Mitochondrial Complex I Inhibition by Metformin: Drug-Exercise Interactions.

Metformin has antidiabetic, anticancer, and prolongevity effects, but seems to interfere with aerobic training mitochondrial adaptations. The primary mechanism of action has been suggested to be the inhibition of mitochondrial complex I. Recent papers (Wang et al. and Cameron et al.), however, provide evidence to deny the hypothesis of a direct action of metformin on complex I.

Muscle mass and inspired oxygen influence oxygen extraction at maximal exercise: Role of mitochondrial oxygen affinity.

AIM: We examined the Fick components together with mitochondrial O2 affinity (p50mito ) in defining O2 extraction and O2 uptake during exercise with large and small muscle mass during normoxia (NORM) and hyperoxia (HYPER). METHODS: Seven individuals performed 2 incremental exercise tests to exhaustion on a bicycle ergometer (BIKE) and 2 on a 1-legged knee extension ergometer (KE) in NORM or HYPER. Leg blood flow and VO2 were determined by thermodilution and the Fick method. Maximal ADP-stimulated mitochondrial respiration (OXPHOS) and p50mito were measured ex vivo in isolated mitochondria. Mitochondrial excess capacity in the leg was determined from OXPHOS in permeabilized fibres and muscle mass measured with magnetic resonance imaging in relation to peak leg O2 delivery. RESULTS: The ex vivo p50mito increased from 0.06 ± 0.02 to 0.17 ± 0.04 kPa with varying substrate supply and O2 flux rates from 9.84 ± 2.91 to 16.34 ± 4.07 pmol O2 ·s-1 ·µg-1 respectively. O2 extraction decreased from 83% in BIKE to 67% in KE as a function of a higher O2 delivery and lower mitochondrial excess capacity. There was a significant relationship between O2 extraction and mitochondrial excess capacity and p50mito that was unrelated to blood flow and mean transit time. CONCLUSION: O2 extraction varies with mitochondrial respiration rate, p50mito and O2 delivery. Mitochondrial excess capacity maintains a low p50mito which enhances O2 diffusion from microvessels to mitochondria during exercise.

Skeletal muscle IL-15/IL-15Rα and myofibrillar protein synthesis after resistance exercise.

In vitro and in vivo studies described the myokine IL-15 and its receptor IL-15Rα as anabolic/anti-atrophy agents, however, the protein expression of IL-15Rα has not been measured in human skeletal muscle and data regarding IL-15 expression remain inconclusive. The purpose of the study was to determine serum and skeletal muscle IL-15 and IL-15Rα responses to resistance exercise session and to analyze their association with myofibrillar protein synthesis (MPS). Fourteen participants performed a bilateral leg resistance exercise composed of four sets of leg press and four sets of knee extension at 75% 1RM to task failure. Muscle biopsies were obtained at rest, 0, 4 and 24 hours post-exercise and blood samples at rest, mid-exercise, 0, 0.3, 1, 2, 4 and 24 hours post-exercise. Serum IL-15 was increased by ~5.3-fold immediately post-exercise, while serum IL-15Rα decreased ~75% over 1 hour post-exercise (P<.001). Skeletal muscle IL-15Rα mRNA and protein expression were increased at 4 hours post-exercise by ~2-fold (P<.001) and ~1.3-fold above rest (P=.020), respectively. At 24 hours post-exercise, IL-15 (P=.003) and IL-15Rα mRNAs increased by ~2-fold (P=.002). Myofibrillar fractional synthetic rate between 0-4 hours was associated with IL-15Rα mRNA at rest (r=.662, P=.019), 4 hours (r=.612, P=.029), and 24 hours post-exercise (r=.627, P=.029). Finally, the muscle IL-15Rα protein up-regulation was related to Leg press 1RM (r=.688, P=.003) and total weight lifted (r=.628, P=.009). In conclusion, IL-15/IL-15Rα signaling pathway is activated in skeletal muscle in response to a session of resistance exercise.

Exercise-mediated modulation of autophagy in skeletal muscle.

Although exercise exerts multiple beneficial health effects, it may also damage cellular structures. Damaged elements are continuously degraded and its constituents recycled to produce renovated structures through a process called autophagy, which is essential for the adaptation to training. Autophagy is particularly active in skeletal muscle, where it can be evaluated using specific molecular markers of activation (unc-51-like kinase 1 [ULK1] phosphorylation) and specific proteins indicating increased autophagosome content (increased total LC3, LC3-II, LC3-II/LC3-I ratio). Studies in humans are technically limited but have provided evidence suggesting the activation of autophagy in skeletal muscle through AMP-activated protein kinase (AMPK) and its downstream target ULK1. Autophagy activation is more likely when the intensity is elevated and the exercise performed in the fasted state. The autophagy-gene program and autophagosome content are upregulated after ultraendurance running competitions. However, autophagosome content is reduced after endurance exercise at moderate intensities (50% and 70% of VO2 max) for 60-120 minutes. Autophagosome content is decreased within the first few hours after resistance training. The effects of regular endurance and strength training on basal autophagy remain to be established in humans. One study has reported that acute severe hypoxia increases autophagosome content in human skeletal muscle, which is reverted by 20 minutes of low-intensity exercise. Experiments with transgenic mice have shown that autophagy is necessary for skeletal muscle adaptation to training. Little is known on how genetic factors, environment, nutrition, drugs and diseases may interact with exercise to modulate autophagy at rest and during exercise in humans.

Effects of velocity loss during resistance training on athletic performance, strength gains and muscle adaptations.

We compared the effects of two resistance training (RT) programs only differing in the repetition velocity loss allowed in each set: 20% (VL20) vs 40% (VL40) on muscle structural and functional adaptations. Twenty-two young males were randomly assigned to a VL20 (n = 12) or VL40 (n = 10) group. Subjects followed an 8-week velocity-based RT program using the squat exercise while monitoring repetition velocity. Pre- and post-training assessments included: magnetic resonance imaging, vastus lateralis biopsies for muscle cross-sectional area (CSA) and fiber type analyses, one-repetition maximum strength and full load-velocity squat profile, countermovement jump (CMJ), and 20-m sprint running. VL20 resulted in similar squat strength gains than VL40 and greater improvements in CMJ (9.5% vs 3.5%, P < 0.05), despite VL20 performing 40% fewer repetitions. Although both groups increased mean fiber CSA and whole quadriceps muscle volume, VL40 training elicited a greater hypertrophy of vastus lateralis and intermedius than VL20. Training resulted in a reduction of myosin heavy chain IIX percentage in VL40, whereas it was preserved in VL20. In conclusion, the progressive accumulation of muscle fatigue as indicated by a more pronounced repetition velocity loss appears as an important variable in the configuration of the resistance exercise stimulus as it influences functional and structural neuromuscular adaptations.

Androgen receptor gene polymorphism influence fat accumulation: A longitudinal study from adolescence to adult age.

To determine the influence of androgen receptor CAG and GGN repeat polymorphisms on fat mass and maximal fat oxidation (MFO), CAG and GGN repeat lengths were measured in 128 young boys, from which longitudinal data were obtained in 45 of them [mean ± SD: 12.8 ± 3.6 years old at recruitment, and 27.0 ± 4.8 years old at adult age]. Subjects were grouped as CAG short (CAGS ) if harboring repeat lengths ≤ 21, the rest as CAG long (CAGL ); and GGN short (GGNS ) if GGN repeat lengths ≤ 23, or long if > 23 (GGNL ). CAGS and GGNS were associated with lower adiposity than CAGL or GGNL (P < 0.05). There was an association between the logarithm of CAG repeats polymorphism and the changes of body mass (r = 0.34, P = 0.03). At adult age, CAGS men showed lower accumulation of total body and trunk fat mass, and lower resting metabolic rate (RMR) and MFO per kg of total lean mass compared with CAGL (P < 0.05). GGNS men also showed lower percentage of body fat (P < 0.05). In summary, androgen receptor CAG and GGN repeat polymorphisms are associated with RMR, MFO, fat mass, and its regional distribution in healthy male adolescents, influencing fat accumulation from adolescence to adult age.

A new equation to estimate temperature-corrected PaCO2 from PET CO2 during exercise in normoxia and hypoxia.

End-tidal PCO2 (PET CO2 ) has been used to estimate arterial pressure CO2 (Pa CO2 ). However, the influence of blood temperature on the Pa CO2 has not been taken into account. Moreover, there is no equation validated to predict Pa CO2 during exercise in severe acute hypoxia. To develop a new equation to predict temperature-corrected Pa CO2 values during exercise in normoxia and severe acute hypoxia, 11 volunteers (21.2 ± 2.1 years) performed incremental exercise to exhaustion in normoxia (Nox, PI O2 : 143 mmHg) and hypoxia (Hyp, PI O2 : 73 mmHg), while arterial blood gases and temperature (ABT) were simultaneously measured together with end-tidal PCO2 (PET CO2 ). The Jones et al. equation tended to underestimate the temperature corrected (tc) Pa CO2 during exercise in hypoxia, with greater deviation the lower the Pa CO2 tc (r = 0.39, P < 0.05). The new equation has been developed using a random-effects regression analysis model, which allows predicting Pa CO2 tc both in normoxia and hypoxia: Pa CO2 tc = 8.607 + 0.716 × PET CO2 [R(2) = 0.91; intercept SE = 1.022 (P < 0.001) and slope SE = 0.027 (P < 0.001)]. This equation may prove useful in noninvasive studies of brain hemodynamics, where an accurate estimation of Pa CO2 is needed to calculate the end-tidal-to-arterial PCO2 difference, which can be used as an index of pulmonary gas exchange efficiency.

Constant infusion transpulmonary thermodilution for the assessment of cardiac output in exercising humans.

To determine the accuracy and precision of constant infusion transpulmonary thermodilution cardiac output (CITT-Q) assessment during exercise in humans, using indocyanine green (ICG) dilution and bolus transpulmonary thermodilution (BTD) as reference methods, cardiac output (Q) was determined at rest and during incremental one- and two-legged pedaling on a cycle ergometer, and combined arm cranking with leg pedaling to exhaustion in 15 healthy men. Continuous infusions of iced saline in the femoral vein (n = 41) or simultaneously in the femoral and axillary (n = 66) veins with determination of temperature in the femoral artery were used for CITT-Q assessment. CITT-Q was linearly related to ICG-Q (r = 0.82, CITT-Q = 0.876 × ICG-Q + 3.638, P < 0.001; limits of agreement ranging from -1.43 to 3.07 L/min) and BTD-Q (r = 0.91, CITT-Q = 0.822 × BTD + 4.481 L/min, P < 0.001; limits of agreement ranging from -1.01 to 2.63 L/min). Compared with ICG-Q and BTD-Q, CITT-Q overestimated cardiac output by 1.6 L/min (≈ 10% of the mean ICG and BTD-Q values, P < 0.05). For Q between 20 and 28 L/min, we estimated an overestimation < 5%. The coefficient of variation of 23 repeated CITT-Q measurements was 6.0% (CI: 6.1-11.1%). In conclusion, cardiac output can be precisely and accurately determined with constant infusion transpulmonary thermodilution in exercising humans.

Mitochondrial coupling and capacity of oxidative phosphorylation in skeletal muscle of Inuit and Caucasians in the arctic winter.

During evolution, mitochondrial DNA haplogroups of arctic populations may have been selected for lower coupling of mitochondrial respiration to ATP production in favor of higher heat production. We show that mitochondrial coupling in skeletal muscle of traditional and westernized Inuit habituating northern Greenland is identical to Danes of western Europe haplogroups. Biochemical coupling efficiency was preserved across variations in diet, muscle fiber type, and uncoupling protein-3 content. Mitochondrial phenotype displayed plasticity in relation to lifestyle and environment. Untrained Inuit and Danes had identical capacities to oxidize fat substrate in arm muscle, which increased in Danes during the 42 days of acclimation to exercise, approaching the higher level of the Inuit hunters. A common pattern emerges of mitochondrial acclimatization and evolutionary adaptation in humans at high latitude and high altitude where economy of locomotion may be optimized by preservation of biochemical coupling efficiency at modest mitochondrial density, when submaximum performance is uncoupled from VO2max and maximum capacities of oxidative phosphorylation.

Maintained peak leg and pulmonary VO2 despite substantial reduction in muscle mitochondrial capacity.

We recently reported the circulatory and muscle oxidative capacities of the arm after prolonged low-intensity skiing in the arctic (Boushel et al., 2014). In the present study, leg VO2 was measured by the Fick method during leg cycling while muscle mitochondrial capacity was examined on a biopsy of the vastus lateralis in healthy volunteers (7 male, 2 female) before and after 42 days of skiing at 60% HR max. Peak pulmonary VO2 (3.52 ± 0.18 L.min(-1) pre vs 3.52 ± 0.19 post) and VO2 across the leg (2.8 ± 0.4L.min(-1) pre vs 3.0 ± 0.2 post) were unchanged after the ski journey. Peak leg O2 delivery (3.6 ± 0.2 L.min(-1) pre vs 3.8 ± 0.4 post), O2 extraction (82 ± 1% pre vs 83 ± 1 post), and muscle capillaries per mm(2) (576 ± 17 pre vs 612 ± 28 post) were also unchanged; however, leg muscle mitochondrial OXPHOS capacity was reduced (90 ± 3 pmol.sec(-1) .mg(-1) pre vs 70 ± 2 post, P < 0.05) as was citrate synthase activity (40 ± 3 µmol.min(-1) .g(-1) pre vs 34 ± 3 vs P < 0.05). These findings indicate that peak muscle VO2 can be sustained with a substantial reduction in mitochondrial OXPHOS capacity. This is achieved at a similar O2 delivery and a higher relative ADP-stimulated mitochondrial respiration at a higher mitochondrial p50. These findings support the concept that muscle mitochondrial respiration is submaximal at VO2max , and that mitochondrial volume can be downregulated by chronic energy demand.

Central and peripheral hemodynamics in exercising humans: leg vs arm exercise.

In humans, arm exercise is known to elicit larger increases in arterial blood pressure (BP) than leg exercise. However, the precise regulation of regional vascular conductances (VC) for the distribution of cardiac output with exercise intensity remains unknown. Hemodynamic responses were assessed during incremental upright arm cranking (AC) and leg pedalling (LP) to exhaustion (Wmax) in nine males. Systemic VC, peak cardiac output (Qpeak) (indocyanine green) and stroke volume (SV) were 18%, 23%, and 20% lower during AC than LP. The mean BP, the rate-pressure product and the associated myocardial oxygen demand were 22%, 12%, and 14% higher, respectively, during maximal AC than LP. Trunk VC was reduced to similar values at Wmax. At Wmax, muscle mass-normalized VC and fractional O2 extraction were lower in the arm than the leg muscles. However, this was compensated for during AC by raising perfusion pressure to increase O2 delivery, allowing a similar peak VO2 per kg of muscle mass in both extremities. In summary, despite a lower Qpeak during arm cranking the cardiovascular strain is much higher than during leg pedalling. The adjustments of regional conductances during incremental exercise to exhaustion depend mostly on the relative intensity of exercise and are limb-specific.

Erythropoietin does not reduce plasma lactate, H⁺, and K⁺ during intense exercise.

It is investigated if recombinant human erythropoietin (rHuEPO) treatment for 15 weeks (n = 8) reduces extracellular accumulation of metabolic stress markers such as lactate, H(+) , and K(+) during incremental exhaustive exercise. After rHuEPO treatment, normalization of blood volume and composition by hemodilution preceded an additional incremental test. Group averages were calculated for an exercise intensity ∼80% of pre-rHuEPO peak power output. After rHuEPO treatment, leg lactate release to the plasma compartment was similar to before (4.3 ± 1.6 vs 3.9 ± 2.5 mmol/min) and remained similar after hemodilution. Venous lactate concentration was higher (P < 0.05) after rHuEPO treatment (7.1 ± 1.6 vs 5.2 ± 2.1 mM). Leg H(+) release to the plasma compartment after rHuEPO was similar to before (19.6 ± 5.4 vs 17.6 ± 6.0 mmol/min) and remained similar after hemodilution. Nevertheless, venous pH was lower (P < 0.05) after rHuEPO treatment (7.18 ± 0.04 vs 7.22 ± 0.05). Leg K(+) release to the plasma compartment after rHuEPO treatment was similar to before (0.8 ± 0.5 vs 0.7 ± 0.7 mmol/min) and remained similar after hemodilution. Additionally, venous K(+) concentrations were similar after vs before rHuEPO (5.3 ± 0.3 vs 5.1 ± 0.4 mM). In conclusion, rHuEPO does not reduce plasma accumulation of lactate, H(+) , and K(+) at work rates corresponding to ∼80% of peak power output.

A time-efficient reduction of fat mass in 4 days with exercise and caloric restriction.

To determine whether a fast reduction in fat mass can be achieved in 4 days by combining caloric restriction (CR: 3.2 kcal/kg body weight per day) with exercise (8-h walking + 45-min arm cranking per day) to induce an energy deficit of ∼5000 kcal/day, 15 overweight men underwent five experimental phases: pretest, exercise + CR for 4 days (WCR), control diet + reduced exercise for 3 days (DIET), and follow-up 4 weeks (POST1) and 1 year later (POST2). During WCR, the diet consisted solely of whey protein (n = 8) or sucrose (n = 7) (0.8 g/kg body weight per day). After WCR, DIET, POST1, and POST2, fat mass was reduced by a mean of 2.1, 2.8, 3.8, and 1.9 kg (P < 0.05), with two thirds of this loss from the trunk; and lean mass by 2.8, 1.0, 0.5, and 0.4 kg, respectively. After WCR, serum glucose, insulin, homeostatic model assessment, total and low-density lipoprotein cholesterol and triglycerides were reduced, and free fatty acid and cortisol increased. Serum leptin was reduced by 64%, 50%, and 33% following WCR, DIET, and POST1, respectively (P < 0.05). The effects were similar in both groups. In conclusion, a clinically relevant reduction in fat mass can be achieved in overweight men in just 4 days by combining prolonged exercise with CR.

Chronic hypoxia increases arterial blood pressure and reduces adenosine and ATP induced vasodilatation in skeletal muscle in healthy humans.

AIMS: To determine the role played by adenosine, ATP and chemoreflex activation on the regulation of vascular conductance in chronic hypoxia. METHODS: The vascular conductance response to low and high doses of adenosine and ATP was assessed in ten healthy men. Vasodilators were infused into the femoral artery at sea level and then after 8-12 days of residence at 4559 m above sea level. At sea level, the infusions were carried out while the subjects breathed room air, acute hypoxia (FI O2 = 0.11) and hyperoxia (FI O2 = 1); and at altitude (FI O2 = 0.21 and 1). Skeletal muscle P2Y2 receptor protein expression was determined in muscle biopsies after 4 weeks at 3454 m by Western blot. RESULTS: At altitude, mean arterial blood pressure was 13% higher (91 ± 2 vs. 102 ± 3 mmHg, P < 0.05) than at sea level and was unaltered by hyperoxic breathing. Baseline leg vascular conductance was 25% lower at altitude than at sea level (P < 0.05). At altitude, the high doses of adenosine and ATP reduced mean arterial blood pressure by 9-12%, independently of FI O2 . The change in vascular conductance in response to ATP was lower at altitude than at sea level by 24 and 38%, during the low and high ATP doses respectively (P < 0.05), and by 22% during the infusion with high adenosine doses. Hyperoxic breathing did not modify the response to vasodilators at sea level or at altitude. P2Y2 receptor expression remained unchanged with altitude residence. CONCLUSIONS: Short-term residence at altitude increases arterial blood pressure and reduces the vasodilatory responses to adenosine and ATP.

Influence of age on leptin induced skeletal muscle signalling.

AIM: Age associated fat mass accumulation could be because of dysregulation of leptin signalling in skeletal muscle. Thus, we investigated total protein expression and phosphorylation levels of the long isoform of the leptin receptor (OB-Rb), and leptin signalling through janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3), insulin receptor substrate 1 (IRS-1), AMP-activated protein kinase (AMPK) and acetyl-coenzyme A carboxylase (ACC), combined with the leptin signalling inhibitors suppressor of cytokine signalling 3 (SOCS3) and protein tyrosine phosphatase 1B (PTP1B) in human skeletal muscle of different age. METHODS: Vastus lateralis muscle biopsies were obtained from 39 men matched for BMI < 30 kg m(-2) and separated into three groups: 13 young (Y, 24 ± 4 years); 14 middle aged (MA, 44 ± 5 years) and 12 aged (A, 58 ± 8 years) subjects. RESULTS: Whole body fat percentage and plasma leptin were higher (P < 0.05), whereas lean mass, plasma free testosterone and total testosterone were lower (P < 0.05) in A compared to Y. Skeletal muscle OB-Rb (170 KDa) protein expression and pTyr(1141) -OB-R170 were comparable between groups, whereas pTyr(985) -OB-R170 was lower in A compared to Y (P < 0.05). pSTAT3 levels tended (P = 0.09) to be lower (50%) in A compared to Y. In A, muscle PTP1B was greater and IRS-1 lower than Y and MA respectively (P < 0.05). PTyr(612) -IRS-1 tended to be lower in A than in Y (P = 0.09). Suppressor of cytokine signalling 3 (SOCS3) protein expression, pJAK2, pSer(1101) -IRS-1, pAMPKα and pACCß were similar between groups. CONCLUSION: Age is associated with dysregulation of the leptin signalling and increased PTP1B protein expression in skeletal muscle.

Low-intensity training increases peak arm VO2 by enhancing both convective and diffusive O2 delivery.

AIM: It is an ongoing discussion the extent to which oxygen delivery and oxygen extraction contribute to an increased muscle oxygen uptake during dynamic exercise. It has been proposed that local muscle factors including the capillary bed and mitochondrial oxidative capacity play a large role in prolonged low-intensity training of a small muscle group when the cardiac output capacity is not directly limiting. The purpose of this study was to investigate the relative roles of circulatory and muscle metabolic mechanisms by which prolonged low-intensity exercise training alters regional muscle VO2 . METHODS: In nine healthy volunteers (seven males, two females), haemodynamic and metabolic responses to incremental arm cycling were measured by the Fick method and biopsy of the deltoid and triceps muscles before and after 42 days of skiing for 6 h day(-1) at 60% max heart rate. RESULTS: Peak pulmonary VO2 during arm crank was unchanged after training (2.38 ± 0.19 vs. 2.18 ± 0.2 L min(-1) pre-training) yet arm VO2 (1.04 ± 0.08 vs. 0.83 ± 0.1 L min(1) , P < 0.05) and power output (137 ± 9 vs. 114 ± 10 Watts) were increased along with a higher arm blood flow (7.9 ± 0.5 vs. 6.8 ± 0.6 L min(-1) , P < 0.05) and expanded muscle capillary volume (76 ± 7 vs. 62 ± 4 mL, P < 0.05). Muscle O2 diffusion capacity (16.2 ± 1 vs. 12.5 ± 0.9 mL min(-1) mHg(-1) , P < 0.05) and O2 extraction (68 ± 1 vs. 62 ± 1%, P < 0.05) were enhanced at a similar mean capillary transit time (569 ± 43 vs. 564 ± 31 ms) and P50 (35.8 ± 0.7 vs. 35 ± 0.8), whereas mitochondrial O2 flux capacity was unchanged (147 ± 6 mL kg min(-1) vs. 146 ± 8 mL kg min(-1) ). CONCLUSION: The mechanisms underlying the increase in peak arm VO2 with prolonged low-intensity training in previously untrained subjects are an increased convective O2 delivery specifically to the muscles of the arm combined with a larger capillary-muscle surface area that enhance diffusional O2 conductance, with no apparent role of mitochondrial respiratory capacity.

A reliable unipedal stance test for the assessment of balance using a force platform.

BACKGROUND: The aim was to develop a unipedal stance test for the assessment of balance using a force platform. METHODS: A single-leg balance test was conducted in 23 students (mean ± SD) age: 23 ± 3 years) in a standard position limiting the movement of the arms and non-supporting leg. Six attempts, with both the jumping (JL) and the contralateral leg (CL), were performed under 3 conditions: 1) eyes opened; 2) eyes closed; 3) eyes opened and executing a precision task. The same protocol was repeated two-week apart. RESULTS: The mean and the best result of the six attempts performed each day were taken as representative of balance. The speed of the centre of pressure (CP-Speed) showed excellent reliability for the "best result" analysis in all tests (ICCs 0.87-0.97), except in the test with the eyes closed performed on the CL (ICC<0.4). The CP-Speed had better reliability with the "best result" than with the "mean result" analysis (P<0.05), whilst no significant differences were observed between the JL and the CL (P=0.71 and P=0.96 for mean and best results analysis, respectively). A lower dispersion in the Bland and Altman graph was observed with the eyes opened than closed, and the dynamic test. CONCLUSION: The single-leg stance balance test proposed is a reliable method to assess balance, especially when performed in a static position, with the eyes opened and using the best result of six attempts as reference, independently of the stance leg.