Theophylline dilates rat diaphragm arterioles via the prostaglandins pathway

1. We investigated by intravital microscopy in rats, the in vivo direct effects of theophylline on the diameters of second and third order diaphragm arterioles.
2. Theophylline (1–100 μM) dilated second and third order diaphragm arterioles significantly, and with an amplitude which was not statistically different from the one obtained with adenosine (1–100 μM). Enprofylline (1–100 μM), a theophylline analogue with poor adenosine-receptor antagonism but with similar or higher phosphodiesterases inhibition properties than theophylline, also dilated diaphragm arterioles, causing however, a significantly smaller dilatation than theophylline.
3. Neither the A1 adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX, 50 nM), nor the A₂ adenosine receptor antagonist 3,7-dimethyl-1-proparglyxanthine (DMPX, 10 μM) reduced significantly theophylline-induced arteriolar dilatation.
4. Theophylline (100 nM) abolished adenosine-induced arteriolar dilatation.
5. The dilatation induced by theophylline was unchanged by the nitric oxide (NO) synthase inhibitor Nω-nitro-L-arginine (NNA, 300 μM).
6. Theophylline-induced arteriolar dilatation was abolished by the prostaglandin synthesis inhibitors mefenamic acid or indomethacin (20 μM).
7. These findings show that theophylline induced a significant dilatation of diaphragm arterioles via the release of prostaglandins.

     

Inhibition of monocyte chemoattractant protein-1 prevents diaphragmatic inflammation and maintains contractile function during endotoxemia

Introduction  

Respiratory muscle weakness is common in sepsis patients. Proinflammatory mediators produced during sepsis have been implicated in diaphragmatic contractile dysfunction, but the role of chemokines has not been explored. This study addressed the role of monocyte chemoattractant protein-1 (MCP-1, also known as CCL2), in the pathogenesis of diaphragmatic inflammation and weakness during endotoxemia.     

Induction of diaphragmatic nitric oxide synthase after endotoxin administration in rats: role on diaphragmatic contractile dysfunction.

Nitric oxide (NO), a free radical that is negatively inotropic in the heart and skeletal muscle, is produced in large amounts during sepsis by an NO synthase inducible (iNOS) by LPS and/or cytokines. The aim of this study was to examine iNOS induction in the rat diaphragm after Escherichia Coli LPS inoculation (1.6 mg/kg i.p.), and its involvement in diaphragmatic contractile dysfunction. Inducible NOS protein and activity could be detected in the diaphragm as early as 6 h after LPS inoculation. 6 and 12 h after LPS, iNOS was expressed in inflammatory cells infiltrating the perivascular spaces of the diaphragm, whereas 12 and 24 h after LPS it was expressed in skeletal muscle fibers. Inducible NOS was also expressed in the left ventricular myocardium, whereas no expression was observed in the abdominal, intercostal, and peripheral skeletal muscles. Diaphragmatic force was significantly decreased 12 and 24 h after LPS. This decrease was prevented by inhibition of iNOS induction by dexamethasone or by inhibition of iNOS activity by N(G)-methyl-L-arginine. We conclude that iNOS was induced in the diaphragm after E. Coli LPS inoculation in rats, being involved in the decreased muscular force.     

Sildenafil and cardiomyocyte-specific cGMP signaling prevent cardiomyopathic changes associated with dystrophin deficiency

We recently demonstrated early metabolic alterations in the dystrophin-deficient mdx heart that precede overt cardiomyopathy and may represent an early "subclinical" signature of a defective nitric oxide (NO)/cGMP pathway. In this study, we used genetic and pharmacological approaches to test the hypothesis that enhancing cGMP, downstream of NO formation, improves the contractile function, energy metabolism, and sarcolemmal integrity of the mdx heart. We first generated mdx mice overexpressing, in a cardiomyocyte-specific manner, guanylyl cyclase (GC) (mdx/GC(+/0)). When perfused ex vivo in the working mode, 12- and 20-week-old hearts maintained their contractile performance, as opposed to the severe deterioration observed in age-matched mdx hearts, which also displayed two to three times more lactate dehydrogenase release than mdx/GC(+/0). At the metabolic level, mdx/GC(+/0) displayed a pattern of substrate selection for energy production that was similar to that of their mdx counterparts, but levels of citric acid cycle intermediates were significantly higher (36 +/- 8%), suggesting improved mitochondrial function. Finally, the ability of dystrophin-deficient hearts to resist sarcolemmal damage induced in vivo by increasing the cardiac workload acutely with isoproterenol was enhanced by the presence of the transgene and even more so by inhibiting cGMP breakdown using the phosphodiesterase inhibitor sildenafil (44.4 +/- 1.0% reduction in cardiomyocyte damage). Overall, these findings demonstrate that enhancing cGMP signaling, specifically downstream and independent of NO formation, in the dystrophin-deficient heart improves contractile performance, myocardial metabolic status, and sarcolemmal integrity and thus constitutes a potential clinical avenue for the treatment of the dystrophin-related cardiomyopathies.     

Effects of etomidate, propofol and thiopental anaesthesia on arteriolar tone in the rat diaphragm

We have assessed, by intravital microscopy in rats, the effects of different anaesthetics on diaphragmatic arteriolar diameter. Rats were anaesthetized with etomidate, propofol or thiopental (groups E, P and T, respectively) and the diameters of the arterioles were measured sequentially at baseline and after topical application of either mefenamic acid (MA, 20 microgramsmol litre-1) or N omega-nitro-L-arginine (NNA, 300 microgramsmol litre-1), inhibitors of prostaglandins and nitric oxide, respectively. In group E, baseline arteriolar diameters were significantly higher than those in the two other groups (P < 0.01). MA and NNA induced significant constriction in the three groups (P < 0.001). However, whereas constriction induced by NNA was similar in the three groups, constriction induced by MA was significantly higher in group E compared with groups P and T (P < 0.05). We conclude that diaphragmatic arteriolar diameters in rats were greater during etomidate than during thiopental or propofol anaesthesia. This phenomenon may be mediated by prostaglandins.      

Expression and regulation of CC class chemokines in the dystrophic (mdx) diaphragm

In the murine (mdx) model of Duchenne muscular dystrophy, dystrophic changes are much more severe in the diaphragm than in limb muscles, and the diaphragm more closely resembles the human disease phenotype. Chemokines could play a central role in governing such phenotypic differences, as inflammation is an important disease modifier. Here we report that CC chemokine receptors (CCRs 1, 2, 3, 5) and ligands (macrophage inflammatory protein-1alpha, RANTES) are expressed at higher levels in dystrophic than in wild-type muscles across age groups (6, 12, and 24 wk). Moreover, chemokine ligand expression and muscle inflammation are significantly higher in dystrophic diaphragms than in limb muscles of the same animals. In vitro, CCR1 is constitutively expressed by cultured primary diaphragmatic myotubes. Stimulation of myotubes by proinflammatory cytokines (tumor necrosis factor-alpha, interleukin-1alpha, interferon-gamma) found within the in vivo dystrophic muscle environment, upregulates CCR1 in mdx and wild-type cultures, and also increases expression of its ligand RANTES to a significantly greater degree in the mdx group. Taken together, our results suggest that CC chemokines may play an important role in sustaining inflammation within the mdx diaphragm, which could help account for its more severe phenotype and also offer a target for therapeutic intervention in Duchenne patients.     

Mechanical ventilation protects against diaphragm injury in sepsis: interaction of oxidative and mechanical stresses.

Overproduction of nitric oxide (NO) with attendant oxidative and nitrosative stress has been implicated in sepsis-induced diaphragm dysfunction. Here we determined the impact of controlled mechanical ventilation (MV) on rat diaphragm sarcolemmal injury, inducible NO synthase (iNOS) expression, and oxidative stress during endotoxemia. At 4 h after injection of endotoxin, impaired sarcolemmal integrity and decreased force production by the diaphragm were observed in spontaneously breathing rats. The use of MV during endotoxemia largely eliminated sarcolemmal damage and significantly improved diaphragm force production. These benefits were not associated with alterations in either iNOS expression or protein carbonyls (marker of oxidation), which remained abnormally elevated in septic diaphragms despite MV. Therefore, we hypothesized that the protection afforded by MV was due to its ability to decrease the level of mechanical stress placed on the sarcolemma, because the latter could be hyperfragile in the setting of increased oxidative stress. Using an in vitro system to independently modulate oxidative and mechanical stresses, we confirmed that these two factors act together in a synergistic fashion to favor sarcolemmal injury. Accordingly, our data suggest that MV protects the diaphragm during sepsis by abrogating an injurious interaction between oxidative and biomechanical stresses imposed on the sarcolemma.     

Sarcolemmal damage in dystrophin deficiency is modulated by synergistic interactions between mechanical and oxidative/nitrosative stresses

Dystrophin deficiency is the cause of Duchenne muscular dystrophy, but the precise physiological basis for muscle necrosis remains unclear. To determine whether dystrophin-deficient muscles are abnormally susceptible to oxidative and nitric oxide (NO)-driven tissue stress, a hindlimb ischemia/reperfusion (I/R) model was used. Dystrophic mdx mice exhibited abnormally high levels of lipid peroxidation and protein nitration, which were preceded by exaggerated NO production during ischemia. Visualization of NO with the fluorescent probe 4,5-diaminofluorescein diacetate suggested that excess NO production during ischemia occurred within a subset of mdx fibers. In mdx muscles only, prior exposure to I/R dramatically increased the level of sarcolemmal damage resulting from stretch-mediated mechanical stress, indicating greatly exacerbated hyperfragility of the dystrophic fiber membrane. Treatment with NO synthase inhibitors (l-N(G)-nitroarginine methyl ester hydrochloride or 7-nitroindazol) effectively blocked the synergistic interaction between I/R and mechanical stress-mediated sarcolemmal damage under these conditions. Taken together, our findings provide direct ex-perimental evidence that several prevailing hy-potheses regarding the cause of muscle fiber damage in dystrophin-deficient muscle can be integrated into a common pathophysiological framework involving interactions between oxidative stress, ab-normal NO regulation, and hyperfragility of the sarcolemma.     

Impact of IL-10 on diaphragmatic cytokine expression and contractility during Pseudomonas Infection

Severe weakness of the respiratory muscles, with attendant respiratory failure and death, has been documented in sepsis. In this study, we show that during murine pulmonary infection with Pseudomonas aeruginosa, multiple proinflammatory genes are up-regulated not only within the lungs, but also within the diaphragm. Significant induction of TNF-alpha, IL-1alpha, IL-1beta, IL-6, and IL-18 gene expression occurred within the diaphragm in a bacterial dose-dependent manner. We determined whether the anti-inflammatory cytokine IL-10 could blunt proinflammatory gene expression within the diaphragm under these conditions. The IL-10 receptor was found to be expressed by the diaphragm in vivo as well as in primary diaphragmatic muscle cell cultures. Transduction of myoblasts with an adenoviral vector (Ad-IL-10) induced strong IL-10 expression, and intramuscular injection of the same vector in vivo produced significant increases in IL-10 serum levels. Ad-IL-10 treatment of mice infected with P. aeruginosa significantly inhibited the induction of proinflammatory cytokines within the diaphragm, but not in the infected lungs. Ad-IL-10 treatment also led to greatly improved diaphragmatic force production in infected mice. These results suggest that pulmonary infection triggers proinflammatory gene expression by the diaphragm along with diaphragmatic weakness. Shifting the balance between pro- and anti-inflammatory mediators in favor of the latter by IL-10 gene delivery was able to restore normal diaphragmatic force-generating capacity under these conditions, suggesting a possible avenue for therapeutic intervention.     

Bcl-2-associated autophagy regulator Naf-1 required for maintenance of skeletal muscle

Nutrient-deprivation autophagy factor-1 (NAF-1) was identified as an endoplasmic reticulum (ER) BCL-2-interacting protein, which functions to mediate the ability of ER BCL-2 to antagonize Beclin 1-dependent autophagy and depress ER calcium stores. In humans, a point mutation in Naf-1 (synonyms: Cisd2, Eris, Miner1 and Noxp70) is responsible for the neurodegenerative disorder Wolfram Syndrome 2. Here, we describe the generation and characterization of the Naf-1 gene deletion in mice. Naf-1 null mice display discernable clinical signs of degeneration at 2-3 months of age, with early evidence of significant defects in the structure and performance of skeletal muscle. Skeletal muscles from Naf-1 knockout mice demonstrate a significant shift towards slow-twitch (type I) fibers and greater resistance to muscle fatigue. Force-generating capacity is dramatically reduced in Naf-1(-/-) muscle. Consistent with its role in ER BCL-2-mediated regulation of autophagy and calcium flux, these physiological deficiencies were accompanied by augmented autophagy and dysregulated calcium homeostasis. In contrast, this also included adaptive enlargement of mitochondria with extensive cristae structures. Thus, NAF-1, a BCL-2-associated autophagy regulator, is required for homeostatic maintenance of skeletal muscle. Our findings uncover a novel pathway that is required for normal muscle maintenance, which may ultimately provide a novel therapeutic target for treating certain muscle pathologies.