Fig 1.
Delta gastrocnemius muscle weights of all the groups in both study cohorts.
Mean values and standard deviation of delta of the variable gastrocnemius weight in the immobilization (top panel) and recovery (bottom panel) cohorts of mice. Definition of abbreviations: g, grams; I, immobilization; R, recovery. Statistical significance is represented as follows: **, p≤0.01, ***, p≤0.001, and n.s., non-significant differences between any of the immobilized animals and the non-immobilized controls; §§§, p≤0.001, and n.s., non-significant differences between any of the R groups of animals and the 7-day I mice.
Fig 2.
Limb strength gain of all the groups in both study cohorts.
Mean values and standard deviation of limb strength gain of the immobilization (top panel) and recovery (bottom panel) cohorts of mice. Definition of abbreviations: I, immobilization; R, recovery. Statistical significance is represented as follows: **, p≤0.01, and ***, p≤0.001 between any of the immobilized animals and the non-immobilized controls; §, p≤0.05, §§§, p≤0.001, and n.s., non-significant differences between any of the R groups of animals and the 7-day I mice.
Fig 3.
Delta of muscle fibers proportions of all the groups in both study cohorts.
Mean values and standard deviation of delta of the variables type I and type II fibers proportions of the immobilization (top panel) and recovery (bottom panel) cohorts of mice. Definition of abbreviations: I, immobilization; R, recovery. Statistical significance is represented as follows: **, p≤0.01, and n.s., non-significant differences in type I fibers proportions between any of the immobilized animals and the non-immobilized controls; ¶¶, p≤0.01, and n.s., non-significant differences in type II fibers proportions between any of the immobilized animals and the non-immobilized controls; n.s., non-significant differences in either type I or type II fibers proportions between any of the R groups of animals and the 7-day I mice.
Fig 4.
Delta of the slow- and fast-twitch fibers cross-sectional area of all the groups in both study cohorts.
Mean values and standard deviation of delta of the variables fiber type I and type II cross sectional areas of the immobilization (top panel) and recovery (bottom panel) cohorts of mice. Definition of abbreviations: μm2, square micrometers; I, immobilization; R, recovery. Statistical significance is represented as follows: **, p≤0.01, ***, p≤0.001, and n.s., non-significant differences in type I cross-sectional area between any of the immobilized animals and the non-immobilized controls; ¶¶, p≤0.01, ¶¶¶, p≤0.001, and n.s., non-significant differences in type II cross-sectional area between any of the immobilized animals and the non-immobilized controls; §§, p≤0.01, and n.s., non-significant differences in type I cross-sectional area between any of the R groups of animals and the 7-day I mice; #, p≤0.05, ##, p≤0.01, and n.s., non-significant differences in type II cross-sectional area between any of the R groups of animals and the 7-day I mice.
Fig 5.
Gastrocnemius slow- and fast-twitch fibers cross-sectional area was reduced by immobilization.
Representative examples of the gastrocnemius muscle in animals of the 7-, 15-, and 30-day immobilized cohorts, and non-immobilized control group. Myofibers positively stained with the anti-MyHC type I antibody are stained in brown color (x 400). Type II fibers were not stained (white color).
Fig 6.
Delta of muscle structural abnormalities of all the groups of the immobilization cohorts.
Mean values and standard deviation of deltas of the variables abnormal fraction (top panel), internal nuclei (middle panel), and inflammatory cells (bottom panel) in the gastrocnemius of the immobilization cohorts of animals. Definition of abbreviations: I, immobilization; R, recovery. Statistical significance is represented as follows: *, p≤0.05, **, p≤0.01, ***, p≤0.001, and n.s., non-significant differences between any of the immobilized animals and the non-immobilized controls.
Fig 7.
Gastrocnemius slow- and fast-twitch fibers cross-sectional area improved after the recovery period.
Representative examples of the gastrocnemius muscle in animals of the 7-, 15-, and 30-day recovery cohorts. Myofibers positively stained with the anti-MyHC type I antibody are stained in brown color (x 400). Type II fibers were not stained (white color).
Fig 8.
Delta of muscle structural abnormalities of all the groups of the recovery cohorts.
Mean values and standard deviation of deltas of the variables abnormal fraction (top panel), internal nuclei (middle panel), and inflammatory cells (bottom panel) in the gastrocnemius of the recovery cohorts of rodents. Definition of abbreviations: I, immobilization; R, recovery. Statistical significance is represented as follows: §, p≤0.05, §§§, p≤0.001, and n.s., non-significant differences between any of the R groups of animals and the 7-day I mice.
Fig 9.
Delta of gastrocnemius tyrosine release of all the groups in both study cohorts.
Mean values and standard deviation of delta of the variable tyrosine release (nmol/mg/2h) in the gastrocnemius of the immobilization (top panel) and recovery (bottom panel) cohorts of mice. Definition of abbreviations: nmol, nanomol; mg, milligram; h, hour; I, immobilization; R, recovery. Statistical significance is represented as follows: ***, p≤0.001, and n.s., non-significant differences between any of the immobilized animals and the non-immobilized controls; §§, p≤0.01, and §§§, p≤0.001 between any of the R groups of animals and the 7-day I mice.
Fig 10.
Delta of gastrocnemius proteasome trypsin-like and chymotrypsin-like activities of all the groups in both study cohorts.
Mean values and standard deviation of delta of the variables trypsin-like and chymotrypsin-like activities (nmol/mg/30’) in the gastrocnemius of the immobilization (top panel) and recovery (bottom panel) cohorts of mice. Definition of abbreviations: nmol, nanomol; mg, milligram; I, immobilization; R, recovery. Statistical significance is represented as follows: **, p≤0.01, ***, p≤0.001 and n.s., non-significant differences in trypsin-like activity between any of the immobilized animals and the non-immobilized controls; ¶¶¶, p≤0.001, and n.s., non-significant differences in chymotrypsin-like activity between any of the immobilized animals and the non-immobilized controls; §, p≤0.05, and n.s., non-significant differences in trypsin-like activity between any of the R groups of animals and the 7-day I mice; ##, p≤0.01, ###, p≤0.001, and n.s., non-significant differences in chymotrypsin-like activity between any of the R groups of animals and the 7-day I mice.
Fig 11.
Gastrocnemius 20S proteasome C8 content of all the groups in both study cohorts.
Mean values and standard deviation of 20S proteasome C8 subunit content in the gastrocnemius of the immobilization (top panel) and recovery (bottom panel) cohorts of mice, as measured by optical densities in arbitrary units (OD, a.u.). Definition of abbreviations: OD, optical densities; a.u., arbitrary units; I, immobilization; R, recovery. Statistical significance is represented as follows: *, p≤0.05, **, p≤0.01, ***, p≤0.001, and n.s., non-significant differences between any of the immobilized animals and the non-immobilized controls; §§, p≤0.01, §§§, p≤0.001, and n.s., non-significant differences between any of the R groups of animals and the 7-day I mice. Note that as arbitrary units were used for the measurement of the optical densities in each set of immunoblots (see methods), scale bars in the ordinate axes differ in the graphs of the immobilization groups from those of the recovery cohorts.
Fig 12.
Total protein ubiquitination content of all the groups in both study cohorts.
Mean values and standard deviation of total ubiquitinated proteins in the gastrocnemius of the immobilization (top panel) and recovery (bottom panel) cohorts of mice, as measured by optical densities in arbitrary units (OD, a.u.). Definition of abbreviations: OD, optical densities; a.u., arbitrary units; I, immobilization; R, recovery. Statistical significance is represented as follows: *, p≤0.05, and n.s., non-significant differences between any of the immobilized animals and the non-immobilized controls; §, p≤0.05, §§§, p≤0.001, and n.s., non-significant differences between any of the R groups of animals and the 7-day I mice. Note that as arbitrary units were used for the measurement of the optical densities in each set of immunoblots (see methods), scale bars in the ordinate axes differ in the graphs of the immobilization groups from those of the recovery cohorts.
Fig 13.
E3 ligase atrogin-1 content of all the groups in both study cohorts.
Mean values and standard deviation of atrogin-1 protein content in the gastrocnemius of the immobilization (top panel) and recovery (bottom panel) cohorts of mice, as measured by optical densities in arbitrary units (OD, a.u.). Definition of abbreviations: OD, optical densities; a.u., arbitrary units; I, immobilization; R, recovery. Statistical significance is represented as follows: *, p≤0.05, **, p≤0.01, ***, p≤0.001, and n.s., non-significant differences between any of the immobilized animals and the non-immobilized controls; §, p≤0.05, §§, p≤0.01, and n.s., non-significant differences between any of the R groups of animals and the 7-day I mice. Note that as arbitrary units were used for the measurement of the optical densities in each set of immunoblots (see methods), scale bars in the ordinate axes differ in the graphs of the immobilization groups from those of the recovery cohorts.
Fig 14.
E3 ligase MURF-1 content of all the groups in both study cohorts.
Mean values and standard deviation of MURF-1 protein content in the gastrocnemius of the immobilization (top panel) and recovery (bottom panel) cohorts of mice, as measured by optical densities in arbitrary units (OD, a.u.). Definition of abbreviations: MURF-1, muscle ring finger protein 1; OD, optical densities; a.u., arbitrary units; I, immobilization; R, recovery. Statistical significance is represented as follows: **, p≤0.01, ***, p≤0.001, and n.s., non-significant differences between any of the immobilized animals and the non-immobilized controls; §, p≤0.05, §§, p≤0.01, §§§, p≤0.001, and n.s., non-significant differences between any of the R groups of animals and the 7-day I mice. Note that as arbitrary units were used for the measurement of the optical densities in each set of immunoblots (see methods), scale bars in the ordinate axes differ in the graphs of the immobilization groups from those of the recovery cohorts.
Fig 15.
E3 ligase TRIM32 content of all the groups in both study cohorts.
Mean values and standard deviation of TRIM32 protein content in the gastrocnemius of the immobilization (top panel) and recovery (bottom panel) cohorts of mice, as measured by optical densities in arbitrary units (OD, a.u.). Definition of abbreviations: TRIM32, tripartite motif containing 32; OD, optical densities; a.u., arbitrary units; I, immobilization; R, recovery. Statistical significance is represented as follows: n.s., non-significant differences between any of the immobilized animals and the non-immobilized controls; n.s., non-significant differences between any of the R groups of animals and the 7-day I mice. Note that as arbitrary units were used for the measurement of the optical densities in each set of immunoblots (see methods), scale bars in the ordinate axes differ in the graphs of the immobilization groups from those of the recovery cohorts.
Fig 16.
GDF15 content of all the groups in both study cohorts.
Mean values and standard deviation of GDF15 protein content in the gastrocnemius of the immobilization (top panel) and recovery (bottom panel) cohorts of mice, as measured by optical densities in arbitrary units (OD, a.u.). Definition of abbreviations: GDF15, growth differentiation factor 15; OD, optical densities; a.u., arbitrary units; I, immobilization; R, recovery. Statistical significance is represented as follows: n.s., non-significant differences between any of the immobilized animals and the non-immobilized controls; n.s., non-significant differences between any of the R groups of animals and the 7-day I mice. Note that as arbitrary units were used for the measurement of the optical densities in each set of immunoblots (see methods), scale bars in the ordinate axes differ in the graphs of the immobilization groups from those of the recovery cohorts.
Fig 17.
Myosin heavy chain content of all the groups in both study cohorts.
Mean values and standard deviation of MyHC protein content in the gastrocnemius of the immobilization (top panel) and recovery (bottom panel) cohorts of mice, as measured by optical densities in arbitrary units (OD, a.u.). Definition of abbreviations: MyHC, Myosin Heavy Chain; OD, optical densities; a.u., arbitrary units; I, immobilization; R, recovery. Statistical significance is represented as follows: **, p≤0.01, and n.s., non-significant differences between any of the immobilized animals and the non-immobilized controls; §§§, p≤0.001, and n.s., non-significant differences between any of the R groups of animals and the 7-day I mice. Note that as arbitrary units were used for the measurement of the optical densities in each set of immunoblots (see methods), scale bars in the ordinate axes differ in the graphs of the immobilization groups from those of the recovery cohorts.
Fig 18.
Actin content of all the groups in both study cohorts.
Mean values and standard deviation of actin protein content in the gastrocnemius of the immobilization (top panel) and recovery (bottom panel) cohorts of mice, as measured by optical densities in arbitrary units (OD, a.u.). Definition of abbreviations: OD, optical densities; a.u., arbitrary units; I, immobilization; R, recovery. Statistical significance is represented as follows: n.s., non-significant differences between any of the immobilized animals and the non-immobilized controls; n.s., non-significant differences between any of the R groups of animals and the 7-day I mice. Note that as arbitrary units were used for the measurement of the optical densities in each set of immunoblots (see methods), scale bars in the ordinate axes differ in the graphs of the immobilization groups from those of the recovery cohorts.
Fig 19.
Plasma Troponin-I levels in both study cohorts.
Mean values and standard deviation of fast troponin I plasma levels (ng/ml) of the immobilization (top panel) and recovery (bottom panel) cohorts of mice. Definition of abbreviations: ng, nanogram; ml, milliliter; I, immobilization; R, recovery. Statistical significance is represented as follows: **, p≤0.01, ***, p≤0.001, and n.s., non-significant differences between any of the immobilized animals and the non-immobilized controls; §§, p≤0.01, and n.s., non-significant differences between any of the R groups of animals and the 7-day I mice.
Fig 20.
Activated Akt content of all the groups in both study cohorts.
Mean values and standard deviation from the p-Akt/Akt ratio is depicted in the gastrocnemius of the immobilization (top panel) and recovery (bottom panel) cohorts of mice, as measured by optical densities in arbitrary units (OD, a.u.). Definition of abbreviations: Akt, RAC-alpha serine/threonine-protein kinase; p-Akt, phosphorylated-Akt; OD, optical densities; a.u., arbitrary units; I, immobilization; R, recovery. Statistical significance is represented as follows: n.s., non-significant differences between any of the immobilized animals and the non-immobilized controls; n.s., non-significant differences between any of the R groups of animals and the 7-day I mice. Note that as arbitrary units were used for the measurement of the optical densities in each set of immunoblots (see methods), scale bars in the ordinate axes differ in the graphs of the immobilization groups from those of the recovery cohorts.
Fig 21.
Activated p70S6K content of all the groups in both study cohorts.
Mean values and standard deviation from the p-p70S6K/p70S6K ratio is depicted in the gastrocnemius of the immobilization (top panel) and recovery (bottom panel) cohorts of mice, as measured by optical densities in arbitrary units (OD, a.u.). Definition of abbreviations: p70S6K, p70 S6 kinase; p-70S6K, phosphorylated p70S6K; OD, optical densities; a.u., arbitrary units; I, immobilization; R, recovery. Statistical significance is represented as follows: *, p≤0.05, and n.s., non-significant differences between any of the immobilized animals and the non-immobilized controls; §, p≤0.05, §§, p≤0.01, and n.s., non-significant differences between any of the R groups of animals and the 7-day I mice. Note that as arbitrary units were used for the measurement of the optical densities in each set of immunoblots (see methods), scale bars in the ordinate axes differ in the graphs of the immobilization groups from those of the recovery cohorts.
Fig 22.
Mitochondrial content of all the groups in both study cohorts.
Mean values and standard deviation of the mitochondrial DNA copy number in the gastrocnemius of the immobilization (top panel) and recovery (bottom panel) cohorts of rodents, as measured by the mtDNA/mDNA ratio in arbitrary units (a.u.). Definition of abbreviations: mt, mitochondrial; n, nuclear; DNA, deoxyribonucleic acid; I, immobilization; R, recovery. Statistical significance is represented as follows: *, p≤0.05, **, p≤0.01, and ***, p≤0.001 between any of the immobilized animals and the non-immobilized controls; §§, p≤0.01, §§§, p≤0.001, and n.s., non-significant differences between any of the R groups of animals and the 7-day I mice.