SIMON MALENFANT1, FRANÇOIS POTUS1, VINCENT MAINGUY1, EVELYNE LEBLANC1, MATHIEU MALENFANT1, FERNANDA RIBEIRO2, DIDIER SAEY2, FRANÇOIS MALTAIS2, SE´BASTIEN BONNET1, and STEEVE PROVENCHER1 1

Pulmonary Hypertension Research Group, Que´bec Heart and Lungs Institute Research Center, Laval University, Que´bec City, Que´bec, CANADA; and 2Que´bec Heart and Lungs Institute Research Center, Laval University, Que´bec City, Que´bec, CANADA

ABSTRACT MALENFANT, S., F. POTUS, V. MAINGUY, E. LEBLANC, M. MALENFANT, F. RIBEIRO, D. SAEY, F. MALTAIS, S. BONNET, and S. PROVENCHER. Impaired Skeletal Muscle Oxygenation and Exercise Tolerance in Pulmonary Hypertension. Med. Sci. Sports Exerc., Vol. 47, No. 11, pp. 2273–2282, 2015. Background: Limb muscle dysfunction is documented in pulmonary arterial hypertension (PAH), but little is known regarding muscle oxygen (O2) supply and its possible effects on exercise tolerance in PAH. Methods: Ten patients with PAH and 10 matched controls underwent progressive maximal cardiopulmonary exercise test, voluntary and nonvolitional dominant quadriceps muscle strength measures, and nondominant quadriceps biopsy to assess maximal oxygen uptake, muscle function, and lower limb fiber type and capillarity, respectively. Both groups then performed normoxic and hyperoxic submaximal intensity exercise protocol at the same absolute workload during which muscle O2 supply was assessed by measuring changes in myoglobin– deoxyhemoglobin level ($[Mb-HHb]) and tissue oxygenation index in the dominant quadriceps using near-infrared spectroscopy. Changes in cardiac output, estimated systemic O2 delivery, and systemic O2 saturation were also assessed noninvasively throughout both submaximal exercises. Results: Patients with PAH displayed lower maximal oxygen uptake (P G 0.01), skeletal muscle strength (P G 0.05), and capillarity (P = 0.01). Throughout the normoxic submaximal exercise protocol, $[Mb-HHb] (P G 0.01) was higher whereas changes in tissue oxygenation index (P G 0.01) and systemic O2 saturation (P = 0.01) were lower in patients with PAH compared with those in controls. Conversely, changes in cardiac output and estimated systemic O2 delivery were similar between groups. Muscle oxygenation remained unchanged with O2 supplementation. Among variables known to influence tissue oxygenation, only quadriceps capillarity density correlated with $[Mb-HHb] (r = j0.66, P G 0.01), which in turn correlated with maximal oxygen uptake (r = j0.64, P G 0.01), 6-min walked distance (r = j0.74, P = 0.01), and both voluntary (r = j0.46, P = 0.04) and nonvolitional (r = j0.50, P = 0.02) quadriceps strength. Conclusions: Capillary rarefaction within the skeletal muscle influences exercise tolerance and quadriceps strength at least partly through impaired muscle oxygen supply in PAH. Key Words: PULMONARY HYPERTENSION, SKELETAL MUSCLE OXYGENATION, EXERCISE INTOLERANCE, SUBMAXIMAL EXERCISE, NEAR-INFRARED SPECTROSCOPY

P

ulmonary arterial hypertension (PAH) is a rare disease characterized by progressive increase in pulmonary vascular resistance ultimately leading to right ventricular failure and premature death. Prime symptoms include dyspnea and fatigue resulting in restricted exercise tolerance and poor health-related quality of life (15). Although pharmacological therapies improve cardiopulmonary

hemodynamics and exercise capacity (33), most patients remain markedly intolerant to exercise (15). Exercise intolerance in PAH has traditionally been attributed to the central limitation associated with the remodeled pulmonary vasculature and the progressively failing right ventricle (28,34). However, in recent years, attention has turned toward other factors, including higher ventilatory demand resulting from complex mismatch between respiratory needs (21), ventilatory constraints (19), and respiratory muscle function (19,20). Alterations in skeletal muscle morphology (3), function (25), metabolism (26), oxygenation at rest (8), and impaired matching of dynamic O2 delivery and ˙ O2) at the onset of heavy exercise (2) oxygen consumption (V have also been consistently observed in PAH. Intriguingly, total systemic O2 extraction at the end of maximal exercise is also impaired in PAH (39), suggesting that global O2 use from exercising muscles is reduced in PAH during peak exercise (22). More recently, we demonstrated that PAH was associated with microRNA-126–dependent impaired angiogenesis, resulting in skeletal muscle microcirculation loss (27). Whether this capillary rarefaction is associated with

Address for correspondence: M. Simon Malenfant, M.Sc., Pulmonary Hypertension Research Group, Que´bec Heart and Lungs Institute Research Center, Laval University, 2725 Chemin Sainte-Foy Que´bec, Que´bec, Canada G1V 4G5; E-mail: [email protected] Submitted for publication January 2015. Accepted for publication April 2015. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.acsm-msse.org). 0195-9131/15/4711-2273/0 MEDICINE & SCIENCE IN SPORTS & EXERCISEÒ Copyright Ó 2015 by the American College of Sports Medicine DOI: 10.1249/MSS.0000000000000696

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Impaired Skeletal Muscle Oxygenation and Exercise Tolerance in Pulmonary Hypertension

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clinically relevant impairments in oxygen delivery and use in skeletal muscles during early and lower intensity of exercise remains unknown. Thus, in addition to the limited increase in maximal cardiac output, abnormalities in skeletal muscle microcirculation could further limit global O2 delivery to ˙ O2peak and exercising myocytes and contribute to decreased V exercise tolerance in PAH. To better characterize the exercise pathophysiology in PAH, the main purpose of this investigation was to examine muscle oxygen supply during normoxic and hyperoxic submaximal exercise and its physiological determinants. We hypothesized that oxygen supply to lower limb muscles would be lower in patients with PAH as a result of reduced skeletal muscle capillarity density and would correlate with exercise intolerance in PAH. We also hypothesized that oxygen supplementation would not correct the lower limb muscle oxygen supply.

METHODS Study Subjects Twenty participants were recruited to test our hypothesis. Ten New York Heart Association (NYHA) functional class II–III idiopathic patients with PAH with hemodynamic assessment G6 months were recruited at the Que´bec Heart and Lung Institute. PAH was defined as a mean pulmonary artery pressure 925 mm Hg at rest with a pulmonary capillary wedge pressure e15 mm Hg, and PAH diagnosis was made according to international guidelines by experienced physicians (15). Only patients with no change in their therapy and in a stable condition over the last 4 months were eligible. Exclusion criteria were as follows: 1) unstable clinical condition (e.g., recent syncope, World Health Organization functional class IV), 2) a 6-min walked distance of G300 m during routine follow-up at the pulmonary hypertension clinic, 3) left ventricular ejection fraction G40%, 4) restrictive (lung fibrosis on computed tomography scan or total lung capacity G80% of predicted) or obstructive (FEV1/FVC G70%) lung disease, 5) obesity, with body mass index 930.0 kgImj2, 6) known locomotor abnormalities, and 7) type 2 diabetes mellitus. Ten healthy sedentary subjects individually matched for age, sex, weight, and height served as controls. All measurements were completed over two visits in our exercise physiology laboratory. At least 1 wk was allowed between visits to maximize muscle healing after biopsy to ensure the participants_ comfort. The institution ethics committee (Comite´ d_Ethique de la Recherche de l_Institut Universitaire de Cardiologie et de Pneumologie de Que´bec; protocol number, CE´R 20643) approved the research protocol, and all patients and healthy controls gave a written consent. Study Design For the first visit, nonfasting blood samples were drawn for hemoglobin and hematocrit analysis. Thereafter, dominant quadriceps maximum voluntary contraction (MVC) and

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twitch tension (TWq) were measured (31), followed by a biopsy of the nondominant quadriceps vastus lateralis muscle, according to the Bergstro¨m technique (4). All biopsies were performed by the same investigator (S.P.) in the vastus lateralis, 12–15 cm above the knee, to concur with the nearinfrared spectroscopy (NIRS) probe placement on the dominant leg. Tissue samples were immediately frozen in liquid nitrogen, embedded in optimum cutting temperature: (OCT) compound, frozen in cooled isopentane, and stored at j80-C. Transverse sections of 10 Km were cut using a cryostat Leica Jung CM3000 (Wetzlar, Germany). Muscle sections were stained to see myofibrillar adenosine triphosphatase activity according to the single-step modified ethanol technique, as routinely performed in our laboratory (25–27). For each subject, the proportion of Type I and Type II muscle fibers was assessed and calculated as the number of fibers of each type divided by the total number of muscle fibers. The surfaces of 40 randomly selected fibers were measured using an image analyzing software (AxioVision 4.8.2; Carl Zeiss MicroImaging, Standort, Germany). Capillarization was determined by quantitative immunohistochemistry microscopy using primary CD31-antibody (1:100, sc-1506-R; Santa Cruz Biotechnology, Santa Cruz, CA). Capillaries were quantified by fiber type and by surface area. Subjects also performed a supine progressive maximal cardiopulmonary exercise test (CPET) on an electrically braked ergocycle (Corival; Lode B.V., Groningen, The Netherlands). Detailed descriptions of the MVC, TWq, and CPET are given in the supplemental digital content (see Document, Supplemental Digital Content 1, Methodology, http://links.lww.com/MSS/A550). For the second visit, two supine submaximal intensity inhouse exercise protocols were realized without and with oxygen supplementation (normoxia and hyperoxia) in a randomized order determined using the Latin square design. Patients remained blinded to oxygen supplementation throughout the tests. Both exercise were separated by 1 h of complete rest, according to the ATS/ACCP statement on cardiopulmonary exercise testing (1) and previous publications (23,24). Additional care was also taken before beginning the second submaximal exercise by questioning patients about their state of fatigue to ensure that they fully recovered before the second exercise was performed. Ambient air (FiO2, 0.21) and oxygen supplementation (FiO2, 1.00) was administrated for both exercise protocols through a 60-L nondiffusing collection Douglas bag (Hans Rudolph, Inc, Kansas City, MI). Each exercise protocol consisted of 3 min of rest and 3 min of unloaded cycling followed by a progressive increase of 5 W in work rate until 70% of maximal workload was reached during the CPET for patients with PAH. This later intensity was maintained for 3 min and was followed by an active 3-min recovery and postexercise rest of 3 min. To minimize the effect of variable cardiac output and exercise workload on skeletal muscle oxygenation, each healthy control performed submaximal exercise protocols at the same absolute workload as that of his respective paired patient with PAH.

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changes in systemic oxygen delivery ($DO2) during exercise were estimated according to the following formula: $DO2 = $CO  changes in arterial oxygen content (estimated as 1.34  baseline hemoglobin  $SpO2) during exercise. The $CO, $SpO2, and estimated $DO2 were all calculated from the last 30 s of the preexercise resting period.

Statistical Analysis Data are presented as mean (SD), unless otherwise specified. Categorical variables were analyzed using the Fisher exact test. Unpaired t-tests were used to compare the characteristics of the two groups. A two-way ANOVA with repeated measures and Tukey–Kramer multiple comparison test was used to compare variables continuously assessed during submaximal exercise protocols (i.e., $[Mb-HHb], $TOI, $CO, $SpO2, and $DO2) after a logarithmic transformation was applied to best normalize the data. Pearson correlation coefficients were used to evaluate the relation between endexercise $[Mb-HHb] and variables that could influence skeletal muscle oxygenation (i.e., capillary density, workload, $CO, $SpO2, and $DO2 achieved during submaximal exercise baseline hemoglobin levels) and resting pulmonary hemodynamics (mean pulmonary arterial pressure, cardiac index, and pulmonary vascular resistance). Pearson correlation coefficients were also used to assess the relation between altered skeletal muscle oxygenation, maximal oxygen consumption, and quadriceps_ function. P G 0.05 was considered statistically significant. Data were analyzed using GraphPad Prism version 6.0f for Mac (GraphPad, La Jolla, CA).

TABLE 1. Clinical characteristics. PAH (n = 10) Demographics Age (yr) 45 (12) Sex (F/M) 9/1 BMI (kgImj2) 24 (5) NYHA functional class II/III 6/4 Resting pulmonary hemodynamics mPAP (mm Hg) 54 (12) 2.9 (0.8) CI (LIminj1Imj2) j5 PVR (dynIsIcm ) 771 (415) PAOP (mm Hg) 10 (3) Medication PDE-5 inh. (n) 2 ERA (n) 2 Prostacyclin analogs (n) 1 PDE-5 inh./ERA (n) 4 None (n) 1 Blood sampling p50 (mm Hg) 24.1 (1.5) 13.8 (0.7) Hemoglobin (gIdLj1) Hematocrit 0.41 (0.02)

Healthy Controls (n = 10)

P Value

48 (13) 9/1 25 (3)

0.58 1.00a 0.63





— — — —

— — — —

— — — — 10 — 13.8 (1.2) 0.40 (0.04)

0.94 0.78

Data are presented as mean (SD). Analyzed by Fisher exact test. BMI, body mass index; CI, cardiac index; ERA, endothelin receptor antagonist; F, female; M, male; mPAP, mean pulmonary artery pressure; PAOP, pulmonary artery occlusion pressure; PDE-5 inh., phosphodiesterase type 5 inhibitors; PVR, pulmonary vascular resistance; p50, partial pressure of O2 to saturate 50% of hemoglobin.

a

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Lower limb oxygen supply. During both normoxia and hyperoxia exercise protocols, oxygenation changes in the dominant quadriceps muscle were continuously assessed by NIRS using a single-distance, continuous-wave light, dual-channel Oxiplex TS (ISS, Champaign, IL). This system exploits the difference in optical absorption spectra between the oxygenated and deoxygenated states of hemoglobin and myoglobin to accurately measure changes in tissue oxygenation characteristics of small vessels e200 Km. Its use and limitations have been extensively reviewed (10,14) and validated in patients with various conditions (35,36,43), including patients with PAH (2). Before the placement of the NIRS probe, the NIRS system was calibrated and the skin was carefully shaved from the thigh of the participants. The adipose tissue thickness was measured using skin calipers (Baseline Skinfold Caliper, NexGen Ergonomics, Canada). NIRS silicon fiber optode holder was placed over the vastus lateralis muscle belly of the quadriceps, 12–15 cm above the knee, and secured with a standard surgical adhesive tape (Transpore Surgical Tape; 3M Canada, Ontario, Canada). NIRS fiber optode consisted of eight light-emitting diodes operating at wavelengths of 690 and 830 nm and one detector fiber bundle with a separated distance of approximately 4 cm, corresponding to a penetration depth of 2 cm. The signal was analyzed with an algorithm using the modified Beer–Lambert law. The variables assessed by NIRS were oxygenated, deoxygenated, and total myoglobin– hemoglobin concentration (respectively, [Mb-HbO2], [MbHHb], and [Mb-Hbtot]). Muscle oxygen saturation was evaluated using changes ($) in [Mb-HbO2]/[Mb-Hbtot] ratio, corresponding to the tissue oxygenation index ($TOI). However, muscle oxygen supply was primarily assessed using the $[Mb-HHb] response to the exercise protocols, as contrary to $TOI, this signal is relatively insensitive to blood volume change (13,30). Indeed, $[Mb-HHb] is considered to be an index of fractional O2 extraction in microcirculation, reflecting the balance between O2 delivery and use (17,18). All NIRS data were collected at 1 Hz and subsequently analyzed offline. [Mb-HHb] and TOI values were presented as percentages of change from the last 30 s of the preexercise resting period. To further demonstrate that impaired oxygenation to the exercising muscle was related to altered muscle perfusion rather than exercise-induced hypoxemia, oxygen supply to an inactive skeletal muscle (right arm triceps) during exercise was investigated. Of note, the supine position was adopted for our maximal and submaximal exercises to reduce external light contamination to the NIRS probes and to favor complete rest of the right triceps muscle. Additional measurements during submaximal exercises. Noninvasive changes in cardiac output ($CO) was measured continuously during both exercise protocols using a continuous, noninvasive finger photoplethysmography monitoring system based on the Modelflow method (Nexfin HD; BMEYE, Academic Medical Centre, Amsterdam, The Netherlands). Systemic oxygen saturation was also continuously measured by pulse oximetry (SpO2). Finally, estimated

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RESULTS The baseline characteristics of the study population and PAH treatments are shown in Table 1. All patients with PAH were in NYHA functional class II (n = 6) or III (n = 4). Note that one patient was recruited at the time of diagnosis and was therefore not receiving PAH-specific therapy at the time of the study. Hemoglobin content and hematocrit were comparable between groups, and hemoglobin affinity for oxygen was normal for patients with PAH. No adverse events were observed during quadriceps strength assessment, muscle biopsy, and exercise tests. Exercise tolerance, quadriceps muscle strength, and capillarity were significantly lower in PAH. Patients with PAH exhibited significant exercise intolerance as well as lower MVC (23.3 (4.5) vs 31.0 (5.4) kg; P G 0.01) and TWq (8.2 (1.5) vs 9.9 (2.2); P = 0.05) (Table 2). Analyses of the capillary density for a determined surface revealed significant difference between patients with PAH and healthy controls (201 (84) vs 304 (69) nImmj2; P = 0.01), and strong trends were observed when the capillarity was expressed as the number of capillaries per fiber Type I (P = 0.07) and II (P = 0.09). Quadriceps muscle oxygenation is impaired in PAH. The adipose tissue thickness was 11.2 (5.9) mm for TABLE 2. Exercise capacity and peripheral muscle characteristics. PAH (n = 10) Exercise capacity CPET V˙O2peak (mLIminj1Ikgj1) V˙O2peak (% predicted) Work rate (W) V˙E/V˙CO2 slope 6MWT Distance (m) % predicted (%) Skeletal muscle strength MVC (kg) TWq (kg) Skeletal muscle morphology Fiber typing (%) Type I Type II Fiber surface area (Km2) Type I Type II Capillarity (n) Capillary density (nImmj2) Capillary/Type I fibers (n) Capillary/Type II fibers (n) Submaximal end-exercise End-exercise work rate (W) Normoxic $[Mb-HHb] (% from baseline) Hyperoxic $[Mb-HHb] (% from baseline) Normoxic $TOI (% from baseline) Hyperoxic $TOI (% from baseline)

Healthy Controls (n = 10)

P Value

24.7 (3.4) 108 (38) 123 (24) 35 (7)

G0.0001 G0.01 G0.0001 G0.001

479 (70) 80 (15)

— —

— —

23.3 (4.5) 8.2 (1.5)

31.0 (5.4) 9.9 (2.2)

G0.01 0.05

40 (13) 60 (13)

41 (10) 59 (10)

0.84 0.85

4389 (1358) 4209 (1532)

4926 (1199) 4145 (924)

0.40 0.91

201 (84) 2.18 (1.11) 1.64 (0.87)

304 (69) 3.11 (0.82) 2.24 (0.43)

0.01 0.07 0.09

41 (10) 29.6 (11.6)

41 (10) 9.7 (8.0)

1.0 G0.0001

26.1 (13.1)

11.2 (13.9)

0.03

j8.7 (j5.8)

j1.9 (j3.2)

G0.001

j8.6 (j5.6)

j2.7 (j3.8)

0.003

14.7 62 58 59

(2.2) (20) (15) (15)

Data are presented as mean (SD). MVC, voluntary strength; TWq, nonvolitional strength; V˙CO2, volume of expired carbon dioxide; V˙E, minute ventilation; 6MWT, 6-min walk test; $[Mb-HHb], change in the myoglobin–deoxyhemoglobin concentration from baseline; $TOI, change in tissue oxygenation index from baseline.

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patients with PAH and 11.6 (4.4) mm for controls (P = 0.88). ˙ O2 values were 0.622 (0.132) Normoxic end-exercise V LIminj1 and 0.539 (0.145) LIminj1 (P = 0.20) for patients with PAH and control subjects, respectively. Patients with PAH presented a rapid and continuous increase in [Mb-HHb] throughout the normoxic exercise (Fig. 1A (left); Table 2). A decrease in TOI was already apparent in the early stage of exercise with further deepening toward the end of exercise (Fig. 1B (left); Table 2). Whereas patients with PAH presented sustained decrease in SpO2 ($SpO2, j5.5% (1.8) vs –0.2% (0.6); P = 0.001) (Fig. 2C (left)), $CO and $DO2 did not differ between groups during the entire exercise protocol (Fig. 2A and B (left)). The addition of oxygen supplementation significantly slowed the fall in SpO2 (P = 0.04 for oxygen vs no oxygen supplementation) throughout the exercise protocol for patients with PAH (Fig. 2C), although $SpO2 remained slightly deeper at the end of the submaximal exercise stage for patients with PAH (j2.6% (0.9) vs 0.3% (0.4); P = 0.001). Conversely, oxygen supplementation had no effect on skeletal muscle oxygenation ($[Mb-HHb] and $TOI) (Fig. 1A and B (right); Table 2) during exercise (all P = not statistically significant for oxygen vs no oxygen supplementation), $CO (Fig. 2A (right)), and $DO2 (Fig. 2B (right)). Interestingly, $[Mb-HHb] and $TOI within inactive skeletal muscle did not differ between patients with PAH and controls (see Figure, Supplemental Digital Content 2, Normoxic $[Mb-HHb] and $TOI throughout submaximal exercise protocol for patients with PAH and healthy controls, http://links.lww.com/MSS/A551; and Figure, Supplemental Digital Content 3, Hyperoxic $[Mb-HHb] and $TOI throughout the in-house submaximal exercise protocol for patients with PAH and healthy controls, http://links.lww.com/MSS/A552). Lower limb capillarity density affects muscle oxygen perfusion. Among the variables that could affect skeletal muscle oxygenation, only quadriceps muscle capillary density correlated with $[Mb-HHb] at the end of submaximal exercise (Fig. 3). Quadriceps muscle capillary density also ˙ O2peak measured during CPET (r = 0.72; correlated with V P G 0.01). Conversely, $[Mb-HHb] did not correlate with the workload, $CO, $SpO2, or $DO2 achieved during submaximal exercise as well as with hemoglobin levels and resting pulmonary hemodynamics (all r G 0.2, all P 9 0.6). On the other hand, $[Mb-HHb] at the end of submaximal ˙ O2peak (Fig. 4A). Explorexercise strongly correlated with V atory analysis also suggested that $[Mb-HHb] at the end of the submaximal exercise correlated with the 6-min walked distance (Fig. 4B) and voluntary (Fig. 4C) and nonvolitional (Fig. 4D) quadriceps strength, suggesting an implication of impaired muscle oxygen perfusion on lower limb function.

DISCUSSION The present study demonstrated that patients with PAH exhibit lower skeletal muscle oxygen supply at the microcirculation level during exercise that may contribute to their

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CLINICAL SCIENCES FIGURE 1—Skeletal muscle oxygenation during normoxic and hyperoxic submaximal exercise in patients with PAH and healthy controls. Patients with PAH (open circles) exhibited early and persistent increases in [$Mb-HHb] (A, left) and decreases in $TOI (B, left) throughout normoxic exercise compared with healthy controls (solid circles). Oxygen supplementation had no effect on skeletal muscle $[Mb-HHb] (A, right) and $TOI (B, right) (all P = NS for oxygen vs no oxygen supplementation). The x-axis represents the different stages of the submaximal exercise protocol. The y-axis represents the relative change in percentage of [$Mb-HHb] and $TOI at 0% (the beginning of the stage), 25%, 50%, 75%, and 100% isotime for each stage of the exercise compared with the last 30 s of the preexercise resting period. The end of submaximal exercise period is shown by the gray area. Results are presented as means (SEM). *P G 0.05; **P G 0.01; and †P G 0.0001 between groups.

exercise intolerance. First, compared with that of controls, patients with PAH presented greater $[Mb-HHb] within the quadriceps muscle, reflecting significant imbalance between O2 delivery and use despite normal cardiac output response and estimated systemic oxygen delivery. Although patients with PAH also exhibited exercise-induced desaturation, quadriceps oxygenation did not correlate with systemic oxygen saturation during exercise and remained unchanged when performing the same exercise in hyperoxic condition. Conversely, reduced skeletal muscle oxygenation markedly correlated with quadriceps muscle capillary density. Finally, ˙ O2peak, skeletal muscle oxygenation correlated with patients_ V walking distance, as well as voluntary and nonvolitional quadriceps muscle strength. Collectively, these results suggest that capillary rarefaction within the skeletal muscle influences exercise tolerance and quadriceps muscle function at least partly through impaired muscle oxygen perfusion to working muscle myocytes. Exercise pathophysiology in PAH is characterized by disproportionate increase in pulmonary artery pressure, low

SKELETAL MUSCLE AND EXERCISE INTOLERANCE

stroke volume, limited chronotropic response, and ventilation– perfusion mismatch (29) resulting in excessive ventilation (19,20), exercise-induced hypoxemia, and overall poor exercise tolerance (34). Interestingly, cardiac output at rest or during submaximal exercise remains in the normal range in moderate PAH (29) and the relative increase in cardiac output during submaximal exercise is normal (40), as observed in the present study. More importantly, there is a major discrepancy between the moderately reduced cardiac output and ˙ O2peak observed in PAH (39). the profoundly decreased V Total systemic oxygen extraction during exercise is also impaired in PAH (39), suggesting that global O2 use from exercising muscles is reduced in PAH (22). Thus, in addition to the limited increase in maximal cardiac output, abnormalities in skeletal muscle microcirculation could further limit global O2 extraction by exercising myocytes and contribute to ˙ O2peak in PAH. decreased V Potential mechanisms of impaired skeletal muscle oxygenation in PAH. Although this could intuitively be attributed to right ventricle dysfunction and exercise-induced

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CLINICAL SCIENCES FIGURE 2—Cardiac output and estimated systemic oxygen delivery during normoxic and hyperoxic submaximal exercise in patients with PAH and healthy controls. Patients with PAH (open circles) presented no change in normoxic $CO (A, left) and estimated $DO2 (B, left) during exercise compared with healthy controls (solid circles). Oxygen supplementation had no impact on $CO (A, right) and $DO2 (B, right) during exercise (all P = NS for oxygen vs no oxygen supplementation), although it was associated with slightly higher $CO and $DO2 in patients with PAH compared with controls. Conversely, patients with PAH presented sustained decrease in $SpO2 (C, left) compared with controls (solid circles) for normoxic exercise while oxygen supplementation significantly reduced $SpO2 decrement (P = 0.04) for patients (C, right). The x-axis represents the different stages of the submaximal exercise protocol. The y-axis represents the relative change in percentage of CO, SpO2, and DO2 at 0% (the beginning of the stage), 25%, 50%, 75%, and 100% isotime for each stage of the exercise compared with the last 30 s of the preexercise resting period. The end of submaximal exercise period is shown by the gray area. Results are presented as means (SEM). *P G 0.05; **P G 0.01; and †P G 0.0001 between groups.

hypoxemia, increases in cardiac output were normal during both submaximal exercises and skeletal muscle oxygenation remained impaired despite near-normalization of systemic oxygen saturation and normalization of systemic oxygen delivery during hyperoxic exercise. In addition, no correlations

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were found between quadriceps muscle oxygen supply and exercise-induced changes in cardiac output, systemic oxygen saturation, O2 delivery, and hemoglobin levels. More recently, we documented that exercise intolerance in PAH was associated with skeletal muscle microcirculation loss and

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impaired angiogenesis secondary to miR-126 downregulation (27). We thus investigated whether microcirculation loss affected quadriceps muscle oxygenation. Interestingly, quadriceps muscle capillarity density tightly correlated with $[Mb-HHb],

FIGURE 4—Relation between [$Mb-HHb] within the quadriceps during normoxic submaximal exercise with maximal exercise tolerance and ˙ O2peak (A), the 6-min quadriceps_ function. Relative change (percentage from baseline) of [$Mb-HHb] at the end of exercise significantly correlated with V walked distance (B), the MVC (C), and the TWq of the dominant quadriceps (D). 6MWT, 6-min walk test.

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FIGURE 3—Relation between relative change (percentage from baseline) of [$Mb-HHb] within the quadriceps during normoxic submaximal exercise and capillary density.

which was associated with lower exercise tolerance and quadriceps strength. This finding is not surprising because coupling between microcirculation and active motor units largely influence O2 ˙ O2peak. Even in healthy uptake by the exercising muscles and V subjects, blood flow distributes heterogeneously within tissues (16) and muscle microcirculation markedly affects oxygen and nutrient delivery and exercise tolerance (32). Importantly, ˙ O2peak induced by training in healthy subjects increases in V are mainly linked to improved skeletal muscle microcirculation, resulting in prolonged peripheral blood transit time and reduced diffusion distance of O2 to the mitochondria and optimized O2 uptake from exercising muscles (9,38). Such phenomenon is also observed in PAH (7). Although controversy exists regarding the relative contribution of convective (bulk delivery of O2) and diffusive (movement of O2 from ˙ O2peak hemoglobin to mitochondria) elements in determining V in patients with congestive heart failure (11) and PAH (39), mismatch in perfusion and oxidative metabolism has been hypothesized as the limiting factor of the total amount of O2 extracted by exercising muscles in PAH (39,42). This explanation is physiologically plausible in PAH because, in addition to capillary rarefaction (27), increases in endothelin 1 levels and reductions in nitric oxide and prostaglandins,

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three major pathways involved in the pathogenesis of PAH (12), influence peripheral endothelium-dependent flowmediated vascular tone (5,44). Abnormalities in diffusive O2 transport may also explain the disconnection between $[Mb-HHb] and $SpO2 during exercise in hyperoxic condition, as previously observed in congestive heart failure (11). The present findings reinforce the multifactorial nature of exercise capacity in PAH. Indeed, it is becoming evident that exercise limitation is a multisystem process in PAH, as in other conditions, involving different components that are likely interconnected. How impaired skeletal muscle oxygenation interplays with other respiratory, cardiac, and neuronal mechanisms involved in exercise limitation in PAH, however, remains unknown. Methodological considerations. Several methodological considerations should be taken into account. First, the NIRS technique is unable to differentiate hemoglobin from myoglobin because the absorbency signals of these two chromophores occur at relatively the same wavelength (6). Moreover, the adipose tissue thickness (6), skin blood flow (37), and intramuscular adipose infiltration specific to PAH (26) may influence absolute NIRS light absorption. We reported relative changes in percentage of [Mb-HHb] to minimize these confounding factors. Thus, increase in $[MbHHb] is representative of altered O2 delivery in the lower limb capillary bed and is expected to be associated with impaired global skeletal muscle extraction, as previously reported in PAH (39). Furthermore, increases in cardiac output are relatively preserved during submaximal exercise in moderate PAH, whereas cardiac output during maximal exercise is generally reduced. To minimize the effect of variable cardiac output and exercise workload on skeletal muscle oxygenation, both PAH and control subjects underwent exercise at matched absolute exercise intensity rather than matched relative exercise intensity. In addition, the hemoglobin affinity for oxygen at rest was normal in patients with PAH. Nevertheless, early dyspnea and early lactic acid accumulation that characterize patients with PAH during exercise could have resulted in a leftward and rightward shift of the oxygen– hemoglobin dissociation curve, respectively (19). However, this is likely to have minimal effect on muscle $[Mb-HHb] because the exercise did not reach maximal intensity. Moreover, lower limb $[Mb-HHb] was already increased in PAH at very low exercise levels (even during unloaded exercise). Thus, differences in the relative exercise intensity or local hemoglobin affinity between patients with PAH and healthy controls were unlikely to explain differences in $[Mb-HHb] observed in the present study. In addition, increases in cardiac output and systemic oxygen delivery were comparable in both groups in normoxic condition and were slightly higher in patients with PAH during hyperoxic exercise. This observation was unexpected. In healthy subjects, exercise-induced increases in cardiac output are almost entirely diverted to exercising skeletal muscles and skin during exercise. Whether this increase in systemic

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oxygen delivery was mainly directed toward exercising muscles in patients with PAH remains to be confirmed. Indeed, heightened sympathetic nervous tone (41) and blood flow redistribution toward respiratory muscles at the expense of lower limb muscles could theoretically explain the enhanced systemic blood flow and oxygen delivery as well as the higher $[Mb-HHb] observed in PAH skeletal muscle during exercise. Nevertheless, this phenomenon is unlikely to fully explain these differences given the absence of significant changes in oxygen delivery within nonexercise muscles (see Figure, Supplemental Digital Content 2, Normoxic $[Mb-HHb] and $TOI throughout submaximal exercise protocol for patients with PAH and healthy controls, http://links.lww.com/MSS/A551; and Figure, Supplemental Digital Content 3, Hyperoxic $[Mb-HHb] and $TOI throughout the in-house submaximal exercise protocol for patients with PAH and healthy controls, http://links.lww.com/MSS/A552) and the O2-delivery-and-use imbalance at very low exercise levels. Importantly, however, our study population had relatively preserved functional capacity, which may have decreased the magnitude of differences between patients with PAH and healthy subjects. Thus, our results may not be representative of oxygenation abnormalities that would have been observed in patients with the most severe condition who may more heavily rely on conductive (cardiac output and O2 delivery) rather than diffusive O2 transport to meet the metabolic demands of exercise. This also likely explains the lack of Type 1 fiber rarefaction in patients with PAH, as previously described in similar PAH populations (7,25,27). Similarly, although our sample size precluded valid subgroup analyses, the influence of specific PAH medications on limb blood flow during exercise could not be ruled out.

CONCLUSIONS The present study documented that PAH results in impaired skeletal muscle oxygenation even during submaximal exercise, which tightly correlated with skeletal muscle capillary rarefaction. More importantly, impaired skeletal muscle oxygenation was associated with exercise intolerance and lower quadriceps strength. Although exercise intolerance in PAH is certainly multifactorial in origin, these results reinforce the increasingly recognized effect of skeletal muscle dysfunction on exercise pathophysiology in PAH and the important role of muscle capillarity density and oxygen supply on exercise intolerance in PAH. The authors wish to thank the contribution of members of the Pulmonary Hypertension Research Group team (http://www. pulmonaryarterialhypertension.ca) as well as the work and contribution of the Respiratory Health Network tissue bank (IUCPQ site) for storing human quadriceps biopsies. We also acknowledge the help of Annie Dube´, Ph.D., Luce Bouffard, Inf., Anne-Sophie Neyron, M.Sc., E´ric Nadreau, M.Sc., Sandra Breuils-Bonnet, M.Sc., and E`ve Tremblay, B.Sc., for their technical assistance with exercise testing and biopsies, respectively, as well as Serge Simard, M.Sc. for statistical assistance. S. M. is a recipient of a doctoral training award from the Fond de Recherche du Que´bec en Sante´ (FRQ-S) (grant number, 27818). F. P.

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intolerance in pulmonary hypertension. S. P. is a FRQ-S senior clinical scientist. Dr. Bonnet is a consultant for Merck & Co., Inc. Dr. Provencher has received research grants from Actelion Pharmaceuticals Ltd., Bayer AG, and GlaxoSmithKline and has received speaker fees from Actelion Pharmaceuticals Ltd. Dr. Maltais has received research grants from Boehringer Ingelheim, GlaxoSmithKline, AstraZeneca, Nycomed, Pfizer, and personal fees from Boehringer Ingelheim, GlaxoSmithKline, and AstraZeneca. The Pulmonary Hypertension Research Group is also supported by the FRQ-S. The remaining authors reported no potential conflicts of interest. The results of the present study do not constitute endorsement by the American College of Sports Medicine.

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is supported by a doctoral training award from the Que´bec Heart and Lungs Institute Research Center. The authors_ contributions are as follows: S. M., V. M., D. S., F. M., and S. P. planned the study design. S. M. recruited the patients, performed data collection and analysis, and wrote the manuscript. F. P., V. M., M. M., E. L., and F. R. contributed to data collection and analysis. D. S., F. M., S. B., and S. P. critically reviewed the manuscript. All authors approved the final version of the manuscript. F. M. holds a CIHR/GSK research chair on COPD at Universite´ Laval. S. B. holds grants from the CIHR and from the Heart and Stroke Foundation of Canada. He also holds a CIHR research chair on vascular biology. S. P. holds a CIHR grant focusing on angiogenesis and exercise

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Impaired Skeletal Muscle Oxygenation and Exercise Tolerance in Pulmonary Hypertension.

Limb muscle dysfunction is documented in pulmonary arterial hypertension (PAH), but little is known regarding muscle oxygen (O2) supply and its possib...
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