title: TGF-β1-mediated differentiation of fibroblasts is associated with increased mitochondrial content and cellular respiration.
abstract: OBJECTIVE
  Cytokine-dependent activation of fibroblasts to myofibroblasts, a key event in fibrosis, is accompanied by phenotypic changes with increased secretory and contractile properties dependent on increased energy utilization, yet changes in the energetic profile of these cells are not fully described. We hypothesize that the TGF-β1-mediated transformation of myofibroblasts is associated with an increase in mitochondrial content and function when compared to naive fibroblasts.
  
  
  METHODS
  Cultured NIH/3T3 mouse fibroblasts treated with TGF-β1, a profibrotic cytokine, or vehicle were assessed for transformation to myofibroblasts (appearance of α-smooth muscle actin [α-SMA] stress fibers) and associated changes in mitochondrial content and functions using laser confocal microscopy, Seahorse respirometry, multi-well plate reader and biochemical protocols. Expression of mitochondrial-specific proteins was determined using western blotting, and the mitochondrial DNA quantified using Mitochondrial DNA isolation kit.
  
  
  RESULTS
  Treatment with TGF-β1 (5 ng/mL) induced transformation of naive fibroblasts into myofibroblasts with a threefold increase in the expression of α-SMA (6.85 ± 0.27 RU) compared to cells not treated with TGF-β1 (2.52 ± 0.11 RU). TGF-β1 exposure increased the number of mitochondria in the cells, as monitored by membrane potential sensitive dye tetramethylrhodamine, and expression of mitochondria-specific proteins; voltage-dependent anion channels (0.54 ± 0.05 vs. 0.23 ± 0.05 RU) and adenine nucleotide transporter (0.61 ± 0.11 vs. 0.22 ± 0.05 RU), as well as mitochondrial DNA content (530 ± 12 μg DNA/106 cells vs. 307 ± 9 μg DNA/106 cells in control). TGF-β1 treatment was associated with an increase in mitochondrial function with a twofold increase in baseline oxygen consumption rate (2.25 ± 0.03 vs. 1.13 ± 0.1 nmol O2/min/106 cells) and FCCP-induced mitochondrial respiration (2.87 ± 0.03 vs. 1.46 ± 0.15 nmol O2/min/106 cells).
  
  
  CONCLUSIONS
  TGF-β1 induced differentiation of fibroblasts is accompanied by energetic remodeling of myofibroblasts with an increase in mitochondrial respiration and mitochondrial content.
authors: Sheikh Khalifa bin Hamad Al Thani Center for Integrative Research on Cardiovascular Aging, Aurora Research Institute, Aurora Health Care, Milwaukee, Wisconsin, 53215, United States of America.
authors: Ulugbek
authors: U
authors: Negmadjanov
authors: Sheikh Khalifa bin Hamad Al Thani Center for Integrative Research on Cardiovascular Aging, Aurora Research Institute, Aurora Health Care, Milwaukee, Wisconsin, 53215, United States of America.
authors: Zarko
authors: Z
authors: Godic
authors: Sheikh Khalifa bin Hamad Al Thani Center for Integrative Research on Cardiovascular Aging, Aurora Research Institute, Aurora Health Care, Milwaukee, Wisconsin, 53215, United States of America.
authors: Farhan
authors: F
authors: Rizvi
authors: Sheikh Khalifa bin Hamad Al Thani Center for Integrative Research on Cardiovascular Aging, Aurora Research Institute, Aurora Health Care, Milwaukee, Wisconsin, 53215, United States of America.
authors: Larisa
authors: L
authors: Emelyanova
authors: Sheikh Khalifa bin Hamad Al Thani Center for Integrative Research on Cardiovascular Aging, Aurora Research Institute, Aurora Health Care, Milwaukee, Wisconsin, 53215, United States of America.
authors: Gracious
authors: G
authors: Ross
authors: Laboratory of Immunology, Aurora Health Care, Milwaukee, Wisconsin, 53215, United States of America.
authors: John
authors: J
authors: Richards
authors: Sheikh Khalifa bin Hamad Al Thani Center for Integrative Research on Cardiovascular Aging, Aurora Research Institute, Aurora Health Care, Milwaukee, Wisconsin, 53215, United States of America.
authors: Ekhson L
authors: EL
authors: Holmuhamedov
authors: Sheikh Khalifa bin Hamad Al Thani Center for Integrative Research on Cardiovascular Aging, Aurora Research Institute, Aurora Health Care, Milwaukee, Wisconsin, 53215, United States of America; Aurora Cardiovascular Services, Aurora Health Care, Milwaukee, Wisconsin, 53215, United States of America.
authors: Arshad
authors: A
authors: Jahangir
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects*; D019069:Cell Respiration / Q000502:physiology*; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D007124:Immunoenzyme Techniques; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D041681:NIH 3T3 Cells; D012333:RNA, Messenger / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
chemical_list: D024101:Mitochondrial Proteins; D012333:RNA, Messenger; D053773:Transforming Growth Factor beta1