Connective tissue cells expressing fibro/adipogenic progenitor markers increase under chronic damage: relevance in fibroblast-myofibroblast differentiation and skeletal muscle fibrosis

Abstract

Fibrosis occurs in skeletal muscle under various pathophysiological conditions such as Duchenne muscular dystrophy (DMD), a devastating disease characterized by fiber degeneration that results in progressive loss of muscle mass, weakness and increased extracellular matrix (ECM) accumulation. Fibrosis is also observed after skeletal muscle denervation and repeated cycles of damage followed by regeneration. The ECM is synthesized largely by fibroblasts in the muscle connective tissue under normal conditions. Myofibroblasts, cells that express α-smooth muscle actin (α-SMA), play a role in many tissues affected by fibrosis. In skeletal muscle, fibro/adipogenic progenitors (FAPs) that express cell-surface platelet-derived growth factor receptor-α (PDGFR-α) and the transcription factor Tcf4 seem to be responsible for connective tissue synthesis and are good candidates for the origin of myofibroblasts. We show that cells positive for Tcf4 and PDGFR-α are expressed in skeletal muscle under normal conditions and are increased in various skeletal muscles of mdx mice, a murine model for DMD, wild type muscle after sciatic denervation and muscle subjected to chronic damage. These cells co-label with the myofibroblast marker α-SMA in dystrophic muscle but not in normal tissue. The Tcf4-positive cells lie near macrophages mainly concentrated in dystrophic necrotic-regenerating foci. The close proximity of Tcf4-positive cells to inflammatory cells and their previously described role in muscle regeneration might reflect an active interaction between these cell types and growth factors, possibly resulting in a muscular regenerative or fibrotic condition.

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Abbreviations

α-SMA:

α-Smooth muscle actin

Ang-1-7:

Angiotensin 1-7

ECM:

Extracellular matrix

DMD:

Duchenne muscular dystrophy

FAPs:

Fibro/adipogenic progenitors

PDGFR-α:

Platelet-derived growth factor receptor-α

TGF-β:

Transforming growth factor-β

TNF-α:

Tumor necrosis factor type α

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Acknowledgments

The authors are grateful to Darling Vera, Lina Correa, Victor Troncoso and Ana Vasquez for their technical support and to Ximena Verges for help with the confocal microscopy.

Author contributions

O.C. conducted most of the experiments. O.C. and E.B. conceived the concepts, designed the study, analyzed the data and wrote the manuscript. D.R. and J.E.O. contributed to the denervation and chronic damage studies, respectively. H.O. helped with the Pax7 and NG2 cell type analyses. All authors read and approved the final version of the manuscript.

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Correspondence to Enrique Brandan.

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The authors declare no competing financial interests.

Additional information

This study received financial support from the Fondecyt 1150106 grant to E.B., the CARE-PFB-12/2007 grant and Fondecyt 3140357 grant to D.R. and the Conicyt Doctoral Fellowship grant to O.C. and J.E.O.

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Fig. S1
figure7

Increase in Tcf4-positive connective tissue fibroblasts in diaphragm of dystrophic skeletal muscle. a–i Representative confocal images showing a significant increase in interstitial Tcf4-positive cells (green) in cross-sections of the mdx muscle compared with the WT. Laminin-α2 (red) and nuclei (blue) are also stained (middle). e, f, k, l Representative confocal images of collagen type I immunostained in a cross-section of a WT and a mdx diaphragm. Nuclei (blue) are also stained. Bars 50 μm (top, middle), 100 μm (bottom). m, n Tcf4-positive cells were measured per field in the gastrocnemius and the tibialis anterior, respectively. Cells were counted at 60× magnification. Values are means ± SEM, n = 3 animals in each group. ***P < 0.001 WT vs mdx; with two-tailed Student’s t-test. o–r Western blot analysis of Tcf4, PDGFR-α and fibronectin in the gastrocnemius and tibialis anterior extracts obtained from the skeletal muscles of WT and mdx mice (5-months-old). The corresponding total Tcf4, PDGFR-α and fibronectin levels were measured by densitometry analysis in WT and mdx mice. Total GAPDH was used as a loading control. *P < 0.05, **P < 0.005, ***P < 0.0005 WT vs mdx; one-tailed Student’s t-test; n = 3 in each experimental group (GIF 107 kb)

Fig. S2
figure8

Architectural changes and fibrosis increase in skeletal muscles of adult mdx mice. Representative images of hematoxylin and eosin (top) and Sirius Red staining of total collagen (bottom) showing muscle architectural features in cryosections of the diaphragm, gastrocnemius and tibialis anterior muscles in WT and mdx skeletal muscle. Bars 50 μm (GIF 190 kb)

Fig. S3
figure9

Architectural changes and fibrosis increase in the gastrocnemius of adult WT mice following sciatic denervation. Cryosections of the gastrocnemius muscle in contralateral and denervated muscle, sampled 2 weeks after surgery. Representative images of hematoxylin and eosin staining (top), Sirius Red staining of total collagen (middle) and collagen type I immunofluorescence showing atrophic skeletal muscle architecture and increase in this fibrotic protein (bottom). Nuclei are stained with Hoechst (blue, bottom). Bars 50 μm (GIF 278 kb)

Fig. S4
figure10

Fibrosis induction in tibialis anterior muscle of adult WT mice following six repeated BaCl2 injections. Representative images of hematoxylin and eosin staining (top), Sirius Red staining of total collagen (middle), and collagen type I immunofluorescence (bottom) of WT tibialis anterior muscles subjected to six consecutive weekly rounds of BaCl2 injections (50 μl 1.2 % BaCl2), compared with six consecutive weekly rounds of saline injections (0.9 % NaCl), sampled 2 weeks after the final injection. Nuclei are stained with Hoechst (blue, bottom). Bars 100 μm (GIF 296 kb)

Fig. S5
figure11

Increase in NG2-positive pericytes in dystrophic and denervated skeletal muscles. a, b Representative confocal images showing that NG2-positive pericytes and interstitial Tcf4-positive cells are spatially located in different zones in WT diaphragm (serial cross-sections; dotted lines extracellular matrix [ECM]). Laminin-α2 (red) and nuclei (blue) are also stained. c, d Representative confocal images showing that NG2-positive pericytes and interstitial Tcf4-positive cells are spatially located in different zones in mdx tibialis anterior serial cross-sections. e–j Representative confocal images showing that NG2-positive pericytes increase in mdx when compared with WT muscles. These increases correlate with the degree of fibrosis found in the diaphragm, gastrocnemius and tibialis anterior muscles. k, l Representative confocal images showing that NG2-positive pericytes increase following denervation (2 weeks after sciatic denervation) when compared with those in contralateral gastrocnemius muscle. e–l Nuclei (blue) are also stained. Bars 50 μm (GIF 157 kb)

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Contreras, O., Rebolledo, D.L., Oyarzún, J.E. et al. Connective tissue cells expressing fibro/adipogenic progenitor markers increase under chronic damage: relevance in fibroblast-myofibroblast differentiation and skeletal muscle fibrosis. Cell Tissue Res 364, 647–660 (2016). https://doi.org/10.1007/s00441-015-2343-0

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Keywords

  • Tcf4
  • PDGFR-α
  • Fibroblasts
  • Muscular dystrophy
  • Fibrosis