Levosimendan affects oxidative and inflammatory pathways in the diaphragm of ventilated endotoxemic mice.

INTRODUCTION: Controlled mechanical ventilation and endotoxemia are associated with diaphragm muscle atrophy and dysfunction. Oxidative stress and activation of inflammatory pathways are involved in the pathogenesis of diaphragmatic dysfunction. Levosimendan, a cardiac inotrope, has been reported to possess anti-oxidative and anti-inflammatory properties. The aim of the present study was to investigate the effects of levosimendan on markers for diaphragm nitrosative and oxidative stress, inflammation and proteolysis in a mouse model of endotoxemia and mechanical ventilation. METHODS: Three groups were studied: (1) unventilated mice (CON, n =8), (2) mechanically ventilated endotoxemic mice (MV LPS, n =17) and (3) mechanically ventilated endotoxemic mice treated with levosimendan (MV LPS + L, n =17). Immediately after anesthesia (CON) or after 8 hours of mechanical ventilation, blood and diaphragm muscle were harvested for biochemical analysis. RESULTS: Mechanical ventilation and endotoxemia increased expression of inducible nitric oxide synthase (iNOS) mRNA and cytokine levels of interleukin (IL)-1ß, IL-6 and keratinocyte-derived chemokine, and decreased IL-10, in the diaphragm; however, they had no effect on protein nitrosylation and 4-hydroxy-2-nonenal protein concentrations. Levosimendan decreased nitrosylated proteins by 10% (P <0.05) and 4-hydroxy-2-nonenal protein concentrations by 13% (P <0.05), but it augmented the rise of iNOS mRNA by 47% (P <0.05). Levosimendan did not affect the inflammatory response in the diaphragm induced by mechanical ventilation and endotoxemia. CONCLUSIONS: Mechanical ventilation in combination with endotoxemia results in systemic and diaphragmatic inflammation. Levosimendan partly decreased markers of nitrosative and oxidative stress, but did not affect the inflammatory response.

Hypercapnia attenuates ventilator-induced diaphragm atrophy and modulates dysfunction.

INTRODUCTION: Diaphragm weakness induced by prolonged mechanical ventilation may contribute to difficult weaning from the ventilator. Hypercapnia is an accepted side effect of low tidal volume mechanical ventilation, but the effects of hypercapnia on respiratory muscle function are largely unknown. The present study investigated the effect of hypercapnia on ventilator-induced diaphragm inflammation, atrophy and function. METHODS: Male Wistar rats (n = 10 per group) were unventilated (CON), mechanically ventilated for 18 hours without (MV) or with hypercapnia (MV + H, Fico2 = 0.05). Diaphragm muscle was excised for structural, biochemical and functional analyses. RESULTS: Myosin concentration in the diaphragm was decreased in MV versus CON, but not in MV + H versus CON. MV reduced diaphragm force by approximately 22% compared with CON. The force-generating capacity of diaphragm fibers from MV + H rats was approximately 14% lower compared with CON. Inflammatory cytokines were elevated in the diaphragm of MV rats, but not in the MV + H group. Diaphragm proteasome activity did not significantly differ between MV and CON. However, proteasome activity in the diaphragm of MV + H was significantly lower compared with CON. LC3B-II a marker of lysosomal autophagy was increased in both MV and MV + H. Incubation of MV + H diaphragm muscle fibers with the antioxidant dithiothreitol restored force generation of diaphragm fibers. CONCLUSIONS: Hypercapnia partly protects the diaphragm against adverse effects of mechanical ventilation.

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.

Titin and diaphragm dysfunction in mechanically ventilated rats.

PURPOSE: Diaphragm weakness induced by mechanical ventilation may contribute to difficult weaning from the ventilator. For optimal force generation the muscle proteins myosin and titin are indispensable. The present study investigated if myosin and titin loss or dysfunction are involved in mechanical ventilation-induced diaphragm weakness. METHODS: Male Wistar rats were either assigned to a control group (n = 10) or submitted to 18 h of mechanical ventilation (MV, n = 10). At the end of the experiment, diaphragm and soleus muscle were excised for functional and biochemical analysis. RESULTS: Maximal specific active force generation of muscle fibers isolated from the diaphragm of MV rats was lower than controls (128 ± 9 vs. 165 ± 13 mN/mm(2), p = 0.02) and was accompanied by a proportional reduction of myosin heavy chain concentration in these fibers. Passive force generation upon stretch was significantly reduced in diaphragm fibers from MV rats by ca. 35%. Yet, titin content was not significantly different between control and MV diaphragm. In vitro pre-incubation with phosphatase-1 decreased passive force generation upon stretch in diaphragm fibers from control, but not from MV rats. Mechanical ventilation did not affect active or passive force generation in the soleus muscle. CONCLUSIONS: Mechanical ventilation leads to impaired diaphragm fiber active force-generating capacity and passive force generation upon stretch. Loss of myosin contributes to reduced active force generation, whereas reduced passive force generation is likely to result from a decreased phosphorylation status of titin. These impairments were not discernable in the soleus muscle of 18 h mechanically ventilated rats.

Plasma from septic shock patients induces loss of muscle protein.

INTRODUCTION: ICU-acquired muscle weakness commonly occurs in patients with septic shock and is associated with poor outcome. Although atrophy is known to be involved, it is unclear whether ligands in plasma from these patients are responsible for initiating degradation of muscle proteins. The aim of the present study was to investigate if plasma from septic shock patients induces skeletal muscle atrophy and to examine the time course of plasma-induced muscle atrophy during ICU stay. METHODS: Plasma was derived from septic shock patients within 24 hours after hospital admission (n = 21) and healthy controls (n = 12). From nine patients with septic shock plasma was additionally derived at two, five and seven days after ICU admission. These plasma samples were added to skeletal myotubes, cultured from murine myoblasts. After incubation for 24 hours, myotubes were harvested and analyzed on myosin content, mRNA expression of E3-ligase and Nuclear Factor Kappa B (NFκB) activity. Plasma samples were analyzed on cytokine concentrations. RESULTS: Myosin content was approximately 25% lower in myotubes exposed to plasma from septic shock patients than in myotubes exposed to plasma from controls (P < 0.01). Furthermore, patient plasma increased expression of E3-ligases Muscle RING Finger protein-1 (MuRF-1) and Muscle Atrophy F-box protein (MAFbx) (P < 0.01), enhanced NFκB activity (P < 0.05) and elevated levels of ubiquitinated myosin in myotubes. Myosin loss was significantly associated with elevated plasma levels of interleukin (IL)-6 in septic shock patients (P < 0.001). Addition of antiIL-6 to septic shock plasma diminished the loss of myosin in exposed myotubes by approximately 25% (P < 0.05). Patient plasma obtained later during ICU stay did not significantly reduce myosin content compared to controls. CONCLUSIONS: Plasma from patients with septic shock induces loss of myosin and activates key regulators of proteolysis in skeletal myotubes. IL-6 is an important player in sepsis-induced muscle atrophy in this model. The potential to induce atrophy is strongest in plasma obtained during the early phase of human sepsis.

Proteasome inhibition improves diaphragm function in an animal model for COPD.

Diaphragm muscle weakness in patients with chronic obstructive pulmonary disease (COPD) is associated with increased morbidity and mortality. Recent studies indicate that increased contractile protein degradation by the proteasome contributes to diaphragm weakness in patients with COPD. The aim of the present study was to investigate the effect of proteasome inhibition on diaphragm function and contractile protein concentration in an animal model for COPD. Elastase-induced emphysema in hamsters was used as an animal model for COPD; normal hamsters served as controls. Animals were either treated with the proteasome inhibitor Bortezomib (iv) or its vehicle saline. Nine months after induction of emphysema, specific force-generating capacity of diaphragm bundles was measured. Proteolytic activity of the proteasome was assayed spectrofluorometrically. Protein concentrations of proteasome, myosin, and actin were measured by means of Western blotting. Proteasome activity and concentration were significantly higher in the diaphragm of emphysematous hamsters than in normal hamsters. Bortezomib treatment reduced proteasome activity in the diaphragm of emphysematous and normal hamsters. Specific force-generating capacity and myosin concentration of the diaphragm were reduced by ~25% in emphysematous hamsters compared with normal hamsters. Bortezomib treatment of emphysematous hamsters significantly increased diaphragm-specific force-generating capacity and completely restored myosin concentration. Actin concentration was not affected by emphysema, nor by bortezomib treatment. We conclude that treatment with a proteasome inhibitor improves contractile function of the diaphragm in emphysematous hamsters through restoration of myosin concentration. These findings implicate that the proteasome is a potential target of pharmacological intervention on diaphragm weakness in COPD.

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.

Protein profiling of B-cell lymphomas using tissue biopsies: A potential tool for small samples in pathology.

Non-Hodgkin's lymphoma comprises many related but distinct diseases and diagnosis and classification is complex. Protein profiling of lymphoma biopsies may be of potential value for use in this lymphoma classification and the discovery of novel markers. In this study, we have optimized a method for SELDI-TOF MS based protein profiling of frozen tissue sections, without dissection of tumour cells. First we have compared chip surfaces and lysis buffers. Also, we have determined the minimal input using laser dissection microscopy. Subsequently, we have analyzed and compared protein profiles of diffuse large B-cell lymphoma (n=8), follicular lymphoma (n=8) and mantle cell lymphoma (n=8). Benign, reactive lymph nodes (n=14) were used as a reference group.CM10 chip surface in combination with urea lysis buffer and an input of approximately 50,000 lymphocytes allowed the detection of many differential peaks. Identification of the diffuse large B-cell lymphoma cases was reliably made in the supervised classification. Unsupervised clustering showed segregation into a benign/indolent cluster predominantly formed by benign, reactive lymph nodes and follicular lymphoma cases and into a more aggressive cluster formed by diffuse large B-cell lymphoma and mantle cell lymphoma cases. In conclusion, our protocol enables protein profiling of protein lysates derived from small histological samples and the subsequent detection of many differentially expressed proteins, without the need of tumour cell dissection. These results support further evaluation of protein profiling of small lymphoma biopsies as an additional tool in pathology.

Plexin D1 expression is induced on tumor vasculature and tumor cells: a novel target for diagnosis and therapy?

We previously reported that during mouse embryogenesis, plexin D1 (plxnD1) is expressed on neuronal and endothelial cells. Endothelial cells gradually loose plxnD1 expression during development. Here we describe, using in situ hybridization, that endothelial plxnD1 expression is regained during tumor angiogenesis in a mouse model of brain metastasis. Importantly, we found PLXND1 expression also in a number of human brain tumors, both of primary and metastatic origin. Apart from the tumor vasculature, abundant expression was also found on tumor cells. Via panning of a phage display library, we isolated two phages that carry single-domain antibodies with specific affinity towards a PLXND1-specific peptide. Immunohistochemistry with these single-domain antibodies on the same tumors that were used for in situ hybridization confirmed PLXND1 expression on the protein level. Furthermore, both these phages and the derived antibodies specifically homed to vessels in brain lesions of angiogenic melanoma in mice after i.v. injection. These results show that PLXND1 is a clinically relevant marker of tumor vasculature that can be targeted via i.v. injections.

Mice lacking leukocyte common antigen-related (LAR) protein tyrosine phosphatase domains demonstrate spatial learning impairment in the two-trial water maze and hyperactivity in multiple behavioural tests.

Leukocyte common antigen-related (LAR) protein is a cell adhesion molecule-like receptor-type protein tyrosine phosphatase. We previously reported that in LAR tyrosine phosphatase-deficient (LAR-Delta P) mice the number and size of basal forebrain cholinergic neurons as well as their innervation of the hippocampal area was reduced. With the hippocampus being implicated in behavioural activity aspects, including learning and memory processes, we assessed possible phenotypic consequences of LAR phosphatase deficiency using a battery of rodent behaviour tests. Motor function and co-ordination tests as well as spatial learning ability assays did not reveal any performance differences between wildtype and LAR-Delta P mice. A spatial learning impairment was found in the difficult variant of the Morris water maze. Exploration, nestbuilding and activity tests indicated that LAR-Delta P mice were more active than wildtype littermates. The observed hyperactivity in LAR-Delta P mice could not be explained by altered anxiety or curiosity levels, and was found to be persistent throughout the nocturnal period. In conclusion, behavioural testing of the LAR-Delta P mice revealed a spatial learning impairment and a significant increase in activity.