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Cited by 172Muscle Physiology and DisordersAdipose Tissue and MetabolismCardiovascular and exercise physiology

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p38γ Mitogen-Activated Protein Kinase Is a Key Regulator in Skeletal Muscle Metabolic Adaptation in Mice

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Pogozelski, Andrew R., Geng, Tuoyu, Li, Ping, Yin, Xinhe, Lira, Vitor A., Zhang, Mei, Chi, Jen-Tsan, Yan, Zhen·2009·PLoS ONE
DOI 10.1371/journal.pone.0007934PMC PMC2775956
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Abstract

Regular endurance exercise induces skeletal muscle contractile and metabolic adaptations, conferring salutary health benefits, such as protection against the metabolic syndrome. The plasticity of skeletal muscle has been extensively investigated, but how the adaptive processes are precisely controlled is largely unknown. Using muscle-specific gene deletion in mice, we now show that p38gamma mitogen-activated protein kinase (MAPK), but not p38alpha and p38beta, is required for endurance exercise-induced mitochondrial biogenesis and angiogenesis, whereas none of the p38 isoforms are required for IIb-to-IIa fiber-type transformation. These phenotypic findings were further supported by microarray and real-time PCR analyses revealing contractile activity-dependent p38gamma target genes, including peroxisome proliferator-activated receptor gamma co-activator-1alpha (Pgc-1alpha) and vascular endothelial growth factor (Vegf), in skeletal muscle following motor nerve stimulation. Gene transfer-mediated overexpression of a dominant negative form of p38gamma, but not that of p38alpha or p38beta, blocked motor nerve stimulation-induced Pgc-1alpha transcription. These findings provide direct evidence for an obligated role of p38gamma MAPK-PGC-1alpha regulatory axis in endurance exercise-induced metabolic adaptation, but not contractile adaptation, in skeletal muscle.

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Figures

Figure 1 Muscle-specific deletion of the p38α, p38β or p38γ gene does not affect endurance exercise-induced fiber-type transformation. Wild type, p38α, p38β, and p38γ MKO mice were subjected to 4 weeks of voluntary running (Ex) or sedentary cage activity (Sed) followed by fiber-type and immunoblot analyses in plantaris muscles. A) PCR of genomic DNA with the appropriate primers (see Materials and Methods) for the loxP flanked p38 alleles in wild type (WT), heterozygous (+/−) and homozygous (−/−) mice with loxP-flanked p38 alleles. Only the homozygous mice with the Cre transgene (not shown) were considered p38 muscle-specific knockout (MKO) mice; B) Immunoblot analysis for p38α and p38γ protein in plantaris muscles of p38 MKO mice in comparison with the wild type littermates. α-tubulin was used as a loading control; and C) Images of immunofluorescence staining of plantaris muscle sections with antibodies against myosin heavy chain IIb (Green), IIa (Blue) and I (Red). Appreciable increases in the percentage of type IIa fibers with concurrent decreases in type IIb fibers are noted in Ex group compared with Sed group among all four genetic backgrounds. The quantitative data is presented in Table 1.
Figure 1 Muscle-specific deletion of the p38α, p38β or p38γ gene does not affect endurance exercise-induced fiber-type transformation. Wild type, p38α, p38β, and p38γ MKO mice were subjected to 4 weeks of voluntary running (Ex) or sedentary cage activity (Sed) followed by fiber-type and immunoblot analyses in plantaris muscles. A) PCR of genomic DNA with the appropriate primers (see Materials and Methods) for the loxP flanked p38 alleles in wild type (WT), heterozygous (+/−) and homozygous (−/−) mice with loxP-flanked p38 alleles. Only the homozygous mice with the Cre transgene (not shown) were considered p38 muscle-specific knockout (MKO) mice; B) Immunoblot analysis for p38α and p38γ protein in plantaris muscles of p38 MKO mice in comparison with the wild type littermates. α-tubulin was used as a loading control; and C) Images of immunofluorescence staining of plantaris muscle sections with antibodies against myosin heavy chain IIb (Green), IIa (Blue) and I (Red). Appreciable increases in the percentage of type IIa fibers with concurrent decreases in type IIb fibers are noted in Ex group compared with Sed group among all four genetic backgrounds. The quantitative data is presented in Table 1.CC BY · PubMed Central
Figure 2 Muscle-specific deletion of the p38γ gene attenuates endurance exercise-induced metabolic adaptation. Wild type, p38α, p38β, and p38γ MKO mice were subjected to 4 weeks of voluntary running (Ex) with sedentary mice (Sed) as control, and plantaris muscles were harvested for immunofluorescence-based capillary density and immunoblot analyses. A) Representative images of COX IV and Cyt c proteins in plantaris muscle. Lines divide the images from different gels. Appreciable increases in COX IV and Cyt c are noted in Ex group compared with Sed group in WT, p38α MKO and p38β MKO mice, but not in p38γ MKO mice; B) Quantitative analysis of COX IV in plantaris muscles (n = 5–8); C) Quantitative analysis of Cyt c in plantaris muscles (n = 5–8). * and *** denote p<0.05 and 0.001, respectively. D) Images of immunofluorescence staining of plantaris muscle sections with antibodies against CD31. Appreciable increases in capillary density are noted in Ex group compared with Sed group in WT, p38α MKO and p38β MKO mice, but not in p38γ MKO mice; and E) Quantitative analysis of capillary density in the whole plantaris muscles (n = 5–8). * denotes p<0.05.
Figure 2 Muscle-specific deletion of the p38γ gene attenuates endurance exercise-induced metabolic adaptation. Wild type, p38α, p38β, and p38γ MKO mice were subjected to 4 weeks of voluntary running (Ex) with sedentary mice (Sed) as control, and plantaris muscles were harvested for immunofluorescence-based capillary density and immunoblot analyses. A) Representative images of COX IV and Cyt c proteins in plantaris muscle. Lines divide the images from different gels. Appreciable increases in COX IV and Cyt c are noted in Ex group compared with Sed group in WT, p38α MKO and p38β MKO mice, but not in p38γ MKO mice; B) Quantitative analysis of COX IV in plantaris muscles (n = 5–8); C) Quantitative analysis of Cyt c in plantaris muscles (n = 5–8). * and *** denote p<0.05 and 0.001, respectively. D) Images of immunofluorescence staining of plantaris muscle sections with antibodies against CD31. Appreciable increases in capillary density are noted in Ex group compared with Sed group in WT, p38α MKO and p38β MKO mice, but not in p38γ MKO mice; and E) Quantitative analysis of capillary density in the whole plantaris muscles (n = 5–8). * denotes p<0.05.CC BY · PubMed Central
Figure 3 Overexpression of dominant negative p38γ, but not that of p38α and p38β, blocks contractility-induced Pgc-1α mRNA expression. Plasmid DNA (100 µg) containing an empty vector (pCI-neo) or FLAG-tagged dominant negative forms of p38α (p38α (AF)), p38β (p38β(AF)), or p38γ (p38γ(AF)) were injected into right and left tibialis anterior muscles followed by electric pulse-mediated gene transfer (described in Materials and Methods). After 10 days of recovery, motor nerve stimulation (10 Hz, 2 hours) was performed for the left tibialis anterior muscle, and both the stimulated and the contralateral control tibialis anterior muscles were harvested for analyses for transgene expression, signaling molecule activation and Pgc-1α mRNA expression. A) Fluorescence image showing the efficacy (∼60%) of gene transfer in tibialis anterior muscle (outline in red). The scale bar equals 200 µ; B) Immunoblot analysis for the tibialis anterior muscles 10 days after gene transfer showing expression of FLAG-tagged p38α (AF), p38β(AF), and p38γ(AF) compared with the tibialis anterior muscle transfected with pCI-neo control plasmid. Lines divide images from different gels. Non-specific bands (NB) were labeled; and C) Real-time PCR analysis showing that motor nerve stimulation results in significant induction of Pgc-1α mRNA in tibialis anterior muscles transfected with pCI-neo, p38α (AF) or p38β(AF), which is blocked by overexpression of p38γ(AF). The data was normalized by 18S ribosomal RNA (n = 6–12). *, ** and *** denote p<0.05, 0.01 and 0.001, respectively.
Figure 3 Overexpression of dominant negative p38γ, but not that of p38α and p38β, blocks contractility-induced Pgc-1α mRNA expression. Plasmid DNA (100 µg) containing an empty vector (pCI-neo) or FLAG-tagged dominant negative forms of p38α (p38α (AF)), p38β (p38β(AF)), or p38γ (p38γ(AF)) were injected into right and left tibialis anterior muscles followed by electric pulse-mediated gene transfer (described in Materials and Methods). After 10 days of recovery, motor nerve stimulation (10 Hz, 2 hours) was performed for the left tibialis anterior muscle, and both the stimulated and the contralateral control tibialis anterior muscles were harvested for analyses for transgene expression, signaling molecule activation and Pgc-1α mRNA expression. A) Fluorescence image showing the efficacy (∼60%) of gene transfer in tibialis anterior muscle (outline in red). The scale bar equals 200 µ; B) Immunoblot analysis for the tibialis anterior muscles 10 days after gene transfer showing expression of FLAG-tagged p38α (AF), p38β(AF), and p38γ(AF) compared with the tibialis anterior muscle transfected with pCI-neo control plasmid. Lines divide images from different gels. Non-specific bands (NB) were labeled; and C) Real-time PCR analysis showing that motor nerve stimulation results in significant induction of Pgc-1α mRNA in tibialis anterior muscles transfected with pCI-neo, p38α (AF) or p38β(AF), which is blocked by overexpression of p38γ(AF). The data was normalized by 18S ribosomal RNA (n = 6–12). *, ** and *** denote p<0.05, 0.01 and 0.001, respectively.CC BY · PubMed Central
Figure 4 Muscle-specific deletion of the p38γ gene results in alterations in endurance exercise-induced gene expression. Wild type and p38γ KO mice were subjected to motor nerve stimulation (10 Hz, 2 hours) via the deep peroneal nerve, which innervates the tibialis anterior muscle. One hour following motor nerve stimulation, both the stimulated tibialis anterior and the contralateral control tibialis anterior muscles were harvested for total RNA isolation and analyzed by real-time PCR and Affymetrix microarray analyses. A) Real-time PCR analysis for Pgc-1α, Pgc-1β, Vegf, Nrf-1, Nrf-2 and Dscr1 mRNA (n = 5–6). *, ** and *** denote p<0.05, 0.01 and 0.001, respectively; B) Hierarchical analysis for the 932 genes with expression variations of at least 2 fold in one sample, showing the effects of motor nerve stimulation; and C) Motor-nerve stimulation-induced changes in gene expression in wild type and p38 KO mice are compared after 601 genes were selected based on expression variations of at least 2 fold in one sample. The Pgc-1α gene is among the genes with reduced induction in response to motor nerve stimulation in MKO mice.
Figure 4 Muscle-specific deletion of the p38γ gene results in alterations in endurance exercise-induced gene expression. Wild type and p38γ KO mice were subjected to motor nerve stimulation (10 Hz, 2 hours) via the deep peroneal nerve, which innervates the tibialis anterior muscle. One hour following motor nerve stimulation, both the stimulated tibialis anterior and the contralateral control tibialis anterior muscles were harvested for total RNA isolation and analyzed by real-time PCR and Affymetrix microarray analyses. A) Real-time PCR analysis for Pgc-1α, Pgc-1β, Vegf, Nrf-1, Nrf-2 and Dscr1 mRNA (n = 5–6). *, ** and *** denote p<0.05, 0.01 and 0.001, respectively; B) Hierarchical analysis for the 932 genes with expression variations of at least 2 fold in one sample, showing the effects of motor nerve stimulation; and C) Motor-nerve stimulation-induced changes in gene expression in wild type and p38 KO mice are compared after 601 genes were selected based on expression variations of at least 2 fold in one sample. The Pgc-1α gene is among the genes with reduced induction in response to motor nerve stimulation in MKO mice.CC BY · PubMed Central
Figure 5 Signaling and transcriptional control of endurance exercise-induced contractile adaptation and metabolic adaptation. This is a schematic presentation of a hypothetical model for endurance exercise-induced signaling/transcriptional control of skeletal muscle adaptation. Endurance exercise on one hand induces activation of the Ca2+-dependent calcineurin (CnA)-NFAT pathway in control of fiber-type transformation and on the other hand activates p38γ, which promotes PGC-1α activity and expression in control of mitochondrial biogenesis and angiogenesis.
Figure 5 Signaling and transcriptional control of endurance exercise-induced contractile adaptation and metabolic adaptation. This is a schematic presentation of a hypothetical model for endurance exercise-induced signaling/transcriptional control of skeletal muscle adaptation. Endurance exercise on one hand induces activation of the Ca2+-dependent calcineurin (CnA)-NFAT pathway in control of fiber-type transformation and on the other hand activates p38γ, which promotes PGC-1α activity and expression in control of mitochondrial biogenesis and angiogenesis.CC BY · PubMed Central

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Metadata

Authors
Pogozelski, A. R., Geng, T., Li, P., Yin, X., Lira, V. A., Zhang, M., Chi, J.-T., & Yan, Z.
Affiliations
  • Duke Medical Center (US)
  • University of Virginia (US)
Corresponding author
Yan, Zhen
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2009-11-20
Volume
4
Issue
11
Pages
e7934
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0007934
PMC
PMC2775956
OpenAlex
W2060618287
Cited by
164
References
49
Citations by year
2026: 6 · 2025: 3 · 2024: 6 · 2023: 7 · 2022: 4 · 2021: 4 · 2020: 12 · 2019: 9 · 2018: 6 · 2017: 7 · 2016: 10 · 2015: 16 · 2014: 17 · 2013: 18 · 2012: 15
FWCI
5.24
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • Howard Hughes Medical Institute
  • Boehringer Ingelheim
  • National Institutes of Health
  • National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01AR050429, AR 050429)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Muscle Physiology and Disorders
  • Health Sciences › Medicine › Physiology (medical) › Adipose Tissue and Metabolism
  • Health Sciences › Medicine › Physiology (medical) › Cardiovascular and exercise physiology
Keywords
p38γ MAPK, endurance exercise, mitochondrial biogenesis, angiogenesis, PGC-1α, vascular endothelial growth factor, muscle fiber type shift, motor nerve stimulation
MeSH terms
Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha, Animals, Genes, Dominant, Male, Mice, Inbred C57BL, Muscle Contraction, Neovascularization, Pathologic, Physical Conditioning, Animal, Transcription Factors, Trans-Activators, Gene Expression Regulation, Enzymologic, Muscle, Skeletal, Protein Isoforms, Oligonucleotide Array Sequence Analysis, MAP Kinase Signaling System, Vascular Endothelial Growth Factor A, p38 Mitogen-Activated Protein Kinases, Mice

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