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

Abstract

Summary 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 identified a previously un-reported bis-phosphorylated proteoform of fast skeletal RLC and localized the sites of decreasing phosphorylation to Ser14/15. Of these sites, Ser14 phosphorylation represents a previously unidentified site 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.

Age affects myosin relaxation states in skeletal muscle fibers of female but not male mice

“…A PTM that is closely associated with myosin function and muscle contractility is phosphorylation, which has also been shown to be modulated by estradiol signaling [21]. Studies in rodents and women have shown that phosphorylated RLC (pRLC) decreases with age while there was no effect of age on pRLC level in males [15,16]. We see the same trend in our current study, where muscle relaxation is affected by age in female mice, but not in males.…”

“…This study examined the impact of the natural aging process on relaxed states of myosin in skeletal muscle fibers from male and female mice. Age-related alterations in myosin function during contraction and relaxation have been reported [15,16,[34][35][36][37][38][39], but ours is the first to report findings for the changes in myosin ATPase activity in the DRX and SRX states with age and sex specificity. Following our previous work that showed altered SRX under relaxed conditions in fibers from an ovariectomy-induced aging model [25], here we measured the single-nucleotide turnover in resting skeletal muscle fibers isolated from naturally aged mice from both sexes.…”

“…Because the decline in intrinsic contractile function of muscle fibers with age could be explained by age-related changes in myosin ATPase activity [16], it is pertinent to examine myosin's basal nucleotide turnover in that context. In a minimalistic model, myosin heads in muscle fibers occupy either the active or relaxed states, the latter of which can be sub-fractionated into DRX or SRX ( Fig.…”

“…SRX can be rapidly mobilized into DRX or the active states in response to regulatory allosteric cues such as increased intracellular [Ca 2+ ] or ionic strength, interaction with myosin binding protein C (MyBP-C), phosphorylation of the myosin regulatory light chain (RLC), passive strain, or decreased temperature [1][2][3]. Among these regulators, RLC phosphorylation is associated with muscle thermogenesis, disease, and aging [14][15][16]. Fig.…”

Simultaneous Quantification of Protein Expression and Modifications by Top-down Targeted Proteomics: A Case of the Sarcomeric Subproteome

“…The area under curve was manually determined for each protein isoform using DataAnalysis. To quantify protein modifications, the relative abundances of specific modifications were calculated as their corresponding percentages among all the detected protein forms in the deconvoluted averaged mass spectra as described previously (24,26,28,29,36). All masses reported are monoisotopic values for both intact and fragment ions.…”