title: Cessation of cyclic stretch induces atrophy of C2C12 myotubes.
abstract: Cyclic stretch of differentiated myotubes mimics the loading pattern of mature skeletal muscle. We tested a cell culture model of disuse atrophy by the cessation of repetitive bouts of cyclic stretch in differentiated C2C12 myotubes. Myotubes were subjected to cyclic strain (12%, 0.7 Hz, 1 h/d) on collagen-I-coated Bioflex plates using a computer-controlled vacuum stretch apparatus (Flexcell Int.) for 2 (2dSTR) or 5 (5dSTR) consecutive days. Control cultures were maintained in the Bioflex plates without cyclic stretch for 2d or 5d. Additionally, some cultures were stretched for 2 d followed by cessation of stretch for 3d (2dSTR3dCES). Cyclic stretching (5dSTR) increased myotube diameter and overall myotube area by ~2-fold (P<0.05) compared to non-stretched controls, while cessation of stretch (2dSTR3dCES) resulted in ~80% smaller myotubes than 5dSTR cells, and 40-50% smaller than non-stretched controls (P<0.05). Further, the calpain-dependent cleavage products of αII-spectrin (150 kDa) and talin increased (3.5-fold and 2.2-fold, respectively; P<0.05) in 2dSTR3dCES myotubes, compared to non-stretched controls. The 1h cyclic stretching protocol acutely increased the phosphorylation of Akt (+4.5-fold; P<0.05) and its downstream targets, FOXO3a (+4.2-fold; P<0.05) and GSK-3β (+1.8-fold; P<0.05), which returned to baseline by 48 h after cessation of stretch. Additionally, nitric oxide production increased during stretch and co-treatment with the NOS inhibitor, l-NAME, inhibited the effects of stretch and cessation of stretch. We conclude that cessation of cyclic stretching causes myotube atrophy by activating calpains and decreasing activation of Akt. Stretch-induced myotube growth, as well as activation of atrophy signaling with cessation of stretch, are dependent on NOS activity.
authors.affiliation: Center for Exercise Science, Department of Applied Physiology & Kinesiology, University of Florida, Gainesville, FL 32611, USA.
authors.forename: Quinlyn A
authors.initials: QA
authors.lastname: Soltow
authors.forename: Elizabeth H
authors.initials: EH
authors.lastname: Zeanah
authors.forename: Vitor A
authors.initials: VA
authors.lastname: Lira
authors.forename: David S
authors.initials: DS
authors.lastname: Criswell
mesh_terms: D000818:Animals; D001696:Biomechanical Phenomena; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D048429:Cell Size; D003593:Cytoplasm / Q000201:enzymology / Q000378:metabolism; D004789:Enzyme Activation; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000378:metabolism; D007150:Immunohistochemistry; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000201:enzymology / Q000378:metabolism / Q000473:pathology*; D009129:Muscle Tonus; D009133:Muscular Atrophy / Q000378:metabolism / Q000473:pathology*; D018995:Myosin Heavy Chains / Q000378:metabolism; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000037:antagonists & inhibitors / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000378:metabolism; D024507:Nonmuscle Myosin Type IIA / Q000378:metabolism; D010766:Phosphorylation; D059748:Proteolysis; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction; D013314:Stress, Mechanical; D013997:Time Factors
chemical_list: D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; C474033:FoxO3 protein, mouse; D009569:Nitric Oxide; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D051057:Proto-Oncogene Proteins c-akt; D024507:Nonmuscle Myosin Type IIA; D018995:Myosin Heavy Chains; D019331:NG-Nitroarginine Methyl Ester