Sequential broadside application of 100 µM FCCP at two separate regions in the same FDB fibre.

<p> FCCP is applied sequentially to two regions of a fibre (<b>A,</b> indicated by the white areas labelled <b>a</b> and <b>b</b> to the left of the fibre) and induces release of TMRE from mitochondria only in the area exposed to FCCP. Panel <b>B</b> shows an enlargement of region <b>a</b> and panel <b>D</b> shows an enlargement of region <b>b</b>. Regions of interest (ROI 1 to 3) used to extract intensity traces for <b>C</b> and <b>E</b> are shown in <b>B</b>_measure and <b>D</b>_measure at rest (Con), at the end of 120 s of FCCP (FCCP) and 130 s after removal of FCCP (Post). Panels <b>C</b> and <b>E</b> are plots of the changes in the TMRE F/F₀ at the three ROI sites before, during and after the application of FCCP to the fibre. In mitochondria at site 1, F/F₀ falls to a minimum of ∼0.3 after 110–120 s. At site 2, F/F₀ also declines with a time lag of about 20 s. Importantly at site 3 that is only 50 µm from the site of FCCP application, the TMRE signal does not decrease but rather increases during and after the period of FCCP application. The coloured bar to the right in <b>A</b> shows the LUT from low (blue) to high (white) values applied to each pixel in the image. Red vertical calibration bar in <b>A</b> represents 50 µm.</p

Typical effect of H₂O₂ application on the MitoSOX Red signal in an intact single FDB muscle fibre.

<p>Each panel <b>A–E</b> shows a magnification of the end of the fibre where H₂O₂ was applied and the white box in the lower right of each panel shows the whole length of the fibre. Importantly, in the two other fibres also seen in the white box, there is no change in the MitoSOX signal during the H₂O₂ application. <b>A</b> shows the FDB fibre at rest, and at two (<b>B</b>) and four minutes (<b>C</b>) after the start of H₂O₂ application. The coloured bar to the left in <b>A</b> shows the LUT from low (black) to high (white) values applied to each pixel in the image. <b>D</b> shows where the H₂O₂ was applied and <b>E</b> shows the sites where the MitoSOX signal was measured. The graph in <b>F</b> shows the time course of the changes in mitochondrial MitoSOX signal in the muscle fibre, H₂O₂ application was continuous from time 0. White horizontal calibration bar in <b>C</b> represents 50 µm.</p

Schematic diagram of the multifunctional pipette used in the experiments.

<p><b>A.</b> Cartoon of the general layout with four wells (Solutions, numbered 1 to 4) which can store separate chemicals for delivery through a single channel in the pipette tip and two wells (Switch) which store the waste sucked through the pair of channels on the side of the pipette tip. <b>B.</b> The key elements in the pipette tip shown in more detail with a solution exiting through the central channel and removed through the paired vacuum suction channels to the side of the central channel. <b>C.</b> View of the pipette placed close to the end of a muscle fibre during an actual experiment. The red and blue arrows indicate delivery and removal of solution that define the delivery zone and the end of the fibre sits in front of the central channel. Black calibration bar in the upper right of <b>C</b> represents 75 µm.</p

Local application of FCCP causes a transient increase in resting cytosolic [Ca²⁺].

<p>Panel <b>A</b> shows typical fluo-3 fluorescence before (Con), during (FCCP) and after (Post) focal application of 100 µM FCCP (Sites, white rectangle) and twitch contractions evoked in the same fibre after FCCP (Twitches) in a single FDB muscle fibre. Note that FCCP induced a clear increase in fluo-3 signal only at site 1 (FCCP) which reversed completely after washout of FCCP (Post). <b>B</b> is a graph of mean F/F₀ at 90 to 120 s exposure to FCCP (black bars) and at 120 s after removal of the FCCP (white bars). Note that the increase in fluo-3 F/F₀ caused by exposure to FCCP is small compared to the increase seen in response to electrically evoked responses (Twitches). The coloured bar to the right in <b>A</b>_Twitches shows the LUT from low (blue) to high (white) values applied to each pixel in the image. Values shown are mean ± sem (n = 9), * indicates significantly different from resting F/F₀, p<0.01. Red horizontal calibration bar in <b>A</b>_Sites represents 50 µm.</p

Additional file 2: Figure S2. of Impaired Ca2+ release contributes to muscle weakness in a rat model of critical illness myopathy

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

Additional file 1: Figure S1. of Impaired Ca2+ release contributes to muscle weakness in a rat model of critical illness myopathy

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 110 kDa refers to the protein marker size in the ladder lane (PDF 5931 kb

Additional file 3: Figure S2. of Intracellular Ca2+-handling differs markedly between intact human muscle fibers and myotubes

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

Additional file 4: Figure S3. of Intracellular Ca2+-handling differs markedly between intact human muscle fibers and myotubes

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 < 0.05 relative to myoblasts, and number sign denotes P < 0.05 relative to myotubes. (B) mRNA quantities of the different MHC isoforms in human muscle fibers and myotubes. Data are presented as the mean ± SEM. Aterisk denotes P < 0.05 relative to MHC IIx, and number sign denotes P < 0.05 relative to MHC IIa

Additional file 2: Figure S1. of Intracellular Ca2+-handling differs markedly between intact human muscle fibers and myotubes

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 < 0.05

Additional file 1: Table S1. of Intracellular Ca2+-handling differs markedly between intact human muscle fibers and myotubes

Patient characteristics