抄録
Human skeletal muscle fibers are classified into three types; Type I (slow muscle) rich in mitochondria and highly aerobic metabolism, Type IIx (fast muscle) highly dependent on glycolysis system, and Type IIa (medium type muscle). It is important to investigate the mechanisms of fiber type-specific skeletal myogenesis in a reliable human system, because of that the muscle fiber composition is closely related to the diseases, such as type 2 diabetes and sarcopenia. We and others have reported that, with the endurance exercise load, miR-494 downregulated in mouse gastrocnemius muscle and in human vastus lateralis. Transcriptional coactivator p300 has been reported to play a role in skeletal myogenesis via the activation of MyoD. We have established human skeletal myogenesis system using human induced pluripotent stem cells (hiPSCs) transefected with muscle-specific transcription factor (MyoD/Tet-ON), as we previously reported. In this study, we investigated the role of miR-494 and p300 in fiber type-specific skeletal myogenesis in hiPS-MyoD cells.
Since miR-494 targets on p300 expression in silico analysis, we have performed the transient expression of miR-494 mimic at 24 hours after the myogenic induction in hiPS-MyoD cells, and found that (1) the expression of p300 protein decreased to 25%, and (2) the efficiency of myotube formation significantly decreased to 70%, (3) with the decreased expression of Myh2, type IIa myofiber specific protein. Furthermore, (4) overexpression of miR-494 in hiPS-MyoD cells significantly decreased the basal oxygen consumption rate, measured by extracellular cell flux analyzer. These results were similarly reproduced by p300 knockdown using specific siRNA, performed at 24 hours after induction of differentiation, without any increase of cell death compared to control cells. To confirm whether the effects of miR-494 on fiber type-specific skeletal myogenesis are dependent on the suppression of p300, we performed the rescue experiments with the overexpression of p300 in hiPS-MyoD cells transfected with miR-494 mimic, and found that p300 overexpression rescued the phenotype of (2)-(4) caused by overexpression of miR-494 mimic. These results suggest that miR-494 regulates fiber type-specific human skeletal myogenesis via the targeting of transcriptional coactivator p300.
