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

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

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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
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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.

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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

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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

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