title: Differences in transcriptional patterns of extracellular matrix, inflammatory, and myogenic regulatory genes in myofibroblasts, fibroblasts, and muscle precursor cells isolated from old male rat skeletal muscle using a novel cell isolation procedure.
abstract: Aged skeletal muscle displays increased fibrosis and impaired regeneration. While it is not well characterized how skeletal muscle fibroblasts contribute to these phenomena, transforming growth factor-β1 (TGF-β1) and Delta/Notch signaling have been implicated to influence muscle regeneration. In this study, a unique combination of aging phenotypes is identified in differentiating fibroblasts (myofibroblasts), proliferating fibroblasts, and muscle precursor cells (MPCs) that characterize an impaired regenerative potential observed in aged skeletal muscle. Using a novel dual-isolation technique, that isolates fibroblasts and MPCs from the same rat skeletal muscle sample, and cell culture conditions of 5 % O(2) and 5 % CO(2), we report for the first time that myofibroblasts from 32-mo-old skeletal muscle, compared to 3-mo-old, display increased levels of mRNA for the essential extracellular matrix (ECM) genes, collagen 4α1 (83 % increase), collagen 4α2 (98 % increase), and laminin 2 (113 % increase), as well as increased levels of mRNA for the inflammatory markers, interleukin-6 (4.3-fold increase) and tumor necrosis factor α (3.2-fold increase), and TGF-β1 (84 % increase), whose protein controls proliferation and differentiation. Additionally, we demonstrate that proliferating fibroblasts from 32-mo-old skeletal muscle display increased levels of mRNA for the Notch ligand, Delta 1 (≥2.0-fold increase). Together, these findings suggest that increased expression of ECM and inflammatory genes in myofibroblasts from 32-mo-old skeletal muscle may contribute to the fibrogenic phenotype that impairs regeneration in aged skeletal muscle. Furthermore, we believe the novel dual-isolation technique developed here may be useful in studies that investigate communications among MPCs, fibroblasts, and myofibroblasts in skeletal muscle.
authors.affiliation: Department of Health, Leisure, and Exercise Science, Appalachian State University, 111 Rivers Street, ASU Box 32071, Boone, NC 28608, USA. zwetslootka@appstate.edu
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Zwetsloot
authors.forename: Anders
authors.initials: A
authors.lastname: Nedergaard
authors.forename: Leigh T
authors.initials: LT
authors.lastname: Gilpin
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Childs
authors.forename: Frank W
authors.initials: FW
authors.lastname: Booth
mesh_terms: D000375:Aging / Q000378:metabolism*; D000818:Animals; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D005109:Extracellular Matrix / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D007249:Inflammation / Q000235:genetics / Q000378:metabolism*; D015850:Interleukin-6 / Q000378:metabolism; D007797:Laminin / Q000378:metabolism; D008297:Male; D023421:Models, Animal; D018482:Muscle, Skeletal / Q000166:cytology* / Q000378:metabolism; D058628:Myofibroblasts / Q000166:cytology / Q000378:metabolism*; D018006:Myogenic Regulatory Factors / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D051880:Receptors, Notch / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D014158:Transcription, Genetic; D053773:Transforming Growth Factor beta1 / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D015850:Interleukin-6; D007797:Laminin; D018006:Myogenic Regulatory Factors; D012333:RNA, Messenger; D051880:Receptors, Notch; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha; D003094:Collagen