Experimental determination of oxidation rate, phosphorylation rate and ADP/ATP concentrations.

<p>Our experimental set-up was composed of an oxygraph, a spectrophotometer and a luminometer. An optic fiber, connected to the spectrophotometer, was inserted in the oxygraphic vessel (picture on the top-left hand corner). Mitochondrial oxidation rate was determined using the Clark electrode of the oxygraph. Phosphorylation rate was assessed, with the help of the optic fiber, by the continuous monitoring of NADPH production in the oxygraphic vessel. Samplings were performed at the onset and the end of the recording to assess both ADP and ATP concentrations using a bioluminescence-based assay with the help of a luminometer (picture on the top-right hand corner). For clarity, all parameters that were measured during each experiment are highlighted by colored circles. HK: hexokinase, G6PDH: glucose 6 phosphate dehydrogenase, G6P: glucose 6 phosphate, OM: outer membrane, IM: inner membrane.</p

Typical recording of oxidation rate, phosphorylation rate and ADP/ATP concentrations.

<p>Oxidation and phosphorylation rates were recorded simultaneously in liver and muscle mitochondria oxidizing glutamate+malate+succinate as substrates. Mitochondrial protein concentration in the oxygraphic vessel was 25 µg.ml⁻¹. Steady states of oxygen consumption and phosphorylation rates were obtained using the coupled enzymatic system composed of Glucose (5 mM) - Hexokinase (2.5 U.ml⁻¹, Sigma-Aldrich, H4502) - Glucose-6-phosphotate dehydrogenase (2.5 U.ml⁻¹, Sigma-Aldrich, G6378) - NADP⁺ (1.6 mM). Dashed arrows correspond to the sampling of measurement medium taken from the oxygraphic vessel during each recording for determination of ADP and ATP concentrations.</p

Dependence of oxidation and phosphorylation rates on ADP concentration in liver and muscle mitochondria.

<p>Oxidation and phosphorylation rates were recorded simultaneously in liver (n = 4) and muscle (n = 5) mitochondria oxidizing glutamate+malate+succinate as substrates. True steady state of oxidation and phosphorylation rates were obtained using coupled enzymatic system composed of Glucose (5 mM) - Hexokinase (2.5 U.ml⁻¹, Sigma-Aldrich, H4502) - Glucose-6-phosphotate dehydrogenase (2.5 U.ml⁻¹, Sigma-Aldrich, G6378) - NADP⁺ (1.6 mM). Data presented in panels A and B correspond to absolute oxidation and phosphorylation rates, respectively. Maximal oxidation and phosphorylation rates obtained for muscle and liver mitochondria were 408.0±42.5 vs. 85.2±5.5 nmolO₂.min⁻¹.mg⁻¹ and 1672.8±134.1 vs. 361.3±33.41 nmolATP.min⁻¹.mg⁻¹, respectively. Panels C and D show normalized oxidation and phosphorylation rates, respectively. Data were fitted using the Michaelis-Menten equation presented in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020709#s2" target="_blank">materials and methods</a> section. Data are presented as mean ± SD. Differences were tested using an unpaired bilateral student's t-test. ** p<0.01 between liver and muscle, # p<0.01 vs. KmVox.</p

Changes in the P/O ratio as a function of ADP concentration in liver and muscle mitochondria.

<p>P/O ratio was determined by calculating the phosphorylation to oxidation rates ratio. Data for liver (n = 4) and muscle (n = 5) are presented as mean ± SD. Differences were tested using an unpaired bilateral student's t-test. ** p<0.01 between liver and muscle.</p

Fiber type-specific PGC-1α content in human vastus lateralis muscle and its relations to mitochondrial and lipid contents.

<p>(A–C) in situ immunolabeling of a muscle cross-section for Myosin Heavy Chain (MHC) type IIx & laminin (A), type I (B), Type IIa (C). (D) Merge of MHC type IIx & laminin (green), type I (Blue) and type IIa (red) immunolabeling. (E–F) in situ immunolabeling for PGC-1α (E), and nuclei (F) obtained on a serial cross-section. A merge image of the PGC-1α and nuclei channels of a control cross-section where the PGC-1α primary antibody was omitted is presented in (G). (H) Merge of PGC-1α and nuclei immunolabelings. (I) in situ stain for Succinate DeHydrogenase activity (SDH) obtained on a serial cross-section. (J) Quantification of the fiber type-specific PGC-1α content (n = 7 subjects, 168±97 fibers analyzed per subject). (K) Quantification of the fiber type-specific SDH stain intensity (n = 6 subjects, 147±74 fibers analyzed per subject). Before being analyzed, SDH images were inverted in ImageJ for the quantified signal to be directly proportional to the SDH activity. (J, K) Values arising from the same subject are connected by a line. (L) Fiber type-specific PGC-1α content relative to type I fibers. (M) Fiber type-specific SDH stain intensity relative to type I fibers. (J–M) Fiber types that do not share the same letter are significantly different (p<0.05). Due to the fact that no type IIx fibers were identified in 3 out of the 7 subjects, statistical comparisons were performed using paired two-tailed t-tests. (N–O) in situ immunolabeling of a muscle cross-section for MHC type IIx & laminin (green), type I (Blue) and type IIa (red) (N) and its corresponding Oil Red O stain (a marker of lipid content performed on a muscle serial cross-section (O). (P) Quantifications of the fiber type-specific Oil Red O stain intensity (N = 11). Fiber types that do not share the same letter are significantly different (p<0.05). Due to the fact that no type IIx fibers were identified in 4 out of the 11 subjects, statistical comparisons were performed using paired two-tailed t-tests. Scale bar: 50 µm.</p

The Relationship between Muscle Fiber Type-Specific PGC-1α Content and Mitochondrial Content Varies between Rodent Models and Humans

<div><p>PGC-1α regulates critical processes in muscle physiology, including mitochondrial biogenesis, lipid metabolism and angiogenesis. Furthermore, PGC-1α was suggested as an important regulator of fiber type determination. However, whether a muscle fiber type-specific PGC-1α content exists, whether PGC-1α content relates to basal levels of mitochondrial content, and whether such relationships are preserved between humans and classically used rodent models are all questions that have been either poorly addressed or never investigated. To address these issues, we investigated the fiber type-specific content of PGC-1α and its relationship to basal mitochondrial content in mouse, rat and human muscles using in situ immunolabeling and histochemical methods on muscle serial cross-sections. Whereas type IIa fibers exhibited the highest PGC-1α in all three species, other fiber types displayed a hierarchy of type IIx>I>IIb in mouse, type I = IIx> IIb in rat, and type IIx>I in human. In terms of mitochondrial content, we observed a hierarchy of IIa>IIx>I>IIb in mouse, IIa >I>IIx> IIb in rat, and I>IIa> IIx in human skeletal muscle. We also found in rat skeletal muscle that type I fibers displayed the highest capillarization followed by type IIa >IIx>IIb. Finally, we found in human skeletal muscle that type I fibers display the highest lipid content, followed by type IIa>IIx. Altogether, our results reveal that (i) the fiber type-specific PGC-1α and mitochondrial contents were only matched in mouse, (ii) the patterns of PGC-1α and mitochondrial contents observed in mice and rats do not correspond to that seen in humans in several respects, and (iii) the classical phenotypes thought to be regulated by PGC-1α do not vary exclusively as a function of PGC-1α content in rat and human muscles.</p></div

Verification of the specificity of the PGC-1α antibody.

<p>Representative images of PGC-1α (approx. 100 KDa) and β-tubulin (approx. 50 KDa - loading control) western blots performed in mouse gastrocniemius (M. Gas) rat soleus (R. SOL) and plantaris (R. PL) muscles and muscle homogenate obtained from PGC-1α^−/− mice (M. PGC-1α^−/−).</p

Fiber type-specific PGC-1α content in the mouse gastrocnemius muscle and its relation to mitochondrial content.

<p>(A–C) in situ immunolabeling of a muscle cross-section for Myosin Heavy Chain (MHC) type IIb & laminin (A), type I (B), Type IIa (C). (D) Immunolabeling for MHC type IIx (green) was performed on a serial cross-section. (E) Merge of MHC type IIb & laminin (yellow), type I (Blue) and type IIa (red) immunolabeling (type IIx fibers appear in black). (F–H) in situ immunolabeling for PGC-1α (F), dystrophin (G) and nuclei (H) obtained on a serial cross-section. A merged image of the PGC-1α, dystrophin and nuclei channels of a control cross-section where the PGC-1α and dystrophin primary antibodies were omitted is presented in (I). (J) Merge of PGC-1α, dystrophin and nuclei immunolabelings. (K) in situ stain for Succinate DeHydrogenase activity (SDH) obtained on a serial cross-section. (L) Quantification of the fiber type-specific PGC-1α content (n = 6 mice, 434±65 fibers analyzed per animal). (M) Quantification of the fiber type-specific SDH stain intensity (n = 5 mice, 155±29 fibers analyzed per animal). Before being analyzed, SDH images were inverted in ImageJ for the quantified signal to be directly proportional to the SDH activity. (N) Fiber type-specific PGC-1α content relative to type I fibers. (O) Fiber type-specific SDH stain intensity relative to type I fibers. (L, M) Values arising from the same animal are connected by a line. (L–O) Fiber types that do not share the same letter are significantly different (p<0.05). Statistical comparisons were performed using a one-way anova with repeated measures and a Tukey’s post hoc test. Scale bar: 50 µm.</p

Fiber type-specific nuclear and cytosolic PGC-1α content in the rat plantaris muscle.

<p>Representative nuclei (A) and PGC-1α (B) immunolabeling of an individual fiber (traced and isolated using ImageJ). (C–D) Surface plot of nuclei (C) and PGC-1α (D) of the images shown in (A) and (B) respectively. (E–F) Quantifications of the fiber type-specific nuclear (E) and cytosolic (F) PGC-1α content (performed on 9 fibers for each fiber type). (G) Nuclear to cytosolic PGC-1α ratio. (E–F–G) Fiber types that do not share the same letter are significantly different (p<0.05). Statistical comparisons were performed using a one-way anova and a Tukey’s post hoc test.</p

Fiber type-specific PGC-1α content in the rat Plantaris muscle and its relation to mitochondrial content and capillarization.

<p>(A–C) in situ immunolabeling of a muscle cross-section for Myosin Heavy Chain (MHC) type IIb & laminin (A), type I (B), Type IIa (C). (D) Immunolabeling for MHC type IIx (green) performed on a serial cross-section. (E) Merge of MHC type IIb & laminin (yellow), type I (Blue) and type IIa (red) immunolabeling (type IIx fibers appearing in black). (F–G) in situ immunolabeling for PGC-1α (F), and nuclei (G) obtained on a serial cross-section. A merge image of the PGC-1α and nuclei channels of a control cross-section where the PGC-1α primary antibody was omitted is presented in (H). (I) Merge of PGC-1α and nuclei immunolabelings. (J) in situ stain for Succinate DeHydrogenase activity (SDH) obtained on a serial cross-section. (K) Quantification of the fiber type-specific PGC-1α content (n = 5 rats, 176±42 fibers analyzed per animal). (L) Quantification of the fiber type-specific SDH stain intensity (n = 5 rats, 151±63 fibers analyzed per animal). Before being analyzed, SDH images were inverted in ImageJ for the quantified signal to be directly proportional to the SDH activity. (M) Fiber type-specific PGC-1α content relative to type I fibers. (N) Fiber type-specific SDH stain intensity relative to type I fibers. (O) Lead-ATPase stain performed on a serial section to visualize capillaries. (P) Quantification of the fiber type-specific capillary number per fiber perimeter (n = 4 rats, 63±16 fibers analyzed per animal). (K, L, P) Values arising from the same animal are connected by a line. (K, L, M, N, P) Fiber types that do not share the same letter are significantly different (p<0.05). Statistical comparisons were performed using a one-way anova with repeated measures and a Tukey’s post hoc test. Scale bar: 50 µm.</p