Characterisation of skeletal development and the use of anabolic agents in murine models of Duchenne muscular dystrophy

Lead Research Organisation: University of Edinburgh
Department Name: The Roslin Institute


Duchenne muscular dystrophy (DMD) is a severe and ultimately fatal disease. It affects up to 1 in 4000 males and there is no cure. Steroids are the only treatment that can help slow down muscle weakness but they have many side effects including growth failure and osteoporosis. It is unclear exactly why, but even boys who are not treated with steroids tend to be short and have weak bones. We do not know whether the bone and growth problems are just caused by the muscle weakness (our bones need to be 'loaded' by muscle activity in order to strengthen and grow efficiently), or whether there is another primary problem. It may be that because DMD is a chronic inflammatory process, this disrupts the growth hormone (GH)/insulin-like growth factor (IGF-1) pathway. Growth hormone is produced by the anterior pituitary gland. It can act directly on the skeleton, but mainly acts indirectly, through IGF-1. Together the GH/IGF-1 pathway plays a crucial role in regulating normal bone growth and bone mass during childhood, and children with either GH or IGF-1 deficiency are short and have osteoporosis. There are no treatments available to help bone growth in DMD.

We will investigate the role of GH and IGF-1 in bone and growth of mice models of DMD. The mouse model commonly used in DMD is the X-linked Muscular Dystrophy (mdx) mouse, but it has significant limitations because the disease process is not as severe and muscle regeneration occurs in the mdx mice, unlike in patients with Duchenne. Very few medications that are effective in the mdx mouse have shown to be useful in patients. Therefore we will use a new mouse model, the Cmah-/-mdx mouse. This mouse is bred from the mdx mouse, but also has an additional gene deletion to make it more 'human' and therefore we hope will behave in more similar way to patients with Duchenne. I will look, for the first time, at bone and growth in the Cmah-/-mdx and compare it to the mdx and a healthy 'wildtype' mouse. I will then give the mice steroids to determine the effects on their growth and skeleton. I will also see if the Cmah-/mdx mouse responds to GH and IGF-1, both alone and in combination. To do this I will use a range of techniques, including micro CT and labeling of the bones and growth plate to monitor the rate of growth. I will also culture metatarsals (feet) bones from the embryos of these mice models to see if they also display reduced growth and bone fragility. If they do this will show us that there is a primary problem relating to the skeleton in addition to the problems caused by steroids and muscle weakness.

This work is very important because both GH and IGF-1 are readily available and are already used in children. If they are found to help bone development and encourage growth, either alone or acting together, in mice models, then this work could be carried out as a clinical trial in patients with DMD. It is also important to further describe the new Cmah-/-mdx mouse model, so that it can be used in other DMD experiments. The data that we collect may also help in the design of new therapies that are directed specifically at the mechanism of the problem that leads to poor bone health and growth in DMD.

Technical Summary

Short stature and osteoporosis are common in Duchenne Muscular Dystrophy (DMD) and its pathophsyiology may include a paracrine as well as an endocrine abnormality of the GH/IGF-1 axis, which is further exacerbated by glucocorticoid (GC) use. The commonly used mdx mouse has a phenotype which is less severe than DMD boys. A new Cmah-/-mdx mouse model has increased disease severity, but its skeletal phenotype has never been investigated.

To test the overall hypothesis that murine models of DMD have an intrinsic abnormality of linear growth and skeletal development that can be rescued by modulation of the GH/IGF-1 axis, I will investigate:-
1. Growth and growth plate (GP) chondrogenesis in mdx and Cmah-/-mdx murine models,expecting impairment in both, and increased severity in Cmah-/-mdx. A cross-sectional study of mice culled at 2,4 and 6 wks will be performed to determine linear bone growth rate at the chondro-osseus junction. Chondrocyte hypertrophy and proliferation will be quantified using image analysis and bromodeoxyuridine uptake. GP cartilage matrix production and GH/IGF receptor, IGF1,2 and IGFBP3 expression will be confirmed by immunohistochemistry. Metatarsal growth in explant culture will also be studied to examine any intrinsic skeletal abnormality.
2. Bone development and fragility in the Mdx and Cmah-/-mdx. A cross-sectional study of bone quality by micro-CT will be performed and the bone marrow studied to understand adipogenesis and osteoblastogenesis.
3. Growth response and bone development following exposure to GC,expecting them to be further reduced by daily dexamethasone.
4. Growth and bone response following treatment with rhGH and/or rhIGF-1, expecting combination therapy to be most effective.
If rhGH or rhIGF-1 rescue bone and growth pre-clinically then this could be translated clinically. These data may also help inform the design of novel therapies directed specifically at the mechanism of insult leading to skeletal fragility in DMD.

Planned Impact

Academia: As detailed in the academic beneficiaries section, there are several ways in which the scientific and clinical communities will benefit from the research output of this project. In addition, by fulfilling the objectives outlined in the proposal, I will also gain significant laboratory experience to complement my clinical research experience which will have a significant impact on my career development, in the short, medium and long term.

NHS: The findings from this study will potentially benefit patients across multiple specialties. Firstly, this project is the first part of a process to improve the management of short stature and osteoporosis in DMD. These are two important and debilitating issues (both physically and psychologically) that patients with Duchenne face, and strategies to manage them would have a large impact on the quality of health and morbidity of this population. One of the main translational impacts of this research would be a pilot clinical trial in DMD to evaluate the effectiveness of combined GH and IGF therapy, assuming that these are proven to be effective in the mouse models. As both agents are already licensed for use in specific situations in the paediatric population, this trial could potentially be set-up within a medium term timeframe.

Patient groups: The John Walton Muscular Dystrophy Research Centre is closely involved with a number of DMD patient groups to identify areas of unmet need. In particular, a recent Parent Project Muscular Dystrophy Conference highlighted the importance of research into strategies to improve bone health in DMD. The letter of support (attached) from Duchenne Now also emphasises the need for preventative strategies to tackle skeletal fragility and short stature.

General Public: Although DMD is a rare disease, there are significant costs associated with the multi-disciplinary care and management of the condition. Until a cure is found, fractures in DMD will continue to have a large cost implication, in terms of health care requirements, reducing independence and the potential for loss of earnings. This research is an early step towards reducing the morbidity, and therefore health care costs, associated with the condition.

Industry: If we can prove the beneficial role of GH and IGF-1 for the skeleton in Cmah-/-mdx mouse then the data and a potential opportunity to be involved in follow-on work would be of interest to pharmaceutical companies. Through the TREAT-NMD network (described in the communications plan) and dissemination of findings at conferences and through publishing, we will be able to communicate our translational research findings to industry and thereby establish a direct pathway to impact of any potential novel observations made through this grant.

Policy Makers: The UK is already heavily involved in the development and ongoing revision of Standards of Care for the multi-disciplinary management of DMD (led by Professor Kate Bushby from the JWMDRC on behalf of the DMD Care Considerations Working Group). This work would potentially have an impact on policy if it becomes clear that GH and IGF-1 may have a role in the holistic management of boys with DMD.


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Description Created new clinical guidelines- Bone health in DMD
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Membership of a guideline committee
Impact Guidelines have led to improved concordance with recently published National standards of care for DMD and optimised patient care.
Description Have initiated a joint muscle endocrine clinic to serve neuromuscular patients in North East England
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Influenced training of practitioners or researchers
Impact New muscle endocrine clinic will facilitate the access to endocrine services for patients in the North East of England with neuromuscular diseases and enhance the standard of care that they receive.
Description British Society of Paediatric Endocrinology Research Award
Amount £10,000 (GBP)
Organisation Royal College of Paediatrics and Child Health (RCPCH) 
Department British Society for Paediatric Endocrinology and Diabetes (BSPED)
Sector Charity/Non Profit
Country United Kingdom
Start 01/2018 
End 01/2019
Description Testosterone Therapy for Pubertal Delay in Duchenne Muscular Dystrophy (clinical trial ref NCT02571205)
Amount £211,422 (GBP)
Organisation Duchenne Now 
Sector Charity/Non Profit
Country Unknown
Start 12/2015 
End 04/2019
Description Invited to give talk entitled, "Growth and development of muscle and bone in neuromuscular disorders at MDUK Scottish Conference in November 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Patients, carers and/or patient groups
Results and Impact I was asked to give a talk at the Scottish Muscular Dystrophy UK about my current research as part of my MDUK/MRC clinical research training fellowship.
Year(s) Of Engagement Activity 2017