978-1-4244-7000-6/11/$26.00 ©2011 IEEE

CAN BREATHING OSCILLATIONS REDUCE ASTHMA MEDICATION?

Mathur, M., Al-Jumaily, A.M., IJpma, G. Alany, R.

Institute of Biomedical Technologies School of Pharmacy Auckland University of Technology

Auckland, New Zealand University of Auckland Auckland, New Zealand

ABSTRACT

Airway hyperresponsiveness and obstruction are the major characteristics of asthma. Current asthma treatments using anti-inflammatory agents and airway smooth muscle (ASM) relaxants are expensive, variable in effectiveness and are associated with several cardiovascular side effects. ASM behaviour in vitro has been studied by various researchers to possibly provide an explanation of its mechanical properties. Previous in vitro experiments conducted on ASM tissues suggest that length perturbations applied to contracted muscle result in a reduction in the contractile ability of the tissue. This study focuses on investigating the combined effects of bronchodilators and length oscillations on the dynamics of contracted ASM. Isolated porcine tracheal smooth muscle tissues are contracted using Acetylcholine. Isoproterenol (Iso), a �-agonist, is used as a bronchodilator to relax the contracted ASM. Our results suggest that the combined effect of Iso and breathing oscillations is noted to be greater than the added effects of Iso and breathing alone. We speculate that, when breathing is combined with Iso, breathing facilitates the detachment of actomyosin bridges, while Iso prevents further phosphorylation of myosin heads, inhibiting the binding of myosin to actin.

I. INTRODUCTION Asthma is a respiratory disease characterized by chronic

airway inflammation, hyperresponsiveness and reversible airway obstruction. Asthma is a huge burden on the world due to its increased global prevalence and associated morbidity & mortality rates. Although many treatments have been developed for the alleviation of asthma symptoms, no cure yet has been found. Airway smooth muscle is regarded as the key effector responsible for bronchoconstriction in asthma. In healthy airways, deep inspiration (DI) is known to act as a bronchodilating and bronchoprotective mechanism [1, 2]. Nadel and colleagues were one of the first to report the bronchodilatory and bronchoprotective properties of the DI [2, 3]. In health airways, the DI has been shown to reverse bronchoconstriction when applied to a contracted airway [4, 5]. DI induced before the administration of a contractile agent, suppresses the subsequent bronchoconstriction providing a protecting effect [4-9]. Over the years, various researchers have proposed different mechanisms responsible for the DI induced bronchodilatation and bronchoprotection such as

neural/hormonal mechanisms, myogenic response, cross-bridge dynamics and length adaptation.

Length perturbations (as they occur in the case of tidal and deep breathing) applied to contracted ASM are known to relax the muscle [4, 10-12]. Fredberg et al. suggested that length perturbations promote the detachment of the actomyosin bridges, thus relaxing the muscle [11, 13].

�-agonists such as Isoproterenol are well known for their ability to relax the airway smooth muscle through the stimulation of cAMP production [14]. Isoproterenol (Iso) promotes the coupling between the �-adrenoceptor and an intermediary stimulatory G protein to activate adenylate cyclase. Adenylate cyclase promotes the conversion of ATP to cAMP. cAMP effects are believed to be mediated via two processes: decrease in intracellular Ca2+ and phosphorylation of MLCK by cAMP-dependent protein kinase (cAMP-PK), in turn causing relaxation [14].

Gump et al. [15] compared the effects of Isoproterenol with that of tidal oscillations on the relaxation of activated airway smooth muscle suggesting similar force inhibition with Isoproterenol (10-5M) and 4% length oscillations. Gump also conducted dose response for Isoproterenol ranging from 10-7 to 10-5M when the muscle is isometric and when the muscle is subjected to 4% length oscillations.

This study examines the effects of oscillations combined with Isoproterenol. The recovery force subsequent to the application of oscillations or Isoproterenol is the main point of interest here. The recovery force subsequent to the application of oscillations or Isoproterenol is the main point of interest here. Recovery force is the stable force subsequent to oscillations expressed as a percentage of the maximal contractile force.

II. METHODS Porcine trachea were obtained from a local abattoir and transferred in physiological saline solution (composition in mM: NaCl: 110.54, KCl: 3.39, MgSO4: 0.82, KH2PO4: 1.2, NaHCO3: 25.68, Glucose: 5.55, CaCl2.2H2O: 2.4; pH 7.4; aerated with 95% O2/5% CO2 to maintain the oxygen supply and pH of the solution). Rectangular strips of trachealis muscle were dissected from the trachea after removal of the epithelium and connective tissue layer. Muscle strips (2-3 mm wide × 5-9 mm long) were mounted vertically in a 5-ml tissue bath containing physiological

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saline solution at 37°C that was bubbled wCO2. One end of each muscle strip was fixglass hook using silk thread, while the attached to a servo-controlled lever (modeScientific). The muscle is contracted using 10-6 M(ACh). To determine the reference length (Llength is increased progressively after eauntil the force of active contraction reachvalue. All experiments are conducted at thIsoproterenol, a general �-agonist, isbronchodilator to relax the contracted ASMThe protocol contained three types of follows:

A. Iso only The tissue was contracted with 10-6M ACh After ~5 minutes as the active force plateawas added to the bath. After 5 minutes, tisswith PSS to completely relax the muscle.

B. Oscillations only The tissue was contracted; as the active fplateau (~5 min), oscillations were then minutes, and the force was allowed to reminutes of recovery, the tissue was relaxed

C. Iso combined with oscillations The tissue was contracted for 3 minsuperimposed oscillations induced for 10 mseconds. While the tissue oscillated, Iso wbath after 100 seconds. The recovery force cases was noted after 5 min. After the relaxed, the PSS was replaced two to three tnext contraction to ensure fully washing out The data was analyzed by conducting a tdata and 1-way ANOVA for comparing avalue less than 0.05 was considered asample size ‘n’ was taken as 4 tissues.

III. RESULTS To resemble asthmatic contraction, in this contracted by ACh. The effect of Iso alocontracted ASM is presented in Fig 1, breathing oscillations is shown in Fig 2. effects of breathing oscillations with however, are presented in Figure 3. These firecovery force normalized with respect tocontractile force vs. time. The recovery forcthe application of oscillations and/or Isopmain focus in this study. These figures areof the 4 samples of experiments conducted.

with 95% O2/5% xed tightly to a other end was

el 300C; Aurora

M Acetylcholine Lref), the muscle ach stimulation hes a maximum his length (Lref). s used as a .

f sequences as

for ~5 minutes. aued, 10-7M Iso sue was washed

force reached a induced for 3

ecover. After 5 with PSS.

, followed by minutes and 100 was added to the

in all the above tissue is fully

times before the t ACh and Iso.

t-test on paired all conditions. P acceptable. The

work ASM are one in relaxing

while that of The combined Isoproterenol,

igures show the o the maximum ce subsequent to roterenol is the e representative

Time (min)

Figure 1. Isoproterenol on contracted ASM

Time (mi Figure 2. Breathing oscillations on contracte

Time (mi Figure 3. Breathing oscillations combcontracted ASM As evident from the results, combined with Isoproterenol abreathing oscillations and Isoprotethe airway smooth muscle. ThIsoproterenol with breathing oapproximately 70.25 ± 6.5 % reduto 23.75 ± 4.5 % and 16 ± 7.37 %with Iso and breathing alone (n=4

10-7M Iso

10-7M Iso

Rec

over

y Fo

rce

(%F m

ax)

Rec

over

y Fo

rce

(%F m

ax)

Rec

over

y Fo

rce

(%F m

ax)

)

in)

ed ASM

in)

ined with Isoproterenol on

breathing oscillations are more potent than erenol alone in relaxing

he combined effect of oscillations causes an uction in force compared % reduction respectively 4). The relaxation due to

~70% reduction

~16% reduction

~24% reduction

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combined Iso and breathing is more than the sum of the Iso and breathing applied individually.

Iso

Breathing

Breathing + Is

o0.0

0.2

0.4

0.6

0.8

1.0

Rec

over

y Fo

rce

(%Fm

ax)

Figure 4. 1-way ANOVA analysis for Iso, breathing and both in

combination.

A t-test conducted on the data suggested a significant difference in the recovery force using breathing oscillations in the presence and absence of Isoproterenol (p = 0.01). 1-way ANOVA conducted on the data (Fig 4) suggested a significant difference in the recovery force in cases of breathing, Iso and in combination (p = 0.03).

IV. DISCUSSION The results compare the effect of tidal oscillations with that of Iso and combined Iso with oscillations on in vitro ASM. The main finding of our research is that oscillations of breathing frequency and amplitude in ASM tissues increase the relaxing effect of Isoproterenol. This combined effect is considerably higher than the sum of the relaxant effects of breathing and Iso individually. Gump et al. [15] observed that when the Iso was applied with or without tidal oscillations, the degree of relaxation was similar between both modalities at all but 10-5M concentration of Iso. This behavior suggested different mechanisms of relaxation for Iso and oscillations. Gump et al. suggested that the effect of Iso combined with tidal strains is multiplicative rather than additive. Contrary to that, our results propose that breathing oscillations facilitate the relaxation by Iso at the concentration of 10-

7M. The combined effect of Iso and breathing oscillations was noted to be greater than the added effects of Iso and breathing. The difference in observations could be due to the choice of porcine ASM in this study as opposed to bovine ASM used by Gump and colleagues. Also this study takes into account the recovery force subsequent to

application of oscillations or Iso, whereas Gump et al. focused on the dynamics during oscillations. Iso relaxes the smooth muscle by reducing the levels of Ca2+ through a range of signaling events, in turn preventing further phosphorylation of myosin heads. Breathing is known to acutely relax the muscle by promoting the detachment of myosin heads from actin. The effect of tidal oscillations is rapid due to the high detachment rates leading to high speed sliding of myosin with regards to the actin filament resulting in a change of the contractile state. We speculate that, when breathing is combined with Iso, breathing facilitates the detachment of actomyosin bridges, while Iso prevents further phosphorylation of myosin heads, inhibiting the binding of myosin to actin. Although the mechanisms of action of Iso and oscillations are different, breathing assists the role of Iso in relaxing the airway smooth muscle. In future research, the effects of Iso and oscillations will be assessed at other concentrations of Iso as well as using different amplitudes and frequencies of length oscillations.

ACKNOWLEDGEMENTS The authors would like to thank Dr Simeon Cairns from the School of Sport and Recreation, AUT University for providing invaluable expertise to this work.

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