Assessment of mitochondrial ADP-stimulated respiratory kinetics in permeabilized skeletal myofibres (PmFB) is increasingly used in clinical diagnostic and basic research settings. However, estimates of the Km for ADP vary considerably (∼20-300 μM) and tend to overestimate respiration at rest. Noting PmFBs spontaneously contract during respiration experiments, we systematically determined the impact of contraction, temperature and oxygenation on ADP-stimulated respiratory kinetics. Blebbistatin (BLEB), a myosin II ATPase inhibitor, blocked contraction under all conditions and yielded high Km values for ADP of >∼250 and ∼80 μM in red and white rat PmFB, respectively. In the absence of BLEB, PmFB contracted and the Km for ADP decreased by ∼2 to 10-fold in a temperature-dependent manner. PmFB were sensitive to hyperoxia (increased Km) in the absence of BLEB (contracted) at 30°C but not 37°C. In PmFB from humans, contraction elicited high sensitivity to ADP (m <100 μM) whereas blocking contraction (+BLEB) and including PCr:Cr = 2 to mimic the resting energetic state yielded a Km for ADP = ∼1560 μM, consistent with estimates of in vivo resting respiratory rates of <1% maximum. These results demonstrate the sensitivity of muscle to ADP varies over a wide range in relation to contractile state and cellular energy charge, providing evidence that enzymatic coupling of energy transfer within skeletal muscle becomes more efficient in the working state.

Maximal O2 delivery and O2 uptake (VO2) per 100 g of active muscle mass are far greater during knee extensor (KE) than during cycle exercise: 73 and 60 ml. min-1. 100 g-1 (2.4 kg of muscle) (R. S. Richardson, D. R. Knight, D. C. Poole, S. S. Kurdak, M. C. Hogan, B. Grassi, and P. D. Wagner. Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H1453-H1461, 1995) and 28 and 25 ml. min-1. 100 g-1 (7.5 kg of muscle) (D. R. Knight, W. Schaffartzik, H. J. Guy, R. Predilleto, M. C. Hogan, and P. D. Wagner. J. Appl. Physiol. 75: 2586-2593, 1993), respectively. Although this is evidence of muscle O2 supply dependence in itself, it raises the following question: With such high O2 delivery in KE, are the quadriceps still O2 supply dependent at maximal exercise? To answer this question, seven trained subjects performed maximum KE exercise in hypoxia [0.12 inspired O2 fraction (FIO2)], normoxia (0.21 FIO2), and hyperoxia (1.0 FIO2) in a balanced order. The protocol (after warm-up) was a square wave to a previously determined maximum work rate followed by incremental stages to ensure that a true maximum was achieved under each condition. Direct measures of arterial and venous blood O2 concentration in combination with a thermodilution blood flow technique allowed the determination of O2 delivery and muscle VO2. Maximal O2 delivery increased with inspired O2: 1.3 +/- 0.1, 1.6 +/- 0.2, and 1.9 +/- 0.2 l/min at 0.12, 0.21, and 1.0 FIO2, respectively (P < 0.05). Maximal work rate was affected by variations in inspired O2 (-25 and +14% at 0.12 and 1.0 FIO2, respectively, compared with normoxia, P < 0.05) as was maximal VO2 (VO2 max): 1.04 +/- 0.13, 1. 24 +/- 0.16, and 1.45 +/- 0.19 l/min at 0.12, 0.21, and 1.0 FIO2, respectively (P < 0.05). Calculated mean capillary PO2 also varied with FIO2 (28.3 +/- 1.0, 34.8 +/- 2.0, and 40.7 +/- 1.9 Torr at 0.12, 0.21, and 1.0 FIO2, respectively, P < 0.05) and was proportionally related to changes in VO2 max, supporting our previous finding that a decrease in O2 supply will proportionately decrease muscle VO2 max. As even in the isolated quadriceps (where normoxic O2 delivery is the highest recorded in humans) an increase in O2 supply by hyperoxia allows the achievement of a greater VO2 max, we conclude that, in normoxic conditions of isolated KE exercise, KE VO2 max in trained subjects is not limited by mitochondrial metabolic rate but, rather, by O2 supply.

We studied six patients with chronic obstructive pulmonary disease (COPD) (FEV1 = 1.1 +/- 0.2 L, 32% of predicted) and six age- and activity level-matched control subjects while performing both maximal bicycle exercise and single leg knee-extensor exercise. Arterial and femoral venous blood sampling, thermodilution blood flow measurements, and needle biopsies allowed the assessment of muscle oxygen supply, utilization, and structure. Maximal work rates and single leg VO2max (control subjects = 0.63 +/- 0.1; patients with COPD = 0.37 +/- 0.1 L/minute) were significantly greater in the control group during bicycle exercise. During knee-extensor exercise this difference in VO2max disappeared, whereas maximal work capacity was reduced (flywheel resistance: control subjects = 923 +/- 198; patients with COPD = 612 +/- 81 g) revealing a significantly reduced mechanical efficiency (work per unit oxygen consumed) with COPD. The patients had an elevated number of less efficient type II muscle fibers, whereas muscle fiber cross-sectional areas, capillarity, and mitochondrial volume density were not different between the groups. Therefore, although metabolic capacity per se is unchanged, fiber type differences associated with COPD may account for the reduced muscular mechanical efficiency that becomes clearly apparent during knee-extensor exercise, when muscle function is no longer overshadowed by the decrement in lung function.

Recently, we observed that muscle capillarization, vascular endothelial growth factor (VEGF) protein, and the VEGF mRNA response to acute exercise were lower in aged compared with young women (Croley AN, Zwetsloot KA, Westerkamp LM, Ryan NA, Pendergast aged men, Hickner RC, Pofahl WE, and Gavin TP. J Appl Physiol 99: 1875-1882, 2005). We hypothesized that similar age-related differences in muscle capillarization and VEGF expression would exist between young and aged men. Skeletal muscle biopsies were obtained from the vastus lateralis before and at 4 h after a submaximal exercise bout for the measurement of morphometry, capillarization, VEGF, KDR, and Flt-1 in seven aged (mean age 65 yr) and eight young (mean age 21 yr) sedentary men. In aged compared with young men, muscle capillary contacts and capillary-to-fiber perimeter exchange index were lower regardless of fiber type. Muscle VEGF mRNA and protein were lower in aged men both at rest and 4 h postexercise. Exercise increased muscle VEGF mRNA and protein and KDR mRNA independent of age group. There were no effects of exercise or age on muscle Flt-1 mRNA or protein or KDR protein. These results confirm that skeletal muscle capillarization and VEGF expression are lower in aged compared with young men.

The skeletal muscle capillary supply is an important determinant of maximum exercise capacity, and it is well known that endurance exercise training increases the muscle capillary supply. The muscle capillary supply and exercise-induced angiogenesis are regulated in part by vascular endothelial growth factor (VEGF). VEGF is produced by skeletal muscle cells and can be secreted into the circulation. We investigated whether there are differences in circulating plasma VEGF between sedentary individuals (Sed) and well-trained endurance athletes (ET) at rest or in response to acute exercise. Eight ET men (maximal oxygen consumption: 63.8 +/- 2.3 ml x kg(-1) x min(-1); maximum power output: 409.4 +/- 13.3 W) and eight Sed men (maximal oxygen consumption: 36.3 +/- 2.1 ml x kg(-1) x min(-1); maximum power output: 234.4 +/- 13.3 W) exercised for 1 h at 50% of maximum power output. Antecubital vein plasma was collected at rest and at 0, 2, and 4 h postexercise. Plasma VEGF was measured by ELISA analysis. Acute exercise significantly increased VEGF at 0 and 2 h postexercise in ET subjects but did not increase VEGF at any time point in Sed individuals. There was no difference in VEGF between ET and Sed subjects at any time point. When individual peak postexercise VEGF was analyzed, exercise did increase VEGF independent of training status. In conclusion, exercise can increase plasma VEGF in both ET athletes and Sed men; however, there is considerable variation in the individual time of the peak VEGF response.