Impaired Mitochondrial Energetics Characterize Poor Early Recovery of Muscle Mass Following Hind Limb Unloading in Old Mice

Abstract

The progression of age-related sarcopenia can be accelerated by impaired recovery of muscle mass following periods of disuse due to illness or immobilization. However, the mechanisms underlying poor recovery of aged muscle following disuse remain to be delineated. Recent evidence suggests that mitochondrial energetics play an important role in regulation of muscle mass. Here, we report that 22- to 24-month-old mice with low muscle mass and low glucose clearance rate also display poor early recovery of muscle mass following 10 days of hind limb unloading. We used unbiased and targeted approaches to identify changes in energy metabolism gene expression, metabolite pools and mitochondrial phenotype, and show for the first time that persistent mitochondrial dysfunction, dysregulated fatty acid β-oxidation, and elevated H2O2 emission occur concomitantly with poor early recovery of muscle mass following a period of disuse in old mice. Importantly, this is linked to more severe whole-body insulin resistance, as determined by insulin tolerance test. The findings suggest that muscle fuel metabolism and mitochondrial energetics could be a focus for mining therapeutic targets to improve recovery of muscle mass following periods of disuse in older animals.

Figure 1.

Old mice do not recover muscle mass or insulin sensitivity following 10 days of hind limb unloading and 3 days of recovery. (A) Soleus weight following unloading, unloading/reloading, and control in adult and old mice, n = 11 per group. (B) Body weight for the adult and old mice following unloading, unloading/reloading, and control periods, n = 11 per group. (C) Average daily food intake for old and adult mice during unloading, unloading/reloading, and control periods, n = 11 per group. (D) Protein synthesis/breakdown signaling: pAKT/AKT, pmTOR/mTOR, pS6/S6, and pFOXO3a/FOXO3a ratios, n = 5 per group. (E) Blood glucose reductions (percent change from baseline) following intraperitoneal insulin injection. The slope of the fall in glucose levels from t = 0 to 15 minutes was used as an index of whole-body insulin action, n = 5 per group. (F) Soleus insulin-stimulated glucose clearance rates in control or unloaded/reloaded adult and old mice, n = 5 per group. (G) Day and night cage ambulatory activity (x, y, z) in control or unloaded/reloaded adult and old mice, n = 5 per group. Data are presented as mean ± SEM. The letters A and B denote significant differences between group/time points (p < 0.05, ANOVA/Tukey Correction). **p <0.05 ANOVA/Bonferroni correction. *p <0.05 versus control ANOVA/Bonferroni Correction. ANOVA = analysis of variance; CON = control; mTOR = mammalian target of rapamycin; RL = reloading recovery; FOXO3a = Forkhead box O3; UN = unloading.

Figure 2.

Mitochondrial function is impaired during unloading and improves following 3 days of recovery only in adult mice. (A) Complex I (Glutamate and Malate) supported LEAK (CI_L or State 4) respiration. (B) Palmitoylcarnitine supported OXPHOS (State 3) respiration. (C) Complex I supported OXPHOS respiration. (D) Complex I & II supported OXPHOS respiration. (E) Mitochondrial maximal H₂O₂ emission as determined with Amplex Red, during oligomycin-induced state 4 respiration. (F) Mitochondrial calcium retention capacity. n = 8–10 per group for permeabilized fiber bundle experiments. (G) Total OXPHOS protein content by immunoblot, n = 5 per group. (H) Total cardiolipin content by tandem mass spectrometry. (I) Heatmap depicting change in individual immature, mature, and remodeled cardiolipin species. The normalized (z-score) mean of each row (cardiolipin species) was visualized in the heatmap, n = 7 per group. (J) Representative immunoblot for OXPHOS proteins. Data are presented as mean ± SEM. **p <0.05 ANOVA/Bonferroni correction. *p <0.05 versus control ANOVA/Bonferroni correction. ANOVA = analysis of variance.

Figure 3.

Acylcarnitine, organic acid, and amino acid profiles during unloading and reloading. (A) Acylcarnitine profile in soleus of adult and old mice during control, unloading, and reloading conditions. Acyl chain length and degree of saturation are denoted by standard nomenclature (C:D, where C is the number of carbon atoms and D is the number of double bonds). (B) Metabolite profiles showing lactate and pyruvate and organic acid intermediates of the tricarboxylic acid (TCA) cycle. (C) Amino acids profile in soleus during control, unloading, and reloading conditions. n = 6 per group. Data are presented as mean ± SEM. **p <0.05 ANOVA/Bonferroni correction. *p <0.05 versus control ANOVA/Bonferroni correction. ANOVA = analysis of variance; BLQ = below limit of quantification.

Figure 4.

Gene regulatory networks for oxidative metabolism are suppressed during unloading and do not recovery following 3 days of reloading. (A) KEGG pathway analysis of RNA-sequencing data set representing significantly downregulated pathways relating to mitochondria, metabolism, and protein metabolism during unloading and after 3 days of reloading. (B) Heatmap depicting IPA Upstream Regulator analysis to identify upstream transcriptional regulators responsible for the transcriptomic profile seen with unloading and reloading, plotted as a centralized Z-score for each transcription factor. The RNA-sequencing data set used was filtered with reads per kilobase per million mapped reads (RPKM) >1, p <0.05. (C) RT-qPCR assay for expression of key nuclear transcription factors that drive mitochondrial energy metabolism. n = 4–9 per group. Data are presented as mean ± SEM. *p <0.05 versus control ANOVA/Bonferroni correction. FOXO = Forkhead box O; KEGG = Kyoto Encyclopedia of Genes and Genomes; mTOR = mammalian target of rapamycin; RT-qPCR = reverse transcription-quantitative polymerase chain reaction; TCA = tricarboxylic acid.

Figure 5.

Comparative analysis of transcriptomic and metabolomics profiles during unloading and reloading. (A) Heatmap containing RNA-sequencing data set representing the level of expression of genes with reads per kilobase per million mapped reads (RPKM) >1, plotted as a centralized Z-score for each gene in fatty acid metabolism, tricarboxylic acid (TCA) cycle, and electron transport chain/oxidative phosphorylation pathways defined by Ingenuity Pathway Analysis. (B) Heatmap containing the levels of acylcarnitines, organic acids, amino acids, pyridine, and adenine nucleotides and total level of free fatty acids (FFA), diacylglycerol (DAG), and triacylglycerol (TAG). For all heatmaps, relative downregulation is in blue and upregulation in red. The intensity of color indicates the magnitude of the change. *The energy charge ratio is defined as ([ATP] + 1/2[ADP])/([AMP] + [ADP] + [ATP]). CON = control; RL = reloading recovery; UN = unloading.