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The purpose of this research was to develop a technique for rapid measurement of O2uptake (V˙o2) kinetics in single isolated skeletal muscle cells. Previous attempts to measure single cellV˙o2have utilized polarographic-style electrodes, thereby mandating large fluid volumes and relatively poor sensitivity. Thus our laboratory has developed an ∼100-μl, well-stirred chamber for the measurement ofV˙o2in isolated Xenopus laevismyocytes using a phosphorescence quenching technique [Ringer solution with 0.05 mM Pd- meso-tetra(4-carboxyphenyl)porphine] to monitor the fall in extracellular Po2(which is proportional to cellular V˙o2within the sealed chamber). V˙o2in single living myocytes dissected from Xenopus lumbrical muscles was measured from rest across a bout of repetitive tetanic contractions (0.33 Hz) and in response to a ramp protocol utilizing an increasing contraction frequency. In response to the square-wave contraction bout, the increase in V˙o2to steady state (SS) was 16.7 ± 1.3 ml · 100 g−1· min−1(range 13.0–21.9 ml · 100 g−1· min−1; n = 6). The rise in V˙o2at contractions onset ( n = 6) was fit with a time delay (2.1 ± 1.2 s, range 0.0–7.7 s) plus monoexponential rise to SS (time constant = 9.4 ± 1.5 s, range 5.2–14.9 s). Furthermore, in two additional myocytes,V˙o2increased progressively as contraction frequency increased (ramp protocol). This technique for measuring V˙o2in isolated, single skeletal myocytes represents a novel and powerful investigative tool for gaining mechanistic insight into mitochondrial function andV˙o2dynamics without potential complications of the circulation and other myocytes.

We tested the hypothesis that the mechanisms involved in the more rapid onset of fatigue when O2availability is reduced in contracting skeletal muscle are similar to those when O2availability is more sufficient. Two series of experiments were performed in isolated, single skeletal muscle fibers from Xenopus laevis. First, relative force and free cytosolic Ca2+concentrations ([Ca2+]c) were measured simultaneously in single fibers ( n = 6) stimulated at increasing frequencies (0.25, 0.33, 0.5, and 1 Hz) at an extracellular[Formula: see text] of either 22 or 159 Torr. Muscle fatigue (force = 50% of initial peak tension) occurred significantly sooner ( P < 0.05) during the low- (237 ± 40 s) vs. high-[Formula: see text]treatments (280 ± 38 s). Relative [Ca2+]cwas significantly decreased from maximal values at the fatigue time point during both the high- (72 ± 4%) and low-[Formula: see text] conditions (78 ± 4%), but no significant difference was observed between the treatments. In the second series of experiments, using the same stimulation regime as the first, fibers ( n = 6) exposed to 5 mM caffeine immediately after fatigue demonstrated an immediate but incomplete relative force recovery during both the low- (89 ± 4%) and high-[Formula: see text] treatments (82 ± 3%), with no significant difference between treatments. Additionally, there was no significant difference in relative [Ca2+]cbetween the high- (100 ± 12% of prefatigue values) and low-[Formula: see text] treatments (108 ± 12%) on application of caffeine. These results suggest that in isolated, single skeletal muscle fibers, the earlier onset of fatigue that occurred during the low-extracellular[Formula: see text] condition was modulated through similar pathways as the fatigue process during the high and involved a decrease in relative peak [Ca2+]c.

We tested the hypothesis that contracting skeletal muscle can rapidly restore force development during reperfusion after brief total ischemia and that this rapid recovery depends on O2 availability and not an alternate factor related to blood flow. Isolated canine gastrocnemius muscle ( n = 5) was stimulated to contract tetanically (isometric contraction elicited by 8 V, 0.2-ms duration, 200-ms trains, at 50-Hz stimulation) every 2 s until steady-state conditions of muscle blood flow (controlled by pump perfusion) and developed force were attained (3 min). While maintaining the same stimulation pattern, muscle blood flow was then reduced to zero (complete ischemia) for 2 min. Normal blood flow was then restored to the contracting muscle; however, two distinct conditions of oxygenation (at the same blood flow) were sequentially imposed: deoxygenated blood (30 s), blood with normal arterial O2content (30 s), a return to deoxygenated blood (30 s), and finally a return to normal arterial O2 content (90 s). During the ischemic period, force development fell to 39 ± 6 (SE)% of normal (from 460 ± 40 to 170 ± 20 N/100 g). When muscle blood flow was restored to normal by perfusion with deoxygenated blood, developed force continued to decline to 140 ± 20 N/100 g. Muscle force rapidly recovered to 310 ± 30 N/100 g ( P < 0.05) during the 30 s in which the contracting muscle was perfused with oxygenated blood and then fell again to 180 ± 30 N/100 g when perfused with blood with low[Formula: see text]. These findings demonstrate that contracting skeletal muscle has the capacity for rapid recovery of force development during reperfusion after a short period of complete ischemia and that this recovery depends on O2availability and not an alternate factor related to blood flow restoration.

The purpose of this study was to examine the development of fatigue in isolated, single skeletal muscle fibers when O2availability was reduced but not to levels considered rate limiting to mitochondrial respiration. Tetanic force was measured in single living muscle fibers ( n = 6) from Xenopus laevis while being stimulated at increasing contraction rates (0.25, 0.33, 0.5, and 1 Hz) in a sequential manner, with each stimulation frequency lasting 2 min. Muscle fatigue (determined as 75% of initial maximum force) was measured during three separate work bouts (with 45 min of rest between) as the perfusate [Formula: see text] was switched between values of 30 ± 1.9, 76 ± 3.0, or 159 Torr in a blocked-order design. No significant differences were found in the initial peak tensions between the high-, intermediate-, and low-[Formula: see text] treatments (323 ± 22, 298 ± 27, and 331 ± 24 kPa, respectively). The time to fatigue was reached significantly sooner ( P < 0.05) during the 30-Torr treatment (233 ± 39 s) compared with the 76- (385 ± 62 s) or 159-Torr (416 ± 65 s) treatments. The calculated critical extracellular [Formula: see text]necessary to develop an anoxic core within these fibers was 13 ± 1 Torr, indicating that the extracellular[Formula: see text] of 30 Torr should not have been rate limiting to mitochondrial respiration. The magnitude of an unstirred layer (243 ± 64 μm) or an intracellular O2diffusion coefficient (0.45 ± 0.04 × 10−5cm2/s) necessary to develop an anoxic core under the conditions of the study was unlikely. The earlier initiation of fatigue during the lowest extracellular[Formula: see text] condition, at physiologically high intracellular [Formula: see text] levels, suggests that muscle performance may be O2dependent even when mitochondrial respiration is not necessarily compromised.

Increased ventilation-perfusion (V˙a/Q˙) inequality is observed in ∼50% of humans during heavy exercise and contributes to the widening of the alveolar-arterial O2 difference (A-[Formula: see text]). Despite extensive investigation, the cause remains unknown. As a first step to more direct examination of this problem, we developed an animal model. Eight Yucatan miniswine were studied at rest and during treadmill exercise at ∼30, 50, and 85% of maximal O2 consumption (V˙o 2 max). Multiple inert-gas, blood-gas, and metabolic data were obtained. The A-[Formula: see text]increased from 0 ± 3 (SE) Torr at rest to 14 ± 2 Torr during the heaviest exercise level, but arterial[Formula: see text]([Formula: see text]) remained at resting levels during exercise. There was normalV˙a/Q˙inequality [log SD of the perfusion distribution (log[Formula: see text]) = 0.42 ± 0.04] at rest, and moderate increases (log[Formula: see text] = 0.68 ± 0.04, P < 0.0001) were observed with exercise. This result was reproducible on a separate day. TheV˙a/Q˙inequality changes are similar to those reported in highly trained humans. However, in swine, unlike in humans, there was no inert gas evidence for pulmonary end-capillary diffusion limitation during heavy exercise; there was no systematic difference in the measured[Formula: see text] and the[Formula: see text] as predicted from the inert gases. These data suggest that the pig animal model is well suited for studying the mechanism of exercise-inducedV˙a/Q˙inequality.

To test the hypothesis that muscle O2 uptake (V˙o 2) on-kinetics is limited, at least in part, by peripheral O2 diffusion, we determined theV˙o 2 on-kinetics in 1) normoxia (Control); 2) hyperoxic gas breathing (Hyperoxia); and 3) hyperoxia and the administration of a drug (RSR-13, Allos Therapeutics), which right-shifts the Hb-O2dissociation curve (Hyperoxia+RSR-13). The study was conducted in isolated canine gastrocnemius muscles ( n = 5) during transitions from rest to 3 min of electrically stimulated isometric tetanic contractions (200-ms trains, 50 Hz; 1 contraction/2 s; 60–70% peakV˙o 2). In all conditions, before and during contractions, muscle was pump perfused with constantly elevated blood flow (Q˙), at a level measured at steady state during contractions in preliminary trials with spontaneous Q˙. Adenosine was infused intra-arterially to prevent inordinate pressure increases with the elevated Q˙. Q˙ was measured continuously, arterial and popliteal venous O2 concentrations were determined at rest and at 5- to 7-s intervals during contractions, andV˙o 2 was calculated asQ˙ ⋅ arteriovenous O2 content difference.[Formula: see text] at 50% Hbo 2saturation (P50) was calculated. Mean capillary [Formula: see text](P [Formula: see text]) was estimated by numerical integration. P50 was higher in Hyperoxia+RSR-13 [40 ± 1 (SE) Torr] than in Control and in Hyperoxia (31 ± 1 Torr). After 15 s of contractions,P [Formula: see text]was higher in Hyperoxia (97 ± 9 Torr) vs. Control (53 ± 3 Torr) and in Hyperoxia+RSR-13 (197 ± 39 Torr) vs. Hyperoxia. The time to reach 63% of the difference between baseline and steady-stateV˙o 2 during contractions was 24.7 ± 2.7 s in Control, 26.3 ± 0.8 s in Hyperoxia, and 24.7 ± 1.1 s in Hyperoxia+RSR-13 (not significant). Enhancement of peripheral O2 diffusion (obtained by increasedP [Formula: see text]at constant O2 delivery) during the rest-to-contraction (60–70% of peakV˙o 2) transition did not affect muscle V˙o 2on-kinetics.

The purpose of this study was to examine the bioenergetics and regulation of O2uptake (V˙o2) and force production in contracting muscle when blood flow was moderately reduced during a steady-state contractile period. Canine gastrocnemius muscle ( n = 5) was isolated, and 3-min stimulation periods of isometric, tetanic contractions were elicited sequentially at rates of 0.25, 0.33, and 0.5 contractions/s (Hz) immediately followed by a reduction of blood flow [ischemic (I) condition] to 46 ± 3% of the value obtained at 0.5 Hz with normal blood flow. TheV˙o2of the contracting muscle was significantly ( P < 0.05) reduced during the I condition [6.5 ± 0.8 (SE) ml ⋅ 100 g−1⋅ min−1] compared with the same stimulation frequency with normal flow (11.2 ± 1.5 ml ⋅ 100 g−1⋅ min−1), as was the tension-time index (79 ± 12 vs. 123 ± 22 N ⋅ g−1⋅ min−1, respectively). The ratio ofV˙o2to tension-time index remained constant throughout all contraction periods. Muscle phosphocreatine concentration, ATP concentration, and lactate efflux were not significantly different during the I condition compared with the 0.5-Hz condition with normal blood flow. However, at comparable rates of V˙o2and tension-time index, muscle phosphocreatine concentration and ATP concentration were significantly less during the I condition compared with normal-flow conditions. These results demonstrate that, in this highly oxidative muscle, the normal balance of O2supply to force output was maintained during moderate ischemia by downregulation of force production. In addition, 1) the minimal disruption in intracellular homeostasis after the initiation of ischemia was likely a result of steady-state metabolic conditions having already been activated, and 2) the difference in intracellular conditions at comparable rates ofV˙o2and tension-time index between the normal flow and I condition may have been due to altered intracellular O2tension.