MuRF1 (TRIM63) is a muscle-specific E3 ubiquitin ligase and component of the ubiquitin proteasome system. MuRF1 is transcriptionally upregulated under conditions that cause muscle loss, in both rodents and humans, and is a recognized marker of muscle atrophy. In this study, we used in vivo electroporation to determine if MuRF1 overexpression alone can cause muscle atrophy and, in combination with ubiquitin proteomics, identify the endogenous MuRF1 substrates in skeletal muscle. Overexpression of MuRF1 in adult mice increases ubiquitination of myofibrillar and sarcoplasmic proteins, increases expression of genes associated with neuromuscular junction instability, and causes muscle atrophy. A total of 169 ubiquitination sites on 56 proteins were found to be regulated by MuRF1. MuRF1-mediated ubiquitination targeted both thick and thin filament contractile proteins, as well as, glycolytic enzymes, deubiquitinases, p62, and VCP. These data reveal a potential role for MuRF1 in not only the breakdown of the sarcomere, but also the regulation of metabolism and other proteolytic pathways in skeletal muscle.
Muscle-specific E3 ubiquitin ligases have been identified in muscle atrophy-inducing conditions. The purpose of the current study was to explore the functional role of Fbxl22, and a newly identified splice variant (Fbxl22-193), in skeletal muscle homeostasis and neurogenic muscle atrophy. In mouse C2C12 muscle cells, promoter fragments of the Fbxl22 gene were cloned and fused with the secreted alkaline phosphatase reporter gene to assess the transcriptional regulation of Fbxl22. The tibialis anterior muscles of male C57/BL6 mice (12-16 weeks old) were electroporated with expression plasmids containing the cDNA of two Fbxl22 splice variants and tissues collected after 7, 14 and 28 days. Gastrocnemius muscles of wild type and MuRF1 knockout mice were electroporated with an Fbxl22 RNAi or empty plasmid, denervated three days post-transfection, and tissues collected 7 days post-denervation. The full-length gene and novel splice variant are transcriptionally induced early (after 3 days) during neurogenic muscle atrophy. In vivo overexpression of Fbxl22 isoforms in mouse skeletal muscle lead to evidence of myopathy/atrophy suggesting that both are involved in the process of neurogenic muscle atrophy. Knockdown of Fbxl22 in MuRF1 KO muscles resulted in significant additive muscle sparing at 7 days of denervation. Targeting two E3 ubiquitin ligases appears to have a strong additive effect on protecting muscle mass loss with denervation and these findings have important implications in the development of therapeutic strategies to treat muscle atrophy.
Muscle-specific E3 ubiquitin ligases have been identified in muscle atrophy-inducing conditions. The purpose of the current study was to explore the functional role of Fbxl22, and a newly identified splice variant , in skeletal muscle homeostasis and neurogenic muscle atrophy. The full-length gene and novel splice variant are transcriptionally induced early (after 3 days) during neurogenic muscle atrophy. In vivo overexpression of Fbxl22 isoforms in mouse skeletal muscle lead to evidence of myopathy/atrophy suggesting that both are involved in the process of neurogenic muscle atrophy. Knockdown of Fbxl22 in MuRF1 KO muscles resulted in significant additive muscle sparing at 7 days of denervation. Targeting two E3 ubiquitin ligases appears to have a strong additive effect on protecting muscle mass loss with denervation and these findings have important implications in the development of therapeutic strategies to treat muscle atrophy.
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