Immobility induced skeletal muscle atrophy results in an age-specific decrease in Akt and ribosomal protein S6 phosphorylation.

<p><b>A.</b> Western blotting of whole muscle protein homogenates of phosphorylated Akt and total Akt. <b>B.</b> Immobility decreased levels of phosphorylated Akt/total Akt ratio (p-Akt/Akt) at the early (2–4 days) phase of immobility in young but not aged skeletal muscle. * Time effect, p<0.05, compared to pre. # Age effect, p<0.001 young compared to old within time point. Due to lack of muscle tissue n = 6 (3 young and 3 old) in these analyses. <b>C.</b> Western blotting of whole muscle protein homogenates of total and phosphorylated S6 ribosomal protein. <b>D.</b> The percentage of the total number of subjects at each time point where p-S6 could be detected. Chi-square: Young p<0.001, Old p = 0.44. In a high number of especially young subjects phosphorylated S6 ribosomal protein (but not total S6 ribosomal protein) became non detectable after immobilization which made an exact quantification impossible.</p

Changes in the transcriptional status of PGC-1α and PGC-1β as a result of immobility-induced disuse-muscle atrophy.

<p>The mRNA level of PGC-1α and PGC-1β was determined using qRT-PCR. <b>A.</b> The results revealed a marked down-regulation of PGC-1α at 24 h in young muscle and a down-regulation in both young and old muscle at the later time points (2 d, 4 d and 14 d). <b>B.</b> The expression levels of PGC-1β mRNA also revealed an age-specific down-regulation after 24 h in young muscle and a down-regulation in both young and old muscle at 2 d and 4 d. In contrast to PGC-1α, the expression levels of PGC-1β mRNA returned to basal levels at 14 days of immobilization, indicating that the two genes may play different roles in the later stages of muscle unloading. * Time effect, p<0.05 compared to pre. Data are geometric means ± back-transformed SEM.</p

Immobility-induced skeletal muscle atrophy causes an age-specific decline in muscle size.

<p><b>A.</b> Scheme of experimental setup, including the time points of muscle biopsy procedure. <b>B</b>. Percentage decreases in muscle size (mean muscle fiber area) after 4 days of immobility in young (n = 11) and old (n = 9) as well as after 14 days of immobilization in young (n = 11) and old (n = 12), respectively. * Time effect, p<0.05 compared to pre, # Age effect, p<0.05 young compared to old within time point. Group mean data ± SEM. Mean myofiber area was assessed in the quadriceps femoris muscle, by muscle biopsy sampling. <b>C.</b> Muscle histology from resting state (pre) and immobility (14 d) was analyzed by myofibrillar ATPase at pH 10.3 preincubations demonstrating type I (white) and type II muscle fibers (black) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051238#pone.0051238-Brooke1" target="_blank">[52]</a>.</p

Changes in the transcriptional status of IGF-1Ea and MGF as a result of immobility induced disuse-muscle atrophy.

<p>Expression levels of IGF-1Ea and MGF mRNA were determined using qRT-PCR. <b>A & B</b>. The data revealed an age-specific (old subjects only) up-regulation of IGF-1Ea and MGF at 1 day and 2 days of immobility while an up-regulation was observed after 14 days of immobility in both age groups. * Time effect, p<0.05 compared to pre. Data are geometric means ± back-transformed SEM.</p

Immobility increases the transcriptional status of Bax, BCL2L1 and p53 and the immunodetection of TUNEL.

<p><b>A–C</b>. The mRNA level of Bax, BCL2L1 and p53 was determined using qRT-PCR and significant increases were found in the early phase (0–4 days) as well as later phase (14 days) of disuse-muscle atrophy <b>D.</b> Skeletal muscle cryosections were immunostained for nNOS (red), DAPI (blue) and TUNEL (green). <b>E.</b> Total numbers of TUNEL-positive nuclei were quantified for both young and old muscle and significant increases of TUNEL-positive nuclei were detected in old muscle in the early phase of immobility (1–2 days) and in both young and old after 4 days of immobility. <b>F.</b> Double immunohistochemical staining for TUNEL and the muscle satellite cell marker Pax7 did not reveal any TUNEL-positive muscle satellite cells. Additional green fluorescent expression on the shown image is due to autofluorescence by lipofusin and this is considered non-specific in our analysis. *Time effect, p<0.05 compared to pre. <u>*</u> Time effect, p<0.05 bar indicates young and old combined compared to pre. Data are means ± SEM.</p

Changes in the transcriptional status of FoxO1, FoxO3 and FoxO4 as a result of immobility-induced muscle disuse.

<p><b>A–C.</b> The mRNA level of FoxO1, FoxO3 and FoxO4 was determined using qRT-PCR. No up-regulation in the mRNA expression levels in any of these three genes was observed during the initial phase of immobility, in contrast, a general down-regulation in all three genes was observed in both young and aged muscle at the 4 d time point, potentially reflecting a negative feedback signal from high presence of active FoxO protein in the muscle cell. However, it is difficult to interpret the role of FoxO in the present study since the phosphorylated forms of FoxO were not measured. <u>*</u> Time effect, p<0.05 bar indicates young and old within time point. Data are geometric means ± back-transformed SEM.</p

Immobility results in an increase in the transcriptional status and immunodetection of Atrogin-1 and MuRF-1 in the early phase of disuse muscle atrophy.

<p><b>A & B</b>. The mRNA level of Atrogin-1 and MuRF-1 was determined using qRT-PCR and significant increases were found in the early phase (2–4 days) of immobility, in both young and old skeletal muscle. At the later time-point (14 d) Atrogin-1 and MuRF-1 expression levels decreased in aged muscle, whereas the expression levels of Atrogin-1 and MuRF-1 mRNA were unchanged from baseline in young muscle. Data are geometric means ± back-transformed SEM. <b>C</b>. Immunodetection of DAPI (blue), and MuRF-1 (red) are shown for 10 µm skeletal muscle cryosections. Potential muscle fiber specificity was analyzed by simultaneous incubation of primary antibodies for MuRF-1 mixed with either anti-skeletal Myosin-Fast or anti-skeletal Myosin-Slow (green), demonstrating an almost 100% affinity of MuRF-1 for type 1 muscle fibers. <b>D</b>. Total numbers of MuRF-1 positive myofibers were quantified for both young and old muscle and a significant increase was detected in the number of MuRF-1 positive fibers after 4 days of immobility. * Time effect, p<0.05 compared to pre. <u>*</u> Time effect, p<0.05 bar indicates young and old combined compared to pre. Data are means ± SEM. <b>E</b>. Immunodetection of DAPI (blue), Laminin (green) and MuRF-1 (red) are shown for 10 µm skeletal muscle cryosections at pre, 1 d, 2 d and 4 d of immobilization.</p