Introduction: Regular physical activity is capable of improving and maintaining human health. However, the molecular mechanisms of exercise-induced adaptations are still not fully understood. Electrical pulse stimulation (EPS) is used to induce visually detectable contractions of differentiated muscle cells, myotubes, to mimic exercise in vitro.
Aim: Our aim was to identify the EPS protocol that can induce significant physiological response in cultured human primary myotubes, by comparing the effectiveness of two EPS protocols, with 1. continuous, and 2. intermittent stimulation.
Materials and methods: Differentiated primary human skeletal muscle cells derived from healthy, lean men (n=3, 31±2.45 yrs, 23,72±0.82 kg/m2) were exposed to two EPS (Ionoptix, USA) protocols: (i) commonly used 24h continuous stimulation (frequency 1Hz, pulse duration 2ms), and (ii) 24h intermittent stimulation, where higher frequency stimulation (5Hz, pulse duration 2ms) is followed by subthreshold stimulation (0.2Hz, pulse duration 4 ms). This cycle was repeated 9 times in 24h period. Oxidation of radioactively labeled 14C-glucose and 14C-palmitate, incorporation of 14C-glucose into glycogen, changes in content of mitochondrial respiratory chain proteins (western blot) and in fiber-type specific gene markers (qPCR) were determined. Control cells were exposed to electrodes without EPS. Data are presented as average±SEM and differences were analyzed with paired T-test (GraphPad Prism 9.4.1).
Results: Both types of EPS led to visually detectable contractions of myotubes and facilitated the incorporation of glucose into glycogen (continuous stimulation: 19.72±5.09 pmol/3h/µg, p=0.0063; intermittent stimulation: 17.97±4.88 pmol/3h/µg, p=0.0051 vs. control: 15.14±4.79 pmol/3h/µg, n=3). However, oxidative glucose utilization tended to increase only after the intermittent stimulation (intermittent stimulation: 9.41±1.76 pmol/3h/µg, vs. control: 7.58±1.77 pmol/3h/µg, p=0.0835, n=3). Intermittent stimulation also led to significantly increased total glucose disposal (intermittent stimulation: 27.38±6.49 pmol/3h/µg, vs. control: 22.72±6.30 pmol/3h/µg, p=0.0031, n=3), while after continuous stimulation we observed only a trend (continuous stimulation: 26.48±6.93 pmol/3h/µg, vs. control: 22.72±6.30 pmol/3h/µg, p=0.0744, n=3). Importantly, we observed a 20% increase in total fatty acid oxidation in cells exposed to intermittent stimulation (intermittent stimulation: 983.5±149.582 pmol/3h/mg, vs. control: 799.6±98.8 pmol/3h/mg, p=0.0669, n=4), while continuous stimulation did not induce significant change due to high response variability (continuous stimulation: 1067.4±249.0 pmol/3h/mg, , vs. control: 799.6±98.8 pmol/3h/mg, p=0.33, n=4). There were no changes in the protein content of respiratory chain complexes in response to two protocols (n=4, p>0.1). However, there was an increase in mRNA for MYH2 (marker of fast IIa fibers) specifically after continuous stimulation (continuous stimulation: 45.33±5.16 AU, vs. control: 27.342±4.34 AU, p=0.0356, n=4).
Conclusion: Electrical pulse stimulation is in vitro model used for studying exercise-related adaptative mechanisms in muscle cells. We identified intermittent EPS as more effective in inducing relevant physiological changes in metabolism, as exemplified by an improvement in glucose and palmitate oxidation.
Ethical approval: The study was approved by the ethics committee of the University Hospital Bratislava, Comenius University Bratislava and the Ethics Committee of the Bratislava Region Office and conforms to the ethical guidelines of the Declaration of Helsinki. All participants provided witnessed written informed consent prior to entering the study.