Abstract
BackgroundMutations in the PYGM gene encoding skeletal muscle glycogen phosphorylase (GP) cause a metabolic disorder known as McArdle's disease. Previous studies in muscle biopsies and cultured muscle cells from McArdle patients have shown that PYGM mutations abolish GP activity in skeletal muscle, but that the enzyme activity reappears when muscle cells are in culture. The identification of the GP isoenzyme that accounts for this activity remains controversial.Methodology/Principal FindingsIn this study we present two related patients harbouring a novel PYGM mutation, p.R771PfsX33. In the patients' skeletal muscle biopsies, PYGM mRNA levels were ∼60% lower than those observed in two matched healthy controls; biochemical analysis of a patient muscle biopsy resulted in undetectable GP protein and GP activity. A strong reduction of the PYGM mRNA was observed in cultured muscle cells from patients and controls, as compared to the levels observed in muscle tissue. In cultured cells, PYGM mRNA levels were negligible regardless of the differentiation stage. After a 12 day period of differentiation similar expression of the brain and liver isoforms were observed at the mRNA level in cells from patients and controls. Total GP activity (measured with AMP) was not different either; however, the active GP activity and immunoreactive GP protein levels were lower in patients' cell cultures. GP immunoreactivity was mainly due to brain and liver GP but muscle GP seemed to be responsible for the differences.Conclusions/SignificanceThese results indicate that in both patients' and controls' cell cultures, unlike in skeletal muscle tissue, most of the protein and GP activities result from the expression of brain GP and liver GP genes, although there is still some activity resulting from the expression of the muscle GP gene. More research is necessary to clarify the differential mechanisms of metabolic adaptations that McArdle cultures undergo in vitro.
Highlights
Glycogen phosphorylase (GP) is a finely regulated key enzyme in the metabolism of glycogen
Conclusions/Significance: These results indicate that in both patients’ and controls’ cell cultures, unlike in skeletal muscle tissue, most of the protein and glycogen phosphorylase (GP) activities result from the expression of brain GP and liver GP genes, there is still some activity resulting from the expression of the muscle GP gene
Differential expression of GP genes in skeletal muscle and cultured cells Total GP activity was measured in skeletal muscle biopsy from patient P1 and was undetectable
Summary
Glycogen phosphorylase (GP) is a finely regulated key enzyme in the metabolism of glycogen. There are three different GP isoenzymes encoded by three different genes, apparently evolved from a common ancestral gene, i.e. PYGB (brain) PYGL (liver), and PYGM (skeletal muscle) [2]. The clinical features of this disorder, known as McArdle’s disease or glycogenolysis type V had first been described a few years earlier by Brian McArdle [5], and encompass exercise intolerance with reversible acute crises of premature fatigue, myalgia and contractures, sometimes accompanied by severe rhabdomyolysis and myoglobinuria; these episodes are triggered by static or isometric muscle contractions as well as by dynamic, strenuous exercises such as running [6]. Mutations in the PYGM gene encoding skeletal muscle glycogen phosphorylase (GP) cause a metabolic disorder known as McArdle’s disease. The identification of the GP isoenzyme that accounts for this activity remains controversial
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