Impaired myoblast differentiation and muscle IGF‐1 receptor signaling pathway activation after N‐glycosylation inhibition

The role of N‐glycosylation in the myogenic process remains poorly understood. Here, we evaluated the impact of N‐glycosylation inhibition by Tunicamycin (TUN) or by phosphomannomutase 2 (PMM2) gene knockdown, which encodes an enzyme essential for catalyzing an early step of the N‐glycosylation pathway, on C2C12 myoblast differentiation. The effect of chronic treatment with TUN on tibialis anterior (TA) and extensor digitorum longus (EDL) muscles of WT and MLC/mIgf‐1 transgenic mice, which overexpress muscle Igf‐1Ea mRNA isoform, was also investigated. TUN‐treated and PMM2 knockdown C2C12 cells showed reduced ConA, PHA‐L, and AAL lectin binding and increased ER‐stress‐related gene expression (Chop and Hspa5 mRNAs and s/uXbp1 ratio) compared to controls. Myogenic markers (MyoD, myogenin, and Mrf4 mRNAs and MF20 protein) and myotube formation were reduced in both TUN‐treated and PMM2 knockdown C2C12 cells. Body and TA weight of WT and MLC/mIgf‐1 mice were not modified by TUN treatment, while lectin binding slightly decreased in the TA muscle of WT (ConA and AAL) and MLC/mIgf‐1 (ConA) mice. The ER‐stress‐related gene expression did not change in the TA muscle of WT and MLC/mIgf‐1 mice after TUN treatment. TUN treatment decreased myogenin mRNA and increased atrogen‐1 mRNA, particularly in the TA muscle of WT mice. Finally, the IGF‐1 production and IGF1R signaling pathways activation were reduced due to N‐glycosylation inhibition in TA and EDL muscles. Decreased IGF1R expression was found in TUN‐treated C2C12 myoblasts which was associated with lower IGF‐1‐induced IGF1R, AKT, and ERK1/2 phosphorylation compared to CTR cells. Chronic TUN‐challenge models can help to elucidate the molecular mechanisms through which diseases associated with aberrant N‐glycosylation, such as Congenital Disorders of Glycosylation (CDG), affect muscle and other tissue functions. N‐glycosylation inhibitions by Tunicamycin (TUN) or by knockdown of phosphomannomutase 2 (PMM2) gene block the C2C12 myoblast fusion and impair the myogenic program. TUN treatment decreased myogenic markers and increased atrophy markers in muscles of WT and MLC/mIgf‐1 mice, which overexpress muscle Igf‐1Ea mRNA isoform. Finally, the production of N‐glycosylated IGF‐1Ea prohormone and IGF1R signaling pathway activation were markedly inhibited after TUN treatment. Our results offer new insights into impairments of myogenic differentiation capacity in the pathological context of disorders of N‐glycosylation.