C9759

C57BL/6J wild type (WT) and Parkin knockout male mice (10-12 weeks old, 20–26 g, N = 35 for each genotype) were used in these studies. The Parkin^tm1Shn (Parkin^−/−) mice were generated by [36 (link)], provided by the Multidisciplinary Center for Biological Research (CEMIB) and transported to the School of Applied Sciences of the University of Campinas in Limeira (São Paulo, Brazil). All animal experiments were approved by the Ethics Committee on the Use of Animals of the University of Campinas (CEUA, protocol no. 4687-1/2017 approved in August, 31th 2017). The mice animals were submitted to surgical procedure to expose the TA muscle and the injury was induced by injection of cardiotoxin in the TA muscle of the posterior left paw. The mice were pre-anesthetized with analgesic (buprenorphine—0.1 mg/kg) and anesthetized (ketamine—80 mg/kg; and xylazine—10 mg/kg), the left tibialis anterior (TA) muscle exposed and CTX from Najamossambicamossambica (C9759, Sigma-Aldrich^®, Saint Louis, MO, USA) in PBS was injected (20 μL of 10 μM) at four longitudinally sites. The mice were harvested at 3dpi, 10 10dpi, and 21dpi. The TA muscle from the posterior right paw of all mice was used as the control group (CTRL) in each group. After euthanasia, the TA muscle was removed, weighed, divided and frozen in isopentane or liquid nitrogen.

To induce muscle regeneration, 100 μl of 10 mM cardiotoxin (CTX; C9759, Sigma-Aldrich) were injected intramuscularly into the tibialis anterior (TA) muscle of anesthetized 10-week-old male mice using a 29 G syringe. Regenerating muscles were isolated 2, 4 and 7 days after CTX injection.

8–12 weeks old male C57Bl/6 J mice (Janvier) were anesthetized with isoflurane and 80 µL of 10 µM cardiotoxin (Sigma, C9759) or 50% glycerol in saline solution (NaCl 0.9%) were injected into the right quadriceps.