test.


Results, Conclusions, and Discussions:: Age of cECM had variable effects on CFs across developmental ages, while CF age appeared to cause consistent trends regardless of ECM coating. Non-TGF-β1 nCFs all had significantly higher α-SMA positive cell percentages than aCFs on all coatings tested (Fig 1a, 1b). AFM data indicated nCF cell stiffness on fECM was elevated versus nCFs on fECM and fCFs on gelatin/fibronectin, and aCFs on aECM were significantly stiffer than all groups (Fig 1c). Scratch assays indicated aECM appeared to amplify nCF migration in culture versus fECM (data not shown). RNA-seq revealed matrix-related and immune modulation gene expression differences between fCF/nCF and aCFs (Fig 1d).

Overall, we found that while ECM compositional differences may partially motivate phenotype of CFs, the developmental age of CFs appears to be a dominant factor in their behavior. It appeared fCFs and nCFs exhibited higher α-SMA expression, suggesting a higher level of activity independent of cECM age, and potentially variable responses to TGF-β1, although further validation of this is required. Current work is focused on applying these results in fibrin-cECM constructs to determine how age-dependent differences in CF/cECM crosstalk impacts engineered tissue properties, and to evaluate these results in a 3D context.


Acknowledgements (Optional): : This work is supported by grants from the US Department of Defense (#W81XWH-16-1-0304 to LDB), NSF (NSF#1603524 to LDB) and AHA (#18PRE33960362 to LRP, #20TPA35500082 to LDB).


References (Optional): : (1) Porter KE, Turner NA. Pharmacology & Therapeutics. 2009;123(2):255-78, (2) Williams C, et al, Acta biomaterialia. 2014;10(1):194-204, (3) Li YA, Huda; Bursac, Nenad. Acta biomaterialia. 2017;55:120-30.