Dosage-Adjusted Resistance Training in Mice with a Reduced Risk of Muscle Damage

Progressive resistance training (PRT), which involves performing muscle contractions against progressively greater external loads, can increase muscle ...

University of Nevada, Reno School of Medicine

Aberrant expression of DUX4, a gene unique to humans and primates, causes Facioscapulohumeral Muscular Dystrophy-1 (FSHD), yet the pathogenic mechanism is unknown. As transgenic overexpression models have largely failed to replicate the genetic changes seen in FSHD, many studies of endogenously expressed DUX4 have been limited to patient biopsies and myogenic cell cultures, which never fully differentiate into mature muscle fibers. We have developed a method to xenograft immortalized human muscle precursor cells from patients with FSHD and first-degree relative controls into the tibialis anterior muscle compartment of immunodeficient mice, generating human muscle xenografts. We report that FSHD cells mature into organized and innervated human muscle fibers with minimal contamination of murine myonuclei. They also reconstitute the satellite cell niche within the xenografts. FSHD xenografts express DUX4 and DUX4 downstream targets, retain the 4q35 epigenetic signature of their original donors, and express a novel protein biomarker of FSHD, SLC34A2. Ours is the first scalable, mature in vivo human model of FSHD. It should be useful for studies of the pathogenic mechanism of the disease as well as for testing therapeutic strategies targeting DUX4 expression.

Muscle xenografts reproduce key molecular features of facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by incomplete silencing of the disease locus, leading to pathogenic misexpression of  in skeletal muscle. Previously, we showed that CRISPR inhibition could successfully target and repress  in FSHD myocytes. However, an effective therapy will require both efficient delivery of therapeutic components to skeletal muscles and long-term repression of the disease locus. Thus, we re-engineered our platform to allow  delivery of more potent epigenetic repressors. We designed an FSHD-optimized regulatory cassette to drive skeletal muscle-specific expression of dCas9 from  fused to HP1α, HP1γ, the MeCP2 transcriptional repression domain, or the SUV39H1 SET domain. Targeting each regulator to the  promoter/exon 1 increased chromatin repression at the locus, specifically suppressing DUX4 and its target genes in FSHD myocytes and in a mouse model of the disease. Importantly, minimizing the regulatory cassette and using the smaller Cas9 ortholog allowed our therapeutic cassettes to be effectively packaged into adeno-associated virus (AAV) vectors for  delivery. By engineering a muscle-specific epigenetic CRISPR platform compatible with AAV vectors for gene therapy, we have laid the groundwork for clinical use of dCas9-based chromatin effectors in skeletal muscle disorders.

Targeted epigenetic repression by CRISPR/dSaCas9 suppresses pathogenic  expression in FSHD.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by misexpression of DUX4 in skeletal myocytes. As DUX4 is the key therapeutic target in FSHD, surrogate biomarkers of DUX4 expression in skeletal muscle are critically needed for clinical trials. Although no natural animal models of FSHD exist, transgenic mice with inducible DUX4 expression in skeletal muscles rapidly develop myopathic phenotypes consistent with FSHD. Here, we established a new, more-accurate FSHD-like mouse model based on chronic DUX4 expression in a small fraction of skeletal myonuclei that develops pathology mimicking key aspects of FSHD across its lifespan. Utilizing this new aged mouse model and DUX4-inducible mouse models, we characterized the DUX4-related microRNA signatures in skeletal muscles, which represent potential biomarkers for FSHD. We found increased expression of miR-31-5p and miR-206 in muscles expressing different levels of DUX4 and displaying varying degrees of pathology. Importantly, miR-206 expression is significantly increased in serum samples from FSHD patients compared with healthy controls. Our data support miR-31-5p and miR-206 as new potential regulators of muscle pathology and miR-206 as a potential circulating biomarker for FSHD. This article has an associated First Person interview with the first author of the paper.

Identification of candidate miRNA biomarkers for facioscapulohumeral muscular dystrophy using DUX4-based mouse models.

The true prevalence of facioscapulohumeral muscular dystrophy (FSHD) is unknown due to difficulties with accurate clinical evaluation and the complexities of current genetic diagnostics. Interestingly, all forms of FSHD are linked to epigenetic changes in the chromosome 4q35 D4Z4 macrosatellite, suggesting that epigenetic analysis could provide an avenue for sequence-based FSHD diagnostics. However, studies assessing DNA methylation at the FSHD locus have produced conflicting results; thus, the utility of this technique as an FSHD diagnostic remains controversial. Here, we critically compared two protocols for epigenetic analysis of the FSHD region using bisulfite genomic sequencing: Jones et al., that contends to be individually diagnostic for FSHD1 and FSHD2, and Gaillard et al., that can identify some changes in DNA methylation levels between groups of clinically affected FSHD and healthy subjects, but is not individually diagnostic for any form of FSHD. We performed both sets of assays on the same genetically confirmed samples and showed that this discrepancy was due strictly to differences in amplicon specificity. We propose that the epigenetic status of the FSHD-associated D4Z4 arrays, when accurately assessed, is a diagnostic for genetic FSHD and can readily distinguish between healthy, FSHD1 and FSHD2. Thus, epigenetic diagnosis of FSHD, which can be performed on saliva DNA, will greatly increase accessibility to FSHD diagnostics for populations around the world.

Takako I. Jones

Department of Pharmacology

University of Nevada, Reno School of Medicine

Dosage-Adjusted Resistance Training in Mice with a Reduced Risk of Muscle Damage

Takako I. Jones

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Pharmacology

University of Nevada, Reno School of Medicine

Dosage-Adjusted Resistance Training in Mice with a Reduced Risk of Muscle Damage

Department of Pharmacology