Final Report Summary - MUSCLE REPAIR MDX (Molecular mechanisms involved in skeletal muscle repair and muscular dystrophy.)
Objectives of this project were to identify the role of Histone Deacetylase 4 (HDAC4) in skeletal muscle during regeneration, in satellite cells and in muscular dystrophy with a loss-of-function approach. To study HDAC4 in skeletal muscle with a genetic approach, we generated different tissue-specific KO mouse lines. In the first one, HDAC4 deletion occurs in adult skeletal muscle. Mice are viable and fertile and do not show an obvious phenotype in skeletal muscle. Satellite cells, the muscle stem cells, are the cellular population mainly responsible for skeletal muscle regeneration. To assess if HDAC4 affects satellite cell behavior, we also generate a mouse line in which HDAC4 is deleted only in muscle stem cells.
To study muscle regeneration, we freeze-injured the muscles of control and HDAC4 KO mice and analyzed muscle regeneration by histological and molecular analyses, over time. We found that HDAC4 KO muscles cannot efficiently regenerate, demonstrating that the lack of HDAC4 in skeletal muscle delays skeletal muscle regeneration.
We have also characterized satellite cell proliferation and differentiation in vitro, from both the KO mouse lines. HDAC4 KO satellite cells proliferate less (with no differences in apoptosis) and cannot efficiently differentiate in muscle lineage, showing aberrant expression of fibrotic, adipose and myeloid marker genes. This phenotype is reflected in vivo with impaired regeneration in HDAC4 KO mice, still evident one month following injury, and striking accumulation of fibrotic and adipose tissues after serial injury experiments, highlighting the importance of HDAC4 for satellite cell pool maintenance.
Searching for the molecular pathways altered by HDAC4, we performed sequencing analysis (RNA-seq) on the transcripts from control and HDAC4 KO samples, followed by chromatin-immunoprecipitation analyses to define the HDAC4 direct targets. From the RNA-seq results, the lack of HDAC4 mostly affects the expression of genes related with cell proliferation and oxidative redox balance in satellite cells.
To determine if HDAC4 affects muscular dystrophy, we crossed HDAC4 KO mice with mdx mice, the murine model of Duchenne muscular dystrophy, and analyzed the disease progression over time in two of the most affected muscles, the diaphragm and the gastrocnemius. For muscular dystrophy, male and female mice were analyzed separately, from 3 weeks to 16 month-old, by assessing muscle mass and by histological and functional analyses. Lack of HDAC4 in skeletal muscle worsened the pathological features of dystrophic musclesWe choose to pursue the analyses on the diaphragm muscles of female mice, and we performed transcriptome sequencing analysis to evaluate the molecular pathways altered by HDAC4 absence in mdx mice. The results highlight the pathways mostly affected by HDAC4 in skeletal muscle in muscular dystrophy.
The expected final results of this proposal were to elucidate the role of HDAC4 in skeletal muscle regeneration, in satellite cells and in muscular dystrophy, and they have been mostly achieved. The lack of HDAC4 in skeletal muscle negatively affects muscle regeneration program, while the absence of HDAC4 in satellite cells blocks their proliferation and compromised their commitment in muscle lineage. Moreover, the absence of HDAC4 in skeletal muscle worsened the pathological features of muscular dystrophy. These findings are important on the light of the fact that pan-HDAC inhibitors are in clinical trial for the treatment of muscular dystrophies, despite showing side effects.
Understanding the role of HDAC4 and the pathways affected by its absence provides the experimental basis for the development of new drugs for the treatment of muscular dystrophies.