PGC-1alpha plays a functional role in exercise-induced mitochondrial biogenesis and angiogenesis but not fiber-type transformation in mouse skeletal muscle

Abstract

Endurance exercise stimulates peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) expression in skeletal muscle, and forced expression of PGC-1alpha changes muscle metabolism and exercise capacity in mice. However, it is unclear if PGC-1alpha is indispensible for endurance exercise-induced metabolic and contractile adaptations in skeletal muscle. In this study, we showed that endurance exercise-induced expression of mitochondrial enzymes (cytochrome oxidase IV and cytochrome c) and increases of platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31)-positive endothelial cells in skeletal muscle, but not IIb-to-IIa fiber-type transformation, were significantly attenuated in muscle-specific Pgc-1alpha knockout mice. Interestingly, voluntary running effectively restored the compromised mitochondrial integrity and superoxide dismutase 2 (SOD2) protein expression in skeletal muscle in Pgc-1alpha knockout mice. Thus, PGC-1alpha plays a functional role in endurance exercise-induced mitochondrial biogenesis and angiogenesis, but not IIb-to-IIa fiber-type transformation in mouse skeletal muscle, and the improvement of mitochondrial morphology and antioxidant defense in response to endurance exercise may occur independently of PGC-1alpha function. We conclude that PGC-1alpha is required for complete skeletal muscle adaptations induced by endurance exercise in mice.

Fig. 1.

Voluntary exercise training in muscle-specific peroxisome proliferator-activated receptor γ coactivator-1α gene (Pgc-1α) knockout (MKO) and wild-type (WT) mice. A: gel images of PCR products amplified from genomic DNA from WT (Pgc-1α +/+:Myog-Cre +/+ or Pgc-1α fl/+:Myog-Cre +/+) and MKO (Pgc-1α fl/fl:Myog-Cre Cre/+) mice. B: total RNA from plantaris muscles of WT and MKO mice were assayed for mRNA expression of Pgc-1α, Pgc-1β, estrogen-related receptor α (Esrra), NADH dehydrogenase (ubiquinone) Fe-S protein 1 (Ndufs1), and vascular endothelial growth factor (Vegf) by real-time PCR. 18S ribosomal RNA was used to normalize the expression (n = 6). *P < 0.05, ***P < 0.001. C: representative running activity recording. Six continuous voluntary running activity recordings are shown for WT and MKO mice during the 4 wk of voluntary running. Each of the clusters of running activities (spikes) that correspond to a duration of half-day represents the nocturnal running activity at night. D: daily voluntary running distance in WT and MKO mice during the 4 wk of voluntary running (P = 0.99). Values are means ± SE (n = 7). E: heart weight (normalized by body weight) in sedentary (Sed) and 4-wk endurance exercise-trained (Ex) WT and MKO mice (n = 6–7). *P < 0.05.

Fig. 2.

Immunofluorescence and immunoblot analyses for fiber-type composition in plantaris muscles of sedentary (Sed) and 4-wk endurance exercise-trained (Ex) WT and MKO mice. A: cross sections of plantaris muscles were immunostained for myosin heavy chains (MHC) I (red), IIa (blue), and IIb (green). Bars, 500 μm. All the fibers were counted and measured for cross-sectional area for calculation of fiber-type composition (please see Table 1). B: immunoblot analysis of MHC I, MHC IIa, and MHC IIb in plantaris muscles from sedentary (S) and endurance exercise-trained (E) mice with β-actin as loading control. Soleus muscle (Sol) was used as a positive control for MHC I. C: quantifications of MHC I, MHC IIa, and MHC IIb protein expression in plantaris muscles. Values are means ± SE (n = 6–7). **P < 0.01.

Fig. 3.

Immunoblot analysis for myoglobin and mitochondrial enzymes and immunofluorescence analysis for capillary density in skeletal muscles of sedentary (Sed) and 4-wk endurance exercise-trained (Ex) WT and MKO mice. A: immunoblot analysis of cytochrome oxidase IV (COX IV), cytochrome c (Cyt c), and myoglobin (Mb) protein expression in plantaris muscles from sedentary (S) and endurance exercise trained mice (E) with β-actin as loading control. Each lane was loaded with 40 μg of muscle protein from whole muscle homogenates. B: quantifications of COX IV, Cyt c and Mb protein expression in plantaris muscle (n = 6–7). *P < 0.05, **P < 0.01, ***P < 0.001. C: immunofluorescence staining of endothelial cells with anti-CD31 antibody in cross sections of plantaris muscle. Bars = 100 μm. D: quantifications of capillary density in plantaris muscle (n = 6–7). *P < 0.05, ***P < 0.001, respectively.

Fig. 4.

Transmission electron microscopy (TEM) analysis for mitochondrial morphology and immunoblot analysis for antioxidant enzyme expression in plantaris muscles of sedentary (Sed) and 4-wk endurance exercise-trained (Ex) WT and MKO mice. A: TEM images of longitudinal plantaris muscle sections of glycolytic and oxidative myofibers. Arrows show degenerative mitochondrion. To show detailed structure of some degenerative mitochondria, cropped images are shown with 2.5× magnification of the original images on one corner. Bars = 1 μm. B: quantification of abnormal mitochondria in ≥14 randomly acquired images as a percentage of total mitochondria. *P < 0.05, ***P < 0.001, respectively. C: immunoblot analysis of superoxide dismutase 2 (SOD2) protein expression in plantaris muscles from sedentary (S) and endurance exercise trained mice (E) with β-actin as loading control. Each lane was loaded with 40 μg of muscle protein from whole muscle homogenates. D: quantifications of SOD2 protein expression in plantaris muscle (n = 5–7). *P < 0.05.