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. 2011;6(12):e28290.
doi: 10.1371/journal.pone.0028290. Epub 2011 Dec 8.

Skeletal muscle-specific expression of PGC-1α-b, an exercise-responsive isoform, increases exercise capacity and peak oxygen uptake

Miki Tadaishi  1 , Shinji Miura, Yuko Kai, Yutaka Kano, Yuichi Oishi, Osamu Ezaki
Affiliations

Skeletal muscle-specific expression of PGC-1α-b, an exercise-responsive isoform, increases exercise capacity and peak oxygen uptake

Miki Tadaishi et al. PLoS One. 2011.

Abstract

Background: Maximal oxygen uptake (VO(2max)) predicts mortality and is associated with endurance performance. Trained subjects have a high VO(2max) due to a high cardiac output and high metabolic capacity of skeletal muscles. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a nuclear receptor coactivator, promotes mitochondrial biogenesis, a fiber-type switch to oxidative fibers, and angiogenesis in skeletal muscle. Because exercise training increases PGC-1α in skeletal muscle, PGC-1α-mediated changes may contribute to the improvement of exercise capacity and VO(2max). There are three isoforms of PGC-1α mRNA. PGC-1α-b protein, whose amino terminus is different from PGC-1α-a protein, is a predominant PGC-1α isoform in response to exercise. We investigated whether alterations of skeletal muscle metabolism by overexpression of PGC-1α-b in skeletal muscle, but not heart, would increase VO(2max) and exercise capacity.

Methodology/principal findings: Transgenic mice showed overexpression of PGC-1α-b protein in skeletal muscle but not in heart. Overexpression of PGC-1α-b promoted mitochondrial biogenesis 4-fold, increased the expression of fatty acid transporters, enhanced angiogenesis in skeletal muscle 1.4 to 2.7-fold, and promoted exercise capacity (expressed by maximum speed) by 35% and peak oxygen uptake by 20%. Across a broad range of either the absolute exercise intensity, or the same relative exercise intensities, lipid oxidation was always higher in the transgenic mice than wild-type littermates, suggesting that lipid is the predominant fuel source for exercise in the transgenic mice. However, muscle glycogen usage during exercise was absent in the transgenic mice.

Conclusions/significance: Increased mitochondrial biogenesis, capillaries, and fatty acid transporters in skeletal muscles may contribute to improved exercise capacity via an increase in fatty acid utilization. Increases in PGC-1α-b protein or function might be a useful strategy for sedentary subjects to perform exercise efficiently, which would lead to prevention of life-style related diseases and increased lifespan.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Skeletal muscle-specific expression of PGC-1α-b.
(A) Total expression of PGC-1α and its target genes, COX2 and COX4, in wild-type (Wt) and PGC-1α-b mice (A line and B line) at 25 weeks of age (n = 3) was measured by quantitative real-time RT-PCR in skeletal muscle (gastrocnemius, Gas) and heart. Values are means ± SE. ***P<0.001 vs. Wt. (B) Total lysates from skeletal muscle (Gas) and heart were subjected to SDS-PAGE followed by Western blot analysis with anti-PGC-1α antibodies (n = 3). Typical blots are shown. For skeletal muscle samples, densitometric analysis was performed on the bands with increased intensity (arrow heads) following skeletal muscle-specific overexpression of PGC-1α-b.
Figure 2
Figure 2. PGC-1α-b induced changes in gene expression in skeletal muscle.
Results of quantitative RT-PCR analysis of transcripts encoding proteins involved in fiber type, glucose metabolism, lipid metabolism, and others in quadriceps from wild-type (Wt) and PGC-1α-b mice (A line and B line) at 8 weeks of age. Values are means ± SE (n = 6–9). * P<0.05, **P<0.01, *** P<0.001 vs. Wt. MHC, myosin heavy chain; GYS, glycogen synthase; PHKA, phosphorylase kinase alpha, PYGM, muscle glycogen phosphorylase; GLUT, glucose transporter; HK, hexokinase; PFKM, muscle phosphofructokinase; PFKFB, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase; PKM, muscle pyruvate kinase; PDK, pyruvate dehydrogenase kinase; LPL, lipoprotein lipase; FATP, fatty acid transport protein; FABP-pm, plasma membrane fatty acid binding protein; FABP, fatty acid binding protein; CPT, carnitine palmitoyltransferase; MCAD, medium chain acyl-CoA dehydrogenase; Mb, myoglobin; VEGF, vascular endothelial growth factor; PDGF, platelet-derived growth factor; nNOS, neuronal nitric oxide synthase; eNOS, endothelial nitric oxide synthase.
Figure 3
Figure 3. Mitochondrial volume and its function in skeletal muscles overexpressing PGC-1α-b.
(A) For estimation of mitochondrial DNA copy number in skeletal muscle (tibialis anterior (TA), extensor digitorum longus (EDL), and soleus (Sol)), the relative mitochondrial DNA copy number from individual mice in each group was calculated as the ratio of COX2 (mitochondrial) to COX4 (nuclear) genes as determined by real-time PCR. The relative mitochondrial DNA copy number was expressed as the percentage of the ratio in wild-type (Wt). CS activity (gastrocnemius (Gas), EDL, and Sol) was normalized to tissue weight. Values are means ± SE of Wt and PGC-1α-b transgenic mice (A-line and B-line) at 10 weeks of age (n = 3–4). *P<0.05, ***P<0.001 vs. Wt. †††P<0.001 vs. Gas. (B) State III respiration rate of the mitochondrial fraction prepared from skeletal muscle (Gas, quadriceps, and TA) was measured in the presence and absence of 2.5 µg/ml oligomycin. CS activity was also measured in the mitochondrial fraction. Each data point is the mean value of the measurements normalized to the protein content of the fraction. Skeletal muscles were sampled from both Wt and PGC-1α-b transgenic mice (A-line and B-line) at 10 weeks of age. Oxidative phosphorylation of the mitochondrial fraction prepared from skeletal muscle was unchanged in mice overexpressing PGC-1α-b in skeletal muscle.
Figure 4
Figure 4. Skeletal muscle-specific expression of PGC-1α-b induced angiogenesis in skeletal muscle.
(A) Transverse frozen sections of TA from wild-type (Wt) and PGC-1α-b transgenic mice at 10–11 weeks of age were immunostained for CD31 (endothelial-specific PECAM). Representative immunostains in the superficial region is shown. (B) Quantification of CD31-positive capillaries/mm2 and capillaries per individual myofiber in superficial and deep regions (n = 3 per group). Data are presented as mean ± SE. *, P<0.05, ****, P<0.001 vs. Wt.
Figure 5
Figure 5. Exercise tolerance, RQ ratio, and calculated glucose and lipid oxidation rate, during exercise.
(A) PGC-1α-b transgenic mice (A-line and B-line, n = 3 and 4) and wild-type littermates (Wt, n = 7) (each 8 weeks old) were exercised by forced running on a treadmill at 10 m/min. The speed increased by 2 m/min every 3 min until exhaustion. Mice ran until exhaustion (exercise tolerance test). Exercise tolerance is shown as a Kaplan-Meier survival curve. A significant difference (P<0.01, log-rank test) was observed between the exercise tolerances of both lines of PGC-1α-b transgenic mice vs. Wt. (B) Oxygen consumption and carbon dioxide production were monitored using an O2/CO2 metabolism measuring system for small animals, which was equipped with an air-tight treadmill chamber. The RQ ratio, and calculated glucose and lipid oxidation rates are shown. Each value is the mean ± SE of 3–7 mice. Statistical significance was calculated as the area under the curve when sedentary and during running (0–36 min). Significant differences were not observed when sedentary for any parameters. P values vs. Wt are shown in the figure in cases where the difference is significant. (C) Peak oxygen uptake as defined by the measurement of oxygen consumption at the point of failure, is shown. *P<0.05 vs. Wt. (D) The RQ ratio, and calculated glucose and lipid oxidation rate are plotted against relative exercise intensity, as estimated by the percentage of mean speed at exhaustion for each genotype. Statistical significance was calculated as the area under the curve using the mean value at rest for each genotype as base line. P values vs. Wt are shown in the figure in cases where the difference is significant.
Figure 6
Figure 6. Skeletal muscle glycogen content and blood lactate concentration before and after exercise.
PGC-1α-b transgenic mice and wild-type littermates (Wt) (each 8 weeks old) were exercised by forced running on a treadmill at 10 m/min. The speed increased 2 m/min every 3 min up to 30 m/min. Mice ran for 30 min. Skeletal muscle (gastrocnemius) glycogen content was measured before (Sed) and after 30 min of exercise (n = 3–5). Values are means ± SE. **P<0.01 vs. Sed. †††P<0.001 vs. Wt. Blood lactate concentration was measured before (Sed) and at 30 min of exercise (n = 4–6). Values are means ± SE. ***P<0.001 vs. Sed. †††P<0.001 vs. Wt.
Figure 7
Figure 7. PGC-1α-b-mediated increase in fatty acid utilization in skeletal muscle improves maximal oxygen uptake and exercise capacity.
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