Factors underlying the early limb muscle weakness in acute quadriplegic myopathy using an experimental ICU porcine model

The basic mechanisms underlying acquired generalized muscle weakness and paralysis in critically ill patients remain poorly understood and may be related to prolonged mechanical ventilation/immobilization (MV) or to other triggering factors such as sepsis, systemic corticosteroid (CS) treatment and administration of neuromuscular blocking agents (NMBA). The present study aims at exploring the relative importance of these factors by using a unique porcine model. Piglets were all exposed to MV together with different combinations of endotoxin-induced sepsis, CS and NMBA for five days. Peroneal motor nerve conduction velocity and amplitude of the compound muscle action potential (CMAP) as well as biceps femoris muscle biopsy specimens were obtained immediately after anesthesia on the first day and at the end of the 5-day experimental period. Results showed that peroneal nerve motor conduction velocity is unaffected whereas the size of the CMAP decreases independently of the type of intervention, in all groups after 5 days. Otherwise, despite a preserved size, muscle fibre specific force (maximum force normalized to cross-sectional area) decreased dramatically for animals exposed to MV in combination with CS or/and sepsis. These results suggest that the rapid declines in CMAP amplitude and in force generation capacity are triggered by independent mechanisms with significant clinical and therapeutic implications.     

In vitro functions of the rat diaphragm during postnatal development.

This study was designed to determine the developmental changes in the functional characteristics of the rat diaphragm. A total of 150 animals were studied at 1, 3, 5, 7 and 9 weeks of postnatal age. Body and diaphragm muscle weights were measured. Diaphragm strips were studied in an in vitro preparation to assess muscle contractile and endurance properties. Total diaphragm weight increased considerably, by a factor of 23 over the 9 week-period of study and was highly correlated with body weight (r = 0.93, P less than 0.01). However, the ratio of diaphragm-to-body weight decreased progressively with age. In comparison with those from older animals, diaphragms from 1 and 3 weeks old animals: (1) generated similar force normalized for muscle weight but a lower force normalized for fibre cross-sectional area (P less than 0.05), (2) had longer time-to-peak tension and one-half relaxation times (P less than 0.01) and (3) were more resistant to fatigue (P less than 0.01). The mechanisms underlying the diaphragm functional development were discussed.     

Peroxynitrite induces contractile dysfunction and lipid peroxidation in the diaphragm.

Peroxynitrite may be generated in and around muscles in several pathophysiological conditions (e.g., sepsis) and may induce muscle dysfunction in these disease states. The effect of peroxynitrite on muscle force generation has not been directly assessed. The purpose of the present study was to assess the effects of peroxynitrite administration on diaphragmatic force-generating capacity in 1) intact diaphragm muscle fiber bundles (to model the effects produced by exposure of muscles to extracellular peroxynitrite) and 2) single skinned diaphragm muscle fibers (to model the effects of intracellular peroxynitrite on contractile protein function) by examining the effects of both peroxynitrite and a peroxynitrite-generating solution, 3-morpholinosydnonimine, on force vs. pCa characteristics. In intact diaphragm preparations, peroxynitrite reduced diaphragm force generation and increased muscle levels of 4-hydroxynonenal (an index of lipid peroxidation). In skinned fibers, both peroxynitrite and 3-morpholinosydnonimine reduced maximum calcium-activated force. These data indicate that peroxynitrite is capable of producing significant diaphragmatic contractile dysfunction. We speculate that peroxynitrite-mediated alterations may be responsible for much of the muscle dysfunction seen in pathophysiological conditions such as sepsis.     

Role of nitric oxide on diaphragmatic contractile failure in Escherichia coli endotoxemic rats

Contractile dysfunction of the respiratory muscles plays an important role in the genesis of respiratory failure during sepsis. Nitric oxide (NO), a free radical that is cytotoxic and negatively inotropic in the heart and skeletal muscle, is produced in large amounts during sepsis by a NO synthase inducible (iNOS) by LPS and/or cytokines. The aim of this study was to investigate whether iNOS was induced in the diaphragm of Escherichia coli endotoxemic rats and whether inhibition of iNOS induction or of NOS synthesis attenuated diaphragmatic contractile dysfunction. Rats were inoculated intravenously (IV) with 10 mg/kg of E. coli endotoxin (LPS animals) or saline (C animals). Six hours after LPS inoculation animals showed a significant increase in diaphragmatic NOS activity (L-citrulline production, P < 0.005). Inducible NOS protein was detected by Western-Blot in the diaphragms of LPS animals, while it was absent in C animals. LPS animals had a significant decrease in diaphragmatic force (P < 0.0001) measured in vitro. In LPS animals, inhibition of iNOS induction with dexamethasone (4 mg/kg IV 45 min before LPS) or inhibition of NOS activity with N(G)-methyl-L-arginine (8 mg/kg IV 90 min after LPS) prevented LPS-induced diaphragmatic contractile dysfunction. We conclude that increased NOS activity due to iNOS was involved in the genesis of diaphragmatic dysfunction observed in E. coli endotoxemic rats.     

Invited review: Mechanisms underlying motor unit plasticity in the respiratory system.

Neuromotor control of skeletal muscles, including respiratory muscles, is ultimately dependent on the function of the motor unit (comprising an individual motoneuron and the muscle fibers it innervates). Considerable diversity exists across diaphragm motor units, yet remarkable homogeneity is present (and maintained) within motor units. In recent years, the mechanisms underlying the development and adaptability of respiratory motor units have received great attention, leading to significant advances in our understanding of diaphragm motor unit plasticity. For example, following imposed inactivity of the diaphragm muscle, there are changes at phrenic motoneurons, neuromuscular junctions, and muscle fibers that tend to restore the ability of the diaphragm to sustain ventilation. The role of activity, neurotrophins, and other growth factors in modulating this adaptability is discussed.     

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.     

Adaptations of the diaphragm in emphysema.

In adult male hamsters the influence of emphysema (EMP) on the in vitro contractile and fatigue properties and the histochemical, morphometric, and metabolic properties of muscle fibers in the costal diaphragm was determined 6 mo after the administration of either elastase or saline (controls, CTL). Isometric contractile properties were determined in vitro using supramaximal direct muscle stimulation. Optimal fiber length for force generation was significantly shorter in the EMP than in the CTL diaphragm. Maximum specific force (i.e., force per unit area) was 25% lower than CTL. Fatigue resistance was significantly improved in the EMP diaphragm compared with CTL. Diaphragm muscle fibers were classified as type I or II on the basis of histochemical staining for myofibrillar adenosinetriphosphatase after alkaline preincubation. The proportions of type I and II fibers were similar between the two groups. Cross-sectional areas of type II fibers were 30% larger in EMP than in CTL diaphragms. Succinate dehydrogenase activities of both type I and II fibers were higher in EMP than in CTL diaphragms. The number of capillaries surrounding both type I and II fibers increased with EMP, but in proportion to the hypertrophy of these fibers. Thus, capillary density (number of capillaries per fiber cross-sectional area) remained unchanged. We postulate that these contractile, morphometric, and metabolic adaptations reflect an increased activation of the diaphragm in response to the loads imposed by EMP.     

Titin-based mechanosensing and signaling: role in diaphragm atrophy during unloading?

The diaphragm, the main muscle of inspiration, is constantly subjected to mechanical loading. One of the very few occasions during which diaphragm loading is arrested is during controlled mechanical ventilation in the intensive care unit. Recent animal studies indicate that the diaphragm is extremely sensitive to unloading, causing rapid muscle fiber atrophy: unloading-induced diaphragm atrophy and the concomitant diaphragm weakness has been suggested to contribute to the difficulties in weaning patients from ventilatory support. Little is known about the molecular triggers that initiate the rapid unloading atrophy of the diaphragm, although proteolytic pathways and oxidative signaling have been shown to be involved. Mechanical stress is known to play an important role in the maintenance of muscle mass. Within the muscle's sarcomere titin is considered to play an important role in the stress-response machinery. Titin is the largest protein known to date and acts as a mechanosensor that regulates muscle protein expression in a sarcomere strain-dependent fashion. Thus, titin is an attractive candidate for sensing the sudden mechanical arrest of the diaphragm when patients are mechanically ventilated, leading to changes in muscle protein expression. Here, we provide a novel perspective on how titin, and its biomechanical sensing and signaling, might be involved in the development of mechanical unloading-induced diaphragm weakness.     

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.     

Diaphragm muscle fiber function and structure in humans with hemidiaphragm paralysis

Recent studies proposed that mechanical inactivity of the human diaphragm during mechanical ventilation rapidly causes diaphragm atrophy and weakness. However, conclusive evidence for the notion that diaphragm weakness is a direct consequence of mechanical inactivity is lacking. To study the effect of hemidiaphragm paralysis on diaphragm muscle fiber function and structure in humans, biopsies were obtained from the paralyzed hemidiaphragm in eight patients with hemidiaphragm paralysis. All patients had unilateral paralysis of known duration, caused by en bloc resection of the phrenic nerve with a tumor. Furthermore, diaphragm biopsies were obtained from three control subjects. The contractile performance of demembranated muscle fibers was determined, as well as fiber ultrastructure and morphology. Finally, expression of E3 ligases and proteasome activity was determined to evaluate activation of the ubiquitin-proteasome pathway. The force-generating capacity, as well as myofibrillar ultrastructure, of diaphragm muscle fibers was preserved up to 8 wk of paralysis. The cross-sectional area of slow fibers was reduced after 2 wk of paralysis; that of fast fibers was preserved up to 8 wk. The expression of the E3 ligases MAFbx and MuRF-1 and proteasome activity was not significantly upregulated in diaphragm fibers following paralysis, not even after 72 and 88 wk of paralysis, at which time marked atrophy of slow and fast diaphragm fibers had occurred. Diaphragm muscle fiber atrophy and weakness following hemidiaphragm paralysis develops slowly and takes months to occur.     

Endurance exercise attenuates ventilator-induced diaphragm dysfunction

Controlled mechanical ventilation (MV) is a life-saving measure for patients in respiratory failure. However, MV renders the diaphragm inactive leading to diaphragm weakness due to both atrophy and contractile dysfunction. It is now established that oxidative stress is a requirement for MV-induced diaphragmatic proteolysis, atrophy, and contractile dysfunction to occur. Given that endurance exercise can elevate diaphragmatic antioxidant capacity and the levels of the cellular stress protein heat shock protein 72 (HSP72), we hypothesized that endurance exercise training before MV would protect the diaphragm against MV-induced oxidative stress, atrophy, and contractile dysfunction in female Sprague-Dawley rats. Our results confirm that endurance exercise training before MV increased both HSP72 and the antioxidant capacity in the diaphragm. Importantly, compared with sedentary animals, exercise training before MV protected the diaphragm against MV-induced oxidative damage, protease activation, myofiber atrophy, and contractile dysfunction. Further, exercise protected diaphragm mitochondria against MV-induced oxidative damage and uncoupling of oxidative phosphorylation. These results provide the first evidence that exercise can provide protection against MV-induced diaphragm weakness. These findings are important and establish the need for future experiments to determine the mechanism(s) responsible for exercise-induced diaphragm protection.     

The JAK–STAT Pathway Is Critical in Ventilator-Induced Diaphragm Dysfunction

Mechanical ventilation (MV) is one of the lynchpins of modern intensive-care medicine and is life saving in many critically ill patients. Continuous ventilator support, however, results in ventilation-induced diaphragm dysfunction (VIDD) that likely prolongs patients’ need for MV and thereby leads to major associated complications and avoidable intensive care unit (ICU) deaths. Oxidative stress is a key pathogenic event in the development of VIDD, but its regulation remains largely undefined. We report here that the JAK–STAT pathway is activated in MV in the human diaphragm, as evidenced by significantly increased phosphorylation of JAK and STAT. Blockage of the JAK–STAT pathway by a JAK inhibitor in a rat MV model prevents diaphragm muscle contractile dysfunction (by ~85%, p < 0.01). We further demonstrate that activated STAT3 compromises mitochondrial function and induces oxidative stress in vivo, and, interestingly, that oxidative stress also activates JAK–STAT. Inhibition of JAK–STAT prevents oxidative stress-induced protein oxidation and polyubiquitination and recovers mitochondrial function in cultured muscle cells. Therefore, in ventilated diaphragm muscle, activation of JAK–STAT is critical in regulating oxidative stress and is thereby central to the downstream pathogenesis of clinical VIDD. These findings establish the molecular basis for the therapeutic promise of JAK–STAT inhibitors in ventilated ICU patients.     

Isolation and purification of a lethal scorpion toxin with neuromuscular blocking activity

Toxin-L a lethal neuromuscular blocking agent was isolated from the venom of the scorpion, Lychas laevifrons (Pocock), by the CM-cellulose ion-exchange chromatography. It was a homogenous, thermolabile and low molecular weight protein. The toxin produced irreversible blockade of indirect stimulation induced twitch responses on innervated rat phrenic nerve-diaphragm and chick biventer cervicis preparation. The toxin did not produce any contractile response on toad rectus abdominis muscle preparation. On chronically denervated rat diaphragm, the toxin failed to alter the responses induced by direct stimulation, exogenous acetylcholine, potassium chloride and caffeine. Acetylcholine and carbachol induced contractions on isolated chick biventer cervicis remained unaltered by the toxin. Neostigmine failed to alter toxin induced neuromuscular blockade on innervated rat diaphragm. The toxin released a significant amount of acetylcholine from innervated rat diaphragm. It may be concluded that the toxin acts presynaptically through the release of acetylcholine, thereby producing neuromuscular blockade.     

Apocynin improves diaphragmatic function after endotoxin administration.

Free radicals are known to play an important role in modulating the development of respiratory muscle dysfunction during sepsis. Moreover, neutrophil numbers increase in the diaphragm after endotoxin administration. Whether or not superoxide derived from infiltrating white blood cells contributes to muscle dysfunction during sepsis is, however, unknown. The purpose of the present study was to examine the effect of apocynin, an inhibitor of the superoxide-generating neutrophil NADPH complex, on endotoxin-induced diaphragmatic dysfunction. We studied groups of rats given saline, endotoxin, apocynin, or both endotoxin and apocynin. Animals were killed 18 h after injection, a portion of the diaphragm was used to assess force generation, and the remaining diaphragm was used for determination of 4-hydroxynonenal (a marker of lipid peroxidation) and nitrotyrosine levels (a marker of free radical-mediated protein modification). We found that endotoxin reduced diaphragm force generation and that apocynin partially prevented this decrease [e.g., force in response to 20 Hz was 23 +/- 1 (SE), 12 +/- 2, 23 +/- 1, and 19 +/- 1 N/cm(2), respectively, for saline, endotoxin, apocynin, and endotoxin/apocynin groups; P < 0.001]. Apocynin also prevented endotoxin-mediated increases in diaphragm 4-hydroxynonenal and nitrotyrosine levels (P < 0.01). These data suggest that neutrophil-derived free radicals contribute to diaphragmatic dysfunction during sepsis.     

Diaphragm atrophy and weakness in cortisone-treated rats

Despite frequent therapeutic use, the potential of corticosteroids to produce respiratory muscle myopathy is unknown. We studied effects of chronic steroid treatment on diaphragm mass and function. Eleven Sprague-Dawley rats were treated with cortisone acetate (100 mg.kg-1.day-1 im) for 10 days. Controls (injected with vehicle) included 11 freely eating rats and 11 animals pair fed to match food intake of cortisone rats. Steroid treatment depressed body weight 30% compared with controls. Mass of diaphragm, gastrocnemius, and extensor digitorum longus showed significant atrophy (30%); heart and soleus were unaffected. Isometric contractile properties of costal diaphragm strips were studied in vitro using direct stimulation. The force-frequency relationship was markedly depressed by steroid treatment, both at low and high frequencies. However, force developed per unit cross-sectional area was similar among all three groups, as were twitch characteristics. When stimulated every minute, forces developed by control strips fell progressively, whereas the forces of cortisone-treated strips remained unchanged. When stimulated every 5 s, the fall in force was not different between groups. We conclude that cortisone weakened the diaphragm by decreasing muscle mass but made the diaphragm more resistant to one form of fatigue in vitro.     

The resilience of the size principle in the organization of motor unit properties in normal and reinnervated adult skeletal muscles

Henneman's size principle relates the input and output properties of motoneurons and their muscle fibers to size and is the basis for size-ordered activation or recruitment of motor units during movement. After nerve injury and surgical repair, the relationship between motoneuron size and the number and size of the muscle fibers that the motoneuron reinnervates is initially lost but returns with time, irrespective of whether the muscles are self- or cross-reinnervated by the regenerated axons. Although the return of the size relationships was initially attributed to the recovery of the cross-sectional area of the reinnervated muscle fibers and their force per fiber, direct enumeration of the innervation ratio and the number of muscle fibers per motoneuron demonstrated that a size-dependent branching of axons accounts for the size relationships in normal muscle, as suggested by Henneman and his colleagues. This same size-dependent branching accounts for the rematching of motoneuron size and muscle unit size in reinnervated muscles. Experiments were carried out to determine whether the daily amount of neuromuscular activation of motor units accounts for the size-dependent organization and reorganization of motor unit properties. The normal size-dependent matching of motoneurons and their muscle units with respect to the numbers of muscle fibers per motoneuron was unaltered by synchronous activation of all of the motor units with the same daily activity. Hence, the restored size relationships and rematching of motoneuron and muscle unit properties after nerve injuries and muscle reinnervation sustain the normal gradation of muscle force during movement by size-ordered recruitment of motor units and the process of rate coding of action potentials. Dynamic modulation of size of muscle fibers and their contractile speed and endurance by neuromuscular activity allows for neuromuscular adaptation in the context of the sustained organization of the neuromuscular system according to the size principle.     

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.     

Increased expression of the nicotinic acetylcholine receptor in stimulated muscle

Chronic low-frequency stimulation has been used as a model for investigating responses of skeletal muscle fibres to enhanced neuromuscular activity under conditions of maximum activation. Fast-to-slow isoform shifting of markers of the sarcoplasmic reticulum and the contractile apparatus demonstrated successful fibre transitions prior to studying the effect of chronic electro-stimulation on the expression of the nicotinic acetylcholine receptor. Comparative immunoblotting revealed that the alpha- and delta-subunits of the receptor were increased in 10-78 day stimulated specimens, while an associated component of the surface utrophin-glycoprotein complex, beta-dystroglycan, was not drastically changed in stimulated fast skeletal muscle. Previous studies have shown that electro-stimulation induces degeneration of fast glycolytic fibres, trans-differentiation leading to fast-to-slow fibre transitions and activation of muscle precursor cells. In analogy, our results indicate a molecular modification of the central functional unit of the post-synaptic muscle surface within existing neuromuscular junctions and/or during remodelling of nerve-muscle contacts.     

Hypoxia-induced skeletal muscle fiber dysfunction: role for reactive nitrogen species

Hypoxia impairs skeletal muscle function, but the precise mechanisms are incompletely understood. In hypoxic rat diaphragm muscle, generation of peroxynitrite is elevated. Peroxynitrite and other reactive nitrogen species have been shown to impair contractility of skinned muscle fibers, reflecting contractile protein dysfunction. We hypothesized that hypoxia induces contractile protein dysfunction and that reactive nitrogen species are involved. In addition, we hypothesized that muscle reoxygenation reverses contractile protein dysfunction. In vitro contractility of rat soleus muscle bundles was studied after 30 min of hyperoxia (Po2 approximately 90 kPa), hypoxia (Po2 approximately 5 kPa), hypoxia + 30 microM N(G)-monomethyl-L-arginine (L-NMMA, a nitric oxide synthase inhibitor), hyperoxia + 30 microM L-NMMA, and hypoxia (30 min) + reoxygenation (15 min). One part of the muscle bundle was used for single fiber contractile measurements and the other part for nitrotyrosine detection. In skinned single fibers, maximal Ca2+-activated specific force (Fmax), fraction of strongly attached cross bridges (alphafs), rate constant of force redevelopment (ktr), and myofibrillar Ca2+ sensitivity were determined. Thirty minutes of hypoxia reduced muscle bundle contractility. In the hypoxic group, single fiber Fmax, alphafs, and ktr were significantly reduced compared with hyperoxic, L-NMMA, and reoxygenation groups. Myofibrillar Ca2+ sensitivity was not different between groups. Nitrotyrosine levels were increased in hypoxia compared with all other groups. We concluded that acute hypoxia induces dysfunction of skinned muscle fibers, reflecting contractile protein dysfunction. In addition, our data indicate that reactive nitrogen species play a role in hypoxia-induced contractile protein dysfunction. Reoxygenation of the muscle bundle partially restores bundle contractility but completely reverses contractile protein dysfunction.