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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.

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)-1β, 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 P P 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.

Contains fulltext : 134064.pdf (Publisher’s version ) (Open Access) 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.

Mechanical ventilation is frequently used in patients under general anesthesia during invasive procedures. Invasive animal experiments similarly require the maintenance of normal hemodynamic and pulmonary parameters during long-term general anesthesia. The authors describe a method for mechanical ventilation of mice. Mice were ventilated and monitored for up to 8 h of general anesthesia during surgery. Hemodynamic and pulmonary parameters remained within the normal ranges. The authors believe that this ventilation technique can be of great value for experimental procedures in mice that require general anesthesia.

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/mm2, 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. Electronic supplementary material The online version of this article (doi:10.1007/s00134-012-2504-5) contains supplementary material, which is available to authorized users.

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.