Published ahead of print on April 29, 2004, doi:10.1164/rccm.200304-578OC
© 2004 American Thoracic Society
Angiotensin Converting Enzyme Genotype and Strength in Chronic Obstructive Pulmonary DiseaseRespiratory Muscle Laboratory, Royal Brompton Hospital; Department of Cardiovascular Genetics, Rayne Institute; and Respiratory Muscle Laboratory, Guy's King's and St Thomas' School of Medicine, King's College Hospital, London, United Kingdom Correspondence and requests for reprints should be addressed to Nicholas Hopkinson, M.A., M.R.C.P., Respiratory Muscle Laboratory, Royal Brompton Hospital, Fulham Road, London, SW3 6NP, United Kingdom. E-mail: n.hopkinson{at}ic.ac.uk
Quadriceps muscle weakness is an important contributor to exercise limitation in patients with chronic obstructive pulmonary disease. The deletion allele of the angiotensin converting enzyme gene polymorphism has previously been associated with a greater response to strength training in healthy subjects and might, therefore, protect against detraining in these patients. In 103 stable outpatients (mean [SD] FEV1 34.4 [16.5] % predicted), the angiotensin deletion allele was associated with greater isometric quadriceps strength; mean (SD) 31.4 (10.8) kg for insertion homozygotes, 34.1 (13.0) kg for heterozygotes, and 38.3 (11.6) kg for deletion homozygotes (p = 0.04 linear trend). Adjusted for fat-free mass, the relationship was stronger (linear trend p = 0.007). There was no correlation between strength and genotype in a group of 101 age-matched healthy control subjects. Twitch quadriceps force in response to magnetic femoral nerve stimulation, measured in 39 patients, was also genotype dependent; 8.3 (2.6) kg for insertion homozygotes, 10.1 (3.6) kg for heterozygotes, and 12.4 (3.5) kg for deletion homozygotes (p = 0.002 linear trend). Body mass index and fat-free mass did not differ significantly between genotypes in either group. There was no association in either patients or control subjects between genotype and inspiratory muscle strength. In chronic obstructive pulmonary disease the deletion allele is associated with greater quadriceps strength independent of confounding factors.
Key Words: gene polymorphism • respiratory muscle • skeletal muscle
Skeletal muscle dysfunction is an important complication of chronic obstructive pulmonary disease (COPD) (1), which is associated with reduced quality of life (2), increased utilization of healthcare resources (3), and increased mortality (2). Indeed, in many patients, exercise is limited by perceived leg effort rather than breathlessness (4). Although a variety of associations have been reported, including disuse atrophy, poor nutrition, and systemic inflammatory mediators, such as tumor necrosis factor- Local renin-angiotensin systems exist in human tissues, including skeletal muscle (5–10). A polymorphism of the human angiotensin converting enzyme (ACE) gene has been identified in which the absence (Deletion, D) rather than the presence (Insertion, I allele) of a 287 base-pair fragment is associated with higher circulating (11) and tissue (12, 13) ACE activity and, consequently, with significantly higher angiotensin II and lower bradykinin levels (14). Angiotensin II is known to be involved in the regulation of production of cytokines, such as interleukin 6 (15), as well as growth factors, and might, therefore, modulate the development of cachexia (16). It has been shown to modulate the action of insulin on skeletal muscle (17) and may also affect skeletal muscle growth, being necessary both for angiogenesis (18) and for an optimal muscle trophic response to loading (19). Moreover, angiotensin II infusion has been shown to increase tetanic tension in rat hind-limb preparations (20). In healthy subjects, the D allele has previously been shown to be associated with a greater gain in skeletal muscle strength in response to isometric quadriceps training (21). Thus, we hypothesized that the D allele might be associated with preservation in muscle strength amongst patients with COPD. We have tested this by genotyping a cohort of patients with COPD and a group of age-matched healthy control subjects, and comparing inspiratory muscle strength and quadriceps maximum voluntary contraction (QMVC). Some of the results of these studies have been reported previously in abstract form (22).
The hospital's research ethics committee approved the study, and subjects gave written informed consent for participation. Consecutive white patients with a diagnosis of COPD consistent with the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria (23) attending clinics at Royal Brompton Hospital were invited to take part in the study. Patients were excluded if they had  -1 antitrypsin deficiency, significant comorbidity, including a clinical diagnosis of left ventricular failure, malignancy, arthritis, neuromuscular disease, or were using ACE inhibitors or angiotensin II receptor antagonists. Clinical information, including treatment, number of exacerbations in the previous year (defined as those requiring antibiotics), and average daily dose of oral prednisolone, was obtained through a structured interview with reference to patients' medical records. Healthy, age-matched controls were recruited for this study through advertisements in local newspapers (see online supplement for details). Data from this population and from some of the patients have been published previously (24). Spirometry was obtained using a pneumotachograph with flow integration, lung volumes by whole body plethysmography, and gas transfer with a single-breath technique (Compact Lab system; Jaeger, Germany). Blood gas tensions were measured in arterialized earlobe capillary samples. Predicted values used are those of the European Coal and Steel Community (25). Fat-free mass (FFM) was determined using bioelectrical impedance analysis (Bodystat 1500; Bodystat, Isle of Man, UK). In the patients, a disease-specific regression equation was used (26) whereas the device's internal algorithms were used for the healthy control subjects. QMVC was measured with subjects seated in a specially adapted chair using the technique of Edwards and coworkers, which required them to try to extend their knee as hard as they could by pulling against an inextensible strap attached above their ankle (Figure E1 in online supplement) (27). Values were normalized for body weight (% QMVC) according to convention (27). Values are also expressed per kg of FFM (QMVC/FFM). In 39 of the patients we also measured twitch quadriceps force in response to magnetic femoral nerve stimulation, as we have previously described (Figure E2 in online supplement) (28). To assess inspiratory muscle strength, maximum sniff nasal pressure (SNiP) was also determined (29). ACE genotype was determined by polymerase chain reaction amplification using a published 3-primer method that included an I-specific oligonucleotide. (30) Two independent technicians blind to subject characteristics confirmed genotypes. Discrepancies were resolved by repeat genotyping. No other genetic analysis has been performed in these subjects, and all clinical and physiological data were obtained blinded to genotype.
Statistical analysis, using StatView software (SAS Institute, NC), was directed toward an association between genotype and quadriceps strength both in absolute terms and as a percent of the predicted value. One-way analysis of variance and test for linear trend were used to look for codominant effects across genotype on continuous variables, and Further details of the methods used for genotyping and the assessment of quadriceps and inspiratory muscle strength are available in the online supplement.
We studied 103 patients with COPD and 101 healthy control subjects. Their clinical characteristics, body composition, and measures of strength are described in Table 1 . The patient group was significantly weaker than the control one, both in terms of quadriceps and inspiratory muscle strength, whether considered as a whole (Table 1), because there was a significantly higher proportion of men in the patient group [  2, p = 0.0001]), or assessed separately by sex (see Table E1 in the online supplement).
In both groups, the ACE genotype distribution was similar to that previously reported in the UK population, consistent with Hardy-Weinberg equilibrium (31). In the healthy controls, no parameter measured was related to genotype (Table 2) .
In the patients, there was no evidence of an association between genotype and age, sex, smoking history, or exacerbation frequency. Twelve patients were on long-term oral prednisolone taking doses of 10 mg or less per day, DD genotype 4%, ID genotype 16.3%, and II genotype 10.3%. Neither this nor the use of long-acting ß2 agonists (n = 54) differed significantly with genotype ( 2, p = 0.3 and 0.2, respectively). Average daily dose of prednisolone received in the preceding 12 months was the same in the three groups. Twenty-eight patients were using oral theophyllines, with a significantly lower proportion in the DD group: DD 8%, ID 39%, II 24%; 2, p = 0.02. No differences between genotypes were observed with respect to lung function. In particular, although there was a trend for more severe hyperinflation in patients with a D allele (Table 3)
, this only achieved significance for the functional residual capacity and not for total lung capacity or residual volume, and only when testing for single rather than multiple end points.
Quadriceps Strength In the patients, QMVC correlated significantly with weight, r2 = 0.27, height, r2 = 0.24, and FFM, r2 = 0.41 (all p < 0.0001). These parameters were not themselves significantly associated with genotype (all p > 0.4). QMVC was not associated with any lung function parameter. Strength varied significantly across genotypes from II (31.4 ± 10.8 kg) to ID (34.1 ± 13.0 kg) to DD (38.3 ± 11.6 kg) (linear trend p = 0.04) (Table 3) (See Figure E3 in the online supplement). Percent predicted QMVC and QMVC/FFM also varied significantly (linear trend, p = 0.012 and 0.007, respectively) as did twitch quadriceps force (linear trend, p = 0.002). Possible modification of the effect of ACE genotype on QMVC/FFM by disease severity (FEV1 and diffusing capacity) was not significant when examined via interaction terms in analysis of covariance models (p = 0.4 and 0.7, respectively). There was no correlation between strength and average daily dose of prednisolone received in the preceding year. The effect of genotype on strength remained statistically significant when the 12 patients on long-term oral prednisolone were excluded.
Inspiratory Muscle Strength
The main finding of this study is that the D allele of the ACE gene polymorphism is associated with greater quadriceps strength in patients with COPD judged by either volitional or nonvolitional techniques. This effect was independent of sex and of confounding clinical variables. In particular, our findings could not be explained by differences in disease severity. In a muscle group not exposed to detraining, the inspiratory muscles, a parallel change was not observed, nor was the effect seen in the quadriceps of healthy control subjects. We acknowledge that a limitation of this study is the absence of a direct measure of quadriceps bulk. It is not, therefore, possible to clarify whether our results are due to relatively preserved regional muscle bulk and/or greater specific tension, i.e., improved strength per unit of muscle. Arguments can be advanced for both mechanisms. It has been shown that the quadriceps response, both to training and detraining in healthy subjects, involves changes in torque per unit of cross-sectional area, which could be due to changes in the degree of neurological activation or an alteration in muscle architecture (32). Angiotensin II has been shown to affect both sympathetic and neuromuscular transmission, as well as increase tetanic force, mechanisms that might produce an increase in specific force (20, 33, 34). Interestingly, it has been shown that the I allele is associated with a greater proportion of slow-twitch fibers in human skeletal muscle (35), which could lead to greater endurance but reduced strength for a given muscle mass. Recent work suggests that it is this fiber-type shift that is the main determinant of impaired quadriceps function in COPD (36). In favor of a D allele-mediated preservation of quadriceps muscle mass is the association of the D allele with exercise-induced cardiac hypertrophy (37) and the dependence of hypertrophy upon local angiotensin II synthesis (19). We note that the ratio of quadriceps strength to whole body FFM differed between patients and controls, being lower in the COPD group, a finding consistent with previous reports (38). By contrast, previous studies have found that strength adjusted for extremity FFM (38) and for quadriceps cross-sectional area (39) is sustained in COPD. This would favor a local effect of a response to detraining modified by ACE genotype. The absence of an effect of genotype on whole body FFM lends support to the idea that skeletal muscle weakness is due predominantly to local effects, i.e., disuse atrophy rather than systemic ones, as do the findings in the control group. The finding that twitch quadriceps strength, a nonvolitional test, was also genotype dependent argues against a genotype-dependent effect on motivation. We did not perform a direct measure of functional capacity, but QMVC has previously been found to correlate with exercise capacity in patients with COPD, supporting the clinical relevance of our measurement (40, 41). In addition, regionally applied therapies, which are aimed specifically at improving quadriceps strength, have been shown to improve walking distance (42). We found no relationship between inspiratory muscle strength and ACE genotype. It is well established that the inspiratory muscles operate under an increased load in COPD (43) and are not subject to the same detraining effect as the quadriceps, and, indeed, allowing for lung volume, the inspiratory muscles are not weaker than those of control subjects (44). In fact, available data suggest that they are, in effect, endurance "trained" with increases in oxidative capacity and a shift toward Type I fibers (45). The absence of a relationship with genotype might be explained by the previous observation that the D allele is associated with a higher proportion of Type II fibers. Alternative cardiovascular mechanisms might, however, be at work. Among male patients with COPD admitted for a rehabilitation program, Van Suylen and colleagues found ACE DD genotype to be negatively associated with electrocardiographic right ventricular hypertrophy (46). In addition, DD genotype has been associated with impaired peripheral tissue oxygenation in patients with COPD (47). However, we would not have anticipated a decrease in tissue oxygenation (or cardiac output) to be mechanistically associated with an increase in skeletal muscle strength. The absence of a relationship between strength and genotype in the control group suggests that the ACE gene modifies the phenotypic response either to inactivity or to some other aspect of COPD. To test this further requires both prospective studies in patients to evaluate changes over time and, theoretically, to study the effect of a detraining intervention in healthy elderly subjects. It is difficult to see how the latter could be undertaken ethically, given the known benefits of exercise in this age group. Pulmonary rehabilitation is an important treatment modality in COPD (48). In this context, our finding that the severity of skeletal muscle weakness in COPD is related to ACE genotype may offer some insight into factors that determine the response to this intervention. We speculate that rehabilitation programs with a greater emphasis on strength training might be beneficial for patients with the I allele. Further studies are needed to clarify the mechanism underlying this association, and to explore the relationship between ACE genotype and muscle strength in other diseases in which skeletal muscle deconditioning occurs.
The authors thank Dr. A. K. Simonds and Professor D. M. Geddes, both of the Royal Brompton Hospital, for permission to study patients under their care.
Supported by the Wellcome Trust, UK (N.H.S.), the British Lung Foundation, UK (A.H.N.), the British Heart Foundation (J.P.), the Medical Research Council, UK (W.D-C.M.), the Portex Endowment at the Institute of Child Health, London, UK (H.M.), and the European Union grant QLK6-CT-2002–02285 (M.I.P.). This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org Conflict of Interest Statement: N.S.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; A.H.N. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; J.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; E.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; W.D.-C.M. has received assistance for travel to international conferences from Schering-Plough, GlaxoSmithKline, Allen & Hanbury's, Boehringer-Ingelheim, and Bayer pharmaceutical companies; J.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; H.M. is a named inventor on a patent concerning the role of RAS in metabolic efficiency, but passed this at no personal gain to a University-based company, and receives a stipend of approximately £10,000 annually and stock options from Ark Therapeutics Ltd. for consulting work relating to the use of RAS manipulation in cancer; M.I.P. has received lecture fees totaling approximately $3,700 between 2000 and 2003 from AstraZeneca, GlaxoSmithKline, Nutricia, and Cephalon, and has also attended professional meetings (ERS, ATS, APSS) as a guest of various companies, including Glaxo Wellcome, AstraZeneca, Cephalon, 3M, and Ciba. The Rayne Institute Department of Cardiovascular Genetics has received unrestricted educational grants from Aventis Pharmaceuticals over the past 3 years. Received in original form April 27, 2003; accepted in final form April 23, 2004
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O2 with small muscle mass exercise. Am J Respir Crit Care Med 2004;169:89–96.
