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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 (~30-40%) and cross-bridge cycling kinetics (~20%) in CHF. Myosin content was ~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. myosin; bortezomib; single fiber contractility; myosin

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, 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.