Streptomycin decreases the functional shift to a slow phenotype induced by electrical stimulation in engineered...

Chronic low-frequency stimulation (CLFS) has long been used to induce a fast-to-slow phenotype shift in skeletal muscle. In this study, we explore the role of frequency (10 and 20 Hz), active time (15-60%), and streptomycin in inducing a fast-to-slow shift in engineered muscle. We found that C2C12 engineered muscle could respond to CLFS with an adult-like active time of 60% and found that a constant 10 Hz train of 0.6 s, followed by 0.4 s rest, induced a partial fast-to-slow phenotype shift. Following 2 weeks of CLFS, time-to-peak tension (TPT) (control [CTL]=40.9±0.2 ms; 10 Hz=58.5±3.5 ms; 20 Hz=48.2±2.7 ms) and half-relaxation time (1/2RT) (CTL=50.4±0.6 ms; 10 Hz=76.1±3.3 ms; 20 Hz=66.6±2.3 ms) slowed significantly in frequency, but not in an active time-dependent manner. Streptomycin significantly blunted the slowing of TPT and 1/2RT induced by CLFS by minimizing the fast-to-slow shift in SERCA isoform. Streptomycin (Nonstim=-42.8%±2.5%; Stim=-38.1%±3.6%) significantly prevented the improvement in fatigue resistance seen in CTL constructs (Nonstim=-58.4%±3.6%; Stim=-27.8%±1.7%). Streptomycin reduced the increase seen in GLUT4 protein following CLFS (CTL=89.4%±6.7%; STREP=41.0%±4.3%) and prevented increases in the mitochondrial proteins succinate dehydrogenase (SDH) and ATP synthase. These data demonstrate that streptomycin significantly blunts the fast-to-slow shift induced by CLFS. In the absence of streptomycin, CLFS induced slowing of contractile dynamics and improved fatigue resistance and suggests that this model can be used to study the mechanisms underlying CLFS-induced adaptations in muscle phenotype.