Muscle Length-Dependent Effects of Neuromuscular Electrical Stimulation and Experimental Modulation Strategies on MyoD, Caspase-3, PCNA, and BDNF Expression

Background Neuromuscular electrical stimulation (NMES) is a cornerstone of clinical rehabilitation, yet the influence of muscle length on structural and molecular remodeling during stimulation remains incompletely understood. This study investigated the early post-stimulation molecular responses and subsequent morphological changes following NMES applied at different muscle lengths, with skeletal muscle ultrastructure and the expression of MyoD, Caspase-3, PCNA, and BDNF assessed in parallel at 2, 5, and 14 days. Methods Male Wistar Albino rats received a single session of supramaximal NMES to the gastrocnemius at long (L-NMES) or medium (M-NMES) muscle length. The potential of pre-stimulation static stretching (NMES + SS) and post-stimulation cold-water therapy (NMES + CWT) to modulate early post-stimulation responses was also evaluated. Tissues were sampled at 2, 5, and 14 days post-stimulation for transmission electron microscopy and immunohistochemical analysis. Results L-NMES induced more pronounced early ultrastructural alterations, including myofibrillar disorganization and mitochondrial abnormalities, compared to M-NMES. Expression of BDNF, Caspase-3, MyoD, and PCNA was significantly higher in L-NMES-2 than M-NMES-2 and control groups (p < 0.05); peak values were [BDNF: 50.92 ± 2.78% vs. 32.72 ± 2.67%; MyoD: 3.40 ± 0.59% vs. 2.10 ± 0.29%; Caspase-3: 3.20 ± 0.59% vs. 2.34 ± 0.48%; PCNA: 3.34 ± 0.56% vs. 2.70 ± 0.59%]. NMES + SS preserved ultrastructural integrity while maintaining elevated MyoD and PCNA expression. NMES + CWT attenuated BDNF and MyoD expression compared to L-NMES-2 (p < 0.05). By Day 14, myofibrillar organization was largely restored across all groups. Conclusion Muscle length is a relevant modulator of mechanical stress and subsequent molecular responses following NMES in a rat model. Stimulation at long muscle length was associated with a more pronounced early molecular response, encompassing apoptotic signaling (Caspase-3) together with early damage-associated and stress-responsive signaling (MyoD, PCNA, BDNF), which was attenuated by pre-stimulation static stretching.