Inflammation participates in tissue repair through multiple mechanisms including directly regulating the cell fate of resident progenitor cells critical for successful regeneration. Upon surveying target cell types of the TNF ligand TWEAK, we observed that TWEAK binds to all progenitor cells of the mesenchymal lineage and induces NF-jB activation and the expression of pro-survival, pro-proliferative and homing receptor genes in the mesenchymal stem cells, suggesting that this pro-inflammatory cytokine may play an important role in controlling progenitor cell biology. We explored this potential using both the established C2C12 cell line and primary mouse muscle myoblasts, and demonstrated that TWEAK promoted their proliferation and inhibited their terminal differentiation. By generating mice deficient in the TWEAK receptor Fn14, we further showed that Fn14-deficient primary myoblasts displayed significantly reduced proliferative capacity and altered myotube formation. Following cardiotoxin injection, a known trigger for satellite cell-driven skeletal muscle regeneration, Fn14-deficient mice exhibited reduced inflammatory response and delayed muscle fiber regeneration compared with wild-type mice. These results indicate that the TWEAK/Fn14 pathway is a novel regulator of skeletal muscle precursor cells and illustrate an important mechanism by which inflammatory cytokines influence tissue regeneration and repair. Coupled with our recent demonstration that TWEAK potentiates liver progenitor cell proliferation, the expression of Fn14 on all mesenchymal lineage progenitor cells supports a broad involvement of this pathway in other tissue injury and disease settings.

Objective Congenital muscular dystrophy type 1A (MDC1A) is an autosomal recessive disease that is caused by loss-of-function mutations in the laminin-α2 gene and results in motor nerve and skeletal muscle dysfunction. In a previous study, we used genetic modifications to show that inappropriate induction of apoptosis was a significant contributor to pathogenesis in a laminin-α2-deficient mouse model of MDC1A. To identify a possible pharmacological therapy for laminin-α2-deficency, this study was designed to determine if treatment with minocycline or doxycycline, which are tetracycline derivatives reported to have anti-apoptotic effects in mammals, would significantly increase lifespan and improve neuromuscular function in laminin-α2-deficient mice. Methods Mice that were homozygous for a targeted, inactivating mutation of the laminin-α2-gene were placed into control, minocycline-treated, or doxycycline-treated groups. Drug treatment began within two weeks of birth and the progression of disease was followed over time using behavioral, growth, histological, and molecular assays. Results We found that treatment with either minocycline or doxycycline increased the median lifespan of laminin-α2-null mice from ∼32 days to ∼70 days. Furthermore, doxycycline improved postnatal growth rate and delayed the onset of hindlimb paralysis. Doxycycline-treated laminin-α2-deficient muscles had increased Akt phosphorylation, decreased inflammation, and decreased levels of Bax protein, TUNEL-positive myonuclei, and activated caspase-3. Interpretation Doxycycline or other drugs with similar functional profiles may be a possible route to improving neuromuscular dysfunction due to laminin-α2-deficiency.

The most common form of human congenital muscular dystrophy (CMD) is caused by mutations in the laminin-α2 gene. Loss of laminin-α2 function in this autosomal recessive type 1A form of CMD results in neuromuscular dysfunction and, often, early death. Laminin-α2-deficient skeletal muscles in both humans and mice show signs of muscle cell death by apoptosis. To examine the significance of apoptosis in CMD1A pathogenesis, we determined whether pathogenesis in laminin-α2-deficient (Lama2 -/-) mice could be ameliorated by inhibiting apoptosis through either (a) inactivation of the proapoptosis protein Bax or (b) overexpression of the antiapoptosis protein Bcl-2 from a muscle-specific transgene. We found that both of these genetic interventions produced a several-fold increase in the lifespan of Lama2 -/-mice. Bax inactivation also improved postnatal growth rate and myofiber histology and decreased fixed contractures of Lama2 -/-mice. Thus, Bcl-2 family-mediated apoptosis contributes significantly to pathogenesis in the mouse model of CMD1A, and antiapoptosis therapy may be a possible route to amelioration of neuromuscular dysfunction due to laminin-α2 deficiency in humans.

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.

The most common form of human congenital muscular dystrophy (CMD) is caused by mutations in the laminin-α2 gene. Loss of laminin-α2 function in this autosomal recessive type 1A form of CMD results in neuromuscular dysfunction and, often, early death. Laminin-α2-deficient skeletal muscles in both humans and mice show signs of muscle cell death by apoptosis. To examine the significance of apoptosis in CMD1A pathogenesis, we determined whether pathogenesis in laminin-α2-deficient (Lama2 -/-) mice could be ameliorated by inhibiting apoptosis through either (a) inactivation of the proapoptosis protein Bax or (b) overexpression of the antiapoptosis protein Bcl-2 from a muscle-specific transgene. We found that both of these genetic interventions produced a several-fold increase in the lifespan of Lama2 -/-mice. Bax inactivation also improved postnatal growth rate and myofiber histology and decreased fixed contractures of Lama2 -/-mice. Thus, Bcl-2 family-mediated apoptosis contributes significantly to pathogenesis in the mouse model of CMD1A, and antiapoptosis therapy may be a possible route to amelioration of neuromuscular dysfunction due to laminin-α2 deficiency in humans.