title: Inhibiting c-Jun N-terminal kinase partially attenuates caffeine-dependent cell death without alleviating the caffeine-induced reduction in mitochondrial respiration in C2C12 skeletal myotubes.
abstract: Caffeine is a widely consumed stimulant that has previously been shown to promote cytotoxic stress and even cell death in numerous mammalian cell lines. Thus far there is little information available regarding the toxicity of caffeine in skeletal muscle cells. Our preliminary data revealed that treating C2C12 myotubes with 5 mM caffeine for 6 h increased nuclear fragmentation and reduced basal and maximal oxygen consumption rate (OCR) in skeletal myotubes. The purpose of this study was to further elucidate the pathways by which caffeine increased cell death and reduced mitochondrial respiration. We specifically examined the role of c-Jun N-terminal kinase (JNK), which has previously been shown to simultaneously increase caspase-dependent cell death and reduce mitochondrial respiration in other mammalian cell lines. We found that caffeine promoted a dose-dependent increase in cell death in multinucleated myotubes but did not in mononucleated myoblasts. The addition of 10 μM Z-DEVD-FMK, a specific inhibitor of executioner caspases, completely inhibited caffeine-dependent cell death. Further, the addition of 400 μM dantrolene, a specific ryanodine receptor (RYR) inhibitor, prevented the caffeine-dependent increase in cell death and the reduction in basal and maximal OCR. We also discovered that caffeine treatment significantly increased the phosphorylation of JNK and that the addition of 30 μM SP600125 (JNKi), a specific JNK inhibitor, partially attenuated caffeine-induced cell death without preventing the caffeine-dependent reduction in basal and maximal OCR. Our results suggest that JNK partially mediates the increase in caspase-dependent cell death but does not contribute to reduced mitochondrial respiration in caffeine-treated skeletal muscle cells. We conclude that caffeine increased cell death and reduced mitochondrial respiration in a calcium-dependent manner by activating the RYR and promoting reticular calcium release.
authors.affiliation: Division of Natural Sciences and Engineering, University of South Carolina Upstate, Spartanburg, SC, USA.
authors.forename: R M
authors.initials: RM
authors.lastname: Downs
authors.affiliation: Division of Natural Sciences and Engineering, University of South Carolina Upstate, Spartanburg, SC, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Hughes
authors.affiliation: Department of Biology and Marine Biology, University of North Carolina Wilmington, Wilmington, NC, USA.
authors.forename: S T
authors.initials: ST
authors.lastname: Kinsey
authors.affiliation: Department of Biology and Marine Biology, University of North Carolina Wilmington, Wilmington, NC, USA.
authors.forename: M C
authors.initials: MC
authors.lastname: Johnson
authors.affiliation: Division of Natural Sciences and Engineering, University of South Carolina Upstate, Spartanburg, SC, USA. Electronic address: bbaumgar@uscupstate.edu.
authors.forename: B L
authors.initials: BL
authors.lastname: Baumgarner
mesh_terms: D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D002110:Caffeine / Q000494:pharmacology* / Q000633:toxicity; D053148:Caspase 3 / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D003620:Dantrolene / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051379:Mice; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D018485:Muscle Fibers, Skeletal / Q000187:drug effects* / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology
chemical_list: D000873:Anthracenes; D047428:Protein Kinase Inhibitors; C432165:pyrazolanthrone; D002110:Caffeine; D048031:JNK Mitogen-Activated Protein Kinases; D053148:Caspase 3; D003620:Dantrolene
keywords: Caffeine; Cell death; Mitochondrial respiration; Skeletal muscle; c-Jun N-terminal kinase