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Abstract: SA-OR031

HIF Stabilizer Decreases Mitochondrial Oxygen Consumption in Skeletal C2C12 Myotube

Session Information

Category: Anemia and Iron Metabolism

  • 201 Anemia and Iron Metabolism: Basic

Authors

  • Takemura, Koji, The University of Tokyo Graduate school of medicine, Tokyo, Japan
  • Nishi, Hiroshi, the University of Tokyo School of Medicine, Tokyo, Japan
  • Higashihara, Takaaki, The University of Tokyo Graduate school of medicine, Tokyo, Japan
  • Nangaku, Masaomi, the University of Tokyo School of Medicine, Tokyo, Japan
Background

Erythropoietin (EPO) and hypoxia-inducible factor (HIF) stabilizers (PH inhibitors) are efficient therapeutic modalities against anemia in CKD. However, extra-renal action of PH inhibitors has not been fully investigated. Previous reports caution us about the actual misuse of PH inhibitors in doped athletes, but nonhematopoietic effects on skeletal muscles remain controversial.

Methods

To study direct pharmacological effects of roxadustat on skeletal muscles, cultured muscle cells were assessed from multiple perspectives including cell viability, myotube differentiation and glucose metabolism. Murine C2C12 myoblasts were cultured in media containing 2% horse serum, for differentiation. Quantitative PCR was applied for expression analysis of myogenin (Myog), differentiation marker, Myh-7, encoding a slow-twitch muscle isoform, Myh-1, 2, and 4, encoding fast-twitch isoforms and glycolytic enzymes such as lactate dehydrogenase (LDH), phosphoinositide-dependent kinase-1 (Pdk1), and glycogen synthase-1 (Gys1). Biochemical quantitative assay of lactate was also utilized. Glucose and mitochondrial metabolic profile was quantified with extracellular flux analyzer.

Results

Culture in the low serum media stimulated C2C12 myoblasts to differentiate and to fuse into multinucleated myotubes with enhanced expression of Myog and Myh-1, 2, 4, and 7. Roxadustat treatment did not affect myotube viability, morphology, or increase differentiation marker expression. However, 2-deoxyglucose uptake was enhanced. The treatment also elevated expression of Ldha, Pdk1, and Gys1, and promoted glycolytic rate with culture lactate concentration increased. By contrast, the treatment decreased the ratio of mitochondrial/nuclear DNA and down-regulated mitochondrial respiration, which lentivirus-mediated genetic silencing of HIF-1α in C2C12 cells reversed. Electron microscopic evaluation showed mitochondrial fragmentation in roxadustat-treated myotubes.

Conclusion

Roxadustat, a PH inhibitor to stabilize HIF that is beneficial for renal anemia treatment, may exert a direct effect on skeletal muscles. Although it did not affect cellular viability or morphology, roxadustat
compromised the ability of cells to undergo oxidative phosphorylation, which action was mediated by HIF-1α, and directed cells to acceleration of glycolytic process and lactic acid generation.