Duchenne muscular dystrophy (DMD) patients suffer from a genetic mutation preventing synthesis of functional dystrophin proteins. In absence of dystrophin, muscle fibers are prone to injury leading to severe muscle weakness and wheelchair dependency. Ultimately due to cardiorespiratory failure DMD patients die between the 2nd and 4th decade of life (1–3). There are several therapeutic approaches resulting in dystrophin restoration in skeletal muscle, while cardiac myocytes are more difficult to target. Dystrophin restoring therapies aim to improve muscle function, leading to increased physical activity and thus to increased cardiac workload, thereby potentially affecting (cardio)myopathy. There are two hypotheses on the effect of exercise on disease progression in DMD, 1: “Use it or lose it”: a beneficial relation between exercise and heart and muscle function, and 2: “Use it and lose it”: increased muscle and cardiac workload accelerates (cardio)myopathy. Therefore, we studied the effect of low intensity exercise on skeletal muscle and cardiac function in DMD mouse models expressing varying dystrophin levels. We subjected 15.5 month old female mdx-Xist∆hs mice expressing low dystrophin levels based on non-random X-inactivation (quadriceps: 3-40%, heart: 3-14%, n=28), C57Bl/10ScSn-mdx/J (mdx) (0% dystrophin, n=8), and their respective wild type strains C57BL/10ScSnJ (Bl10-WT, n=8) and Xist∆hs (Xist-WT, n=8) to voluntary wheel running or no exercise for a period of two months. Mice were housed in individual cages containing a running wheel for eight sessions of 3-4 days for 28 days. Thereafter, muscle function was assessed in a two-week functional test regime consisting of grid and wire hanging tests. At the age of 18 months, a MRI scan was acquired to assess cardiac function. Mice were scanned on a 7 Tesla Bruker Pharmascan with ParaVision 5.1. A retrospective Intragrate FLASH sequence was used to acquire short-axis images, with FA=18°, TE=1.7ms, TR=6ms, FOV=3.4x3.4cm, matrix=256x144, repetitions=256 and 8-9 slices. Images were segmented using MASS for Mice (4) to calculate ejection fraction (EF), stroke volume, cardiac output (CO) and end-diastolic volume of the left and right ventricles. All cardiac parameters were normalized to body mass. Cardiac data presented here are only incomplete for the mdx-Xist∆hs mice (n=10 instead of 28). Fibrosis of the quadriceps and heart was assessed by histological assessments and dystrophin levels of the mdx-Xist∆hs mice by Western blot.

Mdx mice ran 6.7 km, mdx-Xist∆hs 9.4 km and Bl10-WT and Xist-WT wild-type mice 10.1 and 14.3 km respectively per night on the voluntary wheel (figure 1). Muscle function was impaired in mdx mice, but improved for both hanging tests in mdx-Xist∆hs mice. Voluntary wheel exercise improved hanging performance in all strains (figure 2). Fibrosis of the quadriceps was pronounced in mdx mice, while it partly normalized to wild type levels in mdx-Xist∆hs mice. Voluntary wheel exercise did not exacerbate fibrosis.

Cardiac hypertrophy, determined by heart-to-body mass ratio, was only observed in mdx mice, but not in mdx-Xist∆hs or wild type mice and was not affected by the exercise regime (figure 3). EF and CO were reduced in mdx mice compared to Bl10-WT mice. Low amounts of dystrophin (3-14%) in the mdx-Xist∆hs mice improved EF and CO compared to mdx mice. Collagen infiltration was highest in mdx mice, less pronounced in mdx-Xist∆hs mice and did not increase upon exercise in any of the strains.

Exercise improved CO in all strains, but CO was still reduced in mdx mice. In mdx-Xist∆hs mice, exercise renormalized CO to wild type levels and the amount of voluntary exercise for this group was similar to their WT controls (figure 1+4).

Low amounts of dystrophin improved muscle function assessed with hanging tests and decreased muscle pathology in the aged mdx-Xist∆hs mice. This is in line with previous reports in young mdx-Xist∆hs mice (5). The increased cardiac workload improved CO in all strains, even in the mdx mice, where cardiac function is already compromised by phenotype. Cardiomyopathy did not deteriorate further between the age of 10 and 18 months in mdx mice (6).

We observed a significant strain effect, with mice on a Bl6/Bl10 (mdx-Xist∆hs and Xist-WT mice) background exercising much less than mice on a Bl10 background. These strain differences may have had an effect on the disease progression of the mdx-Xist∆hs mice due to the intrinsic behavioral differences between the backgrounds.

These results show that low dystrophin levels improve skeletal muscle and cardiac function and suggest that low intensity exercise is beneficial for both cardiac and muscle function in both dystrophic and wild type mice.