Original article

J.E. SHARMAN, D.P. JOHNS, J. MARRONE, J. WALLS, R. WOOD-BAKER, E.H. WALTERS

CARDIOVASCULAR EFFECTS OF METHACHOLINE-INDUCED AIRWAY OBSTRUCTION IN MAN

Menzies Research Institute Tasmania, University of Tasmania, Hobart, Tasmania, Australia
Cardiovascular disease is the most frequent cause of death in people with chronic respiratory disease. The cause of this association has been attributed to airway obstruction leading to cardiovascular dysfunction (increased central blood pressure (BP) and aortic stiffness). However, this has never been experimentally tested. Methacholine is routinely used to stimulate airway function changes that mimic airway pathology. This study aimed to determine the cardiovascular effects of methacholine-induced airway obstruction. Fifteen healthy young adults (aged 22.9±2.5 years; 4 male; mean±S.D.) underwent a bronchial challenge test (randomized, blinded, cross-over design) in which they received nebulized methacholine inhalation in serially increasing concentrations (from 0.39 to 25 mg/ml) or saline (0.9%; control) on two separate days. Bronchoconstriction was assessed by forced expiratory volume at one second (FEV1) and cardiovascular effects by augmentation index, brachial BP, central BP, heart rate and aortic stiffness. Methacholine significantly decreased FEV1 from baseline to peak inhaled concentration compared with saline (–0.48±0.34 vs. -0.07±0.16 L; p<0.001), but there was no between-group change in augmentation index (1.6±7.0 vs. 3.7±10.2%; p=0.49), brachial systolic BP (–3.3±7.6 vs. –4.7±5.7 mmHg; p=0.59), central systolic BP (–1.1±5.2 vs. –0.3±5.5 mmHg; p=0.73), heart rate (0.4±7.1 vs. -0.8±6.6 bpm; p=0.45) or aortic stiffness (0.2±1.3 vs. 0.8±1.8 m/s; p=0.20; n=12). Thus, methacholine induced airway obstruction does not acutely change brachial BP or central haemodynamics. This finding refutes the notion that airway obstruction per se leads to cardiovascular dysfunction, at least in healthy individuals in the acute setting.
Key words:
airway obstruction, cardiovascular disease, methacholine, forced expiratory volume, chronic obstructive pulmonary disease, aortic stiffness

INTRODUCTION

Several large studies have shown that cardiovascular disease (CVD) morbidity and mortality rates are higher in patients with chronic obstructive pulmonary disease (COPD) (1-6). Indeed, among patients with COPD, the most frequent cause of hospitalization and death has been reported as being heart failure and ischemic heart disease, rather than COPD-related events (7). Both CVD and COPD share common etiological pathways of cigarette smoking and systemic inflammation. However, the increased risk for CVD death associated with COPD is maintained after statistical correction for these variables, as well as other known risk factors such as age, obesity, cholesterol, diabetes, physical activity, social economic status, chest deformity and brachial blood pressure (BP) (8). Beyond these conventional risk factors, some investigators have reported that augmentation index (a marker of central BP and left ventricular loading) and aortic stiffness (9) are significantly higher in patients with varying severity of airway obstruction (10, 11). Furthermore, lung function as determined by the forced expiratory volume at 1 second (FEV1), has been shown to independently predict aortic stiffness in a large prospective cohort (12).

Although the underlying pathophysiology is unclear, taken together these data raise the possibility that airway pathophysiological changes may directly influence large artery function (e.g. aortic stiffness) and central BP. This could have important consequences for CVD risk because a major role of a normal functioning aorta is to buffer the rise in central (ascending aortic) systolic BP (SBP) with each cardiac contraction. In the case of a stiffened aorta (as found in patients with chronic airway disease) there is greater impedance to left ventricular outflow, resulting in augmentation of central SBP (and augmentation index) (13, 14). Central BP and aortic stiffness can be reliably estimated by analysis of arterial pressure waveforms recorded non-invasively with applanation tonometry (15). Using these methods it is now well appreciated that these markers predict CVD events and mortality better and independently of brachial BP (16, 17).

To begin to understand if there is a direct pathophysiological connection between arterial and airway function, we sought to determine the central and peripheral haemodynamic response (augmentation index, brachial BP, central BP, heart rate and aortic stiffness) to acute airway obstruction induced with inhaled methacholine in a healthy young cohort that was not influenced by possible age or disease related risk factors. Bronchoconstriction induced with inhaled methacholine is appropriate to study an acute direct influence on cardiovascular effects by airway obstruction, because the method has been shown to selectively cause airway lumen narrowing via cholinoceptors of bronchial circumferential smooth muscle, but without systemic spill-over effects of absorbed methacholine (18).

MATERIALS AND METHODS

The study was approved by the University of Tasmania Human Research Ethics Committee. All participants provided written, informed consent and the studies conformed to the standards set by the latest revision of the Declaration of Helsinki.

Study participants

Twenty healthy young adults were recruited by local advertisement. Inclusion criteria were: participants aged 18 to 25 years; and able to perform acceptable and repeatable spirometry (see description below for definition). Exclusion criteria were: a clinical history of CVD or renal disease; epilepsy; self-reported history of hypertension or hypercholesterolemia; eye or abdominal surgery within 6 weeks of attendance; taking lipid lowering or antihypertensive medication (including beta-blocker eye drops); pregnancy or breast feeding; baseline FEV1 less than 70% of the predicted value or an FEV1 less than 1.5 litres.

Study protocol

This was a randomized, placebo-controlled, cross-over study in which each participant attended the laboratory for testing on two separate days at similar times. Fig. 1 provides an overview of the study design. On each testing day participants were asked to avoid caffeinated drinks, smoking, heavy meals and strenuous exercise. At each visit, eligibility criteria were checked and anthropometric measures acquired. At the first visit, participants performed spirometry to obtain a baseline measure of FEV1 and were then randomized to receive either active treatment (incremental doses of inhaled nebulized methacholine) or control (inhaled nebulized 0.9% normal saline, the same solution as the diluent used in the preparation for inhaled methacholine), and on the subsequent visit participants received the alternate intervention.

Brachial and central BP, augmentation index, heart rate and spirometry were measured at baseline and immediately after each dose of methacholine and saline up to peak. BP and augmentation index measures were recorded within one minute and FEV1 within three minutes of each dose administered. Due to time limitations, aortic stiffness was only recorded at baseline and after peak dose inhaled. Each participant, as well as the investigator recording cardiovascular measures (JES), were blinded to the treatment allocation. In order to ensure appropriate participant safety, the investigator performing spirometric testing (JM) was not blinded to treatment allocation.

Spirometry and methacholine challenge

Prior to inhalation of methacholine or saline control, each participant performed spirometry in the seated position to establish baseline FEV1 and to screen for the presence of FEV1 exclusion criteria. Spirometry was conducted according to international guidelines (19). Acceptable and repeatable spirometry was defined as being initiated at full lung inflation and with maximum expiratory effort and no evidence of artefact (e.g. cough in the first second) and with less than 150 ml variation between the two highest FEV1 values. The methacholine challenge test involved oral inhalation of increasing concentrations of aerosolised methacholine generated by a breath-activated Mefar dosimeter (Mefar, model MB3, Bovezzo, Milan, Italy). FEV1 was measured between each incremental dose. The next dose of methacholine/saline was administered as soon as possible after completion of successful recording of FEV1. The cumulative doses after stage 1 baseline saline were stage 2 – 0.0156 mg; stage 3 – 0.0625 mg; stage 4 – 0.25 mg; stage 5 – 2.0 mg. Bronchial challenge testing was stopped if the FEV1 fell by >20% from baseline values or at the request of the participant.

Brachial blood pressure

Baseline measures of brachial BP were recorded in duplicate (one minute apart) according to standard guidelines (20) after at least five minutes of seated rest using a validated oscillometric device (Omron HEM 907). Due to time constraint, only one measure of brachial BP was taken immediately after each inhalation dose.

Central blood pressure

Applanation tonometry was used to record radial artery waveforms over a nine second period at each challenge time point. Customized software (SphygmoCor 8.1, AtCor Medical, Sydney, NSW) was used to derive the ascending aortic (central) pressure waveform using a generalized transfer function which we have shown to be valid and reliable (21, 22) in response to hemodynamic perturbations. Baseline measures of central BP were recorded in duplicate immediately after the baseline brachial BP measures, and one measure of central BP was recorded at the end of each respiratory challenge. Heart rate was recorded from the radial tonometric waveforms.

Augmentation index

From the central pressure waveform, augmentation index was calculated from the difference between the second and first systolic peaks as a percentage of central pulse pressure (15).

Aortic stiffness

Aortic pulse wave velocity, the reference standard for determining aortic stiffness (15), was measured by electrocardiogram-gated, sequential applanation tonometry at the carotid and femoral arteries (SphygmoCor 8.1, AtCor Medical, Sydney, New South Wales). Measures were acquired with participants in a semi-recumbent position on a chair using pillows to support the torso. This approach was used in order to rapidly acquire all measures (seated and semi-recumbent) without needing to make substantial positional changes (i.e. seated to supine) that may also affect consistency of physiological responses.

Statistical analysis

Data were analysed using SPSS PASW Statistics 18 software (SPSS Inc., Chicago, Illinois) and are presented as mean ± S.D. except where indicated. Statistical significance was assessed at P<0.05. Paired t-tests were used to compare baseline differences between each visit (saline versus methacholine). The change from baseline to peak saline versus methacholine was assessed by analysis of covariance corrected for body mass index. Mixed between-within analysis of variance and multivariate statistics corrected for body mass index were used to determine the:

1) change of measured variables from baseline to each respiratory challenge time point (main effects for time);

2) comparison between saline and methacholine for each variable (main effects for intervention) and;

3) difference between interventions over time (interaction effects).

Significance was determined with the Wilks’ Lambda test and effect size calculated from the partial Eta squared. If a significant effect was observed by analysis of variance, paired t-tests were undertaken to determine at which time point there were between-intervention differences. From our previous work (21) we determined that a clinically significant (12%) (16) between-group difference in augmentation index could be detected with 15 participants. Moreover, we recently recorded aortic pulse wave velocity in 15 healthy subjects tested on two occasions (28±18 days apart) and found this to average 6.35±1.07 m/s (unpublished data). From these data we calculated that with n=12, a between-group difference of 1.07 m/s in aortic pulse wave velocity could be detected (all calculations with two-sided a=0.05 and β=0.20).

RESULTS

From the 20 participants enrolled in the study, three were excluded due to low % predicted FEV1 and two participants failed to return for the second visit. The characteristics of the 15 participants completing the study are presented in Table 1. At baseline there were no significant differences between intervention and control for any measured variable (P>0.05 for all). One challenge was stopped after the first saline dose due to a drop in FEV1 >20% and all remaining readings for this visit were carried forward for data analysis. Three participants declined to have aortic pulse wave velocity measured. Therefore, full sets of baseline and peak values were available on 12 participants.

Table 1. Participant characteristics.
Table 1
FEV1, Forced expiratory volume at 1 second; FVC, forced vital capacity.

Table 2 shows the changes from baseline to peak challenge for respiratory and haemodynamic variables. As expected, there was a significantly greater fall in FEV1 following inhalation of methacholine compared with saline from baseline to peak challenge. However, there was no significant between-intervention change for any haemodynamic variable. To ensure that the lack of haemodynamic change was not type II error, we conducted the same analysis in a subgroup (n=8) of participants who experienced ≥10% fall in FEV1 (range –11% to –29%) from baseline to peak methacholine dose. In these participants, the mean change in FEV1 was significantly greater with methacholine compared to saline (–21.0±8.4 % vs. –3.3±5.7%; P<0.001), but there were no significant change from baseline to peak dose for aortic pulse wave velocity or any haemodynamic variable (P>0.11 for all).

Table 2. Change in respiratory and haemodynamic variables from baseline to peak challenge of methacholine and saline.
Table 2
Data is mean ± S.D. P value is for the between-intervention change corrected for body mass index. *P<0.001 for comparison with baseline.

Results of the analysis of variance of saline compared with methacholine are presented in Table 3. There was a significant time and interaction effect for FEV1. However, there were no other significant changes over time or between interventions for any variable. Similar results were obtained when the analysis was restricted to the eight participants who had a ≥10% fall in FEV1 from baseline to peak methacholine dose. Results also remained similar when the analysis was restricted to three, instead of six, time points (baseline, stage 4 dose and peak challenge dose). Fig. 2 shows the between-intervention percentage changes over time for FEV1 and brachial SBP. Fig. 3 shows the change in augmentation index and central SBP between-interventions over time.

Table 3. Multivariate statistics for the mixed between-within analysis of variance for the time, intervention and interaction effects of saline compared with methacholine challenge in 15 healthy participants.
Table 3
ES, effect size by partial Eta squared. Data corrected for body mass index.
Figure 1
Fig. 1. Summary of study protocol with cumulative dose of methacholine (METH) or saline control. AIx, augmentation index; BP, blood pressure; FEV1, forced expiratory volume at 1 second.
Figure 2 Fig. 2. Percentage change from baseline for brachial systolic blood pressure (SBP; circles; left y axis) and forced expiratory volume at 1 second (FEV1; squares; right y axis) over five stages of increasing doses of methacholine (black circles and squares) compared with saline control (open circles and squares) in 15 healthy participants. Compared with control, there was a significant fall in FEV1 in the final two stages with methacholine (*P=0.012, **P<0.0001) but no significant change in brachial SBP at the same time points (P=0.91 and P=0.59).
Figure 3 Fig. 3. Augmentation index (panel A) and central systolic blood pressure (SBP; panel B) at baseline and over five stages of increasing doses of methacholine (black circles) compared with saline control (open circles) in 15 healthy participants. There were no significant changes over time or between interventions for either variable.

Univariate correlates of the change in FEV1 from baseline to peak challenge was determined by association with all BP and arterial stiffness variables. There were no significant associations when the data were assessed with all measures (saline plus methacholine changes), or with saline or methacholine independently (P>0.05 for all), or in the complete group or in the subgroup of participants with ≥10% fall in FEV1.

DISCUSSION

To our knowledge this is the first study to report the arterial haemodynamic effects of experimentally controlled bronchoconstriction causing acute airway obstruction in man. The rationale for this investigational approach was based, firstly, on the knowledge that CVD risk is increased in patients with airway disease (1-7) and, secondly that airway obstruction has been implicated in a possible causative role for increasing aortic stiffness and central BP, but this has never been explored mechanistically (11, 12). Prior to studying a potentially more complex ‘model’ of older patients with established disease and airway remodelling (23), we wished to determine the effects in healthy young participants. Our novel findings were that augmentation index, brachial BP, central BP, heart rate and aortic stiffness were unchanged despite significant bronchoconstriction. These data do not support the hypothesis that airway obstruction per se causes large artery dysfunction, at least in healthy individuals in the acute setting.

This study was an experimental design in the acute setting and, as such, chronic factors influencing arterial and respiratory function, such as changes in arterial wall structure or sympathetic activation, could not be addressed. A multitude of factors potentially influencing cardio-respiratory interaction have been identified in animal models. For example, antioxidant depletion through poor nutritional health can affect susceptibility to both vascular and lung damage (24, 25). Furthermore, aortic and airway physiology are dependent on nitric oxide bioavailability (26, 27), and in apparently healthy subjects, aortic stiffness is associated with both impaired brachial artery reactivity (28) and systemic endothelial function (29). In patients with COPD, brachial endothelial function has been shown to be impaired (30) and also inversely associated with FEV1 (31). Importantly, augmentation index and aortic stiffness are sensitive to changes in endothelial function and mean arterial pressure (26, 32, 33). If low FEV1 directly influences arterial function by increasing aortic stiffness, as previous studies suggest (12), then augmentation index (indicative of left ventricular afterload) and aortic pulse wave velocity would be expected to increase at peak methacholine challenge dose when FEV1 was at its lowest. These central haemodynamic responses would not necessarily be evident from conventional brachial BP measurements (34).

Although we determined sample size from our previous published data and this study was powered sufficiently to detect changes in augmentation index, it may be argued that larger effect sizes may be needed to detect significant changes in aortic stiffness. Nonetheless, the absence of any hint of change in aortic stiffness with intervention in the current analysis makes a flawed conclusion unlikely. Our findings of no change in central haemodynamic responses are also consistent with observations in conscious sheep where inhaled methacholine produced airway lumen narrowing but without significant effects on aortic pressure, right atrial pressure or heart rate at similar methacholine doses used in our study (18). However, higher concentrations of inhaled methacholine (i.e. 16–32 mg/ml) were shown to induce a rise in both aortic pressure and heart rate, possibly from mechanical and reflex responses to increased cardiac output as well as sensory input from respiratory muscles and pulmonary stretch receptors (18). Since aortic stiffness is positively associated with both aortic distending pressure (35) and heart rate (36), at high dose methacholine one may expect a functional (reflex) increase in aortic stiffness, through preferential recruitment of ‘stiffer’ collagen fibres at higher aortic pressures (35).

We used inhalation of the cholinergic agonist, methacholine, with the aim of promoting acute bronchoconstriction. Other possible pharmacological effects of this agent are bradycardia and/or vasodilation (by nitric oxide dependent mechanisms), which could have influenced the findings. Arguing against this possibility was the lack of change in heart rate or mean arterial pressure in the immediate period after methacholine administration, which tends to reassure that there was no significant systemic cardiovascular spill-over effects of the drug as previously shown by McLeod et al. (18). Continuous haemodynamic monitoring was not undertaken, so there remains the unlikely possibility that a decrease in heart rate from methacholine could have been quickly reversed by cardiovascular compensatory mechanisms, such as increased sympathetic activity. Furthermore, healthy young subjects were studied, rather than patients with established airway disease in which a different response could potentially occur and the findings cannot be generalized to other populations.

It is now well accepted that patients with chronic airway disease are at greater risk for CVD events and mortality (1-6), but the reasons for these associations are not known. Independent of brachial BP, recent studies implicate the possible causative influence of low FEV1 on increased central BP and aortic stiffness (11, 12). However, this current study found that acute falls in FEV1 provoked by inhaled methacholine had no significant effect on brachial and central BP or aortic stiffness compared to saline control inhalations. While this response in normal young subjects is indicative of a relative disconnection between changed FEV1 and large artery haemodynamics, we speculate that a different reaction may occur in patients with airway disease who have less arterial and lung function reserve capacity. Further studies using similar design to this current investigation may help elucidate the relation between CVD and respiratory disease.

Acknowledgements: This work was supported by a National Health and Medical Research Council of Australia Career Development Award (Dr Sharman; reference 569519).

Conflict of interests: None declared.

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R e c e i v e d : November 15, 2014
A c c e p t e d : March 28, 2014
Author’s address: Dr. James E. Sharman, Menzies Research Institute Tasmania, University of Tasmania, 23 Private Bag, Hobart, 7000, Australia e-mail: James.Sharman@menzies.utas.edu.au