Table 1.
Data on primary antibodies used for detection of different MyHC isoforms.
Table 2.
The basis for the classification of muscle fibre phenotypes based on the staining intensity pattern of the different MyHC mAbs.
Figure 1.
Focal muscle changes and myositis in the exercised and non-exercised leg.
Muscle samples from a control (A), an exercised (B) and a contralateral non-exercised soleus (C–G) muscles after 6w of E/EMS. Sections A–E are stained with hematoxylin-eosin (HTX-eosin), section F is immunolabeled for mAb D33 against desmin and section G for mAb 1891 against fibronectin. Figs. E–G are serial sections. Note the infiltration of inflammatory cells (B, E), presence of internal nuclei (arrows B, D) and atypical formed fibers in both the exercised and non-exercised muscles (B–E). Small angular fibers are shown in Fig C (arrows). Presence of extremely large fiber size variability, split fibers (arrowheads) and increased number of internal nuclei (arrows) are shown in Fig D. A necrotic fiber (asterisk) within in an area with inflammation is shown in E–G. Note the severe infiltration of inflammatory cells in and around the necrotic fibers (E), the lack of desmin staining of the necrotic fibers (F) and the extensive fibrosis in the area with myositis and necrotic fibers (G). Scale bar 50 µm.
Figure 2.
Bilateral regeneration of muscle fibers.
Muscle cross-sections immunolabeled with mAb F1.652 against embryonic MyHC and DAPI (stains nuclei blue) in a control (A), an exercised (B) and a non-exercised (C) gastrocnemius muscles after 6w of E/EMS. Note the lack of staining for embryonic MyHC in the control sample (A) and the high number of small sized fibers stained for embryonic MyHC in areas with inflammation in the exercised and non-exercised muscles (B, D). Note also the higher variability in size as well as the generally larger sizes of fibers expressing embryonic MyHC in the exercised than in the non-exercised side, indicating that the degenerative/regenerative process is earlier initiated in the exercised than in the non-exercised side. Scale bar 50 µm.
Figure 3.
Changes in nerve fascicles in the non-exercised leg.
Serial section of a nerve fascicle in the non-exercised soleus muscle after 6w of exercise immunolabeled for mAb β-tublin III against axons (A, C, E) and mAb S-100β against Schwann cells and DAPI (B, D, F). Nuclei are stained blue or pink with DAPI. The frames 1 and 2 inserted in figs. A and B corresponds to the regions shown in Figs. C, E and D, F, respectively. Axons stained for mAb β-tublin III are enclosed by Schwanns cells stained by mAb S-100β. Certain Schwann cell structures lack staining for mAb β-tublin III, indicating loss of axons (C–F, arrowheads). The group of small sized axons stained for β-tublin III in frame 2 (E,F, arrows) indicates on a process of regeneration by sprouting of axons (E, F). Scale bar 50 µm.
Figure 4.
Blood vessel distribution in normal and pathological muscle areas.
The figure shows an example of blood vessel distribution in areas with normal morphology and in areas with pathological changes in the exercised gastrocnemius muscle after 3w of E/EMS. Section A is stained with HTX-eosin and shows an area with normal morphology (1), an pathological area with fibrosis and fat infiltration (2) and an area with severe mysositis and muscle fiber alterations (3). Sections B1-B3 are immunolabeled for polyclonal Ab PC128 against laminin, a component of the basement membranes of muscle fibers and vessels, and section C1–C3 are immunolabeled for mAb M0823 (CD31) against endothelium in vessels. Sections D1–D3 are merged images of immunolabeling for CD31 and laminin. Note the normal pattern of capillaries around fibers in the unaffected area (frame 1), the low number of capillaries in the area with fibrosis (frame 2), and the high number of comparatively larger vessels (arrows) in the area marked with inflammation (frame 3). Indications M1, M2 and M3 represent a serially sectioned muscle fiber in each frame stained for laminin (B), CD31 (C) and both laminin and CD31 (D). Scale bar 100 µm.
Figure 5.
Bilateral changes in vascular supply in affected areas in the exercised and non-exercised gastrocnemius and soleus muscles.
Serial cross-sections from a control (A, F), an exercised (B, G) and non-exercised (C, H) gastrocnemius (GA) muscle and an exercised (D, I) and non-exercised (D, H) soleus (SOL) muscle. The cross-sections A–E are stained for mAb M0823 (CD31) against endothelium in vessels and the merged sections E–J are stained for CD31 (yellow/green), pAb PC128 against laminin (red) and DAPI (stains nuclei blue). Sections A, F show a control with a normal pattern of capillaries around fibers whereas section B–E, F–J shows different patterns of changes in capillarization in areas with inflammation and fibrosis. The samples from the exercised and non-exercised GA muscles shows examples of low capillary density in an area with inflammation/fibrosis (B, G) and in an area with mainly fibrosis (C, H). The sample from the exercised soleus muscle (D, I) shows an example of increased number of vessels with a larger size than capillaries in an area with fibrosis, inflammation and degenerating/regenerating fibers. The sample from the non-exercised SOL (E, J) shows an area with severe inflammation and increased capillarization. The differences in vascularization patterns probably represent different stages in the degenerative/regenerative process in the muscles. Arrows point at immunoreactions for capillaries and arrowheads against larger vessels in each section. Scale bar 50 µm.
Figure 6.
Muscle fiber phenotypes in the soleus and gastrocnemius muscles.
Serial sections from the soleus (A, B and E, F) and gastronemius (C, D and G, H) muscles stained for mAb A4951 (MyHCI) (A–D) and mAb N2.261 (stains MyHCIIa strongly, MyHCI weakly) (E–H). Sections A, E and C, G are from controls and sections B, F and D, H are from the 6w exercised leg. Note the high frequency of fibers containing MyHCI in the soleus muscle (A, E) and the higher frequency of fibers containing MyHCIIx/b in the gastrocnemius muscle (C, G). Note also the higher number of fibers containing fast MyHCII in both muscles after 6w of E/EMS. Scale bar 50 µm.
Figure 7.
Graphs showing frequency of fiber types in the exercised and non-exercised muscles after 1w, 3w and 6w of unilateral exercise.
Figs. A–C show the magnitude of changes in fiber type frequency (%) in the soleus muscles and Figs. D–H shows corresponding changes in the gastrocnemius muscles. The mean group values in the figure are connected with lines to facilitate interpretation of the direction of changes after E/EMS, but the lines do not imply that the changes between the sample points represent a linear change over this period. Exercised leg is defined by continuous black line, non-exercised leg by dotted line. Significant difference (p<0.05) within the exercised leg is marked with E and significant difference within the non-exercised leg is marked with N. Significant differences relative to controls are marked (c), to 1w group (1w) and to 3w group (3w). Significant differences (p<0.05) between exercised and non-exercised legs are marked with an asterisk (*). Note the shift against a lower proportion of fibers containing slow MyHCI and a higher proportion of fibers containing fast MyHCII in both exercised and non-exercised muscles. Note also the lack of hybrid MyHCI+IIa fibers in the exercised soleus muscle after exercise. Bars indicate SD.
Figure 8.
Graphs showing changes in fiber area and capillary parameters in the exercised and non-exercised muscles after 1w, 3w and 6w of unilateral exercise.
Figures show changes in the exercised and non-exercised legs of soleus (A, C, E, G) and gastrocnemius (B, D, F, H) muscles for the following parameters; muscle fiber cross-sectional area (CSA) (A, B), the number of capillaries around each individual fiber (CAF) (C, D), the number of capillaries around fibers relative to cross-sectional fiber area (CAFA) (E, F), and capillary density (CD) (G, H). The mean group values in the figure are connected with lines to facilitate interpretation of the direction of changes after E/EMS, but the lines do not imply that the changes between the sample points represent a linear change over this period. Exercised leg is defined by continuous black line, non-exercised side by dotted line. Significant difference (p<0.05) within the exercised leg is marked with E and significant difference within the non-exercised leg is marked with N. Significant differences relative to controls are marked (c), to 1w group (1w) and to 3w group (3w). Significant differences (p<0.05) between exercised and non-exercised legs are marked with an asterisk (*). Note the overall similarities in changes in fiber area and in capillarization between the exercised and non-exercised legs in both the soleus and gastrocnemius muscles, except that there was no decrease in capillary supply after 1w and 3w of exercise in the soleus non-exercised muscle. Bars indicate SD.
Figure 9.
Graphs showing fiber area and capillary parameters for pure fiber types in the exercised and non-exercised soleus muscles after 1w, 3w and 6w of unilateral exercise.
Figures show changes for the following parameters related to fibers containing MyHCI and MyHCIIa; muscle fiber cross-sectional area (CSA) (A, D), number of capillaries around each individual fiber (CAF) (B, E), and number of capillaries around fibers relative to cross-sectional fiber area (CAFA) (C, F). The mean group values in the figure are connected with lines to facilitate interpretation of the direction of changes after E/EMS, but the lines do not imply that the changes between the sample points represent a linear change over this period. Exercised side is defined by continuous black line, non-exercised side by dotted line. Significant difference (p<0.05) within the exercised leg is marked with E and significant difference within the non-exercised leg is marked with N. Significant differences relative to controls are marked (c), to 1w group (1w) and to 3w group (3w). Significant differences (p<0.05) between exercised and non-exercised legs are marked with an asterisk (*). Note the overall similarities in fiber area alterations between the exercised and non-exercised muscles. Furthemore, note also the more marked changes in fiber area and capillary supply among fibers containing MyHCII compared with fibers containing MyHCI. Bars indicate SD.
Figure 10.
Graphs showing fiber area and capillary parameters for fiber types in the exercised and non-exercised gastrocnemius muscle after 1w, 3w and 6w of unilateral exercise.
Figure show changes in; muscle fiber cross-sectional area (CSA) (A, D, G), number of capillaries around each individual fiber (CAF) (B, E, H), and number of capillaries around fibers relative to cross-sectional fiber area (CAFA) (C, F, I) for fibers containing MyHCI, MyHCIIa and MyHCIIx/b. The mean group values in the figure are connected with lines to facilitate interpretation of the direction of changes after E/EMS, but they do not imply that the changes between the sample points represent a linear change over this period. Exercised leg is defined by continuous black line, non-exercised leg by dotted line. Significant difference (p<0.05) within the exercised leg is marked with E and significant difference within the non-exercised leg is marked with N. Significant differences relative to controls are marked (c), to 1w group (1w) and to 3w group (3w). Significant differences (p<0.05) between exercised and non-exercised sides are marked with an asterisk (*). Note the overall similarities in changes for CSA and CAF for all fiber types between the exercised and non-exercised muscles. Bars indicate SD.