L-arginine decreases inflammation and modulates the nuclear factor-kappaB/matrix metalloproteinase cascade in mdx muscle fibers.

Duchenne muscular dystrophy (DMD) is a lethal, X-linked disorder associated with dystrophin deficiency that results in chronic inflammation, sarcolemma damage, and severe skeletal muscle degeneration. Recently, the use of L-arginine, the substrate of nitric oxide synthase (nNOS), has been proposed as a pharmacological treatment to attenuate the dystrophic pattern of DMD. However, little is known about signaling events that occur in dystrophic muscle with l-arginine treatment. Considering the implication of inflammation in dystrophic processes, we asked whether L-arginine inhibits inflammatory signaling cascades. We demonstrate that L-arginine decreases inflammation and enhances muscle regeneration in the mdx mouse model. Classic stimulatory signals, such as proinflammatory cytokines interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha, are significantly decreased in mdx mouse muscle, resulting in lower nuclear factor (NF)-kappaB levels and activity. NF-kappaB serves as a pivotal transcription factor with multiple levels of regulation; previous studies have shown perturbation of NF-kappaB signaling in both mdx and DMD muscle. Moreover, L-arginine decreases the activity of metalloproteinase (MMP)-2 and MMP-9, which are transcriptionally activated by NF-kappaB. We show that the inhibitory effect of L-arginine on the NF-kappaB/MMP cascade reduces beta-dystroglycan cleavage and translocates utrophin and nNOS throughout the sarcolemma. Collectively, our results clarify the molecular events by which L-arginine promotes muscle membrane integrity in dystrophic muscle and suggest that NF-kappaB-related signaling cascades could be potential therapeutic targets for DMD management.

Alteration of the piglet diaphragm contractility in vivo and its recovery after acute hypercapnia.

BACKGROUND: The effects of hypercapnic acidosis on the diaphragm and its recovery to normocapnia have been poorly evaluated. The authors studied diaphragmatic contractility facing acute variations of arterial carbon dioxide tension (Paco2) and evaluated the contractile function at 60 min after normocapnia recovery. METHODS: Thirteen piglets weighing 15-20 kg were anesthetized, ventilated, and separated into two groups: a control group (n = 5) evaluated in normocapnia (time-control experiments) and a hypercapnia group (n = 8) in which animals were acutely and shortly exposed to five consecutive ranges of Paco2 (40, 50, 70, 90, and 110 mmHg). Then carbon dioxide insufflation was stopped. Diaphragmatic contractility was assessed by measuring transdiaphragmatic pressure variations obtained after bilateral transjugular phrenic nerve pacing at increased frequencies (20-120 Hz). For each level of arterial pressure of carbon dioxide, pressure-frequency curves were obtained in vivo by phrenic nerve pacing. RESULTS: In the hypercapnia group, mean +/- SD transdiaphragmatic pressure significantly decreased from 41 +/- 3 to 29 +/- 3 cm H2O (P < 0.05) between the first (40 mmHg) and fifth (116 mmHg) stages of capnia at the frequency of 100 Hz stimulation. The observed alteration of the contractile force was proportional to the level of Paco2 (r = 0.61, P < 0.01). Normocapnia recuperation allowed a partial recovery of the diaphragmatic contractile force (80% of the baseline value) at 60 min after carbon dioxide insufflation interruption. CONCLUSION: A short exposure to respiratory acidosis decreased diaphragmatic contractility proportionally to the degree of hypercapnia, and this alteration was only partially reversed at 60 min after exposure.

Role of respiratory system impedance in the difference of ventilatory control between children and adults.

How children are able to adapt their ventilation to the intensity of exercise faster than adults remain unclear. We hypothesized that differences of VE observed between children and adults depend on either peripheral chemoreceptors or central command activity. We examined ventilatory control parameters in either normoxic or hypoxic condition (FI 02 =0.15). We analyzed the adaptability of the respiratory exchanges by (i) the measurement of ventilatory kinetics time-constant and (ii) the central command by the mouth-occlusion pressure (P0.1). A group of nine pre-pubescent children (mean age 9.5+/-1 years) and a group of eight adults (mean age 24+/-3.1 years) performed a constant-load exercise. In normoxia, children had significantly shorter time-constant (tau) VCO2 (respectively, 38.5+/-4.3 and 53.1+/-5.3s; P<0.001), tau VE (respectively, 52.5+/-13.1s vs. 66.1+/-12.3s; P<0.001), and tau P0.1 (57.4+/-15.4 and 61.0+/-12.9s, respectively; P<0.001) than adults. In hypoxia, children exhibited shorter tau P0.1/VT/Ti compare to adults. Reinforced by the significant correlation between tau VE and tau P0.1/VT/Ti for children but not adults, we concluded that ventilatory response differences could be due in part to the respiratory system impedance.

The effect of respiratory muscle training with CO2 breathing on cellular adaptation of mdx mouse diaphragm.

The aim of our study was to investigate the cellular mechanisms induced by hypercapnic stimulation of ventilation, during 6 weeks/30 min per day, in 10 mdx and 8 C57BL10 mice (10+/-0.2 months old). Ten mdx and eight C57BL10 mice served as control group. This respiratory training increases in vitro maximal tetanic tension of the diaphragm only in mdx mice. Western blot analysis of diaphragm showed: (1) an over-expression of alpha-dystrobrevin in mdx and C57BL10 training group compared to control group (8100+/-710 versus 6100+/-520 and 2800+/-400 versus 2200+/-250 arbitrary units); (2) a decrease in utrophin expression only in mdx training group compared to control group (2100+/-320 versus 3100+/-125 arbitrary units). Daily respiratory muscle training in mdx mice, induces a beneficial effect on diaphragm strength, with an over-expression of alpha-dystrobrevin. Further studies are needed to determine if, in absence of dystrophin, the over-expression of alpha-dystrobrevin could be interpreted as a possible pathway to improve function of dystrophic muscle.

Effects of short vs. prolonged mechanical ventilation on antioxidant systems in piglet diaphragm.

OBJECTIVE: Prolonged controlled mechanical ventilation (MV) is known to induce diaphragmatic oxidative stress that seems to be an important factor reducing force-generating capacity. To better understand the cellular mechanisms involved, this work examined the effect of short vs. prolonged MV on antioxidant defense in the diaphragm. DESIGN AND SETTING: Prospective, randomized, controlled animal study in a university laboratory. METHODS: Eleven piglets (15-20 kg) were assigned to one of two groups: a long-MV group (n=6) ventilated for 3 days or a short-MV group (n=5) ventilated for 3 h. Force frequency curves of the transdiaphragmatic pressure (Pdi) were obtained in vivo by phrenic nerve pacing. Oxidative stress was evaluated by thiobarbituric reactive substance (TBARs) content and the enzymatic antioxidant activity of both superoxide dismutase (SOD) and glutathione peroxidase (GPx) in samples of diaphragm. RESULTS: Pdi decreased in the long-MV group by 30-35% over the 3 days at all frequencies compared to the short-MV group. Diaphragm TBARs content was significantly higher and SOD activity lower in long-MV animals than in short-MV animals after 72 h. GPx activity tended to be lower in diaphragms from long-MV animals, but this difference was not significant. CONCLUSIONS: This study shows that 3 days of MV in piglets is associated with a decrease in antioxidant activity which could emphasize oxidative stress and both contribute to the diaphragm dysfunction caused by MV.

Changes in respiratory muscle endurance during puberty.

The evaluation of respiratory muscle endurance provides clinically useful information on muscle function, especially in children with respiratory and neuromuscular diseases. However, endurance may be lower in young children than in older children because of the major physical changes of puberty. We thus compared respiratory muscle endurance in 15 healthy pre- and peripubertal children (S1-S2/P1 group) and 14 healthy children near the end of the pubertal process (S4P4 group). All performed a respiratory muscle endurance test threshold load fixed at 50% of the individual maximal inspiratory pressure; (Pi(max)), using a standardized method with a controlled breathing pattern. No significant difference was found between groups for Pi(max). The mean endurance time limit for the S1-S2/P1 group was 138 +/- 20 sec. The S4P4 group was able to breathe with the threshold valve for more than 20 min (1,200 sec) without task failure, except for one girl (385 sec). This study shows that inspiratory muscle endurance is significantly lower in children in early puberty compared to children at the end of the pubertal process. If the underlying mechanisms are not well-known, the present study revealed that if we use the same inspiratory load in prepubertal children as in adults during clinical testing, we are likely to underestimate the susceptibility to task failure of their respiratory muscles. To define a fatigue threshold for the respiratory muscles, as a function of age, thus appears clinically important in further studies, particularly for the management of children with respiratory diseases.

Dose-dependent effect of individualized respiratory muscle training in children with Duchenne muscular dystrophy.

The aim of this study was to evaluate the effects of low intensity, home inspiratory muscle training on respiratory muscle endurance in children with Duchenne muscular dystrophy, using a double-blind protocol. The originality aspect of this study is the use of a reproducible method of endurance and of the same method for evaluation and training. We studied eight trained children (mean age 14.7+/-4.5 years) and eight control children (mean age, 12.6+/-1.8 years). For 6 weeks, children breathed twice a day for 10 min through a valve with either 30% (training group) or less than 5% (control group) of their maximum inspiratory pressure (P(imax)). The results showed (1) a 46% improvement in the time limit after training in the training group and no change in the control group and (2) a significant correlation between the total time of respiratory muscle training and the percentage of endurance improvement in the training group. We conclude that specific training improves respiratory muscle endurance in Duchenne muscular dystrophy and the effectiveness of training appears to be dependent on the quantity of training.

Comparison of the effects of heat and moisture exchangers and heated humidifiers on ventilation and gas exchange during non-invasive ventilation.

OBJECTIVE: To compare the short-term effects of a heat and moisture exchanger (HME) and a heated humidifier (HH) during non-invasive ventilation (NIV). DESIGN: Prospective, clinical investigation. SETTING: Intensive care unit of a university hospital. PATIENTS: Twenty-four patients with acute respiratory failure (ARF). INTERVENTION: Each patient was studied with a HME and a HH in a random order during two consecutive 20min periods of NIV. MEASUREMENTS AND RESULTS: Respiratory rate (RR), expiratory tidal volume (VTe) and expiratory minute ventilation (VE) were measured during the last 5 min of each period and blood gases were measured. Mean pressure support and positive end-expiratory pressure levels were, respectively, 15+/-4 and 6+/-2 cmH(2)O. VE was significantly greater with HME than with HH (14.8+/-4.8 vs 13.2+/-4.3 l/min; p<0.001). This increase in VE was the result of a greater RR for HME than for HH (26.5+/-10.6 vs 24.1+/-9.8 breaths/min; p=0.002), whereas the VT for HME was similar to that for HH (674+/-156 vs 643+/-148 ml; p=0.09). Arterial partial pressure of carbon dioxide (PaCO(2)) was significantly higher with a HME than with a HH (43.4+/-8.9 vs 40.8+/-8.2 mmHg; p<0.005), without significantly changing oxygenation. CONCLUSION: During NIV the increased dead space of a HME can negatively affect ventilatory function and gas exchange. The effect of HME dead space may decrease efficiency of NIV in patients with ARF.

Effects of controlled mechanical ventilation on respiratory muscle contractile properties in rabbits.

OBJECTIVE: We examined in rabbits the effects of more than 48 h of mechanical ventilation on the contractile properties and fiber type adaptations of the respiratory muscles. DESIGN AND SETTING: Experimental prospective study in a university laboratory. ANIMALS AND INTERVENTIONS: Nineteen rabbits were randomly allocated to two groups: control (n=10) or mechanically ventilated (MV; n=9) for 51+/-3 h. MEASUREMENTS AND RESULTS: Respiratory muscles contractile properties were analyzed before and after a fatigue protocol using in vivo isometric 1-s tetanic contraction characteristics in both muscles: peak tetanic force, contraction time, relaxation time, and total contraction time. Both muscle fiber type proportions, diameter, and cross-sectional areas were measured using ATPase staining. The MV rabbits showed significant weight loss in both muscles, accompanied by a reduced peak tetanic force (9.96+/-3.2 vs. 7.44+/-2.2 N for diaphragm of control and MV animals respectively), fatigue resistance index, and increased relaxation time (57.5+/-8.7 vs. 85.8+/-9.4 ms for diaphragm of control and MV animals) and contraction time. These impairments in the MV group worsened after the fatigue runs. Both muscle showed a significant atrophy of type IIa and IIb fibers but a stability in type I fibers cross-sectional area. CONCLUSIONS: Mechanical ventilation in rabbits produces alterations in contractile properties of the diaphragm and 5th external intercostal muscle, increases both muscles fatigue, and promotes atrophy of type II fibers.

Post-extubation stridor in intensive care unit patients. Risk factors evaluation and importance of the cuff-leak test.

OBJECTIVE: To evaluate the incidence and identify factors associated with the occurrence of post-extubation stridor and to evaluate the performance of the cuff-leak test in detecting this complication. DESIGN: Prospective, clinical investigation. SETTING: Intensive care unit of a university hospital. PATIENTS: Hundred twelve extubations were analyzed in 112 patients during a 14-month period. INTERVENTION: A cuff-leak test before each extubation. MEASUREMENTS AND RESULTS: The incidence of stridor was 12%. When we chose the thresholds of 130 ml and 12% to quantify the cuff-leak volume, the sensitivity and the specificity of the test were, respectively, 85% and 95%. The patients who developed stridor had a cuff leak significantly lower than the others, expressed in absolute values (372+/-170 vs 59+/-92 ml, p<0.001) or in relative values (56+/-20 vs 9+/-13%, p<0.001). Stridor was associated with an elevated Simplified Acute Physiology Score (SAPS II), a medical reason for admission, a traumatic or difficult intubation, a history of self-extubation, an over-inflated balloon cuff at admission to ICU and a prolonged period of intubation. These results provide a framework with which to identify patients at risk of developing a stridor after extubation. CONCLUSION: A low cuff-leak volume (<130 ml or 12%) around the endotracheal tube prior to extubation is useful in identifying patients at risk for post-extubation stridor.

Respiratory pressures in boys from 11-17 years old: a semilongitudinal study.

We hypothesized that in children, maximal respiratory pressures increase with age and more during the pubertal growth spurt. The aim of this work was to follow maximal respiratory pressures, with a semilongitudinal study, in three groups of prepubertal, peripubertal, and postpubertal children in order to specify pressure changes with age and determine the contributions of growth and puberty. A semilongitudinal study was conducted in 44 boys assigned to three overlapping age groups: from 11-13 (prepubertal stage), from 13-15 (pubertal stage), and from 15-17 years (postpubertal stage). The children underwent annual testing for maximal respiratory pressures and spirometric values. Body mass, height, lean body mass assessed from four skinfold thicknesses, and stage of development were also evaluated. Maximal respiratory pressure increased significantly with growth from 11-17 years for maximal inspiratory pressure, and from 11-15 years for maximal expiratory pressure. We observed an increase in the slope of the regression lines between age and lean body mass during the pubertal growth spurt, without an increase in the slopes of the regression lines between age and respiratory pressures. In conclusion, the relationships between age and respiratory pressures are not modified by the growth spurt, indicating that anthropometric characteristics have little role in determining respiratory pressures.

Ventilatory parameters and maximal respiratory pressure changes with age in Duchenne muscular dystrophy patients.

The aim of this longitudinal study was to precise, in children with Duchenne muscular dystrophy, the respective functional interest of ventilatory parameters (Vital capacity, total lung capacity and forced expiratory volume in one second [FEV(1)]) in comparison to maximal inspiratory pressure (Pimax) during growth. In ten boys the mean age of 9.1 +/- 1 years) to mean age of 16 +/- 1.4 years followed over a period of 7 years, we found that: (1) ventilatory parameters expressed in percentage of predicted value, after a normal ascending phase, start to decrease between 11 and 12 years, (2) Pimax presented only a decreasing phase since the beginning of the study and thus was already at 67% of predicted value at 12 years while ventilatory parameters was still normal, (3) after 12 years the mean slopes of decrease per year of vital capacity and FEV1 were higher (10.7 and 10.4%) than that of Pimax (6.9%), (4) at 15 years mean values of vital capacity and FEV1 (53.3 and 49.5% of predicted values) was simlar to that of Pimax (48.3%). In conclusion, if at early stages of the disease, Pimax is a more reliable index of respiratory impaiment than ventilatory parameters, the follow-up of ventilatory parameters, when they start to decrease, is a better indicator of disease progression and, at advanced stages they provided same information about the functional impact of disease.

Ventilation during air breathing and in response to hypercapnia in 5 and 16 month-old mdx and C57 mice.

Previous studies have shown a blunted ventilatory response to hypercapnia in mdx mice older than 7 months. We test the hypothesis that in the mdx mice ventilatory response changes with age, concomitantly with the increased functional impairment of the respiratory muscles. We thus studied the ventilatory response to CO(2) in 5 and 16 month-old mdx and C57BL10 mice (n = 8 for each group). Respiratory rate (RR), tidal volume (VT), and minute ventilation (VE) were measured, using whole-body plethysmography, during air breathing and in response to hypercapnia (3, 5 and 8% CO(2)). The ventilatory protocol was completed by histological analysis of the diaphragm and intercostals muscles. During air breathing, the 16 month-old mdx mice showed higher RR and, during hypercapnia (at 8% CO(2) breathing), significantly lower RR (226 +/- 26 vs. 270 +/- 21 breaths/min) and VE (1.81 +/- 0.35 vs. 3.96 +/- 0.59 ml min(-1) g(-1)) (P < 0.001) in comparison to C57BL10 controls. On the other hand, 5 month-old C57BL10 and mdx mice did not present any difference in their ventilatory response to air breathing and to hypercapnia. In conclusion, this study shows similar ventilation during air breathing and in response to hypercapnia in the 5 month-old mdx and control mice, in spite of significant pathological structural changes in the respiratory muscles of the mdx mice. However in the 16 month-old mdx mice we observed altered ventilation under air and blunted ventilation response to hypercapnia compared to age-matched control mice. Ventilatory response to hypercapnia thus changes with age in mdx mice, in line with the increased histological damage of their respiratory muscles.

L-arginine improves dystrophic phenotype in mdx mice.

A possible treatment for Duchenne muscular dystrophies would be to compensate for dystrophin loss by increasing the expression of utrophin, another cytoskeletal protein of the muscle membrane. We previously found that L-arginine, the substrate for nitric oxide synthase, significantly increased utrophin level in muscle and targeted it to the sarcolemma. Here, we have addressed the expected benefit in the mdx mice. Magnetic resonance imaging of lower limbs revealed a 35% reduction of the necrotic zones, confirmed by histological staining of muscles. This regression of the necrosis was also supported by the drastic reduction of Evans blue incorporation, a cell impermeable dye. The creatine kinase level in the serum decreased by 57%. Utrophin level increased 2- to 3-fold in muscles. Beta-dystroglycan was relocalised with utrophin to the membrane. In the diaphragm, the most affected muscle in mdx mice, the isometric tension increased by 30%, with regression of collagen and of cytoplasmic lipid overloading. Finally, molsidomine, a therapeutic agent that is converted to a NO donor, also attenuated the dystrophic phenotype. Our results suggest that pharmacological activators of the NO pathway may constitute a realistic treatment for Duchenne and Becker muscular dystrophies.

Effect of prolonged undernutrition on rat diaphragm mitochondrial respiration.

Previous studies have shown that undernutrition induces an impairment of the respiratory muscle function in patients with chronic lung disease. To explain this, we hypothesized that undernutrition could decrease oxidative metabolism in the diaphragm. We therefore examined the effect of prolonged undernutrition on diaphragm mitochondrial oxygen uptake with pyruvate and palmitate as substrates in adult rats. Ten rats served as controls (CTL). Ten nutritionally deprived rats (ND) received 40% of their estimated daily nutrition. Five weeks of undernutrition induced a 33% decrease in state 3 respiration with pyruvate plus malate as substrate (993 +/- 171 versus 1488 +/- 167 nmol atomic O/mg/min, P < 0.01) and a 39% decrease with palmitate plus malate (516 +/- 89 versus 850 +/- 165 nmol atomic O/mg/min, P < 0.05). With succinate plus rotenone, there was no significant difference in the respiratory rate between groups. In the ND group, we found a significant decrease in citrate synthase activity (P < 0.01), and also in reduced nicotinamine adenine dinucleotide (NADH) dehydrogenase activity (P < 0.05), which cannot alone induce such a state 3 respiratory decrease. This showed that undernutrition in rat diaphragm does not induce an alteration in protein complexes I, II, III, and IV, or the F complex containing the mitochondrial ATPase of the electron transport chain. In conclusion, the main result of this study was that prolonged undernutrition induced a decrease in mitochondrial respiration secondary to a significant reduction in NADH generation by the Krebs cycle, which may affect respiratory muscle function with implications for patient care.

Electrophysiologic changes during exercise testing in patients with chronic obstructive pulmonary disease.

To determine whether skeletal muscle is involved in the exercise limitation of chronic obstructive pulmonary disease (COPD), we investigated electrical adaptations in muscle during incremental cycling exercise testing. Changes in quadriceps activity were compared using surface electromyography (SEMG) and motor point stimulation in ten COPD patients and ten healthy subjects. Patients showed significantly lower exercise capacity, and M-wave duration was increased from exercise onset (P < 0.05) with a parallel decrease in amplitude (P < 0.05). The SEMG power spectrum median frequency was always higher (P < 0.04) in patients and its decline was earlier (P < 0.01). The ratio of the root mean square of the SEMG to oxygen uptake was decreased (P < 0.001) during exercise in patients, although it remained constant in controls. Electromyographic parameters were significantly more involved in the exercise limitation than ventilatory factors. Thus, modified electrical activity in muscle appeared in COPD patients from exercise onset, indicating that skeletal muscle function is clearly implicated in the exercise intolerance of these patients.