Mitochondrial Adaptations in Elderly and Young Men Skeletal Muscle Following 2 Weeks of Bed Rest and Rehabilitation

Abstract

The aim of the study was to evaluate the expression levels of proteins related to mitochondrial biogenesis regulation and bioenergetics in vastus lateralis muscle biopsies from 16 elderly and 7 young people subjected to 14 days of bed-rest, causing atrophy, and subsequent 14 days of exercise training. Based on quantitative immunoblot analyses, in both groups a reduction of two key regulators of mitochondrial biogenesis/remodeling and activity, namely PGC-1α and Sirt3, was revealed during bed-rest, with a subsequent up-regulation after rehabilitation, indicating an involvement of PGC-1α-Sirt3 axis in response to the treatments. A difference was observed comparing the young and elderly subjects as, for both proteins, the abundance in the elderly was more affected by immobility and less responsive to exercise. The expression levels of TOM20 and Citrate Synthase, assayed as markers of outer mitochondrial membrane and mitochondrial mass, showed a noticeable sensitivity in the elderly group, where they were affected by bed-rest and rehabilitation recalling the pattern of PGC-1α. TOM20 and CS remained unchanged in young subjects. Single OXPHOS complexes showed peculiar patterns, which were in some cases dissimilar from PGC-1α, and suggest different influences on protein biogenesis and degradation. Overall, exercise was capable to counteract the effect of immobility, when present, except for complex V, which was markedly downregulated by bed-rest, but remained unaffected after rehabilitation, maybe as result of greater extent of degradation processes over biogenesis. Phosphorylation extent of AMPK, and its upstream activator LKB1, did not change after bed-rest and rehabilitation in either young or elderly subjects, suggesting that the activation of energy-sensing LKB1-AMPK signaling pathway was "missed" due to its transient nature, or was not triggered under our conditions. Our study demonstrates that, as far as the expression of various proteins related to mitochondrial biogenesis/remodeling, adaptations to bed-rest and rehabilitation in the two populations were different. The impact of bed-rest was greater in the elderly subjects, where the pattern (decrease after bed rest and recovery following rehabilitation) was accompanied by changes of mitochondrial mass. Modifications of protein abundance were matched with data obtained from gene expression analyses of four public human datasets focusing on related genes.

Keywords: aging; exercise; immobility; in silico gene expression data mining; mitochondria-related proteins; skeletal muscle; western blot.

FIGURE 1

Changes in expression levels of PGC-1α (A) and Sirt3 (B) in vastus lateralis muscle biopsies from young and elderly subjects after bed rest and rehabilitation. Panels (A,B) graphs denoted as (1) represent immunoblot data of 7 young (Y; aged 18–30 years) and 16 elderly (E; aged 55–65 years) subjects, under the three conditions: BDC, baseline data collection before bed rest; BR14, after 14 days bed rest; R+14, after 2-week rehabilitation. Gels were loaded with samples from Y and E subjects (20 μg of proteins) along with IS (40 μg), prepared as described in Section “Materials and Methods.” ImageQuant TL values for single immunoreactive bands normalized to Coomassie staining of PVDF membrane and corrected for IS are expressed as arbitrary units (AU). Quantitative data are means ± SD of three different assays for each subject. Red line: Elderly group; black line: Young group. ^∗Represents statistical significance (p < 0.05) vs. BDC condition, # between BR14 and R+14 conditions. Immunoblot images denoted as (2) are representative of experiments with samples from both Y and E subjects loaded on each gel matching the conditions (namely all BDC or BR14 or R+14 samples), and images denoted as (3) are representative of the corresponding Coomassie-stained whole PVDF membranes, used as loading and transferring measurement. Immunoblot images denoted as (4) are representative of experiments where each subject was analyzed individually by loading gel with samples of the three investigated conditions together (BDC, BR 14, and R+14). Gels were loaded with scalar amounts (5, 10, 15, and 20 μg) of Y or E samples to verify the linearity of the band intensity vs. the loaded protein. The central lane of the gel was loaded with molecular weight markers.

FIGURE 2

Changes in OXPHOS complexes’ expression levels in vastus lateralis muscle biopsies from young and elderly subjects after bed rest and rehabilitation. (A) Graphs represent immunoblot data (means ± SD) of OXPHOS complexes I, II, III, IV, V for the Y and E subjects, under BDC, BR14, and R+14 conditions. Red line: Elderly group; black line: Young group. (B) Immunoblot images denoted as (1) are representative of experiments with samples from both Y and E subjects loaded on each gel matching the conditions, along with IS, and images denoted as (2) are representative of the corresponding Coomassie-stained whole PVDF membranes used as loading and transferring measurement. All details of the analysis and quantification are as in Figure 1.

FIGURE 3

Changes in TOM20 (A), Citrate Synthase (B), and GAPDH (C) expression levels in vastus lateralis muscle biopsies from young and elderly subjects after bed rest and rehabilitation. Panels (A–C) graphs denoted as (1) represent immunoblot data (means ± SD) for the Y and E subjects under BDC, BR14, and R+14 conditions. Red line: Elderly group; black line: Young group. Immunoblot images denoted as (2) are representative of experiments with samples from both Y and E subjects loaded on each gel matching the conditions, along with IS, and images denoted as (3) are representative of the corresponding Coomassie-stained whole PVDF membranes, used as loading and transferring measurement. All details of the analysis and quantification are as in Figure 1.

FIGURE 4

p-AMPK/AMPK (A) and p-LKB1/LKB1 (B) ratios in vastus lateralis muscle biopsies from young and elderly subjects after bed rest and rehabilitation. In both panels (A,B), histograms denoted as (1) represent the fold-increase (means ± SD) of the normalized ratio between phosphorylated and total proteins from immunoblot analyses for Y and E subjects under BDC (empty columns), BR14 (gray columns), and R+14 (black columns) conditions. Immunoblot images denoted as (2) refer to total (on the left) and phosphorylated (on the right) proteins and are representative of experiments where samples from both Y and E subjects were loaded together on each gel matching the conditions, along with IS. Images denoted as (3) are representative of the corresponding Coomassie-stained whole PVDF membranes, used as loading and transferring measurement. All details of the analysis and quantification are as in Figure 1.