Novel Sarcopenia-related Alterations in Sarcomeric Protein Post-translational Modifications (PTMs) in Skeletal Muscles Identified by Top-down Proteomics

Liming Wei,Zachery R Gregorich,Ziqing Lin,Wenxuan Cai,Yutong Jin,Susan H Mckiernan,Sean Mcilwain,Judd M Aiken,Richard L Moss,Gary M Diffee,Ying Ge

doi:10.1074/mcp.ra117.000124

Abstract

Sarcopenia, the age-related loss of skeletal muscle mass and strength, is a significant cause of morbidity in the elderly and is a major burden on health care systems. Unfortunately, the underlying molecular mechanisms in sarcopenia remain poorly understood. Herein, we utilized top-down proteomics to elucidate sarcopenia-related changes in the fast- and slow-twitch skeletal muscles of aging rats with a focus on the sarcomeric proteome, which includes both myofilament and Z-disc proteins-the proteins that constitute the contractile apparatuses. Top-down quantitative proteomics identified significant changes in the post-translational modifications (PTMs) of critical myofilament proteins in the fast-twitch skeletal muscles of aging rats, in accordance with the vulnerability of fast-twitch muscles to sarcopenia. Surprisingly, age-related alterations in the phosphorylation of Cypher isoforms, proteins that localize to the Z-discs in striated muscles, were also noted in the fast-twitch skeletal muscle of aging rats. This represents the first report of changes in the phosphorylation of Z-disc proteins in skeletal muscle during aging. In addition, increased glutathionylation of slow skeletal troponin I, a novel modification that may help protect against oxidative damage, was observed in slow-twitch skeletal muscles. Furthermore, we have identified and characterized novel muscle type-specific proteoforms of myofilament proteins and Z-disc proteins, including a novel isoform of the Z-disc protein Enigma. The finding that the phosphorylation of Z-disc proteins is altered in response to aging in the fast-twitch skeletal muscles of aging rats opens new avenues for the investigation of the role of Z-discs in age-related muscle dysfunction.

Highlights

From the ‡Department of Cell and Regenerative Biology, University of Wisconsin-Madison, 1111 Highland Ave., Madison, Wisconsin, 53705; §Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, P
In agreement with findings in human skeletal muscle [16], we recently identified an age-related decrease in the phosphorylation of RLC in fast-twitch skeletal muscles of aging rats that can account for sarcopenic muscle functional impairments [17]
Sarcopenia Affects Both Fast- and Slow-twitch Skeletal Muscles in Aging Rats—Consistent with previous reports of age-related fast-skeletal muscle fiber atrophy [7, 8, 37, 42], GAS muscles from 36-month-old rats displayed characteristic signs of sarcopenia, including significant decreases in muscle mass and cross-sectional area (CSA) at the muscle mid-belly (Fig. 1E–1G). These changes were, in part, attributable to a decrease in the mean fiber diameter (Fig. 1I), which is consistent with the wellestablished sarcopenia-associated atrophy of type II muscle fibers [7, 8, 42]

Summary

The abbreviations used are

Tropomyosin; TnI, troponin I; TnT, troponin T; TnC, troponin C; MHC, myosin heavy chain; PTMs, post-translational modifications; MS, mass spectrometry; MS/MS, tandem MS; ECD, electron capture dissociation; F344BN, Fisher 344 ϫ Brown Norway F1 hybrid; GAS, gastrocnemius; SOL, soleus; CSA, cross-sectional area; LC, liquid chromatography; q-TOF, quadrupole-time-of-flight; LTQ, linear ion trap; FT-ICR, Fourier transform ion cyclotron resonance; Ptotal, total protein phosphorylation; fsTnT, fast skeletal troponin T; fsTnI, fast skeletal troponin I; ␣Tpm, ␣-tropomyosin; MLC-1F, fast skeletal myosin essential light chain 1; MLC3F, fast skeletal myosin essential light chain 3; fsTnC, fast skeletal troponin C; MLC-2F, fast skeletal isoform of the myosin regulatory light chain; nEnigma, novel isoform of the Enigma protein; ssTnT1, slow skeletal troponin T isoform 1; ssTnI, slow skeletal troponin I; MLC-1S, slow skeletal myosin essential light chain; ssTnC, slow skeletal troponin C; MLC-1V, slow skeletal/ventricular myosin essential light chain; MLC-2S, slow skeletal/ventricular myosin regulatory light chain; ␤Tpm, ␤-tropomyosin; pfsTnI, phosphorylated fsTnI; GSSfsTnI, glutathionylated fsTnI; p␣Tpm, phosphorylated ␣Tpm; p␤Tpm, phosphorylated ␤Tpm; pMLC-2F, mono-phosphorylated MLC-2F; ppMLC-2F, bis-phosphorylated MLC-2F; pssTnT1, mono-phosphorylated ssTnT1; pssTnI, mono-phosphorylated ssTnI; GSS-ssTnI, glutathionylated ssTnI; pCypher2s, mono-phosphorylated Cypher2s; pCypher4s, mono-phosphorylated Cypher4s. The analysis of intact proteoforms in top-down proteomics provides a “bird’s eye” view of all protein sequence variations and PTMs, which can subsequently be localized using a variety of tandem MS (MS/MS) techniques, such as collision induced dissociation and electron capture dissociation (ECD) (20 –22). We used quantitative top-down proteomics to gain insights into age-related molecular changes in fast- and slow-twitch skeletal muscles, with a particular emphasis on changes occurring in the sarcomeric proteome. We detected few changes in myofilament protein PTMs in the slow-twitch skeletal muscles of aging rats, a significant increase in the glutathionylation of slow skeletal troponin I (ssTnI), a modification that may help protect against oxidative damage and contribute to differences in the presentation of sarcopenia in. We provide evidence that the phosphorylation of Z-disc proteins is altered with age in fast-twitch skeletal muscles

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION