High phosphate induces skeletal muscle atrophy and suppresses myogenic differentiation by increasing oxidative stress and activating Nrf2 signaling

Lin-Huei Chung,Herng-Sheng Lee,Donald M Salter,Shih-Ming Huang,Yu-Juei Hsu,Shu-Ting Liu

doi:10.18632/aging.103896

Abstract

Skeletal muscle wasting represents both a common phenotype of aging and a feature of pathological conditions such as chronic kidney disease (CKD). Although both clinical data and genetic experiments in mice suggest that hyperphosphatemia accelerates muscle wasting, the underlying mechanism remains unclear. Here, we showed that inorganic phosphate (Pi) dose-dependently decreases myotube size, fusion index, and myogenin expression in mouse C2C12 skeletal muscle cells. These changes were accompanied by increases in reactive oxygen species (ROS) production and Nrf2 and p62 expression, and reductions in mitochondrial membrane potential (MMP) and Keap1 expression. Inhibition of Pi entry, cytosolic ROS production, or Nrf2 activation reversed the effects of high Pi on Nrf2, p62, and myogenin expression. Overexpression of Nrf2 respectively increased and decreased the promoter activity of p62-Luc and myogenin-Luc reporters. Analysis of nuclear extracts from gastrocnemius muscles from mice fed a high-Pi (2% Pi) diet showed increased Nrf2 phosphorylation in sham-operated and 5/6 nephrectomized (CKD) mice, and both increased p62 phosphorylation and decreased myogenin expression in CKD mice. These data suggest that high Pi suppresses myogenic differentiation in vitro and promotes muscle atrophy in vivo through oxidative stress-mediated protein degradation and both canonical (ROS-mediated) and non-canonical (p62-mediated) activation of Nrf2 signaling.

Highlights

Muscle wasting, defined as loss of muscular mass and contractile capacity, is both a physiological consequence of normal aging and a common premature aging phenotype in patients with chronic kidney disease (CKD) [1]
To model the clinical conditions observed in cases of acquired hyperphosphatemia, we assessed the effects of high Pi on myogenic differentiation
The present study demonstrates that exposing C2C12 skeletal muscle cells to high Pi leads to changes consistent with muscle wasting, namely reduced myotube size, increased reactive oxygen species (ROS) generation, decreased protein synthesis, and accelerated protein degradation

Summary

Introduction

Muscle wasting, defined as loss of muscular mass and contractile capacity, is both a physiological consequence of normal aging and a common premature aging phenotype in patients with chronic kidney disease (CKD) [1]. For example, both fibroblast growth factor 23 (FGF23) and the renal transmembrane protein Klotho act as Pi-regulating hormones whose genetic ablation leads to hyperphosphatemia and premature aging phenotypes, including muscle atrophy. Muscle atrophy and other premature aging phenotypes were reversed in both Klotho- and FGF23-knockout mice by either a low-Pi diet or by genetic ablation of renal sodium-dependent Pi transporter solute carrier family 34, member 1 (NaPi2a) [5,6,7]. The molecular mechanisms underlying high Piinduced muscle atrophy are not yet fully understood, but there is increasing evidence that oxidative stress and activation of the Nrf2/Keap1/p62 pathway may be involved

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