In vivo experimental approaches

Mouse lines were generated in C57BL6J congenic and maintained in a pure background. In all experiments, both male and female mice were used. As controls Knock-in littermates from the same strain without the Cre allele were used. Mice were maintained in individually ventilated cages, maintained at 23 °C, 12:12 h light–dark cycle, with specified pathogen-free hygiene levels, free access to water and a regular chow diet ad libitum (Sniff V1554-300), and monitored regularly for signs of suffering. K320E transgenic (point mutation K320E; Rosa26-Stop-construct; downstream EGFP) mice were generated previously by our group²⁴. Mice expressing Cre recombinase under the control of the skeletal muscle-specific MLC1f- promoter or satellite cells Pax7-Cre^ERT were generated by crossing R26-K320EloxP/+ mice with mice expressing Cre recombinase under the control of the skeletal muscle-specific MLC1f- promoter or satellite cells Pax7-Cre^ERT.

All procedures and experimentation with mice were performed according to protocols approved by the local authority (LANUV, Landesamt für Natur, Umwelt und Verbraucherschutz NRW, approval number: 2019-A090). Autophagic flux was tested by intraperitoneal injection of 50 mg/kg chloroquine 4 h prior to euthanasia. Activation of Pax7-Cre^ERT promotor was performed by daily intraperitoneal injection for 5 days, of  2 mg tamoxifen dissolved in miglyol. For muscle regeneration experiments, 2 days after the last tamoxifen injection, mice were anesthetized with 2%Xylazin, 10% Ketamine in NaCl 0.9% and 10 μM Cardiotoxin (Naja Pallida, Latoxan) was injected inside the TA fascia. After 2 days of rest, 2 mg/kg rapamycin dissolved in miglyol was injected intraperitoneally, daily for 5 days.