Figure 3: On sepsis satellite cells display altered but hyperactive mitochondria.

(a–e) Quantification by RT–qPCR of nuclear genes in the time course of sepsis in the control (no injury no CLP) at 6 h, 24 h, 48 h and 4 days (d) post induction of sepsis; n=6 per time point; (a) expression of hypoxia-inducible factor1 (Hif1a); (b) NADPH oxidase 1 (Nox1); (c,d) expression of antioxidant enzymes Sod1 and Sod2; (e) peroxisome proliferator-activated receptor coactivator 1 (PGC1a). (f,g) Quantification of immunofluorescence intensity by confocal microscopy image acquisition and ImageJ analysis. The levels of carbonylated (antibody anti-dinitrophenyl) and nitrated (antibody anti-nitrotyrosine) proteins (resulting essentially from reactive oxidative and nitrosative species, respectively) were measured in controls, 6 h, 24 h, 48 h and 4 days post induction of sepsis. Representative immunostaining (three-dimensional-reconstructed cells) are displayed. Scale bar, 5 μm. (h) Confocal microscopy immunofluorescence intensity quantification of TOM22 (mitochondrial outer membrane protein) and (i) confocal microscopy intensity of MitoTracker Deep Red staining probe in control (healthy) and septic mice. Once acquired by confocal microscopy the images were analysed using the mean grey value ImageJ plugin. (j) Mitochondrial DNA content (mtDNA) in control and septic SC. (k) Agarose gel electrophoresis of long PCR amplifications on sorted SC mtDNA in controls and septic Tg:Pax7nGFP 6 h, 24 h, 48 h and 4 days post sepsis showing mtDNA size alterations. Left panel, amplification of a fragment of expected size (9,898 bp) from positions 7,998–16,299/1 (conventional end/origin of circular mtDNA) and 1–14,400 of the mtDNA; the image on this panel is intentionally highly contrasted to underscore multiple bands, and also the low intensity of the expected band (9,898 bp) in sepsis samples compared with controls; centre panel, schematic representation of genes and regulatory regions in the human mtDNA, with the coordinates of PCR-amplified regions; right panel, amplification of a fragment of expected size 9,677 bp from positions 15,377 to 5,701 of the mtDNA. Note that size alterations in cells from septic mice are present in one (left panel) but not the other PCR-amplified fragment. (l) Tetramethylrhodamine ethylamine (TMRE; mitochondrial membrane potential) levels. (m) Relative percentage of glycolytic and mitochondrial (OXPHOS) ATP in controls (no injury no CLP) and 24 h post sepsis. (n) Percentage of ATP relative to control in controls (n=6) and septic Tg:Pax7nGFP at 24 h (n=6). (o) Basal mitochondrial respiration of SC in non-septic (control) and 24 h post sepsis evaluated with Seahorse XFe96 analyser. Unless specified data are represented as mean±s.d. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; NS, not significant, compared with the respective control (Mann–Whitney test).