Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart
Xing Fu, … , Burns C. Blaxall, Jeffery D. Molkentin
Xing Fu, … , Burns C. Blaxall, Jeffery D. Molkentin
Published April 16, 2018
Citation Information: J Clin Invest. 2018;128(5):2127-2143. https://doi.org/10.1172/JCI98215.
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Research Article Cardiology

Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart

Abstract

Fibroblasts are a dynamic cell type that achieve selective differentiated states to mediate acute wound healing and long-term tissue remodeling with scarring. With myocardial infarction injury, cardiomyocytes are replaced by secreted extracellular matrix proteins produced by proliferating and differentiating fibroblasts. Here, we employed 3 different mouse lineage-tracing models and stage-specific gene profiling to phenotypically analyze and classify resident cardiac fibroblast dynamics during myocardial infarction injury and stable scar formation. Fibroblasts were activated and highly proliferative, reaching a maximum rate within 2 to 4 days after infarction injury, at which point they expanded 3.5-fold and were maintained long term. By 3 to 7 days, these cells differentiated into myofibroblasts that secreted abundant extracellular matrix proteins and expressed smooth muscle α-actin to structurally support the necrotic area. By 7 to 10 days, myofibroblasts lost proliferative ability and smooth muscle α-actin expression as the collagen-containing extracellular matrix and scar fully matured. However, these same lineage-traced initial fibroblasts persisted within the scar, achieving a new molecular and stable differentiated state referred to as a matrifibrocyte, which was also observed in the scars of human hearts. These cells express common and unique extracellular matrix and tendon genes that are more specialized to support the mature scar.

Authors

Xing Fu, Hadi Khalil, Onur Kanisicak, Justin G. Boyer, Ronald J. Vagnozzi, Bryan D. Maliken, Michelle A. Sargent, Vikram Prasad, Iñigo Valiente-Alandi, Burns C. Blaxall, Jeffery D. Molkentin

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Figure 1

Proliferation of Tcf21 lineage–traced fibroblasts after MI.

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Proliferation of Tcf21 lineage–traced fibroblasts after MI. (A) Schemati...
(A) Schematic of the Tcf21 genetic locus with a tamoxifen-regulated MerCreMer cDNA cassette inserted into exon 1 (E1).The MerCreMer-containing Tcf21 locus was introduced into R26EGFP mice containing a loxP site–flanked stop cassette upstream of EGFP to allow for Cre-dependent lineage tracing. (B) Experimental scheme whereby Tcf21MCM/+;R26EGFP mice were given tamoxifen for 4 weeks and rested for 1 week before MI surgery. Mice were treated with a single EdU injection at the indicated time points after MI, and hearts were harvested 4 hours after each EdU injection for IHC analysis. (C–E) Quantification of EdU+ (white) and Ki67+ (red) Tcf21 lineage–traced (EGFP+) fibroblasts (green) in infarct region (C) and border zone (D) after a single EdU injection at the indicated time points after MI by IHC and representative IHC images. The white bar in C represents a 0 time point. (E) Nuclei are shown with DAPI (blue); these same images are shown in Supplemental Figure 1C in a larger temporal array. (F) Experimental scheme of tamoxifen treatment of Tcf21MCM/+;R26EGFP mice before MI surgery and 7 daily EdU injections during indicated time periods after MI. Hearts were harvested 4 hours after the last EdU injection for IHC analysis. (G–I) Quantification of EdU+ (white) and Ki67+ (red) Tcf21 lineage–traced fibroblasts (green) in the infarct region (G) and border zone (H) after 7 daily EdU injections during the indicated time periods after MI by IHC and representative IHC images (I). Nuclei are shown with DAPI (blue). (J) Quantification of Tcf21 lineage–traced fibroblasts in the infarct region at the indicated time points by FACS. Density of cells is presented as the number of cells per mg of infarct tissue. (C, D, G, H, and J) Data are shown as mean ± SD (n = 3). E and I show representative images from 3 separate hearts analyzed. Scale bars: 20 μm.