Short-duration electrical pulse stimulation induces myokines and mitochondrial adaptations in C2C12 myotubes

Physical activity induces rapid myokine and metabolic responses in skeletal muscle, yet the immediate cellular adaptations to short-duration contractile activity remain incompletely defined. C2C12 myotubes were exposed to electrical pulse stimulation (EPS; 40 V, 2 ms, 1 Hz) for 1 h and 2 h to evaluate time-dependent responses. A subset of cells was treated with AICAR (0.5 mM for 24 h or 1 mM for 2 h) to examine whether direct AMPK activation reproduces contraction-like effects. Cell viability, fiber-type markers, IL-6 and brain-derived neurotrophic factor (BDNF) secretion, glucose uptake, mitochondrial membrane potential (ΔΨm), mitochondrial DNA (mtDNA) abundance, and AMPK/PGC-1α signaling were assessed. EPS did not alter cell viability or fiber-type markers within 2 h. EPS increased IL-6 mRNA and IL-6 secretion, and significantly elevated BDNF secretion in the culture supernatants at 2 h. Glucose uptake increased over time (p for trend = 0.001). mtDNA abundance increased at 1 h (p < 0.05) and further at 2 h (p < 0.001), whereas ΔΨm showed a modest linear trend (p for trend = 0.046). AMPK phosphorylation increased progressively (p for trend < 0.001), accompanied by an emerging, non-significant increase in PGC-1α mRNA. AICAR activated AMPK and increased IL-6 secretion but did not alter BDNF levels, indicating partial overlap with EPS-induced signaling. Collectively, short-duration EPS elicits coordinated early myokine, metabolic, and mitochondrial responses in C2C12 myotubes, while pharmacological AMPK activation recapitulates IL-6 but not BDNF secretion, suggesting that contraction-specific signals cooperate with AMPK to regulate BDNF secretion. Short-duration EPS rapidly increases IL-6 secretion and BDNF release in C2C12 myotubes. EPS enhances glucose uptake and increases mtDNA abundance, with a modest increase in ΔΨm concomitant with AMPK activation. Pharmacological AMPK activation (AICAR) reproduces the IL-6 but not the BDNF response, indicating contraction-specific regulation of BDNF secretion.