In vitro muscle contraction: A technical review on electrical pulse stimulation in C2C12 cells

Electrical pulse stimulation (EPS) of skeletal muscle cells is increasingly used to model exercise In vitro. The murine C2C12 myotube system has become a common platform for such studies, yet wide variability in EPS protocols hampers reproducibility and cross-study comparisons. In this technical review, we analysed 54 peer-reviewed studies that employed EPS in C2C12 and extracted used EPS protocols to provide an overview of the most commonly used settings for the EPS parameters (pulse duration, frequency, voltage and stimulation duration). Additionally, we summarized the biological processes investigated in these studies to illustrate the range of research topics typically addressed using this model. The majority of studies used 2 ms pulses at 1 Hz and moderate voltages (10-20 V), often over 24 h of stimulation. Glucose uptake was the most commonly assessed endpoint, followed by AMPK activation, inflammation and mitochondrial adaptations. Correlation analyses revealed interdependence between pulse duration, voltage and EPS duration, indicating that these parameters are often balanced to avoid excessive or suboptimal stimulation. While frequency was largely standardized, voltage and pulse duration showed greater variation. Our findings underscore the need for more detailed parameter reporting and deliberate protocol design aligned with specific experimental objectives, such as mimicking endurance- or resistance-type exercise stimuli. This review serves as a resource for selecting EPS parameters tailored to specific biological processes and encourages standardization to improve translational relevance.