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. 2018 Feb;17(1):e12700.
doi: 10.1111/acel.12700. Epub 2017 Nov 9.

Running-wheel activity delays mitochondrial respiratory flux decline in aging mouse muscle via a post-transcriptional mechanism

Sarah Stolle  1   2 , Jolita Ciapaite  1   2 , Aaffien C Reijne  1   2   3 , Alzbeta Talarovicova  1   2 , Justina C Wolters  1   2   4 , Raúl Aguirre-Gamboa  5 , Pieter van der Vlies  5 , Kim de Lange  5 , Pieter B Neerincx  5   6 , Gerben van der Vries  5   6 , Patrick Deelen  5   6 , Morris A Swertz  5   6 , Yang Li  5 , Rainer Bischoff  4 , Hjalmar P Permentier  4 , Peter L Horvatovitch  4 , Albert K Groen  1   2   7 , Gertjan van Dijk  2   3   8 , Dirk-Jan Reijngoud  1   2 , Barbara M Bakker  1   2
Affiliations

Running-wheel activity delays mitochondrial respiratory flux decline in aging mouse muscle via a post-transcriptional mechanism

Sarah Stolle et al. Aging Cell. 2018 Feb.

Abstract

Loss of mitochondrial respiratory flux is a hallmark of skeletal muscle aging, contributing to a progressive decline of muscle strength. Endurance exercise alleviates the decrease in respiratory flux, both in humans and in rodents. Here, we dissect the underlying mechanism of mitochondrial flux decline by integrated analysis of the molecular network. Mice were given a lifelong ad libitum low-fat or high-fat sucrose diet and were further divided into sedentary and running-wheel groups. At 6, 12, 18 and 24 months, muscle weight, triglyceride content and mitochondrial respiratory flux were analysed. Subsequently, transcriptome was measured by RNA-Seq and proteome by targeted LC-MS/MS analysis with 13 C-labelled standards. In the sedentary groups, mitochondrial respiratory flux declined with age. Voluntary running protected the mitochondrial respiratory flux until 18 months of age. Beyond this time point, all groups converged. Regulation Analysis of flux, proteome and transcriptome showed that the decline of flux was equally regulated at the proteomic and at the metabolic level, while regulation at the transcriptional level was marginal. Proteomic regulation was most prominent at the beginning and at the end of the pathway, namely at the pyruvate dehydrogenase complex and at the synthesis and transport of ATP. Further proteomic regulation was scattered across the entire pathway, revealing an effective multisite regulation. Finally, reactions regulated at the protein level were highly overlapping between the four experimental groups, suggesting a common, post-transcriptional mechanism of muscle aging.

Keywords: Regulation Analysis; integrative data analysis; metabolism; mitochondrial function; skeletal muscle aging; targeted proteomics.

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Figures

Figure 1
Figure 1
Overview of Regulation Analysis and system of interest. (a) The flow of information from gene, via mRNA, to proteins and metabolic flux. Regulation coefficients are indicated in the scheme. (b) Scheme of mitochondrial substrate oxidation. Colour coding of different pathways represented here is also used in Figure 5
Figure 2
Figure 2
Skeletal muscle properties. (a) Running‐wheel (RW) activity. (b) Body weight. (c) Quadriceps muscle weight. (d) Triglyceride content in quadriceps muscle. Data shown as average of n = 7–8 mice per experimental group and time point ± SEM, except for panel A with n = 15–23. **p < .001 and *p < .05 compared to HFS (+)RW group; ## p < .001 and # p < .05 compared to LF (−)RW group; $ p < .05 compared to HFS (−)RW group. LF (−)RW, low‐fat without running wheel; LF (+)RW, low‐fat with running wheel; HFS (−)RW, high‐fat sucrose without running wheel; HFS (+)RW, high‐fat sucrose with running wheel
Figure 3
Figure 3
Skeletal muscle mitochondrial properties. (a, b) Maximal ADP‐stimulated O2 flux (state 3) in isolated skeletal muscle mitochondria oxidizing pyruvate plus malate (PM) or palmitoyl‐CoA plus L‐carnitine plus malate (PCM) in low‐fat and high‐fat sucrose diet groups. (c) Relative mtDNA copy number in quadriceps muscle. (d) Mitochondrial protein content in skeletal muscle determined as citrate synthase activity in tissue homogenate divided by that in isolated mitochondria, both normalized for the protein content of the preparation (Fig. S2C and D, Supporting information). (e, f) Maximum skeletal muscle oxygen flux capacity expressed per total tissue protein, determined as the state 3 O2 flux in isolated mitochondria multiplied by the mitochondrial protein content in skeletal muscle. Data are averages of n = 7–8 mice per experimental group and time point ± SEM. # p < .05 compared to LF (−)RW group; $$ p < .001 and $ p < .05 compared to HFS (−)RW group. Experimental group abbreviations as in Figure 2; PM, pyruvate plus malate; PCM, palmitoyl‐CoA plus L‐carnitine plus malate
Figure 4
Figure 4
The effects of HFS diet, RW activity and age on mitochondrial proteins involved in substrate transport, OXPHOS pathway, fatty acid β‐oxidation, TCA cycle and antioxidant defence. (a) Summary of the linear regression analysis showing numbers of proteins uniquely affected by HFS diet, RW activity, and age with their overlap (Venn diagram), and direction (positive or negative) of the effect (table). (b) The correlation between the concentration of a specific mitochondrial protein and the mitochondrial O2 flux driven by pyruvate plus malate (PM) in state 3. All time points are combined in the same plot. Only six proteins with the highest Pearson correlation coefficient are shown. Data are n = 4 mice per experimental group and time point. Experimental group abbreviations as in Figure 2
Figure 5
Figure 5
Regulation of O2 flux in skeletal muscle mitochondria of aging mice. (a, b) Hierarchical regulation coefficient ρh for LF (−) RW (a) and LF (+) RW mice (b). A coefficient of 1 means that the change in flux during aging can be explained completely by the change in protein concentration, whereas a coefficient of 0 means that the flux is completely metabolically regulated (n = 4 per group). (c, d) Transcriptional regulation coefficient ρm RNA for LF (−) RW and LF (+) RW mice. Only proteins with a ρh > 0 and p < .05 were taken into account. The coefficients are based on n = 3 for mRNA, n = 4 for protein per group. The average ± SD is plotted in ascending order independently for each condition for all regulation coefficients. *(ρh ≠ 0.5), ♦ (ρh > 0) each with adjusted p value < .05. If the same protein was significant for *ρh ≠ 0.5 and ♦ ρh > 0, then only * was indicated. The enzymes that belong to the same metabolic pathway are highlighted in the same colour as pathways represented in Figure 1. (e) Venn diagram showing reactions with a ρh > 0 and p < .05, where regulation of protein concentration contributes significantly and often more than 50% to the change in oxidative flux with age
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