Improvement of muscle strength in a mouse model for congenital myopathy treated with HDAC and DNA methyltransferase inhibitors

To date there are no therapies for patients with congenital myopathies, muscle disorders causing poor quality of life of affected individuals. In approximately 30% of the cases, patients with congenital myopathies carry either dominant or recessive mutations in the ryanodine receptor 1 (RYR1) gene; recessive RYR1 mutations are accompanied by reduction of RyR1 expression and content in skeletal muscles and are associated with fiber hypotrophy and muscle weakness. Importantly, muscles of patients with recessive RYR1 mutations exhibit increased content of class II histone deacetylases and of DNA genomic methylation. We recently created a mouse model knocked-in for the p.Q1970fsX16+ p.A4329D RyR1 mutations, which are isogenic to those carried by a severely affected child suffering from a recessive form of RyR1-related multi-mini core disease. The phenotype of the RyR1 mutant mice recapitulates many aspects of the clinical picture of patients carrying recessive RYR1 mutations. We treated the compound heterozygous mice with a combination of two drugs targeting DNA methylases and class II histone deacetylases. Here, we show that treatment of the mutant mice with drugs targeting epigenetic enzymes improves muscle strength, RyR1 protein content, and muscle ultrastructure. This study provides proof of concept for the pharmacological treatment of patients with congenital myopathies linked to recessive RYR1 mutations.


Sample-size estimation
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Replicates
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Statistical reporting
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Group allocation
The information concerning replicates can be found in the Methods section. All experiments show data on biological replicates (i.e. individual mice); technical replicates are present in figure 4 where we performed calcium measurements on single FDB fibers (n ranging from 63-155) isolated from 4-6 mice. Because of fiber type heterogeneity of FDB fibers, we performed calcium measurments in a large number of single fibers isolated per mouse. Detailed information is given in Table S4. For the results of qPCR experiments shown in Figure 5A, each symbol represents one biological sample and the reaction was performed in duplicate and averaged. This information in included in the Methods section.
No mice were excluded form the data points (i.e we did not remove any outliers).
• Statistical analysis methods are reported in the Statistical analysis section at the end of the Methods section. • The statistical method that was used is also indicated in the figure legend accompanying each figure, in the results section and in the Table footnotes. • Whenever data are not presented as box plot, the reported values indicate the mean ±SD value. Except for Figure 3 and Tables a and 2 where the mean± SEM are given. 3 • Indicate how samples were allocated into experimental groups (in the case of clinical studies, please specify allocation to treatment method); if randomization was used, please also state if restricted randomization was applied • Indicate if masking was used during group allocation, data collection and/or data analysis Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: Additional data files ("source data") • We encourage you to upload relevant additional data files, such as numerical data that are represented as a graph in a figure, or as a summary table • Where provided, these should be in the most useful format, and they can be uploaded as "Source data" files linked to a main figure or table • Include model definition files including the full list of parameters used • Include code used for data analysis (e.g., R, MatLab) • Avoid stating that data files are "available upon request" Please indicate the figures or tables for which source data files have been provided: • For group allocation, mice were genotyped and randomly assigned to vehicle or drug treatment. • For ex vivo muscle force determination and grip strength assessment the experimenter was blinded as to the mouse genotype and drug treatment. This information is included in the text (results section).
The numerical data that are represented in Figures 2, 3 and 4 are given in Supplementary Tables S2, S3 and S4.