## 4 Discussion  
In light of the requirement for experimental models that elicit considerable hypertrophy in a timely manner, we sought to build an improved muscle training protocol employing electrical stimulation that mimics a conventional resistance exercise in humans. In terms of fiber type composition, we have also emphasized a rather homogenous (TA) muscle (predominantly type II fibers). Additionally, the TA muscle may be easily removed for further examination and is rather large compared to muscles employed in specific studies, such as the plantar [24], flexor hallucids longus [30], extensor digitorum longus, and soleus [28], allowing for sufficient tissue for further analysis at multiple levels. In addition, TA is the most common muscle used in investigations employing electroporation models, where it is relatively simple to transfer plasmids containing foreign genes into tissue cells. Notably, the use of a fast-twitch muscle may have created a limiting bias, as the results obtained may not be applicable to a predominately slow-twitch muscle. The major difference in this study is the high intensity of training, in which the volume (10 sets with 12 repetitions), contraction/rest rate between contractions (3 s on and 2 s off), and resting time between sets (1 min) are, to our knowledge, the most intense in the literature [11; 12; 13; 14; 38; 39]. This protocol design caused a rapid and strong hypertrophy (\(\sim\)30% hypertrophy) after only two weeks of training. After 3-6 weeks of exercise, related published programs typically achieve 8-15 percent hypertrophy [2; 11; 37; 39]. Notably, increased time to acquire quantifiable hypertrophy is associated with an increased likelihood of anesthetic events that could greatly increase animal stress levels, hence influencing molecular parameters and exercise response. Our maximal-force measurements reveal  
Figure 6: Phosphorylation and total protein implicated in AKT/mTOR signaling following two weeks of electrical stimulation. (**A**) representative band patterns of AKT-mTOR elements after electrical stimulation for two weeks. AKT (**B–D**), mTOR (**E–G**), P70 (**H–J**), and EIF4E (**K–M**) bands were analyzed by densitometry (n = 5 per group). The data are presented mean \(\pm\) SD. In the statistical analysis, an unpaired \(t\)-test and a Mann-Whitney test were employed. * \(p\) < 0.05 vs. control group. identical increases when compared to the changes in skeletal muscle fiber CSA, indicating that the hypertrophy seen corresponds to an increase in muscle size without a change in specific force. In this study, we examined satellite cell activity as a tissue remodeling marker and observed a one-week increase in Pax-7-positive nuclei that persisted for two weeks following electrical stimulation. As a marker of satellite cell proliferation, these results are expected for Pax-7, and its increases at the beginning of training are required for muscle tissue remodeling and adaptation. We also examined MyoD-positive nuclei, and after two weeks of electrical stimulation, we detected an increase. This supports a subsequent burst of satellite cell differentiation, which is consistent with the anticipated tissue remodeling that occurs after the union of these cells with mechanically stressed fibers. This remodeling was reflected, as expected, in the centralized nuclei that were observed primarily after two weeks of training (Figure 1G), whereas sarclemma was preserved, as observed by IgG and dystrophin measurements (Figure 2). Measuring the expression of key molecules involved in protein synthesis is an additional way to monitor the efficacy of the muscle training described here. Muscle training activates mTOR signaling, which is a critical regulator of protein synthesis in skeletal muscle. Two weeks of electrical stimulation elicit an increase in mTOR phosphorylation and downstream components of this pathway, such as p70, according to our findings.