Physical Activity Associated Proteomics of Skeletal Muscle: Being Physically Active in Daily Life May Protect Skeletal Muscle From Aging

Abstract

Muscle strength declines with aging and increasing physical activity is the only intervention known to attenuate this decline. In order to adequately investigate both preventive and therapeutic interventions against sarcopenia, a better understanding of the biological changes that are induced by physical activity in skeletal muscle is required. To determine the effect of physical activity on the skeletal muscle proteome, we utilized liquid-chromatography mass spectrometry to obtain quantitative proteomics data on human skeletal muscle biopsies from 60 well-characterized healthy individuals (20-87 years) who reported heterogeneous levels of physical activity (not active, active, moderately active, and highly active). Over 4,000 proteins were quantified, and higher self-reported physical activity was associated with substantial overrepresentation of proteins associated with mitochondria, TCA cycle, structural and contractile muscle, and genome maintenance. Conversely, proteins related to the spliceosome, transcription regulation, immune function, and apoptosis, DNA damage, and senescence were underrepresented with higher self-reported activity. These differences in observed protein expression were related to different levels of physical activity in daily life and not intense competitive exercise. In most instances, differences in protein levels were directly opposite to those reported in the literature observed with aging. These data suggest that being physically active in daily life has strong and biologically detectable beneficial effects on muscle.

Keywords: DNA damage; immunity; mass spectrometry; mitochondria; physical activity; proteomics; skeletal muscle; spliceosome.

FIGURE 1

Physical activity associated proteins in human skeletal muscle. (A) Proteins associated with physical activity. Level of significance of each protein was assessed by lmerTest from the mixed model regression results and the protein was reported as significant if the p < 0.05. Red circles are proteins overrepresented with higher physical activity and blue circles are proteins underrepresented with higher physical activity. The proteins that were least affected by physical activity are shown as gray, light blue, and orange. (B) The average raw data protein levels are shown for most significantly overrepresented or underrepresented with higher physical activity, according to self-reported physical activity level. The left axis represents the log2 relative abundance and the x-axis the level of physical activity.

FIGURE 2

Categorization of physical activity associated hallmarks of aging proteins in human skeletal muscle. (A) Categories of proteins which were detected by mass spectrometry are shown, focusing on pathways that emerge from aggregated clusters of proteins and have been associated with aging and/or physical activity. Level of significance of each protein was assessed by lmerTest from the mixed model regression results and the protein was reported as significant if the p < 0.05. (B) Proteins most significantly upregulated with higher physical activity, of which 75% were mitochondrial proteins. (C) 695 proteins were significantly underrepresented with higher physical activity, including those connected with proteostasis, splicing, and immunity/inflammation. (D) Subcategorization of the 48 upregulated “others” category proteins from B, which were not considered in detail in this study. (E) Subcategorization of the downregulated “others” category proteins from C not considered in detail in this study, including protein classes associated with cell proliferation, cytoskeleton, lipid metabolism, and translation. Square bracket on left shows number of proteins associated in each category.

FIGURE 3

Bioenergetics pathways upregulated with higher physical activity in human skeletal muscle. (A) Proteins associated with oxidative phosphorylation. Proteins from complex I, complex II, complex III, complex IV, and complex V in the electron transport chain and assembly complex proteins were overrepresented with higher physical activity. The y-axis shows the log2 abundance change of the protein associated with one higher point in the physical activity score (0–3) and the x-axis shows the gene name associated with each protein. Different complexes are color coded. (B) Many TCA cycle proteins were overrepresented with higher physical activity. All significantly quantified proteins are shown as bar plots. Level of significance of each protein was assessed by lmerTest from the mixed model regression results and the protein was reported as significant if the p < 0.05.

FIGURE 4

DNA damage and DNA repair proteins decreased in abundance with higher physical activity. All significant DNA damage and DNA repair proteins were negatively correlated with physical activity except SOD-2. The level of significance of each DNA damage protein was assessed by lmerTest from the mixed model regression results and the protein was reported as significant if the p < 0.05. Heat map shows four physical activity levels (x axis) and the average abundance of significant DNA damage and DNA repair proteins correlated with physical activity levels (y axis). Dark blue suggests lower relative log2 abundance of the protein and red suggests higher log2 relative protein abundance. The range of log2 abundance of the protein is from –0.1 to 0.2 (legend on top). The similarity between genes and physical activity groups are represented by hierarchical clustering on y- and x-axis, respectively.

FIGURE 5

Spliceosome complex proteins in skeletal muscle and physical activity. All significant spliceosome complex proteins were negatively correlated with physical activity. The level of significance of each spliceosome protein was assessed by lmerTest from the mixed model regression results and the protein was reported as significant if the p < 0.05. The heat map correlates four physical activity levels (x-axis) with the average abundance of spliceosomes genes correlated with physical activity levels (y-axis). Dark blue suggests negative correlation and red suggests positive correlation. The normalized range of the protein abundance for physical activity correlation is shown from red to blue (legend on bottom right). The color-coded “categories” column of the heatmap shows the spliceosome complex category annotated for each gene and the spliceosome category annotation label legend on top right shows the spliceosome major complex categories. Similarity between the spliceosome genes are shown by hierarchical clustering and represented with the dendrogram on the left y-axis.