Aberrant Respiratory Control in the Mdx Mouse Model of Duchenne Muscular Dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is a neuromuscular disease that results in the absence of the dystrophin protein, leading to severe muscle weakness. Although patients suffer from cardio‐respiratory failure, there is a paucity of information regarding the control of breathing in pre‐clinical models of DMD.We measured ventilation, oxygen consumption (VO2) and carbon dioxide production (VCO2) in freely behaving mdx (C57BL/10ScSn‐Dmdmdx/J; n=12) and wild‐type (C57BL/10ScSn; n=13) mice at 8 weeks of age during normoxia (FiO2=0.21) and in response to a graded hypoxic challenge (FiO2=0.15, 0.12, 0.1 & 0.08). Carotid sinus nerve activity was measured ex vivo in an artificially perfused preparation in WT (n=6) and mdx (n=6) mice during normoxia (Po2=100 Torr) and graded hypoxia (Po2=80, 60, 40 Torr). Diaphragm EMG was recorded in urethane (1.8 g/kg) anaesthetised WT (n=9) and mdx (n=7) mice during eupnea and in response to chemostimulation (5% CO2/15%O2). Diaphragm muscle force‐frequency relationship was examined ex vivo (n=7 per group). qRT‐PCR was used to examine gene expression in diaphragm muscle from WT and mdx (n=8 per group) mice. Data were expressed as mean±SD and were statistically compared by unpaired Student t‐test or two‐way ANOVA with Bonferroni post‐hoc test.Minute ventilation (VE) was significantly reduced in mdx mice compared with WT. There was no significant difference in VO2 and VCO2 between WT and mdx when expressed in absolute terms. Carotid sinus nerve unitary discharge was significantly depressed during normoxia in mdx compared with WT ex vivo. Chemostimulation increased diaphragm EMG activity in all animals, with mdx displaying significantly enhanced reflex responsiveness to chemostimulation (amplitude and area under curve of integrated EMG signal; % change from baseline). Diaphragm force was significantly reduced in mdx compared with WT across all stimulation frequencies ex vivo. Immunofluorescence for MHC fibre types revealed decreased type 2B and increased type 2A in mdx diaphragm. In addition, mdx diaphragm had significantly increased central nucleation and inflammatory cell infiltration. NF‐kB and TNF‐α mRNA expression were both significantly increased in mdx diaphragm compared with WT.A significant respiratory phenotype presents at a young age in the mdx mouse. Mdx mice hypoventilate during normoxia (decreased VE/VCO2), with evidence of hypo‐activity of the carotid body during normoxia, and impaired force‐generating capacity. We observed unaltered carotid body and ventilatory responsiveness to hypoxia in mdx but enhanced neural activation of diaphragm muscle (EMG) in response to chemostimulation. Mdx diaphragm muscle has severe mechanical dysfunction, is in a pro‐inflammatory state and has altered fibre type composition and distribution. Our data suggest potentiated neural output for a given chemosensory input but this is insufficient to overcome sensory and muscle deficits and therefore hypoventilation prevails. We reveal that respiratory control is altered in the mdx mouse model of DMD at a young age.Support or Funding InformationDepartment of Physiology (UCC) and Muscular Dystrophy Ireland.