Programme Contents

Choline as a nutritional intervention to alleviate the dystrophic pathology in mdx mice

Duchenne muscular dystrophy (DMD) is a devastating muscle wasting disorder caused by a variety of mutations in the dystrophin gene. It is characterized by progressive muscle wasting and weakness leading to loss of ambulation and premature death from cardiorespiratory complications. A lack of dystrophin protein renders muscle fibres fragile and prone to membrane tears leading to impaired Ca²⁺ homeostasis, excessive inflammation, increased muscle breakdown and altered metabolism in other tissues (Stapleton et al., 2014). A cure for DMD may eventually come from corrective gene or cell therapies, but in the interim, other treatments are needed urgently to counteract the progressive muscle loss and weakness. Choline, an essential water-soluble nutrient, is involved in multiple biological processes, including modulation of inflammation and oxidative stress, and it forms a substrate for membrane phospholipids. Based on these properties, we tested the hypothesis that choline supplementation would ameliorate the dystrophic pathology in mdx mice.

All experiments were approved by the Animal Ethics Committee of The University of Melbourne and conducted in accordance with the Australian code of practice for the care and use of animals for scientific purposes (NHMRC). To assess whether a dietary intervention could slow the progression of the dystrophic pathology, three-week old male mdx mice (n=40) were fed choline-enriched feed containing 5 g/kg choline (MCHL; n=20), or a control purified laboratory feed (MCON; n=20) for four weeks. Rotarod performance, grip strength and running (wheel) distance were assessed during treatment. At the end of treatment, mice were anaesthetized deeply with sodium pentobarbitone (60 mg/kg, i.p.), and selected muscles and the liver were excised. Mice were killed by cardiac excision, while anaesthetized deeply. Maximal sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) activity was measured as a proxy of Ca²⁺ handling capacity (Gehrig et al., 2012). Muscle structure was assessed using (immuno)histochemical analyses, protein expression was assessed from western immunoblotting, while muscle and liver gene expression were analysed by qPCR.

Choline treatment did not improve functional performance in mdx mice but in the severely affected diaphragm, it blunted inflammation [macrophage infiltration (CD68 -33.0%, P<0.05)] and reduced collagen infiltration (-34.0%, P<0.05). In quadriceps muscles, choline treatment increased maximal SERCA activity (37.8%, P<0.05) and reduced markers of inflammation (Tnfα, F4/80 and Cd206 mRNA, P<0.05). Choline treatment reduced Acta2 mRNA expression (-34.0%, P<0.05) but did not alter triglyceride accumulation or other markers of inflammation and fibrosis in livers of mdx mice.

Together these data suggest that choline supplementation slowed progression of the dystrophic pathology, evident from reductions in diaphragm fibrosis and inflammation, and it enhanced maximal SERCA activity in quadriceps muscles. The reduction in fibrosis is clinically relevant for increasing the efficacy of future gene, cell and drug therapies for DMD.

Stapleton DI, Lau X, Flores M, Trieu J, Gehrig SM, Chee A, Naim T, Lynch GS & Koopman R. (2014). PLoS ONE 9, e91514.

Gehrig SM, van der Poel C, Sayer TA, Schertzer JD, Henstridge DC, Church JE, Lamon S, Russell AP, Davies KE, Febbraio MA & Lynch GS. (2012). Nature 484, 394-398.