BackgroundMDC1A is a congenital neuromuscular disorder with developmentally complex and progressive pathologies that results from a deficiency in the protein laminin α2. MDC1A is associated with a multitude of pathologies, including increased apoptosis, inflammation and fibrosis. In order to assess and treat a complicated disease such as MDC1A, we must understand the natural history of the disease so that we can identify early disease drivers and pinpoint critical time periods for implementing potential therapies.ResultsWe found that DyW mice show significantly impaired myogenesis and high levels of apoptosis as early as postnatal week 1. We also saw a surge of inflammatory response at the first week, marked by high levels of infiltrating macrophages, nuclear factor κB activation, osteopontin expression and overexpression of inflammatory cytokines. Fibrosis markers and related pathways were also observed to be elevated throughout early postnatal development in these mice, including periostin, collagen and fibronectin gene expression, as well as transforming growth factor β signaling. Interestingly, fibronectin was found to be the predominant fibrous protein of the extracellular matrix in early postnatal development. Lastly, we observed upregulation in various genes related to angiotensin signaling.MethodsWe sought out to examine the dysregulation of various pathways throughout early development (postnatal weeks 1-4) in the DyW mouse, the most commonly used mouse model of laminin-deficient muscular dystrophy. Muscle function tests (stand-ups and retractions) as well as gene (qRT-PCR) and protein levels (western blot, ELISA), histology (H&E, picrosirius red staining) and immunohistochemistry (fibronectin, TUNEL assay) were used to assess dysregulation of matricelluar protieins.ConclusionsOur results implicate the involvement of multiple signaling pathways in driving the earliest stages of pathology in DyW mice. As opposed to classical dystrophies, such as Duchenne muscular dystrophy, the dysregulation of various matricellular proteins appears to be a distinct feature of the early progression of DyW pathology. On the basis of our results, we believe that therapies that may reduce apoptosis and stabilize the homeostasis of extracellular matrix proteins may have increased efficacy if started at a very early age.

Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is an autosomal recessive disorder caused by mutations in the laminin-α2 gene (OMIM: 607855). Currently, no treatment other than palliative care exists for this disease. In our previous work, genetic interventions in the Lama2(Dy-w) mouse model for MDC1A demonstrated that limited regeneration and uncontrolled apoptosis are important drivers of this disease. However, targeting one of these disease drivers without addressing the other results in only partial rescue of the phenotype. The present study was designed to determine whether utilizing a combinatorial treatment approach can lead to a more profound amelioration of the disease pathology. To accomplish this task, we generated Bax-null Lama2(Dy-w)mice that overexpressed muscle-specific IGF-1 (Lama2(Dy-w)Bax(-/-)+IGF-1tg). Further to test the translational potential of IGF-1 administration in combination with Bax inhibition, we treated Lama2(Dy-w)Bax(-/-) mice postnatally with systemic recombinant human IGF-1 (IPLEX™). These two combinatorial treatments lead to similar, promising outcomes. In addition to increased body and muscle weights, both transgenic overexpression and systemic administration of IGF-1 combined with Bax-inhibition resulted in improved muscle phenotype and locomotory function that were nearly indistinguishable from wild-type mice. These results provide a fundamental proof of concept that justifies the use of a combination therapy as an effective treatment for MDC1A and highlights a compelling argument toward shifting the paradigm in treating multifaceted neuromuscular diseases.

Laminin‐deficient congenital muscular dystrophy (MDC1A) is the second most prevalent form of muscular dystrophy and is caused by a defect in the alpha chain of the muscle specific extracellular matrix protein Laminin‐211. Loss of this protein leads to vast signaling defects leading to failed regeneration, inflammation, fibrosis, and apoptosis. While the genetic cause is known, distinct pathomechanisms have yet to be elucidated. Integrins are heterodimeric membrane proteins that play key roles in many critical cell processes including cell adhesion and signal transduction. However, when dysregulated, they have been shown to be key pathological drivers in many disease scenarios. Using the DyW mouse model of MDC1A, qRT‐PCR, ELISA, and immunohistochemistry, we show for the first time that integrins, and most importantly Integrin‐αV, which is known to activate Transforming Growth Factor‐beta (TGF‐β) in fibrotic scenarios, are dysregulated in dystrophic muscle. Further, we found that protein levels of both latent and active TGF‐β are upregulated in the serum of DyW mice but only active TGF‐β is attenuated in response to treatment with Losartan (Angiotensin II type 1 receptor blocker known to be a potent antifibrotic agent). Interestingly, along with reduced levels of active TGF‐β, we also see a rescue of Integrin‐αV overexpression. Therefore, it is possible that the attenuation of TGF‐β signaling by Losartan could be mediated by downregulation of Integrin‐αV. These results suggest Integrin‐αV could be an important player in the induction and mediation of fibrotic pathology in MDC1A and could serve as a novel therapeutic target in this and other fibrotic, degenerative diseases.

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Laminin‐deficient congenital muscular dystrophy (MDC1A) is the second most prevalent form of muscular dystrophy and is caused by a defect in the alpha chain of the muscle specific extracellular matrix protein Laminin‐211. Loss of this protein leads to vast signaling defects leading to failed regeneration, inflammation, fibrosis, and apoptosis. While the genetic cause is known, distinct pathomechanisms have yet to be elucidated. Integrins are heterodimeric membrane proteins that play key roles in many critical cell processes including cell adhesion and signal transduction. However, when dysregulated, they have been shown to be key pathological drivers in many disease scenarios. Using the DyW mouse model of MDC1A, qRT‐PCR, ELISA, and immunohistochemistry, we show for the first time that integrins, and most importantly Integrin‐αV, which is known to activate Transforming Growth Factor‐beta (TGF‐β) in fibrotic scenarios, are dysregulated in dystrophic muscle. Further, we found that protein levels of both latent and active TGF‐β are upregulated in the serum of DyW mice but only active TGF‐β is attenuated in response to treatment with Losartan (Angiotensin II type 1 receptor blocker known to be a potent antifibrotic agent). Interestingly, along with reduced levels of active TGF‐β, we also see a rescue of Integrin‐αV overexpression. Therefore, it is possible that the attenuation of TGF‐β signaling by Losartan could be mediated by downregulation of Integrin‐αV. These results suggest Integrin‐αV could be an important player in the induction and mediation of fibrotic pathology in MDC1A and could serve as a novel therapeutic target in this and other fibrotic, degenerative diseases.

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