Limb immobilization induces a coordinate down-regulation of mitochondrial and other metabolic pathways in men and women

Abstract

Advancements in animal models and cell culture techniques have been invaluable in the elucidation of the molecular mechanisms that regulate muscle atrophy. However, few studies have examined muscle atrophy in humans using modern experimental techniques. The purpose of this study was to examine changes in global gene transcription during immobilization-induced muscle atrophy in humans and then explore the effects of the most prominent transcriptional alterations on protein expression and function. Healthy men and women (N = 24) were subjected to two weeks of unilateral limb immobilization, with muscle biopsies obtained before, after 48 hours (48 H) and 14 days (14 D) of immobilization. Muscle cross sectional area (approximately 5%) and strength (10-20%) were significantly reduced in men and women (approximately 5% and 10-20%, respectively) after 14 D of immobilization. Micro-array analyses of total RNA extracted from biopsy samples at 48 H and 14 D uncovered 575 and 3,128 probes, respectively, which were significantly altered during immobilization. As a group, genes involved in mitochondrial bioenergetics and carbohydrate metabolism were predominant features at both 48 H and 14 D, with genes involved in protein synthesis and degradation significantly down-regulated and up-regulated, respectively, at 14 D of muscle atrophy. There was also a significant decrease in the protein content of mitochondrial cytochrome c oxidase, and the enzyme activity of cytochrome c oxidase and citrate synthase after 14 D of immobilization. Furthermore, protein ubiquitination was significantly increased at 48 H but not 14 D of immobilization. These results suggest that transcriptional and post-transcriptional suppression of mitochondrial processes is sustained throughout 14 D of immobilization, while protein ubiquitination plays an early but transient role in muscle atrophy following short-term immobilization in humans.

Figure 1. Early (A) and late (B) transcriptional changes during immobilization-induced muscle atrophy.

(A) Two-time point comparisons of Log₂ probe signal between the PRE and 48 H time points were conducted and significant differences were assessed using standard paired T-Tests. Genes with FDR q-value <0.05 were considered to be significantly altered. Genes were then organized along functional lines and the number of genes in each category was plotted. χ² analyses comparing actual versus expected number of genes within each category (as detailed in the Materials and Methods) were conducted to determine whether gene categories were significantly (P≤0.05) up- or down-regulated at the early (48 H) time-point following immobilization. (B) Two-time point comparisons between PRE and 14 D probe signals were conducted as in (A) to identify gene categories that were up- or down-regulated as part of the late (14 D) transcriptional response to immobilization. Red bars are down-regulated genes whereas green bars are up-regulated genes. Arrowheads indicate gene categories that were significantly up- or down-regulated. Gene categories representing fatty acid and carbohydrate metabolism, and mitochondrial function, including oxidative phosphorylation, β-oxidation, and mitochondrial regulation, were significantly down-regulated at both 48 H and 14 D of immobilization. In addition, the gene category representing mRNA synthesis and maturation (e.g. splicing, polyadenelation, 5′ cap binding proteins) was significantly up-regulated at both 48 H and 14 D. The gene category representing metallothioneins was up-regulated at 48 H but not 14 D, while gene categories representing proteases and DNA regulation (e.g. repair enzymes and chromatin remodeling proteins) were up-regulated at 14 D but not 48 H. Lastly, the gene category representing protein synthesis was down-regulated at 14 D but not at 48 H. ECM: extracellular matrix; Prot. Degradation: protein degradation; DNA. Reg.: DNA regulatory factors; RNA Synth.: RNA synthesis and maturation; Ox. Stress: oxidative stress; Carb. Metabolism: carbohydrate metabolism.

Figure 2. Changes in mitochondrial protein during immobilization-induced muscle atrophy.

The protein level of the mitochondrial proteins citrate synthase (CS), cytochrome c oxidase (COX) subunits II and IV were determined by western blotting as detailed in the Materials and Methods. Protein levels were normalized to actin. Only COX II protein was significantly (P≤0.05) reduced following 14 D of immobilization (asterisk).

Figure 3. Changes in mitochondrial enzyme activity during immobilization-induced muscle atrophy.

The enzyme activity of CS and COX was determined spectrophotometrically as detailed in the Materials and Methods. The enzymatic activity of both CS and COX were significantly (P≤0.05) reduced following 14 D of immobilization (asterisk).

Figure 4. Transcriptional status of atrogin-1 and MuRF-1 and global protein ubiquitination during immobilization-induced muscle atrophy.

(A) The mRNA level of both atrogin-1 and MuRF-1 was determined using qRT-PCR and significant (P≤0.05) increases in both genes were detected following 48 H of immobilization but not at 14 D. (B) Overall protein ubiquitination was examined using Western blotting and numerous ubiquitin-positive protein bands were detected (left). The most ubiquitin-dense proteins were greater than 250 kDa in MW. Thus lanes were divided into high MW (>250 kDa) and low MW (<250 kDa) sections and quantified using densitometry. A significant (P≤0.05) increase in protein ubiquitination of high MW proteins was detected following 48 H of immobilization (right, asterisk) but not 14 D. No significant changes in low molecular weight (<250 kDa) protein ubiquitination were detected at any time-point.

Figure 5. Changes in mTOR phosphorylation during immobilization-induced muscle atrophy.

The protein level of phospho-mTOR was measured in muscle samples undergoing immobilization-induced muscle atrophy using Western blotting. Blots were then stripped and re-probed for total mTOR. Protein levels were normalized to tubulin. No significant differences in total mTOR protein levels were detected at any time-points (not shown), however the ratio of phospho-mTOR to mTOR decreased significantly (P≤0.05) at the 48 H time-point (asterisk) indicating decreased mTOR protein phosphorylation during the early phase of immobilization-induced muscle atrophy.