## 3 Results  
Electrical Stimulation Induced Skeletal Muscle Hypertrophy and Enhanced Tibialis Anterior Maximal Strength in Mice  
Initially, we evaluated the effectiveness of ES exercise in generating hypertrophy in TA. By evaluating the fiber's CSA, we were able to detect an increase of about 15% (CTR: 1792 \(\pm\) 21.07 \(\upmu\)m\({}^{2}\) vs. ES1: 2054 \(\pm\) 19.71 \(\upmu\)m\({}^{2}\), \(p\) < 0.0001) in this parameter after only one week of training (Figure 1A,C). After two weeks of ES training, the CSA increased to nearly 30% (CTR: 1758 \(\pm\) 21.69 \(\upmu\)m\({}^{2}\) vs. ES2: 2266 \(\pm\) 25.23 \(\upmu\)m\({}^{2}\), \(p\) < 0.0001) (Figure 1B,C). Likewise, frequency distribution analysis indicated an increase in CSA across both time periods, particularly in fibers ranging from 600 to 1000 \(\upmu\)m\({}^{2}\) and 2200 to 2600 \(\upmu\)m\({}^{2}\) in the ES1 group, and from 600 to 1800 \(\upmu\)m\({}^{2}\) and 2600 to 3800 \(\upmu\)m\({}^{2}\) in the ES2 group (Figure 1D). Histologically, we found that hypertrophied fibers retained their polygonal structure and abundance of extracellular matrix (Figure 1A,B). To determine the effect of additional training time, we exercised animals for four weeks and found a similar degree of hypertrophy, around 30% (CTR: 1573 \(\pm\) 20.76 \(\upmu\)m\({}^{2}\) vs. ES4: 2061 \(\pm\) 23.82 \(\upmu\)m\({}^{2}\), \(p\) < 0.0001) (Supplementary Figure S1). At two weeks of ES training, there was a substantial increase in muscle mass (CTR: 1.6 \(\pm\) 0.055 mg/g vs. ES2: 1.9 \(\pm\) 0.097 mg/g, \(p\) = 0.0205), although there was no difference at one week (Figure 1E). Analysis of the number of centralized nuclei and myonuclei per fiber revealed no significant differences among groups (Figure 1F,G).  
Because we observed a somewhat rapid hypertrophy using the procedure described here, we chose to determine whether tissue damage following the fiber size takes place (Figure 2). In the control and ES groups, dystrophin staining was typical of a regular tissue, reflecting the polygonal form of the fibers. In the positive control group (muscle obtained  
Figure 1: Morphometric and myonuclei counting analyses after one and two weeks of electrical stimulation are depicted. (**A**,**B**) are representative photomicrographs of dystrophin (green) immunofluorescence (DAPI in blue, used to identify nuclei) in TA muscle after one and two weeks of electrical stimulation, respectively (50 \(\upmu\)m scale bar). (**C**) Mean fiber cross-sectional area of the TA muscle following one and two weeks of electrical stimulation (n = 5). (**D**) Distribution of fiber cross-sectional area after one and two weeks of electrical stimulation (n = 5). (**E**) TA muscle mass analysis after one and two weeks of electrical stimulation (n = 5). (**F**) Quantity of myonuclei per fiber following electrical stimulation for one and two weeks. (**G**) Percentage of fibers containing centralized nuclei following electrical stimulation for one and two weeks. The data were displayed as mean \(\pm\) SEM. Two-way ANOVA was followed by Tukey’s post-test in the statistical analysis. Three independent experiments were conducted. \({}^{*}p<0.05\) versus control and # \(p<0.05\) versus ES1.  
four days after injection of cardiotoxin), we noticed altered dystrophin staining (reduced fiber polygonal form) and discontinuity in specific fibers. Similarly, we did not observe IgG staining in the control or ES groups, but we did observe IgG-positive fibers in the cardiotoxin injected group, as expected (Figure 2).  
Next, we measured the tibialis muscle's maximal strength using in vivo function analysis. At one week after muscle training, there was no change in TA force. Alternatively, two weeks of muscle training resulted in almost 30% (CTR: 411 \(\pm\) 27.29 mN vs. ES2: 525 \(\pm\) 35.61 mN, \(p\) = 0.0260) increase in TA force (Figure 3). Specific force was not altered among groups.