Relaxation of tracheal smooth muscle is impaired in innate airway hyperresponsiveness.

The current study was designed to determine whether the nonspecific in vivo airway hyperresponsiveness of the inbred Fisher F-344 rat strain is associated with impaired spontaneous relaxation of airway smooth muscle. Strips of the posterior portion of the trachea from 10 adult Fisher and 10 adult Lewis rats were electrically stimulated at pH 7.4, 2.5 mM Ca(2+)concentration, at 37 degrees C. Both isotonic and isometric relaxations of tracheal smooth muscle (TSM) were investigated. Half time for isotonic relaxation at preload was markedly prolonged in Fisher rats (8.33+/-3.21 s) compared with Lewis rats (3.53+/-0.54 s; p<0.001). Maximum lengthening velocity at preload and peak rate of isometric tension decline were significantly decreased in Fisher rats compared with Lewis rats. The ratio of shortening velocity to lengthening velocity at preload, as well as the ratio of the isometric peak rates of tension development to tension decline were higher in Fisher rat TSM than in Lewis rat TSM. These differences were associated with a six-fold higher expression of myosin light chain kinase in Fisher rats than in Lewis rats. In Fisher rats, these results suggest that innate airway hyperresponsiveness is associated with both a reduced level and a slower rate of TSM spontaneous relaxation, promoting maintenance of airway constriction.

Use of arginine-glycine-aspartic acid adhesion peptides coupled with a new collagen scaffold to engineer a myocardium-like tissue graft.

BACKGROUND: Cardiac tissue engineering might be useful in treatment of diseased myocardium or cardiac malformations. The creation of functional, biocompatible contractile tissues, however, remains challenging. We hypothesized that coupling of arginine-glycine-aspartic acid-serine (RGD+) adhesion peptides would improve cardiomyocyte viability and differentiation and contractile performance of collagen-cell scaffolds. METHODS: Clinically approved collagen scaffolds were functionalized with RGD+ cells and seeded with cardiomyocytes. Contractile performance, cardiomyocyte viability and differentiation were analyzed at days 1 and 8 and/or after culture for 1 month. RESULTS: The method used for the RGD+ cell-collagen scaffold coupling enabled the following features: high coupling yields and complete washout of excess reagent and by-products with no need for chromatography; spectroscopic quantification of RGD+ coupling; a spacer arm of 36 A, a length reported as optimal for RGD+-peptide presentation and favorable for integrin-receptor clustering and subsequent activation. Isotonic and isometric mechanical parameters, either spontaneous or electrostimulated, exhibited good performance in RGD+ constructs. Cell number and viability was increased in RGD+ scaffolds, and we saw good organization of cell contractile apparatus with occurrence of cross-striation. CONCLUSIONS: We report a novel method of engineering a highly effective collagen-cell scaffold based on RGD+ peptides cross-linked to a clinically approved collagen matrix. The main advantages were cell contractile performance, cardiomyocyte viability and differentiation.

Increased entropy production in diaphragm muscle of PPAR alpha knockout mice.

Peroxisome proliferator activated receptor alpha (PPAR alpha) regulates fatty acid beta-oxidation (FAO) and plays a central role in the metabolic and energetic homeostasis of striated muscles. The thermodynamic consequences of the absence of PPAR alpha were investigated in diaphragm muscle of PPAR alpha knockout mice (KO). Statistical mechanics provides a powerful tool for determining entropy production, which quantifies irreversible chemical processes generated by myosin molecular motors and which is the product of thermodynamic force A/T (chemical affinity A and temperature T) and thermodynamic flow (myosin crossbridge (CB) cycle velocity upsilon). The behavior of both wild type (WT) and KO diaphragm was shown to be near-equilibrium and in a stationary state, but KO was farther from equilibrium than WT. In KO diaphragm, a substantial decrease in contractile function was associated with an increase in both A/T and upsilon and with profound histological injuries such as contraction band necrosis. There were no changes in PPAR delta and gamma expression levels or myosin heavy chain (MHC) patterns. In KO diaphragm, a marked increase in entropy production (A/T x upsilon) accounted for major thermodynamic dysfunction and a dramatic increase in irreversible chemical processes during the myosin CB cycle.

Statistical mechanics of myosin molecular motors in skeletal muscles.

Statistical mechanics provides the link between microscopic properties of matter and its bulk properties. The grand canonical ensemble formalism was applied to contracting rat skeletal muscles, the soleus (SOL, n = 30) and the extensor digitalis longus (EDL, n = 30). Huxley's equations were used to calculate force (pi) per single crossbridge (CB), probabilities of six steps of the CB cycle, and peak muscle efficiency (Eff(max)). SOL and EDL were shown to be in near-equilibrium (CB cycle affinity 2.5 kJ/mol) and stationary state (linearity between CB cycle affinity and myosin ATPase rate). The molecular partition function (z) was higher in EDL (1.126+/-0.005) than in SOL (1.050+/-0.003). Both pi and Eff(max) were lower in EDL (8.3+/-0.1 pN and 38.1+/-0.2%, respectively) than in SOL (9.2+/-0.1 pN and 42.3+/-0.2%, respectively). The most populated step of the CB cycle was the last detached state (D3) (probability P(D3): 0.890+/-0.004 in EDL and 0.953+/-0.002 in SOL). In each muscle group, both pi and Eff(max) linearly decreased with z and statistical entropy and increased with P(D3). We concluded that statistical mechanics and Huxley's formalism provided a powerful combination for establishing an analytical link between chemomechanical properties of CBs, molecular partition function and statistical entropy.

Mechanics and crossbridge kinetics of tracheal smooth muscle in two inbred rat strains.

The aim of the study was to determine whether the nonspecific in vivo airway hyperresponsiveness of the inbred Fisher F-344 rat strain was associated with differences in the intrinsic contractile properties of tracheal smooth muscle (TSM) when compared with Lewis rats. Isotonic and isometric contractile properties of isolated TSM from Fisher and Lewis rats (each n=10) were investigated, and myosin crossbridge (CB) number, force and kinetics in both strains were calculated using Huxley's equations adapted to nonsarcomeric muscles. Maximum unloaded shortening velocity and maximum extent of muscle shortening were higher in Fisher than in Lewis rats (approximately 46% and approximately 42%, respectively), whereas peak isometric tension was similar. The curvature of the hyperbolic force/velocity relationship did not differ between strains. Myosin CB number and unitary force were similar in both strains. The duration of CB detachment and attachment was shorter in Fisher than in Lewis rats (approximately -46% and -34%, respectively). In Fisher rats, these results show that inherited, genetically determined factors of airway hyperresponsiveness are associated with changes in crossbridge kinetics, contributing to an increased tracheal smooth muscle shortening capacity and velocity.

Myosin cross-bridge kinetics in airway smooth muscle: a comparative study of humans, rats, and rabbits.

To analyze the kinetics and unitary force of cross bridges (CBs) in airway smooth muscle (ASM), we proposed a new formalism of Huxley's equations adapted to nonsarcomeric muscles (Huxley AF. Prog Biophys Biophys Chem 7: 255-318, 1957). These equations were applied to ASM from rabbits, rats, and humans (n = 12/group). We tested the hypothesis that species differences in whole ASM mechanics were related to differences in CB mechanics. We calculated the total CB number per square millimeter at peak isometric tension (Psi x10(9)), CB unitary force (Pi), and the rate constants for CB attachment (f(1)) and detachment (g(1) and g(2)). Total tension, Psi, and Pi were significantly higher in rabbits than in humans and rats. Values of Pi were 8.6 +/- 0.1 pN in rabbits, 7.6 +/- 0.3 pN in humans, and 7.7 +/- 0.2 pN in rats. Values of Psi were 4.0 +/- 0.5 in rabbits, 1.2 +/- 0.1 in humans, and 1.9 +/- 0.2 in rats; f(1) was lower in humans than in rabbits and rats; g(2) was higher in rabbits than in rats and in rats than in humans. In conclusion, ASM mechanical behavior of different species was characterized by specific CB kinetics and CB unitary force.

Myosin cross bridges in skeletal muscles: "rower" molecular motors.

Different classes of molecular motors, "rowers" and "porters," have been proposed to describe the chemomechanical transduction of energy. Rowers work in large assemblies and spend a large percentage of time detached from their lattice substrate. Porters behave in the opposite way. We calculated the number of myosin II cross bridges (CB) and the probabilities of attached and detached states in a minimal four-state model in slow (soleus) and fast (diaphragm) mouse skeletal muscles. In both muscles, we found that the probability of CB being detached was approximately 98% and the number of working CB was higher than 10(9)/mm(2). We concluded that muscular myosin II motors were classified in the category of rowers. Moreover, attachment time was higher than time stroke and time for ADP release. The duration of the transition from detached to attached states represented the rate-limiting step of the overall attached time. Thus diaphragm and soleus myosins belong to subtype 1 rowers.

Angiotensin-converting enzyme inhibitor therapy improves respiratory muscle strength in patients with heart failure.

STUDY OBJECTIVES: Respiratory muscle strength has been shown to be reduced in patients with chronic heart failure. The purpose of this prospective study was to determine whether long-term therapy with the angiotensin-converting enzyme (ACE) inhibitor perindopril improves respiratory muscle strength in patients with chronic heart failure. PATIENTS AND METHODS: Eighteen patients with stable chronic heart failure were administered perindopril, 4 mg/d, in addition to their standard therapy for a period of 6 months. Fourteen patients completed the study. Maximum inspiratory pressure (PImax) and maximum expiratory pressure (PEmax) expressed in percentage of predicted values, left ventricular ejection fraction (LVEF) determined by means of two-dimensional echocardiography, and pulmonary volumes were obtained before and after therapy. MEASUREMENTS AND RESULTS: As compared to baseline, there was a significant increase in both PImax and PEmax after therapy (57 +/- 27% predicted vs 78 +/- 36% predicted and 62 +/- 20% predicted vs 73 +/- 15% predicted, respectively; each p < 0.05). LVEF increased (34 +/- 5% vs 41 +/- 10%; p < 0.05); functional class improved by > or = 1 New York Heart Association (NYHA) class in five patients. There were no changes in pulmonary volumes. No correlation was found between changes in PImax and PEmax and changes in either LVEF or NYHA functional class. CONCLUSIONS: In patients with chronic heart failure, long-term therapy with the ACE inhibitor perindopril improved respiratory muscle strength, as indicated by significant increases in PImax and PEmax.

Effects of dantrolene on rat diaphragm muscle during postnatal maturation.

BACKGROUND: Dantrolene is the only known effective treatment for malignant hyperthermia. However, its effects on diaphragm muscle during postnatal maturation remain unknown. METHODS: The effects of dantrolene (10(-8) to 10(-4) M) were investigated in vitro on diaphragm muscle strips in adult rats and in postnatal rats aged 3, 10, and 17 days, and compared with those of ryanodine (10(-8) to 10(-6) M). The authors studied contraction and relaxation under isotonic and isometric conditions (29 degrees C, Krebs-Henseleit solution, tetanic stimulation at 50 Hz). Data are mean +/- SD. RESULTS: During postnatal maturation, the authors observed a progressive increase in active force developed per cross-sectional area (from 34 +/- 25 to 69 +/- 32 mN/mm2; P < 0.05) and maximum shortening velocity (from 2.9 +/- 0.5 to 4.9 +/- 1.4 Lmax/s; P < 0.05). Dantrolene induced a negative inotropic effect in diaphragm muscles in isotonic and isometric conditions in all groups, but this effect was significantly less marked in the 3-day-old rats compared with older rats. Dantrolene did not induce significant lusitropic effects during postnatal maturation. Developmental changes in the pharmacologic response to dantrolene were more rapid than those of ryanodine. CONCLUSION: Dantrolene induced less pronounced negative inotropic effects on the diaphragm in neonatal rats as compared with adult rats. Our study suggests that developmental changes in the pharmacologic response to dantrolene are more rapid than those of ryanodine.

Species-dependent changes in mechano-energetics of isolated cardiac muscle during hypoxia.

The present study investigates the mechanical and energetic changes induced by hypoxia in isolated cardiac muscles of different species characterized by different myosin isoforms. Classic mechanical parameters of contraction and energetic parameters derived from the tension-velocity relationship were studied in rat and guinea pig left ventricular papillary muscles and in frog ventricular strips before and after 15 min hypoxia (n = 8 in each group). The isomyosin pattern is predominantly V1 with high ATPase activity in rat and V3 with low ATPase activity in guinea pig and frog heart ventricles. At baseline, cardiac mechanical performance was greater in rat than in guinea pig and frog muscle, but the economy of tension generation did not differ significantly between the three species. Hypoxia significantly decreased myocardial mechanical performance in all three groups. Mechanical impairment was more marked in rat than in the other two species and was intermediate in guinea pig. The energetic consequences of hypoxia differed according to species and in a different manner from the mechanical parameters. Hypoxia decreased the economy of tension generation in rat heart, in contrast to no change in guinea pig and frog muscle. These results suggest that in terms of mechano-energetic properties, cardiac muscles with V1 isomyosin were more sensitive to hypoxia than those containing V3 isomyosin.

[Biomechanics and bio-energetics of smooth muscle contraction. Relation to bronchial hyperreactivity]. / Biomécanique et bioénergétique de la contraction musculaire lisse--Liens avec l'hyperréactivité bronchique.

Mechanical studies of isolated muscle and analysis of molecular actomyosin interactions have improved our understanding of the pathophysiology of airway smooth muscle. Mechanical properties of airway smooth muscle are similar to those of other smooth muscles. Airway smooth muscle exhibits spontaneous intrinsic tone and its maximum shortening velocity (Vmax) is 10-30 fold lower than in striated muscle. Smooth muscle myosin generates step size and elementary force per crossbridge interaction approximately similar to those of skeletal muscle myosin. Special slow cycling crossbridges, termed latch-bridges, have been attributed to myosin light chain dephosphorylation. From a mechanical point of view, it has been shown that airway hyperresponsiveness is characterized by an increased Vmax and an increased shortening capacity, with no significant change in the force-generating capacity.

Severe mechanical dysfunction in pharyngeal muscle from adult mdx mice.

The mdx mouse is a widely used animal model of human muscular dystrophy. Although diaphragm muscle exhibits severe muscle weakness throughout the life of the animal, the limb muscle function of mdx mice spontaneously recovers by 6 mo of age. Pharyngeal dilator muscles such as sternohyoid (SH) contribute to upper airway patency during breathing. We hypothesized that SH muscle function was impaired in 6-mo-old mdx mice. Mechanical properties and myosin heavy chain (MHC) composition were investigated in isolated SH from 6-mo-old control (C, n = 10) and mdx (n = 10) mice. As compared with C, peak tetanic tension (Pmax) and maximum shortening velocity were 50% and 16% lower in mdx mice (p < 0.001 and p < 0.05, respectively). Peak mechanical power was lower in mdx than in C (19.0 +/- 3.2 versus 57.4 +/- 5.1 mW g(-)(1), p < 0.001). Both C and mdx SH were composed exclusively of fast myosin isoforms. As compared with C, mdx SH presented a higher proportion of IIX-MHC and a reduction in IIB-MHC (each p < 0.001). In conclusion, our results demonstrated severe SH muscle dysfunction in 6-mo-old mdx mice, that is, at a time when limb muscle function has recovered. Thus, SH muscle of the mdx mouse may be an excellent muscle for studying Duchenne muscular dystrophy.

Short-term variability of pulse pressure and systolic and diastolic time in heart transplant recipients.

In heart transplant recipients (HTR), short-term systolic blood pressure variability is preserved, whereas heart rate variability is almost abolished. Heart period is the sum of left ventricular ejection time (LVET) and diastolic time (DT). In the present time-domain prospective study, we tested the hypothesis that short-term fluctuations in aortic pulse pressure (PP) in HTR were related to fluctuations in LVET. Seventeen male HTR (age 48 +/- 6 yr) were studied 16 +/- 11 mo after transplantation. Aortic root pressure was obtained over a 15-s period using a micromanometer both at rest (n = 17) and following the cold pressor test (CPT, n = 14). There was a strong positive linear relationship between beat-to-beat LVET and beat-to-beat PP in all patients at rest and in 13 of 14 patients following CPT (each P < 0.01). The slope of this relationship showed little scatter both at rest (0.34 +/- 0.07 mmHg/ms) and following CPT (0.35 +/- 0.09 mmHg/ms, P = not significant). Given the essentially fixed heart period, DT varied inversely with LVET. As a result, in 13 of 17 HTR at rest and in 12 of 14 HTR following CPT, there was a negative linear relationship between beat-to-beat PP and DT. In conclusion, our short-term time-domain study demonstrated a strong positive linear relationship between LVET and blood pressure variability in male HTR. We also identified a subgroup of HTR in whom there was a mismatch between PP and DT.

Isotonic mechanics of a pharyngeal dilator muscle and diaphragm in the rat before and after fatigue.

Pharyngeal and diaphragm muscles contract and relax in synergy, which is why it was decided to compare their mechanical performance throughout the overall load continuum. The effects of fatigue were also studied. The isotonic mechanics of rat sternohyoid (SH; n=10) and diaphragm (D; n=10) were investigated in vitro. Force and length were measured in muscles contracting from zero load up to isometry. Maximum isometric tension (Pmax), peak mechanical work (Wmax), maximum unloaded shortening velocity (vzL) and mechanical efficiency (eff(max)) were recorded. Data were obtained both at baseline and after fatigue. SH muscles had a lower Pmax (96.0+/-13.7 versus 119.5+/-22.7 mN x mm(-2); p<0.05), a lower Wmax (5.5+/-1.2 versus 8.0+/-2.1 mJ x g(-1); p<0.01), a lower eff(max) (56.0+/-6.9 versus 62.6+/-5.8%; p<0.05) and a higher vzL (4.8+/-0.4 versus 3.4+/-0.4 initial length (L0) x s(-1); p<0.001) than D muscles. Wmax occurred at a higher relative load in SH (40% Pmax) than in D (30% Pmax). Fatigue did not modify eff(max) in SH muscles, whereas it significantly improved eff(max) in D muscles. These findings suggest that under control conditions, economy of force generation was less efficient in sternohyoid than in diaphragm muscles. Fatigue in sternohyoid muscles induced unfavourable mechanical behaviour. This may partly explain pharyngeal dilator muscle failure in the presence of increased loads. Whether these findings are relevant to human sleep apnoea syndrome has yet to be determined.

Myosin molecular motor dysfunction in dystrophic mouse diaphragm.

Cross-bridge properties and myosin heavy chain (MHC) composition were investigated in isolated diaphragm from 6-mo-old control (n = 12) and mdx (n = 12) mice. Compared with control, peak tetanic tension fell by 50% in mdx mice (P < 0.001). The total number of cross bridges per square millimeter (x10(9)), the elementary force per cross bridge, and the peak mechanical efficiency were lower in mdx than in control mice (each P < 0.001). The duration of the cycle and the rate constant for cross-bridge detachment were significantly lower in mdx than in control mice. In the overall population, there was a linear relationship between peak tetanic tension and either total number of cross bridges per square millimeter or elementary force per cross bridge (r = 0.996 and r = 0.667, respectively, each P < 0.001). The mdx mice presented a higher proportion of type IIA MHC (P < 0.001) than control mice and a reduction in type IIX MHC (P < 0.001) and slow myosin isoforms (P < 0.01) compared with control mice. We concluded that, in mdx mice, impaired diaphragm strength was associated with qualitative and quantitative changes in myosin molecular motors. It is proposed that reduced force generated per cross bridge contributed to diaphragm weakness in mdx mice.

Relaxation of diaphragm muscle.

Relaxation is the process by which, after contraction, the muscle actively returns to its initial conditions of length and load. In rhythmically active muscles such as diaphragm, relaxation is of physiological importance because diaphragm must return to a relatively constant resting position at the end of each contraction-relaxation cycle. Rapid and complete relaxation of the diaphragm is likely to play an important role in adaptation to changes in respiratory load and breathing frequency. Regulation of diaphragm relaxation at the molecular and cellular levels involves Ca(2+) removal from the myofilaments, active Ca(2+) pumping by the sarcoplasmic reticulum (SR), and decrease in the number of working cross bridges. The relative contribution of these mechanisms mainly depends on sarcomere length, muscle tension, and the intrinsic contractile function. Increased capacity of SR to take up Ca(2+) can arise from increased density of active SR pumping sites or in slow-twitch fibers from phosphorylation of phospholamban, whereas impaired coupling between ATP hydrolysis and Ca(2+) transport into the SR or intracellular acidosis reduces SR Ca(2+) pump activity. In experimental conditions of decreased contractile performance, slowed, enhanced, or unchanged relaxation rates have been reported in vitro. In vivo, a slowing in the rate of decline of the respiratory pressure is generally considered an early reliable index of respiratory muscle fatigue. Impaired relaxation rate may, in turn, favor mismatch between blood flow and metabolic demand, especially at high breathing frequencies.

Impaired load dependence of diaphragm relaxation during congestive heart failure in the rabbit.

The load dependence (LD) of relaxation was studied in the diaphragm of rabbits with congestive heart failure (CHF). CHF (n = 15) was induced by combined chronic volume and pressure overload. Aortic insufficiency was induced by forcing a catheter through the aortic sigmoid valves, followed 3 wk later by abdominal aortic stenosis. Six weeks after the first intervention, animals developed CHF. Sham-operated animals served as controls (C; n = 12). Diaphragm mechanics were studied in vitro on isolated strips, at 22 degrees C, in isotonic and isometric loading conditions. Contractility was lower in the CHF group, as reflected by lower total tension: 1.11 +/- 0.10 in CHF vs. 2.38 +/- 0.15 N/cm(2) in C in twitch (P < 0.001) and 2.46 +/- 0.22 in CHF vs. 4.90 +/- 0.25 N. cm(-2) in C in tetanus (P < 0.001). The index LD was used to quantify the load dependence of relaxation: LD is <1 in load-dependent muscles and tends toward 1 in load-independent muscles. LD was significantly higher in CHF than in C rabbits, in both twitch (0.99 +/- 0.01 vs. 0.75 +/- 0.03; P < 0. 001) and tetanus (0.95 +/- 0.02 vs. 0.84 +/- 0.02; P < 0.001). In the CHF rabbits' diaphragm, the fall in total tension was linearly related to the fall in load dependence of relaxation. The decrease in load dependence of relaxation in CHF animals suggests sarcoplasmic reticulum abnormalities. Impairment of the sarcoplasmic reticulum may also partly account for the decrease in contractile performance of diaphragm in CHF animals.

Impaired skeletal muscle performance in the early stage of cardiac pressure overload in rabbits: beneficial effects of angiotensin-converting enzyme inhibition.

Abnormalities of skeletal muscles are frequently observed in patients with congestive heart failure. In these patients, angiotensin-converting enzyme (ACE) inhibitors improve exercise performance. The present study was designed to assess whether skeletal muscle dysfunction develops in the early stage of cardiac overload and if so, whether such functional alterations can be prevented by ACE inhibition. Mechanical performance, cross-bridge (CB) properties, and myosin heavy chain composition were investigated in respiratory and limb skeletal muscles of rabbits with moderate cardiac hypertrophy, and after single therapy with the ACE inhibitor perindopril (PE). After constriction of the aorta, the rabbits were treated during a 10-week period with either PE (1 mg/kg/day; n = 9) or a placebo (PL; n = 15). A third group of sham-operated animals received PL (n = 10). Analyses were performed on isolated diaphragm and soleus strips. Compared with sham-operated animals (shams), peak tetanic tension in PL fell by 40% in diaphragm and 34% in soleus. There were no significant differences in peak tetanic tension and the maximum shortening velocity between PE and shams. In both muscles, the total number of CBs was significantly lower in PL than in shams, but did not differ between shams and PE. The elementary force per CB did not differ between groups. In both muscles, the myosin heavy chain composition did not differ between groups. The study demonstrated that intrinsic performance of diaphragm and soleus muscles was affected early in the development of chronic pressure overload. Single therapy with PE tended to preserve muscle strength, essentially by limiting the loss of CBs.

The effects of dantrolene on the contraction, relaxation, and energetics of the diaphragm muscle.

UNLABELLED: Dantrolene is used in patients with muscle spasticity and is the only known effective treatment for malignant hyperthermia. However, its effects on muscle relaxation and energetics are unknown and may have important consequences in diaphragmatic function. We studied the effects of dantrolene (10(-8) to 10(-4) M) on diaphragm muscle strips (n = 12) in the hamster in vitro (Krebs-Henseleit solution, 29 degrees C, 95% oxygen/5% carbon dioxide) in response to tetanic stimulation (50 Hz). We studied contraction and relaxation under isotonic and isometric conditions, as well as energetics. Data are mean +/- SD. Dantrolene induced a negative inotropic effect in the hamster diaphragm (active force at 10(-4) M: 34% +/- 7% of baseline; P < 0.05) but did not significantly modify the curvature of the force-velocity relationship. Dantrolene did not significantly modify isotonic relaxation. Dantrolene, up to 10(-5) M, did not significantly impair isometric relaxation. In conclusion, dantrolene induced a marked negative inotropic effect on diaphragm muscle without affecting myothermal efficiency and relaxation. IMPLICATIONS: Dantrolene induced a significant and concentration-dependent negative inotropic effect on diaphragm muscle but did not modify isotonic relaxation, which suggests no alteration of the calcium reuptake by the sarcoplasmic reticulum; up to 10(-5) M dantrolene did not alter isometric relaxation, i.e., myofilament calcium sensitivity. Dantrolene did not modify the energetics of diaphragm muscle.