Chronic hypoxia modulates diaphragm function in the developing rat.

We studied the effect of chronic hypoxia on contractile properties and neuromuscular transmission in the developing rat diaphragm. We hypothesized that chronic hypoxia delays maturation of neuromuscular transmission. Phrenic nerve hemidiaphragm preparations were harvested from 3- to 26-day-old rats and littermates raised in 9.5% oxygen. Specific force, contraction time, and one-half relaxation time were measured. Each diaphragm was stimulated directly or via its nerve with 1-s trains at 10-100 Hz. Contraction time and one-half relaxation time decreased with advancing age in both groups, with a greater rate of decrease in hypoxic diaphragms. Specific force was lower for hypoxic diaphragms compared with controls. Diaphragms from the 3- to 10-day-old control and hypoxic groups generated less force in response to stimulation at frequencies >40 Hz but did so to a greater degree with nerve stimulation. Nerve stimulation of diaphragms from 11- to 18-day-old hypoxic rats showed a greater decrease in force with increasing frequency compared with age-matched controls. Diaphragms from 19- to 26-day-old rats showed no difference between the hypoxic and control groups. We conclude that chronic hypoxia leads to diaphragms that generate lower specific force as well as to a delayed maturation of mechanisms involved in neuromuscular transmission.

Effect of respiratory muscle training on GLUT-4 in the sheep diaphragm.

PURPOSE: Endurance exercise training is associated with enhanced glucose uptake and hence improvement in carbohydrate metabolism. Glucose transport (GLUT) membrane proteins are regulated by a variety of physiological stimuli, including exercise. In limb muscle, both acute exercise and endurance training enhance the expression of the skeletal muscle transporter, GLUT-4. The purpose of this study is to determine whether chronic loading enhances GLUT-4 expression in the diaphragm. METHODS: The effect of chronic inspiratory flow resistive (IFR) loading on diaphragm GLUT-4 was studied in a model of respiratory muscle endurance training in sheep. IFR loads (resistance 50-100 cm H2O x L(-1) x s(-1)) were maintained for 3 h x d(-1), 5-6 d x wk(-1) for 3 wk. Loading was adjusted so that PaO2 was >60 Torr and PaCO2 <45 Torr in room air. Six untrained sheep were used as controls. GLUT-4 protein and mRNA were analyzed by Western and Northern analysis respectively. RESULTS: GLUT-4 protein levels were two-fold greater in trained animals when compared with controls (P < 0.01). GLUT-4 mRNA levels in the trained muscles was not significantly different from controls. CONCLUSIONS: We conclude that in the sheep diaphragm, chronic IFR loading increases GLUT-4 protein levels. This increase may be one of the mechanisms contributing to the improved respiratory muscle endurance previously demonstrated in this animal model of respiratory muscle training.

Developmental differences in endplate response to P-type calcium channel blockade in the rat diaphragm.

Endplate potentials (epps) were recorded intracellularly from single diaphragm fibers of newborn (7-10 days, n = 11) and older (24-30 days, n = 11) rats in the presence of 100 nM omega-agatoxin IVA, a P-type Ca2+ channel blocker. The muscle was stimulated via the phrenic nerve for 1 s at 40 Hz. In both age groups epp amplitude decreased with omega-agatoxin, however the decrease was greater in the older group (mean = 60% of control vs. 40% of control in the younger group). A larger number of fibers in the older group (84% vs. 54% in the young) showed a > or = 50% decrease in epp amplitude. These data suggest that although P-type Ca2+ channels are present in the immature presynaptic nerve terminals at the neuromuscular junction, functional maturation of these channels occurs with development. This may contribute to the susceptibility to neuromuscular transmission failure in the newborn diaphragm.

Rhinovirus infection associated with serious lower respiratory illness in patients with bronchopulmonary dysplasia.

OBJECTIVE: To determine the characteristics of rhinovirus infection in patients with bronchopulmonary dysplasia. SUBJECTS AND METHODS: Between July 1, 1993, and July 1, 1995, 40 patients with bronchopulmonary dysplasia were identified. Viral cultures were obtained in ambulatory patients presenting with an acute respiratory illness requiring hospitalization or in hospitalized patients with a respiratory deterioration. When rhinovirus was isolated epidemiologic data were collected, and the characteristics of the illness, its severity and outcome were noted. Key features of rhinovirus and respiratory syncytial virus (RSV) bronchiolitis were compared. RESULTS: There were 8 cases of lower respiratory tract illness associated with rhinovirus infection in 6 infants (mean age, 7.1 +/- 4.1 months) and 1 child (age, 40 months), an incidence of 0.15 infection/patient year. The mean gestational age and birth weight of these patients were 27.3 (+/- 2.75) weeks and 853 (+/-341) g, respectively. There were 5 males. Four patients needed intensive care unit admission and 1 required mechanical ventilation. By comparison there were 13 cases of RSV bronchiolitis, an incidence of 0.25 infection/patient year. The 2 groups were similar epidemiologically and an equal proportion of patients with rhinovirus and RSV needed intensive care unit admission. A greater percentage of patients with RSV required mechanical ventilation (50% vs. 14%), but this difference was not statistically significant. Three cases of rhinovirus were nosocomial, and 1 infant had a second infection. Four patients required 5 hospitalizations caused by rhinovirus infection, and the mean duration of hospital stay was 11 days. All children had sustained worsening in their respiratory status after rhinoviral illness requiring additional therapy. CONCLUSIONS: Rhinovirus is a common and potentially serious lower respiratory pathogen in bronchopulmonary dysplasia patients. Rhinovirus infection has lasting pulmonary sequelae in these children.

Effect of inspiratory training on mitochondrial DNA and cytochrome-c oxidase expression in the diaphragm.

We have previously shown that respiratory training with inspiratory flow-resistive (IFR) loads improves diaphragm performance and is associated with an increase in cytochrome-c oxidase (COX) activity (1). The present study was conducted to define the level at which the increase in COX activity is controlled. Six sheep were trained with IFR loads for 3 h/day for 3 wk. The diaphragm was sampled from the six trained sheep and from six control sheep. Quantitative DNA and RNA slot-blot analyses with mitochondrially coded COX subunit III and nuclearly coded subunit IV probes and immunoblotting with anti-COX holoenzyme antibodies were performed. We found that in the diaphragm the amount of COX subunit proteins coded in either genetic system was greater in the trained than in the control sheep. Neither the amount of mitochondrial DNA nor mRNA for COX subunits was different between the two groups. We conclude that the increase in COX activity in the diaphragm after chronic respiratory training is determined by the amount of subunit proteins, possibly involving translation/degradation of these proteins.

Developmental changes in rat diaphragm endplate response to repetitive stimulation.

Endplate potentials (epps) were recorded intracellularly from single diaphragm fibers, in vitro, of newborn (< or = 10 days, n = 10) and older (18-29 days, n = 5) rats with glass microelectrodes. The muscle was stimulated via the phrenic nerve for 1 s at 10, 20 or 50 Hz. Muscle action potentials were blocked by mu conotoxin GIIIA, a specific muscle Na+ channel blocker. In diaphragms from older animals, epps followed nerve stimulation at the 3 frequencies, with a gradual decrement in amplitude to 70% of the first epp at 50 Hz. The younger age group showed an initial enhancement of epp amplitude, followed by large variability in amplitude. These data suggest that neuromuscular transmission failure in the newborn diaphragm is secondary to variability in neurotransmitter release as compared to the more mature diaphragm.

Effect of chronic respiratory loading on the subunit composition of cytochrome c oxidase in the diaphragm.

To elucidate in the diaphragm, 1) whether chronic inspiratory loading increases the amount of cytochrome c oxidase (COX) subunit proteins, and 2) how well the regulation of mitochondrially and nuclearly coded COX subunits is coordinated, we have trained six adult sheep with inspiratory flow-resistive loads for 3 h/day for 3 wk. Six other sheep served as controls. Proteins from crude muscle homogenates were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotted, and reacted with polyclonal rabbit anti-bovine COX antibodies. A mitochondrially coded subunit (II) and nuclearly coded subunits (IV and VII) reacted with anti-COX antibodies and were quantified with laser densitometry using purified COX as a standard. In the costal diaphragm and for the equivalent amount of muscle homogenate protein, the integrated optical densities (IOD) for subunits II, IV, and VII were significantly greater in the trained sheep than in the controls. Similarly, the IOD for subunits II and VII were significantly greater in the trained than in the controls in the crural diaphragm. There were no differences between the two groups in the quadriceps, a muscle that was used as an untrained, internal control muscle. The ratios of the IOD for each of the two nuclearly coded subunits to that for mitochondrially coded subunit II were not different between the two groups. These data suggest that chronic inspiratory loading increases both mitochondrially and nuclearly coded COX subunits in the diaphragm and that the subunits coded by the two genetic systems are coordinately regulated.

Effect of hypoxia on neuromuscular transmission in the developing diaphragm.

To study the effects of hypoxia on neuromuscular transmission in the developing diaphragm, phrenic nerve-hemidiaphragm preparations were obtained from newborn (4-9 days) and older (22-30 days) rats. Diaphragms were stimulated directly or indirectly (via the nerve) for 1 s at frequencies of 10-80 Hz. Force generated in response to stimulation was measured during perfusion of oxygenated Ringer solution (control) and Ringer solution bubbled with 95% N2-5% CO2 (hypoxia). After 45 min of hypoxia, the force response of the older diaphragms to direct stimulation had decreased to approximately 50% of control at > or = 40 Hz; however, when stimulation occurred via the nerve at these frequencies only 15-20% of control force was generated. In the newborn diaphragms, the force decrement after similar or longer periods of hypoxia (< or = 90 min) was 30- 40% irrespective of the route or frequency of stimulation. After 15 min of reoxygenation, the force response to both muscle and nerve stimulation recovered completely in the older diaphragms but only partially in the newborn diaphragms (range 77% of control at 50 Hz to 95% of control at 10 Hz). These data suggest that in the newborn diaphragm 1) neuromuscular transmission is more resistant to the effects of hypoxia than the older diaphragm and 2) the predominant effect of hypoxia is peripheral in the diaphragm muscle fibers, whereas in the older diaphragm the effect is before or at the neuromuscular junction.

Diaphragmatic failure during loaded breathing: role of neuromuscular transmission.

To determine whether central or peripheral mechanisms are responsible for diaphragmatic failure during loaded breathing, phrenic nerve activity (iENG), diaphragm muscle electromyogram (iEMG), and transdiaphragmatic pressure (Pdi) were measured in unanesthetized chronically instrumented sheep during inspiratory flow-resistive (IFR) loaded breathing. After placement of the IFR load, Pdi increased initially and remained relatively stable for 10-30 min [Pdi = 69.9 +/- 6.3 (SE) cmH2O, n = 6]; arterial PCO2 also increased from baseline (35.8 +/- 0.9 Torr) to 55.1 +/- 4.7 Torr. During IFR loading, iEMG and iENG also increased from baseline, but during the plateau phase of Pdi, iENG continued to increase at the same time while iEMG was stable, and the M wave, evoked by phrenic nerve stimulation, decreased during this period. After the plateau phase, Pdi decreased and arterial PCO2 increased, at which point the study was terminated (at 82.1 +/- 20.6 min). The observation that iENG increased while Pdi and iEMG were stable demonstrates a reduced efficiency of neuromuscular transmission and suggests that the neuromuscular junction is an important site of diaphragmatic failure in unanesthetized sheep during IFR loaded breathing.

Blood flow to respiratory muscles and major organs during inspiratory flow resistive loads.

To determine whether diaphragmatic fatigue in the intact animal subjected to loaded breathing is associated with a decrease in diaphragmatic blood flow, seven unanesthetized sheep were subjected to severe inspiratory flow resistive (IFR) loads that led to a decrease in transdiaphragmatic pressure (Pdi) and a rise in arterial PCO2 (PaCO2). Blood flow to the diaphragm, other respiratory muscles, limb muscles, and major organs was measured using the radionuclide-labeled microsphere method. With these loads blood flow increased to the diaphragm (621 +/- 242%) and all the other inspiratory and expiratory diaphragm (621 +/- 242%) and all the other inspiratory and expiratory muscles; there was no statistically significant change in blood flow to these muscles at the time when Pdi decreased and PaCO2 rose. Blood flow also increased to the heart (103 +/- 34%), brain (212 +/- 39%), and adrenals (76 +/- 9%), whereas pancreatic flow decreased (-66 +/- 14%). Limb muscle blood flow remained unchanged. We conclude that in unanesthetized sheep subjected to IFR loads 1) we did not demonstrate a decrease in respiratory muscle blood flow associated with diaphragmatic fatigue and ventilatory failure, and 2) there is a redistribution of blood flow among major organs.

Failure to generate action potentials in newborn diaphragms following nerve stimulation.

Action potentials were recorded intracellularly from single diaphragmatic fibers, in vitro, of newborn (3-10 d, n = 18) and older (> or = 21 d, n = 10) rats using flexible microelectrodes. At 20 and 50 Hz phrenic nerve stimulation (1 s duration), action potential transmission failure was significantly higher in the newborn than in the older fibers. During the failure periods, small and highly variable depolarizations were observed which were most likely EPPs. These results show that failure of action potential transmission across the neuromuscular junction is more prevalent in the newborn, and we speculate that this failure is due to inadequate release of neurotransmitter in newborn muscle fibers.

Glycogen content in neonatal diaphragmatic fibers in response to inspiratory flow resistive loads.

To study intramuscular glycogen use with increased work loads in the neonatal diaphragm, unanesthetized lambs were subjected to severe inspiratory flow resistive loads. With these loads, transdiaphragmatic pressure and arterial CO2 tension increased several fold above baseline and then remained stable for a period of 10-30 min. This was followed by a decrease in transdiaphragmatic pressure and a marked increase in arterial CO2 tension with severe acidosis. Intramuscular glycogen content was determined histochemically by the periodic acid-Schiff reaction and was quantified microphotometrically by the fiber types (type I, IIa, or IIc) present in the lamb costal diaphragm. Glycogen content in the control diaphragm was greatest in type IIa fibers and lowest in type I fibers. With severe inspiratory flow resistive loads, the greatest decrease in glycogen content was in type IIa fibers, followed by type IIc and type I fibers. Therefore, our data suggest that in the neonatal lamb subjected to inspiratory flow resistive loads the diaphragm uses intramuscular glycogen during increased work loads. These data do not indicate that the marked depletion of glycogen in the diaphragm is the cause of the decrease in diaphragmatic force.

Effect of chronic respiratory load on cytochrome oxidase activity in diaphragmatic fibers.

To determine whether the increase in oxidative capacity after respiratory muscle training with chronic inspiratory loads in sheep is specific to a particular fiber type, we measured cytochrome c oxidase (COX) activity in type I and type II fibers. COX activity in individual fibers was examined histochemically and measured as relative optical density by use of an image processing system. Fiber types were differentiated by the myosin adenosine-triphosphatase reaction. We found that COX activity was higher in both fiber types in the trained diaphragms than in the control diaphragms (P less than 0.01). The increase with training was greater in type II (39%) than in type I fibers (21%), resulting in relatively homogeneous COX activity in all diaphragmatic fibers. The proportion of type I fibers increased from 43.4 +/- 5.4% in the control diaphragm to 53.1 +/- 2.9% in the trained diaphragm, whereas the proportion of type II fibers decreased (P less than 0.001). We conclude that respiratory muscle training activates oxidative enzyme activity in both diaphragmatic fiber types; this activation is differentially more in type II fibers, which also decrease in proportion, and less in type I fibers, which increase in proportion.

Developmental changes in neuromuscular transmission in the rat diaphragm.

Neuromuscular transmission was studied in diaphragms from rats of three ages, 4-7 days old, 11-12 days old, and adults with the use of an in vitro phrenic nerve-hemidiaphragm preparation. Each hemidiaphragm was stimulated via either muscle or nerve with 1-s stimulus trains at frequencies from 10 to 100 Hz. The patterns of force development obtained in response to the two routes of stimulation were compared for each group. Diaphragms from adults developed maximum force in response to stimulation of approximately 40 Hz with no significant decrease in force at higher frequencies. Within each stimulus train, once peak force was achieved, it was maintained for the remainder of the stimulus and responses to nerve and muscle stimulation were almost identical. In contrast, diaphragms from 4- to 7-day-old rats developed maximum force at approximately 20 Hz; stimulation at greater than or equal to 60 Hz induced significantly less peak force. This decrease in peak force at higher frequencies was significantly larger for nerve than for muscle stimulation. In addition, during each nerve stimulus train diaphragms from 4- to 7-day-old rats were unable to maintain peak force, which decreased at frequencies greater than 20 Hz. The decrease in force reached approximately 50% of peak at stimulation frequencies greater than or equal to 60 Hz. Diaphragms from 11- to 12-day-old rats showed intermediate responses. Based on the responses to phrenic nerve stimulation, we conclude that the neonatal rat diaphragm shows marked neuromuscular transmission failure that is not seen in the adult.(ABSTRACT TRUNCATED AT 250 WORDS)

O2 metabolism of the sheep diaphragm during flow resistive loaded breathing.

To determine whether O2 availability limited diaphragmatic performance, we subjected unanesthetized sheep to severe (n = 11) and moderate (n = 3) inspiratory flow resistive loads and studied the phrenic venous effluent. We measured transdiaphragmatic pressure (Pdi), systemic arterial and phrenic venous blood gas tensions, and lactate and pyruvate concentrations. In four sheep with severe loads, we measured O2 saturation (SO2), O2 content, and hemoglobin. We found that with severe loads Pdi increased to 74.7 +/- 6.0 cmH2O by 40 min of loading, remained stable for 20-30 more min, then slowly decreased. In every sheep, arterial PCO2 increased when Pdi decreased. With moderate loads Pdi increased to and maintained levels of 40-55 cmH2O. With both loads, venous PO2, SO2, and O2 content decreased initially and then increased, so that the arteriovenous difference in O2 content decreased as loading continued. Hemoglobin increased slowly in three of four sheep. There were no appreciable changes in arterial or venous lactate and pyruvate during loading or recovery. We conclude that the changes in venous PO2, SO2, and O2 content may be the result of changes in hemoglobin, blood flow to the diaphragm, or limitation of O2 diffusion. Our data do not support the hypothesis that in sheep subjected to inspiratory flow resistive loads O2 availability limits diaphragmatic performance.

Metabolic and functional adaptation of the diaphragm to training with resistive loads.

To study the metabolic and functional changes that occur during training with inspiratory flow resistive loads, a chronically instrumented unanesthetized sheep preparation was used. Sheep were exposed to resistances ranging from 50 to 100 cmH2O.l-1.s, for 2-4 h/day, 5-6 days/wk, for a total of 3 wk. Load intensity was adjusted to maintain arterial Po2 (PaO2) above 60 Torr and arterial PCO2 (PaCO2) below 45 Torr. Training produced significant (P less than 0.05) increases in citrate synthase, 3-hydroxyacyl-CoA dehydrogenase, and cytochrome oxidase in the costal and crural diaphragm of the trained sheep (n = 9) compared with control sheep (n = 7). Phosphofructokinase did not increase. In the quadriceps, citrate synthase, 3-hydroxyacyl-CoA dehydrogenase, and phosphofructokinase did not change with training but cytochrome oxidase increased significantly (P less than 0.01). Function of the diaphragm was assessed in a subset of five sheep exposed to the same severe load 1 wk before and 2 days after the final training session. After training, sheep had a lower PaCO2 (10-40%), generated a higher transdiaphragmatic pressure (20-40%), and could sustain this level of transdiaphragmatic pressure for 0.5-2 h longer. The respiratory duty cycle was 10-15% lower, whereas minute ventilation and tidal volume were 20-30% higher in the posttraining test. We conclude that 1) training with inspiratory flow resistive loads improves the performance of the respiratory neuromuscular system and 2) the shift in enzyme profile of the diaphragm is at least in part responsible for this improvement.

Respiratory muscle response to load and glycogen content in type I and II fibers.

To study the relation between the response of respiratory muscle to inspiratory loads and glycogen content, we subjected unanesthetized sheep to moderate and severe inspiratory flow resistive (IFR) loads. Only severe IFR loads eventually led to a decrease in transdiaphragmatic pressure (Pdi) and a concomitant rise in PaCO2. Respiratory and nonrespiratory skeletal muscle samples were obtained at necropsy. Glycogen content was determined biochemically in muscle homogenates. Frozen sections were stained with periodic acid-Schiff (PAS) for glycogen and fibers were typed using myosin ATPase stain. Fibers were categorized as full, intermediate, or devoid of glycogen by a subjective scoring system of PAS staining intensity. We found that glycogen content decreased in the costal and crural diaphragm and in the intercostal muscles as the duration of moderate IFR loaded breathing was increased. With severe loads glycogen content decreased significantly, reaching about 40 and 22% of control levels in the costal and crural diaphragm, respectively (P less than 0.01). In addition, with severe IFR loads, a statistically significant proportion of both type I and type II muscle fibers was depleted of glycogen when compared with that of controls (P less than 0.05), but more type II fibers were depleted than type I fibers (50 vs 23%). These data indicate that in sheep subjected to IFR loads: (1) glycogen content in the respiratory muscles decreases as the severity and duration of loaded breathing increases and (2) respiratory muscle fatigue occurs at a time when considerable glycogen is still present in type I fibers in the diaphragm.

Within-breath electromyographic changes during loaded breathing in adult sheep.

To investigate the changes in diaphragm electromyogram (EMG) during the course of severe loaded breathing, we subjected five conscious adult sheep to inspiratory flow resistive breathing (resistance greater than 150 cmH2O X l-1 X s) for up to 2-3 h and studied the total EMG power per breath (iEMG) and the EMG power per unit time after dividing the duration of EMG activity within each breath into three equal parts (iEMG1, iEMG2, and iEMG3). Both total breath iEMG and transdiaphragmatic pressure (Pdi) increased, remained at a high level for a certain period of time, and then started to fall. A change in the pattern of iEMG within a breath was observed during loaded breathing. The increase in total-breath iEMG was associated mostly with an increase in iEMG3, or the last part of the EMG power within each inspiration. Similarly, the decrease in total breath iEMG was primarily due to a decrease in iEMG3. We conclude that, in sheep subjected to severe IFR loads for prolonged periods the marked increase in total-breath iEMG at the beginning of loaded breathing and the marked decrease in this iEMG at the time of decrease in Pdi are largely due to changes in iEMG that occur during the latter third of each breath. We speculate that during loaded breathing the recruitment pattern of diaphragmatic muscle fibers changes during the course of an inspiratory effort.

Increase in electromyogram low-frequency power in nonfatigued contracting skeletal muscle.

We studied the relationship between changing elbow joint angle and the power spectral density of the biceps brachii muscle electromyogram (EMG) during submaximal isometric contractions. For this purpose, we recorded the EMG of the biceps brachii muscle with surface electrodes in 13 subjects. Each subject held a 2.8-kg weight and contracted the biceps isometrically for 30 s at one of two lengths. The length of the muscle was changed by flexing the forearm toward the upper arm to form an angle of 135 degrees (L1) or 45 degrees (L2). We found that the mean centroid frequency (fc) of the EMG power spectral density was 26% lower at L1 than at L2 (P less than 0.01). For each subject there was no significant change in fc during the isometric contraction at either angle. In addition, in nine subjects who sustained fatiguing contractions of the biceps with a 6-kg load, fc decreased by 15% (P less than 0.025). These data suggest that a change in the length at which a muscle contracts isometrically can alter or induce indirectly an alteration in the frequency content of its EMG. This finding may have important implications for the assessment of respiratory muscle EMG especially during loaded breathing.