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Sepsis-induced diaphragm dysfunction is a major contributor to respiratory failure in mechanically ventilated patients. There are no pharmacological treatments for this syndrome, but studies suggest that diaphragm weakness is linked to mitochondrial free radical generation. We hypothesized that administration of mitoquinone mesylate (MitoQ), a mitochondrially targeted free radical scavenger, would prevent sepsis-induced diaphragm dysfunction. We compared diaphragm function in 4 groups of male mice: 1) sham-operated controls treated with saline (0.3 mL ip), 2) sham-operated treated with MitoQ (3.5 mg/kg/day given intraperitoneally in saline), 3) cecal ligation puncture (CLP) mice treated with saline, and 4) CLP mice treated with MitoQ. Forty-eight hours after surgery, we assessed diaphragm force generation, myosin heavy chain content, state 3 mitochondrial oxygen consumption (OCR), and aconitase activity. We also determined effects of MitoQ in female mice with CLP sepsis and in mice with endotoxin-induced sepsis. CLP decreased diaphragm specific force generation and MitoQ prevented these decrements (e.g. maximal force averaged 30.2 ± 1.3, 28.0 ± 1.3, 12.8 ± 1.9, and 30.0 ± 1.0 N/cm(2) for sham, sham + MitoQ, CLP, and CLP + MitoQ groups, respectively, P < 0.001). CLP also reduced diaphragm mitochondrial OCR and aconitase activity; MitoQ blocked both effects. Similar responses were observed in female mice and in endotoxin-induced sepsis. Moreover, delayed MitoQ treatment (by 6 h) was as effective as immediate treatment. These data indicate that MitoQ prevents sepsis-induced diaphragm dysfunction, preserving force generation. MitoQ may be a useful therapeutic agent to preserve diaphragm function in critically ill patients with sepsis. NEW & NOTEWORTHY This is the first study to show that mitoquinone mesylate (MitoQ), a mitochondrially targeted antioxidant, treats sepsis-induced skeletal muscle dysfunction. This biopharmaceutical agent is without known side effects and is currently being used by healthy individuals and in clinical trials in patients with various diseases. When taken together, our results suggest that MitoQ has the potential to be immediately translated into treatment for sepsis-induced skeletal muscle dysfunction.

Sepsis-induced diaphragm dysfunction contributes to respiratory failure and mortality in critical illness. There are no treatments for this form of diaphragm weakness. Studies show that sepsis-induced muscle dysfunction is triggered by enhanced mitochondrial free radical generation. We tested the hypothesis that SS31, a mitochondrially targeted antioxidant, would attenuate sepsis-induced diaphragm dysfunction. Four groups of mice were studied: 1) sham-operated controls, 2) sham-operated+SS31 (10 mg·kg−1·day−1), 3) cecal ligation puncture (CLP), and 4) CLP+SS31. Forty-eight hours postoperatively, diaphragm strips with attached phrenic nerves were isolated, and the following were assessed: muscle-field-stimulated force-frequency curves, nerve-stimulated force-frequency curves, and muscle fatigue. We also measured calpain activity, 20S proteasomal activity, myosin heavy chain (MHC) levels, mitochondrial function, and aconitase activity, an index of mitochondrial superoxide generation. Sepsis markedly reduced diaphragm force generation; SS31 prevented these decrements. Diaphragm-specific force generation averaged 30.2 ± 1.4, 9.4 ± 1.8, 25.5 ± 2.3, and 27.9 ± 0.6 N/cm2 for sham, CLP, sham+SS31, and CLP+SS31 groups ( P < 0.001). Similarly, with phrenic nerve stimulation, CLP depressed diaphragm force generation, effects prevented by SS31. During endurance trials, force was significantly reduced with CLP, and SS31 prevented these reductions ( P < 0.001). Sepsis also increased diaphragm calpain activity, increased 20S proteasomal activity, decreased MHC levels, reduced mitochondrial function (state 3 rates and ATP generation), and reduced aconitase activity; SS31 prevented each of these sepsis-induced alterations ( P ≤ 0.017 for all indices). SS31 prevents sepsis-induced diaphragm dysfunction, preserving force generation, endurance, and mitochondrial function. Compounds with similar mechanisms of action may be useful therapeutically to preserve diaphragm function in patients who are septic and critically ill. NEW & NOTEWORTHY Sepsis-induced diaphragm dysfunction is a major contributor to mortality and morbidity in patients with critical illness in intensive care units. Currently, there is no proven pharmacological treatment for this problem. This study provides the novel finding that administration of SS31, a mitochondrially targeted antioxidant, preserves diaphragm myosin heavy chain content and mitochondrial function, thereby preventing diaphragm weakness and fatigue in sepsis.

Recent work indicates that infections are a major contributor to diaphragm weakness in patients who are critically ill and mechanically ventilated, and that diaphragm weakness is a risk factor for death and prolonged mechanical ventilation. Infections activate muscle calpain, but many believe this is an epiphenomenon and that other proteolytic processes are responsible for infection-induced muscle weakness. We tested the hypothesis that muscle-specific overexpression of calpastatin (CalpOX; an endogenous calpain inhibitor) would attenuate diaphragm dysfunction in cecal ligation puncture (CLP)-induced sepsis. We studied 1) wild-type (WT) sham-operated mice, 2) WT CLP-operated mice, 3) CalpOX sham-operated mice, and 4) CalpOX CLP-operated mice ( n = 9–10/group). Twenty-four hours after surgery, we assessed the diaphragm force-frequency relationship, diaphragm mass, and total protein content and diaphragm levels of talin and myosin heavy chain (MHC). CLP markedly reduced diaphragm-specific force generation (force/cross-sectional area), which was prevented by calpastatin overexpression (force averaged 21.4 ± 0.5, 6.9 ± 0.8, 22.4 ± 1.0, and 18.3 ± 1.3 N/cm2, respectively, for WT sham, WT CLP, CalpOX sham, and CalpOX CLP groups, P < 0.001). Diaphragm mass and total protein content were similar in all groups. CLP induced talin cleavage and reduced MHC levels; CalpOX prevented these alterations. CLP-induced sepsis rapidly reduces diaphragm-specific force generation and is associated with cleavage and/or depletion of key muscle proteins (talin, MHC), effects prevented by muscle-specific calpastatin overexpression. These data indicate that calpain activation is a major cause of diaphragm weakness in response to CLP-induced sepsis.

Diaphragm caspase-8 activation plays a key role in modulating sepsis-induced respiratory muscle dysfunction. It is also known that double-stranded RNA-dependent protein kinase (PKR) is a regulator of caspase-8 activation in neural tissue. We tested the hypothesis that the PKR pathway modulates sepsis-induced diaphragmatic caspase-8 activation. We first evaluated the time course of diaphragm PKR activation following endotoxin administration in mice. We then determined whether administration of a PKR inhibitor (2-aminopurine) prevents endotoxin-induced diaphragm caspase-8 activation and contractile dysfunction in mice. Finally, we investigated if inhibition of PKR (using either 2-aminopurine or transfection with dominant-negative PKR) blocks caspase-8 activation in cytokine treated C2C12 cells. Endotoxin markedly activated diaphragm PKR (with increases in both active phospho-PKR protein levels, P < 0.03, and directly measured PKR activity, P < 0.01) and increased active caspase-8 levels ( P < 0.01). Inhibition of PKR with 2-aminopurine prevented endotoxin-induced diaphragm caspase-8 activation ( P < 0.01) and diaphragm weakness ( P < 0.001). Inhibition of PKR with either 2-aminopurine or transfection with dominant-negative PKR blocked caspase-8 activation in isolated cytokine-treated C2C12 cells. These data implicate PKR activation as a major factor mediating cytokine-induced skeletal muscle caspase-8 activation and weakness.

Loss of functional capacity of skeletal muscle is a major cause of morbidity in patients with a number of acute and chronic clinical disorders, including sepsis, chronic obstructive pulmonary disease, heart failure, uremia, and cancer. Weakness in these patients can manifest as either severe limb muscle weakness (even to the point of virtual paralysis), respiratory muscle weakness requiring mechanical ventilatory support, and/or some combination of these phenomena. While factors such as nutritional deficiency and disuse may contribute to the development of muscle weakness in these conditions, systemic inflammation may be the major factor producing skeletal muscle dysfunction in these disorders. Importantly, studies conducted over the past 15 years indicate that free radical species (superoxide, hydroxyl radicals, nitric oxide, peroxynitrite, and the free radical-derived product hydrogen peroxide) play an key role in modulating inflammation and/or infection-induced alterations in skeletal muscle function. Substantial evidence exists indicating that several free radical species can directly alter contractile protein function, and evidence suggests that free radicals also have important effects on sarcoplasmic reticulum function, on mitochondrial function, and on sarcolemmal integrity. Free radicals also modulate activation of several proteolytic pathways, including proteosomally mediated protein degradation and, at least theoretically, may also influence pathways of protein synthesis. As a result, free radicals appear to play an important role in regulating a number of downstream processes that collectively act to impair muscle function and lead to reductions in muscle strength and mass in inflammatory conditions.

The mechanisms by which infections induce diaphragm dysfunction remain poorly understood. The purpose of this study was to determine which caspase pathways (i.e., the extrinsic, death receptor-linked caspase-8 pathway, and/or the intrinsic, mitochondrial-related caspase-9 pathway) are responsible for endotoxin-induced diaphragm contractile dysfunction. We determined 1) whether endotoxin administration (12 mg/kg IP) to mice induces caspase-8 or -9 activation in the diaphragm; 2) whether administration of a caspase-8 inhibitor ( N-acetyl-Ile-Glu-Thr-Asp-CHO, 3 mg/kg iv) or a caspase-9 inhibitor ( N-acetyl-Leu-Glu-His-Asp-CHO, 3 mg/kg iv) blocks endotoxin-induced diaphragmatic weakness and caspase-3 activation; 3) whether TNF receptor 1-deficient mice have reduced caspase activation and diaphragm dysfunction following endotoxin; and 4) whether cytokines (TNF-α or cytomix, a mixture of TNF-α, interleukin-1β, interferon-γ, and endotoxin) evoke caspase activation in C2C12 myotubes. Endotoxin markedly reduced diaphragm force generation ( P < 0.001) and induced increases in caspase-3 and caspase-8 activity ( P < 0.03), but failed to increase caspase-9. Inhibitors of caspase-8, but not of caspase-9, prevented endotoxin-induced reductions in diaphragm force and caspase-3 activation ( P < 0.01). Mice deficient in TNF receptor 1 also had reduced caspase-8 activation ( P < 0.001) and less contractile dysfunction ( P < 0.01) after endotoxin. Furthermore, incubation of C2C12 cells with either TNF-α or cytomix elicited significant caspase-8 activation. The caspase-8 pathway is strongly activated in the diaphragm following endotoxin and is responsible for caspase-3 activation and diaphragm weakness.

Infections produce significant respiratory muscle weakness, but the mechanisms by which inflammation reduces muscle force remain incompletely understood. Recent work suggests that caspase 3 releases actin and myosin from the contractile protein lattice, so we postulated that infections may reduce skeletal muscle force by activating caspase 3. The present experiments were designed to test this hypothesis by determining 1) diaphragm caspase 3 activation in the diaphragm after endotoxin and 2) the effect of a broad-spectrum caspase inhibitor, Z-Val-Ala-Asp(OCH3)-fluoromethylketone (zVAD-fmk), and a selective caspase 3 inhibitor, N-acetyl-Asp-Glu-Val-Asp-al (DEVD-CHO), on endotoxin-induced diaphragm weakness. Caspase 3 activation was assessed by measuring caspase protein levels and by measuring cleavage of a fluorogenic substrate. Diaphragm force was measured in response to electrical stimulation (1–150 Hz). Caspase-mediated spectrin degradation was assessed by Western blotting. Parameters were compared in mice given saline, endotoxin (12 mg/kg ip), endotoxin plus zVAD-fmk (3 mg/kg iv), zVAD-fmk alone, or endotoxin plus DEVD-CHO (3 mg/kg iv). Endotoxin increased diaphragm active caspase 3 protein ( P < 0.003), increased caspase 3 activity ( P < 0.002), increased diaphragm spectrin degradation ( P < 0.001), and reduced diaphragm force ( P < 0.001). Administration of zVAD-fmk or DEVD-CHO prevented endotoxin-induced weakness (e.g., force in response to 150-Hz stimulation was 23.8 ± 1.4, 12.1 ± 1.3, 23.5 ± 0.8, 22.7 ± 1.3, and 24.4 ± 0.8 N/cm2, respectively, for control, endotoxin, endotoxin plus zVAD-fmk, endotoxin plus DEVD-CHO, and zVAD-fmk alone treated groups, P < 0.001). Caspase inhibitors also prevented spectrin degradation. In conclusion, endotoxin administration elicits significant diaphragm caspase 3 activation and caspase-mediated diaphragmatic weakness.

Heart failure evokes diaphragm weakness, but the mechanism(s) by which this occurs are not known. We postulated that heart failure increases diaphragm free radical generation and that free radicals trigger diaphragm dysfunction in this condition. The purpose of the present study was to test this hypothesis. Experiments were performed using halothane-anesthetized sham-operated control rats and rats in which myocardial infarction was induced by ligation of the left anterior descending coronary artery. Animals were killed 6 wk after surgery, the diaphragms were removed, and the following were assessed: 1) mitochondrial hydrogen peroxide (H2O2) generation, 2) free radical generation in resting and contracting intact diaphragm using a fluorescent-indicator technique, 3) 8-isoprostane and protein carbonyls (indexes of free radical-induced lipid and protein oxidation), and 4) the diaphragm force-frequency relationship. In additional experiments, a group of coronary ligation animals were treated with polyethylene glycol-superoxide dismutase (PEG-SOD, 2,000 units·kg−1·day−1) for 4 wk. We found that coronary ligation evoked an increase in free radical formation by the intact diaphragm, increased diaphragm mitochondrial H2O2 generation, increased diaphragm protein carbonyl levels, and increased diaphragm 8-isoprostane levels compared with controls ( P < 0.001 for the first 3 comparisons, P < 0.05 for 8-isoprostane levels). Force generated in response to 20-Hz stimulation was reduced by coronary ligation ( P < 0.05); PEG-SOD administration restored force to control levels ( P < 0.03). These findings indicate that cardiac dysfunction due to coronary ligation increases diaphragm free radical generation and that free radicals evoke reductions in diaphragm force generation.

Lower thoracic spinal cord stimulation (SCS) may be a useful method to restore an effective cough mechanism. In dogs, two groups of studies were performed to evaluate the mechanism of the expiratory muscle activation during stimulation at the T9-T10 level, which results in the greatest changes in airway pressure. In one group, expiratory muscle activation was monitored by evoked muscle compound action potentials (CAPs) from the internal intercostal muscles in the 10th, 11th, and 12th interspaces and from portions of the external oblique innervated by the L1 and L2 motor roots. SCS, applied with single shocks, resulted in short-latency CAPs at T10 but not at more caudal levels. SCS resulted in long-latency CAPs at each of the more caudal caudal recording sites. Bilateral dorsal column sectioning, just below the T11 spinal cord level, did not affect the short-latency CAPs but abolished the long-latency CAPs and also resulted in a fall in airway pressure generation. In the second group, sequential spinal root sectioning was performed to assess their individual mechanical contribution to pressure generation. Section of the ventral roots from T8 through T10 resulted in negligible changes, whereas section of more caudal roots resulted in a progressive reduction in pressure generation. We conclude that 1) SCS at the T9-T10 level results in direct activation of spinal cord roots within two to three segments of the stimulating electrode and activation of more distal roots via spinal cord pathways, and 2) pathway activation of motor roots makes a substantial contribution to pressure generation.

Recent studies have indicated that free radicals may play an important role in the development of muscle dysfunction in many pathophysiological conditions. Because the degree of muscle dysfunction observed in some of these conditions appears to be both free radical dependent and modulated by extracellular calcium concentrations, we thought that there may be a link between these two phenomena; i.e., the propensity of a muscle to generate free radicals may be dependent on extracellular calcium concentrations. For this reason, we compared formation of reactive oxygen species (ROS; i.e., free radicals) by electrically stimulated rat diaphragms (trains of 20-Hz stimuli for 10 min, train rate 0.25 trains/s) incubated in organ baths filled with physiological solutions containing low (1 mM), normal (2.5 mM), or high (5 mM) calcium levels. Generation of ROS was assessed by measuring the conversion of hydroethidine to ethidium. We found ROS generation with contraction varied with the extracellular calcium level, with low ROS production (3.18 ± 0.40 ng ethidium/mg tissue) for low-calcium studies and with much higher ROS generation for normal-calcium (18.90 ± 2.70 ng/mg) or high-calcium (19.30 ± 4.50 ng/mg) studies ( P < 0.001). Control, noncontracting diaphragms (in 2.5 mM calcium) had little ROS production (3.40 ± 0.80 ng/mg; P < 0.001). To further investigate this issue, we added nimodipine (20 μM), an L-type calcium channel blocker, to contracting diaphragms (2.5 mM calcium bath) and found that nimodipine also suppressed ROS formation (2.56 ± 0.85 ng ethidium/mg tissue). These data indicate that ROS generation by the contracting diaphragm is strongly influenced by extracellular calcium concentrations and may be dependent on calcium transport through L-type calcium channels.

Lower thoracic spinal cord stimulation (SCS) results in the generation of large positive airway pressures (Paw) and may be a useful method of restoring cough in patients with spinal cord injury. The purpose of the present study was to assess the mechanical contribution of individual respiratory muscles to pressure generation during SCS. In anesthetized dogs, SCS was applied at different spinal cord levels by using a 15-lead multicontact electrode before and after sequential ablation of the external and internal obliques, transversus abdominis (TA), rectus abdominis, and internal intercostal muscles. Paw was monitored after tracheal occlusion. SCS at the T9 spinal cord level resulted in maximal changes in Paw (60 ± 3 cmH2O). Section of the oblique muscles resulted in a fall in Paw to 29 ± 2 cmH2O. After subsequent section of the rectus abdominis and TA, Paw fell to 25 ± 2 and 12 ± 1 cmH2O respectively. There was a small remaining Paw (4 ± 1 cmH2O) after section of the internal intercostal nerves. Stimulation with a two-electrode lead system (T9 + T13) resulted in significantly greater pressure generation compared with a single-electrode lead due to increased contributions from the obliques and transversus muscles. In a separate group of animals, Paw generation was monitored after section of the abdominal muscles and again after section of the external intercostal and levator costae muscles. These studies demonstrated that inspiratory intercostal muscle stimulation resulted in only a small opposing inspiratory action (≤3 cmH2O). We conclude that, during SCS, 1) contraction of the obliques and TA muscles makes the largest contribution to changes in Paw, and 2) stimulation with a two-electrode lead system results in more complete abdominal muscle activation and enhanced mechanical actions of the obliques and transversus muscles.

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/cm2, 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.

Recent work indicates that free radical-mediated lipid peroxidation takes place within the diaphragm on strenuous contraction. This phenomenon has only been demonstrated using fairly artificial experimental models and has not been studied during the type of sustained respiratory loading typically seen in patients with lung disease. The purpose of the present study was to measure the levels of several biochemical markers of protein oxidation (protein carbonyl levels) and lipid peroxidation (8-isoprostane, reduced glutathione, and oxidized glutathione levels) in diaphragms of rats subjected to chronic respiratory loading. Respiratory loading was accomplished by tracheal banding; groups of animals were loaded for 4, 8, or 12 days, and a group of sham-operated unloaded animals was used as controls. After loading, animals were killed, diaphragm contractility was assessed in vitro by using a portion of the excised diaphragm, and the remaining diaphragm and the soleus muscles were used for biochemical analysis. We found diminished force generation in diaphragms from all groups of banded animals compared with muscles from controls. For example, twitch force averaged 7.8 ± 0.8 (SE) N/cm2 in unloaded animals and 4.0 ± 0.4, 3.0 ± 0.4, and 3.4 ± 0.4 N/cm2 in animals loaded for 4, 8, and 12 days, respectively ( P < 0.0001). Loading also elicited increases in diaphragmatic protein carbonyl concentrations ( P < 0.001), and the time course of alterations in carbonyl levels paralleled loading-induced alterations in the diaphragm force-frequency relationship. Although loading was also associated with increases in diaphragmatic 8-isoprostane levels ( P< 0.003) and reductions in diaphragm reduced glutathione levels ( P < 0.003), the time course of changes in these latter parameters did not correspond to alterations in force. Soleus glutathione and carbonyl levels were not altered by banding. We speculate that respiratory loading-induced alterations in diaphragmatic force generation may be related to free radical-mediated protein oxidation, but not to free radical-induced lipid peroxidation.

Several studies have shown that loaded breathing elicits an oxidation of reduced glutathione (GSH) to oxidized glutathione (GSSG) within the diaphragm, but the effects of loaded breathing on GSH and GSSG levels in other respiratory muscles have not been examined. The present experiment examined this issue by using decerebrate unanesthetized rats in which a large inspiratory resistive load was applied until respiratory arrest. Subsequently, muscle samples from the triangularis sterni, diaphragm (Dia), parasternal intercostal (PI), upper rib cage lateral intercostal, lower rib cage lateral intercostal, and soleus were assayed for GSH and GSSG. Glutathione levels were also measured on samples from unloaded control animals. We found that the Dia from loaded animals had a lower GSH level than did control animals (i.e., 653 +/- 99 and 928 +/- 40 nmol/gm for loaded and control groups, respectively; P < 0.05), higher GSSG level (68 +/- 14 and 32 &/- 7 nmol/gm for loaded and control groups, respectively; P < 0.05), and higher GSSG-to-GSH ratios (GSSG/GSH; 17.0 +/- 6.0 and 3.7 +/- 0.9% for loaded and control groups, respectively; (P

Recent work has shown that loaded breathing produces alterations in diaphragmatic glutathione metabolism. Moreover, it has been suggested that alterations in glutathione levels may be related to the development of respiratory muscle fatigue and respiratory failure during loading. The purpose of this study was to determine whether it was possible to augment diaphragmatic stores of reduced glutathione (GSH) and thereby delay the development of respiratory failure during loaded breathing by administering N-acetylcysteine (NAC), a glutathione precursor. We compared the effects of massive inspiratory loading on saline- and NAC-treated groups of decerebrate unanesthetized rats with loading continuing until respiratory arrest occurred. As controls, we also studied unloaded saline- and NAC-treated animals. After arrest, diaphragms were excised, measurement was made of diaphragmatic GSH and oxidized glutathione (GSSG) concentrations, and assessment was made of in vitro diaphragmatic contractility (i.e., the force-frequency relationship and in vitro fatigability). We found that loading of saline-treated animals produced reductions in the diaphragmatic force-frequency curve, reductions in GSH, and increases in GSSG levels. NAC administration blunted loading-induced decreases in diaphragmatic GSH levels and reduced the in vitro fatigability of excised diaphragm muscle strips. NAC did not significantly alter the time to respiratory arrest, however, and also failed to alter the effect of loaded breathing on the diaphragmatic force-frequency relationship. These findings suggest that free radical-mediated GSH depletion is not the limiting factor determining the development of respiratory failure in this model of loaded breathing.

Recent studies have suggested that coronary and limb muscle vessels do not maximally vasodilate under conditions in which cardiac and limb muscle contractile function is dependent on the level of blood flow but, rather, maintain a "vasodilator reserve." If a vasodilator reserve is also present in the fatiguing diaphragm, it may be possible to augment flow to this muscle with vasodilator administration, improving muscle function. The purpose of the present study was therefore to examine the effect of administration of a potent vasodilator, nitroprusside, on the blood flow and contractile function of the fatiguing diaphragm. Studies were performed using an in situ canine diaphragmatic strip preparation that permitted direct measurement of force and blood flow; cardiac output was monitored with a thermodilution catheter. The effects of nitroprusside were examined with the diaphragm rhythmically contracting in response to both subfatiguing and fatiguing stimulation paradigms. For both contraction paradigms, nitroprusside infusions elicited appreciable increases in cardiac output. Nitroprusside infusions also produced significant increases in diaphragmatic blood flow during subfatiguing diaphragmatic contractions but had no effect on flow during fatiguing contractions. Nitroprusside also had no effect on the rate of diaphragmatic fatigue. These data suggest that, under the conditions examined, the diaphragm exhausts its vasodilator reserve during the development of fatigue and vasodilator administration has no appreciable effect on diaphragm blood flow and function. Moreover, although vasodilator drugs with actions similar to nitroprusside are used clinically to augment flow to vital organs, our data would indicate that these drugs have no functionally significant effect on blood flow to the fatiguing diaphragm.

Recent studies have shown that diaphragm fatigue can be reversed by mechanical augmentation of phrenic arterial flow. The purpose of the present experiment was to determine whether it was possible to pharmacologically augment diaphragm blood flow and reverse fatigue by the administration of norepinephrine. Four groups of studies were performed, all employing our previously described in situ isometric canine diaphragm strip preparation (Supinski et al., J. Appl. Physiol. 60: 1789-1796, 1986). Group I studies examined the effects of norepinephrine on the contractility of the nonfatigued diaphragm in normotensive dogs, group II studies examined the effects of this drug on the contractility of the fatigued diaphragm in normotensive animals, and group III studies examined the effect of this drug on the contractility of the fatigued diaphragm in hypotensive animals. Group IV studies examined the effect of norepinephrine in normotensive animals in which the phrenic artery was cannulated and pump perfused at constant flow. Fatigue was induced in group II, III, and IV studies by rhythmically stimulating the diaphragm via intramuscular electrodes. Norepinephrine had no effect on the contractility of the nonfatigued diaphragm (group I). In normotensive (group II) and hypotensive animals (group III), norepinephrine elicited dramatic increases in arterial blood pressure and phrenic arterial flow and produced a significant upshift in the force-frequency curve of the fatigued diaphragm. However, when phrenic flow was held constant (group IV experiments), norepinephrine failed to augment the contractility of the fatigued diaphragm. These results indicate that 1) norepinephrine can increase phrenic blood flow and augment the contractility of the fatigued diaphragm in both normotensive and hypotensive conditions and 2) this effect of norepinephrine to partially reverse fatigue is secondary to its action to augment diaphragmatic blood flow.

The interosseous external intercostal (EI) muscles of the upper rib cage are electrically active during inspiration, but the mechanical consequence of their activation is unclear. In 16 anesthetized dogs, we simultaneously measured EI (3rd and 4th interspaces) and parasternal intercostal (PA) (3rd interspace) electromyogram and length. Muscle length was measured by sonomicrometry and expressed as a percentage of resting length (%LR). During resting breathing, each muscle was electrically active and shortened to a similar extent. Sequential EI muscle denervation (3rd and 4th interspaces) followed by PA denervation (3rd interspace) demonstrated significant reductions in the degree of inspiratory shortening for each muscle. Mean EI muscle shortening of the third and fourth interspaces decreased from -3.4 +/- 0.5 and -3.0 +/- 0.4% LR (SE) under control conditions to -0.2 +/- 0.2 and -0.8 +/- 0.3% LR, respectively, after selective denervation of each of these muscles (P less than 0.001 for each). After selective denervation of the PA muscle, its shortening decreased from -3.5 +/- 0.3 to +0.6% LR (SE) (P less than 0.001). PA muscle denervation also caused the EI muscle in the third interspace to change from inspiratory shortening of -0.2% to inspiratory lengthening of +0.2% +/- 0.2 (P less than 0.05). We conclude that during eupneic breathing 1) the EI muscles of the upper rib cage, like the PA muscles, are inspiratory agonists and actively contribute to rib cage expansion and 2) PA muscle contraction contributes to EI muscle shortening.

It has recently been postulated that diaphragm fatigue may be due, at least in part, to a form of low-grade injury to subcellular organelles. Moreover, several studies have shown that thiol-containing compounds can protect cardiac and striated skeletal muscle organelles from the deleterious effects of a number of physiological stresses. The purpose of the present study was to determine whether pretreatment with N-acetylcysteine (NAC), a thiol-containing compound, would attenuate the rate of development of diaphragmatic fatigue. Studies were performed with the use of an in situ rabbit diaphragm strip preparation that permitted direct and continuous measurement of diaphragm tension development. Diaphragm fatigue was induced by rhythmically stimulating strips to contract at 30/min (20-Hz trains) for 20 min. The diaphragm force-frequency relationship (10-, 20-, 50-, and 100-Hz stimuli) was assessed immediately before and after fatigue trials and then again 20 min into the period of recovery. Half the animals were treated with intravenous NAC before fatigue, whereas the remaining animals were given intravenous saline. The rate of development of fatigue was markedly greater in saline-treated control than in NAC-treated animals, with reductions in tension of 55 +/- 3 and 34 +/- 3%, respectively, in these two groups of animals over 20 min (P less than 0.001). Although rhythmic stimulation resulted in a downward shift in the force-frequency relationship in both NAC- and saline-treated animals, the magnitude of this shift was substantially greater in saline-treated animals (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

The purpose of the present study was to determine whether the diaphragm, like limb muscles, generates high levels of intramuscular pressure during contraction. Studies were performed on in situ diaphragm strips in 15 dogs. An isometric force transducer was used to measure strip tension, and intramuscular pressure was assessed using both wick and balloon catheter techniques. Tension and intramuscular pressures were recorded during a series of graded contractions elicited by electrically stimulating strips via intramuscular electrodes. Similar techniques were used to record the tension and intramuscular pressures developed during graded contractions of a limb muscle, the vastus medialis. Intramuscular pressure was linearly related to tension in both the diaphragm and vastus medialis. However, for a given level of tension development, vastus medialis pressures were two- to threefold greater than those developed within the diaphragm. For example, at a tension of 2 kg/cm2 vastus medialis and diaphragm intramuscular pressures were 105 +/- 11 and 25 +/- 4 mmHg, respectively, when assessed with wick catheters, and were 159 +/- 11 and 48 +/- 5 mmHg, respectively, when assessed by the balloon catheter technique. These differences may reflect the fact that thick rounded muscles, like the vastus, may be capable of generating higher intramuscular pressures than the thin sheetlike diaphragm. To determine whether intradiaphragmatic pressure swings were large enough to explain the vascular compression produced by contraction, we measured phrenic arterial impedance during contraction in nine animals. In these animals the inferior phrenic artery was cannulated and pump perfused at a constant flow rate. With contraction, phrenic arterial pressure rose; i.e., phrenic arterial impedance increased.(ABSTRACT TRUNCATED AT 250 WORDS)

We have previously shown that electrical stimulation of the thoracic spinal cord produces near maximal activation of the inspiratory intercostal muscles. In the present investigation, we used this technique to evaluate the relative capacity of separate external (EI) and parasternal intercostal (PA) muscle contraction to produce changes in airway pressure and inspired volume. Studies were performed in 23 anesthetized phrenicotomized dogs. Electrical stimuli were applied to the spinal cord after hyperventilation-induced apnea, before and after sequentially severing either the PA or EI muscles from the first through sixth intercostal spaces. During spinal cord stimulation (SCS), measurements were made of inspired volume (delta V) with the airway open and negative airway pressure (delta P) during tracheal occlusion. Compared with control values, sectioning of the PA muscles resulted in a 40.9% reduction in delta P and 35.7% reduction in delta V during SCS. In other animals, initial sectioning of the EI muscles produced reductions in delta P and delta V of 67.4 and 63.0, respectively, during SCS. After subsequent section of the PA muscles, SCS produced only negligible inspired volumes and changes in airway pressure. We conclude that 1) the EI and PA muscles are each capable of generating substantial changes in airway pressure and large inspired volumes and 2) the ventilatory capacity of the EI muscles exceeds that of the PA muscles.

The purpose of the present study was to examine the effect of amrinone, a drug known to augment cardiac output and dilate peripheral vascular beds, on diaphragm blood flow. Studies were performed on 12 anesthetized mechanically ventilated dogs in which strips of left costal diaphragm were developed in situ. Strip blood flow was assessed with a drop counter attached to a catheter tied into the phrenic veins' draining strips. Strip tension was measured with an isometric force transducer. Amrinone was administered as an intravenous bolus of 2 mg/kg followed by a continuous infusion of 25 micrograms.kg-1.min-1. Amrinone increased cardiac output and resting diaphragm blood flow [from 1.8 +/- 0.1 to 3.2 +/- 3 (SE) l/min and from 13 +/- 2 to 29 +/- 6 (SE) ml.100 g-1.min-1, respectively, P less than 0.001 for both comparisons]. Amrinone also increased blood flow during periods of rhythmic contraction (tension time indexes of 0.1-0.4, P less than 0.05 for comparisons of flow with and without amrinone at each tension time index) and increased the magnitude of the postcontraction hyperemia (P less than 0.02 for comparisons of hyperemic flow with and without amrinone at tension time indexes of 0.3 and 0.4). Graded occlusion of the inferior vena cava produced reductions in arterial pressure, cardiac output, and diaphragm blood flow both before and after amrinone. Both cardiac output and diaphragm blood flow were greater after amrinone, however, at all levels of blood pressure examined. These findings indicate that amrinone can override diaphragm vasoregulatory systems and augment diaphragm blood flow.(ABSTRACT TRUNCATED AT 250 WORDS)

The distribution of motor drive to the costal and crural diaphragm and parasternal intercostal muscles was evaluated during progressive isocapnic hypoxia in anesthetized dogs. Bipolar stainless steel wire electrodes were placed unilaterally into the costal and crural portions of the diaphragm and into the parasternal intercostal muscle in the second or third intercostal space. Both peak and rate of rise of electromyographic activity of each chest wall muscle increased in curvilinear fashion in response to progressive hypoxia. Both crural and parasternal intercostal responses, however, were greater than those of the costal diaphragm. The onset of crural activation preceded that of the costal portion of the diaphragm and parasternal intercostal muscle activation. Despite differences in the degree of activation among the various chest wall muscles, the rate of increase in activation for any given muscle was linearly related to the rate of increases for the other two. This suggests that respiratory drive during progressive hypoxia increases in fixed proportion to the different chest wall inspiratory muscles. Our findings lend further support to the concept that the costal and crural diaphragm are governed by separate neural control mechanisms and, therefore, may be considered separate muscles.

The mechanical interaction of the inspiratory muscles in the generation of changes in airway pressure is unclear. Using upper thoracic spinal cord stimulation to activate the intercostal muscles (IC) and bilateral supramaximal phrenic nerve stimulation to activate the diaphragm (D), we measured the changes in airway pressure produced by separate and combined IC and D activation over a wide range of lung volumes. Changes in parasternal IC and D length were assessed by sonomicrometry. With increasing lung volume, activation of the IC and D resulted in progressive decrements in generated airway pressure. Combined IC and D contraction produced greater negative swings in airway pressure than the arithmetic sum of separate IC and D contraction alone, indicating a synergistic effect. Moreover, synergism increased progressively with increasing lung volume. During combined muscle contraction, both the IC and D shortened less than during contraction of either muscle group alone. The tendency for the parasternal muscle to lengthen for a given change in airway pressure during D contraction alone increased with increasing lung volume, suggesting that the tendency for the rib cage to recoil inward increased progressively with increasing lung volume. Likewise, the tendency of the D to lengthen for a given change in airway pressure during IC contraction alone also increased progressively with increasing lung volume, suggesting that the tendency for the abdomen-D compartment to recoil inward also increased with increasing lung volume. We conclude that the IC and D interact synergistically to produce changes in airway pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Costal strips of diaphragmatic muscle obtained from animals with elastase-induced emphysema generate maximum tension at significantly shorter muscle fiber lengths than muscle strips from control animals. The present study examined the consequences of alterations in the length-tension relationship assessed in vitro on the pressure generated by the diaphragm in vivo. Transdiaphragmatic pressure (Pdi) and functional residual capacity (FRC) were measured in 22 emphysematous and 22 control hamsters 4–5 mo after intratracheal injection of pancreatic elastase or saline, respectively. In 12 emphysematous and 12 control hamsters Pdi was also measured during spontaneous contractions against an occluded airway. To allow greater control over muscle excitation, Pdi was measured during bilateral tetanic (50 Hz) electrical stimulation of the phrenic nerves in 10 emphysematous and 10 control hamsters. Mean FRC in the emphysematous hamsters was 183% of the value in control hamsters (P less than 0.01). During spontaneous inspiratory efforts against a closed airway the highest Pdi generated at FRC tended to be greater in control than emphysematous hamsters. When control hamsters were inflated to a lung volume approximating the FRC of emphysematous animals, however, peak Pdi was significantly greater in emphysematous animals (70 +/- 6 and 41 +/- 8 cmH2O; P less than 0.05). With electrophrenic stimulation, the Pdi-lung volume curve was shifted toward higher lung volumes in emphysematous hamsters. Pdi at all absolute lung volumes at and above the FRC of emphysematous hamsters was significantly greater in emphysematous compared with control animals. Moreover, Pdi continued to be generated by emphysematous hamsters at levels of lung volume where Pdi of control subjects was zero.(ABSTRACT TRUNCATED AT 250 WORDS)