INTRODUCTION

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PHARMACOLOGICAL IMPROVEMENT of diaphragm muscle (Dia_mus) contractility may be of clinical importance in the treatment of chronic obstructive pulmonary disease (COPD) when compromised Dia_mus function is a limiting factor. Recent in vitro studies in the rat Dia_mus have demonstrated an increase in isometric contractile force generation with either subcutaneous or in vitro administration of salbutamol (Slb), a ₂-adrenoceptor agonist (24, 25). The ability of the Dia_mus to shorten during activation is also critically important in the generation of ventilatory pressure; however, to date, no study has examined the effects of Slb treatment on isotonic contractile properties of the Dia_mus. In limb muscles, acute administration of Slb has been reported to increase isometric force in predominantly fast-twitch muscles (type II fibers) and to decrease force production in predominantly slow-twitch muscles (type I fibers) (1). The differential effect of Slb on type I and II fibers may also be relevant in the Dia_mus. A selective effect on type II fibers might be expected to result in an increase in shortening velocity and/or power output of the Dia_mus.

The purpose of the present study was to investigate the effects of Slb treatment on the isotonic contractile properties of the rat Dia_mus. On the basis of the observations cited above, we hypothesized that Slb increases the maximum velocity of shortening (V_max) and power output of the Dia_mus. Furthermore, given the well-known transduction mechanisms associated with the ₂-adrenoceptor pathway in smooth and cardiac muscles, we hypothesized that the effects of Slb on isotonic Dia_mus properties are mediated via an elevation in cAMP.

	METHODS

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Animals, treatment, and surgical procedures. All procedures used in this study were approved by the Institutional Animal Care and Use Committee of the Mayo Clinic and were in strict accordance with the American Physiological Society animal care guidelines. Adult male Sprague-Dawley rats (mean body weight 320 ± 4 g) were divided into two groups: 1) saline-treated controls (Ctl; n = 14); and 2) salbutamol treated (Slb; n = 12). Animals were anesthetized by intramuscular administration of ketamine (60 mg/kg) and xylazine (2 mg/kg). To minimize potential Slb diffusion limitations, animals in the Slb group were intracardially administered a loading dose of 25 µg/kg Slb; Ctl animals were administered an equal volume of 0.9% NaCl (0.5 ml/kg). Within 5 min after intracardial infusion of Slb or NaCl, the Dia_mus was excised and transferred to oxygenated Rees-Simpson solution (Ctl) or Rees-Simpson solution containing 0.05 µM salbutamol (Glaxo-Wellcome). The concentration of Slb was calculated based on the mean human serum concentration after a single oral dose of 4 mg (~10-20 µg/l or 0.03-0.07 µM) (12, 13).

cAMP measurements. In a subset of Ctl (n = 8) and Slb-treated (n = 8) animals, midcostal Dia_mus segments were dissected, weighed, and then incubated, in triplicate, for 15, 30, and 60 min in the presence (Slb group) or absence (Ctl group) of 0.05 µM Slb dissolved in oxygenated Rees-Simpson solution. This Rees-Simpson solution also contained 1 mM of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (Sigma Chemical). Immediately after this incubation period, the muscle segments were frozen in melting isopentane cooled in liquid nitrogen and stored at 70°C.

Measurement of Dia_mus contractile properties. On the basis of the time course of changes in cAMP levels in response to Slb (Fig. 1), all contractile measurements were completed within 30 min after excision of the Dia_mus. Segments (~3 mm wide) of the Dia_mus from the midcostal region were mounted vertically in a glass tissue chamber containing oxygenated Rees-Simpson solution with the following composition (in mM): 135 Na⁺, 5 K⁺, 2 Ca²⁺, 1 Mg²⁺, 120 Cl, 25 HCO₃, 11 glucose, 0.3 glutamic acid, 0.4 glutamate, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid buffer, and 0.012 d-tubocurarine chloride (pH 7.4). The solution was oxygenated with 95% O₂-5% CO₂, and temperature was maintained at 26°C. The origin of the muscle bundle at the costal margin was attached to a metal clamp mounted in series with a micromanipulator at the base of the tissue chamber. The central tendon was glued to a plastic holder that was firmly attached to the lever arm of a dual-mode length-force servo-control system (model 300B, Cambridge Technologies).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Effect of acute administration of salbutamol (Slb) on cAMP levels in rat diaphragm muscle (Diamus). Values are means ± SE. After exposure to 0.5 µM Slb, cAMP levels were significantly elevated (P < 0.05). However, by 1 h after Slb exposure, cAMP levels decreased and were comparable between control (Ctl) and Slb-treated Diamus. * Significant difference between Ctl and Slb groups, P < 0.05.

Statistics. Differences in most contractile parameters between the two treatment groups were analyzed by using a Student's t-test. Repeated measurements during the fatigue test were analyzed by using a two-way ANOVA (repeated measurements design). For the cAMP data, treatment effects were assessed by using a two-way ANOVA with treatment group and incubation time as variables. Statistical significance was accepted at a P < 0.05 level. All values are reported as means ± SE.

	RESULTS

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cAMP measurements. In the presence of 0.05 µM Slb, cAMP levels in the Dia_mus increased significantly compared with Ctl (P < 0.05; Fig. 1). The Slb-induced increase in Dia_mus cAMP was time dependent, being elevated by ~65% compared with Ctl after 15 min incubation but elevated only by ~45% after 30 min. By 60 min after incubation, cAMP levels were not significantly different between Slb and Ctl animals.

Isometric contractile properties. The mean Dia_mus strip weight (Ctl: 27.9 ± 1.1 mg and Slb: 26.3 ± 1.3 mg) and L_o (Ctl: 18.6 ± 0.5 mm and Slb: 19.2 ± 0.3 mm) were not different between Ctl and Slb groups. Between 15 and 30 min after incubation with 0.05 µM Slb, P_t of the Dia_mus increased by ~40% compared with Ctl (P < 0.05; Table 1), and P_o was ~20% greater (P < 0.05; Table 1). As a result, the P_t/P_o ratio was also increased by ~15% in the Slb-treated Dia_mus (P < 0.05; Table 1).

Isotonic contractile properties. In the Slb-treated Dia_mus, the force-velocity relationship was shifted upward and to the right compared with Ctl (Fig. 2). The extrapolated V_max of the Slb-treated Dia_mus was ~15% faster than in Ctl (Fig. 2, Table 1; P < 0.05). Therefore, the proportionate effects of Slb on V_max and P_o were comparable.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effect of Slb on isotonic shortening velocity of rat Diamus. Values are means ± SE. Curves represent shortening velocities at different levels of absolute force at which muscle was clamped. In Slb-treated Diamus, force-velocity relationship was shifted upward and to the right compared with Ctl. Lo, optimal fiber length. Maximum shortening velocity, i.e., shortening velocity at zero load, was also significantly increased in Slb group (P < 0.05).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Effect of Slb on power production in rat Diamus. Values are means ± SE. Po, maximum tetanic force. Slb treatment significantly increased power production at different isotonic force levels as well as maximum power produced (P < 0.05).

Isotonic fatigue. During repetitive isotonic contractions, maximum power output of the Dia_mus in both groups progressively declined (P < 0.05; Fig. 4). Accordingly, the work performed by the Dia_mus also progressively decreased with repetitive contractions. The rate of decrement in power output, and consequently the work performed, was significantly faster in the Slb-treated Dia_mus compared with Ctl (P < 0.05; Fig. 4). Isotonic endurance time was also ~10% shorter in the Slb-treated compared with Ctl (P < 0.05; Fig. 4).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effect of Slb on work performed (A) and power output (B) during repetitive isotonic contractions of the rat Diamus. Values are means ± SE. Load clamp level was set for maximum power output (see Fig. 3), and muscle strips were directly stimulated at 75 Hz for 330 ms repeated every second. Time at which the muscle no longer shortened was defined as the isotonic endurance time. Power output was calculated as the product of force and shortening velocity, and work performed was calculated as the product of force and the time integral of the length curve. Rate of decrement in power output was significantly faster in Slb-treated Diamus compared with Ctl (P < 0.05), but both groups fatigued to same power level. Isotonic endurance time was shorter in the Slb group.

	DISCUSSION

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The present study demonstrated that acute Slb treatment increases both isometric and isotonic contractility of the rat Dia_mus. The improved power output and work performance of the Slb-treated Dia_mus were associated with a more rapid rate of fatigue. However, both Slb-treated and Ctl Dia_mus fatigue to the same levels of optimal work performance and maximum power output. The endurance time during repeated isotonic shortening was slightly shorter in the Slb-treated Dia_mus compared with Ctl. It is likely that these changes in fatigability of the Slb-treated Dia_mus reflected the increased work performance of the muscle. Associated with the improved contractile performance of the Dia_mus, there was also a transient increase in cAMP levels. Although not conclusive, these results are consistent with the perspective that the Slb-induced enhancement of Dia_mus contractility is mediated, at least in part, by elevated cAMP.

The increase in Dia_mus specific force (both P_t and P_o) after acute Slb-treatment is consistent with previous studies on the Dia_mus (24, 25) as well as in limb muscles (1). However, in limb muscles, it was suggested that the positive inotropic effect of Slb was limited to fast-twitch muscles, comprising type II fibers, whereas force decreased in response to Slb treatment in slow-twitch muscles comprising type I fibers (1). In the present study, it was not possible to discern whether the positive inotropic effects of Slb on the Dia_mus were restricted to type II fibers. Yet, the effects of Slb treatment on isotonic contractile properties of the Dia_mus are consistent with a selective effect on type II fibers. Both V_max and maximum power output were increased after Slb treatment. The force-power curve was significantly shifted upward in the Slb-treated Dia_mus, and, consequently, the amount of work performed by the Slb-treated Dia_mus increased. The increase in power output and work would be accompanied by an increase in energy consumption, which could underlie the greater susceptibility of the Slb-treated Dia_mus to isotonic fatigue.

The increase in Dia_mus cAMP levels after Slb treatment is in agreement with previous results in both fast- and slow-twitch limb skeletal muscles (1). These results are also consistent with the elevation of cAMP levels in limb skeletal muscles induced by terbutaline, another ₂-adrenoceptor agonist (5, 7, 8). It is likely that the increase in cAMP levels induced by ₂-adrenoceptor stimulation in the Dia_mus involves G-protein activation and increased adenylate cyclase activity (3, 17). In isolated skeletal muscle fibers, the increase in force induced by terbutaline is mimicked by 8-bromoadenosine cAMP, a membrane-permeable analog of cAMP (5-7).

There are several potential mechanisms by which elevated cAMP might mediate an increase in Dia_mus specific force and a faster cross-bridge cycling rate. For example, it has been suggested that the ₂-adrenoceptor agonist-induced elevation in cAMP in skeletal muscle fibers leads to an improvement of excitation-contraction (EC) coupling and an increase in Ca²⁺ release from the sarcoplasmic reticulum (5, 7, 8). This suggestion is supported by the fact that 1 mM caffeine, which stimulates sarcoplasmic reticulum Ca²⁺ release, prevents the inotropic effect of terbutaline on force generation (5, 7). The effect of cAMP on EC coupling could be mediated via the activation of cAMP-dependent protein kinases and the subsequent phosphorylation of either voltage-dependent dihydropyridine receptors in the T tubules or ryanodine-receptor Ca²⁺-release channels in the sarcoplasmic reticulum (14, 19, 20, 26). Indeed, both -adrenergic receptors and adenylate cyclase activity have been detected in T tubules (9). Intracellular Ca²⁺ levels were not measured in the present study; therefore, it remains unclear to what extent a Slb-induced enhancement of EC coupling might have contributed to the observed improvements in Dia_mus contractility. It is also possible that other cAMP-dependent signaling cascades in skeletal muscle fibers could also have contributed to the Slb-induced improvements in Dia_mus contractility. For example, phosphorylation of the regulatory myosin light chain and/or troponin I can affect Ca²⁺ sensitivity and cross-bridge cycling kinetics.

The present study utilized a low, clinically relevant concentration of Slb. The results may be interpreted as a transient effect of Slb on Dia_mus contractility, especially on type IIx and IIb fibers. During normal ventilatory maneuvers of the Dia_mus, motor units consisting of type I and IIa fibers are predominantly recruited (21). These fiber types produce low amounts of force and are not fatigable (4). Accordingly, the transient effect of Slb on Dia_mus contractility is unlikely to be physiologically significant in the normal animal. However, under conditions such as COPD, increased resistance to breathing may necessitate recruitment of motor units consisting of type IIx and IIb fibers, which produce greater force but are more fatigable. Slb treatment in such situations would enhance contractility and thus add to the inspiratory pressure generating capacity of the Dia_mus. During fatigue, ₂-adrenoceptor agonist treatment may also increase Dia_mus contractility, as other in vivo studies have shown by using terbutaline (2), fenoterol (23), and broxaterol (10).

In conclusion, the present study demonstrated that acute Slb treatment increases cAMP levels and improves both isometric and isotonic contractility of the rat Dia_mus. The rate of fatigue during repeated isotonic contractions was faster in the Slb-treated Dia_mus, but both Slb-treated and Ctl Dia_mus fatigued to the same maximum power output. These results are consistent with the hypothesis that -adrenoceptor stimulation by Slb enhances Dia_mus contractility and that these effects of Slb are likely mediated, at least in part, by cAMP-dependent mechanisms.