The Impact of Varying Caffeine‐Induced Contractile Activity on Metabolic Enzyme Activity

Abstract

Exercise intensity is widely known to differentially affect skeletal muscle metabolic approaches, with a general understanding that short duration, high intensity exercise is associated with an increased reliance on anaerobic glycolysis, while long duration, low to moderate intensity exercise is associated with a greater reliance on aerobic processes. In cell culture, caffeine is a commonly used method to pharmaceutically stimulate contractile activity in skeletal muscle cells; upon exposure to caffeine, ryanodine receptors are stimulated to release calcium, thereby allowing crossbridge cycling and subsequent contractile activity to occur. This technique for exercising muscle cells in culture offers a controlled, experimental platform to study the effect of contractile activity on isolated muscle cells; presumably, varying doses of caffeine should mimic varying levels of exercise intensity, though the degree to which caffeine dose impacts metabolic shifts has yet to be fully elucidated. Here, we exposed C2C12 cells, an immortalized line of mouse skeletal muscle cells, to varying levels of caffeine supplementation throughout myotube differentiation in an attempt to recapitulate metabolic signatures consistent with varying exercise intensities. Following standard proliferation protocols, mature myotubes were differentiated for 7 days. On differentiation days 4‐7, experimental cohorts of cells were exposed to either a low dosage (10mM) or a high dosage (40 mM) of caffeine‐supplemented differentiation media for 20 minutes daily. All cells were harvested for protein on differentiation day seven. Protein concentration across cohorts was measured using a Bradford protein assay. Metabolic changes were characterized by running functional enzyme assays for lactate dehydrogenase (LDH) and citrate synthase (CS) to compare anaerobic and aerobic changes, respectively. Preliminary data suggest that, relative to controls, cells exposed to 10mM caffeine have a decrease in LDH activity, and a trend toward increased CS activity. Conversely, cells exposed to 40mM caffeine show no change in LDH and a trend toward decreased CS activity. Collectively, these preliminary data suggest that C2C12 cells exposed to low doses of caffeine simulating low intensity exercise may have less reliance on anaerobic metabolism with a potential greater reliance on aerobic metabolism, while those exposed to high doses of caffeine simulating high intensity exercise may have a lesser reliance on aerobic metabolism. While more research needs to be done to characterize the precise impact of varying caffeine dosages on C2C12 cells, these initial findings are consistent with previously established trends in exercise intensity and metabolism.