textarchive.ru

Главная > Документ


P21-01

VALIDATION OF A SIMPLE CALIBRATION METHOD FOR HUMAN MUSCLE MICRODIALYSIS

Desvigne N., Barthélémy J.C., Bertholon F., Gay-Montchamp J.P., Freyssenet D., Costes F.

Microdialysis gives a unique opportunity to identify substrates and measure their concentration changes in the human muscle interstitial fluid. A major methodological limitation lies in the difficulty to calibrate the microdialysis probes in order to know the interstitial substance actual concentrations. Indeed, the concentration of a substance in the dialysate is only influenced by its interstitial concentration as well as by the probe recovery of the substance which vary greatly in vivo with changing environmental conditions. The aim of our study was, to validate in vitro a new calibration method, the slope method, based on the determination in individual probe of probe recovery of a substance from that of ethanol, in various dialysis conditions reproduced by changes in substance concentration and recovery. The method feasability and accuracy were confirmed in vivo in a muscle resting study as compared to a reference method, the NO Net Flux method (NNF). Determination of the linear relationship, in a steady state, between recovery of ethanol and that of the substance of interest (lactate (Lac), glucose (Glu)) allows to calculate the concentration of the latter in any conditions. In vitro, we obtained very strong relationships between recovery of ethanol and recovery of Lac or Glu, with a r ranging from 0.986 to 1.000 (all p<0.001); the error of estimation of Lac or Glu concentrations in th dialysis bath was limited to -0.6±5.8% and 0.7±6.2% for, respectively, Lac and Glu, in a large range of probe recovery. In human muscle, the slope method was as accurate as NNF to evaluate interstitial Lac concentration (1.82±0.58 vs 1.83±0.47 mM, respectively). The improved precision of the slope method results from the in situ determination of the individual relationship for each microdialysis catheter. We conclude that, after an initial calibration step, the slope method allows accurate interstitial muscle metabolites measurement and could monitor rapid metabolic changes.

Laboratoire de Physiologie GIP-E2S CHU de Saint-Etienne France

P21-02

QUANTITATIVE POINCARE PLOT ANALYSIS OF HEART RATE VARIABILITY: EFFECT OF ENDURANCE TRAINING

Mourot L., Bouhaddi M., Perrey S., Rouillon J.D., Regnard R.

RHeart rate variability (HRV) provides non-invasive assessment of the autonomic control of heart rate. HRV is expected to decrease from supine rest to upright position and further during exercise. Conversely, endurance training seems to increase HRV. But discrepant results were obtained with linear time- and frequency-domain methods that require stationary R-R intervals time series. The non-linear Poincaré plot of HRV depicts trend changes in heart dynamics independent of R-R stationarity. This study was aimed at showing the usefulness of the Poincaré method to assess the training-induced changes in HRV. Four 10 min manoeuvres were performed (supine lying, standing, steady state exercising and subsequent recovery) by 8 control subjects before and after short-term endurance training and by 8 subjects trained for at least 3 years. HRV was assessed by time- and frequency-domain indexes, in parallel with the Poincaré plot analysis. In the latter each R-R interval is plotted as a function of the previous one, and the standard deviations of the instantaneous and long-term R-R interval variability are calculated. In our subjects, the Poincaré scattergrams became gradually narrower from supine to exercising, with progressive parasympathetic withdrawal. Short- and long-term endurance training led to higher aerobic power and ventilatory threshold shifted towards higher power output. All HRV evaluation methods showed that HRV values were higher after training both when supine lying and standing. The Poincaré scattergrams were wider in the trained state. Standard deviations of the Poincaré plot were significantly correlated with the main parameters of the time- and frequency-domain analyses, especially concerning the parasympathetic indicators. These results suggested that Poincaré plot parameters as well as the "width" of the scattergram could be considered as surrogates of time- and frequency-domain analysis to assess training-induced changes in HRV.

Laboratoires Physiologie Médecine et Sciences du Sport, BESANCON, Faculté des Sciences du Sport MONTPELLIER, FRANCE

P21-03

COLLOID OSMOTIC PRESSURE AND FLUID DISTRIBUTION VOLUMES IN THE DENTAL PULP

Berggreen E., Wiig H.

Interstitial fluid colloid osmotic pressure (COP) is a basic transcapillary fluid balance parameter. In the dental pulp COP and the fluid distribution volumes are unknown. We decided to measure intravascular and interstitial fluid volumes and to test if pulp interstitial fluid could be isolated by centrifugation. Studies were performed in anaesthetized Wistar rats. Incisors pulps were removed in a humidity chamber and sealed in airtight vials. Total extracellular fluid volume measured as the 120 min distribution volume of [51Cr]-labeled EDTA after nephrectomy, averaged 0.631 ± 0.071 (SD) ml/g wet weight (ww). The corresponding intravascular volume, determined as the five min distribution volume of [125I]-labeled human serum albumin, averaged 0.03 ± 0.018 ml/g ww, resulting in an interstitial fluid volume of 0.601 ± 0.077 ml/g ww. To isolate interstitial fluid, pulps were transferred to tubes provided with a basket made from nylon mesh. To examine if fluid derived from the intracellular and intravascular compartment, the ratio for extracellular fluid equilibrated [51Cr]- EDTA and plasma volume equilibrated [125I]- human serum albumin between the fluid and plasma was measured. At 239 G, the centrifugate fluid to plasma ratio of [51Cr]-labeled EDTA and [125I]-labeled human serum albumin averaged 0.864 ± 0.053 and 0.03 ± 0.027, respectively. HPLC of pulp centrifugate showed a pattern resembling that of plasma except for a peak eluting in the ~40 kD MW range. COP was measured in pulp fluid isolated from rats not pretreated with isotopes by use of a colloid osmometer. COP in the incisor pulps interstitial fluid averaged 19.7 ± 3.7 mmHg that was 82 ± 12 % of COP measured in the corresponding plasma. We conclude that dental pulp interstitial fluid can be isolated by centrifugation, and that its interstitial fluid COP is relatively high. The pulp has a high content of extracellular fluid (63%), mainly located outside the vascular compartment (60%).

Department of Physiology, University of Bergen, Norway.

P21-04

AGE-RELATED CHANGES IN MICROVASCULAR FILTRATION CAPACITY

Charles M., Pichot V., Desvignes N., Denis C., Costes F.

The microvascular filtration capacity is an important determinant of local muscle exchange during exercise. It is determined by the product of the surface area available for exchange and the microvascular permeability per unit surface area. A non invasive determination of the microvascular filtration capacity (Kf) in muscle has been proposed by Gamble et al. (J Physiol 1993, 464:407-22). Briefly, mercury-in-silastic strain gauge plethysmography was used to measure volume changes of the limb in response to small cumulative pressure steps applied to produce venous congestion via a cuff inflated proximally from the strain gauge. Each pressure step of 8 mmHg was maintained for 5-7 min to ensure completion of the vascular filling response and to obtain slopes representing fluid filtration only. With elderly, a decrease in capillary density has been demonstrated but its importance is still debated, especially in upper limbs where it could be preserved. In order to test whether microvascular filtration capacity could be altered in older subjects, we performed comparative measurements of Kf of the forearm and the calf in old and young healthy volunteers. We recruited seven old healthy subjects aged 73 ± 4 years and 7 healthy young volunteers aged 29 ± 5 years. All subjects were relatively fit. In the forearm, Kf were not significantly different, 9.01 ± 2.91 and 6.49 ± 1.08 ml/(100ml).min-1.mmHg-1.10-3 in old and young subjects respectively (p=0.064). By contrast, in the calf, Kf was significantly lower in old than in young subjects (1.99 ±0.53 vs 4.78 ±1.42 ml/(100ml).min-1.mmHg-1.10-3; p=0.015). We conclude that, with elderly, microvascular filtration capacity was differently decreased in the upper and the lower limbs. It remains to be determined whether this corresponds to a larger decreased in the capillary density of the calf and whether this is related to the limb exercise capacity of these subjects.

GIP-E2s CHU Saint Etienne France

P21-05

DETERMINATION OF ISOKINETIC MUSCLE STRENGTH IN CHF PATIENTS AND IN COPD PATIENTS

Degache F., Costes F., Calmels P., Garet M., Barthelemy J.C., Roche F.

Objective : To evaluate the relationship between isokinetic strength, and aerobic aptitude in patients with chronic heart failure (CHF) and with chronic obstructive pulmonary disease (COPD).

Methods : Seventeen stable CHF (n=11) and COPD (n=6) patients aged 51.12±9.95 ys old (15 men, 2 women, CHF patients : NYHA class II: n=8 and III: n=3 ) underwent a dynamic isokinetic force evaluation of the quadriceps (IsoK) at an angular velocity of 180°s-1, a symptom-limited cardiopulmonary exercise test with simultaneous monitoring of respiratory gases, and a 6-min walking test. Body composition was assessed by electrical bioimpedance. The relationships between muscle strength, maximal aerobic performance, results of endurance tests and anthropometric datas were assessed by simple regression analysis.

Results : The isokinetic evaluation has been successfully performed in all patients. No cardiac arrhythmic event nor abnormal haemodynamic response was observed in neither group of patients. In CHF patients, individual quadricipital muscle strength was significantly related to fat-free mass (r=0.862; p<0.01). However, neither peak O2 (global: r=0.562, p=NS or weight-adjusted: r=0.083 p=NS) nor endurance capacity (r=0.068, p=NS) were significantly related of the peripheral muscle strength.

In COPD patients, quadricipital muscle strength was not significantly related to fat-free mass (r=0.242; p=NS) nor any other measured factor.

Conclusion : Physiopathological mechanisms of exercise limitation in CHF and COPD patients are complex. Isokinetic strength and O2 peak are not related to each other and are therefore independent. Therefore, a sysrematic evaluation of isokinetic muscle strength may be of great value.

Early degradations in aerobic capacity are probably followed by an alteration in muscle strength.

Keywords : Chronic Heart Failure, Chronic Obstructive Pulmonary Disease, isokinetic, squeletal muscle strength, aerobic capacity.

Service d’Explorations Fonctionnelles Cardio-Respiratoires, Hôpital Nord, CHU de St Etienne, FRANCE

P21-06

DNA-CHIPS REVEAL COORDINATED CHANGE IN EXTRACELLULAR MATRIX-RELATED GENE EXPRESSION IN VARICOSE VEIN

Cario-Toumaniantz C., Boularan C., Loirand G., Pacaud P.

Vascular wall remodeling, resulting from global and coordinated change in gene expression, is a common feature of many vascular diseases affecting arteries or veins such as chronic venous insufficiency (CVI). CVI leads to the development of varicose veins, characterized by extracellular matrix (EM) disorganization and loss in smooth muscle reactivity. However, the molecular mechanisms underlying these processes are unknown. This work was aimed to identify the global pattern of differentially expressed genes in human varicose saphenous vein smooth muscle (SV) in order to reveal genes involved in vein wall dysfunction and remodeling. We used suppression subtractive hybridization to identify differentially expressed genes in healthy SV, obtained from patients undergoing coronary bypass (n=27) and surgically removed varicose SV (n=25). Two cDNA libraries were generated containing up- and down regulated clones in varicose SV. Microarray analysis of these cDNA libraries (n=5) revealed that 16% of clones showed at least a 1.5-fold difference in expression in varicose SV. Half of these clones corresponded to gene coding for EM proteins including Matrix Gla protein (MGP), a known vascular calcification inhibitor, which was up-regulated in varicose SVSM. Using real-time RT-PCR, we confirmed and quantitated the differential expression of these genes in varicose (p<0.05, n=8) compared to healthy SV (n=8). Von Kossa staining which allows detection of calcification areas has been used to assess the functional consequences associated with change in MGP expression. Ectopic calcification process was observed in 80% of varicose SV wall tested. This result suggests that a new phenotypic transition associated with mineralization occured in varicose SV. Furthermore, this work provides the first characterization of a coordinate up-regulation of a set of genes related to the EM in varicose SV that could be directly involved in the alteration of the vein wall functions and organization.

Inserm U533, faculté des Sciences et Techniques, Nantes, France

P21-07

ROLE OF THE MAP KINASE SIGNALLING PATHWAYS IN SKELETAL MUSCLE DEGENERATION AND REGENERATION

Salles J., Yeow K., Ragno M., Dérijard B.

The MAP kinase pathways play important roles in all stages of the cell life cycle: proliferation, differentiation, and death. Dysregulation of these signalling pathways result in a variety of disorders, including cancer and degeneration. The aim of our research is to clarify the role played by the MAP kinase pathways in muscle cell regeneration upon physical wounding and cytokine treatment. Injury to a muscle induces the necrosis of affected myofibers. Subsequently, regeneration occurs as a result of the activation, proliferation, and fusion of myogenic stem cells into myotubes that mature into myofibers. However, little is known about the involvement of the MAP kinase pathways during muscle wounding and recovery.

We have analysed the changes in the levels of p38, JNK, and ERK after muscle wounding. Our results show a rapid increase in the level of phosphorylated p38 and ERK after wounding in C2C12 cells. Treatments of the C2C12 cells with TNFα and IL-1 also result in an increase in activated ERK and p38 levels. In addition, we observed that conditionned medium from wounded cells is able to increase phosphorylated p38 levels but not ERK in unwounded cells. In light of these results, it seems that injured C2C12 cells release certain factor(s) which can act on unwounded cells to trigger p38 (but not ERK) activation. Degradation and synthesis of several macromolecular components of extracellular matrix have also been demonstrated after injury to adult myofibers. We compared the levels of matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) by zymography, as physiological markers of muscle cell wounding and recovery. We observed an MMP-9 overexpression in wounded C2C12 cells against intact cells. Furthermore, using inhibitors of the different MAPK pathways, we demonstrated that this MMP-9 overexpression is p38 pathway-dependent.

In conclusion, our results indicate that the p38 pathway could play an essential role in the muscle skeletal regeneration process.

Laboratoire de Physiologie Cellulaire et Moléculaire, UMR-CNRS 6548, Université de Nice-Sophia Antipolis, Nice

P21-08

ENDOTHELIN RESPONSES TO ENDURANCE EXERCISE AND TO SIX WEEKS TRAINING AFTER HEART TRANSPLANTATION

Doutreleau S., Piquard F., Lonsdorfer E., Lampert E., Mettauer B., Richard R., Epailly E., Lonsdorfer J., Geny B.

Objectives: Short-term survival is no longer the pivotal issue after heart transplantation but most heart-transplant patients (Htx) still present with reduced exercise capacity. Increased endothelin (ET), a potent vasoconstrictive peptide, might mediate important complications and participate in Htx’s exercise capacity limitation. The two objectives of this study are to investigate, for the first time after heart transplantation: 1) whether prolonged exercise increases circulating endothelin and 2) whether training-induced exercise capacity improvement is associated with reduced baseline and exercise ET in Htx.

Methods: After comparing their maximal exercise capacity to that of matched controls, 9 male Htx performed a 45 minutes endurance exercise test (EE). We determined their ET response at rest, at the end of EE and at 30 minutes of recovery. Five Htx also completed a 6 weeks training program, consisting in 3 exercise session per week.

Results: Resting heart rate, systemic blood pressure, creatinine and endotheline (4.8 ± 1.1 vs 1.6 ± 0.2 pmol.l-1) were increased in Htx. Maximal tolerated power was decreased (132 ± 11 vs 205 ± 14 watts) and the the anaerobic threshold occured early in Htx (at 49.5 ± 7.5 % of VO2 max.). EE did not modify ET, even at 30 minutes recovery. Training improved significantly Htx’s submaximal and maximal exercise capacity (12 ± 2 % and 15 ± 6 %, P =0.01), decreased heart rate but only tended to decrease ET.

Conclusion: This study demonstrates that long duration submaximal exercise fails to increase circulating ET after heart transplantation. Further supporting the usefulness of training, it also suggests that training-induced improvement in exercise capacity is not obtained mainly through a decreased ET levels in Htx.

Physiology and Cardiovascular Surgery Departments, EA 3072, Faculté de Médecine et HUS, Strasbourg, France

P21-09

EFFICIENCY OF WORK AT 100% OF MAV : SHORT INTERMITTENT EXERCISE VERSUS CONTINUOUS EXERCISE

Tardieu M., Thevenet D., Prioux J.

Introduction : The aim of this study was to compare the time spent at maximal oxygen uptake (tVO2max) during a 30s-30s realized at 100% of maximal aerobic velocity (MAV) (active recovery : 50% of MAV) with the one measured during maximal continuous run (100% of MAV). Method. 11 endurance-trained runners (16.5 years ± 0.3, VO2max : 62.5 ml.min-1.kg-1 ± 1.3 and MAV : 17.6 km.h-1 ± 0.25) performed 3 field-tests (400-m outdoor tartan track) until exhaustion : 1) an incremental test to determine their VO2max and MAV. 2) an intermittent exercise (IE) alternating 30s at 100% of MAV and 30s at 50% of MAV. 3) a continuous run (CR) at 100% of MAV. Tests IE and CR were randomized. During all tests, respiratory gas exchanges were measured breath-by-breath using a portable system (Cosmed K4b2, Italy). Total exercise duration (tlim), total exercise time at 100% of MAV (tMAV), time spent at VO2max (tVO2max) and blood lactate concentration at the end of exercise ([La]) were measured for IE and CR. Results. tlim and tMAV are significantly (p < 0.001) longer during IE (tlim = 1412.7s ± 147.9 and tMAV = 706.4s ± 74) than during CR (tlim = tMAV = 362.2s ± 93.8). During IE, mean tVO2max, expressed in absolute value (second) is not significantly higher than the one obtain during CR (300s ± 145.2 vs 222.3s ± 32.4). On the other hand, if we express tVO2max in relative value (related to tlim, %tlim) we note that during IE, subjects spend 23.7% (± 35.3) of their total exercise time at VO2max against 60% (± 27) during CR (p < 0.05). [La] obtain at the end of IE and CR are not significantly different (respectively 8.7 mmol.L-1 ± 0.9 vs 10.3 mmol.L-1 ± 0.7). Our results seem to demonstrate that tVO2max during IE is not significantly ifferent from CR. Our hypothesis is not verified. It even seem that continuous run at 100% of MAV is more efficient : related to total exercise time, time spent at VO2max during CR is significantly higher than during IE.

UFR STAPS, 25 bis bd Guy Mollet bp 72206. 44322 Nantes – France

P21-10

EARLY ALTERATIONS OF CARDIAC MITOCHONDRIAL FUNCTION AFTER CORONARY ARTERY LIGATION IN PIGS

Zoll J., Ponsot E., Doutreleau S., Mettauer B., Lampert E., Mazzucotelli JP., Diemunsch P., Piquard F., Geny B.

Objectives: To investigate the characteristics of possible early mitochondrial alterations in ischemic myocardium after coronary artery ligation in vivo in experimental pigs.

Methods : Left descending coronary artery was performed after sternotomy in six White pigs. 45 minutes after ligation the heart was quickly removed. In-situ maximal O2 uptake (Vmax, µmol O2.min-1.g-1 dry weight) of saponin-skinned myofibers were then measured from both ischemic (I) and normal (N) part of the ventricular myocardium, using three different substrates. Increasing amounts of ADP concentration allowed to measure a Km for ADP in the presence (Km+) or absence of creatine (Km-).

Results: Vmax decreased by 20% in ischemic myocardium with both glutamate-malate (18.1±1.3 vs 22.1±1.7 in N, P=0.04) and pyruvate substrates (19.3±1.0 vs 23.3±2.0 in N, P=0.05). No difference was observed with palmityl carnitine (15.6±1.8 in I vs 16.6±0.9 in N). The Km- for ADP decreased in I by 24% (679±79 vs 899±84µM of ADP in N, P= 0.04), reducing the stimulatory effect of creatine (Km-/Km+ = 11.6±2.5 in I vs 18.0±2.2 in N, P=0.04).

Conclusions: Thus, ischemia lasting 45 minutes is sufficient to reduce significantly the maximal oxidative capacity of ventricular myocardium and to alter the mitochondrial respiration control, through a decrease in the functional role of mitochondrial creatine kinase. Interestingly, palmityl carnitine utilization was not affected, supporting that cardiac ischemia results in specific alterations of the mitochondrial function related to the level of energy demand.

Physiology, Cardiovascular Surgery and Anesthesiology Departments, EA 3072, Faculté de Médecine and HUS 67085 Strasbourg - France

P21-11

BTBD1, A NOVEL TOPO1 INHIBITOR, IS IN VITRO INVOLVED IN SKELETAL MUSCLE CELL DIFFERENTIATION.

Pisani D.F., Cabane C., Yeow K., Piétu G., Derijard B., Dechesne C.

ABTBD1 is a recently cloned novel protein homologous to the topoisomerase 1 (TOPO1) inhibitor BTBD2. BTBD1 is able to bind to TOPO1 and is expressed in many tissues with a preferential expression in skeletal muscle. Our objectives were to determine BTBD1 involvement in proliferation and differentiation of skeletal muscle cell and its interaction type with TOPO1.

C2C12 murine myoblasts were used for this study. These cells are able to differentiate in myotubes and have a BTBD1 basal expression.

First we studied BTBD1 gene expression and we showed that : 1/ The BTBD1 gene was highly up-regulated during C2C12 differentiation. 2/ The BTBD1 gene was down-regulated in cell wounding experiments, where confluent scratched cells proliferated again to regenerate. 3/ A small BTBD1 up-regulation was found in modified C2C12 cells blocked at confluence without differentiation. These results suggest that BTBD1 is involved in the proliferation arrest step that precedes the cellular differentiation process, and showed that BTBD1 gene expression is inversely correlated with TOPO1 activity.

Secondly, we studied BTBD1 interaction with TOPO1. In C2C12 cells we stably expressed a non-functional BTBD1 dominant (nfBTBD1) able to bind to TOPO1 and localised in both nucleus and cytoplasm. nfBTBD1-C2C12 cell proliferation was largely affected and no differentiation was observed. These alterations are characteristic of TOPO1 up-activation. This hypothesis was confirmed by nuclear TOPO1 (= active TOPO1) western blot analysis that showed a nuclear TOPO1 decrease during wild type but not nfBTBD1-C2C12 cell differentiation. Moreover, we showed by immunofluorescence analysis that TOPO1 is sequestered in nfBTBD1-C2C12 nucleus.

In conclusion we have determined that BTBD1 is a negative regulator of TOPO1 and that BTBD1 activity is essential for skeletal muscle cell differentiation.

Laboratoire de Physiologie Cellulaire et Moléculaire, CNRS-UMR6548, Université de Nice Sophia-Antipolis, Nice, France.

P21-12

NANDROLONE DECANOATE AFFECTS Ca2+ AND Sr2+ SENSITIVITY OF CONTRACTILE PROTEINS IN RAT MUSCLES

Bouhlel A., Joumaa W., Léoty C.

It is generally accepted that anabolic-androgenic steroids treatment improve skeletal muscle performance. However, a few works have been devoted to the analysis at the cellular level of the change on the skeletal muscle excitation-contraction coupling mechanism. Therefore, the effects of nandrolone decanoate (ND) treatment on the properties of contractile proteins were investigated in Triton-skinned fibres isolated from rat edl and soleus muscles.

One group of male rats received weekly (for 8 weeks) an intramuscular injection of ND (15 mg Kg-1) and the other group received weekly the similar doses of vehicle (sterile peanut oil). Small bundles of two to five fibres were dissected and after skinning mounted in an experimental chamber as already described by Joumaa and Léoty (2002). All procedures accord with the Declaration of Helsinki.

Following ND treatment, the maximal-activated tension recorded in pCa 4.5 and pSr 4.5 solution was increased in edl by 34% and 29%, respectively and in soleus muscles by 28% and 23%, respectively. The tension-pCa or -pSr relationship was significantly shifted leftwards in both types of muscle. In control edl, the pCa50 and the pSr50 were 6.14 ± 0.04 and 5.17 ± 0.03, respectively and in treated edl were 6.26 ± 0.04 and 5.45 ± 0.02, respectively. Furthermore, in control soleus, the pCa50 and the pSr50 were 6.28 ± 0.03 and 5.86 ± 0.01, respectively and in treated soleus 6.45 ± 0.02 and 6.22 ± 0.03, respectively. The hill coefficient for Ca2+ and Sr2+ sensitivity of the contractile apparatus was decreased in edl by 34% and 24%, respectively and in soleus fibres by 26% and 25%, respectively.

The present results show that the contractile apparatus of skeletal muscles were similarly affected by ND treatment and that the sensitivity of contractile proteins in treated edl was similar to that found in untreated soleus muscle.

Joumaa WH, Serrurier B, Bigard X, Léoty C (2002). Acta Physiol Scand 175(3):189-199

Laboratoire de Physiologie Générale, CNRS UMR 6018, Faculté des Sciences et des Techniques, 44322 Nantes France

P21-13

EXPIRATORY FLOW LIMITATION UNDER NEGATIVE PRESSURE DURING EXERCISE: ROLE OF EXPIRATORY MUSCLES

Montandon G., deBisschop C., Guénard H.

Introduction: Expiratory flow limitation (EFL) promotes dynamic hyperinflation and limits exercise tolerance both in healthy subjects and particularly in chronic obstructive pulmonary disease patients. Negative pressure at the mouth during tidal expiration (NEP) allows the detection of EFL. However, patients are differently susceptible to this technique.

Objectives: To assess why subjects respond differently to NEP, electromyographic activity of expiratory muscles (EMG) was measured.

Hypotheses: (1) During exercise, NEP has no effect on EFL below a critical pressure (Pc) which varies among subjects. (2) High NEP is needed for subjects who don’t use expiratory muscles during exercise, whereas low Pc results in the use of these muscles.

Methods: Eight healthy subjects (24-58 years old) were tested under different NEP (3, 5, 7 and 9hPa) at rest and during an exercise performed on a cycle ergometer at 30% and at 60% of maximal aerobic power. EFL and average rectified value (ARV) of surface EMG activities of transversus abdominal muscle were evaluated.

Results: Subjects were classified according to 3 profiles: no limitation during exercise (P0), limitation up to a 3-5 hPa Pc (P1) and limitation up to a 7 hPa Pc (P2). For P0 and P1 subjects (n=3), ARV with NEP was higher than without NEP at rest and during exercise. On the other hand, for P2 subjects (n=5), ARV with NEP was lower than without during exercise. NEP did not increased muscular activity in a given exercise condition and even decreased in some subjects.

Conclusion: For P2 subjects, NEP must be sufficiently high to remove EFL, because the muscle was less active than in P0 and P1 subjects. These results suggest that, by an unknown mechanism, P0 and P1 subjects increase their muscular activity to reduce the effect of NEP. The difference observed among subjects in response to NEP during exercise warrants further investigations.

Laboratory of Physiology, University Victor Segalen Bordeaux 2, Bordeaux, France

P21-14

MITOGEN ACTIVATED PROTEIN KINASE SIGNALLING IN DM1 SKELETAL MUSCLE

Coldefy A.S., Pisani D., Ragno M., Desnuelle C., Dechesne C., Dérijard B.

Myotonic dystrophy (DM1) is an autosomal dominant disorder caused by an instable CTG repeat sequence in the 3’-untranslated region of a protein kinase gene (dmpk).

DM1 is the most common adult onset neuromuscular disease. It involves myotonia, progressive weakness and wasting of skeletal muscle. The congenital form of DM1 is also associated with evidence of delayed or arrested muscle maturation. Furthermore, DMPK is a serine / threonine kinase but its cellular function is still unknown.

Research in our group focuses on the function of Mitogen Activated Protein Kinase (MAPK) signalling pathways in skeletal muscle growth and differentiation. There are three MAPK pathways : the ERK (ERK1 / ERK2) pathway acts primarily to positively regulate the cell cycle, while the p38 and JNK pathways may oppose these function and elicit stress-induced cell cycle arrest and differentiation. It has been recently shown that ERK and p38 pathways play important roles in the process of myogenesis.

Therefore we wanted to know if ERK, p38 or JNK might be involved in the DM1 muscular deficiency. In collaboration with several different groups, we studied MAPK activation in patients skeletal muscle biopsies, in muscles from a myotonic dystrophy mouse model, in human DM1 satellite cells and in C2C12 myoblasts clones.

Considering the Ser / Thr kinase nature of DMPK, cell signalling and regulation of gene expression are very likely to be affected in this disease. Our preliminary results show a variation in the activation of the three MAPK in all our DM1 "studying models", although they are still confusing regarding JNK. Nonetheless, our overall conclusion is that the activation of the MAPK signalling pathways is likely to be impaired in DM1 disease.

Laboratoire de Physiologie Cellulaire et Moléculaire, UMR CNRS 6548, 28 Avenue Valrose, 06108 Nice cedex 2, France.

P21-15

EFFECTS OF CLENBUTEROL TREATMENT AND EXERCISE ON MUSCLE METABOLISM IN RATS

Cavalié H., Mounier R., Clottes E., Bricout V., Lac G.

The aim of this study was to test the effects of a selective beta-2 adrenergic receptor agonist : clenbuterol, on muscle performance and metabolism in growing 3-month old male Wistar rats treated over 8 weeks.

Méthods - Thirty-two Wistar rats weighing 234 ± 2 g were assigned for 8 weeks, five days each week, to a progressive isometric strength training exercise program plus oral clenbuterol (2 mg/kg body weight/day, Exercised + Clenbuterol group : ECL), exercise program without clenbuterol (Exercised group : E), no exercise program plus oral clenbuterol (2 mg/kg/day, Untrained + Clenbuterol : UCL), or no exercise without clenbuterol (Untrained : U). At the end of the 8 weeks, body composition was measured by Hologic QDR 4500A DEXA technique. On day 58, plasma, soleus (Sol) and plantaris (Plan) were harvested after sacrifice until analyses.

Results - We found that fat mass was decreased in E, and decreased further in ECL. Lean mass was increased in UCL and ECL. Insulin-like growth factor-1 (IGF-1) was increased in E, and decreased in UCL. Sol and Plan phenotype, based on myosin heavy chain (MHC) composition, was both affected by treatment or/and training with a shift to a faster phenotype in both Sol and Plan. Strength endurance capacity was decreased in ECL. Muscular metabolism was also altered by clenbuterol administration and/or training in Sol and Plan with a decrease in citrate synthase (CS) and lactate deshydrogenase (LDH) activity in both muscle and an increase in creatine kinase (CK) activity.

Conclusion - This study suggests that clenbuterol-induced muscular hypertrophy treatment is independent of plasma IGF-1 and that the decrease of muscular strength endurance capacity is mainly caused by the decrease of oxydative and glycolytic metabolism in muscle.

BAPS, Univ Clermont, Labo Bio B, Les Cézeaux, 63177 Aubière - FRANCE

P21-16

X-RAY INTERFERENCE MEASURES THE WORKING STROKE OF MUSCLE MYOSIN WITH SUBNANOMETRE PRECISION.

Reconditi M., Linari M., Lucii L., Sun Y.-B., Boesecke P., Narayanan T., Stewart A., Irving T., Fischetti R. F., Piazzesi G., Irving M., Lombardi V.

Force and shortening in muscle are generated by ATP driven cyclic interactions of the myosin head domains, extending from the myosin filament, with the actin filament. According to crystallographic models, in each interaction an interdomain tilting of the myosin head accounts for ~10 nm filament sliding. However, the function of the myosin motor depends on the interaction between conformational changes and external forces, and this cannot be reproduced in crystallographic studies. In the skeletal muscle, due to the quasi crystalline arrangement of the motor proteins in the three-dimensional lattice, small-angle X-ray scattering (SAXS) can be used to record the conformational changes of the myosin motor in the native structure. The improved brightness and focusing of synchrotron X-ray beamlines like ID2 at ESRF (Grenoble, France) and Bio-CAT at APS (Argonne, IL, USA) led to the development of a new technique that can measure the motions of myosin heads in muscle fibres with Å-scale sensitivity. The method depends on X-ray interference between the two arrays of myosin heads in each bipolar myosin filament, which superimposes a finely spaced fringe pattern onto the ~14.5 nm reflection originating from the axial repeat of the myosin heads in each array. We used this method to study the motions of myosin heads associated to the early length transient (1-10 ms duration) that follows a 0.1 ms step in force superimposed on the isometric contraction of single fibres from frog skeletal muscle at 4 °C and 2.1 um sarcomere length. The results provide evidence for a mechanism of force generation based on a working stroke in the myosin heads attached to actin and show that under isotonic contraction the amount of filament sliding accounted for by the elementary step increases from 6 to 10 nm as the force reduces from 75% to 25% the isometric force.

University of Florence, Italy; King's College London, UK; ESRF Grenoble, France; BioCAT - IIT, Chicago, USA.

P21-17

THE P38 PATHWAY REGULATES AKT AT THE PROTEIN AND TRANSCRIPTIONAL ACTIVATION LEVELS DURING MYOGENESIS

Cabane C., Yeow K., Ragno M., Dérijard B.

Muscle cell differentiation is controlled by extracellular growth factors whose signals are transduced into the nucleus. Stress Activated Protein Kinase cascades are involved in this process and members of these pathways are potently expressed in skeletal muscle.

We use the mouse skeletal muscle C2C12 cell line as a model system. This cell line is able to differentiate in vitro such that the major steps in myotube formation are identifiable. Several stable clones expressing various members of the stress-induced MAPK cascades have been isolated, including a C2C12 clone expressing a dominant negative form of the MAPKK MKK3 (p38 pathway).

This stable clone was unable to undergo myotube formation. These cells have been used side by side with control cells in a cDNA grid array screening in order to study gene regulation involving the p38 signalling pathway during differentiation. One of these genes, c-Akt (PKBalpha), is down-regulated during differentiation in the MKK3 dominant negative stable clone.

In addition to the MKK3 dominant negative stable clone, we also show that C2C12 cells stably transfected with a dominant negative form of Akt do not differentiate. Since both the MKK3/p38 and PI3K/c-Akt pathways are required for myogenesis, we decided to study the interaction between these two pathways in C2C12 cells. Our results suggest that Akt acts downstream of p38 in myogenic cell differentiation. Activating the p38 pathway results in the concurrent activation of Akt ; conversely, inhibiting the p38 pathway prevents Akt activation. C2C12 cells stably expressing dominant negative MKK3 express reduced levels of Akt messenger RNA and total protein. Interestingly, levels of phosphorylated Akt are less regulated in these cells. Our results show for the first time that p38 can directly affect Akt at the transcriptional level as well as at the protein activation level during myogenic differentiation.

UMR 6548 CNRS LPCM, Nice, France

P21-18

CHOLINERGIC DRUGS CHANGE THE SYNAPTIC DELAYS OF QUANTAL RELEASE AT THE FROG ENDPLATE

Nikolsky E.E., Samigullin D., Bukharaeva E.A., Vyskocil F.

Acetylcholine (Ach) can hinder the neuromuscular transmission by lowering end-plate current (EPC) amplitude. This lowering is caused by decreasing the number of quanta forming EPC and by postsynaptic receptor desnsitization. However, the decrease of the EPC might also be caused by greater dispersion in synaptic delays of individual quanta1,2. The delay distribution of quanta release differs in the proximal and distal parts of the 100-200 mm long frog endplate nerve terminal and there also exists progressive slowing down of nerve spike conduction velocity in the proximal-distal direction. The minimal synaptic delays are longest (0.50 ms) in proximal part and shortest in distal part (0.31 ms), but the delay dispersion is highest in the former parts and decreases in the distal parts3. Frog endplate therefore provides a unique possibility of studying the sections of the same synapse with more compact or more dispersed quanta release. In the distal parts, the uni-quantal EPCs with long delays increased in number when 5x10-4 M Ach was applied. P90 (an interval at which 90 % of all uni-quantal EPCs had occurred), was reversibly increased by 66% from 0.51 ms to 0.85 ms. In the distal parts of the endplate, stimulation-evoked EPCs with long release latencies increased in number also when non-hydrolysable carbacholine (CB) was applied. This increased asynchrony leads to a substantial drop in the size of reconstructed multiquantal EPCs which was lower by 28% than control one. The presynaptic action of Ach, CB (and also other cholinergic drugs) includes the modulation of the quantal secretion time course and by this the efficacy of the synaptic transmission. Grants: MSMT 113100003, GAÈR 305021333, 202021213, RFBR 000448901.

1. Bukharaeva et al. J. Physiol. 517:879, 1999.

2. Bukharaeva et al. J. Physiol. (Lond.) 538:837, 2002.

3. Samigullin et al. Neurochem. Res. 28:507, 2003 (in press)

State Med. Univ. Kazan, Inst. Biochem. Biophys. Kazan, Dept. Physiol. ASCR, Dept. Physiol, Fac.Sci, Charles Univ.,Prague – Czech Republic

P21-19

STRUCTURAL ASPECTS OF FORCE GENERATION BY MYOSIN HEADS PROBED BY X-RAY INTERFERENCE IN FROG MUSCLE

Piazzesi G., Brunello E., Linari M., Reconditi M., Sun Y.-B., Narayanan T., Panine P., Irving M., Lombardi V.

Force generation in muscle is thought to be due to transition between states with different degree of tilting of the light chain domain of the myosin head. We tested this idea by using X-ray diffraction interference to measure the changes in axial position of the heads associated to changes in isometric force with temperature, a factor that is known to change the force per myosin head. During the isometric contraction of single muscle fibres of Rana temporaria, X-ray interference between the two arrays of myosin heads in each myosin filament splits the M3 reflection from the 14.5 nm axial repeat of the heads into two main peaks. In the experiments reported here patterns were collected at ID2 (European Synchrotron Radiation Facility, ESRF, France) on an image intensified FReLoN CCD detector placed at either 10 m (to collect intensity and fine structure of the low order meridional reflections) or 3 m (to collect intensity of the higher order meridional reflections and of the actin layer lines). Increasing the temperature from 0 °C to 17 °C increased the isometric tetanic force by 44%, decreased the relative intensity between the high angle peak and the low angle peak of the M3 reflection from 0.9 ± 0.02 (mean ± SD, n= 12 fibres) to 0.75 ± 0.01 and increased the spacing of the M3 reflection (14.568 nm at 0 °C) in proportion to the isometric force with a slope about twice that estimated during the elastic response to rapid length changes. The intensity of the first actin layer line, measured in the same range of temperatures, increased by 56 ± 18% (6 fibres). Changes in the fine structure of M3 reflection that accompany temperature dependent changes in the isometric force are interpreted using the tilting lever arm model of the myosin head and a structural model of the sarcomere with distributed filament compliance.

Università di Firenze, Italy; King’s College London, UK; ESRF, France

P21-20

THE FORCE RESPONSE TO STRETCH IN HUMAN SKELETAL MUSCLE FIBRES WITH DIFFERENT MYOSIN ISOFORMS

Linari M., Bottinelli R., Pellegrino M.A., Reggiani C., Lombardi V.

The functional heterogeneity of skeletal muscle in mammalians is based mainly on the existence of multiple isoforms of the myosin heavy chain subunit (MHC) of the molecular motor myosin II: in fibres containing fast MHC isoforms, the unloaded shortening velocity, the maximum power output and the ATPase activity are three-to tenfold larger than in fibres containing slow MHC isoforms. Force enhancement during lengthening of an active muscle, a condition that normally occurs during locomotion in vivo, is accompanied by decrease in ATP consumption below the isometric level, while the rate of cross-bridge detachment/attachment increases with lengthening velocity: the myosin head under strain is prevented for completing the conventional cycle and detach from actin through a step that does not imply ATP splitting. We investigated the kinetics and mechanical features of this cycle in Ca2+ activated single skinned fibres from human skeletal muscle containing different MHC isoforms, identified with single fibre gel electrophoresis. Fibre were activated by using a new set-up that allows development of most of tension following a jump from 0-1°C to test temperature (~12°C). In this way we could prevent the development of sarcomere non-uniformities and record sarcomere length changes with a striation follower in any phase of the mechanical protocol. We found that (i) at any pCa fibres with fast MHC isoforms develop larger isometric forces (~60%) than those with slow isoforms, due to both increased fraction of force generating myosin heads and, to a lesser extent, increased force per head; (ii) independent of the MHC isoform lengthening of maximally activated fibres elicits similar steady forces indicating that mechanical and kinetic properties of the actin-myosin interaction under stretch are similar; in both fibre types force enhancement by stretch is due to recruitment of myosin attachments, without increase in strain per head above the value generated by isometric heads.

Università di Firenze, Firenze, Italy; Università di Pavia, Pavia, Italy; Università di Padova, Padova, Italy

P21-21

Ca2+ INFLUX AND RESTING Ca2+ DECREASE DURING SLOW-TO-FAST TRANSITION OF UNLOADED RAT SOLEUS MUSCLE

Fraysse B., Desaphy J-F., Pierno S., De Luca A., Liantonio A., Rolland J-F., Conte Camerino D.

The present study was designed to determine the muscle-type specificity of passive Ca2+ entry. Using the fura-2 Ca2+ probe and the Mn2+ quenching technique, we observed in control rats that the slow-twitch soleus muscle (SOL) presented a higher [Ca2+]i and a higher Ca2+ influx at rest with respect to the fast-twitch extensor digitorum longus muscle (EDL). By comparing effects of nifedipine and gadolinium on the quench rate, we concluded that the muscle-type specificity of the passive Ca2+ entry was related to difference in expression and/or activity of Ca2+ permeable stretch-activated channels (SAC) and that leak Ca2+ channels activity was similar in both muscle types. During hindlimb unloading (HU), it is well known that rat SOL undergoes a slow-to-fast phenotype transition. In line with this, the resting [Ca2+]i and the passive Ca2+ entry were drastically decreased in rat SOL after 14 days of HU, becoming, together with the response to caffeine, similar to that of control EDL. For a shorter period of HU, 3 days, the caffeine-induced Ca2+ release assessed in SOL was still typical of a slow-twitch muscle. In contrast, the resting [Ca2+]i and the passive Ca2+ influx were already significantly decreased in SOL fibres as compared to control, due to a reduced SAC activity. On the other hand, no correlation between HU-induced muscle wasting and resting [Ca2+]i changes was found in SOL. These results clearly show that SOL resting [Ca2+]i decrease is an early event during suspension, which precedes most of the slow-to-fast functional changes, but could rely on an early reduced Ca2+ entry trough SAC. Accordingly to the crucial role of [Ca2+]i in the regulation of muscle phenotype, our results suggest that the slow-to-fast transition taking place in rat SOL during unloading is a Ca2+-dependent process in contrast to the HU-induced muscle atrophy (grants ASI I/R/040/01, ASI I/R/305/02).

Università di BARI, Sezione di Farmacologia, Dipartimento Farmaco-Biologico, Via Orabona, 4 70125 BARI ITALIA

P21-22

FATIGUE-RELATED CHANGES OF TRICEPS SURAE MOTONEURONE ACTIVITY INDUCED BY STRETCH IN DECEREBRATE CATS

Ljubisavljevic M., Bugaychenko L., Kalezic I., Kostyukov A., Pilyavskii A., Windhorst U., Johansson H.

We investigated fatigue-related changes of the triceps sure muscle (TS) motoneurones activity in decerebrate cats in response to muscle stretch of the homonumous muscle. Long-lasting (1.5–2 hours) intrasomatic recordings were produced from 11 motoneurones. The animals were subjected to bilateral pneumothorax and artificially ventilated while additional rigid fixation of the spinal cord enabled lasting intrasomatic recording during strong long-lasting muscle contractions. Right hindlimb was completely denervated except for the TS. Fatiguing muscle contractions of TS were evoked by electrical stimulation of the otherwise cut S1 ventral root (current strength 1.4 threshold, rate 40 s -1 and duration ranging from 5 to 15 min). Microelectrodes were filled with 2 M solution of potassium acetate and 0.6 M potassium chloride with resistance 1.8 – 2.3 MOhms. Standard slow bell-wise muscle stretches of different amplitude and velocity were applied; maximal amplitude of the stretches did not exceed 8 mm. The stretches evoked very stable and reproducible waves of membrane depolarization reflecting summation of a great number of low-amplitude individual EPSPs. The depolarization in many cases was accompanied by generation of a steady spike discharge with instantaneous rate related closely to temporal course of the muscle stretches. After cessation of the fatiguing stimulation both the Ia EPSPs evoked by TS nerve stimulation and the stretch-evoked depolarization were noticeable depressed reaching 20%. The depolarization decline could increase with duration of the fatiguing stimulation and amplitude of the fatiguing muscle contractions. The stretch-related spike activity was also suppressed; the post-fatigue decrease reaching 5-7 s-1 with following restoration to a pre-fatigue level during 5-10 min.

The results further delineate the central processes controlling effectiveness of afferent inflow regulation of motoneurone activity during muscle fatigue.

Faculty of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates



Скачать документ

Похожие документы:

  1. The Ministry of Education and Science of the Republic of Latvia

    Документ
    ... during the 3 years period, Congress ofFederationofEuropeanPhysiologySocieties, Ljubljana, Slovenia, 12.-15.November ... PhysiologicalSociety and FEPS delegate since 1994; Member of the Board of Latvian Malacological Society ...
  2. Univerzita komenského v bratislave prehľad publikačnej činnosti vykazovaný k 31 10 2007 skupina a1 - knižné publikácie charakteru vedeckej monografie (aaa aab aba abb) počet záznamov 87

    Документ
    ... ), s. 82 [Joint Meeting of The Slovak PhysiologicalSociety, The PhysiologicalSociety and The FederationofEuropeanPhysiologicalSocieties. Bratislava, Slovakia ...
  3. Univerzita komenského v bratislave – lekárska fakulta prehľad publikačnej činnosti vykazovaný k 31 10 2007 skupina a1 - knižné publikácie charakteru vedeckej monografie (aaa aab aba abb) počet záznamov 18

    Документ
    ... ), s. 82 [Joint Meeting of The Slovak PhysiologicalSociety, The PhysiologicalSociety and The FederationofEuropeanPhysiologicalSocieties. Bratislava, Slovakia ...
  4. Publikace pracovníků 3 lf od roku 1993 - 2000 podle jmen vybraných autorů

    Документ
    ... , roč. 48, (Suppl. 1), s. S81. Konference: FederationofEuropienPhysiologicalSocieties (FEPS). Číslo VZ: MSM 111200005 ... , roč. 48, (Suppl. 1), s. S81. Konference: FederationofEuropienPhysiologicalSocieties (FEPS). Číslo VZ: MSM 111200005 ...
  5. Přehled publikační činnosti podle typů dokumentů od 1993 do 2000

    Документ
    ... , roč. 48, (Suppl. 1), s. S81. Konference: FederationofEuropienPhysiologicalSocieties (FEPS). Číslo VZ: MSM 111200005 ... , roč. 48, (Suppl. 1), s. S27. Konference: FederationofEuropienPhysiologicalSocieties (FEPS). Číslo VZ: MSM 111200005 ...

Другие похожие документы..