Contractile and fatigue properties of the rat diaphragm musculature during the perinatal period.

The following two hypotheses regarding diaphragm contractile properties in the perinatal rat were tested. First, there is a major transformation of contractile and fatigue properties during the period between the inception of inspiratory drive transmission in utero and birth. Second, the diaphragm muscle properties develop to functionally match changes occurring in phrenic motoneuron electrophysiological properties. Muscle force recordings and intracellular recordings of end-plate potentials were measured by using phrenic nerve-diaphragm muscle in vitro preparations isolated from rats on embryonic day 18 and postnatal days 0-1. The following age-dependent changes occurred: 1) twitch contraction and half relaxation times decreased approximately two- and threefold, respectively; 2) the tetanic force levels increased approximately fivefold; 3) the ratio of peak twitch force to maximum tetanic force decreased 2.3-fold; 4) the range of forces generated by the diaphragm in response to graded nerve stimulation increased approximately twofold; 5) the force-frequency curve was shifted to the right; and 6) the propensity for neuromuscular transmission failure decreased. In conclusion, the diaphragm contractile and phrenic motoneuron repetitive firing properties develop in concert so that the full range of potential diaphragm force recruitment can be utilized and problems associated with diaphragm fatigue are minimized.     

Effects of emphysema and training on glutathione oxidation in the hamster diaphragm.

Loading of skeletal muscles is associated with increased generation of oxidants, which in turn may impair muscle contractility. We investigated whether the load on the hamster diaphragm imposed by pulmonary emphysema induces oxidative stress, as indicated by glutathione oxidation, and whether the degree of glutathione oxidation is correlated with contractility of the diaphragm. In addition, the effect of 12 wk of treadmill exercise training on contractility and glutathione content in the normal (NH) and emphysematous hamster (EH) diaphragm was investigated. Training started 6 mo after elastase instillation. After the training period, glutathione content and in vitro contractility of the diaphragm were determined. Twitch force and maximal tetanic force were significantly reduced (by approximately 30 and approximately 15%, respectively) in EH compared with NH. In sedentary hamsters, the GSSG-to-GSH ratio was significantly elevated in the EH compared with the NH diaphragm. A significant inverse correlation was found between GSSG-to-GSH ratio and twitch force in the diaphragm (P < 0. 01). Training improved maximal tetanic force and reduced fatigability of the EH diaphragm but did not alter its glutathione content. In conclusion, 1) emphysema induces oxidative stress in the diaphragm, 2) training improves the contractile properties of the EH diaphragm, and 3) this improvement is not accompanied by changes in glutathione redox status.     

Adverse effects of myasthenia gravis on rat phrenic diaphragm contractile performance.

Myasthenia gravis has variable effects on the respiratory system, ranging from no abnormalities to life-threatening respiratory failure. Studies characterized diaphragm muscle contractile performance in rat autoimmune myasthenia gravis. Rats received monoclonal antibody that recognizes acetylcholine receptor determinants (or inactive antibody); 3 days later, phrenic nerve and diaphragm were studied in vitro. Myasthenic rats segregated into two groups, those with normal vs. impaired limb muscle function when tested in intact animals ("mild" and "severe" myasthenic). Baseline diaphragm twitch force was reduced for both severe (P < 0.01) and mild (P < 0.05) myasthenic compared with control animals (twitch force: normal 1,352 +/- 140, mild myasthenic 672 +/- 99, severe myasthenic 687 +/- 74 g/cm2). However, only severe myasthenic diaphragm had impaired diaphragm endurance, based on significantly (P < 0.05) accelerated rate of peak force decline during the initial period of stimulation (0.02 + 0.02, 0.03 +/- 0.01, and 0.09 +/- 0.01%/pulse for normal, mild myasthenic, and severe myasthenic, respectively, during continuous stimulation) and intratrain fatigue (up to 30.5 +/- 7.4% intratrain force drop in severe myasthenic vs. none in normal and mild myasthenic, P < 0.01). Furthermore, compared with continuous stimulation, intermittent stimulation had a protective effect on force of severe myasthenic diaphragm (force after 2,000 pulses was 31.4 +/- 2.0% of initial during intermittent stimulation vs. 13.0 +/- 2.1% of initial during continuous stimulation, P < 0.01) but not on normal diaphragm. These data indicate that baseline force and fatigue may be affected to different extents by varying severity of myasthenia gravis and furthermore provide a mechanism by which alterations in breathing pattern may worsen respiratory muscle function in neuromuscular diseases.     

Effects of chronic nicotine exposure on electrogenic activity of the Na+, K(+)-ATPase and contractility in the rat diaphragm muscle

Rats were chronically treated with nicotine via subcutaneous injections up to a dose 6 mg/kg/day during 2-3 weeks. After this period, resting membrane potential and action potentials of muscle fibres as well as isometric twitch and tetanic (20 s(-1) and 50(-1)) contractions of isolated rat diaphragm were studied. To estimate electrogenic contribution of the alpha2 isoform of the Na+, K(+)-ATPase ouabain in concentration 1 microM was used. Chronic nicotine exposure induced depolarization of resting membrane potential of 2.2 +/- 0.6 mV (p < 0.01). In rats chronically exposed to nicotine, electrogenic contribution of the Na+, K(+)-ATPase alpha2 isoform was twofold lesser than in control animals (3.7 +/- 0.6 mV and 6.4 +/- 0.6 mV, respectively, p < 0.01). Chronic nicotine exposure did not affect force of twitch and tetanic contractions in response to direct or indirect stimulation. A decrease in the twitch contraction time as well as in the rise time of tetanic contractions was observed. Fatigue dynamics was unchanged. The results suggest that chronic nicotine exposure leads to decrease of the Na+, K(+)-ATPase alpha2 isoform electrogenic activity, and as a consequence to damage of the rat diaphragm muscle electogenesis.     

Effect of nifedipine on the contractile function of the rat diaphragm in vitro

The isometric contractile response of the directly-stimulated rat diaphragm was studied before and following addition of the calcium channel blocker, nifedipine. Nifedipine (10 micrograms/ml and 30 micrograms/ml bath concentrations) significantly increased isometric force output during twitch and unfused tetanic stimulation. Force potentiation during unfused tetanic stimulation was equivalent during either high or low voltage stimulation. Nifedipine had no effect on the time to peak force, half relaxation time, or relaxation time during twitch stimulation; thus, both activation and relaxation rates were increased. The force potentiating actions of nifedipine persisted in a calcium-free bathing solution and were enhanced by d-tubocurarine. In contrast to the force enhancing effects found with twitch and unfused tetanic stimulation, nifedipine caused a small but significant reduction in isometric force during maximal fused tetanic stimulation. It is concluded that the force potentiating effects of nifedipine on rat diaphragm are not due to fiber recruitment, enhancement of neuromuscular excitation, or altered inward trans-sarcolemmal calcium flux, but may result from a direct effect of the drug on the rate of activation of the contractile apparatus.     

罗库溴铵和顺式阿曲库铵持续输注对机械通气SD 大鼠膈肌功能 的影响

目的:比较罗库溴铵或顺式阿曲库铵24h持续输注对机械通气SD大鼠膈肌功能的影响.方法:雄性SD大鼠随机分为四组:一组作为对照组,另外三组用来进行24h的机械通气.其中一组为罗库溴铵组持续输注罗库溴铵,一组顺式阿曲库铵和一组生理盐水.结果:24h机械通气后,与生理盐水组相比,罗库溴铵组的膈肌最大强直张力下降29％,膈肌MuRF-1的mRNA水平增加30％,calpain活性增加约57％,而等效剂量的顺式阿曲库铵组则没有明显改变.结论:与顺式阿曲库铵相比,罗库溴铵可引起Calpain活性的增强和泛素-蛋白酶体系统的激活,对膈肌功能的损害和蛋白水解活性的增强作用更明显.     

Theophylline does not increase maximal tetanic force or diaphragm endurance in vitro

These experiments tested the capacity of theophylline to improve diaphragm strength (maximal force development) and endurance (maintenance of force output during repeated contractions). Rodent diaphragm strips were mounted at optimal length in oxygenated Krebs-Ringer solution (37 degrees C, pH 7.37). Direct stimuli used supramaximal current density, 0.2-ms pulses, and 250-ms tetanic trains. Theophylline (500 mg/ml) increased force development at low stimulation frequencies but did not increase maximal force [25.7 +/- 0.5 for theophylline vs. 26.0 +/- 0.4 (SE) N/cm2 for control (n = 34)]. During repeated submaximal (25-36 Hz) tetanic contractions, theophylline did not affect endurance. During repeated maximal (160 Hz) tetanic contractions theophylline reduced endurance, accelerating the fall of developed force. Theophylline also inhibited recovery of force after endurance trials ended. We conclude that theophylline does not increase maximal tetanic force and can reduce diaphragm endurance in vitro.     

Selected Contribution: Improved anoxic tolerance in rat diaphragm following intermittent hypoxia.

Intermittent hypoxia (IH), associated with obstructive sleep apnea, initiates adaptive physiological responses in a variety of organs. Little is known about its influence on diaphragm. IH was simulated by exposing rats to alternating 15-s cycles of 5% O2 and 21% O2 for 5 min, 9 sets/h, 8 h/day, for 10 days. Controls did not experience IH. Diaphragms were excised 20-36 h after IH. Diaphragm bundles were studied in vitro or analyzed for myosin heavy chain isoform composition. No differences in maximum tetanic stress were observed between groups. However, peak twitch stress (P < 0.005), twitch half-relaxation time (P < 0.02), and tetanic stress at 20 or 30 Hz (P < 0.05) were elevated in IH. No differences in expression of myosin heavy chain isoforms or susceptibility to fatigue were seen. Contractile function after 30 min of anoxia (95% N2-5% CO2) was markedly preserved at all stimulation frequencies during IH and at low frequencies after 15 min of reoxygenation. Anoxia-induced increases in passive muscle force were eliminated in the IH animals (P < 0.01). These results demonstrate that IH induces adaptive responses in the diaphragm that preserve its function in anoxia.     

Long-term effects of clenbuterol on diaphragm morphology and contractile properties in emphysematous hamsters

The aim of thepresent study was to investigate the effect of chronic long-termclenbuterol treatment (1 mg/kg subcutaneously twice a day for 12 wk) ondiaphragm morphology and function in emphysematous (EH) and normalhamsters (NH). Clenbuterol increased body weight, diaphragm weight, andskeletal muscle weight in both EH and NH to a similarextent. In the diaphragm, clenbuterol significantly increased myosin heavy chain type I, IIa, and IIx muscle fiber cross-sectional areas by ~35-55% in both EH and NH. Thisresponse to clenbuterol treatment was not significantly differentbetween EH and NH diaphragm. In EH, twitch force(P_t), maximal tetanic force, andforce-frequency curve were significantly reduced compared with NH. InEH, clenbuterol increased P_t by~10%, restoring P_t to NH level.A similar improvement was observed in the force-frequency characteristics. Clenbuterol did not alter contractile properties inNH. In conclusion, long-term clenbuterol treatment resulted in anincreased size of all diaphragm muscle fiber types in both NH and EH.Clenbuterol completely abolished the reduced force generation inducedby emphysema.

     

Autophagy-Associated Atrophy and Metabolic Remodeling of the Mouse Diaphragm after Short-Term Intermittent Hypoxia

Background

Short-term intermittent hypoxia (IH) is common in patients with acute respiratory disorders. Although prolonged exposure to hypoxia induces atrophy and increased fatigability of skeletal muscle, the response to short-term IH is less well known. We hypothesized that the diaphragm and limb muscles would adapt differently to short-term IH given that hypoxia stimulates ventilation and triggers a superimposed exercise stimulus in the diaphragm.

Methods

We determined the structural, metabolic, and contractile properties of the mouse diaphragm after 4 days of IH (8 hours per day, 30 episodes per hour to a FiO2 nadir=6%), and compared responses in the diaphragm to a commonly studied reference limb muscle, the tibialis anterior. Outcome measures included muscle fiber size, assays of muscle proteolysis (calpain, ubiquitin-proteasome, and autophagy pathways), markers of oxidative stress and mitochondrial function, quantification of intramyocellular lipid and lipid metabolism genes, type I myosin heavy chain (MyHC) expression, and in vitro contractile properties.

Results

After 4 days of IH, the diaphragm alone demonstrated significant atrophy (30% decrease of myofiber size) together with increased LC3B-II protein (2.4-fold) and mRNA markers of the autophagy pathway (LC3B, Gabarapl1, Bnip3), whereas active calpain and E3 ubiquitin ligases (MuRF1, atrogin-1) were unaffected in both muscles. Succinate dehydrogenase activity was significantly reduced by IH in both muscles. However, only the diaphragm exhibited increased intramyocellular lipid droplets (2.5-fold) after IH, along with upregulation of genes linked to activated lipid metabolism. In addition, although the diaphragm showed evidence for acute fatigue immediately following IH, it underwent an adaptive fiber type switch toward slow type I MyHC-expressing fibers, associated with greater intrinsic endurance of the muscle during repetitive stimulation in vitro.

Conclusions

Short-term IH induces preferential atrophy in the mouse diaphragm together with increased autophagy and a rapid compensatory metabolic adaptation associated with enhanced fatigue resistance.     

Diaphragm muscle fatigue resistance during postnatal development.

Changes in the contractile and fatigue properties of the cat diaphragm muscle were examined during the first 6 wk of postnatal development. Both twitch contraction time and half-relaxation time decreased progressively with age. Correspondingly, the force-frequency curve was shifted to the left early in development compared with adults. The ratio of peak twitch force to maximum tetanic force decreased with age. Fatigue resistance of the diaphragm was highest at birth and then progressively decreased with age. At birth, most diaphragm muscle fibers stained darkly for myofibrillar adenosinetriphosphatase after alkaline preincubation and thus would be classified histochemically as type II. During subsequent postnatal development, the proportion of type I fibers (lightly stained for adenosinetriphosphatase) increased while the number of type II fibers declined. At birth, type I fibers were larger than type II fibers. The size of both fiber types increased with age, but the increase in cross-sectional area was greater for type II fibers. On the basis of fiber type proportions and mean cross-sectional areas, type I fibers contributed 15% of total muscle mass at birth and 25% in adults. Thus postnatal changes in diaphragm contractile and fatigue properties cannot be attributed to changes in the relative contribution of histochemically classified type I and II fibers. However, the possibility that these developmental changes in diaphragm contractile and fatigue properties correlated with the varying contractile protein composition of muscle fibers was discussed.     

Morphological and functional recovery from diaphragm injury: an in vivo rat diaphragm injury model.

Our objective was to develop an in vivo model to study the timing and mechanisms underlying diaphragm injury and repair. Diaphragm injury was induced in anesthetized rats by the application of a 100 mM caffeine solution for a 10-min period to the right abdominal diaphragm surface. Diaphragms were removed 1, 4, 6, 12, 24, 48, 72, and 96 h and 10 days after the injury, with contractile function being assessed in strips in vitro by force-frequency curves. The extent of caffeine-induced membrane injury was indicated by the percentage of fibers with a fluorescent cytoplasm revealed by inward leakage of the procion orange dye. One hour after caffeine exposure, 32.9 +/- 3.1 (SE) % of fibers showed membrane injury that resulted in 70% loss of muscle force. Within 72-96 h, the percentage of fluorescent cells decreased to control values. Muscle force, however, was still reduced by 30%. Complete muscle strength recovery was observed 10 days after the injury. Whereas diaphragmatic fiber repair occurred within 4 days after injury induction, force recovery took up to 10 days. We suggest that the caffeine-damaged rat diaphragm is a useful model to study the timing and mechanisms of muscle injury and repair.     

Adaptations of diaphragm and medial gastrocnemius muscles to inactivity.

The effects of 2 wk of inactivity on in vitro contractile properties of diaphragm and medial gastrocnemius (MG) muscles were examined in adult hamsters. In addition, inactivity effects on fiber-type proportions and cross-sectional areas were studied. Inactivity of the right hemidiaphragm or MG muscle was induced by either tetrodotoxin (TTX) blockade of nerve impulses or denervation (DNV). Inactivity effects on diaphragm or MG were compared with corresponding sham (saline-treated or untreated control) muscles. After both TTX- and DNV-induced inactivity, isometric twitch contraction and half-relaxation times were prolonged, maximum tetanic force decreased, and fatigue resistance improved. Proportions of type I and II fibers in both diaphragm and MG were unaffected by TTX- and DNV-induced inactivity. However, in both muscles, type I fibers hypertrophied, whereas type II fibers atrophied. In diaphragm, contractile and morphometric adaptations after DNV were generally more pronounced than those induced by TTX. In addition, compared with corresponding untreated or saline-treated control groups, inactivity effects (both TTX and DNV) on MG were generally greater than those induced in diaphragm, with the exception of hypertrophy of type I fibers. We conclude that inactivity exerts differential effects on type I and II fibers in both diaphragm and MG. Yet, these morphometric adaptations cannot completely account for the adaptations in muscle contractile and fatigue properties after inactivity.     

Theophylline minimally alters contractile properties of canine diaphragm in vitro

We examined the effects of theophylline on contractile properties and high-frequency fatigue of canine diaphragm in vitro. Eighteen diaphragm muscle bundles were obtained from 10 anesthetized dogs and equilibrated in oxygenated Krebs solution to 100, 200, or 300 mg/l theophylline. These bundles were compared with 18 matched control bundles from the contralateral hemidiaphragm. No statistically significant differences in twitch tension, tetanic tension, twitch-to-tetanus ratio, time to peak tension, or half-relaxation time were observed. Concentrations of 300 mg/l theophylline, however, significantly (P less than 0.05) increased force production at 10 Hz by 32%. A similar tendency was present at lower concentrations and exhibited a clear dose-response behavior. High-frequency fatigue was similar in control and theophylline-treated bundles. We conclude that supratherapeutic in vitro concentrations of theophylline do not increase maximal tetanic tension and do not protect against muscle fatigue but potentiate relative force production at low stimulation frequencies. This relatively small effect cannot be explained by poor diffusion of the drug in the muscle bundle, because theophylline concentrations in the muscle bath and in the muscle bundle were virtually identical. Moreover, it remains unclear whether this potentially beneficial effect can be achieved at in vivo attainable serum concentrations.     

Granulocyte-colony stimulating factor enhances muscle proliferation and strength following skeletal muscle injury in rats.

Insufficiency of skeletal muscle regeneration often impedes the healing process with functional deficiencies and scar formation. We tested the hematopoietic growth factor granulocyte-colony stimulating factor (G-CSF) with respect to its efficacy to improve functional muscle regeneration following skeletal muscle injury in Wistar rats. After crush injury to the left soleus muscle, animals received daily G-CSF (20 mug/kg ip) or vehicle solution (n = 30 per group each). Sham-operated animals without muscle injury served as controls (n = 15). After in vivo assessment of the fast-twitch and tetanic contraction capacity of the soleus muscles at days 4, 7, and 14 post-injury, sampling of muscle tissue served for analysis of satellite cell proliferation [bromodeoxyuridine (BrdU)/laminin and BrdU/desmin double immunohistochemistry] and cell apoptosis (transferase nick-end labeling analysis). Muscle strength analysis revealed recovery of contraction forces to 26 +/- 2, 35 +/- 3, and 53 +/- 3% (twitch force) and to 20 +/- 3, 24 +/- 2, and 37 +/- 2% (tetanic force) within the 14-day observation period in vehicle-treated animals. In contrast, G-CSF increased contractile forces with markedly higher values at day 7 (twitch force: 42 +/- 2%; tetanic force: 34 +/- 2%) and day 14 (twitch force: 62 +/- 3%; tetanic force: 43 +/- 3%). This enhancement of muscle function was preceded by a significant increase of satellite cell proliferation (BrdU-positive cells/mm(2): 27 +/- 6 vs. vehicle: 12 +/- 3) and a moderate decrease of cell apoptosis (transferase nick-end labeling-positive cells/mm(2): 11 +/- 2 vs. vehicle: 16 +/- 3) at day 4. In conclusion, G-CSF histologically promoted viability and proliferation of muscle cells and functionally enhanced recovery of muscle strength. Thus G-CSF might represent a therapeutic option to optimize the posttraumatic course of muscle tissue healing.     

Influence of tenotomy on posttetanic responses of the rat fast and slow muscle

The effect of two weeks of tenotomy on posttetanic isometric contractile responses of the rat fast: Extensor digitorum longus and slow: soleus muscles was studied in experiments on isolated muscle preparations. Direct tetanic stimulation (100 impulses, 50 Hz) increased the force of contractions by 20-25% (p < 0.05) of both, control and tenotomized fast muscles. Identical to above tetanic stimulation of control, slow muscle resulted in posttetanic depression, a decrease in the amplitude of contractile responses. Tenotomized slow muscles did not develop posttetanic depression. Caffeine (4 mM) increased and dandrolene (10 microM) decreased the force of unitary and tetanic contractions of control and tenotomized muscles. Neither drug, however, affected development of posttetanic phenomena in ether fast or slow muscles. The fact that in extensor digitorum longus, posttetanic potentiation is preserved for at least forty days of tenotomy but disappears after only 2 weeks of denervation suggests important role of neurotrophic influences in regulation of posttetanic responses of fast muscles.     

The effect of head-down hypokinesia on functional state of diaphragm in rats

The effects of long-term (21 days) head-down (-30 degrees) hypokinesia (HDH) on respiratory system and a functional state of diaphragm were investigated in rats. Minute ventilation, oesophageal and abdominal pressure, integrated electrical activity of diaphragm were measured in control and experimental group (after 21 days of HDH) of animals. The measurements were made in several body positions atresting and in occlusion breathing. The results indicate that HDH causes reduction in minute ventilation of lungs, decrease in orthostatic stability and functional reserve of the respiratory system capacity. It was established that the basic mechanism of HDH respiratory effects is contractile failure of diaphragm related to damage in excitation-contraction coupling in its muscular fibres.     

Myofibrillar protein oxidation and contractile dysfunction in hyperthyroid rat diaphragm.

The purpose of the present study was to test the hypothesis that administration of thyroid hormone [3,5,3'-triiodo-L-thyronine (T(3))] could result in oxidation of myofibrillar proteins and, in turn, induce alterations in respiratory muscle function. Daily injection of T(3) for 21 days depressed isometric forces of diaphragm fiber bundles across a range of stimulus frequencies (1, 10, 20, 40, 75, and 100 Hz) (P < 0.05). These reductions in force production were accompanied by a remarkable increment (104%; P < 0.05) in carbonyl groups of myofibrillar proteins. In contrast, T(3) treatment has no effects on the carbonyl content in myosin heavy chain. In additional experiments, we have also tested the efficacy of carvedilol, a nonselective beta(1)- beta(2)-blocker that possesses antioxidative properties. Treatment with carvedilol dramatically improved isometric tetanic force production at stimulus frequencies from 40 to 100 Hz (P < 0.05). Carvedilol also prevented T(3)-induced contractile protein oxidation (P < 0.05). These data suggest that the oxidative modification of myofibrillar proteins may account, at least in part, for an impairment of diaphragm in hyperthyroidism.     

Effect of nitric oxide synthase inhibitor on diaphragmatic function after resistive loading

We studied the effect of a nitric oxide synthase inhibitor, Nomega-Nitro-L-arginine-methyl-ester (L-NAME), on in vitro diphragmatic function both at rest (control) or after inspiratory resistive loading (IRL). Sprague-Dawley rats were anesthetized, instrumented, and then the following experimental groups: (1) controls; (2) L-NAME (100 mg/kg/body weight intravenously alone); (3) IRL alone; and (4) L-NAME + IRL. The IRL protocol consisted of applying a variable resistor to the inspiratory limb of a two-way valve at 70% of maximal airway pressure until apnea. After the experiment, the animals were sacrificed and diaphragmatic strips were obtained for activity of constitutive nitric oxide synthase (cNOS) and measurements of in vitro contractile properties: tetanic (Po) and twitch tensions (Pt). cNOS activity was significantly decreased in the L-NAME and L-NAME + IRL groups (P < or = 0.05) as compared with control and IRL groups. L-NAME alone did not affect Po or Pt. However, in both IRL groups, with and without was a significant decrease in Po and Pt. This reduction was comparable in both groups. In summary, our data showed that L-NAME resulted in a significant decrease cNOS activity, but in vitro contractility was impaired.     

Chronic inflation of ferret lungs with CPAP reduces airway smooth muscle contractility in vivo and in vitro.

The mechanical stress imposed on the lungs during breathing is an important modulator of airway responsiveness in vivo. Our recent study demonstrated that continuous positive airway pressure applied to the lungs of nonanesthetized, tracheotomized rabbits for 4 days decreased lower respiratory system responsiveness to challenge with ACh (Xue Z, Zhang L, Ramchandani R, Liu Y, Antony VB, Gunst SJ, Tepper RS. J. Appl Physiol 99: 677-682, 2005). In addition, airway segments excised from the lungs of these animals and studied in vitro exhibited reduced contractility. However, the mechanism for this reduction in contractility was not determined. The stress-induced decrease in airway responsiveness could have resulted from alterations in the excitation-contraction coupling mechanisms of the smooth muscle cells, or it might reflect changes in the structure and/or composition of the airway wall tissues. In the present study, we assessed the effect of prolonged chronic stress of the lungs in vivo on airway smooth muscle force generation, myosin light chain phosphorylation, and airway wall structure. To enhance the potential development of stress-induced structural changes, we applied mechanical stress for a prolonged period of time of 2-3 wk. Our results demonstrate a direct connection between the decreased airway responsiveness caused by chronic mechanical stress of the lungs in vivo and a persistent decrease in contractile protein activation in the airway smooth muscle isolated from those lungs. The chronic stress also caused an increase in airway size but no detectable changes in the composition of the airway wall.