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Typical examples of the negative controls obtained for Na+ channels, DHPR, RyR and SERCA1 immunohistochemical staining (PDF 871 kb)

Coomassie protein staining. Membranes used for western blot analysis of Na+ channels, DHPR (left panel), RyR1and SERCA1 (right panel). Lanes are marked as SHAM (S) or ICU (I); the value 110Â kDa refers to the protein marker size in the ladder lane (PDF 5931 kb)

mRNA expression of slow type skeletal muscle/β-cardiac, type IIa and type IIx MHCs in human muscle fibers, myotubes and myoblasts. The mRNA quantity of MHC I, MHC IIa, and MHC IIx differs markedly between human muscle fibers, myotubes and myoblasts. (A) mRNA quantities of MHC I, MHC IIa, and MHC IIx in human muscle fibers, myotubes, and myoblasts. Data are arbitrary units where the values have been adjusted so that the mRNA quantities of the different MHC isoforms are approximately equal in muscle fibers so to better visualize the differences within each MHC isoform between the different cell types. Data are presented as the mean ± SEM. Aterisk denotes P

Confirmation of reproducibility of [Ca2+]i transients and force production in human muscle fibers. Typical example of reproducibility of [Ca2+]i transients and force production following electrical stimulation in a human intercostal muscle fiber. (A) Percentage of peak ΔR/R0 (representing [Ca2+]i) and (B) force production in a human intercostal muscle fiber following a single 70 Hz 350 ms train of current pulses. Blue lines represent recordings following the first tetanus 30 min after Indo-1 injection, and red lines represent recordings 8 min thereafter.

Confirmation of myotube formation. (A) Typical example of desmin staining (red) and DAPI (blue) showing myotube formation at 8 days of differentiation. Scale bars indicate 200 μm. (B) mRNA quantity of myogenin, a myogenic transcription factor involved in the terminal differentiation of myotubes, in myoblasts (dashed bar) and myotubes (black bar). Data are presented as the mean ± SEM. Asterisk denotes P

Patient characteristics.

The contractile performance of skeletal muscle declines during intense activities, i.e. fatigue develops. Fatigued muscle can enter a state of prolonged low-frequency force depression (PLFFD). PLFFD can be due to decreased tetanic free cytosolic [Ca(2+) ] ([Ca(2+) ]i ) and/or decreased myofibrillar Ca(2+) sensitivity. Increases in reactive oxygen and nitrogen species (ROS/RNS) may contribute to fatigue-induced force reductions. We studied whether pharmacological ROS/RNS inhibition delays fatigue and/or counteracts the development of PLFFD. Mechanically isolated mouse fast-twitch fibres were fatigued by sixty 150 ms, 70 Hz tetani given every 1 s. Experiments were performed in standard Tyrode solution (control) or in the presence of: NADPH oxidase (NOX) 2 inhibitor (gp91ds-tat); NOX4 inhibitor (GKT137831); mitochondria-targeted antioxidant (SS-31); nitric oxide synthase (NOS) inhibitor (l-NAME); the general antioxidant N-acetylcysteine (NAC); a cocktail of SS-31, l-NAME and NAC. Spatially and temporally averaged [Ca(2+) ]i and peak force were reduced by ∼20% and ∼70% at the end of fatiguing stimulation, respectively, with no marked differences between groups. PLFFD was similar in all groups, with 30 Hz force being decreased by ∼60% at 30 min of recovery. PLFFD was mostly due to decreased tetanic [Ca(2+) ]i in control fibres and in the presence of NOX2 or NOX4 inhibitors. Conversely, in fibres exposed to SS-31 or the anti ROS/RNS cocktail, tetanic [Ca(2+) ]i was not decreased during recovery so PLFFD was only caused by decreased myofibrillar Ca(2+) sensitivity. The cocktail also increased resting [Ca(2+) ]i and ultimately caused cell death. In conclusion, ROS/RNS-neutralizing compounds did not counteract the force decline during or after induction of fatigue.

Double discharges (doublets) of motor neurones at the onset of contractions increase both force and rate of force development during voluntary submaximal contractions. The purpose of this study was to examine the role of doublet discharges on force and myoplasmic free [Ca(2+)] ([Ca(2+)]i) during repeated fatiguing contractions, using a stimulation protocol mimicking the in vivo activation pattern during running. Individual intact fibres from the flexor digitorum brevis muscle of mice were stimulated at 33°C to undergo 150 constant-frequency (five pulses at 70 Hz) or doublet (an initial, extra pulse at 200 Hz) contractions at 300 ms intervals. In the unfatigued state, doublet stimulation resulted in a transient (∼10 ms) approximate doubling of [Ca(2+)]i, which was accompanied by a greater force-time integral (∼70%) and peak force (∼40%) compared to constant frequency contractions. Moreover, doublets markedly increased force-time integral and peak force during the first 25 contractions of the fatiguing stimulation. In later stages of fatigue, addition of doublets increased force production but the increase in force production corresponded to only a minor portion of the fatigue-induced reduction in force. In conclusion, double discharges at the onset of contractions effectively increase force production, especially in early stages of fatigue. This beneficial effect occurs without additional force loss in later stages of fatigue, indicating that the additional energy cost induced by doublet discharges to skeletal muscle is limited.

Calcineurin, a Ca(2+)-calmodulin-dependent protein phosphatase (PP2B) is one of the links between Ca(2+) signals and regulation of gene transcription in cardiac muscle. We studied the Ca(2+) signal specificity of calcineurin activation experimentally and with modelling. In the rat atrial preparation, an increase in pacing frequency increased nuclear activity of the calcineurin-sensitive transcription factor, nuclear factor of activated T-cells (NFAT), 2-fold in a cyclosporin A (CsA)-sensitive manner. In line with this, modelling results predicted that the frequency of cardiac Ca(2+) transients encodes the stimulus for calcineurin activation. We further observed experimentally that calcineurin inhibition by CsA modulated Ca(2+) release in a Ca(2+)-dependent manner. CsA had no effect on [Ca(2+)](i) at a pacing frequency of 1 Hz but it significantly suppressed the amplitude of Ca(2+) transients, systolic [Ca(2+)](i) and time averaged [Ca(2+)](i) at 6 Hz. Calcineurin had a differential role in the expression of immediate-early genes B-type natriuretic peptide (BNP) and c-fos. CsA inhibited the pacing-induced BNP gene expression, whereas pacing alone had no effect on the expression of c-fos. However, in the presence of CsA, c-fos mRNA levels were significantly augmented by increased pacing frequency. These results show that frequency-dependent calcineurin activation has a specific role in [Ca(2+)](i) regulation and gene expression, constantly recruited by varying cardiac Ca(2+) signals.