Electrical stimulation influences satellite cell proliferation and apoptosis in unloading-induced muscle atrophy in mice

Abstract

Muscle atrophy caused by disuse is accompanied by adverse physiological and functional consequences. Satellite cells are the primary source of skeletal muscle regeneration. Satellite cell dysfunction, as a result of impaired proliferative potential and/or increased apoptosis, is thought to be one of the causes contributing to the decreased muscle regeneration capacity in atrophy. We have previously shown that electrical stimulation improved satellite cell dysfunction. Here we test whether electrical stimulation can also enhance satellite cell proliferative potential as well as suppress apoptotic cell death in disuse-induced muscle atrophy. Eight-week-old male BALB/c mice were subjected to a 14-day hindlimb unloading procedure. During that period, one limb (HU-ES) received electrical stimulation (frequency: 20 Hz; duration: 3 h, twice daily) while the contralateral limb served as control (HU). Immunohistochemistry and western blotting techniques were used to characterize specific proteins in cell proliferation and apoptosis. The HU-ES soleus muscles showed significant improvement in muscle mass, cross-sectional area, and peak tetanic force relative to the HU limb (p<0.05). The satellite cell proliferative activity as detected within the BrdU+/Pax7+ population was significantly higher (p<0.05). The apoptotic myonuclei (detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) and the apoptotic satellite cells (detected by cleaved Poly [ADP-ribose] polymerase co-labeled with Pax7) were reduced (p<0.05) in the HU-ES limb. Furthermore the apoptosis-inducing factor and cleaved caspase-3 were down-regulated while the anti-apoptotic Bcl-2 protein was up-regulated (p<0.05), in the HU-ES limb. These findings suggest that the electrical stimulation paradigm provides an effective stimulus to rescue the loss of myonuclei and satellite cells in disuse muscle atrophy, thus maintaining a viable satellite cell pool for subsequent muscle regeneration. Optimization of stimulation parameters may enhance the outcome of the intervention.

Figure 1. Force-length and force-frequency relation of the soleus muscle in the weight-bearing (WB), hindlimb-unloaded (HU), and electrically stimulated (HU-ES) groups.

(A) Curves showing the force-length relationship obtained for one soleus muscle. (B) Force-frequency relations of the muscles normalized to 100-Hz stimulation under each condition. *p<0.05, significant difference between the WB and HU group. Values are means ± SEM.

Figure 2. Satellite cell proliferation.

(A) Immunohistochemically stained cross-sections of soleus muscle from weight-bearing (WB), hindlimb-unloaded (HU), and electrically stimulated (HU-ES) groups showing expression of Pax7 (red) and BrdU (green) incorporation counterstained with DAPI (blue) for nuclei identification. Yellow staining represents Pax7 and BrdU double-positive nuclei. Scale bar = 25 µm. (B) Co-immunostaining of Pax7 and dystrophin on muscle cross-section for satellite cell localization. A Pax7⁺ nucleus (green, arrow) located outside the dystrophin⁺ (red) plasma membrane of a myofibre. DAPI (blue) shows the nuclei. Scale bar = 20 µm. (C) Total Pax7 immunoreactive nuclei; BrdU⁺/Pax7⁺ double-positive nuclei per 10³ myofibers; and ratio of BrdU⁺/Pax7⁺ nuclei within the total Pax7⁺ population. *p<0.05, significant difference compared to WB group; ^#p<0.05, significant difference compared to HU group. Values are means ± SEM.

Figure 3. Satellite cell apoptosis.

(A) Immunohistochemically stained cross-sections of soleus muscle from weight-bearing (WB), hindlimb-unloaded (HU), and electrically stimulated (HU-ES) groups showing expression of Pax7 (red) and cleaved PARP (c-PARP, green) counterstained with DAPI (blue) for nuclei identification. Yellow staining represents Pax7 and cleaved PARP double-positive nuclei. Scale bar = 25 µm. (B) c-PARP⁺/Pax7⁺ double-positive nuclei per 10³ myofibers for different groups. *p<0.05, significant difference compared to WB group; ^#p<0.05, significant difference compared to HU group. Values are means ± SEM.

Figure 4. TUNEL analysis in soleus myofibers.

(A) Representative images showing cross-sections of soleus muscle from weight-bearing (WB), hindlimb-unloaded (HU), and electrically stimulated (HU-ES) groups with TUNEL labeling (green, arrows) counterstained with DAPI (blue) for nuclei identification. Antibodies stained against dystrophin (red) for myofiber visualization. Scale bar = 25 µm. (B) Apoptotic index is expressed as the number of apoptotic myonuclei per 10³ myofibers. *p<0.05, significant difference compared to WB group; ^#p<0.05, significant difference compared to HU group. Values are means ± SEM.

Figure 5. Bcl-2, Bax, cytochrome c, and AIF of the soleus muscle in the weight-bearing (WB), hindlimb-unloaded (HU), and electrically stimulated (HU-ES) groups.

(A) Immunohistochemically stained cross-sections showing expression of Bcl-2, Bax, cytochrome c, and AIF (all in green) counterstained with antibodies against dystrophin (red) for myofiber identification and DAPI (blue) for nuclei visualization. Scale bar = 25 µm. (B) Western blot analysis of expression of Bcl-2, Bax, cytochrome c, and AIF relative protein levels normalized to WB group. Representative immunoreactive bands were shown. *p<0.05, significant difference compared to WB group; ^#p<0.05, significant difference compared to HU group. Values are means ± SEM.

Figure 6. Expression levels of cleaved forms of caspase-3 and PARP in the soleus muscle obtained from weight-bearing (WB), hindlimb-unloading (HU), and electrically stimulated (HU-ES) groups.

(A) Western blot analysis of expression of cleaved caspase-3. (B) Western blot analysis of expression of cleaved PARP. The relative protein levels were normalized to WB group. Representative immunoreactive bands were shown. *p<0.05, significant difference compared to WB group; ^#p<0.05, significant difference compared to HU group. Values are means ± SEM.