Therapeutic potential of miRNAs in Duchenne muscular dystrophy and other neuromuscular diseases

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder that results from mutations in the dystrophin gene, which leads to muscle degeneration and atrophy (Birnkrant et al. 2018). Patients with DMD begin to show symptoms in the first few years of life, which progresses to severe atrophy of skeletal and cardiac muscle. There is no cure for DMD and current treatments such as corticosteroids, physical therapy or breathing assistance are primarily for symptomatic relief. The severity of DMD is due to the absence of functional dystrophin, a cytoskeletal structural protein that protects muscles during contraction. Loss of dystrophin increases the susceptibility of muscle to damage and stress during repeated contractions, which is further exacerbated by inflammation and subsequent replacement of muscle by fat or fibrotic tissue. In addition, satellite cells or muscle stem cells become depleted during multiple cycles of repair, which further accounts for the loss in total muscle mass in DMD. One potential molecular target for DMD therapy is to directly promote muscle regeneration by targeting microRNAs (miRNAs). miRNAs are short non-coding RNA sequences of 22 amino acids that can regulate the expression of other genes during transcription. In DMD patients, muscle-specific miRNAs such as miR-1, miR-133, and miR-206, are elevated in blood in response to muscle damage and have been useful as serum biomarkers that indicate disease progression and severity (Cacchiarelli et al. 2011). A previous study showed that local delivery of muscle-specific miR-1, miR-133, and miR-206 into the anterior tibialis muscle can improve muscle regeneration and recovery after acute injury in a rat model (Nakasa et al. 2010). However, it is unclear how these miRNAs lead to muscle regeneration and many studies have not investigated the molecular mechanisms of individual miRNAs. In a paper published in this issue of The Journal of Physiology, Taetzsch et al. (2021) sought to examine the downstream molecular effects of miR-133b isolated from other miRNAs in the mdx mouse model of DMD. The mdx mouse model has a natural nonsense mutation in the dystrophin gene and develops DMD pathology with a shorter lifespan (Duddy et al. 2015). In mdx mice, some pathological changes and muscle damage are noticeable during the first 30 postnatal days (P30), but a period of recovery and remission occurs after the initial damage at 60 and 90 postnatal days (P60 and P90). First, Taetzsch et al. showed that, similar to DMD patients (Cacchiarelli et al. 2011), the mdx mice had increased miRNA levels including miR-133b at P30 when muscle damage occurred, which decreased at P60 and P90 after muscle recovery. Different from miR-133a and other miRNAs, miR-133b was elevated before muscle damage and reduced after recovery has occurred. To specifically study the role of miR-133b in DMD, Taetzsch et al. (2021) genetically deleted miR-133b in mdx mice by crossing mdx mice with miR-133b−/− knockout mice to generate mdx;miR-133b−/− mice. At P30, the deletion of miR-133b in mdx mice resulted in a more severe DMD phenotype and pathological changes: hindlimb muscles showed reduced muscle fibre size, matrix remodelling from increased laminin in the interstitial space, and increased inflammatory infiltrate. At later ages of P60 and P90, these pathological changes were diminished during muscle recovery and remission. This suggests that miR-133b may be important in slowing the initial muscle damage and its absence led to enhanced muscle damage and inflammation. Taetzsch et al. (2021) further established the definitive roles of miR-133b in stimulating satellite cells via myogenesis and promoting muscle regeneration. During muscle injury, satellite cells or muscle stem cells can become activated and proliferate to replenish skeletal muscle cells. In the mdx;miR-133b−/− mice, satellite cells were tracked with the Pax7 marker, which showed normal levels of satellite cells at P30 during the early stages of muscle damage. However, the total number of satellite cells become progressively depleted at the older ages of P60 and P90, suggesting that loss of miR-133b impairs satellite cell proliferation. They also demonstrated that miR-133b is involved in muscle regeneration in an acute injury model obtained by local muscle injection of cardiotoxin. At one-week post-injection, miR-133b−/− mice had smaller muscle fibres with decreased cross-sectional area and distribution compared to miR-133b+/+ mice. miR-133b is likely to be important for stimulation of satellite cells during muscle regeneration in the context of both acute and chronic injury. Through transcriptomic analysis of muscle-specific RNA-sequencing, Taetzsch et al. (2021) identified novel functions of miR-133b as a direct regulator of multiple downstream genes involved in inflammation and fibrosis. Deletion of miR-133b in mdx mice led to upregulation of genes and pathways implicated in inflammation and fibrosis. There was increased expression of inflammation-related TNF-alpha signalling pathway and downstream targets like TNF-alpha induced protein 2 (TNFAIP2). Additionally, there was upregulation of the TGF-beta signalling pathway that promotes fibrosis, which includes members such as TGF-beta receptor and other signalling molecules like SMAD3 and SMAD5. These results suggest that miR-133b normally suppresses both inflammatory and fibrotic pathways. miR-133b may act specifically in the context of muscle damage to downregulate pathways involved in inflammation and fibrosis, because both wild-type miR-133b+/+ and miR-133b−/− mice with normal muscle mass did not show significant changes in these pathways. Overall, this study established a definitive role for miR-133b as a part of the body's compensatory mechanism during muscle repair after acute injury (Taetzsch et al. 2021). miR-133b is directly involved in muscle repair by stimulating satellite cells and can suppress pathways involved in inflammation and fibrosis. These results raise the possibility that miRNAs can be used in therapy in DMD; they could be locally injected into muscles using a viral vector from either adeno-associated virus or lentivirus. This approach is attractive, because miRNAs, like miR-133b, are naturally occurring molecules in muscle tissue, which will probably have few side effects and low toxicity. An additional consideration is that a cocktail of multiple miRNAs may be effective at simultaneously targeting multiple pathways. miRNAs such as miR-133b seem to generally promote muscle regeneration and may be effective in a range of neuromuscular disorders beyond DMD and other muscular dystrophies. While this study is an important first step at providing evidence that miR-133b deletion worsens DMD pathology, it also raises the question of whether the opposite is true: would miR-133b overexpression improve DMD pathology and outcomes? As a follow-up experiment, it would be interesting to rescue mdx mice with either genetic overexpression of miR-133b or locally delivered miR-133b molecules. Beyond muscle pathology, it may also be important to track the ages of these mice to determine if miR-133b could directly extend their lifespan. Since DMD patients have a diversity of disease phenotypes due to different progression levels, we should also test dose dependent effects of miRNA therapies in different preclinical models of DMD. It is unclear if excessive miRNA-133b delivery could lead to unwanted muscle hypertrophy or undesired immunosuppression that can lead to increased infections. However, this may be a minor concern because both the wild-type miR-133b−/− and miR-133b+/+ mice have no major changes in body weight, skeletal muscle mass, muscle fibre size, or number of satellite cells. This suggests that, at least in mice, most of the effects of miR-133b are in the context of muscle damage. Nonetheless, rigorous testing of miRNA therapies would be required to evaluate the efficacy of miR-133b and other miRNAs before use in clinical trials. In summary, data from Taetzsch et al. (2021) have elucidated important functions of miR-133b and established muscle-specific mi-RNAs like miR-133b as natural modulators that can be used to slow the progression of DMD. miR-133b may directly promote muscle regeneration by stimulating satellite cells as well as downregulating inflammation and fibrosis that exacerbate muscle loss. This provides the foundation for examining other muscle-relevant mi-RNAs and is promising for developing new miRNA-based therapies that can directly treat DMD and other neuromuscular diseases. None. Sole author. Funding from the Karen Toffler Charitable Trust and McKnight Brain Institute at University of Florida.