Quantitative analysis of the kinetics of intramitochondrial membrane potential (ΔΨ_m) and mitochondrial [Ca²⁺]_m changes in C2C12 WT and PV-clones after KCl-induced depolarization.

<p>Results are from at least 3 independent myotube preparations and number of cells analyzed ranged from 6–16 per genotype (WT and PV-clones) and conditions (100^# and 300^# mM KCl; *p<0.05, **p<0.01).</p>#<p>The concentrations represent values of the added solutions to the perfusion chamber. The actual concentration sensed by the cells is assumed to be at least 2-fold lower.</p

Effect of KCl-induced depolarization and thapsigargin treatment in C2C12 WT and PV-clones on mitochondrial membrane potential, [Ca²⁺]_m and

<p><b>[Ca²⁺]_c.</b> A) Representative (normalized) traces of rhod123-loaded C2C12 cells (WT, black line and PV-clone, gray line) exposed to nominally 100 mM KCl for 15 s starting at 30 s. Higher values indicate a decrease in intramitochondrial membrane potential (Δψ_m). The average basal fluorescence before treatment (0–20 s) was subtracted. B) A similar experiment with cells exposed to nominally 300 mM KCl for 60 s starting at 40 s. The actual KCl concentration sensed by the cells is estimated to be less than half of the nominal concentration due to dilution with basal medium. C) Representative traces of rhod-2-loaded C2C12 cells (WT, black line and PV-clone, gray line) exposed to nominally 300 mM KCl for 60 s starting at 40 s. Higher values indicate an increase in [Ca²⁺]_m. Changes in fluorescence are reported as F/F₀, where F₀ is the baseline reached before drug application. Quantitative data is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044837#pone-0044837-t002" target="_blank">Table 2</a>. D) WT and PV-clones were treated with thapsigargin (1 µM) and [Ca²⁺]_c was recorded with Fura-2; representative traces are shown, quantitative data (n = 9 cells, both from WT and PV-clones from 3 independent experiments) is presented in the Results section. Note the different time scales in A, B&C and D.</p

Expression levels of SIRT1 and SIRT1-dependent regulation of PV expression in C2C12 PV-clones.

<p>Exposure of WT C2C12 to Br-A23187 for 48 h increased SIRT1 expression (arrow) in WT C2C12 cells exposed to Br-A23187 determined by Western blot analysis; quantitative results are shown in the histographs (*: p<0.05) SIRT1 levels in PV-clones were lower, even under control conditions and ionophore treatment didn’t cause a change in SIRT1 expression in PV-clones (*; p<0.05 compared to WT, basal conditions). Only the upper band (arrow) is the specific band corresponding to the expected size of SIRT1 and was used for the quantitative analysis. B) At 48 h post-treatment PV expression levels in C2C12 PV-clones were increased by the ionophores Br-A2319987 and ferutinin or by blocking of SIRT1 activity by sirtinol. On the contrary, addition of resveratrol activating the SIRT1/PGC-1α signaling axis led to a decrease in PV expression (*, p<0.05 vs. untreated C2C12 PV-clones).</p

Volumetric analysis of mitochondrial content in WT and PV-transfected C2C12 cells.

<p>A–C: Confocal images of WT C2C12 cell loaded with Mito Tracker Red (A), Calcein-AM (B), DAPI (C): x–y image Z-stack projection (large image), x–z (bottom) and y-z (right) image. Scale bar is 40 µm. These images were transferred to Imaris 3D to calculate relative mitochondrial volume. D–F: Bar histographs of relative mitochondrial volume (D), cytoplasmic volume (E) and relative nuclear volume (F) in C2C12 WT (light bars) and PV-clones (dark bars) either in control medium (left bars) or incubated for 48 h with Br-A23187 (1 µM; right bars). D) The relative mitochondrial volume was increased in WT C2C12 after ionophore treatment (*; p<0.05). The mitochondrial volume in PV-clones, both in control medium or after 48 h ionophore treatment was lower than in untreated WT C2C12 cells; * p<0.05 vs. WT; n>30 cells). The decrease in mitochondrial volume in PV-clones after ionophore treatment was not significant (n.s.) E) No differences in cytoplasmic volumes were detected. F) The nuclear volume in ionophore-treated WT C2C12 was slightly larger than in untreated WT cells (*; p<0.05).</p

Effect of mild ionophore treatment on PV expression levels in PV-transfected C2C12 cells.

<p>A) Detection of mRNA for PV and GAPDH (for normalization) in control (WT) and PV-transfected myotubes by RT-PCR. No signal was detected in WT myotubes before and after ionophore treatment. B) Ionophore treatment did not significantly affect PV mRNA levels (striped bars) of PV-clones at 24–72 h after treatment in comparison to untreated cells (black bars). C) Protein expression levels of PV and GAPDH in WT and PV-clones subjected to Ca²⁺ ionophore treatment (1 µM Br-A23187 for 48 h (+) or untreated cells (−) determined by Western blot analysis. D) Semi-quantitative analysis of PV Western blot signals in C2C12 PV-clones exposed to Br-A23187 for 24–72 h (striped bars) in comparison to untreated cells (black bars). A significant increase in PV expression levels was observed at 48 and 72 h (*; p<0.05; n = 8 values at each time point). E–F: Relative GFP protein expression in GFP-positive C2C12 clones in total protein extracts. Cells were treated for 48 h with the differentiation medium alone (black bar) or with 1 µM Br-A23187 (striped bar). A representative Western blot from clone PV1 is shown in E. For the normalization, GAPDH and/or the Ponceau Red-staining of the nitrocellulose membrane were used. Values were from 2 or 3 clones (PV1–3) and ≥2 independent experiments/clone. G: Stably-transfected C2C12 cells differentiated to myotubes for 6 days were fixed and immunostained for PV expression. PV in multinucleated myotubes is homogenously expressed throughout the cytoplasm.</p

Expression levels of molecules implicated in excitation/transcription coupling and mitochondria biogenesis in mouse skeletal muscle.

<p>A) Differences in mRNA levels in <i>tibialis anterior</i> (TA) from adult PV+/+ and PV−/− mice determined by RT-PCR. Significant increases were observed for PGC-1α, AMPK, CaN and CaMKII (*; p<0.05). Semi-quantitative Western blot analyses with whole muscle TA protein extracts (B) and TA nuclear extracts from PV+/+ and PV−/− mice (D). Significant increases in PV−/− nuclear PGC-1α and CaN were detected compared to PV+/+ mice (*p<0.05). C) Representative Western blots for PGC-1α and CaN from three PV+/+ and PV−/− mice. Note the stronger signals in extracts from PV−/− mice. E) SIRT1 expression levels in TA from adult mice were increased in PV−/− mice; representative SIRT1 Western blot signals from 2 mice of each genotype (upper panel).</p

Expression levels of PGC-1α and CaN in C2C12 cells treated with Ca²⁺ ionophores.

<p>A) Semi-quantitative analysis for PGC-1α. The immunoreactive band used for the quantification migrated with an apparent M_r of ∼90 kDa (calculated M_r of PGC-1α is 90,588 Da), at an identical position as PGC-1α from TA extracts (Fig. 7C). Mild ionophore treatment (1 µM; 48 h) led to an increase in PGC-1α in WT C2C12 cells (*; p<0.05). In untreated PV-clones, PGC-1α levels were decreased compared to control C2C12 cells (*; p<0.05); ionophore treatment didn’t additionally affect PGC-1α levels (n.s.). In the lower part representative Western blot signals used for the calculations are shown. B) Analysis of CaN expression, other details as in A). CaN expression levels were determined in untreated cells (C) or after incubation with the Ca²⁺ ionophores Br-A23187 (Br-) or ferutinin (Fe) for 48 h. CaN signals were slightly decreased in WT clones (n.s.) and were unchanged in the PV-clones after ionophore treatment. Lower part: Representative CaN Western blot signals for C2C12 WT and PV-clones ± Br-A23187.</p

PCR primer sequences used for RT-PCR.

<p>In order to increase the reliability of PCR results, for certain genes 2 sets of primer pairs were used, either 2 different reverse primers (CaN, CaMKIIδ) or forward primers (PGC-1α).</p

Increased mitochondrial mass determined by FACS analysis in control (WT) C2C12 clones exposed to Br-A23187.

<p>A) FACS histogram of WT C2C12 cells treated for 48 h with Br-A23187 (1 µM) and loaded with Mito Tracker Green. A shift to the right (increased relative fluorescence units (RFU)) was observed in WT C2C12 cells. B) Quantitative analysis of relative mitochondrial mass in WT (light bars) and PV-clones (dark bars), before (left bars) and after ionophore treatment (right bars). After ionophore treatment, the mitochondrial mass was significantly increased in the WT clones (*; p<0.05). In ionophore-treated PV-clones, the relative mitochondrial mass was essentially unchanged, the small decrease was not significant (n.s.). In untreated WT and PV-clones, the small difference was also not significant. Each FACS count was of 20,000 particles (n ≥4 experiments).</p

Expression of TRPV1 and SERCA1 in skeletal muscle.

<p>Left panel: Western blot analysis of total protein extracts (25 µg) with anti-SERCA1 (top panel) and anti-TRPV1 antibodies (low panel) from the following tissues: flexus digitorum brevis/interosseal (FDB/IO), soleus, extensor digitorum longus (EDL), red and white gastrocnemius (R Ga and W Ga) and brain. Right panel: Localization of TRPV1 in sarcotubular membrane fractions (R1, fraction enriched in light SR; R2, fraction enriched in longitudinal SR; R3, fraction containing a mixture of longitudinal SR and terminal cisternae; R4, fraction enriched in terminal cisternae): 30 µg of protein from each fraction was separated on SDS/PAGE.</p