Myoblasts were seeded into 12-well plates at a concentration of 10^4 cells/well. Stable transfection was performed by the use of the jetPEI transfection reagent and a total of 2 mg linearized plasmid DNA per well.

Skeletal muscles show a high plasticity to cope with various physiological demands. Different muscle types can be distinguished by the force, endurance, contraction/relaxation kinetics (fast-twitch vs. slow-twitch muscles), oxidative/glycolytic capacity, and also with respect to Ca(2)(+)-signaling components. Changes in Ca(2)(+) signaling and associated Ca(2)(+)-dependent processes are thought to underlie the high adaptive capacity of muscle fibers. Here we investigated the consequences and the involved mechanisms caused by the ectopic expression of the Ca(2)(+)-binding protein parvalbumin (PV) in C2C12 myotubes in vitro, and conversely, the effects caused by its absence in in fast-twitch muscles of parvalbumin null-mutant (PV(-)/(-)) mice in vivo. The absence of PV in fast-twitch muscle tibialis anterior (TA) resulted in an increase in the peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha) and of its positive regulator, the deacetylase sirtuin 1 (SIRT1). TA muscles from PV(-)/(-) mice also have an increased mitochondrial volume. Mild ionophore treatment of control (PV-devoid) C2C12 myotubes causing a moderate elevation in [Ca(2)(+)](c) resulted in an increase in mitochondrial volume, together with elevated PGC-1alpha and SIRT1 expression levels, whilst it increased PV expression levels in myotubes stably transfected with PV. In PV-expressing myotubes the mitochondrial volume, PGC-1alpha and SIRT1 were significantly lower than in control C2C12 myotubes already at basal conditions and application of ionophore had no effect on either one. SIRT1 activation causes a down-regulation of PV in transfected myotubes, whilst SIRT1 inhibition has the opposite effect. We conclude that PV expression and mitochondrial volume in muscle cells are inversely regulated via a SIRT1/PGC-1alpha signaling axis.