Intercostal muscle blood flow limitation during exercise in chronic. obstructive pulmonary disease

Transcription

1 Page 1 of 43 AJRCCM Articles in Press. Published on July 9, 2010 as doi: /rccm oc Media embargo until 2 weeks after above posting date; see thoracic.org/go/embargo Intercostal muscle blood flow limitation during exercise in chronic obstructive pulmonary disease Ioannis Vogiatzis 1,2, Dimitris Athanasopoulos 1,2, Helmut Habazettl 3,4, Andrea Aliverti 5, Zafiris Louvaris 1,2, Evgenia Cherouveim,2, Harrieth Wagner 6, Charis Roussos 1, Peter David Wagner 6 and Spyros Zakynthinos 1 1 Department of Critical Care Medicine and Pulmonary Services, Evangelismos Hospital, "M. Simou, and G.P. Livanos Laboratories", National and Kapodistrian University of Athens, Greece 2 Department of Physical Education and Sport Sciences, National and Kapodistrian University of Athens, Greece 3 Institute of Physiology, Charité Campus Benjamin Franklin, Berlin, Germany 4 Institute of Anesthesiology, German Heart Institute, Berlin, Germany 5 Dipartimento di Bioingegneria, Politecnico di Milano, Milano, Italy 6 Department of Medicine, University of California San Diego, La Jolla, CA, USA Corresponding author: Dr. Ioannis Vogiatzis, Thorax Foundation, 3 Ploutarhou Str Athens, Greece. Phone: ; Fax: ; gianvog@phed.uoa.gr. 1 Copyright (C) 2010 by the American Thoracic Society.

2 Page 2 of 43 Financial support: This work was supported by Thorax Foundation and by grants from the A. Perotti visiting Professorship fund of the Thorax Foundation. Running title: Respiratory muscle blood flow in COPD Manuscript word count: At a Glance Commentary Scientific knowledge on the subject: In healthy subjects restriction of blood flow to the intercostal muscles during heavy exercise reflects the inability of the circulatory system to meet the increasing energy demands of both respiratory and locomotor muscles. Whether the same mechanism would also apply to patients with chronic obstructive pulmonary disease (COPD), normally experiencing greater work of breathing than healthy subjects during exercise, remains currently unknown. What the study adds to the field: The study demonstrates that intercostal muscle blood flow is restricted during high-intensity exercise, whilst quadriceps muscle perfusion is preserved. This happens under conditions of restricted cardiac output, possibly occurring secondary to the increase in expiratory abdominal muscle recruitment and challenges the concept of blood flow redistribution in favour of the respiratory muscles during exercise in patients with COPD. This article has an online data supplement, which is accessible from this issue's table of content online at 2

3 Page 3 of 43 Abstract Rationale: It has been hypothesized that owing to the high work of breathing sustained by patients with chronic obstructive pulmonary disease (COPD) during exercise, blood flow may increase in favour of the respiratory muscles, thereby compromising locomotor muscle blood flow. Objectives: To test the above hypothesis by investigating whether at the same work of breathing, intercostal muscle blood flow during exercise is as high as during resting isocapnic hyperpnoea when respiratory and locomotor muscles do not compete for the available blood flow. Methods: Intercostal and vastus lateralis muscle perfusion was measured simultaneously in ten patients with COPD (FEV 1 =50.5±5.5% predicted) by near-infrared spectroscopy using indocyanine green dye. Measurements and Main Results: Measurements were made at several exercise intensities up to peak work rate (WRpeak) and subsequently during resting hyperpnoea at minute ventilation levels up to those at WRpeak. During resting hyperpnoea, intercostal muscle blood flow increased with the power of breathing to 11.4±1.6 ml. min gr -1 at the same ventilation recorded at WRpeak. Conversely, during graded exercise, intercostal muscle blood flow remained unchanged from rest up to 50% WRpeak (6.8±1.3 ml. min gr -1 ), and then fell to 4.5±0.8 ml. min gr -1 at WRpeak (p=0.003). Cardiac output plateaued above 50% WRpeak (8.4±0.1 l. min -1 ), whilst vastus lateralis muscle blood flow increased progressively, reaching 39.8±7.1 ml. min gr -1 at WRpeak. Conclusions: During intense exercise in COPD, restriction of intercostal muscle perfusion but preservation of quadriceps muscle blood flow along with attainment of a plateau in cardiac output, represent the inability of the circulatory system to satisfy the energy demands of both locomotor and respiratory muscles. 3

4 Page 4 of 43 Word count: 253 words Key words: COPD, exercise, intercostal muscle blood flow, quadriceps muscle blood flow, NIRS. 4

5 Page 5 of 43 Introduction In patients with chronic obstructive pulmonary disease (COPD) exercise intolerance is multifactorial, involving ventilatory, gas exchange, cardiovascular and peripheral muscle abnormalities that ultimately prevent the increased oxygen and carbon dioxide transport demands of the peripheral muscles to be adequately met (1-3). In addition, in the majority of COPD patients, abnormal lung mechanics and expiratory flow limitation during exercise increase the work of breathing several-fold, thereby exacerbating the competition between the respiratory and the locomotor muscles for the available energy/oxygen supplies (4). In this context, unloading the respiratory muscles during exercise (by means of heliox breathing or by proportional assisted ventilation) is associated with improved peripheral muscle oxygenation and exercise performance in COPD (5, 6). These findings have been interpreted to indicate that a significant fraction of the cardiac output, otherwise devoted to the respiratory muscles during exercise, can be redirected to the peripheral muscles. However, actual respiratory or locomotor muscle blood flow measurements were not performed (5, 6). In healthy fit subjects (7) measuring intercostal muscle perfusion (8) revealed that at essentially the same work of breathing blood flow to the intercostal muscles during maximal exercise was lower compared to that during voluntary resting hyperpnoea (i.e.: when the locomotor muscles were inactive). This finding was taken as evidence against the concept of blood flow redistribution in favour of the respiratory muscles during exercise in healthy subjects (9-10). Whether the same finding would apply to patients with COPD, normally experiencing greater work of breathing than healthy subjects both at rest and during exercise is currently unknown. Accordingly, the purpose of the present study was to investigate in patients with COPD whether during exercise intercostal muscle blood flow is different from that recorded 5

6 Page 6 of 43 at the same respiratory muscle load during resting hyperpnoea (i.e.: when respiratory and locomotor muscles do not compete for the available blood flow and thus respiratory muscle blood flow is expected to be the maximal attainable). Simultaneous measurements of intercostal and vastus lateralis muscle blood flow were performed during graded exercise up to maximal levels and during resting hyperpnoea at the same levels of minute ventilation as during exercise. It was reasoned that if intercostal muscle blood flow during exercise was similar to that recorded during resting hyperpnoea and locomotor muscle blood flow was decreased at maximal levels of exercise, this would support the notion that the work of breathing normally incurred during exercise causes preferential redistribution of blood flow from the locomotor to the respiratory muscles (9-10). If however, intercostal muscle blood flow during exercise was lower than during resting hyperpnoea and locomotor muscle blood flow was preserved, this would challenge the concept of blood flow redistribution in favour of the respiratory muscles during exercise in patients with COPD. Methods Subjects Ten patients with clinically stable COPD participated in the study according to certain inclusion criteria (see online supplement) that are detailed in the online data supplement. The study was approved by the University Hospital Ethics Committee. Experimental design Experiments were conducted in two visits. In visit 1, patients underwent a preliminary incremental exercise test to the limit of tolerance (WR peak ). In visit 2, patients initially undertook a graded exercise test to WR peak (protocol 1) which was followed two hours later 6

7 Page 7 of 43 by resting isocapnic hyperpnoea trials (protocol 2) sustained at the same tidal volume, breathing frequency and thus minute ventilation recorded at rest and during exercise. During both protocols, intercostal and vastus lateralis muscle blood flow [assessed by near-infrared spectroscopy (NIRS) and the light-absorbing tracer indocyanine green dye (ICG)] as well as cardiac output, were measured during the final minute of each of the exercise or hyperpnoea trials. In addition, NIRS was utilized to continuously record muscle oxygen saturation (StO 2 ) that is commonly adopted as an index of tissue oxygen availability reflecting the balance between muscle oxygen supply and demand (11). End-inspiratory and end-expiratory compartmental (rib cage and abdominal) chest wall volumes (measured by optoelectronic plethysmography) as well as oesophageal and gastric pressures [to measure tidal excursions in pleural ( Ppl) and gastric ( Pga) pressures, and calculate transdiaphragmatic pressure (Pdi), power of breathing, and the pressure-time product for the diaphragm and expiratory abdominal muscles] were continuously recorded during the exercise and the hyperpnoea trails (details are provided in the online data supplement). Subject preparation Subjects were prepared first with arterial and venous catheters and then with oesophageal and gastric balloons. The catheters were used to inject ICG and sample blood after each injection for cardiac output and muscle blood flow measurements (see online supplement). Protocol 1: graded exercise During graded exercise testing patients completed four bouts of constant-load exercise corresponding to the following targeted intensities: (i) 25% WR peak for 5 min; (ii) 50% WR peak for 5 min; (iii) 75% WR peak for 3-4 min and (iv) 100% WR peak for 2-3 min. Pulmonary gas exchange and ventilatory variables were recorded breath-by-breath, whereas arterial blood pressure was measured every minute (see online supplement). 7

8 Page 8 of 43 Protocol 2: resting isocapnic hyperpnoea trials Patients were asked to maintain targeted ventilation equal to their own mean ventilation recorded at rest and during exercise at 25, 50, 75 and 100% WR peak. Experimenters provided verbal guidance to adjust the rate and depth of their breathing such that the target ventilation was obtained and held constant to within 5%. Isocapnia was maintained by having subjects inspire from a Douglas bag containing 5% CO 2, 21% O 2, balance N 2. Statistical analysis Data are reported as means ± S.E.M, unless otherwise stated. SEM was chosen rather than SD because we were interested in the variance of the mean values rather than the intersubject variance. The minimum sample size was calculated based on 80% power, 5% alpha error and a two-sided 0.05 significance level. Sample size capable of detecting betweencondition (i.e., exercise, hyperpnoea) difference of 30% was estimated for the change in intercostal muscle blood flow at WRpeak using the standard deviations from our previous study (7). The critical sample size was estimated to be nine patients. Two-way ANOVA with repeated measures was used to identify statistically significant differences across the exercise and the hyperpnoea tests. When ANOVA detected statistical significance, pair-wise differences were identified using Tukey s honestly significant difference (HSD) post hoc procedure. Pearson s product moment correlation was used to assess the level of association between continuous variables. The level of significance for all analyses was set at p < Results Subject characteristics and exercise capacity As anticipated from the inclusion criteria, patients had moderate to severe airflow obstruction with increased static lung volumes, moderate reductions in diffusion capacity, and 8

9 Page 9 of 43 mildly altered resting blood gases (Table 1). Four patients were GOLD stage II, three patients were GOLD stage III and the remaining three were GOLD stage IV. Subjects exhibited reduced maximal exercise capacity with moderate oxyhaemoglobin desaturation (Table 2). Power of breathing and respiratory kinematics Power of breathing during exercise and hyperpnoea trials at equivalent rates of minute ventilation was similar (Figure 1A). End-inspiratory and end-expiratory rib cage volumes significantly increased compared to resting values throughout the exercise and hyperpnoea trials (Figure 1C). Rib cage volume changes at the end of inspiration and expiration (reflecting in part, due to the contribution of other respiratory muscles, the activity of the external and internal intercostal muscles, respectively), were not different between the exercise and the resting hyperpnoea trials (Figure 1C). Similarly, volume changes of the abdominal compartment at the end of inspiration (reflecting the activity of the diaphragm) were not different between exercise and hyperpnoea trials (Figure 1D). However, endexpiratory abdominal volume changes (reflecting the activity of the expiratory abdominal muscles) were greater (p=0.037) during exercise than hyperpnoea (Figure 1D). Consequently, end-expiratory total chest wall volume changes were greater (p=0.022) during exercise compared to hyperpnoea (Figure 1B). Peak expiratory gastric pressure (Figure 2A) and pressure-time product for expiratory abdominal muscles (Figure 2D) were also significantly greater (p=0.031 and p=0.022, respectively) during exercise compared to hyperpnoea. Tidal Pdi excursion ( Pdi), pressure-time product for the diaphragm and tidal excursions of gastric over pleural pressure ( Pga/ Ppl) expressing the relative contribution of the diaphragm to the pressure generated by the whole inspiratory muscles, were not different between exercise and hyperpnoea (Figure 2B, 2D and 2C, respectively). Breathing pattern (tidal volume, frequency, inspiratory and expiratory times, and duty cycle), was not different between exercise and hyperpnoea sustained by definition at identical levels of minute ventilation (Figure 3). 9

10 Page 10 of 43 Cardiorespiratory and hemodynamic responses Whole body oxygen uptake and systemic haemodynamics are shown in Figure 4. During exercise whole body oxygen uptake increased linearly (r 2 = 0.92) with work rate up to WR peak (Figure 4A), whereas cardiac output increased up to a load corresponding to 50% WR peak and leveled off thereafter (Figure 4B). The plateau in cardiac output above 50% WR peak was due to a fall in stroke volume (Figure 4C) because heart rate continued to increase linearly (r 2 = 0.95) with work rate up to WR peak (Figure 4D). The pattern of change in intercostal muscle blood flow was very different (p = 0.003) between the exercise and the hyperpnoea tests (Figure 5A). During exercise, intercostal muscle blood flow remained unchanged from rest with increasing rates of work up to 50% WR peak, but significantly decreasing thereafter (Figure 5A). In contrast, during isocapnic hyperpnoea, intercostal muscle blood flow significantly increased up to 75% WR peak (Figure 5A); the change from rest in intercostal muscle blood flow was linear with respect to the power of breathing (r 2 =0.92). During exercise, quadriceps muscle blood flow increased with increasing work rate up to 100% WR peak (r 2 = 0.94), whereas during hyperpnoea, quadriceps muscle blood flow did not change with increasing minute ventilation (Figure 5C). During both exercise and hyperpnoea trials intercostal muscle oxygen saturation significantly decreased from baseline (Figure 5B), albeit the magnitude of decrease was greater during exercise. Similarly, during exercise quadriceps oxygen saturation significantly decreased from baseline but remained unchanged during resting hyperpnoea (Figure 5D). During exercise, mean arterial pressure and systemic vascular conductance increased with increasing work rate up to a load corresponding to 50% WR max. During isocapnic hyperpnoea neither mean arterial pressure nor systemic vascular conductance changed from baseline with increasing minute ventilation (Figure 6 A&C). 10

11 Page 11 of 43 The pattern of change in intercostal muscle vascular conductance was different between the exercise and the hyperpnoea tests (p = 0.003) (Figure 6B). During exercise, intercostal muscle vascular conductance remained unchanged with increasing work rate up to 50% WR peak, significantly decreasing thereafter. In contrast, during isocapnic hyperpnoea intercostal muscle vascular conductance significantly increased up to 75% WR peak leveling off thereafter. During exercise quadriceps muscle vascular conductance increased linearly with increasing work rate peaking at up to 100% WR peak (Figure 6D). During hyperpnoea, quadriceps muscle vascular conductance did not change with increasing ventilation (Figure 6D). Discussion The present study investigated, in patients with COPD, whether there is redistribution of blood flow to the intercostal muscles during graded exercise. Since we were unable to directly measure diaphragmatic blood flow and as with disease progression the act of breathing become gradually more dependent on the inspiratory intercostal muscles (12), respiratory muscle blood flow was assessed by measuring intercostal muscle perfusion using indocyanine green dye and a NIRS probe positioned over the 7 th intercostal space (8). Accordingly, intercostal muscle blood flow during resting hyperpnoea (when respiratory muscle perfusion was presumably the maximal attainable and not limited by competition from the locomotor muscles) was compared to that recorded during graded exercise where limb and respiratory muscles had to compete for the available blood flow. There are two novel findings of this study. Firstly, intercostal muscle blood flow did not rise above resting levels, but fell progressively with increasing effort such that during exercise above 50% of peak work capacity perfusion to the intercostal muscles was significantly lower than during resting hyperpnoea (Figure 5). Secondly, although cardiac output, blood pressure and systemic 11

12 Page 12 of 43 vascular conductance reached a plateau at exercise intensities above 50% of peak work capacity (Figures 4 & 6, respectively), quadriceps muscle vascular conductance and perfusion continued to increase reaching maximal values at peak exercise (Figures 5 & 6). Collectively, these observations suggest that during high-intensity exercise in COPD, restriction of intercostal muscle blood flow, but preservation of quadriceps muscle perfusion along with the attainment of a plateau in cardiac output, represent the inability of the circulatory system to satisfy the energy demands of both locomotor and respiratory muscles. Blood flow distribution during exercise The concept of blood flow redistribution from the locomotor to the respiratory muscles during exercise was originally evolved from studies in healthy subjects showing reductions in limb blood flow with respiratory muscle loading and an increase in locomotor muscle blood flow with respiratory muscle unloading (13, 14). In COPD, unloading the respiratory muscles by means of heliox breathing or proportional assisted ventilation has been shown to improve peripheral muscle oxygenation (5, 6) and exercise performance (15-20). These findings were interpreted as evidence of redistribution of energy supplies from the respiratory to locomotor muscles. However, none of these studies (5, 6, 15-20) directly assessed the interaction between locomotor and respiratory muscle blood flow during exercise in COPD. Accordingly, the novelty of the present investigation is that simultaneous measurements of both intercostal and quadriceps muscle blood flow and vascular conductance were performed under conditions where the amount of both respiratory and leg muscle work concurrently increased during graded discontinuous exercise to the limit of tolerance and was compared to conditions where the respiratory muscles produced similar work whilst the legs remained inactive. As intercostal muscle blood flow was lower during exercise than hyperpnoea, whilst the increase in quadriceps muscle blood flow was preserved throughout incremental exercise (Figure 5), our findings in patients with COPD do not support the notion 12

13 Page 13 of 43 of blood flow redistribution in favour of the respiratory muscles during exercise that was originally proposed from studies in healthy subjects (13, 14). In this context, our results differ from those of previous studies in healthy individuals utilizing either inspiratory resistances (13, 14) or expiratory loading techniques (21, 22) to artificially increase the work of breathing. The most likely reason for this discrepancy is that by artificially increasing either the work of the inspiratory or the expiratory muscles in healthy subjects is an acute procedure which causes abrupt adjustments in the regulation of systemic blood flow (14, 21, 22) possibly forcing redistribution of blood flow from the locomotor to the respiratory muscles (13). In contrast in COPD, the phenomenon of increased breathing work is chronic and as such certain central and/or peripheral muscle adjustments in blood flow regulation may take place to accommodate the energy supply according to demand. Our findings are in agreement with those in healthy subjects in that at essentially the same work of breathing intercostal muscle blood flow during high-intensity exercise was lower compared to that during voluntary resting hyperpnoea (7). In that study (7) following an initial significant increase from rest, intercostal muscle blood flow and vascular conductance fell at intensities exceeding 80% of maximal capacity, whilst cardiac output and quadriceps muscle blood flow reached a plateau. This finding was interpreted as a reflection of the inability of the circulatory system to meet the increasing energy demands of both locomotor and intercostal muscles at exercise intensities exceeding 80% of maximal (7). In the present study, however, intercostal muscle blood flow and vascular conductance during sub-maximal exercise did not rise above baseline value, whilst at higher exercise intensities ( 50% of peak work capacity) both intercostal muscle blood flow and vascular conductance significantly decreased below baseline levels (Figures 5 & 6) most likely reflecting increased sympathetic vasoconstrictor activity to the intercostal muscles (7, 23-25). 13

14 Page 14 of 43 There are several lines of evidence suggesting that in patients with COPD exhibiting significant hyperinflation and expiratory flow limitation, there may be a secondary cardiovascular defect which limits the increase in cardiac output during exercise (15, 26-29). Our results are in agreement with those of earlier studies in COPD (15, 26-29) showing a blunted increase in cardiac output during incremental exercise. In fact, in the present study the reduction in stroke volume and the compensatory increase in heart rate at intensities greater than 50% of peak work capacity (Figure 4) are in tandem with the results on healthy individuals mimicking important pathopysiological features of COPD during exercise either by increasing expiratory loading (21) or by limiting expiratory flow through a Starling resistor (22). Both strategies produced excessive expiratory muscle recruitment and adverse effects on the increase in stroke volume and cardiac output (21-22). Interestingly, our patients showed evidence of significant expiratory abdominal muscle recruitment at intensities greater that 50% of peak work capacity compared to resting hyperpnoea i.e.: decrease in end-expiratory abdominal volume and increase in peak expiratory gastric pressure and pressure-time product for the abdominal muscles (Figures 1 & 2) which most likely hindered the increase in cardiac output beyond that intensity. Indeed, elevations in gastric pressure during expiration caused by increased abdominal muscle recruitment have been shown to act as a Valsava manoeuvre, decreasing cardiac output and producing blood shifts from trunk to extremities (1, 4, and 22). In the present study it is possible that in the face of a blunted cardiac output, blood flow to the intercostal muscles was reduced in favour of the blood flow to the locomotor muscles which exhibited a linear increase up to peak work capacity, thereby confirming, earlier studies (30-, 32) that the peripheral muscle hemodynamic and metabolic capacity is, to some extend, preserved during maximal exercise in COPD. Nevertheless, the progressive reduction in quadriceps muscle oxygen saturation (Figure 5) against the background of a steadily increased muscle blood flow during exercise suggests that the increase in quadriceps muscle blood flow 14

15 Page 15 of 43 was insufficient to prevent the mismatch between oxygen delivery to, and demand of the quadriceps muscles, ultimately making them vulnerable to muscle fatigue (32). Regulation of blood flow within the respiratory muscles The peripheral muscles were possibly not the only ones having a preferential share of the cardiac output during high-intensity exercise. Volume variations of the rib cage and abdominal chest wall compartments and pressure measurements revealed that whilst the activation of the external and internal intercostal muscles (primarily represented by volume changes of the rib cage compartment) as well as activation of the diaphragm (reflected by volume changes of the abdominal compartment at end of inspiration, and evidenced by tidal Pdi excursions and pressure-time product for the diaphragm) were similar between exercise and hyperpnoea, volume variations of the abdominal compartment, peak expiratory gastric pressures and pressure-time product of the expiratory abdominal muscles (all indicating abdominal muscle recruitment) were significantly greater during exercise compared to hyperpnoea (Figures 1 & 2). It is thus plausible that blood flow to the intercostal muscles and the diaphragm may have been redirected to support the increased energy demands of the abdominal muscles. Measurement of respiratory muscle blood flow in COPD In healthy subjects, blood flow measured over the 7 th intercostal space using NIRS and ICG has been considered as perfusing mostly the external and internal intercostal muscles and to a lesser extent the costal segment of the diaphragm (8). This is due to the substantial distance between the sampling point of NIRS on the skin and the diaphragmatic appositional area (8) compared to the shorter distance to the intercostals. In COPD, monitoring intercostal muscle blood flow during exercise has additional important implications as there is evidence to indicate that chronic hyperinflation makes these patients use their intercostal muscles more 15

16 Page 16 of 43 vigorously than normal subjects (12) during exercise, whilst the diaphragm makes a relatively limited contribution to the generation of maximal levels of ventilation (33, 34), likely due to its flattened shape. Intersestingly, our patients exhibited a delayed pattern of exercise-induced dynamic hyperinflation (35) secondary to a significant reduction in end-expiratory abdominal volume and increased abdominal muscle activity. Nevertheless, the progressive reduction in intercostal muscle oxygen saturation (Figure 5) against the background of initially stable and later decreasing intercostal muscle blood flow during graded exercise, is consistent with a mismatch between oxygen delivery to, and demand of, the intercostal muscles, thereby suggesting that intercostal muscle blood flow limitation may have rendered them vulnerable to muscle fatigue (36). In contrast, the decrease from baseline in intercostal muscle oxygen saturation was less pronounced across increasing levels of respiratory muscle work during resting hyperpnoea (Figure 5), thus suggesting that increasing oxygen demand of the intercostal muscles was more adequately met by proportionally increased blood flow and oxygen delivery. On the other hand, abdominal muscle blood flow could not be measured to provide evidence of redistribution in respiratory muscle perfusion as placing the NIRS optodes on the abdomen generally provides a poor signal that underestimates blood flow due to the contribution of often substantial subcutaneous adipose tissue to the light-absorption signal. Study limitations The invasive, intensive and exhausting nature of the present study precluded repeated testing across the different work rates so as to establish reproducibility of cardiac output and regional muscle blood flow measurements in patients with COPD. Furthermore, inclusion of an age-matched, healthy control group would have allowed comparisons between healthy subjects and patients with COPD with respect to the regulation of intercostal muscle blood 16

17 Page 17 of 43 flow during the exercise and hyperpnoea protocols. In addition, performing the exercise protocol first and the hyperpnoea protocol afterwards on the same day may have influenced respiratory muscle oxygen uptake and delivery kinetic responses during the hyperpnoea tests owing to muscle warm-up (37). However, the finding that baseline intercostal muscle blood flow was not different between the two protocols suggests that the time elapsed (2 hours) between the two protocols eliminated any effect of prior exercise on intercostal muscle blood flow regulation during hyperpnoea. In conclusion, during exercise in patients with moderate COPD, the circulatory system appears unable to meet the increasing demands of both locomotor and intercostal muscles. Surprisingly, intercostal muscle blood flow does not rise above resting levels, and indeed falls progressively with increasing locomotor muscle effort, whereas quadriceps muscle blood flow is preserved even in the presence of a plateau in cardiac output. Accordingly, we suggest that the reduction in vascular conductance and blood flow of the intercostal, but not the quadriceps muscles, appears to be the result of a blunted cardiac output response to exercise in an attempt to maintain blood flow to the locomotor and possibly also the expiratory abdominal muscles. Furthermore, the findings of the present study reinforce the impact of interactions between respiratory and cardiovascular systems and the need for an integrative approach of their function during exercise which would potentially reveal the factors limiting exercise performance in patients with COPD. 17

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23 Page 23 of 43 Table 1. Pulmonary function data Age (years) 60 ± 7 Height (cm) 172 ± 6 Weight (kg) 77 ± 18 BMI (kg/m 2 ) 26.1 ± 5.7 FFMI (kg/m 2 ) 18.4 ± 1.8 FEV 1 (l) 1.6 ± 0.6 FEV 1 (% predicted) 50.5 ± 17.5 FVC (l) 3.1 ± 0.5 FVC (% predicted) 75 ± 8 TLC (l) 8.6 ± 1.0 TLC (% predicted) 135 ± 12 RV (l) 4.9 ± 0.9 RV (% predicted) 215 ± 35 FRC (l) 6.1 ± 1.1 FRC (% predicted) 179 ± 21 DL CO (% predicted) 39 ± 13 PaO 2 (mm Hg) 80 ± 5 PaCO 2 (mm Hg) 39 ± 3 SaO 2 (%) 93 ± 4 ph 7.4 ± 0.1 Values are means ± S.D. for 10 subjects. BMI, body mass index; FFMI, fat free mass index; FEV1: forced expiratory volume in one second; FVC, forced vital capacity; TLC, total lung capacity; RV, residual volume; FRC, functional residual capacity; DL CO, diffusing factor of the lung for carbon monoxide; PaO 2, partial pressure of arterial O 2 ; PaCO 2, partial pressure of arterial carbon dioxide; SaO 2 (%), percentage of arterial oxygen saturation. 23

24 Page 24 of 43 Table 2. Peak exercise data WR peak (W) 73 ± 42 VO 2peak (ml kg -1 min -1 ) 15 ± 4 HR peak (beats min -1 ) 117 ± 14 V Epeak (l min -1 ) 49 ± 16 V Tpeak (l min -1 ) 1.6 ± 0.4 f peak (breaths min -1 ) 32 ± 8 SpO 2 (%) 92 ± 3 Borg dyspnoea scores 7 ± 2 Borg leg effort scores 5 ± 2 Values are means ± S.D. for 10 subjects. Exercise data depict the results of the incremental exercise test. WR peak, peak work rate; VO 2peak, peak oxygen uptake; FEV 1, forced expiratory volume in one second; HR peak, peak heart rate;; V Epeak, peak minute ventilation; V Tpeak, peak tidal volume; f peak, peak breathing frequency; SpO 2 (%), percentage arterial oxygen saturation measured by pulse oximetry. 24

25 Page 25 of 43 Figure Legends Figure 1. Power of breathing and chest wall volume regulation. A: power of breathing, B: chest wall, C: rib cage and D: abdominal volume regulation at the end of inspiration and expiration recorded at different fractions of peak work rate (WRpeak) during exercise (open triangles) and during isocapnic hyperpnoea trials (filled triangles) that were sustained at levels of minute ventilation similar to those recorded during exercise. Values are means ± S.E.M. for 10 subjects. Asterisks denote significant differences between the exercise and hyperpnoea tests at different fractions of peak work rate, whereas crosses denote significant differences across the two protocols. Figure 2. Mechanics of breathing. A: peak expiratory gastric pressure, B: tidal excursion in transdiaphragmatic pressure ( Pdi), and C: tidal excursion in gastric pressure ( Pga) to tidal excursion in pleural pressure ( Ppl) ratio (expressing the relative contribution of the diaphragm to the pressure generated by the whole inspiratory muscles) measured at different fractions of peak work rate (WRpeak) during exercise (open triangles) and during resting isocapnic hyperpnoea trials (filled triangles) that were sustained at levels of minute ventilation similar to those recorded during exercise. D: pressure-time product (PTP) of respiratory muscles. Filled squares correspond to PTP for expiratory abdominal muscles (PTPab) during the isocapnic hyperpnoea trials and open squares refer to PTPab during exercise. Similarly, filled triangles correspond to PTP for the diaphragm (PTPdi) during isocapnic resting hyperpnoea and open triangles refer to PTPdi during exercise. Values are means ± S.E.M. for 10 subjects. Asterisks denote significant differences between the exercise and hyperpnoea tests at different fractions of peak work rate, whereas crosses denote significant differences across the two protocols. 25

26 Page 26 of 43 Figure 3. Breathing pattern. A: time of inspiration (T i ), B: time of expiration (T e ), C: duty cycle (T i /T tot ), D: minute ventilation (V E ), E: tidal volume (V T ), and F: breathing frequency (f) recorded at different fractions of peak work rate (WRpeak) during exercise (open triangles) and during isocapnic hyperpnoea trials (filled triangles) that were sustained at levels of minute ventilation similar to those recorded during exercise. Values are means ± S.E.M. for 10 subjects. Figure 4 Metabolic and central hemodynamic responses. A: oxygen uptake, B: cardiac output, C: stroke volume and D: heart rate recorded at different fractions of peak work rate (WRpeak) during exercise (open triangles) and during isocapnic hyperpnoea trials (filled triangles) that were sustained at levels of minute ventilation similar to those recorded during exercise. Values are means ± S.E.M. for 10 subjects. Asterisks denote significant differences between the exercise and hyperpnoea tests at different fractions of WRpeak, whereas crosses denote significant differences across the two trials. Figure 5. Intercostal and quadriceps muscle blood flow and tissue oxygen saturation responses. A: intercostal muscle blood flow, B: intercostal muscle oxygen saturation, C: quadriceps muscle blood flow, and D: quadriceps muscle oxygen saturation recorded at different fractions of peak work rate (WRpeak) during exercise (open triangles) and during isocapnic hyperpnoea trials (filled triangles) that were sustained at levels of minute ventilation similar to those recorded during exercise. Values are means ± S.E.M. for 10 subjects. Asterisks denote significant differences between the two conditions at different fractions of WRpeak,. whereas crosses denote significant differences across the two protocols. 26

27 Page 27 of 43 Figure 6. Central and peripheral vascular responses. A: mean arterial blood pressure, B: intercostal muscle vascular conductance, C: systemic vascular conductance and D: quadriceps muscle vascular conductance recorded at different fractions of peak work rate (WRpeak) during exercise (open triangles) and during isocapnic hyperpnoea trials (filled triangles) that were sustained at levels of minute ventilation similar to those recorded during exercise. Values are means ± S.E.M. for 10 subjects. Asterisks denote significant differences between exercise and hyperpnoea tests at different fractions of WRpeak, whereas crosses denote significant differences across the two trials 27

28 Page 28 of 43 A C End-inspiration Power of breathing (cal.min -1 ) Rib cage volume (l) End-expiration Β End-inspiration D End-inspiration Chest wall volume (l) * * * Abdominal volume (l) * * * End-expiration End-expiration REST 25% 50% 75% 100% WRpeak REST 25% 50% 75% 100% WRpeak Figure 1.

29 Page 29 of 43 A C Peak expiratory gastric pressure (cmh 2 O) * * Pga/ Ppl B D Pdi (cmh 2 O) PTP (cmh 2 O.s. min -1 ) * * * REST 25% 50% 75% 100% WRpeak REST 25% 50% 75% 100% WRpeak Figure 2.

30 Page 30 of 43 A D Β E C F Ti/ Ttot (%) f (breaths.min -1 ) Te (sec) V T (l) Ti (sec) V E (l.min -1 ) REST 25% 50% 75% 100% WRpeak REST 25% 50% 75% 100% WRpeak Figure 3.

31 Page 31 of 43 A C Oxygen uptake (l.min -1 ) * * * * Stroke volume (ml.beat -1 ) * * * B D Cardiac output (l.min -1 ) * * * * Heart rate (beats.min -1 ) * * * REST 25% 50% 75% 100% WRpeak REST 25% 50% 75% 100% WRpeak Figure 4.

32 Page 32 of 43 INTERCOSTAL MUSCLES QUADRICEPS MUSCLES A C Muscle blood flow (ml.min g -1 ) * * Muscle blood flow (ml.min g -1 ) B D Muscle tissue saturation (%) Muscle tissue saturation (%) * * REST 25% 50% 75% 100% WRpeak REST 25% 50% 75% 100% WRpeak Figure 5.

33 Page 33 of 43 A C Mean arterial pressure (mm Hg) * * * * Systemic vascular conductance (ml.min -1 mm Hg -1 ) * * * B D Intercostal muscle vascular conductance (ml.min g -1 mm Hg -1 ) * * Quadriceps muscle vascular conductance (ml.min g -1 mm Hg -1 ) * * * * REST 25% 50% 75% 100% WRpeak REST 25% 50% 75% 100% WRpeak Figure 6.

34 Page 34 of 43 1 ON-LINE DATA SUPPLEMENT Intercostal muscle blood flow limitation during exercise in chronic obstructive pulmonary disease Ioannis Vogiatzis, Dimitris Athanasopoulos, Helmut Habazettl, Andrea Aliverti, Zafiris Louvaris, Evgenia Cherouveim, Harrieth Wagner, Charis Roussos, Peter D. Wagner and Spyros Zakynthinos

35 Page 35 of 43 2 Methods Subjects Ten patients (1 female) with clinically stable COPD participated in the study according to the following inclusion criteria: 1) a post-bronchodilator forced expiratory volume in one second (FEV 1 ) <80% predicted without significant reversibility (<12% change of the initial FEV 1 value or <200 ml); 2) optimal medical therapy according to GOLD (E1); and 3) the absence of other significant diseases that could contribute to exercise limitation. The study was approved by the University Hospital Ethics Committee and was conducted in accordance with the guidelines of the Declaration of Helsinki. Prior to participation in the study, all patients were informed of any risks and discomforts associated with the experiments and gave written, signed, informed consent. Experimental design Experiments were conducted in two visits. In visit 1, patients underwent an incremental preliminary exercise test to the limit of tolerance (WR peak ). In visit 2, patients initially undertook a graded exercise test (protocol 1), which was followed by resting isocapnic hyperpnoea trials (protocol 2). During the graded exercise test patients completed four bouts of constant-load exercise corresponding to the following targeted intensities: (i) 25% WR peak for 5 min; (ii) 50% WR peak for 5 min; (iii) 75% WR peak for 3-4 min and (iv) 100% WR peak for 2-3 min. Prior to imposing the target load on the bicycle ergometer, patients were asked to perform unloaded cycling for 60 seconds reaching and maintaining a cadence of approximately 50 rpm. Between exercise bouts at 25 and 50% WR peak patients rested for 15 min, whereas after completion of exercise bouts at 50 and 75% WR peak patients rested for 15 and 30 min, respectively. Two hours after completion of the exercise tests, patients performed five 5-min bouts of isocapnic hyperpnoea at the same tidal volume, breathing frequency and thus minute ventilation recorded at rest and during exercise at 25, 50, 75 and 100% WR peak.

36 Page 36 of 43 3 Blood flow over the 7 th intercostal space and over the vastus lateralis muscles as well as cardiac output were measured during the final minute of each of the exercise and the hyperpnoea bouts (Figure 1). Preliminary testing In visit 1, the incremental exercise tests were performed on an electromagnetically braked cycle ergometer (Ergoline 800; Sensor Medics, Anaheim, CA, USA) with a ramp increase of load increments of 5 or 10 W.min -1. Throughout the exercise tests, patients were encouraged to maintain a pedaling frequency of revolutions.min -1. Tests were preceded by a 3 min rest period, followed by 3 min of unloaded pedaling. The following pulmonary gas exchange and ventilatory variables were recorded breath by breath (Vmax 229; Sensor Medics, Anaheim, CA, USA): oxygen uptake (VO 2 ), carbon dioxide elimination (VCO 2 ), minute ventilation (VE), tidal volume (VT), breathing frequency (f), and respiratory exchange ratio (RER). Heart rate (HR) and percentage oxygen saturation (% SpO 2 ) were determined using the R-R interval from a 12-lead on-line electrocardiogram (Marquette Max; Marquette Hellige GmbH, Germany) and a pulse oximeter (Nonin 8600; Nonin Medical, North Plymouth, MN, USA), respectively. Subject preparation Subjects were prepared first with arterial and venous catheters for blood flow measurements, and then with oesophageal and gastric balloons for assessment of esophageal and gastric pressures and of the power of breathing. Using local anaesthesia (2% lidocaine) and sterile technique, identical catheters were introduced percutaneously into the left femoral vein and the right radial artery, both oriented in the proximal direction. The catheters were used to collect arterial and venous blood samples and also to inject indocyanine green dye (ICG) into the venous line and sample blood after each injection from the arterial line for