Leo M. A. Heunks

Xanthine oxidase is involved in exercise-induced oxidative stress in chronic obstructive pulmonary disease

In the present study, we hypothesized that exhaustive exercise in patients with chronic obstructive pulmonary disease (COPD) results in glutathione oxidation and lipid peroxidation and that xanthine oxidase (XO) contributes to free radical generation during exercise. COPD patients performed incremental cycle ergometry until exhaustion with (n = 8) or without (n = 8) prior treatment with allopurinol, an XO inhibitor. Reduced (GSH) and oxidized glutathione (GSSG) and lipid peroxides [malondialdehyde (MDA)] were measured in arterial blood. In nontreated COPD patients, maximal exercise (approximately 75 W) resulted in a significant increase in the GSSG-to-GSH ratio (4. 6 +/- 0.9% at rest vs. 9.3 +/- 1.7% after exercise). In nontreated patients, MDA increased from 0.68 +/- 0.08 nmol/ml at rest up to 1. 32 +/- 0.13 nmol/ml 60 min after cessation of exercise. In contrast, in patients treated with allopurinol, GSSG-to-GSH ratio did not increase in response to exercise (5.0 +/- 1.2% preexercise vs. 4.6 +/- 1.1% after exercise). Plasma lipid peroxide formation was also inhibited by allopurinol pretreatment (0.72 +/- 0.15 nmol/ml preexercise vs. 0.64 +/- 0.09 nmol/ml 60 min after exercise). We conclude that strenuous exercise in COPD patients results in blood glutathione oxidation and lipid peroxidation. This can be inhibited by treatment with allopurinol, indicating that XO is an important source for free radical generation during exercise in COPD.In the present study, we hypothesized that exhaustive exercise in patients with chronic obstructive pulmonary disease (COPD) results in glutathione oxidation and lipid peroxidation and that xanthine oxidase (XO) contributes to free radical generation during exercise. COPD patients performed incremental cycle ergometry until exhaustion with ( n = 8) or without ( n = 8) prior treatment with allopurinol, an XO inhibitor. Reduced (GSH) and oxidized glutathione (GSSG) and lipid peroxides [malondialdehyde (MDA)] were measured in arterial blood. In nontreated COPD patients, maximal exercise (∼75 W) resulted in a significant increase in the GSSG-to-GSH ratio (4.6 ± 0.9% at rest vs. 9.3 ± 1.7% after exercise). In nontreated patients, MDA increased from 0.68 ± 0.08 nmol/ml at rest up to 1.32 ± 0.13 nmol/ml 60 min after cessation of exercise. In contrast, in patients treated with allopurinol, GSSG-to-GSH ratio did not increase in response to exercise (5.0 ± 1.2% preexercise vs. 4.6 ± 1.1% after exercise). Plasma lipid peroxide formation was also inhibited by allopurinol pretreatment (0.72 ± 0.15 nmol/ml preexercise vs. 0.64 ± 0.09 nmol/ml 60 min after exercise). We conclude that strenuous exercise in COPD patients results in blood glutathione oxidation and lipid peroxidation. This can be inhibited by treatment with allopurinol, indicating that XO is an important source for free radical generation during exercise in COPD.

Low-tidal-volume mechanical ventilation induces a toll-like receptor 4-dependent inflammatory response in healthy mice.

Background:Mechanical ventilation (MV) can induce ventilator-induced lung injury. A role for proinflammatory pathways has been proposed. The current studies analyzed the roles of Toll-like receptor (TLR) 4 and TLR2 involvement in the inflammatory response after MV in the healthy lung. Methods:Wild-type (WT) C57BL6, TLR4 knockout (KO), and TLR2 KO mice were mechanically ventilated for 4 h. Bronchoalveolar lavage fluid was analyzed for presence of endogenous ligands. Lung homogenates were used to investigate changes in TLR4 and TLR2 expression. Cytokines were measured in lung homogenate and plasma, and leukocytes were counted in lung tissue. Results:MV significantly increased endogenous ligands for TLR4 in bronchoalveolar lavage fluid and relative messenger RNA expression of TLR4 and TLR2 in lung tissue. In lung homogenates, MV in WT mice increased levels of keratinocyte-derived chemokine, interleukin (IL)-1α, and IL-1β. In TLR4 KO mice, MV increased IL-1α but not IL-1β, and the increase in keratinocyte-derived chemokine was less pronounced. In plasma, MV in WT mice increased levels of IL-6, keratinocyte-derived chemokine, and tumor necrosis factor α. In TLR4 KO mice, MV did not increase levels of IL-6 or tumor necrosis factor α, and the response of keratinocyte-derived chemokine was less pronounced. MV in TLR2 KO mice did not result in different cytokine levels compared with WT mice. In WT and TLR2 KO mice, but not in TLR4 KO mice, MV increased the number of pulmonary leukocytes. Conclusions:The current study supports a role for TLR4 in the inflammatory reaction after short-term MV in healthy lungs. Increasing the understanding of the innate immune response to MV may lead to future treatment advances in ventilator-induced lung injury, in which TLR4 may serve as a therapeutic target.

Monitoring of the Respiratory Muscles in the Critically Ill

Evidence has accumulated that respiratory muscle dysfunction develops in critically ill patients and contributes to prolonged weaning from mechanical ventilation. Accordingly, it seems highly appropriate to monitor the respiratory muscles in these patients. Today, we are only at the beginning of routinely monitoring respiratory muscle function. Indeed, most clinicians do not evaluate respiratory muscle function in critically ill patients at all. In our opinion, however, practical issues and the absence of sound scientific data for clinical benefit should not discourage clinicians from having a closer look at respiratory muscle function in critically ill patients. This perspective discusses the latest developments in the field of respiratory muscle monitoring and possible implications of monitoring respiratory muscle function in critically ill patients.

The Calcium Sensitizer Levosimendan Improves Human Diaphragm Function

RATIONALE Acquired diaphragm muscle weakness is a key feature in several chronic conditions, including chronic obstructive pulmonary disease, congestive heart failure, and difficult weaning from mechanical ventilation. No drugs are available to improve respiratory muscle function in these patients. Recently, we have shown that the calcium sensitizer levosimendan enhances the force-generating capacity of isolated diaphragm fibers. OBJECTIVES To investigate the effects of the calcium sensitizer levosimendan on in vivo human diaphragm function. METHODS In a double-blind, randomized, crossover design, 30 healthy subjects performed two identical inspiratory loading tasks. After the first loading task, subjects received levosimendan (40 μg/kg bolus followed by 0.1/0.2 μg/kg/min continuous infusion) or placebo. Transdiaphragmatic pressure, diaphragm electrical activity, and their relationship (neuromechanical efficiency) were measured during loading. Magnetic phrenic nerve stimulation was performed before the first loading task and after bolus administration to assess twitch contractility. Center frequency of diaphragm electrical activity was evaluated to study the effects of levosimendan on muscle fiber conduction velocity. MEASUREMENTS AND MAIN RESULTS The placebo group showed a 9% (P=0.01) loss of twitch contractility after loaded breathing, whereas no loss in contractility was observed in the levosimendan group. Neuro-mechanical efficiency of the diaphragm during loading improved by 21% (P<0.05) in the levosimendan group. Baseline center frequency of diaphragm electrical activity was reduced after levosimendan administration (P<0.05). CONCLUSIONS The calcium sensitizer levosimendan improves neuromechanical efficiency and contractile function of the human diaphragm. Our findings suggest a new therapeutic approach to improve respiratory muscle function in patients with respiratory failure.

Diaphragm Muscle Fiber Weakness and Ubiquitin–Proteasome Activation in Critically Ill Patients

RATIONALE The clinical significance of diaphragm weakness in critically ill patients is evident: it prolongs ventilator dependency, and increases morbidity and duration of hospital stay. To date, the nature of diaphragm weakness and its underlying pathophysiologic mechanisms are poorly understood. OBJECTIVES We hypothesized that diaphragm muscle fibers of mechanically ventilated critically ill patients display atrophy and contractile weakness, and that the ubiquitin-proteasome pathway is activated in the diaphragm. METHODS We obtained diaphragm muscle biopsies from 22 critically ill patients who received mechanical ventilation before surgery and compared these with biopsies obtained from patients during thoracic surgery for resection of a suspected early lung malignancy (control subjects). In a proof-of-concept study in a muscle-specific ring finger protein-1 (MuRF-1) knockout mouse model, we evaluated the role of the ubiquitin-proteasome pathway in the development of contractile weakness during mechanical ventilation. MEASUREMENTS AND MAIN RESULTS Both slow- and fast-twitch diaphragm muscle fibers of critically ill patients had approximately 25% smaller cross-sectional area, and had contractile force reduced by half or more. Markers of the ubiquitin-proteasome pathway were significantly up-regulated in the diaphragm of critically ill patients. Finally, MuRF-1 knockout mice were protected against the development of diaphragm contractile weakness during mechanical ventilation. CONCLUSIONS These findings show that diaphragm muscle fibers of critically ill patients display atrophy and severe contractile weakness, and in the diaphragm of critically ill patients the ubiquitin-proteasome pathway is activated. This study provides rationale for the development of treatment strategies that target the contractility of diaphragm fibers to facilitate weaning.

Proteasome inhibition improves diaphragm function in congestive heart failure rats.

In congestive heart failure (CHF), diaphragm weakness is known to occur and is associated with myosin loss and activation of the ubiquitin-proteasome pathway. The effect of modulating proteasome activity on myosin loss and diaphragm function is unknown. The present study investigated the effect of in vivo proteasome inhibition on myosin loss and diaphragm function in CHF rats. Coronary artery ligation was used as an animal model for CHF. Sham-operated rats served as controls. Animals were treated with the proteasome inhibitor bortezomib (intravenously) or received saline (0.9%) injections. Force generating capacity, cross-bridge cycling kinetics, and myosin content were measured in diaphragm single fibers. Proteasome activity, caspase-3 activity, and MuRF-1 and MAFbx mRNA levels were determined in diaphragm homogenates. Proteasome activities in the diaphragm were significantly reduced by bortezomib. Bortezomib treatment significantly improved diaphragm single fiber force generating capacity (approximately 30-40%) and cross-bridge cycling kinetics (approximately 20%) in CHF. Myosin content was approximately 30% higher in diaphragm fibers from bortezomib-treated CHF rats than saline. Caspase-3 activity was decreased in diaphragm homogenates from bortezomib-treated rats. CHF increased MuRF-1 and MAFbx mRNA expression in the diaphragm, and bortezomib treatment diminished this rise. The present study demonstrates that treatment with a clinically used proteasome inhibitor improves diaphragm function by restoring myosin content in CHF.

Levosimendan Enhances Force Generation of Diaphragm Muscle from Patients with Chronic Obstructive Pulmonary Disease

RATIONALE Levosimendan is clinically used to improve myocardial contractility by enhancing calcium sensitivity of force generation. The effects of levosimendan on skeletal muscle contractility are unknown. Patients with chronic obstructive pulmonary disease (COPD) suffer from diaphragm weakness, which is associated with decreased calcium sensitivity. OBJECTIVES To investigate the effects of levosimendan on contractility of diaphragm fibers from patients with COPD. METHODS Muscle fibers were isolated from diaphragm biopsies obtained from thoracotomized patients with and without COPD (both groups n = 5, 10 fibers per patient). Diaphragm fibers were skinned and activated with solutions containing incremental calcium concentrations and 10 microM levosimendan or vehicle (0.02% dimethyl sulfoxide). Developed force was measured at each step and force versus calcium concentration relationships were derived. Results were grouped per myosin heavy chain isoform, which was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). MEASUREMENTS AND MAIN RESULTS At sub-maximal activation levosimendan improved force generation of COPD and non-COPD diaphragm fibers by approximately 25%, both in slow and fast fibers. Levosimendan increased calcium sensitivity of force generation (P < 0.01) in both slow and fast diaphragm fibers from patients with and without COPD, without affecting maximal force generation. CONCLUSIONS Levosimendan enhances force generating capacity of diaphragm fibers from patients with and without COPD patients by increasing calcium sensitivity of force generation. These results provide a strong rationale for testing the effect of calcium sensitizers on respiratory muscle dysfunction in patients with COPD.

Toll-like Receptor 4 Signaling in Ventilator-induced Diaphragm Atrophy

Background: Mechanical ventilation induces diaphragm muscle atrophy, which plays a key role in difficult weaning from mechanical ventilation. The signaling pathways involved in ventilator-induced diaphragm atrophy are poorly understood. The current study investigated the role of Toll-like receptor 4 signaling in the development of ventilator-induced diaphragm atrophy. Methods: Unventilated animals were selected for control: wild-type (n = 6) and Toll-like receptor 4 deficient mice (n = 6). Mechanical ventilation (8 h): wild-type (n = 8) and Toll-like receptor 4 deficient (n = 7) mice. Myosin heavy chain content, proinflammatory cytokines, proteolytic activity of the ubiquitin-proteasome pathway, caspase-3 activity, and autophagy were measured in the diaphragm. Results: Mechanical ventilation reduced myosin content by approximately 50% in diaphragms of wild-type mice (P less than 0.05). In contrast, ventilation of Toll-like receptor 4 deficient mice did not significantly affect diaphragm myosin content. Likewise, mechanical ventilation significantly increased interleukin-6 and keratinocyte-derived chemokine in the diaphragm of wild-type mice, but not in ventilated Toll-like receptor 4 deficient mice. Mechanical ventilation increased diaphragmatic muscle atrophy factor box transcription in both wild-type and Toll-like receptor 4 deficient mice. Other components of the ubiquitin-proteasome pathway and caspase-3 activity were not affected by ventilation of either wild-type mice or Toll-like receptor 4 deficient mice. Mechanical ventilation induced autophagy in diaphragms of ventilated wild-type mice, but not Toll-like receptor 4 deficient mice. Conclusion: Toll-like receptor 4 signaling plays an important role in the development of ventilator-induced diaphragm atrophy, most likely through increased expression of cytokines and activation of lysosomal autophagy.

Diaphragm fiber strength is reduced in critically ill patients and restored by a troponin activator

To the Editor: Diaphragm weakness in the intensive care unit (ICU) plays an important role in difficult weaning from mechanical ventilation. Diaphragm strength in mechanically ventilated (MV) critically ill patients has been assessed indirectly using phrenic nerve stimulation, which demonstrated that the pressure-generating capacity of the diaphragm was reduced in these patients (1–3). However, this technique cannot distinguish between impaired phrenic nerve function, abnormal neuromuscular transmission, and intrinsic abnormalities in the diaphragm muscle itself. Consequently, it is unknown whether intrinsic contractile weakness of diaphragm muscle fibers occurs in MV critically ill patients. If so, targeted treatment strategies that enhance contractility may improve the success of weaning. Such treatment strategies may include the administration of a novel class of small-molecule drugs, named fast skeletal troponin activators, which improve the contractile strength of skeletal muscle fibers (4). In this study, we obtained diaphragm biopsy specimens from critically ill patients (n = 10; MV for 28–603 h) undergoing laparotomy or thoracotomy, and compared them with control patients undergoing elective lung surgery (n = 10; MV 1–2 h, see Table E1 in the online supplement). The size and the contractile performance of isolated diaphragm muscle fibers were determined. In addition, we tested the ability of the fast skeletal troponin activator, CK-2066260, to improve contractile strength. Diaphragm fiber cross-sectional area (CSA) was determined by means of immunohistochemical analyses with myosin heavy chain antibodies performed on cryosections of the biopsy specimens (5, 6). Figure 1A demonstrates atrophy of slow- and fast-twitch diaphragm fibers in critically ill patients (CSA slow-twitch fibers: control patients, 3,284 ± 793 μm2 vs. critically ill patients, 2,328 ± 763 μm2, P = 0.004; fast-twitch fibers: control patients, 2,766 ± 606 μm2 vs. critically ill patients, 1,819 ± 527 μm2, P < 0.0001). Figure 1. (A) Severe diaphragm muscle fiber atrophy in mechanically ventilated (MV) critically ill patients. Typical examples of serial diaphragm cross-sections stained with antibodies against slow-twitch myosin heavy chain (green). Wheat germ agglutinin (WGA) ... We measured the contractile performance of permeabilized single diaphragm fibers isolated from the biopsy specimens. Fibers were mounted between a force transducer and a length motor, and exposed to activating calcium solutions. Maximal contractile strength was markedly lower in critically ill patients (absolute force slow-twitch fibers: control patients, 0.44 ± 0.16 mN vs. critically ill patients, 0.19 ± 0·07 mN, P < 0.0001; fast-twitch fibers: control patients, 0.49 ± 0.21 mN vs. critically ill patients, 0.24 ± 0.09 mN, P = 0.0002; Figure 1B). After normalization of force to the CSA of these fibers (i.e., specific force), a deficit remained in diaphragm fibers of critically ill patients (see Figure E1). This suggests that, in these critically ill patients, there is not only a loss of contractile proteins, but also dysfunction of the remaining ones. In addition, we measured the sensitivity of force to calcium. The negative logarithm of the calcium concentration needed to obtain 50% of maximal force (pCa50) was unaffected in slow-twitch fibers (control patients, 5.64 ± 0.03 vs. critically ill patients, 5.61 ± 0.08, P = 0.30), whereas, in fast-twitch fibers, the pCa50 was significantly lower in critically ill patients (control patients, 5.76 ± 0.07 vs. critically ill patients, 5.70 ± 0.06, P = 0.036) (Figure 1B). Thus, fast-twitch diaphragm fibers from critically ill patients not only have reduced maximal force, but also require more calcium to generate force. We exposed diaphragm fibers of a representative subset of control patients (nos. I, IV, VI) and critically ill patients (nos. 1, 3, 4, 5) to the fast skeletal troponin activator, CK-2066260, which improves the sensitivity of the calcium sensor in the muscle sarcomere. Compared with vehicle, 5 μM of CK-2066260 significantly increased the calcium sensitivity of diaphragm fibers both in control patients (pCa50: 5.75 ± 0.04 vs. 6.18 ± 0.1, respectively; P < 0.001) and in critically ill patients (5.70 ± 0.07 vs. 6.00 ± 0.13, respectively; P < 0.01) (Figure 1C). Importantly, at physiological calcium concentrations, CK-2066260 restored the contractile force of fast-twitch diaphragm fibers of critically ill patients back to levels observed in untreated fibers from control patients (force at pCa 5.8: untreated control patients, 0.22 ± 0.05 vs. treated critically ill patients, 0.22 ± 0.07 mN; P = 0.954). See the online supplement for details. The current study is the first to show that atrophy and contractile weakness of diaphragm muscle fibers develop in a clinically relevant group of MV critically ill patients. Interestingly, the reduction in the contractile force of diaphragm fibers of these critically ill patients is comparable to the reduction in diaphragm strength estimated previously by phrenic nerve pacing (1, 2), indicating that the reduction in diaphragm strength in these patients largely results from muscle fiber weakness. To date, no drug is approved to improve respiratory muscle function in MV critically ill patients. We made a step toward such a strategy by testing the ability of the fast skeletal troponin activator, CK-2066260, to restore diaphragm fiber strength. We observed that, upon exposure to CK-2066260, fast-twitch diaphragm fibers from critically ill patients regained strength at calcium concentrations that reflect activation during daily live activities to levels found in untreated fibers from control patients (Figure 1C). Because approximately 50% of fibers and total fiber area in the human diaphragm consists of fast-twitch fibers (Figure E3), fast skeletal troponin activators might significantly improve in vivo diaphragm strength. The potential of fast troponin activators is further strengthened by the notion that these drugs do not affect cardiac function (4), which would be an undesirable side effect in critically ill patients. The analog of CK-2066260, tirasemtiv (formerly CK-2017357), is currently under study in patients with amyotrophic lateral sclerosis (clinical trial no. NCT01709149). What causes weakness of diaphragm muscle fibers in critically ill patients? It seems plausible that the observed diaphragm weakness was acquired during ICU stay, as we used strict exclusion criteria to rule out that our study patients had pre-existing diaphragm weakness. Also, during their stay in the ICU, patients received nutrition according to an optimized nutrition algorithm (7). A commonly suggested concept is that mechanical ventilation per se rapidly induces weakness and atrophy of muscle fibers due to contractile inactivity of the diaphragm (8–12). The critically ill patients we studied received MV for 28–603 hours before biopsy, a time frame that was associated with significant reductions in the CSA of diaphragm fibers in braindead organ donors (8, 13). Thus, the diaphragm muscle fiber atrophy and weakness that we observed may, at least partly, be explained by mechanical ventilation per se. Other ICU-related phenomena that could contribute to diaphragm muscle weakness include underlying disease, such as sepsis (14, 15). Clearly, to elucidate the main factors that contribute to the observed diaphragm muscle fiber weakness requires studies with larger cohorts of various patient groups.

beta2-adrenoceptor agonists reduce the decline of rat diaphragm twitch force during severe hypoxia.

The aim of the present study was to investigate the in vitro effects of the short-acting beta2-adrenoceptor agonist salbutamol and the long-acting beta2-adrenoceptor agonist salmeterol on hypoxia-induced rat diaphragm force reduction. In vitro diaphragm twitch force (Pt) and maximal tetanic force (Po) of isolated diaphragm muscle strips were measured for 90 min during hyperoxia (tissue bath PO2 83.8 +/- 0.9 kPa and PCO2 3.9 +/- 0.1 kPa) or severe hypoxia (PO2 7.1 +/- 0.3 kPa and PCO2 3.9 +/- 0.1 kPa) in the presence and absence of 1 microM salbutamol or 1 microM salmeterol. During hyperoxia, salbutamol and salmeterol did not significantly alter the time-related decreases in Pt and Po (to approximately 50% of initial values). Salbutamol had no effects on Po or the Pt-to-Po ratio. Salmeterol treatment significantly reduced Po and increased the Pt-to-Po ratio during hyperoxia (P < 0.05 compared with control value). Hypoxia resulted in a severe decrease in Pt (to approximately 30% of initial value) and Po after 90 min. Both salbutamol and salmeterol significantly reduced the decline in Pt during hypoxia (P < 0.05). The reduction in Po was not prevented. Salbutamol increased Pt rapidly but transiently. Salmeterol had a delayed onset of effect and a longer duration of action. Addition of 1 microM propranolol (a nonselective beta-adrenoceptor antagonist) did not alter Pt, Po, or the Pt-to-Po ratio during hypoxia but completely blocked the inotropic effects of salbutamol and salmeterol, indicating that these effects are dependent on beta2-adrenoceptor agonist-related processes.The aim of the present study was to investigate the in vitro effects of the short-acting β2-adrenoceptor agonist salbutamol and the long-acting β2-adrenoceptor agonist salmeterol on hypoxia-induced rat diaphragm force reduction. In vitro diaphragm twitch force (Pt) and maximal tetanic force (Po) of isolated diaphragm muscle strips were measured for 90 min during hyperoxia (tissue bath [Formula: see text] 83.8 ± 0.9 kPa and [Formula: see text] 3.9 ± 0.1 kPa) or severe hypoxia ([Formula: see text] 7.1 ± 0.3 kPa and [Formula: see text] 3.9 ± 0.1 kPa) in the presence and absence of 1 μM salbutamol or 1 μM salmeterol. During hyperoxia, salbutamol and salmeterol did not significantly alter the time-related decreases in Pt and Po (to ∼50% of initial values). Salbutamol had no effects on Po or the Pt-to-Poratio. Salmeterol treatment significantly reduced Po and increased the Pt-to-Poratio during hyperoxia ( P < 0.05 compared with control value). Hypoxia resulted in a severe decrease in Pt (to ∼30% of initial value) and Po after 90 min. Both salbutamol and salmeterol significantly reduced the decline in Pt during hypoxia ( P < 0.05). The reduction in Po was not prevented. Salbutamol increased Pt rapidly but transiently. Salmeterol had a delayed onset of effect and a longer duration of action. Addition of 1 μM propranolol (a nonselective β-adrenoceptor antagonist) did not alter Pt, Po, or the Pt-to-Poratio during hypoxia but completely blocked the inotropic effects of salbutamol and salmeterol, indicating that these effects are dependent on β2-adrenoceptor agonist-related processes.