The C57BL/10ScSn‐Dmdmdx/J (BL10‐mdx) mouse has been the most commonly used model for Duchenne muscular dystrophy (DMD) for decades. Their muscle dysfunction and pathology is, however, less severe than in patients with DMD, which complicates preclinical studies. Recent discoveries indicate that disease severity is exacerbated when muscular dystrophy mouse models are generated on a DBA2/J genetic background. Knowledge on the natural history of animal models is pivotal for high‐quality preclinical testing. However, for BL10‐mdx mice on a DBA2/J background (D2‐mdx), limited data are available. We addressed this gap in the natural history knowledge. First, we compared histopathological aspects in skeletal muscles of young D2‐mdx, BL10‐mdx, and wild‐type mice. Pathology was more pronounced in D2‐mdx mice and differed in severity between muscles within individuals. Secondly, we subjected D2‐mdx mice to a functional test regime for 34 weeks and identified that female D2‐mdx mice outperform severely impaired males, making females less useful for functional preclinical studies. Direct comparisons between 10‐ and 34‐wk‐old D2‐mdx mice revealed that disease pathology ameliorates with age. Heart pathology was progressive, with some features already evident at a young age. This natural history study of the D2‐mdx mouse will be instrumental for experimental design of future preclinical studies.—Van Putten, M., Putker, K., Overzier, M., Adamzek, W. A., Pasteuning‐Vuhman, S., Plomp, J. J., Aartsma‐Rus, A. Natural disease history of the D2‐mdx mouse model for Duchenne muscular dystrophy. FASEB J. 33, 8110–8124 (2019). http://www.fasebj.org

Limb-girdle muscular dystrophy types 2D and 2F (LGMD 2D and 2F) are autosomal recessive disorders caused by mutations in the alpha- and delta sarcoglycan genes, respectively, leading to severe muscle weakness and degeneration. The cause of the disease has been well characterized and a number of animal models are available for pre-clinical studies to test potential therapeutic interventions. To facilitate transition from drug discovery to clinical trials, standardized procedures and natural disease history data were collected for these mouse models. Implementing the TREAD-NMD standardized operating procedures, we here subjected LGMD2D (SGCA-null), LGMD2F (SGCD-null) and wild type (C57BL/6J) mice to five functional tests from the age of 4 to 32 weeks. To assess whether the functional test regime interfered with disease pathology, sedentary groups were taken along. Muscle physiology testing of tibialis anterior muscle was performed at the age of 34 weeks. Muscle histopathology and gene expression was analysed in skeletal muscles and heart.Muscle histopathology and gene expression was analysed in skeletal muscles and heart. Mice successfully accomplished the functional tests, which did not interfere with disease pathology. Muscle function of SGCA- and SGCD-null mice was impaired and declined over time. Interestingly, female SGCD-null mice outperformed males in the two and four limb hanging tests, which proved the most suitable non-invasive tests to assess muscle function. Muscle physiology testing of tibialis anterior muscle revealed lower specific force and higher susceptibility to eccentric-induced damage in LGMD mice. Analyzing muscle histopathology and gene expression, we identified the diaphragm as the most affected muscle in LGMD strains. Cardiac fibrosis was found in SGCD-null mice, being more severe in males than in females. Our study offers a comprehensive natural history dataset which will be useful to design standardized tests and future pre-clinical studies in LGMD2D and 2F mice.

Limb girdle muscular dystrophy (LGMD) types 2D and 2F are caused by mutations in the genes encoding for α- and δ-sarcoglycan, respectively, leading to progressive muscle weakness. Mouse models exist for LGMD2D ( Sgca -/- ) and 2F ( Sgcd -/- ). In a previous natural history study, we described the pathology in these mice at 34 weeks of age. However, the development of muscle pathology at younger ages has not been fully characterised yet. We therefore performed a study into age-related changes in muscle function and pathology by examining mice at different ages. From 4 weeks of age onwards, male mice were subjected to functional tests and sacrificed at respectively 8, 16 or 24 weeks of age. Muscle histopathology and expression of genes involved in muscle pathology were analysed for several skeletal muscles, while miRNA levels were assessed in serum. In addition, for Sgcd -/- mice heart pathology was assessed. Muscle function showed a gradual decline in both Sgca -/- and Sgcd -/- mice. Respiratory function was also impaired at all examined timepoints. Already at 8 weeks of age, muscle pathology was prominent, and fibrotic, inflammatory and regenerative markers were elevated, which remained relatively constant with age. In addition, Sgcd -/- mice showed signs of cardiomyopathy from 16 weeks of age onwards. These results indicate that Sgca -/- and Sgcd -/- are relevant disease models for LGMD2D and 2F.

Merosin deficient congenital muscular dystrophy 1A (MDC1A) is a very rare autosomal recessive disorder caused by mutations in the LAMA2 gene leading to severe and progressive muscle weakness and atrophy. Although over 350 causative mutations have been identified for MDC1A, no treatment is yet available. There are many therapeutic approaches in development, but the lack of natural history data of the mouse model and standardized outcome measures makes it difficult to transit these pre-clinical findings to clinical trials. Therefore, in the present study, we collected natural history data and assessed pre-clinical outcome measures for the dy2J/dy2J mouse model using standardized operating procedures available from the TREAT-NMD Alliance. Wild type and dy2J/dy2J mice were subjected to five different functional tests from the age of four to 32 weeks. Non-tested control groups were taken along to assess whether the functional test regime interfered with muscle pathology. Respiratory function, body weights and creatine kinase levels were recorded. Lastly, skeletal muscles were collected for further histopathological and gene expression analyses. Muscle function of dy2J/dy2J mice was severely impaired at four weeks of age and all mice lost the ability to use their hind limbs. Moreover, respiratory function was altered in dy2J/dy2J mice. Interestingly, the respiration rate was decreased and declined with age, whereas the respiration amplitude was increased in dy2J/dy2J mice when compared to wild type mice. Creatine kinase levels were comparable to wild type mice. Muscle histopathology and gene expression analysis revealed that there was a specific regional distribution pattern of muscle damage in dy2J/dy2J mice. Gastrocnemius appeared to be the most severely affected muscle with a high proportion of atrophic fibers, increased fibrosis and inflammation. By contrast, triceps was affected moderately and diaphragm only mildly. Our study presents a complete natural history dataset which can be used in setting up standardized studies in dy2J/dy2J mice.

Duchenne muscular dystrophy is a severe muscle wasting disease, characterized by a severely reduced lifespan in which cardiomyopathy is one of the leading causes of death. Multiple therapies aiming at dystrophin restoration have been approved. It is anticipated that these therapies will maintain muscle function for longer and extend the ambulatory period, which in turn will increase the cardiac workload which could be detrimental for cardiac function. We investigated the effects of voluntary running exercise in combination with low dystrophin levels on function and pathology of skeletal muscle and heart. We divided 15.5-month old female mdx (no dystrophin), mdx-Xist Δhs (varying low dystrophin levels) and wild type mice (BL10-WT and Xist Δhs -WT) to either a sedentary or voluntary wheel running regime and assessed muscle function at 17.5 months of age. Thereafter, a cardiac MRI was obtained, and muscle and heart histopathology were assessed. We show that voluntary exercise is beneficial to skeletal muscle and heart function in dystrophic mice while not affecting muscle pathology. Low amounts of dystrophin further improve skeletal muscle and cardiac function. These findings suggest that voluntary exercise may be beneficial for skeletal muscle and heart in DMD patients, especially in conjunction with low amounts of dystrophin. key characteristic of cardiomyopathy is the dilated left ventricle, found in the majority of older DMD patients [3][4][5], while studies on right ventricular function are less coherent [6][7][8]. Early detection is challenging, since cardiac involvement does not always result into clinical symptoms due to exercise intolerance resulting from skeletal muscle weakness.Not only DMD patients, but also Becker muscular dystrophy (BMD) patients suffer from cardiac myopathy. BMD patients have in-frame mutations in the DMD gene leading to synthesis of reduced levels of truncated, but partially functional dystrophin proteins. Consequently, their muscle pathology is less severe than DMD patients. However, despite the expression of low dystrophin levels, about two thirds of BMD patients develop cardiomyopathy [9][10][11]. Similarly, DMD and BMD carriers, expressing 50% of dystrophin, also have an increased risk for cardiomyopathy [12,13].Currently there are two therapeutic strategies approved aimed to