Background: All types of facioscapulohumeral muscular dystrophy (FSHD) are caused by the aberrant activation of the somatically silent DUX4 gene, the expression of which initiates a cascade of cellular events ultimately leading to FSHD pathophysiology. Typically, progressive skeletal muscle weakness becomes noticeable in the second or third decade of life, yet there are many individuals who are genetically FSHD but develop symptoms much later in life or remain relatively asymptomatic throughout their lives. Conversely, FSHD may clinically present prior to 5-10 years of age, ultimately manifesting as a severe early-onset form of the disease. These phenotypic differences are thought to be due to the timing and levels of DUX4 misexpression. Methods: FSHD is a dominant gain-of-function disease that is amenable to modeling by DUX4 overexpression. We have recently created a line of conditional DUX4 transgenic mice, FLExDUX4, that develop a myopathy upon induction of human DUX4-fl expression in skeletal muscle. Here, we use the FLExDUX4 mouse crossed with the skeletal muscle-specific and tamoxifen-inducible line ACTA1-MerCreMer to generate a highly versatile bi-transgenic mouse model with chronic, low-level DUX4-fl expression and cumulative mild FSHD-like pathology that can be reproducibly induced to develop more severe pathology via tamoxifen induction of DUX4-fl in skeletal muscles. Results: We identified conditions to generate FSHD-like models exhibiting reproducibly mild, moderate, or severe DUX4-dependent pathophysiology and characterized progression of pathology. We assayed DUX4-fl mRNA and protein levels, fitness, strength, global gene expression, and histopathology, all of which are consistent with an FSHD-like myopathic phenotype. Importantly, we identified sex-specific and muscle-specific differences that should be considered when using these models for preclinical studies.

Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is a lethal muscle-wasting disease that is caused by mutations in the LAMA2 gene, resulting in the loss of laminin-α2 protein. MDC1A patients exhibit severe muscle weakness from birth, are confined to a wheelchair, require ventilator assistance, and have reduced life expectancy. There are currently no effective treatments or cures for MDC1A. Laminin-α2 is required for the formation of heterotrimeric laminin-211 (ie, α2, β1, and γ1) and laminin-221 (ie, α2, β2, and γ1), which are major constituents of skeletal muscle basal lamina. Laminin-111 (ie, α1, β1, and γ1) is the predominant laminin isoform in embryonic skeletal muscle and supports normal skeletal muscle development in laminin-α2-deficient muscle but is absent from adult skeletal muscle. In this study, we determined whether treatment with Engelbreth-Holm-Swarm-derived mouse laminin-111 protein could rescue MDC1A in the dy(W-/-) mouse model. We demonstrate that laminin-111 protein systemically delivered to the muscles of laminin-α2-deficient mice prevents muscle pathology, improves muscle strength, and dramatically increases life expectancy. Laminin-111 also prevented apoptosis in laminin-α2-deficient mouse muscle and primary human MDC1A myogenic cells, which indicates a conserved mechanism of action and cross-reactivity between species. Our results demonstrate that laminin-111 can serve as an effective protein substitution therapy for the treatment of muscular dystrophy in the dy(W-/-) mouse model and establish the potential for its use in the treatment of MDC1A.

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease caused by mutations in the dystrophin gene, leading to the loss of a critical component of the sarcolemmal dystrophin glycoprotein complex. Galectin-1 is a small 14 kDa protein normally found in skeletal muscle and has been shown to be a modifier of immune response, muscle repair, and apoptosis. Galectin-1 levels are elevated in the muscle of mouse and dog models of DMD. Together, these findings led us to hypothesize that Galectin-1 may serve as a modifier of disease progression in DMD. To test this hypothesis, recombinant mouse Galectin-1 was produced and used to treat myogenic cells and the mdx mouse model of DMD. Here we show that intramuscular and intraperitoneal injections of Galectin-1 into mdx mice prevented pathology and improved muscle function in skeletal muscle. These improvements were a result of enhanced sarcolemmal stability mediated by elevated utrophin and α7β1 integrin protein levels. Together our results demonstrate for the first time that Galectin-1 may serve as an exciting new protein therapeutic for the treatment of DMD.

The leading candidate gene responsible for facioscapulohumeral muscular dystrophy (FSHD) is FRG1 (FSHD region gene 1). However, the correlation of altered FRG1 expression levels with disease pathology has remained controversial and the precise function of FRG1 is unknown. Here, we carried out a detailed analysis of the normal expression patterns and effects of FRG1 misexpression during vertebrate embryonic development using Xenopus laevis. We show that frg1 is expressed in and essential for the development of the tadpole musculature. FRG1 morpholino injection disrupted myotome organization and led to inhibited myotome growth, while elevated FRG1 led to abnormal epaxial and hypaxial muscle formation. Thus, maintenance of normal FRG1 levels is critical for proper muscle development, supportive of FSHD disease models whereby misregulation of FRG1 plays a causal role underlying the pathology exhibited in FSHD patients. Developmental Dynamics 238:1502-1512, 2009.

SUMMARYThe genetic lesion that is diagnostic for facioscapulohumeral muscular dystrophy (FSHD) results in an epigenetic misregulation of gene expression, which ultimately leads to the disease pathology. FRG1 (FSHD region gene 1) is a leading candidate for a gene whose misexpression might lead to FSHD. Because FSHD pathology is most prominent in the musculature, most research and therapy efforts focus on muscle cells. Previously, using Xenopus development as a model, we showed that altering frg1 expression levels systemically leads to aberrant muscle development, illustrating the potential for aberrant FRG1 levels to disrupt the musculature. However, 50-75% of FSHD patients also exhibit retinal vasculopathy and FSHD muscles have increased levels of vascular-and endothelial-related FRG1 transcripts, illustrating an underlying vascular component to the disease. To date, no FSHD candidate gene has been proposed to affect the vasculature. Here, we focus on a role for FRG1 expression in the vasculature. We found that endogenous frg1 is expressed in both the developing and adult vasculature in Xenopus. Furthermore, expression of FRG1 was found to be essential for the development of the vasculature, as a knockdown of FRG1 resulted in decreased angiogenesis and reduced expression of the angiogenic regulator DAB2. Conversely, tadpoles subjected to frg1 overexpression displayed the pro-angiogenic phenotypes of increased blood vessel branching and dilation of blood vessels, and developed edemas, suggesting that their circulation was disrupted. Thus, the systemic upregulation of the FRG1 protein shows the potential for acquiring a disrupted vascular phenotype, providing the first link between a FSHD candidate gene and the vascular component of FSHD pathology. Overall, in conjunction with our previous analysis, we show that FRG1 overexpression is capable of disrupting both the musculature and vasculature, recapitulating the two most prominent features of FSHD.