Proteomic signatures of in vivo muscle oxidative capacity in healthy adults

Abstract

Adequate support of energy for biological activities and during fluctuation of energetic demand is crucial for healthy aging; however, mechanisms for energy decline as well as compensatory mechanisms that counteract such decline remain unclear. We conducted a discovery proteomic study of skeletal muscle in 57 healthy adults (22 women and 35 men; aged 23-87 years) to identify proteins overrepresented and underrepresented with better muscle oxidative capacity, a robust measure of in vivo mitochondrial function, independent of age, sex, and physical activity. Muscle oxidative capacity was assessed by ³¹ P magnetic resonance spectroscopy postexercise phosphocreatine (PCr) recovery time (τ_PCr ) in the vastus lateralis muscle, with smaller τ_PCr values reflecting better oxidative capacity. Of the 4,300 proteins quantified by LC-MS in muscle biopsies, 253 were significantly overrepresented with better muscle oxidative capacity. Enrichment analysis revealed three major protein clusters: (a) proteins involved in key energetic mitochondrial functions especially complex I of the electron transport chain, tricarboxylic acid (TCA) cycle, fatty acid oxidation, and mitochondrial ABC transporters; (b) spliceosome proteins that regulate mRNA alternative splicing machinery, and (c) proteins involved in translation within mitochondria. Our findings suggest that alternative splicing and mechanisms that modulate mitochondrial protein synthesis are central features of the molecular mechanisms aimed at maintaining mitochondrial function in the face of impairment. Whether these mechanisms are compensatory attempt to counteract the effect of aging on mitochondrial function should be further tested in longitudinal studies.

Keywords: 31P MRS; bioenergetic; mitochondria; proteomic; skeletal muscle.

Figure 1

³¹P MRS measurement of in vivo muscle oxidative capacity. (a) Schematic diagram depicting energetic fluxes associated with acute muscle contraction (left) and during postexercise recovery (right). (b) Representative ³¹P spectra collected before, during, and after exercise showing phosphocreatine (PCr) depletion during exercise and concomitant increase in inorganic phosphate (Pi), corresponding to these underlying bioenergetic processes. (c) Time course of PCr changes before, during, and after exercise. Red line corresponds to the fitted mono‐exponential recovery model (see Experimental Procedures section)

Figure 2

Skeletal muscle proteins associated with in vivo muscle oxidative capacity. (a) Protein association with postexercise phosphocreatine (PCr) recovery time (see statistical analysis in Experimental Procedures section). Blue circles denote proteins that are significantly associated with better muscle oxidative capacity (smaller τ_PCr), while red circles correspond to proteins significantly associated with poorer oxidative capacity (bigger τ_PCr). Proteins that did not significantly correlate with τ_PCr (p > .05) are shown in black. (b) Categorization of all proteins that were quantified using mass spectrometry as a function of their subcellular location (upper pie chart) and categorization of proteins that were significantly associated with τ_PCr, that is, significantly associated with better or poorer oxidative capacity (lower pie charts). (c) Top enriched reactome pathways identified by STRING analysis

Figure 3

Schematic representation of oxidative phosphorylation pathways involved in muscle bioenergetics. Complex I (green), electron transport chain (red), fatty acid β‐oxidation (gray), and tricarboxylic acid cycle (TCA; blue). Mapping of significant proteins with p < .05 are shown in both the TCA and fatty acid cycles. For clarity, complex I and electron transport chain proteins are listed in Table 1

Figure 4

Scatter plot of ATP‐binding cassette sub‐family B (ABC transporters) proteins versus. postexercise recovery time of phosphocreatine (τ_PCr) measured in second. The location of ABC transporters is indicated (a) ABCB8, ABCBA, and ABCB7 in the inner mitochondrial membrane from left to right, respectively, (b) ABCB6 in the outer mitochondrial membrane. Linear regression line of log₂τ_PCr versus. log₂ of protein abundance is shown for the ABC transporters, without adjusting for covariates (It is worth noting that all other statistical results, that is, p and β coefficient in the text, are reported after accounting for age and all covariates, see Experimental procedure). (c) Schematic representation of the location of the ABC transporters within mitochondria

Figure 5

Network representation of the 253 proteins associated with better muscle oxidative capacity as organized by molecular action. Nodes correspond to proteins while edges connecting nodes represent molecular action (e.g., binding, catalysis). STRING network construction was performed using high confidence values (0.7), and the robustness of major protein cluster detection was ascertained by reproducing the results even after allowing for higher number of cluster formation. Color nodes highlight enriched pathways according to reactome analysis, that is, mRNA splicing, respiratory electron transport chain, and mitochondrial protein translation. Nonconnected proteins are not represented