Proceedings of The Physiological Society

Physiology 2016 (Dublin, Ireland) (2016) Proc Physiol Soc 37, PCB059

Poster Communications

Chronic Tempol supplementation restores diaphragm muscle force-generating capacity in the dystrophin-deficient mdx mouse

D. P. Burns1, C. Rieux1, I. Ali1, J. Healy1, D. Edge2, K. D. O' Halloran1

1. Physiology, University College Cork, Cork, County Cork, Ireland. 2. Physiology, Trinity Biomedical Sciences Institute, Trinity College Dublin, the University of Dublin, Dublin, Ireland.

Duchenne muscular dystrophy (DMD) is a neuromuscular disease characterised by skeletal muscle weakness (limb & respiratory muscle). DMD is caused by disruption to the dystrophin gene, leading to the absence of the structural protein - dystrophin. Patients die due to respiratory and cardiac failure. Reactive oxygen species (ROS) are important modulators of respiratory muscle function in health and disease. There is evidence of aberrant redox signalling in DMD and a pre-clinical model of DMD, the mdx mouse. We sought to examine the effects of acute and chronic antioxidant (Tempol; superoxide scavenger) treatment on diaphragm muscle dysfunction in the mdx mouse. Fourteen week old mdx (C57BL/10ScSn-Dmdmdx/J; n=22) and wild-type (WT; C57BL/10ScSn; n=7) mice were studied. Ex vivo diaphragm muscle preparations were examined in four groups: WT (n=7), mdx (n=7), mdx & Tempol in vitro (n=7) and mdx & Tempol in vivo (n=8). Diaphragm muscle mechanical properties were examined using a dual-mode lever transducer system. For mdx & Tempol in vitro, diaphragm muscle was incubated in Tempol (10mM). Mdx & Tempol in vivo received Tempol (1mM) supplementation in the drinking water for 2 weeks (12-14 weeks of age). The enzymatic activity of citrate synthase (CS), lactate dehydrogenase (LDH) and phosphofructokinase (PFK) was examined in diaphragm muscle from the WT, mdx and mdx & Tempol in vivo groups by use of commercial spectrophotometric assays. Data are reported as mean±SD and were statistically compared by unpaired Student t-test. Peak specific force (Fmax) was significantly reduced in mdx (12.0±1.8 N/cm2; p<0.0001) compared with WT (22.4±4.1). Application of 10mM Tempol to mdx diaphragm had no effect on mechanical force (12.1±1.4). Chronic tempol supplementation significantly increased mdx diaphragm force (20.1±5.8; p=0.006; mdx Tempol in vivo vs mdx). CS activity was significantly reduced in mdx (0.5±0.1 µmol/min/mg protein; p=0.0003) compared with WT (0.8±0.1); in vivo Tempol significantly increased CS activity in mdx (0.7±0.1; p=0.005). LDH activity was significantly reduced in mdx (0.5±0.2; p=0.001) compared with WT (1.0±0.3); in vivo Tempol significantly increased LDH activity in mdx (0.9±0.2; p=0.002). PFK activity was equivalent in all groups. Pronounced muscle weakness and redox stress is evident in the mdx mouse diaphragm at 14 weeks of age. Acute treatment with Tempol did not rescue force in mdx diaphragm suggestive of structural modelling due to chronic oxidative stress. Chronic treatment with Tempol in vivo restored mdx diaphragm muscle force and metabolic enzyme activity indicating that antioxidant intervention prevents aberrant muscle plasticity secondary to oxidative stress. Antioxidants could serve as useful adjunctive therapies in the treatment of DMD.

Where applicable, experiments conform with Society ethical requirements