Top-down Targeted Proteomics Reveals Decrease in Myosin Regulatory Light Chain Phosphorylation that Contributes to Sarcopenic Muscle Dysfunction

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Zachery R. Gregorich a,b, Ying Peng a, Wenxuan Cai a,b, Yutong Jin a,c, Liming Wei a,d, Albert J. Chen a, Susan H. McKiernan e, Judd M. Aiken f, Richard L. Moss a,g,h, Gary M. Diffee e,h,*, Ying Ge a,b,c,g,h,*
J. Proteome Res., Just Accepted Manuscript
Sarcopenia, the loss of skeletal muscle mass and function with advancing age, is a significant cause of disability and loss of independence in the elderly, and, thus, represents a formidable challenge for the aging population. Nevertheless, the molecular mechanism(s) underlying sarcopenia-associated muscle dysfunction remain poorly understood. In this study, we employed an integrated approach combining top-down targeted proteomics with mechanical measurements to dissect the molecular mechanism(s) in age-related muscle dysfunction. Top-down targeted proteomic analysis uncovered a progressive age-related decline in the phosphorylation of myosin regulatory light chain (RLC), a critical protein involved in the modulation of muscle contractility, in the skeletal muscle of aging rats. Top-down tandem mass spectrometry analysis enabled localization of the sites of decreasing phosphorylation to Ser14/15, two previously uncharacterized sites of phosphorylation in RLC from fast-twitch skeletal muscle. Subsequent mechanical analysis of single fast-twitch fibers isolated from the muscles of rats of different ages revealed that the observed decline in RLC phosphorylation can account for age-related decreases in the contractile properties of sarcopenic fast-twitch muscles. These results strongly support a role for decreasing RLC phosphorylation in sarcopenia-associated muscle dysfunction, and suggest therapeutic modulation of RLC phosphorylation may represent a new avenue for the treatment of sarcopenia.

http://pubs.acs.org/doi/abs/10.1021/acs.jproteome.6b00244
a. Department of Cell and Regenerative Biology, University of Wisconsin-Madison, 1111 Highland Ave., Madison, WI, 53705. b. Molecular and Cellular Pharmacology Training Program, University of Wisconsin-Madison, 1111 Highland Ave., Madison, WI, 53705. c. Department of Chemistry, University of Wisconsin-Madison, 1101 University Ave., Madison, WI, 53706. d. Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, P. R. China. e. Department of Kinesiology, University of Wisconsin-Madison, 2000 Observatory Dr., Madison, WI, 53705. f. Departments of Agriculture, Food, and Nutritional Sciences, University of Alberta Edmonton, Edmonton, AB, Canada. g. Human Proteomics Program, University of Wisconsin Madison, 1111 Highland Ave., Madison, WI, 53705. h. UW Cardiovascular Research Center, University of Wisconsin-Madison, 1111 Highland Ave., Madison, WI, 53705.