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. 2015 May;8(5):467-72.
doi: 10.1242/dmm.020230. Epub 2015 Mar 11.

Sodium Valproate Increases the Brain Isoform of Glycogen Phosphorylase: Looking for a Compensation Mechanism in McArdle Disease Using a Mouse Primary Skeletal-Muscle Culture in Vitro

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Free PMC article

Sodium Valproate Increases the Brain Isoform of Glycogen Phosphorylase: Looking for a Compensation Mechanism in McArdle Disease Using a Mouse Primary Skeletal-Muscle Culture in Vitro

Noemí de Luna et al. Dis Model Mech. .
Free PMC article

Abstract

McArdle disease, also termed 'glycogen storage disease type V', is a disorder of skeletal muscle carbohydrate metabolism caused by inherited deficiency of the muscle-specific isoform of glycogen phosphorylase (GP-MM). It is an autosomic recessive disorder that is caused by mutations in the PYGM gene and typically presents with exercise intolerance, i.e. episodes of early exertional fatigue frequently accompanied by rhabdomyolysis and myoglobinuria. Muscle biopsies from affected individuals contain subsarcolemmal deposits of glycogen. Besides GP-MM, two other GP isoforms have been described: the liver (GP-LL) and brain (GP-BB) isoforms, which are encoded by the PYGL and PYGB genes, respectively; GP-BB is the main GP isoform found in human and rat foetal tissues, including the muscle, although its postnatal expression is dramatically reduced in the vast majority of differentiated tissues with the exception of brain and heart, where it remains as the major isoform. We developed a cell culture model from knock-in McArdle mice that mimics the glycogen accumulation and GP-MM deficiency observed in skeletal muscle from individuals with McArdle disease. We treated mouse primary skeletal muscle cultures in vitro with sodium valproate (VPA), a histone deacetylase inhibitor. After VPA treatment, myotubes expressed GP-BB and a dose-dependent decrease in glycogen accumulation was also observed. Thus, this in vitro model could be useful for high-throughput screening of new drugs to treat this disease. The immortalization of these primary skeletal muscle cultures could provide a never-ending source of cells for this experimental model. Furthermore, VPA could be considered as a gene-expression modulator, allowing compensatory expression of GP-BB and decreased glycogen accumulation in skeletal muscle of individuals with McArdle disease.

Keywords: Glycogen phosphorylase; Glycogenolysis; McArdle disease; Myotubes; Sodium valproate.

Conflict of interest statement

Competing interests

The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Differential glycogen phosphorylase expression in mouse primary skeletal-muscle cultures. Only WT myotubes expressed Pygm mRNA (A). Myoblasts and myotubes from WT and KI mice expressed Pygb mRNA with a high variation and no statistically significant differences between WT and KI (B). Presence of both Pygm transcript and protein (GP-MM) was observed only in WT myotubes (C). PAS staining of WT myoblasts (D), WT myotubes (F), KI myoblasts (E) and KI myotubes (G): only KI myotubes accumulated high glycogen levels (G). KI, knock-in; WT, wild type; RQ, relative quantification. *P<0.05 for the comparison KI versus WT, **P<0.001 for the comparison of KI versus WT. Scale bar: 50 µm.
Fig. 2.
Fig. 2.
VPA treatment increase Pygb expression in mice skeletal muscle cultures. (A) After myotubes were formed, muscle cultures were treated with different concentrations of VPA for 72 h. (B) In both cell cultures (WT and KI), Pygb mRNA increased in treated cultures (**P<0.01, *P<0.05). (C) GP-BB (Pygb) protein was also detected with western blot analysis in WT and in KI cell cultures. Abbreviations: DM, Dulbecco's modified Eagle's medium; KI, knock-in; VPA, valproic acid; WT, wild type; RQ, relative quantification.
Fig. 3.
Fig. 3.
Reduced glycogen accumulation in KI myotubes after treatment with VPA. (A,B) We observed a gradual reduction in glycogen content as myotubes were treated with increasing VPA concentrations. (B) Red areas correspond to sites of major glycogen accumulation, whereas ‘cold’ colors (green, blue) represent sites of low PAS staining in myotubes. Scale bars: 50 µm.
Fig. 4.
Fig. 4.
Long-term VPA treatment of muscle cultures. After 12 days of treatment, only cultures treated with 2 mM VPA showed healthy myotubes.

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