Assessing Autophagy in Muscle Stem Cells.

Silvia Campanario,Ignacio Ramírez-Pardo,Pura Muñoz-Cánoves,Joan Isern,Xiaotong Hong

doi:10.3389/fcell.2020.620409

Abstract

The skeletal muscle tissue in the adult is relatively stable under normal conditions but retains a striking ability to regenerate by its resident stem cells (satellite cells). Satellite cells exist in a quiescent (G0) state; however, in response to an injury, they reenter the cell cycle and start proliferating to provide sufficient progeny to form new myofibers or undergo self-renewal and returning to quiescence. Maintenance of satellite cell quiescence and entry of satellite cells into the activation state requires autophagy, a fundamental degradative and recycling process that preserves cellular proteostasis. With aging, satellite cell regenerative capacity declines, correlating with loss of autophagy. Enhancing autophagy in aged satellite cells restores their regenerative functions, underscoring this proteostatic activity’s relevance for tissue regeneration. Here we describe two strategies for assessing autophagic activity in satellite cells from GFP-LC3 reporter mice, which allows direct autophagosome labeling, or from non-transgenic (wild-type) mice, where autophagosomes can be immunostained. Treatment of GFP-LC3 or WT satellite cells with compounds that interfere with autophagosome-lysosome fusion enables measurement of autophagic activity by flow cytometry and immunofluorescence. Thus, the methods presented permit a relatively rapid assessment of autophagy in stem cells from skeletal muscle in homeostasis and in different pathological scenarios such as regeneration, aging or disease.

Highlights

Skeletal muscle is formed by multinucleated myofibers and exhibits a remarkable capacity to regenerate thanks to its resident stem cells, called satellite cells (SCs) (Mauro, 1961)
The proper isolation of SCs is crucial for characterizing the mechanisms involved in stem-cell quiescence maintenance and/or regenerative functions
Several methods based on fluorescence-activated cell sorting (FACS) using different cell surface markers for positive and negative cell selection have been optimized to isolate SCs (Sherwood et al, 2004; Joe et al, 2010; Pasut et al, 2012; Liu et al, 2013)

Summary

INTRODUCTION

Skeletal muscle is formed by multinucleated myofibers and exhibits a remarkable capacity to regenerate thanks to its resident stem cells, called satellite cells (SCs) (Mauro, 1961). Autophagy was described in the beginning as a cellular process induced by stress, it functions at baseline under quiescence in resting SCs, and this constitutive autophagic activity appears to be indispensable for maintaining stemness (García-Prat et al, 2016). In contrast to young SCs, old SCs show defective autophagic activity This autophagy failure ends up in a progressive accumulation of harmful intracellular waste, mainly composed of altered mitochondrial material, which produces high oxidative stress and DNA damage, leading to muscle stem cell senescence in very old (geriatric) mice (Sousa-Victor et al, 2014; García-Prat et al, 2016). We show different methods to study autophagy in SCs, focusing on their quiescence state

MATERIALS AND METHODS

RESULTS AND DISCUSSION

ETHICS STATEMENT

CONCLUSION

DATA AVAILABILITY STATEMENT