title: Neutral sphingomyelinase 2 is required for cytokine-induced skeletal muscle calpain activation.
abstract: Calpain contributes to infection-induced diaphragm dysfunction but the upstream mechanism(s) responsible for calpain activation are poorly understood. It is known, however, that cytokines activate neutral sphingomyelinase (nSMase) and nSMase has downstream effects with the potential to increase calpain activity. We tested the hypothesis that infection-induced skeletal muscle calpain activation is a consequence of nSMase activation. We administered cytomix (20 ng/ml TNF-α, 50 U/ml IL-1β, 100 U/ml IFN-γ, 10 μg/ml LPS) to C2C12 muscle cells to simulate the effects of infection in vitro and studied mice undergoing cecal ligation puncture (CLP) as an in vivo model of infection. In cell studies, we assessed sphingomyelinase activity, subcellular calcium levels, and calpain activity and determined the effects of inhibiting sphingomyelinase using chemical (GW4869) and genetic (siRNA to nSMase2 and nSMase3) techniques. We assessed diaphragm force and calpain activity and utilized GW4869 to inhibit sphingomyelinase in mice. Cytomix increased cytosolic and mitochondrial calcium levels in C2C12 cells (P < 0.001); addition of GW4869 blocked these increases (P < 0.001). Cytomix also activated calpain, increasing calpain activity (P < 0.02), and the calpain-mediated cleavage of procaspase 12 (P < 0.001). Procaspase 12 cleavage was attenuated by either GW4869 (P < 0.001), BAPTA-AM (P < 0.001), or siRNA to nSMase2 (P < 0.001) but was unaffected by siRNA to nSMase3. GW4869 prevented CLP-induced diaphragm calpain activation and diaphragm weakness in mice. These data suggest that nSMase2 activation is required for the development of infection-induced diaphragm calpain activation and muscle weakness. As a consequence, therapies that inhibit nSMase2 in patients may prevent infection-induced skeletal muscle dysfunction.
authors.affiliation: Division of Pulmonary, Critical Care and Sleep Medicine, Department of Internal Medicine, University of Kentucky, Lexington, Kentucky; and Center for Muscle Biology, University of Kentucky, Lexington, Kentucky.
authors.forename: Gerald S
authors.initials: GS
authors.lastname: Supinski
authors.affiliation: Division of Pulmonary, Critical Care and Sleep Medicine, Department of Internal Medicine, University of Kentucky, Lexington, Kentucky; and Center for Muscle Biology, University of Kentucky, Lexington, Kentucky.
authors.forename: Alexander P
authors.initials: AP
authors.lastname: Alimov
authors.affiliation: Division of Pulmonary, Critical Care and Sleep Medicine, Department of Internal Medicine, University of Kentucky, Lexington, Kentucky; and Center for Muscle Biology, University of Kentucky, Lexington, Kentucky.
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
authors.affiliation: Division of Pulmonary, Critical Care and Sleep Medicine, Department of Internal Medicine, University of Kentucky, Lexington, Kentucky; and Center for Muscle Biology, University of Kentucky, Lexington, Kentucky.
authors.forename: Xiao-Hong
authors.initials: XH
authors.lastname: Song
authors.affiliation: Division of Pulmonary, Critical Care and Sleep Medicine, Department of Internal Medicine, University of Kentucky, Lexington, Kentucky; and Center for Muscle Biology, University of Kentucky, Lexington, Kentucky lacall2@email.uky.edu.
authors.forename: Leigh A
authors.initials: LA
authors.lastname: Callahan
mesh_terms: D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D001597:Benzylidene Compounds / Q000494:pharmacology; D002154:Calpain / Q000378:metabolism*; D002460:Cell Line; D003964:Diaphragm / Q000201:enzymology / Q000503:physiopathology; D004789:Enzyme Activation; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D053580:Muscle Strength; D018908:Muscle Weakness / Q000201:enzymology / Q000276:immunology / Q000382:microbiology; D018482:Muscle, Skeletal / Q000201:enzymology*; D059748:Proteolysis; D018805:Sepsis / Q000201:enzymology / Q000503:physiopathology; D013108:Sphingomyelin Phosphodiesterase / Q000502:physiology*
chemical_list: D000814:Aniline Compounds; D001597:Benzylidene Compounds; C468773:GW 4869; D008070:Lipopolysaccharides; C491483:Smpd3 protein, mouse; D013108:Sphingomyelin Phosphodiesterase; D002154:Calpain
keywords: calpain; cytokines; diaphragm; sepsis; sphingomyelinase