Signatures of Cysteine Oxidation on Muscle Structural and Contractile Proteins Are Associated with Physical Performance and Muscle Function in Older Adults: Study of Muscle, Mobility and Aging (SOMMA)

Abstract

Oxidative stress is considered a contributor to declining muscle function and mobility during aging; however, the underlying molecular mechanisms remain poorly described. We hypothesized that greater levels of cysteine (Cys) oxidation on muscle proteins are associated with decreased measures of mobility. Herein, we applied a novel redox proteomics approach to measure reversible protein Cys oxidation in vastus lateralis muscle biopsies collected from 56 subjects in the Study of Muscle, Mobility and Aging (SOMMA), a community-based cohort study of individuals aged 70 years and older. We tested whether levels of Cys oxidation on key muscle proteins involved in muscle structure and contraction were associated with muscle function (leg power and strength), walking speed, and fitness (VO₂ peak on cardiopulmonary exercise testing) using linear regression models adjusted for age, sex, and body weight. Higher oxidation levels of select nebulin Cys sites were associated with lower VO₂ peak, while greater oxidation of myomesin-1, myomesin-2, and nebulin Cys sites was associated with slower walking speed. Higher oxidation of Cys sites in key proteins such as myomesin-2, alpha-actinin-2, and skeletal muscle alpha-actin were associated with lower leg power and strength. We also observed an unexpected correlation (r = 0.48) between a higher oxidation level of 8 Cys sites in alpha-actinin-3 and stronger leg power. Despite this observation, the results generally support the hypothesis that Cys oxidation of muscle proteins impair muscle power and strength, walking speed, and cardiopulmonary fitness with aging.

Keywords: Cysteine oxidation; Fitness; Muscle; Post-translational modifications; Power; Redox.

Figure 1.

Study design and workflow. Participants undergo baseline physical examinations (age, height, weight, blood pressure, etc.) and measures of muscle function (leg strength and power), walking speed, and fitness (VO₂ peak) as well as specimen collections (blood, urine, muscle, etc.) over multiple days (Cummings et al., 2023). Muscle biopsies were collected from vastus lateralis muscle. A deep redox proteomics profiling workflow was used to quantitatively measure Cys oxidation (i.e., all forms of reversible thiol PTMs). Analysis of redox proteomics data was prioritized for a select group of muscle contraction proteins (Uniprot ID format) to test the hypothesis that protein Cys oxidation is associated with functional measures.

Figure 2.

Selected signatures of Cys oxidation in significant association with fitness, muscle function, and physical performance. Cys sites on muscle proteins with oxidation levels significantly associated with a functional measure (Supplemental File S2) were clustered and plotted in heatmaps (Supplemental Figures S3, S5, S7, S9). A representative cluster of Cys sites (“signature”) from each phenotype-specific heatmap are shown in panels A-D. Each row represents a Cys site with oxidation levels that are significantly associated with a phenotype, while each column represents a participant. Columns are ranked in descending order from left to right based on phenotypic measurement and the phenotypic values are represented by median-centered Z-scores, while Cys oxidation levels were scaled by median-centering. Note that the row-wise hierarchical clustering of Cys sites is reordered from the original corresponding clusters in Supplemental Figures S3, S5, S7, S9. Protein identities are in UniProt format. Sites passing an adjusted p value < 0.05 cutoff are denoted by ‘*’. A) Peak volume of oxygen consumption (VO₂ peak; mL/min) as a measure of cardiopulmonary fitness. B) Walking speed from a 400m walking test (m/s). C) Leg strength as measured by one repetition maximum (kg). D) Leg power measured as the highest peak power (watts) generated.

Figure 3.

Selected signatures of Cys oxidation are negatively associated with fitness, muscle function, and physical performance in older adults. Signatures (sub-clusters) within each phenotype-specific heatmap (Supplemental Figures S3, S5, S7, S9) were plotted for correlation analysis (Supplemental Figures S4, S6, S8, S10). Representative correlations of signatures of highlighted in Fig. 2 are shown in panels A-D, where the mean level of oxidation across all Cys sites in a signature were plotted for each participant against their phenotypic measurement. R and p values are derived from Pearson correlation. A-D: Representative Pearson correlations for A) VO₂ peak, B) walking speed, C) leg strength, and D) leg power.

Figure 4.

Unique, significant positive association between ACTN3 and leg power. A) Representative heatmap of ACTN3 signatures identified as significantly associated with the leg power phenotype. Each row represents a Cys site with oxidation levels that are significantly associated with a phenotype, while each column represents a participant. Columns are ranked in descending order from left to right based on phenotypic measurement and the phenotypic values are represented by median-centered Z-scores, while Cys oxidation levels were scaled by median-centering. Protein identities are in UniProt format. B) Cys sites from the ACTN3 signature in panel A were plotted for correlation analysis, where the mean level of oxidation across all Cys sites in a signature were plotted for each participant against their phenotypic measurement. R and p values are derived from Pearson correlation. C) AlphaFold-predicted structure of ACTN3 with all significantly associated Cys sites denoted by magenta colored text. The actin binding region (AA1-262), neck and spectrin repeat region (AA263-758), and calmodulin-like domain containing 2 pairs of EF hands (AA759-901) are colored in blue, gray, and green, respectively.