Salbutamol enhances isotonic contractile properties of rat diaphragm muscle

The effects of the₂-adrenoceptor agonistsalbutamol (Slb) on isometric and isotonic contractile properties ofthe rat diaphragm muscle(Dia_mus) were examined. Aloading dose of 25 µg/kg Slb was administered intracardially beforeDia_mus excision to ensure adequatediffusion. Studies were then performed with 0.05 µMSlb in the in vitro tissue chamber. cAMP levels were determined by radioimmunoassay. Compared with controls (Ctl), cAMP levels were elevated after Slb treatment. In Slb-treated rats, isometric twitch andmaximum tetanic force were increased by ~40 and ~20%,respectively. Maximum shortening velocity increased by ~15% afterSlb treatment, and maximum power output increased by ~25%. Duringrepeated isotonic activation, the rate of fatigue was faster in theSlb-treated Dia_mus, but bothSlb-treated and Ctl Dia_musfatigued to the same maximum power output. Still, endurance time duringrepetitive isotonic contractions was ~10% shorter in the Slb-treatedDia_mus. These results areconsistent with the hypothesis that -adrenoceptor stimulation by Slbenhances Dia_mus contractility andthat these effects of Slb are likely mediated, at least in part, byelevated cAMP.

     

Isotonic contractile and fatigue properties of developing rat diaphragm muscle

Postnatal transitions in myosin heavy chain (MHC) isoformexpression were found to be associated with changes in both isometric and isotonic contractile properties of rat diaphragm muscle(Dia_m). Expression of MHC_neo predominated inneonatal Dia_m fibers but was usually coexpressed withMHC_slow or MHC_2A isoforms. Expression ofMHC_neo disappeared by day 28. Expression ofMHC_2X and MHC_2B emerged at day 14 andincreased thereafter. Associated with these MHC transitions in theDia_m, maximum isometric tetanic force (P_o), maximum shortening velocity, and maximum power output progressively increased during early postnatal development. Maximum power output ofthe Dia_m occurred at ~40% P_o at days0 and 7 and at ~30% P_o in older animals.Susceptibility to isometric and isotonic fatigue, defined as a declinein force and power output during repetitive activation, respectively,increased with maturation. Isotonic endurance time, defined as the timefor maximum power output to decline to zero, progressively decreasedwith maturation. In contrast, isometric endurance time, defined as thetime for force to decline to 30-40% P_o, remained>300 s until after day 28. We speculate that with thepostnatal transition to MHC_2X and MHC_2Bexpression energy requirements for contraction increase, especiallyduring isotonic shortening, leading to a greater imbalance betweenenergy supply and demand.

     

Zinc-induced alterations in contractile properties of rat diaphragm muscle in vitro

The effect of zinc ions on the isometric contraction of rat diaphragm muscles in the presence and in the absence of external calcium was studied. Using a transducer, the isometric force was measured as a function of supramaximal electrical stimulation, either directly or indirectly applied to the muscle. The following parameters were measured: peak twitch tension, PT, twitch contraction time, CT, relaxation half-time, RT-1/2, and peak rates of tension increase and decrease, +dP/dt and -dP/dt. The following zinc-induced alterations were observed: an increase of the PT; a decrease of the RT-1/2; an increase in the +dP/dt and -dP/dt. The CT was not changed significantly. Our results suggest that zinc ions have a positive inotropic effect on isolated diaphragm muscle. The increase in PT may be explained by a zinc-activated Ca²⁺ uptake by sarcoplasmic reticulum. This was followed by an increase in the rate of rise of tension development, which was secondary to increased -dP/dt. The mechanism(s) by which extracellular Ca²⁺ contributes to this action of zinc is not known.     

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.     

Effect of contractile activity on rat skeletal muscle beta-adrenoceptor properties

The effect of fiber type and endurance exercise training on skeletal muscle beta-adrenoceptor properties were assessed using a direct radioligand binding technique. Six separate muscles, composed of a variety of different fiber types, were examined in treadmill trained and sedentary rats. In trained animals, sarcolemmal preparations from heart and slow twitch soleus muscle exhibited a significantly greater receptor concentration than membranes from white fast twitch glycolytic fibers of the vastus lateralis. No significant changes were observed between trained and sedentary rat muscle beta-adrenoceptor density (beta max, fmole/mg protein) or affinity (Kd, nM) within each muscle type, despite significantly increased myocardial/body weight ratios and skeletal muscle enzyme adaptations associated with the exercise program. These results suggest that muscle beta-adrenoceptor properties may be influenced in part by the motor nerve innervation to that muscle, and are further discussed with respect to a possible relationship between exercise intensity and receptor regulation.     

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.

     

Fatigue of mouse diaphragm muscle in isometric and isotonic contractions

Fatiguabilities of mouse diaphragm muscle in vitro in isometric and isotonic contractions were compared in this study. Isolated mouse diaphragm muscle was stimulated repetitively to induce fatigue during both isometric and isotonic contractions. The supramaximal electrical stimulation used was a train of 100-Hz, 0.5-ms pulses delivered to the muscle every 2 s for 0.5 s. The percentage decrease in isometric tension from beginning to end of the fatiguing process was used as the index of fatigue. The experiments were carried out at different PO2 levels in both normal and zero-glucose Ringer solutions. It was found that fatigue developed more rapidly in isotonic contractions than in isometric ones. Also, the extracellular glucose level demonstrated little effect on the muscle's short-term fatiguability, whereas reductions in the extracellular PO2 exerted a profound effect, especially in the case of isotonic fatigue.     

In vivo contractile properties of fatigued diaphragm

The effects of fatigue on diaphragmatic contractility in vivo are unknown. In this study we used sonomicrometry to examine the velocity of shortening and lengthening and the amount of shortening in the fresh and fatigued canine hemidiaphragm (8 dogs) including the force generated. Fatigue was produced by epiphrenic stimulation of the left phrenic nerve; the right hemidiaphragm acted as the control. We found that 1) hemidiaphragmatic fatigue caused an increase in frequency with reduced tidal volume; 2) fatigue resulted in a near complete cessation of tidal shortening during spontaneous breathing; 3) there was an initial decrease in central activation (electromyogram) to the fatigued hemidiaphragm, an indication of central fatigue; 4) force-frequency curves showed a considerable and prolonged loss of the amount of shortening, velocity, and force generated by the fatigued hemidiaphragm during supramaximal stimulation, an indication of peripheral fatigue; and 5) during spontaneous breathing in the fatigued hemidiaphragm, tidal shortening remained reduced for up to 3 h, whereas in the right right hemidiaphragm tidal shortening and electromyographic activity did not change. We conclude that fatigue of a hemidiaphragm alters the spontaneous breathing pattern and produces profound modifications in its contractile properties without altering contralateral hemidiaphragmatic performance.     

Thermal dependence of isotonic contractile properties of skeletal muscle and sprint performance of the lizardDipsosaurus dorsalis

Contractile properties of the fast-twitch glycolytic (FG) portion of the iliofibularis muscle and sprint running performance were studied at approximately 5 degrees C intervals from 15-44 degrees C in the lizard Dipsosaurus dorsalis. Maximal running velocity (VR) and stride frequency (f) were both greatest when body temperature (Tb) was 40 degrees C, the field-active Tb in Dipsosaurus. At 40 degrees C VR was 4.3 +/- 0.2 m/s and f was 13.5 +/- 0.5 s-1. Between 25 and 40 degrees C, the thermal dependencies of VR and f were approximately constant (Q10's of 1.31 and 1.36 got VR and f, respectively). Below 25 degrees C performance declined more markedly with decreasing temperature. At 20 degrees C strides were qualitatively normal, but VR was only half of the value at 25 degrees C. At 15 degrees C the lizards were substantially incapacitated, and VR was 10% of the value at 20 degrees C. Stride length was approximately 0.33 m and changed very little with Tb from 20-44 degrees C. The time dependent contractile properties of FG muscle were affected more by temperature than was sprint performance. The maximal velocity of shortening at zero load (VO) was 18.7 0/s at 40 degrees C and had a Q10 of 1.7 from 25-40 degrees C. Maximal power output (Wmax) determined from the force-velocity curve was 464 W/kg at 40 degrees C. Below 40 degrees C max varied with temperature with a Q10 of 2-3. The shape of the force-velocity curve changed little with temperature (Wmax/POVO = 0.11). Between 25 and 40 degrees C a relatively temperature-independent process must modulate the effects of temperature on the contractile properties of the muscles that supply the power for burst locomotion. Storage and recovery of elastic energy appears to be a likely candidate for such a process. Below 25 degrees C, however, the contraction time is prolonged to such an extent that the f attainable is limited by the minimum time taken to contract and relax the muscles.     

Procedures for rat in situ skeletal muscle contractile properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation. To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37°C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship. With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.     

Differential effects of peroxynitrite on contractile protein properties in fast- and slow-twitch skeletal muscle fibers of rat

Oxidative modification of contractile proteins is thought to be a key factor in muscle weakness observed in many pathophysiological conditions. In particular, peroxynitrite (ONOO(-)), a potent short-lived oxidant, is a likely candidate responsible for this contractile dysfunction. In this study ONOO(-) or 3-morpholinosydnonimine (Sin-1, a ONOO(-) donor) was applied to rat skinned muscle fibers to characterize the effects on contractile properties. Both ONOO(-) and Sin-1 exposure markedly reduced maximum force in slow-twitch fibers but had much less effect in fast-twitch fibers. The rate of isometric force development was also reduced without change in the number of active cross bridges. Sin-1 exposure caused a disproportionately large decrease in Ca(2+) sensitivity, evidently due to coproduction of superoxide, as it was prevented by Tempol, a superoxide dismutase mimetic. The decline in maximum force with Sin-1 and ONOO(-) treatments could be partially reversed by DTT, provided it was applied before the fiber was activated. Reversal by DTT indicates that the decrease in maximum force was due at least in part to oxidation of cysteine residues. Ascorbate caused similar reversal, further suggesting that the cysteine residues had undergone S-nitrosylation. The reduction in Ca(2+) sensitivity, however, was not reversed by either DTT or ascorbate. Western blot analysis showed cross-linking of myosin heavy chain (MHC) I, appearing as larger protein complexes after ONOO(-) exposure. The findings suggest that ONOO(-) initially decreases maximum force primarily by oxidation of cysteine residues on the myosin heads, and that the accompanying decrease in Ca(2+) sensitivity is likely due to other, less reversible actions of hydroxyl or related radicals.     

Isometric contractile properties of diaphragm strips from alcoholic rats

Chronic ethanol consumption alters the structure and function of human respiratory muscle. We have examined its effect on the active and passive mechanical properties of rat diaphragm strips in vitro. We conditioned eight rats using a liquid diet containing ethanol as 36% of calories. Eight control rats were pair-fed an isocaloric, ethanol-free liquid diet. Rats were killed after 23 wk. Two strips from the left hemidiaphragm were suspended in Krebs-Ringers solution at 25 degrees C, equilibrated with 5% CO2-95% O2. Isometric stresses were calculated from force transducer measurements. Strips were stimulated directly at supramaximal voltage. Twitch stress (Pt), measured at optimal length (Lo), was greater in ethanol-conditioned strips: 5.1 vs. 3.8 N/cm2. Times to peak Pt and twitch half-relaxation times were equal. Tetanic stress at Lo (Po) was also greater after ethanol conditioning: 17.2 vs. 12.8 N/cm2. Pt/Po ratios were equal. Expressed as %Po, tetanic stress-stimulation frequency curves and tetanic stress-length curves were identical. Ethanol-conditioned strips were marginally less compliant when passively stretched to lengths between Lo and 130% Lo. We postulate that ethanol may have increased active stress development by reducing intracellular free water.     

Effects of modulation of nitric oxide on rat diaphragm isotonic contractility during hypoxia.

Nitric oxide (NO) is essential for optimal myofilament function of the rat diaphragm in vitro during active shortening. Little is known about the role of NO in muscle contraction under hypoxic conditions. Hypoxia might increase the NO synthase (NOS) activity within the rat diaphragm. We hypothesized that NO plays a protective role in isotonic contractile and fatigue properties during hypoxia in vitro. The effects of the NOS inhibitor N(G)-monomethyl-l-arginine (l-NMMA), the NO scavenger hemoglobin, and the NO donor spermine NONOate on shortening velocity, power generation, and isotonic fatigability during hypoxia were evaluated (Po(2) approximately 7 kPa). l-NMMA and hemoglobin slowed the shortening velocity, depressed power generation, and increased isotonic fatigability during hypoxia. The effects of l-NMMA were prevented by coadministration with the NOS substrate l-arginine. Spermine NONOate did not alter isotonic contractile and fatigue properties during hypoxia. These results indicate that endogenous NO is needed for optimal muscle contraction of the rat diaphragm in vitro during hypoxia.     

Effects of marinobufagenin on electrophysiological and contractile characteristics of the rat diaphragm

Effects of Na+,K(+)-ATPase inhibitor: marinobufagenin, on contractile and electric characteristics of isolated rat diaphragm were studied for the first time. Marinobufagenin induced dose-dependent (EC50 = 0.3 +/- 0.1 nM) increase in the contraction force (positive inotropic effect). At 1-2 nM, it slowed down the fatigue induced by continuous direct stimulation (2/s) of the muscle. Marinobufagenin at the same concentrations did not affect resting membrane potential or parameters of action potentials of muscle fibers, while at 10 and 20 nM it induced hyperpolarization by approximately 2 mV. Marinobufagenin blocked dose-dependently (IC50 = 2.9 +/- 2.0 nM) hyperpolarizing effect of acetylcholine (100 nM) mediated by increase in electrogenic contribution of alpha2 isoform of the Na+,K(+)-ATPase. This result suggests a capability of marinobufagenin to inhibit this isoform of the Na+,K(+)-ATPase. Possible mechanisms of marinobufagenin effects in skeletal muscle are discussed.     

Altered diaphragm contractile properties with controlled mechanical ventilation.

This study shows that, over time, diaphragm inactivity with controlled mechanical ventilation (CMV) decreases diaphragm force and produces myofibril damage contributing to the reduced force. We measured in vivo and in vitro diaphragm contractile and morphological properties in 30 sedated rabbits grouped (n = 6) as follows: 1 or 3 days of CMV, 1 or 3 days of 0 cmH(2)O continuous positive airway pressure, and control. The CMV rate was set sufficient to suppress diaphragm electrical activity. Compared with the control group, phrenic-stimulated maximum transdiaphragmatic pressure did not decrease with continuous positive airway pressure but decreased to 63% after 1 day of CMV and to 49% after 3 days of CMV. The in vitro tetanic force decreased to 86% after 1 day of CMV and to 44% after 3 days of CMV. After 3 days of CMV, significant myofibril damage occurred in the diaphragm but not in the soleus. The decrease in tetanic force correlated with the volume density of abnormal myofibrils. We conclude that CMV had a detrimental effect on diaphragm contractile properties.