Electrical Pulse Stimulation Induced Changes On Lipid, Mitochondrial, Glut4 And Ampk Content In Human Myotubes

Abstract

It has been previously shown that human myotubes retain certain in vivo characteristics of the donors. Furthermore, we have shown that Electrical pulse stimulation (EPS), an in vitro exercise mimetic, increases mitochondrial and lipid content in cultured human myotubes after 24 hr. of stimulation. PURPOSE: We aimed to examine the EPS induced adaptations to lipid, mitochondrial, Glucose transporter 4 (GLUT4) and total 5’ Adenosine Monophosphate Activated Protein Kinase (AMPK) content using human myotubes from lean donors. Additionally, we investigated the impact of cell harvest time (immediate vs. 24 hr. after the EPS cessation) on the above-mentioned parameters. METHODS: EPS was applied to myotubes for 60 min (bipolar pulses of 100 Hz, 30V, every 5 seconds), 24 hr. or 48 hr. (single bipolar pulses of 1 Hz for 2 ms; 30V) and were harvested at two different time points: immediately after (early harvest) and 24hr after (late harvest) the end of stimulation. Mitochondrial and lipid content were measured by immunohistochemistry using primary antibody for complex IV and bodipy green dye, respectively. A microplate fluorometer was used to quantify fluorescence signals. GLUT4 and AMPK were measured using western immunoblotting. RESULTS: Lipid in myotubes was significantly higher (p<0.05) after 24 hr early harvest EPS compared to unstimulated control. There was no significant changes in lipid content with late harvest EPS. Mitochondrial content was significantly higher (p<0.05) after acute, 24 hr. as well as 48 hr. early harvest EPS, but was significantly lower (p<0.05) after acute late harvest EPS, compared to unstimulated controls. GLUT4 protein content was higher after acute and 24 hr. early harvest and 48 hr. late harvest EPS. Total AMPK was higher after 24 hr. early harvest and 48 hr. late harvest. AMPK was lower after 24 hr. late harvest. CONCLUSION: These findings suggest 24 hours of EPS treatment lead to increase in lipid, mitochondria, GLUT4 and AMPK protein content in human myotubes cultured from lean donors. Additionally, cell-harvesting time a reflection of post-exercise recovery is a key factor for these adaptations.