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

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-1 alpha. 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.