Mechanical unloading is associated with detrimental changes to the structure and function of skeletal muscles, characterized by reduction in muscle mass, myofiber cross-sectional area, contractile strength and speed, as well as slow-to-fast fiber type transformation (for review, see 
). It has also been reported that satellite cells in unloaded muscles exhibited an impaired regenerative capacity 
. We previously observed a reduction in the number of quiescent and mitotically active satellite cells during unloading, and electrical stimulation partially attenuated muscle atrophy by enhancing satellite cell proliferation and activation 
. In that study, we reported a reduction in the myonuclear domain and number during unloading. Electrical stimulation partially prevented the loss in the myonuclear domain but failed to restore the myonuclear number. Regulation of the myonuclei turnover is associated with addition (satellite cell-mediated myonuclei accretion) and loss (by apoptosis) of myonuclei, depending on the muscle activity and ongoing physiological process 
. In the present study, we sought to investigate the contribution of electrical stimulation in regulating the myonuclei turnover through improving proliferation of satellite cells and inhibiting cell apoptosis during the muscle atrophic process.
In this study, electrical stimulation resulted in a significant improvement in soleus muscle mass and cross-sectional area compared to that observed in the unloaded muscles, and restored muscle mass to approximately 80% of the WB controls. This is similar to our previous observation 
. To further characterize the processes that might be associated with the observed electrical stimulation-induced changes, we evaluated the contractile properties of the soleus muscle by measuring changes in: (i) Po
; (ii) length-tension relation; and (iii) force-frequency relation. Following unloading, we observed a reduction in Po
, and the peak length-tension relation was generated at a shorter muscle length. This phenomenon has been shown in disuse-related atrophy as a consequence of loss in sarcomere number in series 
. Furthermore, we observed a greater relative reduction in force at low frequencies of stimulation compared to high. This phenomenon, first described by Edwards and colleagues 
, is known as low-frequency fatigue and is suggested to be caused by a defect in excitation-contraction coupling. We found that this paradigm of electrical stimulation produced some improvement in the mechanical properties of the muscles.
It has been reported that hindlimb suspension resulted in myonuclei undergoing apoptosis 
. We examined the extent of apoptosis in myonuclei and found a substantial increase in the number of apoptotic myonuclei. According to the myonuclei domain hypothesis, such a dramatic increase in apoptotic myonuclei should accompany an increase in muscle fibers exhibiting pro-apoptotic features. Apoptosis can be triggered through two major pathways: the death receptor pathway, which involves Fas, TNF-related apoptosis-inducing ligand, and TNF receptors; or the mitochondrial pathway, where Bax and Bcl-2 are the center of focus 
. Since limited evidence has been demonstrated regarding the involvement of the death receptor pathway on eliciting apoptosis in atrophic muscles, focus was given to the mitochondrial pathway. Indeed, we observed a significant increase in pro-apoptotic Bax and AIF protein expression, as well as in the effector proteins cleaved caspase-3 and c-PARP, in unloaded myofibers. It is suggested that during the onset of apoptosis, Bax-Bax oligomerization takes place and the homomers translocate into the outer mitochondrial membrane, and through modulating the mitochondrial membrane permeabilization, mediates the release of cytochrome c (caspase dependent) and AIF (caspase independent) from the mitochondrial intermembrane space into the cells via the transitional pores 
. Depending on the involvement of caspase, cytochrome c and AIF separately trigger the subsequent signaling molecules and eventually lead to DNA fragmentation. The process of apoptosis can be further fine-tuned, in which Bax-Bax oligomerization can be counteracted by an anti-apoptotic Bcl-2 protein, thereby blocking the formation of transitional pores and thus inhibiting apoptosis 
. The interactions between Bax and Bcl-2, therefore, determine the onset of apoptosis. As expected, a corresponding decrease in anti-apoptotic Bcl-2 protein expression was also detected in myofibers upon unloading. The differential expression of Bax and Bcl-2 protein clearly indicated that the mitochondrial-specific apoptotic pathway was activated by unloading. Electrical stimulation rescued the myonuclei from undergoing apoptosis, apparently by enhancing the Bcl-2 protein expression that suppressed Bax oligomerization without altering the expression of Bax, thus accounting for the partial recovery of the myonuclei domain.
Since satellite cells are the predominant source of muscle regeneration, it seems that the loss of myonuclei would be replenished by the activation and myogenic differentiation of satellite cells. We mapped the quiescent and activated satellite cells using Pax7, a specific marker expressed only in quiescent and proliferating satellite cells but absent in newly regenerated myonuclei 
. In addition to the existing satellite cells exhibiting defective myogenesis as previously shown upon unloading 
, we observed that satellite cells also committed into apoptosis, as indicated by the increase in satellite cell population expressing c-PARP. The cleavage of PARP is suggested to be an early event in apoptosis, in which the detection of PARP cleavage is earlier than other apoptosis-related events; e.g., DNA fragmentation 
. Furthermore, the proliferative potential of satellite cells was also impaired during unloading, as seen in the diminished BrdU+
subpopulation in hindlimb unloaded muscles. The underlying mechanism of extensive apoptosis in satellite cells is not entirely clear. One of the possible explanations might be the alteration in the environment of satellite cells during the atrophic process. Oxidative stress plays an important role in the development of apoptosis and is implicated in muscle atrophy. An increase in lipid peroxidation was observed in mice following hindlimb unloading 
, and furthermore the level of lipid peroxidation was linearly related to the percentage of muscle atrophy 
. It was speculated that unloading-induced lesions observed in the central core of the soleus muscle was similar to the human central core disease 
, which is linked to mutations of muscle-specific Ca2+
-releasing channels, and resulted in excessive release of Ca2+
that led to Ca2+
. Reactive oxygen species (ROS) accumulation has been correlated Ca2+
release from the sarcoplasmic reticulum and lead to activation of the Ca2+
-dependent pathways. The increase in the activity of Ca2+
-dependent protease (i.e. calpain) induced proteolysis in muscles 
. A recent study in human satellite cells has demonstrated that oxidative stress-induced calpain-dependent pathway resulted in the depletion of the satellite cell pool 
. Together, increasing commitment of myonuclei and satellite cells into apoptosis as well as the deficient proliferative capacity of satellite cells may be the crucial if not sole factor that limits muscle repair, thus accounting for the atrophic features in the unloaded muscles.
Passive stretch of the unloaded soleus muscle have been shown to increase satellite cell proliferation 
and maintenance of the satellite cell numbers 
. It was speculated that stretch might prevent Ca2+
accumulation by reducing the overlap of thick and thin filaments, and consequently promote diffusion and removal of Ca2+ 
. Cross talk between the mitochondrial pathway and calpain has been documented, in which calpain promoted apoptosis by inducing cleavage of the Bcl-2 protein 
. Both stretching and electrical stimulation worked similarly by generating tension required by the slow-twitch muscle, an explanation of our finding that electrical stimulation improved muscle function might be the suppression of satellite cells apoptosis through inhibition of Ca2+
accumulation and hence blocked the calpain-mediated Bcl-2 cleavage. The effect of electrical stimulation on the regulation of calpain activity needs further investigation.
Although there was a significant improvement of electrical stimulation in attenuating the atrophic features upon unloading, the functional and morphological parameters did not reach normal levels. Given that numerous mechanisms and complex interplay of signal transduction pathways are involved in regulating muscle atrophy 
, it is likely that a combined (rather than a single) intervention approach should be considered. Interestingly, It has been demonstrated that satellite cell proliferation increased dramatically in the first 6 hours after unloading followed by a slight decline at 12 hours, the proportion of proliferating satellite cells was comparable to control by 48 hours after unloading. From that onwards, the population of proliferating satellite cells decreased far below that of the control group 
. It was believed that a sudden loss of microgravity initially triggered a signal to initiate satellite activation and proliferation ready for subsequent muscle repair. However, such proliferation could not be sustained without appropriate mechanical stimuli. In the present study, the tension generated by electrical stimulation, together with the stimulation frequency which matched the motor unit firing pattern of slow-twitch soleus muscle, provided the required mechanical signal to promote satellite cell proliferation and protect the cells from undergoing apoptosis. It is possible that the favorable effect of electrical stimulation on satellite cell proliferation, as evidenced by the recovery of the entire Pax7-immunopositive satellite cell pool, secures a healthy and sufficient pool of reserve (i.e. satellite cells) for subsequent muscle regeneration when appropriate mechanical stimuli is provided (i.e. reloading).
In conclusion, the present study describes the effect of electrical stimulation on maintaining the satellite cell pool through regulation of cell proliferation and apoptosis during hindlimb unloading-induced atrophy. Electrical stimulation may prove a useful adjunct intervention in controlling skeletal muscle mass. Further optimization of the stimulation parameters may help to improve the regenerative capacity of the muscles.