Kinetic control of oxygen consumption during contractions in self-perfused skeletal muscle.

Fast kinetics of muscle oxygen consumption (VO2) is characteristic of effective physiological systems integration. The mechanism of VO2 kinetic control in vivo is equivocal as measurements are complicated by the twin difficulties of making high-frequency direct measurements of VO2 and intramuscular metabolites, and in attaining high [ADP]; complexities that can be overcome utilising highly aerobic canine muscle for the investigation of the transition from rest to contractions at maximal VO2. Isometric tetanic contractions of the gastrocnemius complex of six anaesthetised, ventilated dogs were elicited via sciatic nerve stimulation (50 Hz; 200 ms duration; 1 contraction s(−1)). Muscle VO2 and lactate efflux were determined from direct Fick measurements. Muscle biopsies were taken at rest and every ∼10 s during the transient and analysed for [phosphates], [lactate] and pH. The temporal VO2 vs. [PCr] and [ADP] relationships were not well fitted by linear or classical hyperbolic models (respectively), due to the high sensitivity of VO2 to metabolic perturbations early in the transient. The time course of this apparent sensitisation was closely aligned to that of ATP turnover, which was lower in the first ∼25 s of contractions compared to the steady state. These findings provide the first direct measurements of skeletal muscle VO2 and [PCr] in the non-steady state, and suggest that simple phosphate feedback models (which are adequate for steady-state observations in vitro) are not sufficient to explain the dynamic control of VO2 in situ. Rather an allosteric or 'parallel activation' mechanism of energy consuming and producing processes is required to explain the kinetic control of VO2 in mammalian skeletal muscle.

Faster O2 uptake kinetics in canine skeletal muscle in situ after acute creatine kinase inhibition.

Creatine kinase (CK) plays a key role both in energy provision and in signal transduction for the increase in skeletal muscle O2 uptake () at exercise onset. The effects of acute CK inhibition by iodoacetamide (IA; 5 mm) on kinetics were studied in isolated canine gastrocnemius muscles in situ (n = 6) during transitions from rest to 3 min of electrically stimulated contractions eliciting ∼70% of muscle peak , and were compared to control (Ctrl) conditions. In both IA and Ctrl muscles were pump-perfused with constantly elevated blood flows. Arterial and venous [O2] were determined at rest and every 5-7 s during contractions. was calculated by Fick's principle. Muscle biopsies were obtained at rest and after ∼3 min of contractions. Muscle force was measured continuously. There was no fatigue in Ctrl (final force/initial force (fatigue index, FI) = 0.97 ± 0.06 (x ± s.d.)), whereas in IA force was significantly lower during the first contractions, slightly recovered at 15-20 s and then decreased (FI 0.67 ± 0.17). [Phosphocreatine] was not different in the two conditions at rest, and decreased during contractions in Ctrl, but not in IA. at 3 min was lower in IA (4.7 ± 2.9 ml 100 g-1 min-1) vs. Ctrl (16.6 ± 2.5 ml 100 g-1 min-1). The time constant (τ) of kinetics was faster in IA (8.1 ± 4.8 s) vs. Ctrl (16.6 ± 2.6 s). A second control condition (Ctrl-Mod) was produced by modelling a response that accounted for the 'non-square' force profile in IA, which by itself could have influenced kinetics. However, τ in IA was faster than in Ctrl-Mod (13.8 ± 2.8 s). The faster kinetics due to IA suggest that in mammalian skeletal muscle in situ, following contractions onset, temporal energy buffering by CK slows the kinetics of signal transduction for the activation of oxidative phosphorylation.

Myocyte vascular endothelial growth factor is required for exercise-induced skeletal muscle angiogenesis.

We have previously shown, using a Cre-LoxP strategy, that vascular endothelial growth factor (VEGF) is required for the development and maintenance of skeletal muscle capillarity in sedentary adult mice. To determine whether VEGF expression is required for skeletal muscle capillary adaptation to exercise training, gastrocnemius muscle capillarity was measured in myocyte-specific VEGF gene-deleted (mVEGF(-/-)) and wild-type (WT) littermate mice following 6 wk of treadmill running (1 h/day, 5 days/wk) at the same running speed. The effect of training on metabolic enzyme activity levels and whole body running performance was also evaluated in mVEGF(-/-) and WT mice. Posttraining capillary density was significantly increased by 59% (P < 0.05) in the deep muscle region of the gastrocnemius in WT mice but did not change in mVEGF(-/-) mice. Maximal running speed and time to exhaustion during submaximal running increased by 20 and 13% (P < 0.05), respectively, in WT mice after training but were unchanged in mVEGF(-/-) mice. Training led to increases in skeletal muscle citrate synthase (CS) and phosphofructokinase (PFK) activities in both WT and mVEGF(-/-) mice (P < 0.05), whereas ß-hydroxyacyl-CoA dehydrogenase (ß-HAD) activity was increased only in WT mice. These data demonstrate that skeletal muscle capillary adaptation to physical training does not occur in the absence of myocyte-expressed VEGF. However, skeletal muscle metabolic adaptation to exercise training takes place independent of myocyte VEGF expression.

The O2 cost of the tension-time integral in isolated single myocytes during fatigue.

One proposed explanation for the Vo(2) slow component is that lower-threshold motor units may fatigue and develop little or no tension but continue to use O(2), thereby resulting in a dissociation of cellular respiration from force generation. The present study used intact isolated single myocytes with differing fatigue resistance profiles to investigate the relationship between fatigue, tension development, and aerobic metabolism. Single Xenopus skeletal muscle myofibers were allocated to a fast-fatiguing (FF) or a slow-fatiguing (SF) group, based on the contraction frequency required to elicit a fall in tension to 60% of peak. Phosphorescence quenching of a porphyrin compound was used to determine Delta intracellular Po(2) (Pi(O(2)); a proxy for Vo(2)), and developed isometric tension was monitored to allow calculation of the time-integrated tension (TxT). Although peak DeltaPi(O(2)) was not different between groups (P = 0.36), peak tension was lower (P < 0.05) in SF vs. FF (1.97 +/- 0. 17 V vs. 2. 73 +/- 0.30 V, respectively) and time to 60% of peak tension was significantly longer in SF vs. FF (242 +/- 10 s vs. 203 +/- 10 s, respectively). Before fatigue, both DeltaPi(O(2)) and TxT rose proportionally with contraction frequency in SF and FF, resulting in DeltaPi(O(2))/TxT being identical between groups. At fatigue, TxT fell dramatically in both groups, but DeltaPi(O(2)) decreased proportionately only in the FF group, resulting in an increase in DeltaPi(O(2))/TxT in the SF group relative to the prefatigue condition. These data show that more fatigue-resistant fibers maintain aerobic metabolism as they fatigue, resulting in an increased O(2) cost of contractions that could contribute to the Vo(2) slow component seen in whole body exercise.

Continued artificial selection for running endurance in rats is associated with improved lung function.

Previous studies found that selection for endurance running in untrained rats produced distinct high (HCR) and low (LCR) capacity runners. Furthermore, despite weighing 14% less, 7th generation HCR rats achieved the same absolute maximal oxygen consumption (Vo(2max)) as LCR due to muscle adaptations that improved oxygen extraction and use. However, there were no differences in cardiopulmonary function after seven generations of selection. If selection for increased endurance capacity continued, we hypothesized that due to the serial nature of oxygen delivery enhanced cardiopulmonary function would be required. In the present study, generation 15 rats selected for high and low endurance running capacity showed differences in pulmonary function. HCR, now 25% lighter than LCR, reached a 12% higher absolute Vo(2max) than LCR, P < 0.05 (49% higher Vo(2max)/kg). Despite the 25% difference in body size, both lung volume (at 20 cmH(2)O airway pressure) and exercise diffusing capacity were similar in HCR and LCR. Lung volume of LCR lay on published mammalian allometrical relationships while that of HCR lay above that line. Alveolar ventilation at Vo(2max) was 30% higher, P < 0.05 (78% higher, per kg), arterial Pco(2) was 4.5 mmHg (17%) lower, P < 0.05, while total pulmonary vascular resistance was (insignificantly) 5% lower (30% lower, per kg) in HCR. The smaller mass of HCR animals was due mostly to a smaller body frame rather than to a lower fat mass. These findings show that by generation 15, lung size in smaller HCR rats is not reduced in concert with their smaller body size, but has remained similar to that of LCR, supporting the hypothesis that continued selection for increased endurance capacity requires relatively larger lungs, supporting greater ventilation, gas exchange, and pulmonary vascular conductance.

Muscle-specific VEGF deficiency greatly reduces exercise endurance in mice.

Vascular endothelial growth factor (VEGF) is required for vasculogenesis and angiogenesis during embryonic and early postnatal life. However the organ-specific functional role of VEGF in adult life, particularly in skeletal muscle, is less clear. To explore this issue, we engineered skeletal muscle-targeted VEGF deficient mice (mVEGF-/-) by crossbreeding mice that selectively express Cre recombinase in skeletal muscle under the control of the muscle creatine kinase promoter (MCKcre mice) with mice having a floxed VEGF gene (VEGFLoxP mice). We hypothesized that VEGF is necessary for regulating both cardiac and skeletal muscle capillarity, and that a reduced number of VEGF-dependent muscle capillaries would limit aerobic exercise capacity. In adult mVEGF-/- mice, VEGF protein levels were reduced by 90 and 80% in skeletal muscle (gastrocnemius) and cardiac muscle, respectively, compared to control mice (P < 0.01). This was accompanied by a 48% (P < 0.05) and 39% (P < 0.05) decreases in the capillary-to-fibre ratio and capillary density, respectively, in the gastrocnemius and a 61% decrease in cardiac muscle capillary density (P < 0.05). Hindlimb muscle oxidative (citrate synthase, 21%; beta-HAD, 32%) and glycolytic (PFK, 18%) regulatory enzymes were also increased in mVEGF-/- mice. However, this limited adaptation to reduced muscle VEGF was insufficient to maintain aerobic exercise capacity, and maximal running speed and endurance running capacity were reduced by 34% and 81%, respectively, in mVEGF-/- mice compared to control mice (P < 0.05). Moreover, basal and dobutamine-stimulated cardiac function, measured by transthoracic echocardiography and left ventricular micromanomtery, showed only a minimal reduction of contractility (peak +dP/dt) and relaxation (peak -dP/dt, tau(E)). Collectively these data suggests adequate locomotor muscle capillary number is important for achieving full exercise capacity. Furthermore, VEGF is essential in regulating postnatal muscle capillarity, and that adult mice, deficient in cardiac and skeletal muscle VEGF, exhibit a major intolerance to aerobic exercise.

Peripheral oxygen transport and utilization in rats following continued selective breeding for endurance running capacity.

Untrained rats selectively bred for either high (HCR) or low (LCR) treadmill running capacity previously demonstrated divergent physiological traits as early as the seventh generation (G7). We asked whether continued selective breeding to generation 15 (G15) would further increase the divergence in skeletal muscle capillarity, morphometry, and oxidative capacity seen previously at G7. At G15, mean body weight was significantly lower (P < 0.001) in the HCR rats (n = 11; 194 +/- 3 g) than in LCR (n = 12; 259 +/- 9 g) while relative medial gastrocnemius muscle mass was not different (0.23 +/- 0.01 vs. 0.22 +/- 0.01% total body weight). Normoxic (Fi(O(2)) = 0.21) Vo(2max) was 50% greater (P < 0.001) in HCR despite the lower absolute muscle mass, and skeletal muscle O(2) conductance (measured in hypoxia; Fi(O(2)) = 0.10) was 49% higher in HCR (P < 0.001). Muscle oxidative enzyme activities were significantly higher in HCR (citrate synthase: 16.4 +/- 0.4 vs. 14.0 +/- 0.6; beta-hydroxyacyl-CoA dehydrogenase: 5.2 +/- 0.2 vs. 4.2 +/- 0.2 mmol.kg(-1).min(-1)). HCR rats had approximately 36% more total muscle fibers and also 36% more capillaries in the medial gastrocnemius. Because average muscle fiber area was 35% smaller, capillary density was 36% higher in HCR, but capillary-to-fiber ratio was the same. Compared with G7, G15 HCR animals showed 38% greater total fiber number with an additional 25% decrease in mean fiber area. These data suggest that many of the skeletal muscle structural and functional adaptations enabling greater O(2) utilization in HCR at G7 continue to progress following additional selective breeding for endurance capacity. However, the largest changes at G15 relate to O(2) delivery to skeletal muscle and not to the capacity of skeletal muscle to use O(2).

Glycolytic activation at the onset of contractions in isolated Xenopus laevis single myofibres.

Intracellular pH (pHi) was measured in isolated Xenopus laevis single myofibres at the onset of contractions, with and without glycolytic blockade, to investigate the time course of glycolytic activation. Single myofibres (n=8; CON) were incubated in 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoyxmethyl ester (10 microM; for fluorescence measurement of pHi) and stimulated for 15 s at 0.67 Hz in anoxia in the absence (control condition; CON) and presence of a glycolytic inhibitor (1 mM iodoacetic acid; IAA). Intracellular pHi and tension were continuously recorded, and the differences in pHi between conditions were used to estimate the activation time of glycolysis. An immediate and steady increase in pHi (initial alkalosis) at the onset of contractions was similar between CON and IAA trials for the first 9 s of the contractile bout. However, from six contractions (approximately 10 s) throughout the remainder of the bout, IAA demonstrated a continued rise in pHi, in contrast to a progressive decrease in pHi in CON (P<0.05). These results demonstrate, with high temporal resolution, that glycolysis is activated within six contractions (10 s at 0.67 Hz) in single Xenopus skeletal muscle fibres.

HIF-1alpha in endurance training: suppression of oxidative metabolism.

During endurance training, exercising skeletal muscle experiences severe and repetitive oxygen stress. The primary transcriptional response factor for acclimation to hypoxic stress is hypoxia-inducible factor-1alpha (HIF-1alpha), which upregulates glycolysis and angiogenesis in response to low levels of tissue oxygenation. To examine the role of HIF-1alpha in endurance training, we have created mice specifically lacking skeletal muscle HIF-1alpha and subjected them to an endurance training protocol. We found that only wild-type mice improve their oxidative capacity, as measured by the respiratory exchange ratio; surprisingly, we found that HIF-1alpha null mice have already upregulated this parameter without training. Furthermore, untrained HIF-1alpha null mice have an increased capillary to fiber ratio and elevated oxidative enzyme activities. These changes correlate with constitutively activated AMP-activated protein kinase in the HIF-1alpha null muscles. Additionally, HIF-1alpha null muscles have decreased expression of pyruvate dehydrogenase kinase I, a HIF-1alpha target that inhibits oxidative metabolism. These data demonstrate that removal of HIF-1alpha causes an adaptive response in skeletal muscle akin to endurance training and provides evidence for the suppression of mitochondrial biogenesis by HIF-1alpha in normal tissue.

Effect of hypoxia on fatigue development in rat muscle composed of different fibre types.

This study investigated the relationship between hypoxia and the rate of fatigue development in contracting rat hindlimb muscles composed primarily of different fibre types. Hindlimb muscles of 11 rats were exposed, and the soleus (SOL) and gastrocnemius/plantaris (GP) were each isolated with circulation intact and attached to individual force transducers. Rats were then equilibrated with either normoxic (N; arterial partial pressure of O(2) 87.7 +/- 1.5 mmHg) or hypoxic conditions (H; arterial partial pressure of O(2) 30.0 +/- 2.4 mmHg) using an inspired O(2) fraction of 0.21 and 0.10, respectively. The stimulation protocol consisted of 2 min each at 0.125, 0.25, 0.33 and 0.5 tetanic contractions s(-1) sequentially for both conditions. Following the 8 min stimulation period, relative developed muscle tension (% of maximal) was nearly identical for both H and N in SOL (54.2 +/- 3.5 versus 54.3 +/- 4.2%), but was significantly (P < 0.05) lower in H than N (10.8 +/- 0.9 versus 43.0 +/- 8.9%) in GP, indicating a greater amount of fatigue during hypoxia only in the GP. Soleus phosphocreatine (PCr) content fell to similar levels (24.1 +/- 1.6 versus 21.1 +/- 4.9 mmol (kg dry weight (dw))(-1)) during both H and N, but in the white portion of the gastrocnemius (WG), PCr was significantly lower following H than N (14.3 +/- 1.5 versus 34.0 +/- 6.0 mmol (kg dw)(-1)). Similarly, muscle lactate increased in both fibre types at fatigue, but only in WG was the increase significantly greater with H (SOL 7.1 +/- 2.0 versus 5.3 +/- 1.1 mmol (kg dw)(-1); WG 13.7 +/- 4.5 versus 5.3 +/- 2.2 mmol (kg dw)(-1)). Increases in calculated muscle [H(+)], free ADP and free AMP were similar between N and H in SOL but were significantly greater during H compared with N in WG. These data demonstrate that hypoxia induces greater fatigue and disruption of cellular homeostasis in rat hindlimb muscle composed primarily of fibres with low oxidative capacity compared with those of a more oxidative type.

Intracellular PO2 kinetics at different contraction frequencies in Xenopus single skeletal muscle fibers.

Increasing contraction frequency in single skeletal muscle fibers has been shown to increase the magnitude of the fall in intracellular Po(2) (Pi(O(2))), reflecting a greater metabolic rate. To test whether Pi(O(2)) kinetics are altered by contraction frequency through this increase in metabolic stress, Pi(O(2)) was measured in Xenopus single fibers (n = 11) during and after contraction bouts at three different frequencies. Pi(O(2)) was measured via phosphorescence quenching at 0.16-, 0.25-, and 0.5-Hz tetanic stimulation. The kinetics of the change in Pi(O(2)) from resting baseline to end-contraction values and end contraction to rest were described as a mean response time (MRT) representing the time to 63% of the change in Pi(O(2)). As predicted, the fall in Pi(O(2)) from baseline following contractions was progressively greater at 0.5 and 0.25 Hz than at 0.16 Hz (32.8 +/- 2.1 and 29.3 +/- 2.0 Torr vs. 23.6 +/- 2.2 Torr, respectively) since metabolic demand was greater. The MRT for the decrease in Pi(O(2)) was progressively faster at the higher frequencies (0.5 Hz: 45.3 +/- 4.5 s; 0.25 Hz: 63.3 +/- 4.1 s; 0.16 Hz: 78.0 +/- 4.1 s), suggesting faster accumulation of stimulators of oxidative phosphorylation. The MRT for Pi(O(2)) off-kinetics (0.5 Hz: 84.0 +/- 11.7 s; 0.25 Hz: 79.1 +/- 8.4 s; 0.16 Hz: 81.1 +/- 8.3 s) was not different between trials. These data demonstrate in single fibers that the rate of the fall in Pi(O(2)) is dependent on contraction frequency, whereas the rate of recovery following contractions is independent of either the magnitude of the fall in Pi(O(2)) from baseline or the contraction frequency. This suggests that stimulation frequency plays an integral role in setting the initial metabolic response to work in isolated muscle fibers, possibly due to temporal recovery between contractions, but it does not determine recovery kinetics.

Continued divergence in VO2max of rats artificially selected for running endurance is mediated by greater convective blood O2 delivery.

We previously showed that after seven generations of artificial selection of rats for running capacity, maximal O2 uptake (VO2max) was 12% greater in high-capacity (HCR) than in low-capacity runners (LCR). This difference was due exclusively to a greater O2 uptake and utilization by skeletal muscle of HCR, without differences between lines in convective O2 delivery to muscle by the cardiopulmonary system (QO2max). The present study in generation 15 (G15) female rats tested the hypothesis that continuing improvement in skeletal muscle O2 transfer must be accompanied by augmentation in QO2max to support VO2max of HCR. Systemic O2 transport was studied during maximal normoxic and hypoxic exercise (inspired PO2 approximately 70 Torr). VO2max divergence between lines increased because of both improvement in HCR and deterioration in LCR: normoxic VO2max was 50% higher in HCR than LCR. The greater VO2max in HCR was accompanied by a 41% increase in QO2max: 96.1 +/- 4.0 in HCR vs. 68.1 +/- 2.5 ml stpd O2 x min(-1) x kg(-1) in LCR (P < 0.01) during normoxia. The greater G15 QO2max of HCR was due to a 48% greater stroke volume than LCR. Although tissue O2 diffusive conductance continued to increase in HCR, tissue O2 extraction was not significantly different from LCR at G15, because of the offsetting effect of greater HCR blood flow on tissue O2 extraction. These results indicate that continuing divergence in VO2max between lines occurs largely as a consequence of changes in the capacity to deliver O2 to the exercising muscle.

Nitric oxide synthase inhibition reduces O2 cost of force development and spares high-energy phosphates following contractions in pump-perfused rat hindlimb muscles.

The purpose of the present experiments was to test the hypotheses that: (i) nitric oxide synthase (NOS) inhibition reduces the O2 cost of force development across a range of contractile demands; and (ii) this reduced O2 cost of force development would be reflected in a sparing of intramuscular higher energy phosphates. Rat distal hindlimb muscles were pump perfused in situ and electrically stimulated (200 ms trains with pulses at 100 Hz, each pulse 0.05 ms duration) for 1 min each at 15, 30 and 60 tetani min(-1) and for 2 min at 90 tetani min(-1) in three groups: 0.01 mM adenosine; 1 mM D-NAME and 0.01 mM adenosine (D-NAME); and 1 mM L-NAME and 0.01 mM adenosine (L-NAME). The gastrocnemius-plantaris-soleus muscle group was freeze clamped post-contractions for metabolite analyses. Force was 19% higher and oxygen uptake (VO2) was 20% lower with L-NAME versus adenosine, and there was a 35% reduction in VO2/time-integrated tension versus adenosine and 24% versus D-NAME that was independent of contraction frequency. L-NAME treatment produced a 33% sparing of muscle phosphocreatine (PCr), and intramuscular lactate was no different between groups. In contrast, D-NAME reduced force by 30%, VO2 by 29% and the O2 cost of force development by 15% compared with adenosine, but had no effect on the degree of intramuscular ATP and PCr depletion. These results show that NOS inhibition improved the metabolic efficiency of force development, either by improving the ATP yield for a given O2 consumption or by reducing the ATP cost of force development. In addition, these effects were independent of contraction frequency.

Systemic oxygen transport in rats artificially selected for running endurance.

The relative contribution of genetic and environmental influences to individual exercise capacity is difficult to determine. Accordingly, animal models in which these influences are carefully controlled are highly useful to understand the determinants of intrinsic exercise capacity. Studies of systemic O(2) transport during maximal treadmill exercise in two diverging lines of rats artificially selected for endurance capacity showed that, at generation 7, whole body maximal O(2) uptake ((.)V(O(2)(max)) was 12% higher in high capacity (HCR) than in low capacity runners (LCR) during normoxic exercise. The difference in (.)V(O(2)(max) between HCR and LCR was larger during hypoxic exercise. Analysis of the linked O(2) conductances of the O(2) transport system showed that the higher (.)V(O(2)(max) was not due to a higher ventilatory response, a more effective pulmonary gas exchange, or an increased rate of O(2) delivery to the tissue by blood. The main reason for the higher (.)V(O(2)(max) of HCR was an increased tissue O(2) extraction, due largely to a higher tissue diffusive O(2) conductance. The enhanced tissue O(2) diffusing capacity was paralleled by an increased capillary density of a representative locomotory skeletal muscle, the gastrocnemius, in HCR. Activities of skeletal muscle oxidative enzymes citrate synthase and beta-HAD were also higher in HCR than LCR. Thus, the functional characteristics observed during exercise are consistent with the structural and biochemical changes observed in skeletal muscle that imply an enhanced capacity for muscle O(2) uptake and utilization in HCR. The results indicate that the improved (.)V(O(2)(max) is solely due to enhanced muscle O(2) extraction and utilization. However, the question arises as to whether it is possible to maintain a continually expanding capacity for O(2) extraction at the tissue level with successive generations, without a parallel improvement in the capacity to deliver O(2) to the exercising muscles.

Determinants of oxidative phosphorylation onset kinetics in isolated myocytes.

At the onset of constant-load exercise, pulmonary oxygen uptake (VO(2)) exhibits a monoexponential increase, following a brief time delay, to a new steady state. To date, the specific factors controlling VO(2) onset kinetics during the transition to higher rates of work remain largely unknown. To study the control of respiration in the absence of confounding factors such as blood flow heterogeneity and fiber type recruitment patterns, the onset kinetics of mitochondrial respiration were studied at the start of contractions in isolated single myocytes. Individual myocytes were microinjected with a porphyrin compound to allow phosphorescent measurement of intracellular PO(2) (P(i)O(2), an analog of VO(2)). Peak tension and P(i)O(2) were continuously monitored under a variety of conditions designed to test the role of work intensity, extracellular PO(2), cellular metabolites, and enzyme activation on the regulation of VO(2) onset kinetics.

Effects of nitric oxide synthase inhibition by L-NAME on oxygen uptake kinetics in isolated canine muscle in situ.

Nitric oxide (NO) has an inhibitory action on O2 uptake (VO2) at the level of the mitochondrial respiratory chain. The aim of this study was to evaluate the effects of NO synthase (NOS) inhibition on muscle (VO2) kinetics. Isolated canine gastrocnemius muscles in situ (n = 6) were studied during transitions from rest to 4-min of electrically stimulated contractions corresponding to approximately 60% of the muscle peak . Two conditions were compared: (i) Control (CTRL) and (ii) L-NAME, in which the NOS inhibitor L-NAME (20 mg kg(-1)) was administered. In both conditions the muscle was pump-perfused with constantly elevated blood flow (Q), at a level measured during a preliminary contraction trial with spontaneous self-perfused (Q). A vasodilatory drug was also infused. Arterial and venous O2 concentrations were determined at rest and at 5-7 s intervals during the transition. VO2 was calculated by Fick's principle. Muscle biopsies were obtained at rest and during contractions. Muscle force was measured continuously. Phosphocreatine hydrolysis and the calculated substrate level phosphorylation were slightly (but not significantly) lower in L-NAME than in CTRL. Significantly (P < 0.05) less fatigue was found in L-NAME versus CTRL. The time delay (TD(f)) and the time constant (tau(f)) of the 'fundamental' component of VO2 kinetics were not significantly different between CTRL (TD(f) 7.2 +/- 1.2 s; and tau(f) 10.6 +/- 1.3, +/- s.e.m.) and L-NAME (TD(f) 9.3 +/- 0.6; and tau(f) 10.4 +/- 1.0). Contrary to our hypothesis, NOS inhibition did not accelerate muscle VO2 kinetics. The down-regulation of mitochondrial respiration by NO does not limit the kinetics of adjustment of oxidative metabolism at exercise onset.

Caffeine administration results in greater tension development in previously fatigued canine muscle in situ.

In isolated single skeletal myocytes undergoing long-term fatiguing contractions, caffeine (CAF) can result in nearly immediate restoration of generated tension to near-prefatigue levels by increasing Ca2+ release via activation of sarcoplasmic reticulum release channels. This study tested whether arterial CAF infusion (>5 mm) would cause a similar rapid restoration of tetanic isometric tension during contractions to fatigue in perfused canine hindlimb muscle in situ. Tetanic contractions were elicited by electrical stimulation (200 ms trains, 50 Hz, 1 contraction s(-1)), and biopsies were taken from the muscle at rest and during contractions: (1) following the onset of fatigue (tension approximately 60% of initial value); and (2) following CAF administration. Resting muscle ATP, PCr and lactate contents were 25.2 +/- 0.4, 76.9 +/- 3.3 and 14.4 +/- 3.3 mmol (kg dry weight)(-1), respectively. At fatigue, generated tetanic tension was 61.1 +/- 6.9% of initial contractions. There was a small but statistically significant recovery of tetanic tension (64.9 +/- 6.6% of initial value) with CAF infusion, after which the muscle showed incomplete relaxation. At fatigue, muscle ATP and PCr contents had fallen significantly (P < 0.05) to 18.1 +/- 1.1 and 18.9 +/- 2.1 mmol (kg dry weight)(-1), respectively, and lactate content had increased significantly to 27.7 +/- 5.4 mmol (kg dry weight)(-1). Following CAF, skeletal muscle ATP and PCr contents were significantly lower than corresponding fatigue values (15.0 +/- 1.3 and 10.9 +/- 2.2 mmol (kg dry weight)(-1), respectively), while lactate was unchanged (22.2 +/- 3.9 mmol (kg dry weight)(-1)). These results demonstrate that caffeine can result in a small, but statistically significant, recovery of isometric tension in fatigued canine hindlimb muscle in situ, although not nearly to the same degree as seen in isolated single muscle fibres. This suggests that, in this in situ isolated whole muscle model, alteration of Ca2+ metabolism is probably only one cause of fatigue.

Relationship between intracellular PO2 recovery kinetics and fatigability in isolated single frog myocytes.

In single frog skeletal myocytes, a linear relationship exists between "fatigability" and oxidative capacity. The purpose of this investigation was to study the relationship between the intracellular Po(2) (Pi(O(2))) offset kinetics and fatigability in single Xenopus laevis myocytes to test the hypothesis that Pi(O(2)) offset kinetics would be related linearly with myocyte fatigability and, by inference, oxidative capacity. Individual myocytes (n = 30) isolated from lumbrical muscle were subjected to a 2-min bout of isometric peak tetanic contractions at either 0.25- or 0.33-Hz frequency while Pi(O(2)) was measured continuously via phosphorescence quenching techniques. The mean response time (MRT; time to 63% of the overall response) for Pi(O(2)) recovery from contracting values to resting baseline was calculated. After the initial square-wave constant-frequency contraction trial, each cell performed an incremental contraction protocol [i.e., frequency increase every 2 min from 0.167, 0.25, 0.33, 0.5, 1.0, and 2.0 Hz until peak tension fell below 50% of initial values (TTF)]. TTF values ranged from 3.39 to 10.04 min for the myocytes. The Pi(O(2)) recovery MRT ranged from 26 to 146 s. A significant (P < 0.05), negative relationship (MRT = -12.68TTF + 168.3, r(2) = 0.605) between TTF and Pi(O(2)) recovery MRT existed. These data demonstrate a significant correlation between fatigability and oxidative phosphorylation recovery kinetics consistent with the notion that oxidative capacity determines, in part, the speed with which skeletal muscle can recover energetically to alterations in metabolic demand.

Effect of contractile duration on intracellular PO2 kinetics in Xenopus single skeletal myocytes.

It has been suggested that skeletal muscle O(2) uptake (Vo(2)) kinetics follow a first-order control model. Consistent with that, Vo(2) should show both 1) similar onset kinetics and 2) an on-off symmetry across submaximal work intensities regardless of the metabolic perturbation. To date, consensus on this issue has not been reached in whole body studies due to numerous confounding factors associated with O(2) availability and fiber-type recruitment. To test whether single myocytes demonstrate similar intracellular Po(2) (Pi(O(2))) on- and off-transient kinetics at varying work intensities, we studied Xenopus laevis single myocyte (n = 8) Pi(O(2)) via phosphorescence quenching during two bouts of electrically induced isometric muscle contractions of 200 (low)- and 400 (high)-ms contraction duration (1 contraction every 4 s, 15 min between trials, order randomized). The fall in Pi(O(2)), which is inversely proportional to the net increase in Vo(2), was significantly greater (P < 0.05) during the high (24.1 +/- 3.2 Torr) vs. low (17.4 +/- 1.6 Torr) contraction bout. However, the mean response time (MRT; time to 63% of the overall change) for the fall in Pi(O(2)) from resting baseline to end contractions was not different (high, 77.8 +/- 11.5 vs. low, 76.1 +/- 13.6 s; P > 0.05) between trials. The initial rate of change at contraction onset, defined as DeltaPi(O(2))/MRT, was significantly greater (P < 0.05) in high compared with low. Pi(O(2)) off-transient MRT from the end of the contraction bout to initial baseline was unchanged (high, 83.3 +/- 18.3 vs. low, 80.4 +/- 21.6 s; P > 0.05) between high and low trials. These data revealed that Pi(O(2)) dynamics in frog isolated skeletal myocytes were invariant despite differing contraction durations and, by inference, metabolic demands. Thus these findings demonstrate that mitochondria can respond more rapidly at the initial onset of contractions when challenged with an augmented metabolic stimulus in accordance with an apparent first-order rate law.

Effects of acute creatine kinase inhibition on metabolism and tension development in isolated single myocytes.

This study investigated the effects of acute creatine kinase (CK) inhibition (CKi) on contractile performance, cytosolic Ca2+ concentration ([Ca2+]c), and intracellular PO2 (PIO2) in Xenopus laevis isolated myocytes during a 2-min bout of isometric tetanic contractions (0.33-Hz frequency). Peak tension was similar between trials during the first contraction but was significantly (P < 0.05) attenuated for all subsequent contractions in CKi vs. control (Con). The fall in PIO2 (DeltaPIO2) from resting values was significantly greater in Con (26.0 +/- 2.2 Torr) compared with CKi (17.8 +/- 1.8 Torr). However, the ratios of Con to CKi end-peak tension (1.53 +/- 0.11) and DeltaPO2 (1.49 +/- 0.11) were similar, suggesting an unaltered aerobic cost of contractions. Additionally, the mean response time (MRT) of DeltaPIO2was significantly faster in CKi vs. Con during both the onset (31.8 +/- 5.5 vs. 49.3 +/- 5.7 s; P < 0.05) and cessation (21.2 +/- 4.1 vs. 68.0 +/- 3.2 s; P < 0.001) of contractions. These data demonstrate that initial phosphocreatine hydrolysis in single skeletal muscle fibers is crucial for maintenance of sarcoplasmic reticulum Ca2+ release and peak tension during a bout of repetitive tetanic contractions. Furthermore, as PIO2 fell more rapidly at contraction onset in CKi compared with Con, these data suggest that CK activity temporally buffers the initial ATP-to-ADP concentration ratio at the transition to an augmented energetic demand, thereby slowing the initial mitochondrial activation by mitigating the energetic control signal (i.e., ADP concentration, phosphorylation potential, etc.) between sites of ATP supply and demand.