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Asthma is a chronic inflammatory disorder of the lower airways characterized by modulation of airway smooth muscle (ASM) function. Infiltration of smooth muscle by inflammatory mediators is partially regulated by transmembrane integrins and the major smooth muscle laminin receptor α7β1 integrin plays a critical role in the maintenance of ASM phenotype. The goal of the current study was to investigate the role of α7 integrin in asthma using smooth muscle-specific α7 integrin transgenic mice (TgSM-Itgα7) using both acute and chronic OVA sensitization and challenge protocols that mimic mild to severe asthmatic phenotypes. Transgenic over-expression of the α7 integrin in smooth muscle resulted in a significant decrease in airway resistance relative to controls, reduced the total number of inflammatory cells and substantially inhibited the production of crucial Th2 and Th17 cytokines in airways. This was accompanied by decreased secretion of various inflammatory chemokines such as eotaxin/CCL11, KC/CXCL3, MCP-1/CCL2, and MIP-1β/CCL4. Additionally, α7 integrin overexpression significantly decreased ERK1/2 phosphorylation in the lungs of TgSM-Itgα7 mice and affected proliferative, contractile, and inflammatory downstream effectors of ERK1/2 that drive smooth muscle phenotype in the lung. Taken together, these results support the hypothesis that enhanced expression of α7 integrin in vivo inhibits allergic inflammation and airway resistance. Moreover, we identify ERK1/2 as a potential target by which α7 integrin signals to regulate airway inflammation. We conclude that identification of therapeutics targeting an increase in smooth muscle α7 integrin expression could serve as a potential novel treatment for asthma., Competing Interests: The authors have no competing or financial interests to declare., (© 2022 The Authors. FASEB BioAdvances published by Wiley Periodicals LLC on behalf of The Federation of American Societies for Experimental Biology.)

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., Conclusions: The ACTA1-MCM;FLExDUX4 bi-transgenic mouse model has mild FSHD-like pathology and detectable muscle weakness. The onset and progression of more severe DUX4-dependent pathologies can be controlled via tamoxifen injection to increase the levels of mosaic DUX4-fl expression, providing consistent and readily screenable phenotypes for assessing therapies targeting DUX4-fl mRNA and/or protein and are useful to investigate certain conserved downstream FSHD-like pathophysiology. Overall, this model supports that DUX4 expression levels in skeletal muscle directly correlate with FSHD-like pathology by numerous metrics.

Duchenne muscular dystrophy (DMD) is a devastating X-linked disease affecting ~1 in 5000 males. DMD patients exhibit progressive muscle degeneration and weakness, leading to loss of ambulation and premature death from cardiopulmonary failure. We previously reported that mouse Laminin-111 (msLam-111) protein could reduce muscle pathology and improve muscle function in the mdx mouse model for DMD. In this study, we examined the ability of msLam-111 to prevent muscle disease progression in the golden retriever muscular dystrophy (GRMD) dog model of DMD. The msLam-111 protein was injected into the cranial tibial muscle compartment of GRMD dogs and muscle strength and pathology were assessed. The results showed that msLam-111 treatment increased muscle fiber regeneration and repair with improved muscle strength and reduced muscle fibrosis in the GRMD model. Together, these findings support the idea that Laminin-111 could serve as a novel protein therapy for the treatment of DMD., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Duchenne muscular dystrophy (DMD) is a lethal, muscle degenerative disease causing premature death of affected children. DMD is characterized by mutations in the dystrophin gene that result in a loss of the dystrophin protein. Loss of dystrophin causes an associated reduction in proteins of the dystrophin glycoprotein complex, leading to contraction-induced sarcolemmal weakening, muscle tearing, fibrotic infiltration and rounds of degeneration and failed regeneration affecting satellite cell populations. The α7β1 integrin has been implicated in increasing myogenic capacity of satellite cells, therefore restoring muscle viability, increasing muscle force and preserving muscle function in dystrophic mouse models. In this study, we show that a Food and Drug Administration (FDA)-approved small molecule, Sunitinib, is a potent α7 integrin enhancer capable of promoting myogenic regeneration by stimulating satellite cell activation and increasing myofiber fusion. Sunitinib exerts its regenerative effects via transient inhibition of SHP-2 and subsequent activation of the STAT3 pathway. Treatment of mdx mice with Sunitinib demonstrated decreased membrane leakiness and damage owing to myofiber regeneration and enhanced support at the extracellular matrix. The decreased myofiber damage translated into a significant increase in muscle force production. This study identifies an already FDA-approved compound, Sunitinib, as a possible DMD therapeutic with the potential to treat other muscular dystrophies in which there is defective muscle repair., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Duchenne muscular dystrophy (DMD) is a devastating disease caused by mutations in the dystrophin gene that result in the complete absence of dystrophin protein. We have shown previously that recombinant mouse Galectin-1 treatment improves physiological and histological outcome measures in the mdx mouse model of DMD. Because recombinant human Galectin-1 (rHsGal1) will be used to treat DMD patients, we performed a dose-ranging study and intraperitoneal or intravenous delivery to determine the efficacy of rHsGal1 to improve preclinical outcome measures in mdx mice. Our studies showed that the optimal dose of rHsGal1 delivered intraperitoneally was 20 mg/kg and that this treatment improved muscle strength, sarcolemma stability, and capillary density in skeletal muscle. We next examined the efficacy of intravenous delivery and found that a dose of 2.5 mg/kg rHsGal1 was well tolerated and improved outcome measures in the mdx mouse model. Our studies identified that intravenous doses of rHsGal1 exceeding 2.5 mg/kg resulted in toxicity, indicating that dosing using this delivery mechanism will need to be carefully monitored. Our results support the idea that rHsGal1 treatment can improve outcome measures in the mdx mouse model and support further development as a potential therapeutic agent for DMD.

Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by mutations in the dystrophin gene, resulting in a complete loss of the dystrophin protein. Dystrophin is a critical component of the dystrophin glycoprotein complex (DGC), which links laminin in the extracellular matrix to the actin cytoskeleton within myofibers and provides resistance to shear stresses during muscle activity. Loss of dystrophin in DMD patients results in a fragile sarcolemma prone to contraction-induced muscle damage. The α7β1 integrin is a laminin receptor protein complex in skeletal and cardiac muscle and a major modifier of disease progression in DMD. In a muscle cell-based screen for α7 integrin transcriptional enhancers, we identified a small molecule, SU9516, that promoted increased α7β1 integrin expression. Here we show that SU9516 leads to increased α7B integrin in murine C2C12 and human DMD patient myogenic cell lines. Oral administration of SU9516 in the mdx mouse model of DMD increased α7β1 integrin in skeletal muscle, ameliorated pathology, and improved muscle function. We show that these improvements are mediated through SU9516 inhibitory actions on the p65-NF-κB pro-inflammatory and Ste20-related proline alanine rich kinase (SPAK)/OSR1 signaling pathways. This study identifies a first in-class α7 integrin-enhancing small-molecule compound with potential for the treatment of DMD., (Copyright © 2017 The American Society of Gene and Cell Therapy. All rights reserved.)

Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is a dramatic neuromuscular disease in which crippling muscle weakness is evident from birth. Here, we use the dyW mouse model for human MDC1A to trace the onset of the disease during development in utero. We find that myotomal and primary myogenesis proceed normally in homozygous dyW-/- embryos. Fetal dyW-/- muscles display the same number of myofibers as wildtype (WT) muscles, but by E18.5 dyW-/- muscles are significantly smaller and muscle size is not recovered post-natally. These results suggest that fetal dyW-/- myofibers fail to grow at the same rate as WT myofibers. Consistent with this hypothesis between E17.5 and E18.5 dyW-/- muscles display a dramatic drop in the number of Pax7- and myogenin-positive cells relative to WT muscles, suggesting that dyW-/- muscles fail to generate enough muscle cells to sustain fetal myofiber growth. Gene expression analysis of dyW-/- E17.5 muscles identified a significant increase in the expression of the JAK-STAT target gene Pim1 and muscles from 2-day and 3-week old dyW-/- mice demonstrate a dramatic increase in pSTAT3 relative to WT muscles. Interestingly, myotubes lacking integrin α7β1, a laminin-receptor, also show a significant increase in pSTAT3 levels compared with WT myotubes, indicating that α7β1 can act as a negative regulator of STAT3 activity. Our data reveal for the first time that dyW-/- mice exhibit a myogenesis defect already in utero. We propose that overactivation of JAK-STAT signaling is part of the mechanism underlying disease onset and progression in dyW-/- mice., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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.

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease for which there is no cure and limited treatment options. Prednisone is currently the first line treatment option for DMD and studies have demonstrated that it improves muscle strength. Although prednisone has been used for the treatment of DMD for decades, the mechanism of action of this drug remains unclear. Recent studies have shown that the α7β1 integrin is a major modifier of disease progression in mouse models of DMD and is therefore a target for drug-based therapies. In this study we examined whether prednisone increased α7β1 integrin levels in mdx mouse and GRMD dog models and myogenic cells from humans with DMD. Our results show that prednisone promotes an increase in α7 integrin protein in cultured myogenic cells and in the muscle of mdx and GRMD animal models of DMD. The prednisone-mediated increase in α7 integrin was associated with increased laminin-α2 in prednisone-treated dystrophin-deficient muscle. Together, our results suggest that prednisone acts in part through increased merosin in the muscle basal lamina and through sarcolemmal stabilization of α7β1 integrin in dystrophin-deficient muscle. These results indicate that therapies that target an increase in muscle α7β1 integrin, its signaling pathways and/or laminin could be therapeutic in DMD.

MG53 promotes sarcolemmal repair in the mdx mouse model of Duchenne muscular dystrophy (Weisleder et al., this issue) and identifies a new protein therapeutic for muscle disease.

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., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Merosin-deficient congenital muscular dystrophy 1A (MDC1A) is a devastating neuromuscular disease that results in children being confined to a wheelchair, requiring ventilator assistance to breathe and premature death. MDC1A is caused by mutations in the LAMA2 gene, which results in the partial or complete loss of laminin-211 and laminin-221, the major laminin isoforms found in the basal lamina of skeletal muscle. MDC1A patients exhibit reduced α7β1 integrin; however, it is unclear how the secondary loss of α7β1 integrin contributes to MDC1A disease progression. To investigate whether restoring α7 integrin expression can alleviate the myopathic phenotype observed in MDC1A, we produced transgenic mice that overexpressed the α7 integrin in the skeletal muscle of the dy(W⁻/⁻) mouse model of MDC1A. Enhanced expression of the α7 integrin restored sarcolemmal localization of the α7β1 integrin to laminin-α2-deficient myofibers, changed the composition of the muscle extracellular matrix, reduced muscle pathology, maintained muscle strength and function and improved the life expectancy of dy(W⁻/⁻) mice. Taken together, these results indicate that enhanced expression of α7 integrin prevents muscle disease progression through augmentation and/or stabilization of the existing extracellular matrix in laminin-α2-deficient mice, and strategies that increase α7 integrin in muscle might provide an innovative approach for the treatment of MDC1A.

The genetic lesion leading to facioscapulohumeral muscular dystrophy (FSHD) is a dominant deletion at the 4q35 locus. The generally accepted disease model involves an epigenetic dysregulation in the region resulting in the upregulation of one or more proximal genes whose overexpression specifically affects skeletal muscle. However, multiple FSHD candidate genes have been proposed without clear consensus. Using Xenopus laevis as a model for vertebrate development our lab has studied the effects of overexpression of the FSHD candidate gene ortholog, frg1 (FSHD region gene 1), showing that increased levels of frg1 systemically led specifically to an abnormal musculature and increased angiogenesis, the two most prominent clinical features of FSHD. Here we studied the overexpression effects of three other promising FSHD candidate genes, DUX4, DUX4c, and PITX1 using the same model system and methods for direct comparison. Expression of even very low levels of either DUX4 or pitx1 early in development led to massive cellular loss and severely abnormal development. These abnormalities were not muscle specific. In contrast, elevated levels of DUX4c resulted in no detectable adverse affects on muscle and DUX4c levels did not alter the expression of myogenic regulators. This data supports a model for DUX4 and PITX1 in FSHD only as pro-apoptotic factors if their expression in FSHD is confined to cells within the myogenic pathway; neither could account for the vascular pathology prevalent in FSHD. Taken together, increased frg1 expression alone leads to a phenotype that most closely resembles the pathophysiology observed in FSHD patients.

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. (c) 2008 Wiley-Liss, Inc.

The 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.

DNA mutations and aberrations are a problem for all forms of life. Eukaryotes specifically have developed ways of identifying and repairing various DNA mutations in a complex and refractory chromatin environment. The chromatin structure is much more than a packaging unit for DNA; it is dynamic. Cells utilize and manipulate chromatin for gene regulation, genome organization and maintenance of genome integrity. Once a DNA aberration has occurred, the various DNA repair machineries interact with chromatin proteins, such as the histone variant H2A.X, and chromatin remodeling machines of the SWI/SNF family to gain access and repair the lesion in a timely manner. Recent studies have thus begun to address the roles of chromatin proteins in DNA repair as well as to dissect the functions of DNA repair machinery in vitro on more physiological, nucleosomal templates.