Calpain 6 inhibits autophagy in inflammatory environments: A preliminary study on myoblasts and a chronic kidney disease rat model

Int J Mol Med. 2021 Oct;48(4):194. doi: 10.3892/ijmm.2021.5027. Epub 2021 Aug 26.

Abstract

A non‑classical calpain, calpain 6 (CAPN6), can inhibit skeletal muscle differentiation and regeneration. In the present study, the role of CAPN6 in the regulation of the autophagy of myoblasts in vitro was investigated. The underlying molecular events and the CAPN6 level in atrophic skeletal muscle in a rat model of chronic kidney disease (CKD) were also investigated. In vitro, CAPN6 was overexpressed, or knocked down, in rat L6 myoblasts to assess autophagy and related gene expression and co‑localization. Subsequently, myoblasts were treated with a mixture of cytokines, and relative gene expression and autophagy were assessed. A rat model of CKD for muscle atrophy was established, and blood chemical level and the expression of CAPN6 in muscle were assessed. The data revealed that the knockdown of CAPN6 in rat myoblasts resulted in increased microtubule‑associated protein 1 light chain 3 (LC3) levels, while its overexpression decreased LC3 levels and impaired autophagy. Additionally, it was observed that the co‑localization of mammalian target of rapamycin (mTOR) and lysosomal‑associated membrane protein 1 (LAMP1), a lysosomal marker, proteins was increased. In addition, mTOR, Raptor and α‑tubulin (a marker of microtubules) increased in the CAPN6 overexpression group. However, inflammatory cytokines, such as interleukin (IL)‑6, tumor necrosis factor (TNF)‑α, interferon (INF)‑γ and lipopolysaccharides upregulated CAPN6 expression, inhibited L6 myoblast autophagy and stabilized mTOR activity. Furthermore, the animal model successfully mimicked human disease as regards an increase in body weight, and a reduction in muscle mass, cross‑sectional area and blood biomarker concentrations; a slight increase in CAPN6 mRNA and protein levels in muscles was observed. Finally, the data of the present study suggested that CAPN6 reduced autophagy via the maintenance of mTOR signaling, which may play a role in CKD‑related muscle atrophy. However, future studies are required to determine whether CAPN6 may be used as an intervention target for CKD‑related skeletal muscle atrophy.

Keywords: autophagy; calpain 6; chronic kidney disease; inflammation; mTOR; muscle atrophy.

MeSH terms

  • Animals
  • Apoptosis / physiology
  • Autophagy / physiology*
  • Calpain / metabolism*
  • Cell Differentiation / physiology
  • Cytokines / metabolism
  • Disease Models, Animal
  • Humans
  • Inflammation / metabolism
  • Male
  • Microtubules / metabolism
  • Muscle Development / physiology
  • Muscle, Skeletal / metabolism
  • Muscular Atrophy / drug therapy
  • Muscular Atrophy / metabolism
  • Myoblasts / metabolism*
  • Rats
  • Rats, Sprague-Dawley
  • Renal Insufficiency, Chronic / metabolism*
  • Signal Transduction / physiology

Substances

  • Cytokines
  • Calpain

Grants and funding

The present study was supported in part by a grant from the Shanghai Youth Science and Technology Excellence Plan Project 2019 (grant no. 19YF1439100), and in part by the National Natural Science Foundation of China (grant no. 81900671).