title: Mild stress of caffeine increased mtDNA content in skeletal muscle cells: the interplay between Ca2+ transients and nitric oxide.
abstract: Caffeine increases mitochondrial biogenesis in myotubes by evoking Ca(2+) transients. Nitric oxide (NO) also induces mitochondrial biogenesis in skeletal muscle cells via upregulation of AMP-activated protein kinase (AMPK) activity and PGC-1α. However, the interplay and timing sequence between Ca(2+) transients and NO releases remain unclear. Herein, we tested the hypothesis that caffeine-evoked Ca(2+) transients triggered NO production to increase mtDNA in skeletal muscle cells. Ca(2+) transients were recorded with Fura-2 AM and confocal microscopy; mtDNA staining, mitochondrial membrane potential and NO level were determined using fluorescent probes PicoGreen, tetramethylrhodamine methyl ester (TMRM) and DAF-FM, respectively. In primary cultured myotubes, a subtle and moderate stress of caffeine increased mtDNA exclusively. Mitochondrial membrane potential and mtDNA were increased by 1 mM as well as 5 mM caffeine, whereas 10 mM caffeine did not change the fluorescence intensity of PicoGreen and TMRM. NO level in myocytes increased gradually following the first jump of Ca(2+) transients evoked by caffeine (5 mM) till the end of recording, when Fura-2 indicated that Ca(2+) transients recovered partly and even disappeared. Importantly, nitric oxide synthase (NOS) inhibitor (L-NAME) suppressed caffeine-induced mtDNA biogenesis, whereas NO donor (DETA-NO) increased mtDNA content. These data strongly suggest that caffeine-induced mtDNA biogenesis is dose-sensitive and dependent on a certain level of stress. Further, an increasing level of NO following Ca(2+) transients is required for caffeine-induced mtDNA biogenesis. Additionally, Ca(2+) transients, a usual and first response to caffeine, was either suppressed or attenuated by L-NAME, DETA-NO, AICAR and U0126, suggesting an inability to control [Ca(2+)](i) in these treated cells. There may be an important interplay between NO and Ca(2+) transients in intracellular signaling system involving NOS, AMPK and MEK.
authors.affiliation: The Key Laboratory of Adolescent Health Assessment and Exercise Intervention, Ministry of Education of China, East China Normal University, Shanghai 200241, China. qzht79@163.com
authors.forename: Shuzhe
authors.initials: S
authors.lastname: Ding
authors.forename: Joanna
authors.initials: J
authors.lastname: Riddoch-Contreras
authors.forename: Joanna R
authors.initials: JR
authors.lastname: Contrevas
authors.forename: Andrey Y
authors.initials: AY
authors.lastname: Abramov
authors.forename: Zhengtang
authors.initials: Z
authors.lastname: Qi
authors.forename: Michael R
authors.initials: MR
authors.lastname: Duchen
mesh_terms: D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D002070:Butadienes / Q000494:pharmacology; D002110:Caffeine / Q000494:pharmacology*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D004272:DNA, Mitochondrial / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects / Q000502:physiology; D018485:Muscle Fibers, Skeletal / Q000378:metabolism; D018482:Muscle, Skeletal / Q000187:drug effects* / Q000378:metabolism*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000032:analysis / Q000096:biosynthesis*; D009570:Nitriles / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D012265:Ribonucleotides / Q000494:pharmacology; D013312:Stress, Physiological / Q000187:drug effects*; D014226:Triazenes / Q000494:pharmacology
chemical_list: C105351:1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene; D002070:Butadienes; D004272:DNA, Mitochondrial; D004791:Enzyme Inhibitors; D009570:Nitriles; D012265:Ribonucleotides; D014226:Triazenes; C113580:U 0126; D009569:Nitric Oxide; D000620:Aminoimidazole Carboxamide; D002110:Caffeine; C031143:AICA ribonucleotide; D019331:NG-Nitroarginine Methyl Ester