ABSTRACT
BACKGROUND: Mitochondrial biogenesis is crucial for myogenic differentiation and regeneration of skeletal muscle tissue and is tightly controlled by the peroxisome proliferator-activated receptor-γ co-activator 1 (PGC-1) signaling network. In the present study, we hypothesized that inactivation of glycogen synthase kinase (GSK)-3ß, previously suggested to interfere with PGC-1 in non-muscle cells, potentiates PGC-1 signaling and the development of mitochondrial biogenesis during myogenesis, ultimately resulting in an enhanced myotube oxidative capacity. METHODS: GSK-3ß was inactivated genetically or pharmacologically during myogenic differentiation of C2C12 muscle cells. In addition, m. gastrocnemius tissue was collected from wild-type and muscle-specific GSK-3ß knock-out (KO) mice at different time-points during the reloading/regeneration phase following a 14-day hind-limb suspension period. Subsequently, expression levels of constituents of the PGC-1 signaling network as well as key parameters of mitochondrial oxidative metabolism were investigated. RESULTS: In vitro, both knock-down as well as pharmacological inhibition of GSK-3ß not only increased expression levels of important constituents of the PGC-1 signaling network, but also potentiated myogenic differentiation-associated increases in mitochondrial respiration, mitochondrial DNA copy number, oxidative phosphorylation (OXPHOS) protein abundance and the activity of key enzymes involved in the Krebs cycle and fatty acid ß-oxidation. In addition, GSK-3ß KO animals showed augmented reloading-induced increases in skeletal muscle gene expression of constituents of the PGC-1 signaling network as well as sub-units of OXPHOS complexes compared to wild-type animals. CONCLUSION: Inactivation of GSK-3ß stimulates activation of PGC-1 signaling and mitochondrial biogenesis during myogenic differentiation and reloading of the skeletal musculature.
Subject(s)
Glycogen Synthase Kinase 3 beta/physiology , Muscle Development/physiology , Muscle, Skeletal/physiology , Organelle Biogenesis , Animals , Cell Differentiation/physiology , Cell Line , Disease Models, Animal , Female , Glycogen Synthase Kinase 3 beta/antagonists & inhibitors , Glycogen Synthase Kinase 3 beta/genetics , Hindlimb Suspension/adverse effects , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle, Skeletal/cytology , Muscular Atrophy/etiology , Muscular Atrophy/pathology , Myoblasts/cytology , Myoblasts/physiology , Oxidative Phosphorylation/drug effects , Pyridines/pharmacology , Pyrimidines/pharmacology , Signal Transduction/drug effects , Signal Transduction/physiology , Transcription Factors/metabolismABSTRACT
BACKGROUND: Aberrant skeletal muscle mitochondrial oxidative metabolism is a debilitating feature of chronic diseases such as chronic obstructive pulmonary disease, type 2 diabetes and chronic heart failure. Evidence in non-muscle cells suggests that glycogen synthase kinase-3ß (GSK-3ß) represses mitochondrial biogenesis and inhibits PPAR-γ co-activator 1 (PGC-1), a master regulator of cellular oxidative metabolism. The role of GSK-3ß in the regulation of skeletal muscle oxidative metabolism is unknown. AIMS: We hypothesized that inactivation of GSK-3ß stimulates muscle oxidative metabolism by activating PGC-1 signaling and explored if GSK-3ß inactivation could protect against physical inactivity-induced alterations in skeletal muscle oxidative metabolism. METHODS: GSK-3ß was modulated genetically and pharmacologically in C2C12 myotubes in vitro and in skeletal muscle in vivo. Wild-type and muscle-specific GSK-3ß knock-out (KO) mice were subjected to hind limb suspension for 14days. Key constituents of oxidative metabolism and PGC-1 signaling were investigated. RESULTS: In vitro, knock-down of GSK-3ß increased mitochondrial DNA copy number, protein and mRNA abundance of oxidative phosphorylation (OXPHOS) complexes and activity of oxidative metabolic enzymes but also enhanced protein and mRNA abundance of key PGC-1 signaling constituents. Similarly, pharmacological inhibition of GSK-3ß increased transcript and protein abundance of key constituents and regulators of mitochondrial energy metabolism. Furthermore, GSK-3ß KO animals were protected against unloading-induced decrements in expression levels of these constituents. CONCLUSION: Inactivation of GSK-3ß up-regulates skeletal muscle mitochondrial metabolism and increases expression levels of PGC-1 signaling constituents. In vivo, GSK-3ß KO protects against inactivity-induced reductions in muscle metabolic gene expression.
Subject(s)
Glycogen Synthase Kinase 3 beta/genetics , Glycogen Synthase Kinase 3 beta/metabolism , Muscle, Skeletal/metabolism , Animals , Cell Line , Cell Respiration/physiology , Enzyme Activation , Gene Expression Profiling , Gene Knockdown Techniques , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitochondria/genetics , Mitochondria/metabolism , Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/enzymology , Oxidative Phosphorylation , Signal Transduction , Transcription Factors/metabolism , Up-RegulationABSTRACT
Myogenic differentiation involves myoblast fusion and induction of muscle-specific gene expression, which are both stimulated by pharmacological (LiCl), genetic, or IGF-I-mediated GSK-3ß inactivation. To assess whether stimulation of myogenic differentiation is common to ligand-mediated GSK-3ß inactivation, myoblast fusion and muscle-specific gene expression were investigated in response to Wnt-3a. Moreover, crosstalk between IGF-I/GSK-3ß/NFATc3 and Wnt/GSK-3ß/ß-catenin signaling was assessed. While both Wnt-3a and LiCl promoted myoblast fusion, muscle-specific gene expression was increased by LiCl, but not by Wnt-3a or ß-catenin over-expression. Furthermore, LiCl and IGF-I, but not Wnt-3a, increased NFATc3 transcriptional activity. In contrast, ß-catenin-dependent transcriptional activity was increased by Wnt-3a and LiCl, but not IGF-I. These results for the first time reveal a segregated regulation of myoblast fusion and muscle-specific gene expression following stimulation of myogenic differentiation in response to distinct ligand-specific signaling routes of GSK-3ß inactivation.
