CoChat
Sign Up
Cited by 265Muscle Physiology and DisordersTissue Engineering and Regenerative MedicineHippo pathway signaling and YAP/TAZ

The DOI matches its Crossref record; 52 of the 52 references it cites were found in a registry or an index.

Aging of the skeletal muscle extracellular matrix drives a stem cell fibrogenic conversion

Open access
Stearns-Reider, Kristen M., D'Amore, Antonio, Beezhold, Kevin, Rothrauff, Benjamin, Cavalli, Loredana, Wagner, William R., Vorp, David A., Tsamis, Alkiviadis, Shinde, Sunita, Zhang, Changqing, Barchowsky, Aaron, Rando, Thomas A., Tuan, Rocky S., Ambrosio, Fabrisia·2017·Aging Cell
DOI 10.1111/acel.12578PMC PMC5418187
Open this paper in CoChat — free accountOpen PDF

Abstract

Age-related declines in skeletal muscle regeneration have been attributed to muscle stem cell (MuSC) dysfunction. Aged MuSCs display a fibrogenic conversion, leading to fibrosis and impaired recovery after injury. Although studies have demonstrated the influence of in vitro substrate characteristics on stem cell fate, whether and how aging of the extracellular matrix (ECM) affects stem cell behavior has not been investigated. Here, we investigated the direct effect of the aged muscle ECM on MuSC lineage specification. Quantification of ECM topology and muscle mechanical properties reveals decreased collagen tortuosity and muscle stiffening with increasing age. Age-related ECM alterations directly disrupt MuSC responses, and MuSCs seeded ex vivo onto decellularized ECM constructs derived from aged muscle display increased expression of fibrogenic markers and decreased myogenicity, compared to MuSCs seeded onto young ECM. This fibrogenic conversion is recapitulated in vitro when MuSCs are seeded directly onto matrices elaborated by aged fibroblasts. When compared to young fibroblasts, fibroblasts isolated from aged muscle display increased nuclear levels of the mechanosensors, Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ), consistent with exposure to a stiff microenvironment in vivo. Accordingly, preconditioning of young fibroblasts by seeding them onto a substrate engineered to mimic the stiffness of aged muscle increases YAP/TAZ nuclear translocation and promotes secretion of a matrix that favors MuSC fibrogenesis. The findings here suggest that an age-related increase in muscle stiffness drives YAP/TAZ-mediated pathogenic expression of matricellular proteins by fibroblasts, ultimately disrupting MuSC fate.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1111/acel.12578

Cites

52 of 52 references verified0 citation errors · 0 not checkable

Statistics

Figures

Figure 1 Collagen fiber orientation, tortuosity, and mechanical properties of young and old skeletal muscle. (A) Representative images from young and old skeletal muscle from multiphoton imaging and second harmonic generation (red = collagen, green = muscle fibers). For image processing, only the red channel was selected (B). While there was no significant difference in collagen fiber orientation between old and young samples, collagen fibers in old skeletal muscle were significantly less tortuous than those in young. (*P = 0.04; n = 6/group) (C). Biaxial mechanical testing indicated a reduction in compliance in the circumferential direction in old skeletal muscle, as compared to young, with increasing stress (n = 7/group) (D). Muscle from old mice displays a significantly lower anisotropy ratio (AR) (AR = (λXD−1)/(λPD−1); PD = preferred fiber direction; XD = cross‐preferred fiber direction) as compared to that from young mice (*P = 0.017) (E). Data are displayed as mean ± SD. Bar = 100 μm.
Figure 1 Collagen fiber orientation, tortuosity, and mechanical properties of young and old skeletal muscle. (A) Representative images from young and old skeletal muscle from multiphoton imaging and second harmonic generation (red = collagen, green = muscle fibers). For image processing, only the red channel was selected (B). While there was no significant difference in collagen fiber orientation between old and young samples, collagen fibers in old skeletal muscle were significantly less tortuous than those in young. (*P = 0.04; n = 6/group) (C). Biaxial mechanical testing indicated a reduction in compliance in the circumferential direction in old skeletal muscle, as compared to young, with increasing stress (n = 7/group) (D). Muscle from old mice displays a significantly lower anisotropy ratio (AR) (AR = (λXD−1)/(λPD−1); PD = preferred fiber direction; XD = cross‐preferred fiber direction) as compared to that from young mice (*P = 0.017) (E). Data are displayed as mean ± SD. Bar = 100 μm.CC BY · PubMed Central
Figure 2 Cytoskeletal phenotype of young and old skeletal muscle fibroblasts. Fibroblasts isolated from old skeletal muscle demonstrate a contracted phenotype and increased cytoskeletal alignment as compared to those isolated from young skeletal muscle (green = tubulin; red = F‐actin; blue = DAPI). Bar = 100 μm for first column only and 50 μm for the remaining images.
Figure 2 Cytoskeletal phenotype of young and old skeletal muscle fibroblasts. Fibroblasts isolated from old skeletal muscle demonstrate a contracted phenotype and increased cytoskeletal alignment as compared to those isolated from young skeletal muscle (green = tubulin; red = F‐actin; blue = DAPI). Bar = 100 μm for first column only and 50 μm for the remaining images.CC BY · PubMed Central
Figure 3 Evaluation of the direct effect of the aged ECM on MuSC fate. Immunofluorescence analysis of desmin (green) and ERTR7 (red) expression of hMuSCs seeded onto decellularized ECM for 3 days. There was a significantly greater percentage of cells stained positive for ERTR7 (young = 23.8 ± 4.8%; old = 66.3 ± 6.5%; *P < 0.001) when seeded onto aged, as compared to young, ECM constructs. Conversely, there was a significantly lower percentage of cells stained positive for desmin (young = 14.8 ± 4.5%; old = 8.5 ± 3.9%; *P = 0.049) when seeded onto aged ECM constructs, as compared to young. Bar = 100 μm.
Figure 3 Evaluation of the direct effect of the aged ECM on MuSC fate. Immunofluorescence analysis of desmin (green) and ERTR7 (red) expression of hMuSCs seeded onto decellularized ECM for 3 days. There was a significantly greater percentage of cells stained positive for ERTR7 (young = 23.8 ± 4.8%; old = 66.3 ± 6.5%; *P < 0.001) when seeded onto aged, as compared to young, ECM constructs. Conversely, there was a significantly lower percentage of cells stained positive for desmin (young = 14.8 ± 4.5%; old = 8.5 ± 3.9%; *P = 0.049) when seeded onto aged ECM constructs, as compared to young. Bar = 100 μm.CC BY · PubMed Central
Figure 4 The effect of age and substrate stiffness on YAP/TAZ signaling in fibroblasts and its influence on MuSC fate. MuSCs cultured on matrices derived from old fibroblasts demonstrated a significant decrease in the percentage of desmin‐positive cells (young = 26.8 ± 1.5%; old = 11.9 ± 1.9%; *P < 0.0001) and a significant increase in the percentage of Tcf4‐positive cells (young = 27.8 ± 1.4%; old = 74.5 ± 1.2%; *P < 0.0001), relative to MuSCs cultured on matrices derived from young fibroblasts (A‐C). Representative immunofluorescence images of YAP/TAZ expression from old and young fibroblasts, and from young fibroblasts cultured on soft (8 kPA) and stiff (32 kPA) silicone gel substrates. Compared to young fibroblasts, old fibroblasts demonstrate a significant increase in the nuclear translocation of both YAP (young = 214.7 ± 9.5; old = 271.1 ± 16.5; *P = 0.0003 and TAZ (young = 152.0 ± 5.3; old = 204.5 ± 10.3; *P = 0.0008). Similarly, young fibroblasts cultured on stiff substrates exhibited a significant increase in YAP/TAZ nuclear translocation, relative to those cultured on soft substrates. When these preconditioned fibroblasts were allowed to elaborate a matrix for 2 days, cells previously cultured on the stiff substrate demonstrated a significant decrease in the percentage of desmin‐positive cells (soft surface = 14.0 ± 1.4; stiff surface 7.3 ± 0.6; P = 0.0004), and a significant increase in the percentage of Tcf4‐positive cells (soft surface = 10.78 ± 1.0; stiff surface 32.3 ± 1.4; P = 0.0008), consistent with that observed in fibroblasts cultured on young as compared to old ECM (E, F). Bar = 100 μm.
Figure 4 The effect of age and substrate stiffness on YAP/TAZ signaling in fibroblasts and its influence on MuSC fate. MuSCs cultured on matrices derived from old fibroblasts demonstrated a significant decrease in the percentage of desmin‐positive cells (young = 26.8 ± 1.5%; old = 11.9 ± 1.9%; *P < 0.0001) and a significant increase in the percentage of Tcf4‐positive cells (young = 27.8 ± 1.4%; old = 74.5 ± 1.2%; *P < 0.0001), relative to MuSCs cultured on matrices derived from young fibroblasts (A‐C). Representative immunofluorescence images of YAP/TAZ expression from old and young fibroblasts, and from young fibroblasts cultured on soft (8 kPA) and stiff (32 kPA) silicone gel substrates. Compared to young fibroblasts, old fibroblasts demonstrate a significant increase in the nuclear translocation of both YAP (young = 214.7 ± 9.5; old = 271.1 ± 16.5; *P = 0.0003 and TAZ (young = 152.0 ± 5.3; old = 204.5 ± 10.3; *P = 0.0008). Similarly, young fibroblasts cultured on stiff substrates exhibited a significant increase in YAP/TAZ nuclear translocation, relative to those cultured on soft substrates. When these preconditioned fibroblasts were allowed to elaborate a matrix for 2 days, cells previously cultured on the stiff substrate demonstrated a significant decrease in the percentage of desmin‐positive cells (soft surface = 14.0 ± 1.4; stiff surface 7.3 ± 0.6; P = 0.0004), and a significant increase in the percentage of Tcf4‐positive cells (soft surface = 10.78 ± 1.0; stiff surface 32.3 ± 1.4; P = 0.0008), consistent with that observed in fibroblasts cultured on young as compared to old ECM (E, F). Bar = 100 μm.CC BY · PubMed Central
Figure 5 The effect of fibroblast YAP/TAZ and TGF‐B1 modulation on MuSC fate. Pharmacologic inhibition of F‐actin polymerization in young myofibroblasts with latrunculin A resulted in a significant increase in the percentage of desmin‐positive cells (*P < 0.0001), and a significant decrease in the percentage of Tcf4‐positive cells (*P < 0.0001), relative to untreated cells (A, B). No significant difference in the percentage of desmin‐ or Tcf4‐positive cells was observed following treatment with leptomycin B (C, D). As with latrunculin A treatment, pharmacologic inhibition of TGF‐β1 resulted in a significant increase in the percentage of desmin‐positive cells (*P < 0.0001), and a significant decrease in the percentage of Tcf4‐positive cells (*P < 0.0001), relative to untreated cells (E, F).
Figure 5 The effect of fibroblast YAP/TAZ and TGF‐B1 modulation on MuSC fate. Pharmacologic inhibition of F‐actin polymerization in young myofibroblasts with latrunculin A resulted in a significant increase in the percentage of desmin‐positive cells (*P < 0.0001), and a significant decrease in the percentage of Tcf4‐positive cells (*P < 0.0001), relative to untreated cells (A, B). No significant difference in the percentage of desmin‐ or Tcf4‐positive cells was observed following treatment with leptomycin B (C, D). As with latrunculin A treatment, pharmacologic inhibition of TGF‐β1 resulted in a significant increase in the percentage of desmin‐positive cells (*P < 0.0001), and a significant decrease in the percentage of Tcf4‐positive cells (*P < 0.0001), relative to untreated cells (E, F).CC BY · PubMed Central
Figure 6 Proposed hypothesis schematic. Tortuous collagen fibrils in the young myomatrix are compliant and experience deformation (εy) with loading during muscle contraction. The compliance of the young ECM triggers mechanotransductive signaling in fibroblasts via integrins to promote the secretion of biochemical matrix factors that are more favorable for muscle stem cell myogenesis. Conversely, in aging, collagen fibrils become more taut and display a decreased deformation in response to contractile activity (εo; εy > εo), thereby rendering the muscle more stiff. The resulting mechanotransductive cascade in fibroblasts increases nuclear translocation of YAP/TAZ and triggers the expression of matrix‐associated biochemical factors that promote muscle stem cell fibrogenesis.
Figure 6 Proposed hypothesis schematic. Tortuous collagen fibrils in the young myomatrix are compliant and experience deformation (εy) with loading during muscle contraction. The compliance of the young ECM triggers mechanotransductive signaling in fibroblasts via integrins to promote the secretion of biochemical matrix factors that are more favorable for muscle stem cell myogenesis. Conversely, in aging, collagen fibrils become more taut and display a decreased deformation in response to contractile activity (εo; εy > εo), thereby rendering the muscle more stiff. The resulting mechanotransductive cascade in fibroblasts increases nuclear translocation of YAP/TAZ and triggers the expression of matrix‐associated biochemical factors that promote muscle stem cell fibrogenesis.CC BY · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Cited by

Sorted by citations

See all 265

Related

See all

Metadata

Authors
Stearns-Reider, K. M., D'Amore, A., Beezhold, K., Rothrauff, B., Cavalli, L., Wagner, W. R., Vorp, D. A., Tsamis, A., Shinde, S., Zhang, C., Barchowsky, A., Rando, T. A., Tuan, R. S., & Ambrosio, F.
Affiliations
  • University of Pittsburgh (US)
  • Department of Surgery; University of Pittsburgh; 450 Technology Drive, Suite 300 Pittsburgh PA 15219 USA
  • University of Leicester (GB)
  • VA Palo Alto Health Care System (US)
Corresponding author
Ambrosio, Fabrisia
ORCID
Venue
Aging Cell
Publisher
Wiley
Published
2017-03-30
Volume
16
Issue
3
Pages
518-528
ISSN
1474-9718 (ISSN-L)
DOI
10.1111/acel.12578
PMC
PMC5418187
OpenAlex
W2601343417
Cited by
265
References
52
Citations by year
2026: 41 · 2025: 30 · 2024: 31 · 2023: 31 · 2022: 25 · 2021: 38 · 2020: 29 · 2019: 22 · 2018: 13 · 2017: 5
FWCI
7.71
Type
journal article
Language
en
Open access
Licence
Locations
  • Aging Cell (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Figshare (submittedVersion, cc-by)
  • Leicester Research Archive (University of Leicester) (submittedVersion, cc-by)
Funders
  • National Institutes of Health
  • National Institute on Aging (K01AG039477)
  • National Institute of Environmental Health Sciences (F32ES022134, R01ES023696, R01ES025529)
  • University of Pittsburgh Medical Center Rehabilitation Institute
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Muscle Physiology and Disorders
  • Health Sciences › Medicine › Surgery › Tissue Engineering and Regenerative Medicine
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Hippo pathway signaling and YAP/TAZ
Keywords
skeletal muscle aging, muscle stem cells, extracellular matrix, muscle fibrosis, muscle stiffness, matricellular proteins, muscle regeneration
MeSH terms
YAP-Signaling Proteins, Acyltransferases, Aging, Animals, Biomechanical Phenomena, Cell Differentiation, Extracellular Matrix, Fibroblasts, Fibrosis, Gene Expression Regulation, Male, Mice, Inbred C57BL, Phosphoproteins, Stem Cells, Transcription Factors, Muscle, Skeletal, Cell Cycle Proteins, Muscle Development, Myoblasts, Adaptor Proteins, Signal Transducing, Mice, Torsion, Mechanical, Primary Cell Culture

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review