The effects of electrical stimulation on the satellite cell activity in muscles undergoing disuse-induced atrophy were determined using morphological and immunohistochemical analyses. Our hypothesis was that the daily application of electrical stimulation would attenuate muscle atrophy, particularly in the slow-twitch soleus muscles, by enhancing satellite cell activity. The major findings were that (i) the loss of muscle CSA and myonuclear domain in the soleus muscle were attenuated by the application of electrical stimulation; and (ii) the number of quiescent (M-cadherin+), and proliferating (BrdU+ and myoD+) satellite cells were higher in the electrically stimulated, unloaded soleus.
Consistent with previous studies [6, 10, 18], slow-twitch soleus muscle showed greater atrophy than fast-twitch EDL muscles following unloading. Slow muscle plays a strong role in antigravity function and is highly dependent on gravity for the normal expression of protein mass and slow phenotype. Conversely, fast-twitch EDL muscle is physically active and does not have an antigravity function. The fiber-type specific atrophic responses can be attributed to many factors such as differences in neuronal recruitment pattern, fiber diameter, SR calcium release function  and the recently reported differences in the levels of capillarisation . We analyzed the CSA of the soleus and EDL muscle fibers in the age-matched WB, HU, and HU-ES groups to ascertain the effects of electrical stimulation on muscle fiber size. The mean CSA of the soleus muscle in the HU-ES group was 34% larger than that of the HU group after 28 d of unloading. ES has no significant influence on the muscle size of the unloaded EDL muscles. This may be because the fiber atrophy was very mild in the EDL muscle. Furthermore, the pattern of stimulation resembles the motor unit firing frequency of slow-twitch muscle fibers. Whether a higher stimulation frequency would benefit the EDL muscle fibers need to be examined in future studies.
In this study, we used a panel of antibodies that recognize proteins uniquely expressed in satellite cells and their progeny during the quiescent, proliferating, or differentiating stage. M-cadherin, a calcium-dependent transmembrane glycoprotein, anchors satellite cells to the sarcolemma and is regarded as one of the markers for quiescent satellite cells [21, 22]. We also examined mitotically active satellite cells upon electrical stimulation during unloading, using antibodies specific for BrdU and myoD that identify the status of satellite cell proliferation [19, 22]. ES induced a higher number of quiescent and proliferating satellite cells in the unloaded soleus muscles. Further, there was a trend for higher number of satellite cells positively stained for myogenin, a marker of myogenic differentiation [15, 22], in the stimulated soleus muscles, although the difference did not reach statistical significance. Satellite cells proliferate to provide myonuclei for growing myofibers. We have conducted this study in growing animals due to the higher mitotic activity and greater abundance of satellite cells [10, 12]. It constitutes a good model in which to study the influence of different interventions on satellite cell proliferation and differentiation since they are less detectable in weight stable animals. Our findings agree with previous studies [10, 11, 26] that have shown that unloading results in a dramatic loss in the satellite cell number and mitotic activity in growing muscles. This suggests that the loss of satellite cells was closely related to a decrease in mechanical load. The decline in satellite cell activity resulted in impaired muscle growth capacity. In fact, the reduction in the number of quiescent and mitotically active satellite cells may reflect activation of the apoptotic process during mechanical unloading . In contrast, electrical stimulation of hindlimb-unloaded soleus muscles maintained the satellite cell mitotic activity to some extent. Although electrical stimulation provides some protection against muscle atrophy by influencing the satellite cell activity, the results showed only a modest benefit, since the reduction in the number of satellite cells was not restored to similar levels in the WB control conditions. In this study, we chose to apply the electrical stimulation at a frequency of 20 Hz, because this protocol resembles the motor unit firing frequency of slow-twitch muscle fibers . Future studies are needed to optimize the electrical stimulation parameters and the optimal duration in order to achieve the desired responses.
Satellite cell proliferation and incorporation are closely associated with changes in the myonuclear number . It has been reported that the number of myonuclei decreases in muscles undergoing atrophy in several different conditions, such as spinal cord injury, microgravity, and hindlimb suspension [3, 29, 30]. In this study, we observed that the myonuclear number and domain were lower (-29% and -34%, respectively) in the soleus muscle fibers following HU when compared to the age-matched WB muscles. The HU-induced loss of myonuclei in the adult rat muscle has been suggested to be a consequence of apoptosis . However, a previous study  suggested that in the unloaded, growing rat muscles, inhibition of myonuclear accretion, and not apoptosis-related loss of myonuclei, was the major cause of the smaller number of myonuclei.
Although electrical stimulation during 28 d of unloading induced a higher number (~50%) of quiescent and proliferating satellite cells in the soleus muscle, there was no significant increase in the number of myonuclei. These results indicate that the activated satellite cells from electrical stimulation were insufficient to restore the myonuclear loss during unloading. There are several points to consider for these observations. First, these results may be associated with an inability of satellite cells to incorporate into the muscle fibers. A previous study showed that satellite cell adhesion was inhibited by mechanical unloading, this may inhibit myonuclear accretion . Second, the stimulation parameters used was not effective enough to induce a significant increase in terminally differentiated satellite cells, these parameters may need to be further modified in order to achieve a more optimized effect. In this study, we observed a significantly larger myonuclear domain in the HU-ES soleus muscles. Since the whole protein-to-DNA ratio in muscle has been accepted as a measure of the myonuclear domain , increased protein content can be accomplished by increasing the myonuclear domain of each myonucleus without changing the total number of myonuclei. As such, a third possibility is that electrical stimulation plays a role in cellular protein metabolism, rather than through satellite cell-related pathways. In fact, the positive effects of electrical stimulation on protein synthesis  and degradation  have been reported in several conditions that lead to atrophy. While a causal relationship cannot be inferred from the present data, the mechanism underlying the recruitment of satellite cells with electrical stimulation in preventing muscle atrophy may be achieved from experiments in which the satellite cells are ablated.
Reduction in satellite cell activity was not observed in unloaded EDL muscles. These data may be associated with an unequal distribution of satellite cells in different fiber types where a higher number of satellite cells is associated with slow-twitch muscles . This may indicate that a larger number of satellite cells is required to maintain the muscle growth and repair in the slow-twitch muscles. We did not determine muscle fiber types in this study, and further investigation is required to elucidate the muscle fiber type and specific adaptation of satellite cells with electrical stimulation.
One of the limitations of the study was the use of M-cadherin staining to quantify satellite cell numbers, which could have led us to underestimate the absolute number. Pax7 staining is used to determine the satellite cell pool size . Detection of Pax7+ satellite cells and/or co-localization of Pax7 with myoD or myogenin may provide more valuable information on the overall satellite cell number regardless of the activation state. Another possible limitation was the use of surface electrodes to deliver electrical currents to the muscles, in contrast to surgically-implanted electrodes, which has been used in other studies [14–16, 19]. However, this is an important point to consider, since satellite cells would be activated in response to injury . Furthermore, the use of surface electrodes is compatible with future research and application in human subjects. To ensure that electrical currents (2 × 3 h day-1) were efficiently delivered to the muscles, we shaved the hindlimb of the animals and replaced the electrodes every 2-3 d in order to prevent resistance caused by hair growth and to ensure that the electrodes were well-attached. The general morphology and satellite cell properties differed in stimulated and contralateral unstimulated muscles following unloading, suggesting that the electrical currents were successfully delivered to the target muscles and had little influence on the contralateral unstimulated muscles.
Several studies have demonstrated that chronic, low-frequency electrical stimulation results in skeletal muscle angiogenesis through the up-regulation of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) [36, 37]. In addition, it has been shown that vessel-related progenitors possess high myogenic potential and may provide a secondary source of myogenesis as well as contribute to neoangiogenesis . It is therefore possible that electrical stimulation induces angiogenesis in the muscles that promote myogenic regeneration and the recovery of lost muscle mass. A recent study demonstrated that the dual delivery of VEGF and insulin-like growth factor-1 (IGF1) led to parallel angiogenesis, myogenesis with activation and proliferation of satellite cells, and a decrease in cell apoptosis . The possible role of electrical stimulation in the regulation of spatial and temporal patterns of myogenic and angiogenic events remains to be established.
Although muscle atrophy was not fully recovered with electrical stimulation in this study, we did observe some promising results that electrical stimulation can induce the proliferation and activation of satellite cells. A variety of strategies have previously been used to countermeasure muscle atrophy, but no strategy has yet to completely prevent the atrophic response. Electrical stimulation may possibly serve as a valuable adjunct to facilitate the recovery of muscle atrophy. It also has a practical implication in clinical situations, where it is either difficult or impossible for patients to return to weight-bearing conditions.