Strong fluid movement near mammalian skeletal muscle improves their stability for force and metabolic studies at 37°C

Studying mammalian skeletal muscles at 37°C is challenging because of a high metabolic rate requiring rapid O2 diffusion and more importantly a high production rate of reactive oxygen species (ROS) requiring fast removal. Both problems can be circumvented when small muscles up to 10 mg wet weight are immersed in physiological solutions and superfused near the surface with rapid solution flow (10–15 ml/min). However, rapid superfusion is not feasible when using expensive drugs and radioactive markers representing conditions that require muscle incubation in small volume. The objective of this study was therefore to optimize the experimental conditions that will give rise to stable muscle preparations at 37°C when a rapid flow of physiological saline solution is not an option. We tested the hypothesis that in the absence of superfusion, one must create fluid movement in order to replace the convection effect of superfusion. Tetanic force can be maintained constant for hours when mouse FDB bundles (1–2 mg wet weight) were superfused whereas a continuous loss of force reaching 10% force by 25 min was observed when superfusion was interrupted. The decrease in tetanic force during fatigue, elicited with one contraction every sec for 3 min, was significantly faster and the final extent of the decrease greater in the absence than in the presence of superfusion. There was, on the other hand, no difference in the decrease in tetanic force between superfusion and gas bubbling both prior to fatigue and during fatigue. A lack of superfusion was also important for the glycogen content. Glycogen levels prior to fatigue were 119 μmoles glucose equivalent/g dry weight when superfusion was maintained while interrupting superfusion for 10 min reduced glycogen levels to 82 μmoles/g dry weight; representing a decrease of 37 μmoles/g dry weight. Following fatigue, glycogen levels were 77 μmoles/g dry muscle with superfusion and 43 μmoles/g dry weight in the absence of superfusion. Notably, the amount of glycogen depletion during fatigue was not different with and without superfusion, the values being 42 and 39 μmoles/g dry weight, respectively. The effect of interrupting superfusion on tetanic force was much greater following a first fatigue bout and 50 min recovery. That is, interrupting superfusion resulted in 35% decrease in tetanic force within 10 min, which was again prevented by gas bubbling or the addition of NAC or tiron, two known ROS scavengers. So, a lack of fluid movement near mammalian skeletal muscle muscles at 37°C results in large loss of tetanic force and glycogen. The loss of tetanic force can be prevented by applying gas bubbling near the muscle or by scavenging ROS. Support or Funding Information Study was supported by the National Science and Engineering Research Council of Canada (NRC)

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