Wessel Theeuwes

Skeletal muscle tissue has a remarkably high plasticity. This is reflected by its large capacity to adapt its mass and oxidative phenotype (OXPHEN; defined as the proportion of oxidative muscle fibers and mitochondrial oxidative capacity) in response to, amongst others, changes in physical activity or muscle loading. In Chapter 1, the molecular pathways regulating skeletal muscle OXPHEN are introduced. This primarily encompasses the peroxisome proliferator-activated receptor-γ co-activator-1 (PGC-1)α signaling network which has been convincingly shown to be a pivotal pathway in the regulation of mitochondrial biogenesis not only in skeletal muscle but in a wide variety of cell types. In addition, this chapter introduces glycogen synthase kinase (GSK)-3β and its role in the regulation of PGC-1α in non-muscle cells. This led to the main objective of this thesis: ‘To investigate the role of GSK-3β in oxidative substrate metabolism and mitochondrial biogenesis in skeletal muscle’. The models that were used to investigate this are presented and physical (in)activity is introduced as one of the most potent external triggers affecting muscle OXPHEN. Furthermore, Chapter 1 introduces the involvement of GSK-3β in the regulation of skeletal muscle mass, e.g. muscle protein turnover and post-natal myogenesis.

In Chapter 2, we investigated if GSK-3β inactivation during muscle reloading (after a period of muscle unloading) is essential for improvements in myogenesis, protein turnover signaling, PGC-1α signaling and subsequent expression of oxidative phosphorylation (OXPHOS) sub-units. We address this hypothesis using whole-body constitutively active (C.A.) GSK‑3α/β knock-in and wild-type mice and investigated the soleus muscle of the hind-limb under baseline conditions, directly after 14-days of hind-limb suspension (HLS; a disuse-induced atrophy model) and during reloading of the hind-limbs. No consistent or significant alterations in reloading-induced changes in muscle mass, protein turnover, post-natal myogenesis nor in the regulation of muscle OXPHEN were observed between the two genotypes. In this chapter, we conclude that GSK-3 inactivation is dispensable for the above-mentioned processes during muscle reloading after a period of inactivity. However, subtle but consistent differences are observed at baseline between the two genotypes suggesting suppression of protein turnover signaling, PGC-1α signaling and mRNA expression of several OXPHOS sub-units resulting from constitutive activation of GSK-3.

These findings led to the hypothesis that inactivation of GSK-3β increases gene expression of PGC-1α, which subsequently induces mitochondrial biogenesis and gene expression of OXPHOS sub-units in adult muscle (Chapter 3). To address this hypothesis, we used pharmacological and genetic approaches to inhibit GSK-3β in fully differentiated C2C12 murine myotubes, as an in vitro analogue of myofibers. In addition, we used muscle-specific GSK-3β knock-out (KO) mice at baseline and directly after 14-days of HLS. Largely in line with our findings in the C.A. GSK-3β knock-in mice (Chapter 2), overexpression of GSK-3β did not alter the expression of PGC-1α or OXPHOS sub-units in C2C12 myotubes. However, pharmacologic and genetic inhibition of GSK-3β potently increases mRNA and protein content of PGC-1α and OXPHOS sub-units in C2C12 myotubes. This is accompanied by increased levels of mitochondrial (mt)DNA. Furthermore, we reveal that increased gene expression of OXPHOS sub-units mediated by inhibition of GSK-3β requires PGC-1α by deploying double knock-down experiments. In addition, muscle-specific GSK-3β KO protects against unloading-induced loss of gene expression of components of the PGC-1α signaling cascade and OXPHOS sub-units. In conclusion, in this chapter we identified that GSK-3β inactivation increases PGC-1α levels in muscle cells and subsequently induces mitochondrial biogenesis and expression of sub-units of OXPHOS complexes.

Several molecular mechanisms control the oxidative capacity of the muscle during both maintenance as well as regeneration of adult skeletal muscle. Impairments in these regulatory mechanisms contribute to the development of skeletal muscle abnormalities. In Chapter 4, we therefore investigated if inactivation of GSK-3β also potentiates the PGC-1α signaling cascade during myogenic differentiation and during recovery of inactivity-induced muscle atrophy. We report that GSK-3β inhibition increases the abundance of key constituents of the PGC-1α signaling pathway and increases expression of OXPHOS sub-units during myogenic differentiation of C2C12 myoblast into myotubes, which is in line with the effects of inhibition of GSK-3β in fully differentiated myotubes that we describe in Chapter 3. Ultimately, knock down of GSK-3β during myogenic differentiation results in enhanced mitochondrial respiration at the end of the myogenic differentiation program. In addition to these findings, in vivo experiments reveals that muscle-specific GSK-3β KO potentiates reloading-induced inductions in gene expression of components of the PGC-1α signaling and OXPHOS sub-units after a period of inactivity. Overall, we conclude that inactivation of GSK-3β potentiates the PGC-1α signaling, resulting in mitochondrial biogenesis and mitochondrial respiration during myogenic differentiation and muscle recovery after a period of physical inactivity.

In Chapter 5, we aimed to elucidate the molecular mechanism by which inactivation of GSK-3β increases Pgc-1α mRNA abundance in skeletal muscle cells. We used fully differentiated C2C12 myotubes to fundamentally underpin this underlying molecular mechanism. PGC-1α promoter activity enhances following inactivation of GSK-3β while chromatin accessibility of the PGC-1α promoter remained unaltered, indicating a transcriptionally-controlled mechanism. Subsequently, several transcription factors known to be involved in the transcriptional control of PGC-1α were pharmacologically or genetically inhibited in C2C12 myotubes in order to investigate their possible involvement in transcriptional regulation of PGC-1α mediated by inactivation of GSK-3β. Inhibition of GSK-3β did not alter myocyte enhancer factor 2 (MEF2) transcriptional activity, indicating that MEF2 isoforms are likely not involved in enhanced Pgc-1α transcription mediated by inactivation of GSK-3β. Furthermore, while knock-down of GSK-3β increased estrogen-related receptor (ERR) expression levels and potentiated transcription of its down-stream targets, ERR transcription factors are not essential for increased Pgc-1α levels mediated by inhibition of GSK-3β. Interestingly, inhibition of GSK-3 activity results in nuclear translocation of transcription factor EB (TFEB). Furthermore, increased Pgc-1α gene expression and activation of the PGC-1α promoter mediated by inactivation of GSK-3β require TFEB. In addition, mutation of a TFEB binding sequence located on the proximal PGC-1α promoter blocked PGC-1α promoter activation induced by inactivation of GSK-3. Overall, we report that inactivation of GSK-3β causes nuclear translocation of TFEB, which is required for induction of PGC-1α promoter activation and subsequent transcription of the Pgc-1α gene.

Finally, in Chapter 6, the overall results of this thesis are discussed and placed in a broader perspective. We review the potential role of GSK-3β as key node in the regulation of skeletal muscle mass and OXPHEN in the context of disease-related factors that might be associated with increased activity of GSK-3β in skeletal muscle including physical inactivity, malnutrition and hypoxia. From a clinical perspective, the therapeutic potential of GSK-3β inhibitors or TFEB agonists is highlighted to treat or prevent muscle abnormalities during ageing and in chronic diseases such as chronic obstructive pulmonary disease, chronic heart failure, chronic kidney disease and type II diabetes (diseases that are often characterized by both loss of muscle mass and deterioration of muscle oxidative capacity). Furthermore, we highlight our novel finding that inactivation of GSK-3β phosphorylates TFEB, resulting in its nuclear translocation, which results in increased promoter activation and gene expression of PGC-1α. Ultimately, inactivation of GSK-3β potentiates skeletal muscle oxidative substrate metabolism and mitochondrial biogenesis via this process.