No abstract

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 (Po(2) approximately 6.5 kPa) and hyperoxic (Po(2) approximately 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(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 nitrotyrosine level. These studies show that hypoxia-induced impairment in diaphragm contractility is associated with increased diaphragm protein nitration, but no causal relationship was found between diaphragm nitrotyrosine formation and in vitro force generation.

Hypoxia disturbs Ca(2+) regulation and increases the intracellular Ca(2+) concentration ([Ca(2+)](i)), which may in turn activate the nitric oxide synthase (NOS) regulated by [Ca(2+)](i). Since nitric oxide (NO) reduces the isometric contractility of rat diaphragm in vitro, we hypothesized that NO contributes to the impaired force generation of an hypoxic diaphragm. The effects of different concentrations of the NOS inhibitor, N(G)-monomethyl-L-arginine (L-NMMA), the NO scavenger haemoglobin (150 micro mol.l(-1)) and the NO donor spermine NONOate (Sp-NO; 1 mmol.l(-1)) were determined on isometric contractility during hypoxia [partial pressure of oxygen, PO(2), about 7 kPa (about 54 mmHg)] and hyperoxia [ PO(2) about 83 kPa (about 639 mmHg)]. Hypoxia significantly reduced maximal twitch force ( F(t)), and submaximal tetanic force (30 Hz, F(30)) in all L-NMMA groups. A low concentration of L-NMMA (30 micromol.l(-1)) increased F(30) but a high concentration (1,000 micromol.l(-1)) reduced F(30) during hypoxia. The effects of L-NMMA on force generation were more pronounced during hypoxia compared to hyperoxia. Peak increases in F(30) and F(t) were observed at a concentration of 30 micromol.l(-1) L-NMMA during hypoxia, but with 10 micromol.l(-1) L-NMMA during hyperoxia. The same concentration of haemoglobin increased F(30) and F(t) less during hypoxia compared to hyperoxia. The Sp-NO reduced F(t), F(30) and maximal tetanic force (F(0)) during hypoxia; these effects were abolished in the presence of haemoglobin. The Sp-NO did not alter F(t), F(30) and F(0)during hyperoxia. We conclude that NO plays a more prominent role during hypoxia and that NO contributes to the depression of force generation in the hypoxic rat diaphragm in vitro. This change may be related to an elevated NO generation within the hypoxic diaphragm.

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.

Effects of modulation of nitric oxide on rat diaphragm isotonic contractility during hypoxia. J Appl Physiol 94: 612-620, 2003. First published October 18, 2002 10.1152/ japplphysiol.00441.2002 is essential for optimal myofilament function of the rat diaphragm in vitro during active shortening. Little is known about the role of NO in muscle contraction under hypoxic conditions. Hypoxia might increase the NO synthase (NOS) activity within the rat diaphragm. We hypothesized that NO plays a protective role in isotonic contractile and fatigue properties during hypoxia in vitro. The effects of the NOS inhibitor N G -monomethyl-L-arginine (L-NMMA), the NO scavenger hemoglobin, and the NO donor spermine NONOate on shortening velocity, power generation, and isotonic fatigability during hypoxia were evaluated (PO 2 ϳ 7 kPa). L-NMMA and hemoglobin slowed the shortening velocity, depressed power generation, and increased isotonic fatigability during hypoxia. The effects of L-NMMA were prevented by coadministration with the NOS substrate L-arginine. Spermine NONOate did not alter isotonic contractile and fatigue properties during hypoxia. These results indicate that endogenous NO is needed for optimal muscle contraction of the rat diaphragm in vitro during hypoxia.N G -monomethyl-L-arginine; hemoglobin; spermine NONOate SKELETAL MUSCLE, INCLUDING the diaphragm, continuously produces nitric oxide (NO) (3,26). NO is produced in biological systems via the enzymatic action of NO synthase (NOS) (32). NO generation by the constitutive NOS isoforms (neuronal NOS and endothelial NOS) is calcium dependent (16). NO production within skeletal muscle is enhanced during contractile activity (5).NO has been shown to modulate contractile properties of skeletal muscle in vitro. The overall effect depends on the experimental paradigm. Inhibition of NOS increases twitch and submaximal tetanic force of the rat diaphragm. These alterations are reversed by NO donors (26), suggesting that NO inhibits excitation-contraction coupling in unfatigued muscle. On the other hand, NO is essential for optimal myofilament function in the rat diaphragm during active shortening in vitro (36). Inhibition of NOS decreases the shortening velocity and power generation of the rat diaphragm under hyperoxic conditions (36). These findings of NOS inhibition on force generation and velocity of shortening indicate multiple possible targets for NO in skeletal muscle. In fatiguing contractions, supplementation with exogenous NO slowed the decline of maximal force in mouse soleus muscle (38). This finding suggests that NO preserves skeletal muscle function in vitro during strenuous contractile activity.Hypoxia impairs force generation and accelerates skeletal muscle fatigue in vitro (21, 48). To date, no study has investigated the role of NO in shortening velocity or isotonic fatigability during hypoxia. This is of particular interest because NO affects excitationcontraction coupling in striated muscle (26), and O 2 tension is an important factor in regulating NOS in an...