Plantaris Muscle Overload Protocol

Plantaris muscle mechanical overload was induced via unilateral ablation of synergist muscles using methods previously described by our lab [24 (link),41 (link)]. Mice were anaesthetized with isoflurane (2–3%) and the distal two-thirds of the gastrocnemius and soleus surgically ablated. A sham surgery was performed on the contralateral leg. After 5 days, mice were fasted overnight, anesthetized with pentobarbital sodium (90–100 mg/kg body weight) for 40 min and euthanized by cervical dislocation. Muscles were excised and weighed to ±0.1 g using an analytical balance (Mettler-Toledo XS105, Columbus, OH, USA), prior to additional experimental perturbations as described below.