Journal of Muscle Research and Cell Motility

, Volume 33, Issue 5, pp 327–337 | Cite as

Mild stress of caffeine increased mtDNA content in skeletal muscle cells: the interplay between Ca2+ transients and nitric oxide

  • Shuzhe Ding
  • Joanna R. Contrevas
  • Andrey Y. Abramov
  • Zhengtang Qi
  • Michael R. Duchen
Original Paper


Caffeine increases mitochondrial biogenesis in myotubes by evoking Ca2+ 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 Ca2+ transients and NO releases remain unclear. Herein, we tested the hypothesis that caffeine-evoked Ca2+ transients triggered NO production to increase mtDNA in skeletal muscle cells. Ca2+ 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 Ca2+ transients evoked by caffeine (5 mM) till the end of recording, when Fura-2 indicated that Ca2+ 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 Ca2+ transients is required for caffeine-induced mtDNA biogenesis. Additionally, Ca2+ 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 [Ca2+]i in these treated cells. There may be an important interplay between NO and Ca2+ transients in intracellular signaling system involving NOS, AMPK and MEK.


mtDNA Mitochondrial biogenesis Nitric oxide AMPK Ca2+ transients Caffeine 



The authors would like to acknowledge the other colleagues in Department of Physiology, University College London for the technical support and reagents. This work was also supported by grants from the National Natural Science Foundation of China (Grant No. 30871212 & 31171142).


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Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Shuzhe Ding
    • 1
    • 2
  • Joanna R. Contrevas
    • 3
  • Andrey Y. Abramov
    • 4
  • Zhengtang Qi
    • 1
    • 2
  • Michael R. Duchen
    • 4
  1. 1.The Key Laboratory of Adolescent Health Assessment and Exercise InterventionMinistry of Education of China, East China Normal UniversityShanghaiChina
  2. 2.College of Physical Education and HealthEast China Normal UniversityShanghaiChina
  3. 3.Institute of NeurologyUniversity College LondonLondonUK
  4. 4.Department of Physiology, Medical Science FacultyUniversity College LondonLondonUK

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