Figure 1.
Muscle fiber size of wild-type and PTN-overexpressing mice after 14 days HU or 91 days space flight.
A) representative Sol muscle cryosections stained with antibodies specific for laminin. The area inside the laminin staining was utilized to measure muscle fiber CSA. B) Effect of HU and spaceflight on CSA of Sol fibers and CSA-to-body weight ratio. C) Effect of HU and spaceflight on CSA of EDL fibers and CSA-to-body weight ratio. Each bar is the mean ± SEM from 2 to 9 mice. Statistical analysis, performed with two-tailed unpaired Student’s t test, indicates significant atrophy in soleus muscle of PTN mice after HU or spaceflight (* indicates P<0.05 versus WT-ground mice, and # indicates P<0.05 versus PTN-ground mice).
Figure 2.
Muscle fiber typing of wild-type and PTN-overexpressing mice after 14 days HU or 91 days space flight.
A) Representative soleus muscle sections immunostained by means of monoclonal antibodies specific for the different myosin heavy chain isoforms. B–C) Fiber type composition of Sol (B) and EDL (C) muscles determined as shown in A. Each bar is the mean ± SEM from 2-to-10 mice. Statistical analysis performed with unpaired Student’s t test indicates significant change (at least P<0.05) versus WT-G (*), WT-HU (§), PTN-G (#), and PTN-HU (°).
Figure 3.
Muscle MyHC isoform composition of wild-type and PTN-overexpressing mice after 14 days HU or 91 days space flight.
A) MyHC isoforms were separated by SDS-PAGE and stained with Coomassie blue. Densitometric analysis on MyHC isoforms is reported for Sol (B) and EDL (C) muscles. Each bar is the mean ± SEM from 2-to-7 mice. Statistical analysis performed with unpaired Student’s t test indicates significant change (at least P<0.05) versus WT-G (*), WT-HU (§), PTN-G (#), PTN-HU (°).
Figure 4.
Muscle vascularization in wild-type and PTN-overexpressing mice after 14 days HU or 91 days space flight.
A) Representative Sol muscle sections stained for alkaline phosphatase activity. Pictures of spaceflown samples (right column) were slightly modified using Photoshop software (Adobe) to increase contrast. B) The number of capillary was counted in Sol and EDL muscle sections stained as shown in A and normalized to the number of muscle fibers. Each bars is the mean capillary-to-fiber ratio ± SEM calculated from WT-ground (n = 6), WT-HU (n = 3), WT-spaceflown (n = 1), PTN-ground (n = 6), PTN-HU (n = 3), and PTN-spaceflown (n = 2) mice. Statistical analysis performed with two-tailed unpaired Student’s t test indicates significant change (P<0.05) versus WT-G (*) and PTN-G (#).
Figure 5.
Resting potassium and chloride conductance of Sol and EDL muscle fibers of wild-type and PTN-overexpressing mice after 14 days HU.
A) The chloride and potassium conductance of sarcolemma at rest were measured in current-clamp mode with the two-intracellular microelectrodes technique. Each bar is the mean conductance ± SEM calculated from 16-to-53 muscle fibers of 3-to-6 mice. Statistical analysis performed with unpaired Student’s t test indicates significant change (P<0.05) versus WT-G (*), WT-HU ($), and PTN-G (#). B) Selective inhibitors of ATP-sensitive potassium channels (50 µM glibenclamide) and large conductance, Ca2+-activated potassium channels (50 nM iberiotoxin) were applied in vitro to soleus muscles fibers to evaluate the contribution of these channels to the resting potassium conductance of sarcolemma. Statistical analysis performed with paired Student’s t test indicates significant change (P<0.05) versus CTRL (a) or glibenclamide (b).
Figure 6.
Cytosolic Ca2+ concentration at rest in Sol (A) and EDL (B) muscle fibers of wild-type and PTN-overexpressing mice after 14 days HU.
The cytosolic Ca2+ concentration was determined in mechanically-dissociated muscle fibers with cytofluorescent imaging technique using the Ca2+ dye FURA-2. Each bar is the mean ± SEM calculated from 13-to-47 muscle fibers of 2-to-3 mice. Statistical analysis performed with unpaired Student’s t test indicates significant change (P<0.05) versus WT-G (*), WT-HU ($), and PTN-G (#).
Figure 7.
Variations in gene expression in Sol muscle induced by hindlimb-unloading and spaceflight.
Transcript levels were determined by real-time PCR for selected genes, classified on the basis of the functional role of the protein they encode, and normalized for the housekeeping HPRT-1 gene. The bars indicate the fold change in gene expression between A) HU and ground conditions in WT mice, B) PTN and WT mice in ground conditions, C) PTN-HU versus WT-G mice, and D) spaceflown PTN mice versus ground WT mice. Significant fold changes, according to two-tailed unpaired Student’s t-test, are indicated with* (at least P<0.05).
Figure 8.
Variations in gene expression in EDL muscle induced by hindlimb-unloading and spaceflight.
Transcript levels were determined by real-time PCR for selected genes, classified on the basis of the functional role of the protein they encode, and normalized for the housekeeping HPRT-1 gene. The bars indicate the fold change in gene expression between A) HU and ground conditions in WT mice, B) PTN and WT mice in ground conditions, C) PTN-HU versus WT-G mice, and D) spaceflown PTN mice versus ground WT mice. Significant fold changes, according to two-tailed unpaired Student’s t-test, are indicated with* (at least P<0.05).