Recovery of respiratory function in mdx mice co-treated with neutralizing interleukin-6 receptor antibodies and urocortin-2.

KEY POINTS: Impaired ventilatory capacity and diaphragm muscle weakness are prominent features of Duchenne muscular dystrophy, with strong evidence of attendant systemic and muscle inflammation. We performed a 2-week intervention in young wild-type and mdx mice, consisting of either injection of saline or co-administration of a neutralizing interleukin-6 receptor antibody (xIL-6R) and urocortin-2 (Ucn2), a corticotrophin releasing factor receptor 2 agonist. We examined breathing and diaphragm muscle form and function. Breathing and diaphragm muscle functional deficits are improved following xIL-6R and Ucn2 co-treatment in mdx mice. The functional improvements were associated with a preservation of mdx diaphragm muscle myosin heavy chain IIx fibre complement. The concentration of the pro-inflammatory cytokine interleukin-1ß was reduced and the concentration of the anti-inflammatory cytokine interleukin-10 was increased in mdx diaphragm following drug co-treatment. Our novel findings may have implications for the development of pharmacotherapies for the dystrophinopathies with relevance for respiratory muscle performance and breathing. ABSTRACT: The mdx mouse model of Duchenne muscular dystrophy shows evidence of hypoventilation and pronounced diaphragm dysfunction. Six-week-old male mdx (n = 32) and wild-type (WT; n = 32) mice received either saline (0.9% w/v) or a co-administration of neutralizing interleukin-6 receptor antibodies (xIL-6R; 0.2 mg kg-1 ) and corticotrophin-releasing factor receptor 2 agonist (urocortin-2; 30 µg kg-1 ) subcutaneously over 2 weeks. Breathing and diaphragm muscle contractile function (ex vivo) were examined. Diaphragm structure was assessed using histology and immunofluorescence. Muscle cytokine concentration was determined using a multiplex assay. Minute ventilation and diaphragm muscle peak force at 100 Hz were significantly depressed in mdx compared with WT. Drug treatment completely restored ventilation in mdx mice during normoxia and significantly increased mdx diaphragm force- and power-generating capacity. The number of centrally nucleated muscle fibres and the areal density of infiltrates and collagen content were significantly increased in mdx diaphragm; all indices were unaffected by drug co-treatment. The abundance of myosin heavy chain (MyHC) type IIx fibres was significantly decreased in mdx diaphragm; drug co-treatment preserved MyHC type IIx complement in mdx muscle. Drug co-treatment increased the cross-sectional area of MyHC type I and IIx fibres in mdx diaphragm. The cytokines IL-1ß, IL-6, KC/GRO and TNF-α were significantly increased in mdx diaphragm compared with WT. Drug co-treatment significantly decreased IL-1ß and increased IL-10 in mdx diaphragm. Drug co-treatment had no significant effect on WT diaphragm muscle structure, cytokine concentrations or function. Recovery of breathing and diaphragm force in mdx mice was impressive in our studies, with implication for human dystrophinopathies.

Restoration of pharyngeal dilator muscle force in dystrophin-deficient (mdx) mice following co-treatment with neutralizing interleukin-6 receptor antibodies and urocortin 2.

NEW FINDINGS: What is the central question of this study? We previously reported impaired upper airway dilator muscle function in the mdx mouse model of Duchenne muscular dystrophy (DMD). Our aim was to assess the effect of blocking interleukin-6 receptor signalling and stimulating corticotrophin-releasing factor receptor 2 signalling on mdx sternohyoid muscle structure and function. What is the main finding and its importance? The interventional treatment had a positive inotropic effect on sternohyoid muscle force, restoring mechanical work and power to wild-type values, reduced myofibre central nucleation and preserved the myosin heavy chain type IIb fibre complement of mdx sternohyoid muscle. These data might have implications for development of pharmacotherapies for DMD with relevance to respiratory muscle performance. The mdx mouse model of Duchenne muscular dystrophy shows evidence of impaired pharyngeal dilator muscle function. We hypothesized that inflammatory and stress-related factors are implicated in airway dilator muscle dysfunction. Six-week-old mdx (n = 26) and wild-type (WT; n = 26) mice received either saline (0.9% w/v) or a co-administration of neutralizing interleukin-6 receptor antibodies (0.2 mg kg-1 ) and corticotrophin-releasing factor receptor 2 agonist (urocortin 2; 30 µg kg-1 ) over 2 weeks. Sternohyoid muscle isometric and isotonic contractile function was examined ex vivo. Muscle fibre centronucleation and muscle cellular infiltration, collagen content, fibre-type distribution and fibre cross-sectional area were determined by histology and immunofluorescence. Muscle chemokine content was examined by use of a multiplex assay. Sternohyoid peak specific force at 100 Hz was significantly reduced in mdx compared with WT. Drug treatment completely restored force in mdx sternohyoid to WT levels. The percentage of centrally nucleated muscle fibres was significantly increased in mdx, and this was partly ameliorated after drug treatment. The areal density of infiltrates and collagen content were significantly increased in mdx sternohyoid; both indices were unaffected by drug treatment. The abundance of myosin heavy chain type IIb fibres was significantly decreased in mdx sternohyoid; drug treatment preserved myosin heavy chain type IIb complement in mdx muscle. The chemokines macrophage inflammatory protein 2, interferon-γ-induced protein 10 and macrophage inflammatory protein 3α were significantly increased in mdx sternohyoid compared with WT. Drug treatment significantly increased chemokine expression in mdx but not WT sternohyoid. Recovery of contractile function was impressive in our study, with implications for Duchenne muscular dystrophy. The precise molecular mechanisms by which the drug treatment exerts an inotropic effect on mdx sternohyoid muscle remain to be elucidated.

Overcoming challenges in the palladium-catalyzed synthesis of electron deficient ortho-substituted aryl acetonitriles.

Highly electron deficient monoaryl, di-aryl and bis-diaryl acetonitriles were effectively synthesized using either a nucleophilic aromatic substitution (NAS) or a palladium-mediated coupling pathway. Synthesis of di-aryl acetonitriles most conveniently proceeded via NAS--palladium-mediated coupling was not required. This reaction, however, results in a product that is more acidic than the reactants. Facile deprotonation of the product prevents efficient formation of the bis-diaryl acetonitrile through a NAS pathway. Thus, palladium-mediated coupling is required to prepare the bis-diaryl acetonitrile efficiently. In the palladium-catalyzed coupling, choice of base and solvent (and thus the counter cation for the benzylic anion nucleophile) is important. Also, choice of the supporting ligand is important, indicating the sensitivity of the reaction to steric and ligand electronic effects.