Overexpression of Multiple E3 Ubiquitin Ligases in Gastrocnemius Muscles from Mice

Abstract

E3 ubiquitin ligases are commonly studied in skeletal muscle under atrophy‐inducing conditions. MuRF1 is a recognized marker of muscle atrophy and we have recently identified Fbxl22 to be involved in the process of denervation‐induced muscle atrophy. Thus, we sought to investigate the role of MuRF1 and Fbxl22 together in the process of skeletal muscle atrophy. Male C57BL/6 mice (16 to 20‐week old) were subjected to in vivo electroporation with either a mouse specific MuRF1 or Fbxl22 plasmid (20ug) in the gastrocnemius (GA) muscles with the contralateral leg serving as a vector control (EV). An additional cohort of mice was electroporated with MuRF1 and Fbxl22 plasmids simultaneously. After 7 days, lateral (LGA) and medial (MGA) gastrocnemius muscles were collected, weighed and sectioned for cross‐sectional area (CSA) and GFP visualization. Western blotting was performed on sarcoplasmic and myofibrillar fractions to determine levels of ubiquitin and sarcomeric proteins in the transfected GA muscles.MuRF1 overexpression (OE) resulted in significant loss of total GA muscle mass (‐8.8%) after 7 days alone. In comparison, Fbxl22 OE led to no loss in total GA muscle mass, although significant reductions in LGA mass (‐3.4%) were observed. Combined OE of MuRF1 and Fbxl22 together resulted in significant loss of GA muscle mass (‐5.5%) but the level of atrophy was not as great as compared to MuRF1 OE alone. In transfected LGA muscles, total ubiquitin levels were significantly elevated in the Fbxl22 OE and combined E3 ligase group for the sarcoplasmic fraction. In the myofibrillar fraction of LGA transfected muscles, only the combined E3 ligase group displayed increased total ubiquitin levels after 7 days. In transfected Fbxl22 LGA muscles, we observed significant increases in vimentin, alpha‐actinin and Troponin T in the sarcoplasmic fractions. Gene manipulation of the E3 ubiquitin ligases, MuRF1 and Fbxl22 in basal skeletal muscle resulted in skeletal muscle atrophy and divergent changes in ubiquitination and sarcomeric proteins were observed following E3 overexpression. Future studies will seek to identify potential unique and shared substrates targeted by Fbxl22 and MuRF1 for ubiquitination.