Background: Hyperphosphatemic conditions such as chronic kidney disease are associated with muscle wasting and impaired life quality. While muscle regeneration relies on myogenic progenitor recruitment, the effects of high phosphate loads on this process has not been investigated in detail. This study aims to clarify the direct effectsof hyperphosphatemic conditions on skeletal myoblast differentiation in an murine cell model system.
Material and methods: C2C12 murine muscle progenitor cells were differentiated with equivalents to physiological and pathological phosphate loads. Phosphate-induced changes in marker gene expression were quantified by RT-PCR. Furthermore, immunohistochemistry was performed to investigate nuclear positive cell counts under treatment. Cell viability and metabolic activity were measured by XTT and BrdU incorporation assays. All experiments were performed in ≥3 independent runs.
Results: Inorganic phosphate directly induces ERK-phosphorylation in pre-differentiated C2C12 myoblast cells. Phosphate concentrations resembling moderate and severe hyperphosphatemia (1.42.9 mmol/l) impaired the expression ***of differentiation markers Myogenin (−61.0%, P<0.0001) and MyoD (−51.0%; P<0.0001). While higher phosphate loads showed more pronounced effects, even moderately hyperphosphatemic conditions could significantly reduce Myogenin (−22.5%, P=0.015) and Myf5 (−33.3%, P=0.039) expression. Analogue effects were found on the protein level, where a significantly decreased count of Myogenin (−42.0%, P=0.004) and MyoD positive cells (−25,7%, P=0.002) was found in phosphate enriched medium. Increased phosphate concentration left metabolic activity and cellular proliferation rate unaltered.
Conclusion: Our data point to a phosphate-induced inhibition of myoblast differentiation without effects on cell viability. Strikingly, phosphate levels corresponding to the upper normal range significantly impaired marker gene and protein expression. Investigation of cellular responses during hyperphosphatemia may help to define serum phosphate cutoffs and modify existing treatment approaches of phosphate binders, especially in patients at risk for sarcopenia.
27 - 29 Sep 2018
European Society for Paediatric Endocrinology