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.     

Effect of acute nutritional deprivation on diaphragm structure and function

The influence of 90 h of acute nutritional deprivation (ND) on the cross-sectional areas of muscle fibers and the contractile and fatigue properties of the adult rat diaphragm were determined. Isometric contractile properties and fatigue resistance of the diaphragm were measured by means of an in vitro nerve-muscle strip preparation. Contractions were evoked by using phrenic nerve stimulation (left hemidiaphragm) or direct muscle stimulation (right hemidiaphragm) in the presence of curare. Acute ND resulted in a 20% reduction in body weight. No significant decrements in diaphragm or soleus weights were noted in the ND animals compared with controls (CTL), whereas the weight of the medial gastrocnemius was reduced by 20% in the ND animals. Peak twitch and tetanic tensions (normalized for the weight of the diaphragm strip) were not reduced in ND compared with CTL animals after either nerve or muscle stimulation. The fatigue index of the diaphragm was significantly reduced in ND animals only after nerve stimulation. After the fatigue test, there was rapid recovery of the additional fatigue noted with nerve stimulation. The proportions of type I and II muscle fibers of the diaphragm were similar in the CTL and ND animals. No differences in diaphragm cross-sectional areas were noted for either type I or II muscle fibers in the CTL and ND animals. It is concluded that acute ND has no effect on diaphragm contractility or morphometry and only an inconsequential influence on diaphragm fatigue.     

Inhibition by nifedipine of the indirectly induced contractile response of the rat diaphragm

The effects of nifedipine (30 micrograms/ml) on isometric force production of in-vitro rat diaphragm were studied during direct and indirect modes of muscle activation. During direct muscle stimulation, nifedipine potentiated isometric force during twitch and unfused tetanic stimulation. Indirectly elicited responses, evoked by stimulation of the phrenic nerve, were uniformly depressed following nifedipine. Inhibition of indirect force production increased with time, while force potentiation with direct activation remained constant. We conclude that inhibitory effects of nifedipine on the phrenic nerve-diaphragm preparation are specific for nerve or neuromuscular junction.     

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.     

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.     

Contractile properties of the human diaphragm in vivo

The mechanical properties of the human diaphragm have been studied at fractional residual capacity in normal seated subjects with closed glottis. The transdiaphragmatic pressure (Pdi) developed in response to single shocks or to trains of stimuli at increasing frequency was approximately 3 times greater during bilateral than unilateral stimulation. During unilateral phrenic nerve stimulation the Pdi twitches increased as the interval (0-200 ms) of a preceding conditioning stimulus to the contralateral phrenic nerve was decreased suggesting that the two hemidiaphragms are mechanically coupled in series. The contraction time and half-relaxation time of single bilateral twitches as well as the Pdi-frequency relationship (5-35 Hz) during bilateral tetanic stimulation indicate that the contractile properties of the human diaphragm are intermediate between those of fast- and slow-twitch muscle fibers. The results suggest that the contractile properties of the human diaphragm are well illustrated by single bilateral twitches recorded from the relaxed muscle, but that the responses to unilateral stimulation are misleading due to distortion by abnormal changes in the muscle geometry.     

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.     

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.     

Effect of nutritional deprivation on diaphragm contractility and muscle fiber size

The influence of nutritional deprivation on the contractile and fatigue properties of the diaphragm was studied in adult rats. Food access was restricted to one-third of normal daily intake until the body weight of nutritionally deprived (ND) animals was approximately 50% of controls (CTL). Isometric contractile properties were studied in an in vitro nerve muscle strip preparation. Both twitch (Pt) and tetanic (Po) tensions of diaphragms from the ND animals were markedly reduced compared with CTL; however, Pt/Po was higher for the ND group. The shape of the force-frequency curve (normalized to Po) was generally similar between the two groups, except at 5 and 10 pulses/s stimulation, where greater relative tensions were produced in diaphragms from the ND animals. Diaphragm fatigue was induced by repetitive stimulation at either 20 or 100 pulses/s. Endurance time (defined as the time required for tension to fall to 50% of initial) of diaphragms from ND animals was prolonged at both 20 and 100 pulses/s. Immediately after induction of fatigue, force-frequency curves for both ND and CTL diaphragms were shifted to the right. However, this rightward shift was attenuated in the ND group compared with CTL. Nutritional deprivation had no effect on the proportions of different fiber types within the diaphragm but did result in a significant decrease in the cross-sectional area of both fast-and slow-twitch fibers. This decrease in cross-sectional area was significantly greater for fast-twitch fibers. We conclude that these changes in diaphragm contractile and fatigue properties occur as a result of the influence of malnutrition on muscle fiber cross-sectional area.     

Diaphragmatic fatigue in the rat

We studied fatigue of rat diaphragm in response to repetitive brief and prolonged electrical stimulation of the phrenic nerve, at 0.2, 1-100 Hz. Low and high frequency of stimulation produced twitch and tetanic contractions in the rat diaphragm. A mean maximum twitch tension of 1.4 +/- 0.1 g was produced at 1 Hz, and a mean maximum tetanic tension of 5.6 +/- 0.3 g was obtained at 100 Hz (means +/- S.E., n = 8). Twitch and tetanic fatigue was produced at all frequencies of stimulations, but with different time scale, or duration, and with different number of stimuli delivered to the muscle. At low rates of stimulation, e.g. 10 Hz, fewer stimuli were needed to fatigue the muscle (3000 in 5 min), whereas at high rates of stimulation, e.g. 50 Hz, more stimuli were needed to fatigue the muscle (6600 in 2.2 min). The amplitude of the tetanic tensions elicited at 10 and 50 Hz, at the end of 5 or 2 min fatiguing stimulation, was 39 +/- 2.7% and 80 +/- 3.1% of their respective control tensions (2.8 +/- 0 2 g and 5.3 +/- 0.5 g, n = 8, P 0.001). It was concluded that fatigue in the rat diaphragm depended on the frequency and duration of stimulation as well as on the number of stimuli delivered to the muscle. Various mechanisms of muscle fatigue are described in the discussion to explain the observations made in the present investigation.     

Chronic exposure to a 60-Hz electric field: Effects on neuromuscular function in the rat

Neuromuscular function in adult male rats was studied following 30 days of exposure to a 60-Hz electric field at 100 kV/m (unperturbed field strength). Isometric force transducers were attached to the tendons of the plantaris (predominantly fast twitch), and soleus (predominantly slow twitch) muscles in the urethan-anesthetized rat. Square-wave stimuli were delivered to the distal stump of the transected sciatic nerve. Several measurements were used to characterize neuromuscular function, including twitch characteristics, chronaxie, tetanic and posttetanic potentiation, and fatigue and recovery. The results from three independent series of experiments are reported. Only recovery from fatigue in slow-twitch muscles was consistently and significantly affected (enhanced) by electrifield exposure. This effect does not appear to be mediated by field-induced changes in either neuromuscular transmission, or in the contractile mechanism itself. It is suggested that the effect may be mediated secondary to an effect on mechanisms regulating muscle blood flow or metabolism.     

Fatigue and recovery contractile properties of young and elderly men

The 24 h recovery pattern of contractile properties of the triceps surae muscle, following a period of muscle fatigue, was compared in physically active young (25 years, n = 10) and elderly (66 years, n = 7) men. The fatigue test protocol consisted of 10 min of intermittent submaximal 20 Hz tetani. The maximal twitch (Pt) and tetanic force at 3 frequencies (10, 20 and 50 Hz) were determined at baseline and at 15 min, 1, 4 and 24 h after fatiguing the muscle. Maximal voluntary contraction (MVC) and vertical jump (MVJ) were also assessed. The loss of force during the fatigue test was not significantly different between the young (18 +/- 13%) and elderly (22 +/- 15%). Both groups showed similar and significant reductions of Pt (15%), tetanic force (10 to 35%) and rate of force development (dp/dt) (20%) 15 min and 1 h into recovery. The loss of force was greater at the lower stimulation frequencies of 10 and 20 Hz. Time-to-peak tension was unchanged from baseline during recovery in either group. The average rate of relaxation of twitch force (-dPt/dt) was decreased (p less than 0.05) and half-relaxation time significantly increased at 15 min and 1 h in the elderly but not the young. The findings indicate that after fatiguing contractions, elderly muscle demonstrates a slower return to resting levels of the rate and time course of twitch relaxation compared to the young.     

Effects of vitamin E and alpha-lipoic acid on skeletal muscle contractile properties.

Initial experiments were conducted using an in situ rat tibialis anterior (TA) muscle preparation to assess the influence of dietary antioxidants on muscle contractile properties. Adult Sprague-Dawley rats were divided into two dietary groups: 1) control diet (Con) and 2) supplemented with vitamin E (VE) and alpha-lipoic acid (alpha-LA) (Antiox). Antiox rats were fed the Con rats' diet (AIN-93M) with an additional 10,000 IU VE/kg diet and 1.65 g/kg alpha-LA. After an 8-wk feeding period, no differences existed (P > 0.05) between the two dietary groups in maximum specific tension before or after a fatigue protocol or in force production during the fatigue protocol. However, in unfatigued muscle, maximal twitch tension and tetanic force production at stimulation frequencies < or = 40 Hz were less (P < 0.05) in Antiox animals compared with Con. To investigate which antioxidant was responsible for the depressed force production, a second experiment was conducted using an in vitro rat diaphragm preparation. Varying concentrations of VE and dihydrolipoic acid, the reduced form of alpha-LA, were added either individually or in combination to baths containing diaphragm muscle strips. The results from these experiments indicate that high levels of VE depress skeletal muscle force production at low stimulation frequencies.     

An overview of phrenic nerve and diaphragm muscle development in the perinatal rat.

In this overview, we outline what is known regarding the key developmental stages of phrenic nerve and diaphragm formation in perinatal rats. These developmental events include the following. Cervical axons emerge from the spinal cord during embryonic (E) day 11. At approximately E12.5, phrenic and brachial axons from the cervical segments merge at the brachial plexi. Subsequently, the two populations diverge as phrenic axons continue to grow ventrally toward the diaphragmatic primordium and brachial axons turn laterally to grow into the limb bud. A few pioneer axons extend ahead of the majority of the phrenic axonal population and migrate along a well-defined track toward the primordial diaphragm, which they reach by E13.5. The primordial diaphragmatic muscle arises from the pleuroperitoneal fold, a triangular protrusion of the body wall composed of the fusion of the primordial pleuroperitoneal and pleuropericardial tissues. The phrenic nerve initiates branching within the diaphragm at approximately E14, when myoblasts in the region of contact with the phrenic nerve begin to fuse and form distinct primary myotubes. As the nerve migrates through the various sectors of the diaphragm, myoblasts along the nerve's path begin to fuse and form additional myotubes. The phrenic nerve intramuscular branching and concomitant diaphragmatic myotube formation continue to progress up until E17, at which time the mature pattern of innervation and muscle architecture are approximated. E17 is also the time of the commencement of inspiratory drive transmission to phrenic motoneurons (PMNs) and the arrival of phrenic afferents to the motoneuron pool. During the period spanning from E17 to birth (gestation period of approximately 21 days), there is dramatic change in PMN morphology as the dendritic branching is rearranged into the rostrocaudal bundling characteristic of mature PMNs. This period is also a time of significant changes in PMN passive membrane properties, action-potential characteristics, and firing properties.     

Genetic ablation of acetylcholinesterase alters muscle function in mice

Although acetylcholinesterase (AChE) knockout mice survive, they have abnormal neuromuscular function. We analysed further the effects of the mutation on hind limb muscle contractile properties. Tibialis anterior muscle from AChE KO mice is unable to maintain tension during a short period of repetitive nerve stimulation (tetanic fade) and has an increased twitch tension in response to a single nerve electric stimulation. In response to direct muscle stimulation, we found that maximal velocity of shortening of soleus muscle is increased and maximum tetanic force is decreased in AchE KO mice versus control animals. As the contractile properties of the soleus muscle were altered by AChE ablation, our results suggest cellular and molecular changes in AChE ablated muscle containing both fast and slow muscle fibres.     

Fatigue of isolated rat diaphragm: role of impaired neuromuscular transmission

We compared the contributions of impaired neuromuscular transmission (transmission fatigue) and impaired muscle contractility (contractile fatigue) to fatigue of the isolated rat diaphragm. To make this comparison, we measured the differences in active tension elicited by direct muscle stimulation and by indirect (phrenic nerve) stimulation before and after fatigue induced by indirect supramaximal stimulation at varying frequencies and durations. Transmission fatigue was observed after all experimental protocols. Although significant contractile fatigue was not demonstrated after brief periods of low-frequency stimulation (6 min, 15 Hz, 25% duty cycle), it was present after longer or higher frequency stimulation. We repeated the direct stimulation in the presence of neuromuscular blockade with 6 microM d-tubocurarine to demonstrate that a reduced response to stimulation of intramuscular branches of the phrenic nerve during direct stimulation was not responsible for the apparent contractile fatigue. Since we found significant decreases in the response to direct stimulation even after neuromuscular blockade, we could verify the presence of contractile fatigue. We conclude that both contractile and transmission fatigue can occur in the isolated rat diaphragm and that transmission fatigue is a much more important factor after brief periods of fatiguing contractions.     

Nitric oxide synthase expression and effects of nitric oxide modulation on contractility of rat extraocular muscle.

Extraocular muscles (EOMs) are specialized skeletal muscles that are constantly active, generate low levels of force for cross sectional area, have rapid contractile speeds, and are highly fatigue resistant. The neuronal isoform of nitric oxide synthase (nNOS) is concentrated at the sarcolemma of fast-twitch muscles fibers, and nitric oxide (NO) modulates contractility. This study evaluated nNOS expression in EOM and the effect of NO modulation on lateral rectus muscle's contractility. nNOS activity was highest in EOM compared with diaphragm, extensor digitorum longus, and soleus. Neuronal NOS was concentrated to the sarcolemma of orbital and global singly innervated fibers, but not evident in the multi-innervated fibers. The NG-nitro-L-arginine methyl ester (L-NAME, a NOS inhibitor), increased submaximal tetanic and peak twitch forces. The NO donors S-nitroso-N-acetylcysteine (SNAC) and spermineNONOate reduced submaximal tetanic and peak twitch forces. The effect of NO on the contractile force of lateral rectus muscle is greater than previously observed on other skeletal muscle. NO appears more important in modulating contraction of EOM compared with other skeletal muscles, which could be important for the EOM's specialized role in generation of eye movements.     

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 acute nutritional deprivation on diaphragm structure and function in adolescent rats.

The influence of 90 h of acute nutritional deprivation (ND; water ad libitum only) on in vitro contractile and fatigue properties, muscle fiber type proportions, and cross-sectional areas (CSA) of the adolescent rat diaphragm was determined. Diaphragm muscle properties in the ND rats were compared with those in control rats (CTL; food and water ad libitum). Acute ND resulted in a 32% reduction in body mass, whereas the body mass of CTL rats increased by 29%. Acute ND resulted in a significant reduction in the mass of the diaphragm (costal, 36%; crural, 43%), soleus (36%), and medial gastrocnemius (45%) muscles. Isometric twitch characteristics of the diaphragm muscle (contraction and half-relaxation times) were prolonged in the ND animals. Peak twitch and maximum tetanic forces were unaffected by ND. Fatigue resistance of the diaphragm muscle was improved in ND animals. Diaphragm muscle fiber type proportions were similar in ND and CTL groups. The CSA of type I and II diaphragm muscle fibers were reduced by 22 and 40%, respectively, in ND animals compared with CTL. We conclude that, whereas an identical protocol of acute ND had no significant effects on diaphragm muscle structure and function in adult rats, adolescent animals exhibit significantly less nutritional reserve. These differences may be due to curtailment of the rapid anabolic rate in growing animals.     

去负荷小鼠比目鱼肌收缩特性和纤维类型转化

目的:探讨去负荷后小鼠比目鱼肌的收缩特性与骨骼肌纤维类型转化之间的关系。方法:采用离体肌肉灌流技术和电刺激方法,在小鼠后肢去负荷28 d引起骨骼肌萎缩后,观察比目鱼肌单收缩、强直收缩能力和肌疲劳指标等收缩特性的改变,同时利用组织免疫荧光染色和实时定量聚合酶链式反应(real-time PCR)等技术检测去负荷后比目鱼肌快慢肌纤维组成和纤维类型转化的变化。结果:去负荷28 d后,小鼠比目鱼肌单收缩力、强直收缩能力和疲劳指数(fatigue index)均有显著性下降,同时伴有快肌纤维亚型的增加和慢肌纤维亚型的减少。结论:去负荷28 d后小鼠比目鱼肌收缩特性的改变和快慢肌纤维类型的转化有关。