Intracellular pH during sequential, fatiguing contractile periods in isolated singleXenopusskeletal muscle fibers

Abstract

-The purpose of the present study was to test the hypothesis that a preceding contractile period in isolated single skeletal muscle fibers would attenuate the decrease in pH during an identical, subsequent contractile period, thereby reducing the rate of fatigue. Intact single skeletal muscle fibers (n ϭ 9) were isolated from Xenopus lumbrical muscle and incubated with the fluorescent cytosolic H ϩ indicator 2Ј,7Ј-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) AM for 30 min. Two identical contractile periods were performed in each fiber, separated by a 1-h recovery period. Force and intracellular pH (pH i) fluorescence were measured simultaneously while fibers were stimulated (tetanic contractions of 350-ms trains with 70-Hz stimuli at 9 V) at progressively increasing frequencies (0.25, 0.33, 0.5, and 1 contraction/s) until the development of fatigue (to 60% initial force). No significant difference (P Ͻ 0.05) was observed between the first and second contractile periods in initial force development, resting pH i, or time to fatigue (5.3 Ϯ 0.5 vs. 5.1 Ϯ 0.6 min). However, the relative decrease in the BCECF fluorescence ratio (and therefore pH i) from rest to the fatigue time point was significantly greater (P Ͻ 0.05) during the first contractile period (to 65 Ϯ 4% of initial resting values) compared with the second (77 Ϯ 4%). The results of the present study demonstrated that, when preceded by an initial fatiguing contractile period, the rise in cytosolic H ϩ concentration in contracting single skeletal muscle fibers during a second contractile period was significantly reduced but did not attenuate the fatigue process in the second contractile period. These results suggest that intracellular factors other than H ϩ accumulation contribute to the fall in force development under these conditions. anaerobic metabolism; glycolysis; oxidative phosphorylation; phosphocreatine; fluorescence; onset kinetics; exercise IT HAS BEEN PREVIOUSLY DEMONSTRATED in whole skeletal muscle that a preceding bout of exercise increases the onset kinetics of oxidative phosphorylation during the rest-to-work transition (7,19). Whether the increase in the rate of O 2 utilization (V O 2 ) was due to cardiovascular (global) and/or microvascular (local) responses resulting in an increased O 2 availability, or to "priming" of intracellular regulators of oxidative metabolism remains controversial (24). However, we have previously demonstrated in isolated whole muscle that increases in O 2 delivery do not positively modulate the V O 2 onset kinetics during submaximal exercise (9, 10). Furthermore, we have recently demonstrated in isolated single skeletal muscle fibers (thereby eliminating vascular O 2 delivery considerations) that a more rapid increase in the onset kinetics of oxidative metabolism occurs during a second contractile period at the same extracellular PO 2 (12). These results suggest that regulation of the onset kinetics of oxidative phosphorylation during submaximal exercise is likely determined by intracellular enzymatic pro...

Phosphocreatine recovery kinetics following low- and high-intensity exercise in human triceps surae and rat posterior hindlimb muscles

“…This was similar to the effect of prior exercise on subsequent bouts of exercise reported previously (6,29,34). In the present study, for example, during the human high-intensity protocol there was a significant time effect (P ϭ 0.015) observed when comparing the lowest pH obtained during each repeated cycle, with the first cycle having a lower pH compared with the subsequent repeated bouts (cycle 1: pH 6.77 Ϯ 0.05; cycles 2-5: pH range 6.86 Ϯ 0.02 to 6.89 Ϯ 0.02).…”

Skeletal Muscle Fatigue: Cellular Mechanisms

“…Importantly, in cases where pH i does drop to low levels in a fatigued muscle, upon ceasing the exercise or stimulation, force typically recovers much faster than pH i (22,78,391,438), indicating that the low pH per se was not responsible for all of the force reduction. Furthermore, attenuating the decline in pH during a stimulation period did not reduce the extent of fatigue in frog muscle fibers (421).…”

Mitochondria: isolation, structure and function

“…Oxygen consumption can nevertheless be measured in intact cultured cells using high sensitivity systems (such as the Seahorse (Gerencser et al 2009), or Oroboros systems (Gnaiger, 2009)), with the compromise being that these systems do not permit simultaneous microscopic measurements. Alternatively, oxygen consumption has also been monitored using a standard electrode in single intact myocytes (Elzinga & van der Laarse, 1988), and this method can probably be adapted to permit monitoring of other aspects of cellular function simultaneously using confocal imaging methods (Stary & Hogan, 2000, 2005).…”