Continued Activation of Hypoxia Inducible Factor 1α by Roxadustat and Inhibition of Differentiation in Skeletal Muscles

Background: Patients with chronic kidney disease are prone to develop renal anemia. Hypoxia-inducible factor prolyl hydroxylase (HIF-PH) inhibitors, such as roxadustat, have recently been recognized as a viable treatment option for this condition by stabilizing hypoxia-inducible factor-1α (HIF-1α). Nonetheless, the consequences of continued HIF-1α activation on skeletal muscle differentiation are not fully understood. This study explores the effects of continued HIF-1α activation on the differentiation of skeletal muscles. Methods: We cultured mouse C2C12 myoblasts to differentiate into myotubes using horse serum, with or without 100 µM roxadustat. We evaluated the extent of muscle differentiation and measured the expression of muscle differentiation-related proteins and genes (MyoD and myogenin) and muscle constituent proteins (myosin heavy chain, MHC). Additionally, two groups of nine-week-old male C57BL/6 mice were treated with either roxadustat or a vehicle control via intraperitoneal injections three times a week for four weeks. We analyzed the expression of HIF-1α, muscle differentiation and constituent proteins in the gastrocnemius muscle. Results: Addition of roxadustat to C2C12 myoblast cultures results in an increased expression of HIF-1α protein. Treatment with roxadustat for 72 hours suppressed myotube formation in C2C12 cells, reducing both the differentiation and fusion indices compared to untreated controls. Gene and protein expression levels of MyoD, myogenin, and protein expression level of MHC were also lower in the roxadustat-treated C2C12 myotubes than untreated C2C12 myotubes. The decrease in MHC protein expression induced by roxadustat was attenuated by siRNA targeting HIF-1α. In the in vivo experiments, roxadustat treatment led to an increase in HIF-1α protein levels and a decrease in the protein expression of MyoD, myogenin, and MHC in the gastrocnemius muscle of the treated mice compared to controls. Conclusion: The results indicate that continued activation of HIF-1α by roxadustat inhibits skeletal muscle differentiation.