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Nontuberculous mycobacteria (NTM) are common in the environment and have been isolated worldwide (1). They are increasingly recognized as pathogens in humans. Pulmonary disease is the most common manifestation (2) [...]

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

Diaphragm weakness commonly occurs in patients with congestive heart failure (CHF) and is an independent predictor of mortality. However, the pathophysiology of diaphragm weakness is poorly understood. We hypothesized that CHF induces diaphragm weakness at the single-fiber level by decreasing myosin content. In addition, we hypothesized that myofibrillar [Ca.sup.2+] sensitivity is decreased and cross-bridge kinetics are slower in CHF diaphragm fibers. Finally, we hypothesized that loss of myosin in CHF diaphragm weakness is associated with increased proteolytic activities of caspase-3 and the proteasome. In skinned diaphragm single fibers of rats with CHF, induced by left coronary artery ligation, maximum force generation was reduced by ~35% (P < 0.01) compared with sham-operated animals for slow, 2a, and 2x fibers. In these CHF diaphragm fibers, myosin heavy chain content per half-sarcomere was concomitantly decreased (P < 0.01). [Ca.sup.2+] sensitivity of force generation and the rate constant of tension redevelopment were significantly reduced in CHF diaphragm fibers compared with sham-operated animals for all fiber types. The cleavage activity of the proteolytic enzyme caspase-3 and the proteasome were ~30% (P < 0.05) and ~60% (P < 0.05) higher, respectively, in diaphragm homogenates from CHF rats than from sham-operated rats. The present study demonstrates diaphragm weakness at the single-fiber level in a myocardial infarct model of CHF. The reduced maximal force generation can be explained by a loss of myosin content in all fiber types and is associated with activation of caspase-3 and the proteasome. Furthermore, CHF decreases myofibrillar [Ca.sup.2+] sensitivity and slows cross-bridge cycling kinetics in diaphragm fibers. skinned muscle single fibers; cross-bridge cycling kinetics; [Ca.sup.2+] sensitivity; proteasome; caspase-3 doi:10.1152/ajpregu.00085.2007

During chronic obstructive pulmonary disease (COPD) diaphragm and peripheral muscle weakness occur. Muscle remodeling and wasting may be a result of apoptosis and changes in muscle-specific transcription factors, such as MyoD, altering muscle-specific gene transcription and muscle regenerative capacity. To investigate this, we instilled under ketamine/ xylazine anesthesia porcine elastase in the lungs of hamsters to induce emphysema. The emphysematous hamster is an accepted model for COPD. In the diaphragm and peripheral muscles we assessed the occurrence of apoptosis, and in the diaphragm and soleus also the expression of MyoD and inhibitor of differentiation protein 2 (Id2). There was no significant muscle atrophy in emphysematous hamsters. The mRNA levels of TNF-[alpha] and markers of apoptosis were significantly elevated in the diaphragm and soleus muscles during emphysema. This was accompanied by an increased presence of nucleosomes in the cytosol. Caspase 3 activity and the DNA-binding activity of the p65 subunit of NF-[kappa]B, however, were unaltered in all muscles. The protein expression of MyoD and Id2 were decreased and increased in the diaphragm and the soleus muscle, respectively. Thus, despite the absence of muscle atrophy in emphysematous hamsters, there was evidence of increased TNF-[alpha] expression, apoptosis, and altered muscle-specific transcriptional regulation as reflected by decreased MyoD and elevated Id2 levels at least in the soleus and diaphragm muscle. These alterations may impair the regenerative capacity of skeletal muscles and ultimately contribute to muscle wasting. apoptosis inducing factor; BAX; inhibitor of differentiation; MyoD; nuclear factor-[kappa]B doi:10.1152/ajpregu.00046.2006

Hypoxia impairs skeletal muscle function, but the precise mechanisms are incompletely understood. In hypoxic rat diaphragm muscle, generation of peroxynitrite is elevated. Peroxynitrite and other reactive nitrogen species have been shown to impair contractility of skinned muscle fibers, reflecting contractile protein dysfunction. We hypothesized that hypoxia induces contractile protein dysfunction and that reactive nitrogen species are involved. In addition, we hypothesized that muscle reoxygenation reverses contractile protein dysfunction. In vitro contractility of rat soleus muscle bundles was studied after 30 min of hyperoxia (P[O.sub.2] ~90 kPa), hypoxia (P[O.sub.2] ~5 kPa), hypoxia + 30 [micro]M [N.sup.G]-monomethyl-L-arginine (L-NMMA, a nitric oxide synthase inhibitor), hyperoxia + 30 [micro]M L-NMMA, and hypoxia (30 min) + reoxygenation (15 min). One part of the muscle bundle was used for single fiber contractile measurements and the other part for nitrotyrosine detection. In skinned single fibers, maximal [Ca.sup.2+]-activated specific force ([F.sub.max]), fraction of strongly attached cross bridges ([[alpha].sub.fs]), rate constant of force redevelopment ([k.sub.tr]), and myofibrillar [Ca.sup.2+] sensitivity were determined. Thirty minutes of hypoxia reduced muscle bundle contractility. In the hypoxic group, single fiber [F.sub.max], [[alpha].sub.fs], and [k.sub.tr] were significantly reduced compared with hyperoxic, L-NMMA, and reoxygenation groups. Myofibrillar [Ca.sup.2+] sensitivity was not different between groups. Nitrotyrosine levels were increased in hypoxia compared with all other groups. We concluded that acute hypoxia induces dysfunction of skinned muscle fibers, reflecting contractile protein dysfunction. In addition, our data indicate that reactive nitrogen species play a role in hypoxia-induced contractile protein dysfunction. Reoxygenation of the muscle bundle partially restores bundle contractility but completely reverses contractile protein dysfunction. nitrotyrosine; denitration; reoxygenation; skinned single fiber; peroxynitrite

Oxidants may play a role in hypoxia-induced respiratory muscle dysfunction. In the present study we hypothesized that hypoxia-induced impairment in diaphragm contractility is associated with elevated peroxynitrite generation. In addition, we hypothesized that strenuous contractility of the diaphragm increases peroxynitrite formation. In vitro force-frequency relationship, isotonic fatigability, and nitrotyrosine levels were assessed under hypoxic (P[O.sub.2] ~6.5 kPa) and hyperoxic (P[O.sub.2] ~88.2 kPa) control conditions and also in the presence of authentic peroxynitrite (60 min), ebselen (60 min), and the nitric oxide synthase inhibitor [N.sup.G]-monomethyl-L-arginine acetate (L-NMMA) (90 min). A hypoxia-induced downward shift of the force-frequency relationship was associated with elevated nitrotyrosine level in the diaphragm. During hypoxia, both ebselen and L-NMMA decreased nitrotyrosine levels but did not affect force generation. Strenuous contractions impaired force generation but did not affect nitrotyrosine levels in the diaphragm during hypoxia. But under hyperoxic conditions, fatiguing contractions were associated with elevated diaphragm nitrotyrosine levels. Under hyperoxic conditions exogenous peroxynitrite impaired force generation and increased nitrotymsine level. These studies show that hypoxia-induced impairment in diaphragm contractility is associated with increased diaphragm protein nitration, but no causal relationship was round between diaphragm nitrotyrosine formation and in vitro force generation. nitrotyrosine; respiratory muscles; rat; contractile properties; nitric oxide

Heunks, Leo M. A., Herwin A. Machiels, Ronney de Abreu, Xiao Ping Zhu, Henricus F. M. van der Heijden, and P. N. Richard Dekhuijzen. Free radicals in hypoxic rat diaphragm contractility: no role for xanthine oxidase. Am J Physiol Lung Cell Mol Physiol 281: L1402-L1412, 2001.Recent evidence indicates that hypoxia enhances the generation of oxidants. Little is known about the role of free radicals in contractility of the rat diaphragm during hypoxia. We hypothesized that antioxidants improve contractility of the hypoxic rat diaphragm and that xanthine oxidase (XO) is an important source of free radicals in the hypoxic diaphragm. The effects of N-acetylcysteine (NAC; 18 mM), Tiron (10 mM), and the XO inhibitor allopurinol (250 [micro]M) were studied on isometric and isotonic force generation during hypoxia (P[O.sub.2] ~7 kPa). NAC and Tiron decreased maximal force generation, slowed the shortening velocity, and decreased the power output. Fatigue rate was decreased in the presence of either NAC or Tiron. Allopurinol did not alter the contractility or fatigability of the diaphragm. During hyperoxia (P[O.sub.2] ~85 kPa), neither NAC nor allopurinol affected the contractility or fatigability of the diaphragm. Thus free radicals play a significant role in diaphragm contractility during hypoxia. Whether antioxidants exert a beneficial or harmful effect on muscle performance depends on the contraction pattern of the muscle. Free radicals generated by XO do not play a role in diaphragm contractility during either hypoxia or hyperoxia. isotonic contractions; allopurinol; Tiron; N-acetylcysteine; respiratory muscles Received 8 September 2000; accepted in final form 2 August 2001

Treatment with a bronchodilator in combination with an inhaled corticosteroid may be more effective than treatment in combination with an anticholinergic-drug or with a bronchodilator alone for patients with obstructive lung diseases, such as asthma and chronic obstructive pulmonary disease (COPD). Among 274 patients with obstructive lung disease who were treated with a beta2-agonist bronchodilator, 91 were also given a corticosteroid drug, 92 were given an anticholinergic drug and 91 were given a placebo, or an inactive substance. Patients taking corticosteroids improved more than those taking anticholinergics or those taking a placebo. Patients treated with corticosteroids were less likely to develop pulmonary (lungs) complications than those treated with anticholinergics or a placebo.

Little is known about the clinical relevance of nontuberculous mycobacteria (NTM) in the Arabian Peninsula. We assessed the prevalence and studied a random sample of isolates at a reference laboratory [...]