INTRODUCTION

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We have a long-standing interest in the response of double-lung recipients to whole-body exercise. Exercise is an integral component of postoperative therapy, and exercise testing is routinely used to monitor the clinical progress of these patients. Maximal exercise capacity generally is diminished in double-lung recipients (1). Within the limited exercise range that double-lung recipients achieve, however, their ventilatory response to these levels of exercise is near-normal (1) despite denervation of central airways and lung parenchyma. Thus, it appears that pulmonary inputs to the respiratory centers are not involved in regulating the ventilatory response to exercise or that their role can be subserved by other ventilatory control mechanisms (2).

We became interested in the role of lung denervation during exercise. To the extent that pulmonary mechanoreceptors modulate ventilatory control, we reasoned that double-lung recipients and intact subjects would respond differently if respiratory mechanics were perturbed. The present study tested this postulate. We examined the acute ventilatory response of double-lung recipients to an expiratory threshold load that was abruptly imposed during exercise. Our postulate predicted that transplant recipients and intact subjects would respond to expiratory loading differently. Abnormalities in the ventilatory response of transplant recipients would demonstrate a persistent influence of lung denervation on neural control of breathing in these patients.

	METHODS

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Subjects

The study was conducted on eight patients (three women, five men) who had undergone double-lung transplant at least 6 mo before the test. Nine healthy subjects (two women, seven men) served as controls. Anthropometric data are shown in Table 1. Two transplant recipients and four healthy subjects were current smokers. The diseases of the recipients were cystic fibrosis (four patients), emphysema associated with -1 antitrypsin deficiency (three patients), and bronchiolitis obliterans (one patient). All transplant recipients were receiving chronic immunosuppressive therapy comprising oral cyclosporine adjusted to maintain serum levels of 200 to 250 ng/ml combined with either depomedrol 40 to 80 mg/wk by intramuscular injection (cystic fibrosis patients) or oral prednisone 10 to 15 mg/d (all other patients). The protocol was approved by the Institutional Review Board for Human Subject Research for Baylor College of Medicine and Affiliated Hospitals. All participants gave informed, written consent prior to the study.

Protocol

On a first occasion (screening day), each subject visited the laboratory for clinical history and examination, spirometry, electrocardiogram (ECG), and measurement of maximal voluntary ventilation (MVV). FVC and FEV₁ were calculated according to guidelines of the American Thoracic Society. MVV was measured by asking the subjects to breathe as much as they could for 12 s; predicted values later were computed according to Miller and colleagues (3). Informed consent was obtained on this initial visit.

On the study day, the subject reached the laboratory at least 1 h before testing. Blood pressure and heart rate were measured. In a subset of subjects (five transplant recipients, five control subjects), a thin latex balloon 10 cm long attached to a polyethylene catheter was positioned in the esophagus 42 to 45 cm from the nostril in order to measure esophageal pressure. The balloon was connected to a pressure transducer (Model PM131; Statham Instruments, Gould, Oxnard, CA) via the catheter and was filled with 1 ml of air. Position of the balloon in the esophagus was checked according to the method of Milic-Emili and colleagues (4). Transpulmonary pressure (Ptp) was estimated during tidal breathing as the difference between mouth and esophageal pressures.

A symptom-limited exercise test was performed on a cycle ergometer (Ergometrics 800S; SensorMedics, Yorba Linda, CA) by incrementing the load 20 Watts every 2 min. The ECG was monitored continuously. Subjects wore noseclips and breathed through mouthpieces attached to a low-resistance Hans Rudolph two-way valve (deadspace approximately 90 ml) (Hans Rudolph, Kansas City, MO). Oxygen uptake (O₂) and carbon dioxide output (CO₂) were continuously measured for the first minute of each exercise level using an inline gas analyzer (Model 2900; SensorMedics). Inspiratory and expiratory flows were measured at the mouth using two Fleisch no. 3 pneumotachographs coupled to differential pressure transducers (Model LCVR, 0-5 cm H₂O; Celesco Instruments, Canoga Park, CA). Volumes were obtained by integrating the flow signals; drift was suppressed by adjusting a potentiometer. Resistances of the inspiratory and expiratory circuits (1.1 cm H₂O/L/s) were linear up to a flow of 3 L/s. Flow, volume, mouth pressure, and Ptp were displayed on a strip chart recorder and stored in real time (sampling frequency, 66 Hz) using a computer (Model 11/73; Digital Electronics Corp., Maynard, MA).

Expiratory threshold loading was imposed for five to six tidal breaths during the second minute of each step of the test. A positive expiratory pressure of 4 cm H₂O was applied by diverting expiratory flow through a Starling resistor composed of collapsible latex tubing within a rigid, pressurized chamber. This device did not alter the pressure-flow relationship of the expiratory circuit when upstream pressures exceeded threshold.

All measurements were made before (C1), during (L), and after (C2) applying the threshold load. The two control phases (C1 and C2) allowed us to detect and correct drift in the volume signal. Each phase lasted approximately 20 s or five to six breaths. O₂ and CO₂ were not measured during Phase L. At the end of Phase C2, the subject performed a forced expiration from end-tidal inspiration to residual volume and then performed a maximal inspiration to TLC. This maneuver allowed for changes of expiratory flow reserve (i.e., the difference between forced and tidal expiratory flow near end-expiration) and inspiratory capacity (IC) to be calculated at each step of the exercise test.

Data Analysis

Irregular breaths and swallows were discarded from the analysis. Breathing pattern was computed for C1, L, and C2 conditions by averaging tidal volume (VT), breathing frequency (f), inspiratory and expiratory time (TI, TE), mean inspiratory and expiratory flow (VT/TI, VT/ TE), ratio of inspiratory time to total respiratory cycle duration (TI/ Ttot), and expiratory minute ventilation (E) over five to six tidal breaths. Changes induced by expiratory loading at each step of exercise were averaged to obtain a single mean value for each subject; individual means were averaged to obtain a group mean; differences between groups were tested by comparing the average responses measured during maximal exercise. Changes in breathing pattern during exercise were compared between groups using a normalization strategy similar to that of Sciurba and coworkers (5); VT, E, and IC were divided by FVC to obtain values normalized for lung volume (5); regression analyses were used to assess changes in f, TI, TE, TI /Ttot, VT/ FVC, and IC/FVC relative to E/FVC. As observed by Sciurba and coworkers (5), all relationships were well fitted by a linear model except VT/FVC versus E  /FVC; we therefore limited this regression to values of E /FVC < 11 or to data measured before the plateau of VT/ FVC, whichever occurred first. Normalized values for end-inspiratory lung volume (VE I) were computed as (IC-VT)/FVC.

Statistical Analysis

Student's paired and unpaired tests were used to compare individual variables within and between groups, respectively. Correlations were assessed using Pearson's test; p < 0.05 was considered statistically significant. All values are expressed as means ± SE.

	RESULTS

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Pulmonary Function

Among individual transplant recipients, three had FEV₁ > 80% predicted and FEV₁/FVC > 75%; four had a FEV₁ < 80% predicted with FEV₁/FVC > 75%; one had a FEV₁/FVC < 70%. Among control subjects, two current smokers had a FEV₁/FVC < 70%; all others had normal spirometric parameters. The averaged data are shown in Table 1. Transplant recipients had systematically lower values for FVC and FEV₁, whereas mean FEV₁/FVC exceeded that measured in control subjects.

Cardiopulmonary Responses to Exercise

Double-lung recipients had poor exercise tolerance. Maximal power output and O₂max averaged less than half the values measured in healthy subjects (Table 2). All patients complained of leg fatigue and pain at the end of exercise, suggesting that deconditioning likely was the main limiting factor. Healthy subjects showed normal exercise tolerance as assessed by O₂max. Three control subjects complained of dyspnea at the end of the test; six complained of leg muscle fatigue. HRmax was significantly less than predicted in both groups (p < 0.001) suggesting that the cardiovascular system did not limit exercise in our 2-min incremental protocol.

The ventilatory response to exercise was comparable between groups. At submaximal levels, transplant recipients and control subjects increased ventilation using similar strategies. Both breathing frequency (Figure 1) and tidal volume (Figure 2) were elevated progressively; tidal volume reached a plateau near the end of exercise in three double-lung recipients and in five control subjects. No differences were detected between groups in either the slopes or the intercepts of linear regressions fitted to f versus E/FVC, VT/FVC versus E/FVC, IC versus E/FVC, TI versus E/FVC, TE versus E/FVC, or VT/ TI versus E/FVC (data not shown). During maximal exercise, transplant recipients breathed less than control subjects did (Table 2); minute ventilation was depressed in proportion to the low O₂max measured in this group. The decrement in ventilation was due primarily to smaller tidal volumes in transplant recipients; ventilatory timing (f, TI/Ttot) was similar to that in control subjects. End-inspiratory lung volume (VEI) was nearly identical between groups; differences in Ptp were not significant.

View larger version (17K): [in this window] [in a new window]   Figure 1.   Adjusted tidal volume (VT/FVC) is plotted against adjusted minute ventilation ( E/FVC) for each intact subject (upper panel ) and double-lung recipient (lower panel ) during incremental exercise. The relationship was nonlinear in five intact subjects and in three transplant recipients because VT did not increase at higher exercise levels.

View larger version (16K): [in this window] [in a new window]   Figure 2.   Breathing frequency (BF, in breaths/min) is plotted against adjusted minute ventilation ( E/FVC) in intact subjects (upper panel ) and double-lung recipients (lower panel ) during incremental exercise.

Ventilatory Loading during Maximal Exercise

The response to expiratory threshold loading was assessed during maximal exercise. Both groups defended tidal volume and adopted slower breathing frequencies, thereby decreasing minute ventilation (Table 3); both groups also lengthened expiration to accommodate slower expiratory flow rates. The two groups regulated inspiration quite differently, however. Control subjects maintained inspiratory time and inspiratory flow rate constant such that TI/Ttot decreased. In contrast, transplant recipients lengthened inspiratory time and exaggerated the overall drop in breathing frequency. This response was evident in the first breath after load imposition; TI was significantly prolonged in transplant recipients compared with that in control subjects (p < 0.001), whereas changes in VT and TE did not differ between groups (data not shown). Several other responses to loading also were group-specific. Transplant recipients increased end-inspiratory Ptp, whereas control subjects terminated expiration at higher lung volumes (0.22 ± 0.06 L > unloaded lung volume).

	DISCUSSION

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The main finding of this study is that double-lung transplant recipients responded abnormally to expiratory loading during exercise. Control subjects retarded expiration only, a response predicted by previous studies (6). In contrast, double-lung recipients lengthened inspiration as well as expiration, which exaggerated the overall drop in minute ventilation caused by loading.

Double-Lung Recipients

Double-lung recipients were not different from the control group in height, age, or sex, but they did exhibit differences in pulmonary function. Pulmonary function values measured in the transplant recipients were within normal limits and were virtually identical to those measured in a separate population of double-lung recipients by Levy and coworkers (1). Within our study, transplant recipients had lower FVC and FEV₁ than did control subjects and FEV₁/FVC was greater than in control subjects. The most likely explanation for these differences is that our transplant recipients may have experienced a mild degree of respiratory muscle weakness as observed in previous studies of lung allograft recipients (10).

As also reported by Levy and coworkers (1), the exercise tolerance of our transplant recipients was severely limited. Peak power and maximal oxygen consumption were less than half that of the control group. Maximal heart rates achieved by both groups were less than 85% predicted, which suggests that exercise was not limited by cardiovascular function. Cardiac denervation can occur during double-lung transplantation, however, and loss of autonomic innervation would have limited the cardiac response to exercise in transplant recipients. Nor did ventilation appear to limit exercise. Spirometric values measured in transplant recipients were in the normal range and these patients retained as much ventilatory reserve during maximal exercise (67% MVV) as did the control subjects (58% MVV). Rather, it is likely that task performance was limited by limb muscle endurance. All transplant recipients reported that they stopped exercise because their legs were tired. This is consistent with previous reports that transplant recipients have lower exercise tolerance because their muscles are relatively deconditioned (11, 12).

Breathing during Exercise

The double-lung recipients studied in these experiments used near-normal breathing strategies during incremental exercise. The pattern by which transplant recipients increased ventilationprogressive increases in both tidal volume and breathing frequencywas indistinguishable from that of the control subjects. Breathing during maximal exercise also was similar between groups, except that transplant recipients took shallower breaths and had lower minute ventilation than did control subjects. The decrement in minute ventilation of transplant recipients (52%) was proportional to reductions in maximal oxygen consumption (53%) and power output (60%). These findings agree with a similar comparison of double-lung recipients and intact control subjects by Levy and coworkers (1) who also observed decrements in tidal volume and minute ventilation of transplant recipients during maximal exercise. Relative to intact control subjects, reductions in ventilation of the double-lung recipients studied by Levy and coworkers (46%) reflected decreases in maximal oxygen consumption (54%) and peak power output (55%). Thus, the ventilatory response of double-lung recipients to incremental exercise appears to be proportional to the metabolic challenge and is not obviously distorted by pulmonary denervation.

Heart-lung transplant recipients also have been studied to assess the ventilatory response to exercise after lung denervation. Banner and coworkers (13) compared heart-lung recipients, heart-only recipients, and intact control subjects; the ventilatory response to the onset of exercise was not different among groups; they concluded that afferent neural information from the lungs and heart were not essential for this response. Sciurba and coworkers (5) compared heart-lung versus heart-only transplant recipients; both groups attained similar maximal exercise levels and maintained adequate ventilation; heart-lung recipients used a different pattern of breathing, which was attributed to loss of pulmonary afferents. Kimoff and colleagues (14) compared heart-lung recipients with intact control subjects; transplant recipients achieved lower maximal oxygen consumptions but had a higher ventilatory response to increasing CO₂ production. Theodore and coworkers (15) reported longitudinal measurements on heart-lung transplant recipients; maximal oxygen consumption increased after transplant, and the ventilatory responses to CO₂ production and oxygen consumption were blunted. Each of these studies used a different protocol and each reached a different conclusion. Thus, data from heart-lung recipients do not identify a robust effect of lung denervation on exercise ventilation, although subtle perturbations may be detectable under specific experimental conditions.

Response to Expiratory Loading

The ventilatory response to expiratory loading is thought to depend on compensatory reflexes and the impedance of the respiratory system (8, 14). The acute response typically includes a slowing of expiratory flow and a lengthening of expiratory time such that breathing frequency and minute ventilation fall (8). The intact subjects in our study exhibited this response.

The ventilatory response to expiratory loading has not been tested in humans after lung transplantation. Double-lung recipients decreased expiratory flow and prolonged expiratory time in the same manner as intact subjects. This suggests that adjustments to expiration did not depend critically on information from lung receptors. Unlike intact subjects, double-lung recipients also altered inspiration when expiratory loading was imposed. Transplant recipients lengthened inspiratory time and tended to slow inspiratory flow, thereby diminishing breathing frequency more markedly than intact subjects. The observation that intact subjects defended inspiratory timing, whereas double-lung recipients did not, suggests that lung receptors mediate this response.

What other pathway may have mediated inspiratory slowing in double-lung recipients? One stimulus unique to this group was an increase in inspiratory impedance. Unlike intact subjects, the expiratory load caused double-lung recipients to develop higher transpulmonary pressures during inspiration. Higher pressures required greater tension development by the diaphragm and inspiratory intercostal muscles. Afferent pathways from these muscles remain intact after lung transplantation, and an increase in developed tension would have been sensed by mechanoreceptors in the muscles, e.g., Golgi tendon organs (16). Increased mechanoreceptor output cannot explain the aberrant response of transplant recipients, however. The reflex response to inspiratory elastic loading is a fall in tidal volume and a rise in breathing frequency (8); transplant recipients had the opposite response.

Differences in inspiratory strategy also may have been corticogenic, i.e., transplant recipients may have consciously chosen to use a longer inspiratory time than intact subjects. Several factors in the present experiments strongly argue against a conscious response. First, the increase in inspiratory time occurred in the first loaded breath. First-breath responses are typical of proprioceptive reflexes; chemoreceptor-mediated reflexes and conscious responses usually require more time. Moreover, subjects could not consciously prepare for a first-breath response because they were unaware that the load was about to be imposed; a screen shielded the expiratory resistor and its operator from view. Second, subjects were blinded to the purpose of the study and did not discuss the experiment with other participants. The likelihood that these two groups would have randomly altered inspiratory timing in the observed manner is two chances in 100. Third, differences between transplant recipients and control subjects were tested during maximal exercise, a condition in which automatic mechanisms predominate in the neural control of breathing and conscious control is minimized.

Conclusion

The ventilatory response of double-lung transplant recipients to incremental exercise is nearly identical to that of intact subjects. Despite the appearance of normality, aberrations in the neural control of breathing can be demonstrated by the abrupt imposition of expiratory threshold loading during exercise. Double-lung recipients and intact subjects both respond to loading by slowing expiration; however, unlike intact subjects who defend inspiratory timing, double-lung recipients prolong inspiration during loading. These findings suggest that afferent information from pulmonary mechanoreceptors is essential for normal regulation of inspiratory timing. Bilateral denervation disrupts these pathways, compromising inspiratory control and exaggerating losses in minute ventilation caused by resistive loading.