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Modeling the Effect of TNF-α upon Drug-Induced Toxicity in Human, Tissue-Engineered Myobundles

  • Brittany N. J. Davis
  • Jeffrey W. Santoso
  • Michaela J. Walker
  • Catherine E. Oliver
  • Michael M. Cunningham
  • Christian A. Boehm
  • Danielle Dawes
  • Samantha L. Lasater
  • Kim Huffman
  • William E. Kraus
  • George A. TruskeyEmail author
Article
  • 39 Downloads

Abstract

A number of significant muscle diseases, such as cachexia, sarcopenia, systemic chronic inflammation, along with inflammatory myopathies share TNF-α-dominated inflammation in their pathogenesis. In addition, inflammatory episodes may increase susceptibility to drug toxicity. To assess the effect of TNF-α-induced inflammation on drug responses, we engineered 3D, human skeletal myobundles, chronically exposed them to TNF-α during maturation, and measured the combined response of TNF-α and the chemotherapeutic doxorubicin on muscle function. First, the myobundle inflammatory environment was characterized by assessing the effects of TNF-α on 2D human skeletal muscle cultures and 3D human myobundles. High doses of TNF-α inhibited maturation in human 2D cultures and maturation and function in 3D myobundles. Then, a tetanus force dose–response curve was constructed to characterize doxorubicin’s effects on function alone. The combination of TNF-α and 10 nM doxorubicin exhibited a synergistic effect on both twitch and tetanus force production. Overall, the results demonstrated that inflammation of a 3D, human skeletal muscle inflammatory system alters the response to doxorubicin.

Keywords

Tissue engineering Human skeletal muscle Inflammation TNF-α Drug testing Muscle regeneration 

Notes

Acknowledgments

We gratefully acknowledge Chris Jackman and Alastair Khodabukus for excellent technical discussions; Megan Kondash for cell isolation; Ringo Yen for project support. This study was supported by NIH Grants UH2TR000505, 4UH3TR000505, UG3TR002142, from NCATS and NIAMS to GAT, as well as an NSF Graduate Research Fellowship to BNJD.

Supplementary material

10439_2019_2263_MOESM1_ESM.tif (13.9 mb)
Supplemental Figure S1 TNF-α inhibits myogenesis in engineered human myobundles. Shown are representative pictures of myotubes differentiated for 5 days, in the absence [(A) vehicle control (0.1% BSA)] or presence of TNF-α [(B) 100 U/mL, (C) 1000 U/mL, (D) 10,000 U/mL)], and immunostained with anti-myosin heavy chain (MHC) and Hoechst. Scale bar=200µm. (TIFF 14229 kb)
10439_2019_2263_MOESM2_ESM.tif (40.1 mb)
Supplemental Figure S2 Cellular cytotoxicity does not contribute to TNF-α enhancement of doxorubicin-induced contractile force depression. Myobundles were differentiated for 5 days either in the absence [vehicle (0.1% BSA)] or presence of TNF-α (100 U/mL), then doxorubicin was added every 48 hours for 7 days either in the absence or presence of 100 U/mL TNF-α. (A,B) Measurement of myobundle LDH release into the media suggests that doxorubicin dose and time did not have an effect on cytotoxicity of vehicle or TNF-α treated myobundles. Data are expressed as mean±SEM of four donors with four biological replicates. (TIFF 41093 kb)
10439_2019_2263_MOESM3_ESM.tif (157 kb)
Supplemental Figure S3 NF-κB activation trends upward with time in a 2D setting but there is no difference after 120 hours in myobundles. Either 0.1% BSA (vehicle control) or 1000 U/mL TNF-α was added daily at the onset of differentiation to (A) 2D human myoblasts over a span of 96 hours and (B) human myobundles differentiated for 120 hours. Translocation to the nucleus was measure by immunofluorescence. NF-κB is held in an inactive state in the cytoplasm and upon activation is shuttled to the nucleus. Here, it can bind to promoter sites to regulate transcription of target genes. Therefore, nuclear localization is an indicator of NF-κB activation. Data are expressed as mean±SEM of four donors with four biological replicates. (TIFF 156 kb)
10439_2019_2263_MOESM4_ESM.pdf (40 kb)
Supplementary material 4 (PDF 40 kb)

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Copyright information

© Biomedical Engineering Society 2019

Authors and Affiliations

  • Brittany N. J. Davis
    • 1
  • Jeffrey W. Santoso
    • 1
  • Michaela J. Walker
    • 1
  • Catherine E. Oliver
    • 1
  • Michael M. Cunningham
    • 2
  • Christian A. Boehm
    • 3
  • Danielle Dawes
    • 1
  • Samantha L. Lasater
    • 1
  • Kim Huffman
    • 4
    • 5
  • William E. Kraus
    • 4
    • 5
    • 6
  • George A. Truskey
    • 1
    • 7
    Email author
  1. 1.Department of Biomedical EngineeringDuke UniversityDurhamUSA
  2. 2.Department of MedicineUniversity of North Carolina at Chapel HillChapel HillUSA
  3. 3.Department of Textile TechnologyRWTH Aachen UniversityAachenGermany
  4. 4.Duke Molecular Physiology InstituteDuke University Medical CenterDurhamUSA
  5. 5.Department of MedicineDuke University Medical CenterDurhamUSA
  6. 6.Department of CardiologyDuke University Medical CenterDurhamUSA
  7. 7.DurhamUSA

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