Increasing O-GlcNAcylation Level on Organ Culture of Soleus Modulates the Calcium Activation Parameters of Muscle Fibers

Abstract

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.

O‐GlcNAcylation as a regulator of the functional and structural properties of the sarcomere in skeletal muscle: An update review

“…These results were also reproduced on human skeletal muscle fibres, and confirmed the reversible decrease in sarcomere sensitivity and affinity to calcium . Additional experiments were done on rat soleus fibres after an increase in global O‐GlcNAc level using PUGNAc and Thiamet‐G, both potent inhibitors of O‐GlcNAcase (OGA) . Unlike the excess GlcNAc conditions, T/pCa relationship was shifted to higher pCa values (Figure ).…”

“…Unlike the excess GlcNAc conditions, T/pCa relationship was shifted to higher pCa values (Figure ). Indeed, pCa 50 value was significantly increased (but not pCa threshold value) meaning that calcium affinity of sarcomere was increased, whereas Hill coefficient (n H ) and P 0 value were not significantly altered (Figure ) . This first story with evidences revealed that calcium activation properties of sarcomere were modulated according to O‐GlcNAcylation.…”

“…In addition, O‐GlcNAcylation is highly dynamic and reversible (such as phosphorylation) because of a unique pair of antagonist enzymes: the OGT or O‐GlcNAc transferase, which transfers the monosaccharide onto a protein, and the OGA or O‐GlcNAcase, which removes it . In the overall skeletal muscle cell, over a thousand of O‐GlcNAc‐modified proteins have been identified since 2004 . The nature of these proteins is diverse, including contractile, sarcolemmal, structural and cytoskeletal proteins, which are involved in sarcomeric cytoarchitecture; an updated and summarized schematic cartoon of the O‐GlcNAcylated skeletal muscle proteome is presented in Figure .…”

Thick Filament Protein Network, Functions, and Disease Association

“…MyHC, ELC, and RLC are also modified by O-GlcNAcylation (102, 229, 461). Accordingly, several sites of O-GlcNAcylation have been reported on Myh6, Myl2, and Myl3 (461, 462).…”

Dexamethasone‐Induced Skeletal Muscle Atrophy Increases O‐GlcNAcylation in C2C12 Cells

“…Moreover, there is an interplay between O‐GlcNAcylation and phosphorylation: in particular, O‐GlcNAcylation seems to be reciprocal to phosphorylation with mutual occupancy at the same side on a polypeptide and with antagonistic effects in regulation of many cellular processes, such as transcription, cell signaling, metabolism, and many others [Hu et al, ]. It has been shown that many skeletal muscle proteins (myosin heavy and light chains, actin, tropomyosin) are O‐GlcNAc modified and this alteration is able to modulate muscle contraction by acting on calcium affinity of fibres [Cieniewski‐Bernard et al, , ]. Furthermore, it has been recently described that the O‐GlcNAcylation process is involved in the development of muscle disuse atrophy.…”