Background

Skeletal muscle regeneration is a series of highly regulated processes mainly executed by muscle stem cells (MuSCs) (Hawke and Garry, 2001), including necrosis of damaged myofibers, immune cell infiltration, clearance of the damaged myofibers, MuSC activation and proliferation, myofiber formation and maturation, and muscle reconstruction. MuSCs, also named satellite cells, were first discovered by Dr. Alexander Mauro in 1961 (Mauro 1961). In response to muscle injuries, the quiescent MuSCs are activated to enter the cell cycle, proliferate, and differentiate into new myofibers or fuse to the injured myofibers to repair muscle injury. After activation and proliferation, part of the MuSCs will return to quiescence and replenish the in vivo stem cell pool (Bischoff, 1975; Konigsberg et al., 1975; Fu et al., 2015 and 2022).

Induction of acute muscle injury is a critical step to study muscle regeneration in vivo. Several methods have been applied to induce consistent acute muscle injury, including freezing, barium chloride injection, glycerol injection, notexin injection, and cardiotoxin (CTX) injection (Mahdy et al., 2015; Hardy et al., 2016). Among the reagents to induce acute muscle injury, CTX is the most commonly used. CTX disrupts the membrane integrity of neuronal and skeletal muscle and cardiac muscle cells (Dufton and Hider 1988); it has been speculated to form pores on the cell membrane that result in membrane depolarization and influx of Ca²⁺, causing overwhelming muscle contraction, myofiber lysis, and acute muscle injury (Harvey et al., 1982). This protocol describes the step-by-step procedure to induce skeletal muscle injury via intramuscular injection of CTX. Following CTX injection, mice can be sacrificed at different time points for further analysis, according to variable experimental requirements.