ULK2 Regulates Autophagic Cargo Recognition Impacting Contractile Function In Skeletal Muscle

Abstract

Autophagy is an anciently conserved pathway responsible for the degradation of long-lived proteins, protein aggregates, and organelles, thereby contributing to efficient protein homeostasis. Autophagy is stimulated by nutrient deprivation and is required for certain beneficial adaptations of exercise. Insufficient autophagy is a common feature of muscle diseases, obesity, type 2 diabetes, and aging. However, regulation of autophagy is incompletely understood at the molecular level. PURPOSE: Define the role of unc-51 like autophagy activating kinase 2 (ULK2), and contrast with that of its close homolog ULK1, in regulation of autophagy and contractile function in skeletal muscle. METHODS: 1) DNA plasmids encoding either Ulk1 or Ulk2 pre-micro RNAs (miR) were electroporated into the tibialis anterior (TA) muscle of one leg, and a control miR plasmid into the contralateral leg of wild type mice. Muscles were harvested 7-8 days afterwards, either at basal conditions or after 24h of starvation. 2) ULK2fl/fl differentiated primary mouse myotubes were infected with Ad-Cre-GFP or Ad-GFP (control) viruses, and harvested up to 96h afterwards. 3) Maximal force of hindlimb dorsiflexors was assessed in adult ULK2 skeletal muscle knockout mice (ULK2fl/fl, Myogenin-Cre+/-; ULK2mKO) via stimulation of the fibular nerve, and compared to control littermates (ULK2fl/fl, Cre-/-). RESULTS: ULK2 is expressed at ~2-fold higher levels than its close homolog ULK1 in skeletal muscle. ULK2 deficiency, but not ULK1, leads to ubiquitin and autophagy receptor protein accumulation (p62, NBR-1), suggesting impaired cargo recognition in adult skeletal muscle and primary myotubes, independent of lysosomal function. Preliminary findings indicate that maximal force is reduced in adult ULK2mKO. CONCLUSION: Here, we demonstrate a novel and fundamental role for ULK2 in regulating cargo recognition, an essential aspect of selective autophagy, which is commonly impaired in conditions of muscle dysfunction. These results reveal ULK2 as a potential therapeutic target for skeletal muscle contractile and metabolic dysfunction, and serve as basis for future studies dissecting the mechanisms of autophagic cargo recognition in skeletal muscle. Supported by AHA (16SDG30360001) and Dept. of Health & Human Physiology, University of Iowa.