Thursday, May 9, 2024
5/9/2024
SUMMARY Skeletal muscle atrophy continues to be a serious consequence of many diseases and conditions for which there is no effective treatment. The E3 ubiquitin ligase, MuRF1 (Trim63), is selectively expressed in striated muscle and has been shown to be elevated in a myriad of atrophy-inducing conditions in both rodent and human muscle and thus is routinely used as a marker of muscle atrophy. Given that MuRF1 is associated with multiple forms of atrophy, and deletion can lead to partial muscle sparing, MuRF1 has been considered a strong candidate for drug development for the treatment of muscle atrophy. However, targeting specific E3 ligases has been difficult to achieve and thus a greater understanding of the downstream targets of MuRF1 could identify new targets for drug development. We have recently shown that overexpression of a full length MuRF1 plasmid into multiple hindlimb muscles of the mouse using in vivo electroporation induces significant atrophy of muscle fibers by 14 days. In an initial discovery effort, we used unbiased ubiquitin modification site-mapping (di-glycine (di-Gly) remnants proteomics followed by liquid chromatography and tandem mass spectrometry and identified 169 lysine sites on 56 proteins that were differentially ubiquitinated in response to MuRF1 overexpression in skeletal muscle. The list of putative MuRF1 substrates includes many proteins not previously linked to MuRF1. Further, many of the putative MuRF1 substrates were ubiquitinated on a single lysine residue and were not immediately targeted for degradation. Our recent data challenges the deeply entrenched dogma that thick filament proteins are the sole targets of MuRF1 and that the role of MuRF1-mediated ubiquitylation is solely protein degradation. The overall objective of the current proposal is to determine the mechanisms by which MuRF1 induces muscle atrophy. We will use diGly remnant affinity proteomics and deuterium oxide labeling in wild type and MuRF1 knock out mice at rest and following a time-course of denervation to test the hypothesis that upregulation of MuRF1 results in the disassembly of the sarcomere through targeted ubiquitylation of specific proteins resulting in increased turnover of the myofibrillar proteins and a loss of fiber cross-sectional area and muscle atrophy. Further, we hypothesize that ubiquitylation of myofibrillar proteins occurs in a stepwise manner with ubiquitylation by MuRF1 being a priming step for further ubiquitylation by other E3 ligases. The deliverables from this investigation will likely generate a paradigm shift in our understanding of how MuRF1 regulates muscle atrophy and may lead to the identification of new therapeutic targets for the treatment and prevention of muscle wasting.
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