Involvement of O-GlcNAcylation in the Skeletal Muscle Physiology and Physiopathology: Focus on Muscle Metabolism

Skeletal muscle represents around 40% of whole body mass. The principal function of skeletal muscle is the conversion of chemical energy toward mechanic energy to ensure the development of force, provide movement and locomotion, and maintain posture. This crucial energy dependence is maintained by the faculty of the skeletal muscle for being a central place as a “reservoir” of amino acids and carbohydrates in the whole body. A fundamental post-translational modification, named O-GlcNAcylation, depends, inter alia, on these nutrients; it consists to the transfer or the removal of a unique monosaccharide (N-acetyl-D-glucosamine) to a serine or threonine hydroxyl group of nucleocytoplasmic and mitochondrial proteins in a dynamic process by the O-GlcNAc Transferase (OGT) and the O-GlcNAcase (OGA), respectively. O-GlcNAcylation has been shown to be strongly involved in crucial intracellular mechanisms through the modulation of signaling pathways, gene expression, or cytoskeletal functions in various organs and tissues, such as the brain, liver, kidney or pancreas, and linked to the etiology of associated diseases. In recent years, several studies were also focused on the role of O-GlcNAcylation in the physiology and the physiopathology of skeletal muscle. These studies were mostly interested in O-GlcNAcylation during muscle exercise or muscle-wasting conditions. Major findings pointed out a different “O-GlcNAc signature” depending on muscle type metabolism at resting, wasting and exercise conditions, as well as depending on acute or long-term exhausting exercise protocol. First insights showed some differential OGT/OGA expression and/or activity associated with some differential stress cellular responses through Reactive Oxygen Species and/or Heat-Shock Proteins. Robust data displayed that these O-GlcNAc changes could lead to (i) a differential modulation of the carbohydrates metabolism, since the majority of enzymes are known to be O-GlcNAcylated, and to (ii) a differential modulation of the protein synthesis/degradation balance since O-GlcNAcylation regulates some key signaling pathways such as Akt/GSK3β, Akt/mTOR, Myogenin/Atrogin-1, Myogenin/Mef2D, Mrf4 and PGC-1α in the skeletal muscle. Finally, such involvement of O-GlcNAcylation in some metabolic processes of the skeletal muscle might be linked to some associated diseases such as type 2 diabetes or neuromuscular diseases showing a critical increase of the global O-GlcNAcylation level

Phospho-GlcNAc modulation of slow MLC2 during soleus atrophy through a multienzymatic and sarcomeric complex.

Although calcium is the major regulator of excitation-contraction coupling, myofilament function can also be modulated through post-translational modifications. In particular, phosphorylation and O-GlcNAcylation are key modulators of calcium activation parameters. Among the regulatory proteins of skeletal muscle contraction, the myosin light chain 2 (MLC2) can undergo both types of post-translational modification. During aging or physical inactivity, the phosphorylation status of the slow isoform of MLC2 (sMLC2) does not correlate with calcium sensitivity, suggesting that the O-GlcNAcylation might modulate sMLC2 activity. To increase understanding of the contractile dysfunction associated with muscle atrophy, we studied the phosphorylation/O-GlcNAcylation interplay on the sMLC2. We demonstrate a two-fold decrease of O-GlcNAcylation level on sMLC2 in a rat model of skeletal muscle atrophy (hindlimb unloading), while phosphorylation increased. Both post-translational modifications were mutually exclusive. Their interplay reversed during reloading. The expression of enzymes involved in the phosphorylation and O-GlcNAcylation interplay on sMLC2 was modified on whole protein pattern as well as on myofilament, and was load-dependent. All enzymes were colocalized on the contractile apparatus. Finally, we describe a multienzymatic complex which might finely modulate the phosphorylation/dephosphorylation and O-GlcNAcylation/de-O-GlcNAcylation of sMLC2 that could be involved in the contractile dysfunction of atrophied muscle. Importantly, this complex was localized at the Z-disk, a nodal point of signalling in skeletal muscle

Increasing O-GlcNAcylation level on organ culture of soleus modulates the calcium activation parameters of muscle fibers.

O-N-acetylglucosaminylation is a reversible post-translational modification which presents a dynamic and highly regulated interplay with phosphorylation. New insights suggest that O-GlcNAcylation might be involved in striated muscle physiology, in particular in contractile properties such as the calcium activation parameters. By the inhibition of O-GlcNAcase, we investigated the effect of the increase of soleus O-GlcNAcylation level on the contractile properties by establishing T/pCa relationships. We increased the O-GlcNAcylation level on soleus biopsies performing an organ culture of soleus treated or not with PUGNAc or Thiamet-G, two O-GlcNAcase inhibitors. The enhancement of O-GlcNAcylation pattern was associated with an increase of calcium affinity on slow soleus skinned fibers. Analysis of the glycoproteins pattern showed that this effect is solely due to O-GlcNAcylation of proteins extracted from skinned biopsies. We also characterized the O-GlcNAcylated contractile proteins using a proteomic approach, and identified among others troponin T and I as being O-GlcNAc modified. We quantified the variation of O-GlcNAc level on all these identified proteins, and showed that several regulatory contractile proteins, predominantly fast isoforms, presented a drastic increase in their O-GlcNAc level. Since the only slow isoform of contractile protein presenting an increase of O-GlcNAc level was MLC2, the effect of enhanced O-GlcNAcylation pattern on calcium activation parameters could involve the O-GlcNAcylation of sMLC2, without excluding that an unidentified O-GlcNAc proteins, such as TnC, could be potentially involved in this mechanism. All these data strongly linked O-GlcNAcylation to the modulation of contractile activity of skeletal muscle

Oxygen supplementation to limit hypoxia-induced muscle atrophy in C2C12 myotubes: comparison with amino acid supplement and electrical stimulation.

International audienceIn skeletal muscle, chronic oxygen depletion induces a disturbance leading to muscle atrophy. Mechanical stress (physical exercise) and nutritional supplement therapy are commonly used against loss of muscle mass and undernutrition in hypoxia, while oxygenation therapy is preferentially used to counteract muscle fatigue and exercise intolerance. However, the impact of oxygenation on skeletal muscle cells remains poorly understood, in particular on signalling pathways regulating protein balance. Thus, we investigated the effects of each separated treatment (mechanical stress, nutritional supplementation and oxygenation therapy) on intracellular pathways involved in protein synthesis and degradation that are imbalanced in skeletal muscle cells atrophy resulting from hypoxia. Myotubes under hypoxia were treated by electrical stimulation, amino acids supplement or oxygenation period. Signalling pathways involved in protein synthesis (PI3K-Akt-mTOR) and degradation (FoxO1 and FoxO3a) were investigated, so as autophagy, ubiquitin-proteasome system and myotube morphology. Electrical stimulation and oxygenation treatment resulted in higher myotube diameter, myogenic fusion index and myotubes density until 48 h post-treatment compared to untreated hypoxic myotubes. Both treatments also induced inhibition of FoxO3a and decreased activity of ubiquitin-proteasome system; however, their impact on protein synthesis pathway was specific for each one. Indeed, electrical stimulation impacted upstream proteins to mTOR (i.e., Akt) while oxygenation treatment activated downstream targets of mTOR (i.e., 4E-BP1 and P70S6K). In contrast, amino acid supplementation had very few effects on myotube morphology nor on protein homeostasis. This study demonstrated that electrical stimulation or oxygenation period are two effective treatments to fight against hypoxia-induced muscle atrophy, acting through different molecular adaptations

Multiplexed Detection of O-GlcNAcome, Phosphoproteome, and Whole Proteome within the Same Gel

The cellular diversity of proteins results in part from their post-translational modifications. Among all of them, the O-GlcNAcylation is an atypical glycosylation, more similar to phosphorylation than classical glycosylations. Highly dynamic, reversible, and exclusively localized on cytosolic, nuclear and mitochondrial proteins, O-GlcNAcylation is known to regulate almost all if not all cellular processes. Fundamental for the cell life, O-GlcNAcylation abnormalities are involved in the etiology of several inherited diseases. Assessing to O-GlcNAcylation pattern will permit to get relevant data about the role of O-GlcNAcylation in cell physiology. To get understanding about the role of O-GlcNAcylation, as also considering its interplay with phosphorylation, the O-GlcNAc profiling remains a real challenge for the community of proteomists/glycoproteomists. Due to development of multiplexed proteomics based on fluorescent detection of proteins, there is growing body of evidence about the proteome knowledge’s. We propose herein a multiplexed proteomic strategy to detect O-GlcNAcylated proteins, phosphoproteins, and the whole proteome within the same bidimensional gel. In particular, we investigated the phosphoproteome through the ProQ Diamond staining, while the whole proteome was visualized through Sypro Ruby staining, or after the labeling of proteins with a T-Dye fluorophore. The O-GlcNAcome was revealed by the way of the Click chemistry and the azide-alkyne cycloaddition of a fluorophore on GlcNAc moieties. This method permits, after sequential image acquisition, the direct in-gel detection of O-GlcNAcome, phosphoproteome and whole proteome

Western blot analysis of regulatory protein expression in slow and fast fibers (B) corresponds to western blot characterization of slow and fast isoforms of MLC2 (above) or TnT (below) for slow fiber isolated from untreated soleus (1), for slow fiber isolated from PUGNAc-treated soleus (2) or fast fiber isolated from epitroclearis muscle (3).

<p>Western blot analysis of regulatory protein expression in slow and fast fibers (B) corresponds to western blot characterization of slow and fast isoforms of MLC2 (above) or TnT (below) for slow fiber isolated from untreated soleus (1), for slow fiber isolated from PUGNAc-treated soleus (2) or fast fiber isolated from epitroclearis muscle (3).</p

Glycopattern analysis of proteins extracted from skinned biopsies comparing with contractile proteins and whole proteins extracts.

<p>Soleus contractile proteins (lanes 1 and 2), proteins extracted from skinned biopsies (lanes 3 and 4) and whole proteins extracted from soleus (lanes 5 and 6) were separated on 12.5% SDS-PAGE and transferred onto nitrocellulose sheet for western blot analysis. Lanes 2, 4 and 6 corresponds to PNGaseF-treated proteins. (A) corresponds to Ponceau staining, (B) to AP-revelation for MAA binding on glycoproteins, (C) to AP-revelation of SNA binding on glycoproteins. Fet (fetuin) and Tf (transferin) correspond to positive glycoprotein markers.</p

O-GlcNAcylation, contractile protein modifications and calcium affinity in skeletal muscle.

O-GlcNAcylation, a generally undermined atypical protein glycosylation process, is involved in a dynamic and highly regulated interplay with phosphorylation. Akin to phosphorylation, O-GlcNAcylation is also involved in the physiopathology of several acquired diseases, such as muscle insulin resistance or muscle atrophy. Recent data underline that the interplay between phosphorylation and O-GlcNAcylation acts as a modulator of skeletal muscle contractile activity. In particular, the O-GlcNAcylation level of the phosphoprotein myosin light chain 2 seems to be crucial in the modulation of the calcium activation properties, and should be responsible for changes in calcium properties observed in functional atrophy. Moreover, since several key structural proteins are O-GlcNAc-modified, and because of the localization of the enzymes involved in the O-GlcNAcylation/de-O-GlcNAcylation process to the nodal Z disk, a role of O-GlcNAcylation in the modulation of the sarcomeric structure should be considered