Natural disease history of mouse models for limb girdle muscular dystrophy types 2D and 2F

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 miceFunctional Genomics of Muscle, Nerve and Brain Disorder

New function of the myostatin/activin type I receptor (ALK4) as a mediator of muscle atrophy and muscle regeneration

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

Towards therapies for muscular dystrophies : targeting TGF-beta and myostatin signalling to improve muscle quality and development of reliable outcome measures for preclinical mouse models

The muscular dystrophies are a heterogeneous collection of inherited and progressive myogenic disorders sharing similar clinical “dystrophic” characteristics. Many therapies are currently being tested in clinical trials and several of them achieved marketing authorisation. Additionally, many promising treatments are currently being tested in preclinical animal models. At a pathological level, patients show chronic inflammation and degeneration of muscle fibers, which eventually are replaced by adipose and fibrotic tissue leading to loss of muscle tissue and function. Targeting pathways involved in regulation of muscle regeneration and fibrosis can improve muscle quality and function. This would be beneficial for many patients with neuromuscular disorders, which share similar pathology. There is a great demand to accelerate the translation of potential new treatments from the bench to the clinic. Therefore, natural history studies and reliable outcome measures are required for preclinical mouse models improving study design and comparison between studies. To address the lack of a therapy targeting muscle pathology, the thesis focuses on evaluating therapeutic potential of antisense oligonucleotides inhibition of TGF-β and myostatin type I receptors and elucidating their function in muscles. Additionally, this thesis contains a natural history dataset that can be useful to design preclinical studies in dystrophic mouse models. </div

Natural disease history of the D2-mdx mouse model for Duchenne muscular dystrophy

Functional Genomics of Muscle, Nerve and Brain Disorder

Efficient downregulation of Alk4 in skeletal muscle after systemic treatment with conjugated siRNAs in a mouse model for Duchenne muscular dystrophy

Downregulation of genes involved in the secondary pathology of Duchenne muscular dystrophy, for example, inflammation, fibrosis, and adiposis, is an interesting approach to ameliorate degeneration of muscle and replacement by fibrotic and adiposis tissue. Small interfering RNAs (siRNAs) are able to downregulate target genes, however, delivery of siRNAs to skeletal muscle still remains a challenge. We investigated delivery of fully chemically modified, cholesterol-conjugated siRNAs targeting Alk4, a nontherapeutic target that is expressed highly in muscle. We observed that a single intravenous or intraperitoneal (IP) injection of 10 mg/kg resulted in significant downregulation of Alk4 mRNA expression in skeletal muscles in both wild-type and mdx mice. Treatment with multiple IP injections of 10 mg/kg led to an overall reduction of Alk4 expression, reaching significance in tibialis anterior (39.7% +/- 6.2%), diaphragm (32.7% +/- 5.8%), and liver (41.3% +/- 29.9%) in mdx mice. Doubling of the siRNA dose did not further increase mRNA silencing in muscles of mdx mice. The chemically modified conjugated siRNAs used in this study are very promising for delivery to both nondystrophic and dystrophic muscles and could have major implications for treatment of muscular dystrophy pathology.</p

Efficient Downregulation of Alk4 in Skeletal Muscle After Systemic Treatment with Conjugated siRNAs in a Mouse Model for Duchenne Muscular Dystrophy

Downregulation of genes involved in the secondary pathology of Duchenne muscular dystrophy, for example, inflammation, fibrosis, and adiposis, is an interesting approach to ameliorate degeneration of muscle and replacement by fibrotic and adiposis tissue. Small interfering RNAs (siRNAs) are able to downregulate target genes, however, delivery of siRNAs to skeletal muscle still remains a challenge. We investigated delivery of fully chemically modified, cholesterol-conjugated siRNAs targeting Alk4, a nontherapeutic target that is expressed highly in muscle. We observed that a single intravenous or intraperitoneal (IP) injection of 10 mg/kg resulted in significant downregulation of Alk4 mRNA expression in skeletal muscles in both wild-type and mdx mice. Treatment with multiple IP injections of 10 mg/kg led to an overall reduction of Alk4 expression, reaching significance in tibialis anterior (39.7% +/- 6.2%), diaphragm (32.7% +/- 5.8%), and liver (41.3% +/- 29.9%) in mdx mice. Doubling of the siRNA dose did not further increase mRNA silencing in muscles of mdx mice. The chemically modified conjugated siRNAs used in this study are very promising for delivery to both nondystrophic and dystrophic muscles and could have major implications for treatment of muscular dystrophy pathology.Functional Genomics of Muscle, Nerve and Brain Disorder

Natural disease history of mouse models for limb girdle muscular dystrophy types 2D and 2F

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</p

Natural disease history of the dy2J mouse model of laminin α2 (merosin)-deficient congenital muscular dystrophy.

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

New function of the myostatin/activin type I receptor (ALK4) as a mediator of muscle atrophy and muscle regeneration.

Functional Genomics of Muscle, Nerve and Brain Disorder

Impaired muscle function and integrity in SGCA- and SGCD-null mice.

<p><b>(a)</b> Normalized four limb grip strength was significantly lower in SGCD-null mice than in wild type and SGCA-null mice <b>(b)</b> Maximum hanging time with two limbs was significantly shorter in SGCA- and SGCD-null mice when compared to wild type mice. SGCD-null mice outperformed SGCA-null mice. Maximum hanging time was significantly shorter in male SGCD-null mice than in female SGCD-null mice. <b>(c)</b> Maximum hanging time with four limbs was significantly shorter in SGCA- and SGCD-null mice when compared to wild type mice. SGCD-null mice outperformed SGCA-null mice. Maximum hanging time was significantly shorter in male SGCD-null mice than in female SGCD-null mice. <b>(d)</b> Creatine kinase levels were significantly elevated in both LGMD strains. <b>(e)</b> No significant differences were detected in respiration amplitude between the mouse models. <b>(f)</b> Respiration rate was significantly decreased in SGCA- and SGCD-null mice at 15 and 34 weeks of age compared to wild type mice. At 15 and 34 weeks of age, SGCA-null mice showed significantly lower respiration rate than SGCD-null mice. No differences were found between males (in blue) and females (in pink). <b>(g)</b> Force frequency relationship of the tibialis anterior muscle. Each data point represents the force measured at each frequency. Muscles of both SGCA- and SGCD-null mice showed a significantly lower specific force than those of wild type mice. SGCD-null muscles generated a significantly lower specific force than those of SGCA-null mice. <b>(h)</b> Relative changes in tetanic force during eleven cycles of eccentric contraction in tibialis anterior muscle. The tetanic tension developed during the first cycle was taken as 100%. The isometric force significantly dropped by 10–15% in SGCA- and SGCD-null mice, while it remained unchanged in wild type mice. For e and f * Indicates a significant difference from wild type (WT) controls. # Indicates a significant difference from SGCA-null mice. Error bars represent standard error of the mean (± SEM).</p