Table 1.
Average number of fibers analyzed per muscle and per species.
Figure 1.
Verification of the specificity of the PGC-1α antibody.
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−/−).
Figure 4.
Fiber type-specific PGC-1α content in rat soleus muscle and its relation to mitochondrial content and capillarization.
(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) Merge of MHC type IIb & laminin (yellow), 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 = 6 rats, 189±28 fibers analyzed per animal). (K) Quantification of the fiber type-specific SDH stain intensity (n = 6 rats, 173±32 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. (L) Fiber type-specific PGC-1α content relative to type I fibers. (M) Fiber type-specific SDH stain intensity relative to type I fibers. (N) Lead-ATPase stain performed on a serial section to visualize capillaries. (O) Quantification of the fiber type-specific capillary number per fiber perimeter (n = 4 rats, 57±19 fibers analyzed per animal). (K, L, P) Values arising from the same animal are connected by a line. (J–M) **: p<0.01. Statistical comparisons were performed using a paired two-tailed t-test. Scale bar: 50 µm.
Figure 2.
Fiber type-specific PGC-1α content in the mouse gastrocnemius muscle and its relation to mitochondrial content.
(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.
Figure 3.
Fiber type-specific PGC-1α content in the rat Plantaris muscle and its relation to mitochondrial content and capillarization.
(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.
Figure 5.
Fiber type-specific PGC-1α content in human vastus lateralis muscle and its relations to mitochondrial and lipid contents.
(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.
Figure 6.
Fiber type-specific nuclear and cytosolic PGC-1α content in the rat plantaris muscle.
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.