Mass Spectrometry-Based Multiomics Identifies Metabolic Signatures of Sarcopenia in Rhesus Monkey Skeletal Muscle

Abstract

Sarcopenia is a progressive disorder characterized by age-related loss of skeletal muscle mass and function. Although significant progress has been made over the years to identify the molecular determinants of sarcopenia, the precise mechanisms underlying the age-related loss of contractile function remains unclear. Advances in "omics" technologies, including mass spectrometry-based proteomic and metabolomic analyses, offer great opportunities to better understand sarcopenia. Herein, we performed mass spectrometry-based analyses of the vastus lateralis from young, middle-aged, and older rhesus monkeys to identify molecular signatures of sarcopenia. In our proteomic analysis, we identified proteins that change with age, including those involved in adenosine triphosphate and adenosine monophosphate metabolism as well as fatty acid beta oxidation. In our untargeted metabolomic analysis, we identified metabolites that changed with age largely related to energy metabolism including fatty acid beta oxidation. Pathway analysis of age-responsive proteins and metabolites revealed changes in muscle structure and contraction as well as lipid, carbohydrate, and purine metabolism. Together, this study discovers new metabolic signatures and offers new insights into the molecular mechanisms underlying sarcopenia for the evaluation and monitoring of a therapeutic treatment of sarcopenia.

Keywords: bottom-up proteomics; multiomics; nonhuman primate; sarcopenia; skeletal muscle; untargeted metabolomics.

Figure 1.. Overview of the integrated functional and multi-omic analysis of aging rhesus monkey skeletal muscle.

Biometric analysis including segmental bioelectrical impedance spectroscopy (S-BIS) was performed on the vastus lateralis from young (median age 7.2y), middle-age (median age 15.1y) and old (median age 28.1y) rhesus monkeys (n=4 per group). Histological measurements including fiber type and non-contractile area characterization was performed. After protein extraction and enzymatic digestion, proteome changes were measured using LC-data independent parallel accumulation serial fragmentation mass spectrometry (LC-diaPASEF MS). An untargeted metabolomics study was performed to measure altered metabolites between groups by LC-Q-TOF tandem MS (MS/MS) analysis after extraction of metabolites. An integrated multi-omics analysis was performed of significantly altered proteins and metabolites to identify concerted biological changes. Figure created with BioRender.com.

Figure 2.. Sarcopenia is associated with changes in muscle composition.

(A) Schematic showing the age of rhesus monkeys used in this study relative to a generalized survival curve. (B) Body weight and body mass index (BMI) for young (red) middle-age (purple), and old (blue) monkeys (n=4 per group). (C) Electrical properties of the upper leg (determined by S-BIS), extracellular to intracellular water ratios, phase angle, and reactance curves. (D) Distribution of fiber types vastus laterals biopsies detected using antibodies specific to type I or type II myosin heavy chain. (E) Quantification of non-contractile content (black arrows) in hematoxylin and eosin-stained sections (left) and fibrotic content (right) as detected by picrosirius expressed as percent area of tissue section (4–5 images per animal). Data shown as median +/− IQR, #p<0.1, * p>0.05, ** p<0.01.

Figure 3.. Proteomic analysis of aging rhesus monkey skeletal muscle.

Proteins where extracted from the vastus lateralis from young, middle-age, and old rhesus monkeys (n=4 per group), enzymatically digested, and subjected to mass spectrometry analysis (Table S1). Volcano plots showing significantly (adjusted p-value < 0.05) altered proteins for ((A) old relative to young (left,) middle-age relative to young (middle), and old relative to middle-age animals (right). Relative levels of muscle structure proteins including (B) actins, (C) myosins, (D) integrins, and (E) collagens. *p<0.01, **p<0.001, ***p<0.0001, and ****p<0.0001. ACTN1=alpha-actinin-1, ACTN4=alpha-actinin-4, MYL9=myosin regulatory light polypeptide 9, MYH11=myosin-11, ITA6=integrin alpha-6, ITA7= integrin alpha-6, COL6A3=collagen alpha-3 (VI) chain, and COL6A1=collagen alpha-1 (VI) chain.

Figure 4.. Gene Ontology (GO) term enrichment analysis of altered proteins from aging rhesus monkey skeletal muscle.

(A) Significantly altered proteins (Table S1) were subject to a GO term analysis to identify associated biological processes (Table S2). Shown are relative peak intensities for selected proteins related to selected biological functions for young, middle, and old rhesus monkeys (n=4 per group): (B) ATP metabolic process (GO: 0046034), (C) AMP metabolic process (GO: 0046033), and (D) fatty acid beta oxidation (GO:0006635). *p<0.01, ** p<0.001, ***p<0.0001, and ****p<0.0001. ITA6=integrin alpha-6, ITA7= integrin alpha-6, COL6A1=collagen alpha-1 (VI) chain, COL6A3=collagen alpha-3 (VI) chain, MYL9=myosin regulatory light polypeptide 9, MYH11=myosin-11, ACTN1=alpha-actinin-1, ACTN4=, ATPD = ATP synthase subunit d, mitochondrial, DHSD=succinate dehydrogenase [ubiquinone] cytochrome b small subunit, mitochondrial, QCR8 = cytochrome b-c1 complex subunit 8 (QCR8), AMPD= AMP deaminase 1, PUR8 = adenylosuccinate, PURA1 = adenylosuccinate synthetase isozyme 1, ECI1 = enoyl-CoA delta isomerase 1, mitochondrial, THIM = acetyl-coenzyme A carboxylase carboxyl transferase subunit alpha 2, and ECHD1=ethylmalonyl-CoA decarboxylase.

Figure 5.. Metabolomic analysis of aging rhesus monkey skeletal muscle.

Metabolites were extracted from the vastus lateralis from young, middle-age, and old rhesus monkeys (n=4 per group) and subject to mass spectrometry analysis (Table S3). Volcano plots showing significantly (adjusted p-value < 0.05) altered metabolites for (A) old relative to young (left,) middle-age relative to young (middle), and old relative to middle-age animals (right). Shown are the relative peak areas for selected metabolites related to (B) energy metabolism (carnitine (left), trimethyllysine (middle), and phosphocarnitine (right)), (C) lipid metabolism (phosphatidylinositol 38:4 (PI 38:4)), (D) fatty acid beta oxidation (palmitoylcarnitine (left) and arachidonoylcarnitine (right). (E) Summary of fatty acid beta oxidation changes observed with age. *p<0.01, **p<0.001, ***p<0.0001, and ****p<0.0001.

Figure 6.. Integrated protein and metabolite pathway analysis.

Altered proteins and metabolites were subject to KEGG pathway analysis (Table S4). Shown are bubble plots depicting top-ranked up (top) and downregulated (bottom) pathways for (A) old relative to young, (B) middle-age relative to young and (C) old relative to middle-age animals. Bubble size is directly related to the number of significantly altered protein and metabolite (features). Color gradient is related to the significance of pathway enrichment (low (yellow) to high (red)). (D) Schematic summarizing the most prominent age-responsive pathway changes.