Deletion of osteopontin or bone sialoprotein induces opposite bone responses to mechanical stimulation in mice.

Osteopontin (OPN) and Bone Sialoprotein (BSP) are co-expressed in bone and display overlapping and complementary physiological properties. Both genes show a rapid expression response to mechanical stimulation. We used mice with single and double deletions (DKO) of BSP and OPN to assess the specificity of their roles in skeletal adaptation to loading. Two-month-old Wild-Type (WT), BSP knockout (BSP-/-), OPN-/- and DKO male mice were submitted to two mechanical stimulation regimen (n = 10 mice/group) respectively impacting trabecular bone (Hypergravity, HG) and cortical bone (Whole Body Vibration, WBV). HG increased trabecular bone volume (BV/TV) in WT femur through reduced resorption, and in BSP-/- mice femur and vertebra through increased bone formation. In contrast, HG increased the turnover of OPN-/- bone, resulting in reduced femur and vertebra BV/TV. HG did not affect DKO bones. Similarly, WBV increased cortical thickness in BSP-/- mice and decreased it in OPN-/-, without affecting structurally WT and DKO bone. Vibrated BSP-/- mice displayed increased endocortical bone formation with a drop in Sclerostin expression, and reduced periosteal osteoclasts with lower Rankl and Cathepsin K expression. In contrast, vibrated OPN-/- endocortical bone displayed decreased formation and increased osteoclast coverage. Therefore, under two regimen (HG and WBV) targeting distinct bone compartments, absence of OPN resulted in bone loss while lack of BSP induced bone gain, reflecting divergent structural adaptations. Strikingly, absence of both proteins led to a relative insensitivity to either mechanical challenge. Interplay between OPN and BSP thus appears as a key element of skeletal response to mechanical stimulation.

Apatite content of collagen materials dose-dependently increases pre-osteoblastic cell deposition of a cement line-like matrix.

Bone matrix, mainly composed of type I collagen and apatite, is constantly modified during the bone remodeling process, which exposes bone cells to various proportions of mineralized collagen within bone structural units. Collagen-mineralized substrates have been shown to increase osteoblast activities. We hypothesized that such effects may be explained by a rapid secretion of specific growth factors and/or deposition of specific matrix proteins. Using MC3T3-E1 seeded for 32h on collagen substrates complexed with various apatite contents, we found that pre-osteoblasts in contact with mineralized collagen gave rise to a dose-dependent deposit of Vascular Endothelial Growth Factor-A (VEGF-A) and RGD-containing proteins such as osteopontin (OPN) and fibronectin (FN). This RGD-matrix deposition reinforced the cell adhesion to collagen-mineralized substrates. It was also observed that, on these substrates, this matrix was elaborated concomitantly to an increased cell migration, allowing a homogeneous coverage of the sample. This particular surface activation was probably done firstly to reinforce cell survival (VEGF-A) and adhesion (OPN, FN) and secondly to recruit and prepare surfaces for subsequent bone cell activity.

CC-chemokine receptor five gene polymorphism in primary IgA nephropathy: the 32 bp deletion allele is associated with late progression to end-stage renal failure with dialysis.

The chemokine (CK) receptor 5 (CCR5) is necessary for two adjacent cysteines (CC)-CKs such as Regulated upon Activation Normal T cell Expressed and Secreted, a/o Macrophage Inflammatory Protein 1alpha/beta to mediate their inflammatory properties. The CCR5 gene polymorphism with 32-basepair deletion (d32) leads to receptor inactivation/dysfunction in homo/heterozygous individuals. We have evaluated its role in both initiation and/or progression of primary immunoglobulin A (IgA) nephropathy (IGAN) in a case-control study involving a prospective cohort of 318 IGAN patients and a matched group of 294 controls. Genotyping was performed by a two-specific primers single polymerase chain reaction technique: normal allele (nl) vs d32 allele. The d32 allele frequency was not different in patients (11.0%) vs controls (8.3%), indicating no significant influence on IGAN initiation. Genotype to clinical phenotype correlation demonstrated that progression to renal/patient death was associated with the d32 allele: 18.2% (12 out of 66 with d32) vs 8.3% (21 out of 252); chi(2)=6.73; P=0.017. The Kaplan-Meier survival without renal/patient death was worse in d32-positive patients (log-rank test; P=0.002). The Cox regression analyses confirmed that the nl/nl genotype was a significant and independent protective factor for progression to end-stage renal failure (ESRF)/dialysis: beta/standard error (s.e.)=-3.1; chi(2)=9.5; relative risk=0.31 (95% confidence interval 0.15-0.65); P=0.002. The d32-CCR5 polymorphism played a significant role in the progression of primary IGAN, with the nl/nl genotype being an independent protective factor for late progression towards ESRF/dialysis. These data raise question about the usefulness of systematic CCR5 genotyping in IGAN patients.

Blood lactate exchange and removal abilities after relative high-intensity exercise: effects of training in normoxia and hypoxia.

The effects of 4 weeks of endurance training in conditions of normoxia or hypoxia on muscle characteristics and blood lactate responses after a 5-min constant-load exercise (CLE) at 90% of the power corresponding to the maximal oxygen uptake were examined at sea-level in 13 sedentary subjects. Five subjects trained in normobaric hypoxia (HT group, fraction of oxygen in inspired gas = 13.2%), and eight subjects trained in normoxia at the same relative work rates (NT group). The blood lactate recovery curves from the CLE were fitted to a biexponential time function: La(t) = La(0) + A1(1 - e- gamma 1.t) + A2(1 - e- gamma 2.t), where the velocity constants gamma 1 and gamma 2 denote the lactate exchange and removal abilities, respectively, A1 and A2 are concentration parameters that describe the amplitudes of concentration variations in the space represented by the arterial blood, La(t) is the lactate concentration at time t, and La(0) is the lactate concentration at the beginning of recovery from CLE. Before training, the two groups displayed the same muscle characteristics, blood lactate kinetics after CLE, and gamma 1 and gamma 2 values. Training modified their muscle characteristics, blood lactate kinetics and the parameters of the fits in the same direction, and proportions among the HT and the NT subjects. Endurance training increased significantly the capillary density (by 31%), citrate synthase activity (by 48%) and H isozyme proportion of lactate dehydrogenase (by 24%), and gamma 1 (by 68%) and gamma 2 (by 47%) values. It was concluded that (1) endurance training improves the lactate exchange and removal abilities estimated during recovery from exercises performed at the same relative work rate, and (2) training in normobaric hypoxia results in similar effects on lactate exchange and removal abilities to training in normoxia performed at the same relative work rates. These results, which were obtained non-invasively in vivo in humans during recovery from CLE, are comparable to those obtained in vitro or by invasive methods during exercise and subsequent recovery.

Effect of hyperoxia on aerobic and anaerobic performances and muscle metabolism during maximal cycling exercise.

The hyperoxia-improved tolerance to maximal aerobic performance was studied in relation to exercising muscle metabolic state. Five students were submitted to four different tests on a cycle ergometer, each being conducted under normoxia and hyperoxia (60% FiO2) on separate days: Test 1, a progressive exercise until exhaustion to determine the maximal work load (Wmax) which was unchanged by hyperoxia; Test 2, an exercise at Wmax (287 +/- 12 W) until exhaustion to determine the performance time (texh) which was elevated by 38% under hyperoxia but exhaustion occurred at the same arterial proton and lactate concentrations; Test 3 (S-Exercise test) consisted of cycling at Wmax for 90% normoxic-texh (4.8 +/- 0.5 min under both O2 conditions) then followed by a 10-s sprint bout during which the total work output (Wtot) was determined; Wtot was elevated by 15% when exercising under hyperoxia; Test 4 (M-Exercise test) consisted also of cycling at Wmax for 4.8 +/- 0.5 min with blood and muscle samples taken at rest and at the end of the exercise to compare the level of different metabolites. During hyperoxic M-Exercise test, glycogen was twice more depleted whereas glucose-6-phosphate and lactate were less accumulated when compared with normoxia. No significant differences were observed for pyruvate, phosphocreatine and muscle/blood lactate ratio between the two conditions. Conversely to normoxia, levels of ATP, ADP and total NADH were maintained at their resting level under 60% FiO2. These data lead us to suppose a higher oxidation rate for pyruvate and NADH in mitochondria, thereby lowering the metabolic acidosis and allowing a better functioning of the glycolytic and contractile processes to delay the time to exhaustion.

Effect of sodium citrate on performance and metabolism of human skeletal muscle during supramaximal cycling exercise.

The aim of this study was to examine whether the alkalosis-induced improvement in supramaximal performance could be explained by a less-altered muscle metabolic status. Eight subjects first performed exhausting exercise at 120% peak oxygen uptake after ingesting either a placebo (PLC) or sodium citrate (CIT) at a dose of 0.5 g.kg-1 body mass to determine exhaustion time (texh). They then, performed exercise (Lim-EX) at the same relative intensity lasting PLCtexh minus 20 s in both treatments. Samples were taken from vastus lateralis muscle at rest (90-min after the ingestion) and at the end of Lim-EX. Arterial blood samples were obtained at rest (immediately prior to and 90 min after ingesting the drug) and during the 20-min post-exercise recovery. The texh was significantly increased by CIT [PLC 258 (SD 29) s, CIT 297 (SD 45) s]. The CIT raised the rest [citrate] in blood [PLC 0.11 (SD 0.01) mmol.l-1, CIT 0.34 (SD 0.07) mmol.l-1] and in muscle [PLC 0.78 (SD 0.23) mmol.kg-1 dry mass, CIT 1.00 (SD 0.21) mmol.kg-1 dry mass]. Resting muscle pH and buffering capacity were unchanged by CIT. The same fall in muscle pH was observed during Lim-EX in the two conditions. This was associated with similar variations in both the cardio-respiratory response and muscle energy and metabolism status in spite of a better blood acid-base status after CIT. Thus, CIT would not seem to allow the alkalinization of the muscle cytosolic compartment. Though sodium citrate works in a similar way to NaHCO3 on plasma alkalinization and exercise performance, the exact nature of the mechanisms involved in the delay of exhaustion could be different and remains to be elucidated.

Performance and fibre characteristics of human skeletal muscle during short sprint training and detraining on a cycle ergometer.

The ergometric effect of sprint training and detraining was studied in relation to muscle fibre changes in seven students trained during 9 weeks on a cycle ergometer. Before and after training and after 7-week detraining, they performed a force-velocity test on a friction-loaded cycle ergometer. On these three occasions, muscle samples were taken from vastus lateralis muscle at rest for histochemical analysis. The training-induced shift of the force-velocity relationship was such that the increase in maximal velocity (vmax) was greatest against high braking forces (FB) with unchanged vmax with no load. This was associated with higher maximal power output (28%) and peak force (16%). The increased maximal mean power output to reach a maximal velocity during a short sprint was obtained against a 23% higher optimal FB (FB,Wmax). At the same time, a considerable hypertrophy in fast twitch b (FTb) fibres was observed. All these changes were maintained after detraining. The training-induced changes in vmax reached against FB1Wmax(vm2Wmax) allowed us to produce evidence for two particular sub-groups in which inverse fibre conversions were observed. In subgroup A, the lowered post-training vm,Wmax was associated with a decrease in both FTa and FTb fibres. Conversely, the vm,Wmax, increase in subgroup B was associated with a higher percentage of FT fibres as the result of increased FTa fibres and decreased FTb fibres. Thus, the fibre hypertrophy associated with a unidirectional fibre translation [FTb-->FTa-->slow twitch (ST)] toward fibres with a high thermodynamic efficiency would result mainly in increased force qualities, whereas the bidirectional fibre translation (ST-->FTa<--FTb) would allow enhancement of both force and velocity properties.

Enzyme adaptations of human skeletal muscle during bicycle short-sprint training and detraining.

The effect of sprint training and detraining on supramaximal performances was studied in relation to muscle enzyme adaptations in eight students trained four times a week for 9 weeks on a cycle ergometer. The subjects were tested for peak oxygen uptake (VO2peak), maximal aerobic power (MAP) and maximal short-term power output (Wmax) before and after training and after 7 weeks of detraining. During these periods, biopsies were taken from vastus lateralis muscle for the determination of creatine kinase (CK), adenylate kinase (AK), glycogen phosphorylase (PHOS), hexokinase (HK), phosphofructokinase (PFK), lactate dehydrogenase (LDH) and its isozymes, 3-hydroxy-acyl-CoA dehydrogenase (HAD) and citrate synthase (CS) activities. Training induced large improvements in Wmax (28%) with slight increases (3%) in VO2peak (P < 0.10). This was associated with a greater glycolytic potential as shown by higher activities for PHOS (9%), PFK (17%) and LDH (31%) after training, without changes in CK and oxidative markers (CS and HAD). Detraining induced significant decreases in VO2peak (4%), MAP (5%) and oxidative markers (10-16%), while Wmax and the anaerobic potential were maintained at a high level. This suggests a high level in supramaximal power output as a result of a muscle glycogenolytic and glycolytic adaptation. A long interruption in training has negligible effects on short-sprint ability and muscle anaerobic potential. On the other hand, a persistent training stimulus is required to maintain high aerobic capacity and muscle oxidative potential. This may contribute to a rapid return to competitive fitness for sprinters and power athletes.

Use of the force-velocity test to determine the optimal braking force for a sprint exercise on a friction-loaded cycle ergometer.

A group of 15 untrained male subjects pedalled on a friction-loaded cycle ergometer as fast as possible for 5-7 s to reach the maximal velocity (vmax) against different braking forces (FB). Power was averaged during a complete crank rotation by adding the power dissipated against FB to the power necessary to accelerate the flywheel. For each sprint, determinations were made of peak power output (Wpeak), power output attained at vmax (Wvmax) calculated as the product of vmax and FB and the work performed to reach vmax expressed in mean power output (Wvmax). The relationships between these parameters and FB were examined. A biopsy taken from the vastus lateralis muscle and tomodensitometric radiographs of both thighs were taken at rest to identify muscle metabolic and morphometric properties. The Wpeak value was similar for all FB. Therefore, the average of values was defined as corrected maximal power (Wmax). This value was 11% higher than the maximal power output uncorrected for the acceleration. Whereas the Wmax determination did not require high loads, the highest Wvmax value (Wmax) was produced when loading was heavy, as evidenced by the Wvmax-FB parabolic relationship. For each subject, the braking force (FB,Wmax) giving Wmax was defined as optimal. The FB,Wmax, equal to 0.844 (SD 0.108) N.kg-1 bodymass, was related to thigh muscle area (r = 0.78, P < 0.05). The maximal velocity (vm,Wmax) reached against this force seemed to be related more to intrinsic fibre properties (% fast twitch b fibre area and adenylate kinase activity). Thus, from the Wmax determination, it is suggested that it should be possible to predict the conditions for optimal exercise on a cycle ergometer.

Optimal velocity for maximal power production in non-isokinetic cycling is related to muscle fibre type composition.

To determine whether power-velocity relationships obtained on a nonisokinetic cycle ergometer could be related to muscle fibre type composition, ten healthy specifically trained subjects (eight men and two women) performed brief periods of maximal cycling on a friction loaded cycle ergometer. Frictional force and flywheel velocity were recorded at a sampling frequency of 200 Hz. Power output was computed as the product of velocity and inertial plus frictional forces. Force, velocity and power were averaged over each down stroke. Muscle fibre content was determined by biopsy of the vastus lateralis muscle. Maximal down stroke power [14.36 (SD 2.37)W.kg-1] and velocity at maximal power [120 (SD 8) rpm] were in accordance with previous results obtained on an isokinetic cycle ergometer. The proportion of fast twitch fibres expressed in terms of cross sectional area was related to optimal velocity (r = 0.88, P < 0.001), to squat jump performance (r = 0.78, P < 0.01) and tended to be related to maximal power expressed per kilogram of body mass (r = 0.60, P = 0.06). Squat jump performance was also related to cycling maximal power. expressed per kilogram of body mass (r = 0.87, P < 0.01) and to optimal velocity (r = 0.86, P < 0.01). All these data suggest that the nonisokinetic cycle ergometer is a good tool with which to evaluate the relative contribution of type II fibres to maximal power output. Furthermore, the strong correlation obtained demonstrated that optimal velocity, when related to training status, would appear to be the most accurate parameter to explore the fibre composition of the knee extensor muscle.

Effects of training in normoxia and normobaric hypoxia on human muscle ultrastructure.

The adaptive response of skeletal muscle to training in normoxia and in severe normobaric hypoxia was studied. The first group of five male subjects trained for 3 weeks on a bicycle (2 h/day, 6 days/week) in normoxia (Control training, Con T). A second group of five subjects trained in an ambient FIO2 decreasing progressively from 12.7% to a final level of 10.0% (hypoxic training, Hyp T). Fourteen months later, these subjects trained in normoxia at the same absolute power (normoxic training, Nor T). Peak oxygen consumption (VO2 max) was measured in normoxic and hypoxic conditions. Biopsies from the vastus lateralis muscle were analysed for fibre size, capillary and ultrastructural composition. Nor T had no effect on muscle tissue or VO2 max. Con T increased volume density of total mitochondria and lipids by 36 and 135% respectively (P < 0.05). Hyp T induced a 10% increase (P < 0.05) in peak VO2 max measured in hypoxia. Mean fibre cross-sectional area, interfibrillar mitochondrial volume density and capillary-to-fibre ratio were increased (P < 0.05) by 10, 42 and 13% respectively in the Hyp T group. These results suggest that training at the same relative workload in normoxia and hypoxia have similar, but not identical, effects on muscle tissue. If training in normoxia is carried out at the same absolute workload as in severe hypoxia, no significant effects are observed.

Ergometric and metabolic adaptation to a 5-s sprint training programme.

The effects of 7 weeks of sprint training (repeated 5-s all-out sprints) on maximal power output (Wv,max) determined during a force-velocity test and a 30-s Wingate test (Wpeak) were studied in ten students [22 (SD 2) years] exercising on a cycle ergometer. Before and after training, muscle biopsies were taken from vastus lateralis muscle at rest for the ten subjects and immediately after a training session for five of them. Sprint training induced an improvement both in peak performances by 25% (Wv,max and Wpeak) and in the 30-s total work by 16%. Before sprint training, the velocity reached with no load (v0) was related to the resting muscle phosphocreatine (PCr) stores (r = 0.87, P < 0.001). The training-induced changes in v0 were observed only when these PCr stores were lowest. This pointed to a possible limiting role of low PCr concentrations in the ability to reach a high velocity. The improvement in performances was linked to an increase in the energy production from anaerobic glycolysis. This result was suggested in muscle by the increase in lactate production measured after a training session associated with the 20% higher activity of both phosphofructokinase and lactate dehydrogenase. The sprint training also increased the proportion of slow twitch fibres closely related to the decrease in fast twitch b fibres. This result would appear to demonstrate an appropriate adaptive reaction following high-intensity intermittent training for the slow twitch fibres which exhibit a greater oxidative capacity.

Effect of arginine aspartate on the exercise-induced hyperammoniemia in humans: a two periods cross-over trial.

To investigate the effect of the ingestion of arginine aspartate (AA) in the decrease of the exercise-induced accumulation of ammonia in plasma, 11 voluntary subjects took part in a cross-over study where AA effect was tested against placebo. Both treatments were randomly administered in a double-blind procedure. To ensure the subjects would be able to present reproducible exercise-testing results during repetitive sessions, they were involved before the experiment in a cycle ergometer training program during 8 weeks. This training determined a significant 14% increase (P less than 0.001) in maximal oxygen uptake (VO2 max). The treatments were administered during 10 days and the two treatments were separated by a 10 day-wash-out period. A 45 min-cycle ergometer test was performed at 80% VO2 max during the 10th day of each treatment to measure plasma ammonia (p[NH4+]) and total blood lactate (b[lact]) concentrations at rest and at the 15th, 30th and 45th min of exercise (determinations of changes from rest; delta p[NH4+] and delta b[lact]). Both concentrations were unchanged between AA and placebo at rest but a significant lesser delta p[NH4+] was found under AA at the 15th min of exercise only (P less than 0.05). On the other hand, an order effect was found for delta p[NH4+] between the two periods of randomized treatment that was interpreted as a remaining training effect. This effect was highly significant at the 30th and 45th min of exercise (P less than 0.001). It was concluded that AA effect was minor with regard to the training effect. As it was not located at the same time of exercise, AA effect would not consequently have the same functional origin (postulated increase in the peripheral clearance of ammonia) than those of training (decrease in muscle production of ammonia).

Effects of endurance training on hyperammonaemia during a 45-min constant exercise intensity.

Eleven laboratory-pretrained subjects (initial VO2max = 54 ml.kg-1.min-1) took part in a study to evaluate the effect of a short endurance training programme [8-12 sessions, 1 h per session, with an intensity varying from 60% to 90% maximal oxygen consumption (VO2max)] on the responses of blood ammonia (b[NH+4]) and lactate (b[la]) concentrations during progressive and constant exercise intensities. After training, during which VO2max did not increase, significant decreases in b[NH+4], b[la] and muscle proton concentration were observed at the end of the 80% VO2max constant exercise intensity, although b[NH+4] and b[la] during progressive exercise were unchanged. On the other hand, no correlations were found between muscle fibre composition and b[NH+4] in any of the exercise procedures. This study demonstrated that a constant exercise intensity was necessary to reveal the effect of training on muscle metabolic changes inducing the decrease in b[NH+4] and b[la]. At a relative power of exercise of 80% VO2max, there was no effect of muscle fibre composition on b[NH+4] accumulation.

Effects of endurance training on capillary supply of human skeletal muscle on two age groups (20 and 60 years).

Two groups of human subjects were submitted to a 20-week endurance training program (1 h a day, 4 days a week, 70-80% max VO2). The first group (G20) consisted of eight 22 +/- 3 years male students, the second group (G60) was composed of seven still very physically active elderly male subjects (62 +/- 4 years). Training significantly increased max VO2 by 15% in G20 and 7% in G60. Muscle samples taken from the vastus lateralis muscle before and after training were histochemically stained for fibre-typing (myofibrillar ATPase), capillary supply and fibre area measurements (amylase PAS and NADH-TR). Fibre-type distribution was unchanged with training. Capillary density (cap X mm-2) increased significantly in both groups from 316 +/- 42 to 396 +/- 73 in G20 and from 308 +/- 48 to 409 +/- 55 in G60. This enhancement of capillary supply was linked to the proliferation of capillaries in G20 where the number of capillaries in contact with ST and FTa fibres (CC) significantly increased from 4.6 to 5.9 and from 4.8 to 6.1 respectively. No significant changes in fibre areas were found in G20. On the contrary, G60 did not show any significant sign of capillary growth (CC unchanged) whereas fibre areas significantly decreased in ST (6,410 to 5,520 micron 2) and FTa fibres (5,830 to 5,090 micron 2). A methodological evaluation of fibre-area measurement was described, with confirmation of the data. It was concluded that this study may illustrate the trainability of skeletal muscle of elderly men in a possibly different way to that seen in a younger age group.

[Use of the LA 640 for a simple method of measuring the concentration of muscle lactate]. / Utilisation du LA 640 pour une méthode simple de mesure de la concentration du lactate musculaire.

A simple technique was elaborated for the measurement of muscle lactate concentration. It was tested on samples of muscle vastus lateralis taken by the Bergström (1962) needle biopsy technique at the end of 20 min exercise bouts corresponding to 60-70% of Vo2 max. The biopsies were freshly frozen in liquid nitrogen, powdered and weighed in a cryostat at -20 degrees C. The extraction were made by saponine and the lactate measured in the saponine solution by an electrochemical-enzymatic method (LA 640). The results concern: the time of taking the biopsies and the freezing time (27 +/- 11 s and 34 +/- 9 s respectively); the accuracy of weighing (inducing a 1% uncertainty in the final result); a comparison of the saponine extraction with the perchloric acid extraction and a checking of the extraction capacity of the former; the accuracy of the whole measurement (the mean relative confidence limits are +/- 8-10%; the reproducibility of the technique through measurement of the variation coefficient (18%) calculated on measurements performed at a 15 days interval in 6 trained subjects. The discussion of the results and their comparison with those of the literature lead to the conclusion that the described method is suitable for muscle lactate measurements.