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.

Skeletal muscle fibrosis and impaired muscle regeneration are major contributors to muscle wasting in Duchenne muscular dystrophy (DMD). Muscle growth is negatively regulated by myostatin (MSTN) and activins. Blockage of these pathways may improve muscle quality and function in DMD. Antisense oligonucleotides (AONs) were designed specifically to block the function of ALK4, a key receptor for the MSTN/activin pathway in skeletal muscle. AON-induced exon skipping resulted in specific Alk4 down-regulation, inhibition of MSTN activity, and increased myoblast differentiation in vitro. Unexpectedly, a marked decrease in muscle mass (10%) was found after Alk4 AON treatment in mdx mice. In line with in vitro results, muscle regeneration was stimulated, and muscle fiber size decreased markedly. Notably, when Alk4 was down-regulated in adult wild-type mice, muscle mass decreased even more. RNAseq analysis revealed dysregulated metabolic functions and signs of muscle atrophy. We conclude that ALK4 inhibition increases myogenesis but also regulates the tight balance of protein synthesis and degradation. Therefore, caution must be used when developing therapies that interfere with MSTN/activin pathways.—Pasteuning-Vuhman, S., Boertje-van der Meulen, J. W., van Putten, M., Overzier, M., ten Dijke, P., Kiełbasa, S. M., Arindrarto, W., Wolterbeek, R., Lezhnina, K. V., Ozerov, I. V., Aliper, A. M., Hoogaars, W. M., Aartsma-Rus, A., Loomans, C. J. M. New function of the myostatin/activin type I receptor (ALK4) as a mediator of muscle atrophy and muscle regeneration.

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.