Targeting IL‐6 and CRFR2 Improves Diaphragm Muscle Function and Restores Ventilation in Mdx Mice

Abstract

Duchenne muscular dystrophy (DMD) is a genetic disease caused by a mutation in the dystrophin gene, leading to the absence of the structural protein ‐ dystrophin. Patients have severe muscle weakness and die prematurely due to respiratory and cardiac failure. The mdx mouse model of DMD shows evidence of reduced normoxic ventilation and impaired respiratory muscle function at 8 weeks of age.6 week old mdx (C57BL/10ScSn‐Dmdmdx/J; n=24) and wild‐type (WT; C57BL/10ScSn; n=23) mice received either saline (0.9% w/v) or a co‐treatment of neutralizing IL‐6 receptor antibodies (xIL‐6R; 0.2 mg/kg) and corticotrophin releasing factor receptor 2 agonist (Urocortin‐2; 30μg/kg). Animals received 6 subcutaneous injections over 2 weeks. Following this, ventilation was assessed in animals by use of whole body plethysmography. Diaphragm muscle contractile function was examined ex vivo. Muscle fibre nucleation and inflammatory cell infiltration were examined by haematoxylin and eosin staining. Immunofluorescence was used to visualize collagen deposition in muscle. Fibre type distribution and cross‐sectional area (CSA) was determined by myosin heavy chain (MHC) immunofluorescence. Values are reported as mean±SD and data were statistically compared by two‐way ANOVA with Bonferroni post‐hoc test.Peak specific force (Fmax) was significantly reduced in mdx (8.7±2.8 N/cm2) compared with WT (19.3±5.5). Co‐treatment increased Fmax for mdx (15.6±2.7) towards WT values. Minute ventilation was significantly reduced in mdx (0.9±0.2 ml/min/g) compared with WT (1.2±0.2). Co‐treatment restored ventilation in mdx (1.3±0.2) to values equivalent to WT. The percentage of centrally‐nucleated muscle fibres was significantly increased in mdx (15.9±2.8%) compared with WT (0.8±0.5%). In addition, the areal density of inflammatory cell infiltrates was significantly increased (6.6±1.5% vs. 2.2±0.7%; mdx vs. WT). Similarly, percentage of collagen deposition was significantly increased in mdx compared with WT (12.2±1.7% vs. 7.0 ± 2.2%). All indices were unaffected by drug co‐treatment. Altered fibre type distribution was apparent in the mdx diaphragm muscle. The areal and numerical density of MHCIIa fibres was significantly increased, whereas MHCIIx and IIb fibres were significantly decreased compared with WT. Differences in fibre type complement were ameliorated by drug co‐treatment.Co‐treatment restored ventilation in mdx and had a positive inotropic effect, increasing mechanical force in dystrophic diaphragm muscle. Co‐treatment restored fibre distribution in mdx but did not affect the infiltration of inflammatory cells, the proportion of fibres with evidence of central nucleation and enhanced collagen type I deposition. Preservation of MHCIIx fibres may underpin, at least in part, recovery of breathing and force production in the co‐treated mdx mice. These data may have implications for the development of pharmacotherapies for DMD with relevance to respiratory muscle performance and ventilatory status.Support or Funding InformationDepartment of Physiology (UCC) and Muscular Dystrophy Ireland. The IL‐6R antibodies were kindly gifted by Chugai Pharmaceuticals, Japan.