Mahasweta Girgenrath

TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration

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‐κB 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.

Inhibition of apoptosis improves outcome in a model of congenital muscular dystrophy

The most common form of human congenital muscular dystrophy (CMD) is caused by mutations in the laminin-alpha2 gene. Loss of laminin-alpha2 function in this autosomal recessive type 1A form of CMD results in neuromuscular dysfunction and, often, early death. Laminin-alpha2-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-alpha2-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-alpha2 deficiency in humans.

Pathology is alleviated by doxycycline in a laminin-α2-null model of congenital muscular dystrophy

Congenital muscular dystrophy type 1A 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 congenital muscular dystrophy type 1A. To identify a possible pharmacological therapy for laminin‐α2 deficiency, we designed this study to determine whether treatment with minocycline or doxycycline, which are tetracycline derivatives reported to have antiapoptotic effects in mammals, would significantly increase lifespan and improve neuromuscular function in laminin‐α2–deficient mice.

Muscle-specific expression of insulin-like growth factor 1 improves outcome in Lama2Dy-w mice, a model for congenital muscular dystrophy type 1A

MDC1A, the second most prevalent form of congenital muscular dystrophy, results from laminin-α2 chain deficiency. This disease is characterized by extensive muscle wasting that results in extremely weak skeletal muscles. A large percentage of children with MDC1A are faced with respiratory as well as ambulatory difficulties. We investigated the effects of overexpressing insulin-like growth factor-1 (IGF-1) as a potential therapeutic target for the disease in the Lama2(Dy-w) mouse, a model that closely resembles human MDC1A. IGF-1 transgenic Lama2(Dy-w) mice showed increased survivability, body weight and muscle weight. In addition, these mice showed better ability to stand up on their hind limbs: a typical exploratory behavior seen in healthy mice. Histology and immunohistochemistry analyses revealed increased regenerative capacity and proliferation in IGF-1 transgenic Lama2(Dy-w) muscles. Western blot analysis showed increased phosphorylation of Akt and ERK1/2, both known to enhance myogenesis. Additionally, we saw increases in the expression of the regeneration markers MyoD, myogenin and embryonic myosin (myosin heavy chain 3, MYH3). We conclude that overexpression of IGF-1 in Lama2(Dy-w) mice increases lifespan and improves their overall wellbeing mainly through the restoration of impaired muscle regeneration, as fibrosis or inflammation was not impacted by IGF-1 in this disease model. Our results demonstrate that IGF-1 has a promising therapeutic potential in the treatment of MDC1A.

Dysregulation of matricellular proteins is an early signature of pathology in laminin-deficient muscular dystrophy

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.

Diseased muscles that lack dystrophin or laminin-α2 have altered compositions and proliferation of mononuclear cell populations

BackgroundMultiple types of mononucleate cells reside among the multinucleate myofibers in skeletal muscles and these mononucleate cells function in muscle maintenance and repair. How neuromuscular disease might affect different types of muscle mononucleate cells had not been determined. In this study, therefore, we examined how two neuromuscular diseases, dystrophin-deficiency and laminin-α2-deficiency, altered the proliferation and composition of different subsets of muscle-derived mononucleate cells.MethodsWe used fluorescence-activated cell sorting combined with bromodeoxyuridine labeling to examine proliferation rates and compositions of mononuclear cells in diseased and healthy mouse skeletal muscle. We prepared mononucleate cells from muscles of mdx (dystrophin-deficient) or Lama2-/- (laminin-α2-deficient) mice and compared them to cells from healthy control muscles. We enumerated subsets of resident muscle cells based on Sca-1 and CD45 expression patterns and determined the proliferation of each cell subset in vivo by BrdU incorporation.ResultsWe found that the proliferation and composition of the mononucleate cells in dystrophin-deficient and laminin-α2-deficient diseased muscles are different than in healthy muscle. The mdx and Lama2-/- muscles showed similar significant increases in CD45+ cells compared to healthy muscle. Changes in proliferation, however, differed between the two diseases with proliferation increased in mdx and decreased in Lama2-/- muscles compared to healthy muscles. In particular, the most abundant Sca-1-/CD45- subset, which contains muscle precursor cells, had increased proliferation in mdx muscle but decreased proliferation in Lama2-/- muscles.ConclusionThe similar increases in CD45+ cells, but opposite changes in proliferation of muscle precursor cells, may underlie aspects of the distinct pathologies in the two diseases.

Triggering regeneration and tackling apoptosis: a combinatorial approach to treating congenital muscular dystrophy type 1 A

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.

Prdm1 (Blimp-1) and the Expression of Fast and Slow Myosin Heavy Chain Isoforms during Avian Myogenesis In Vitro

Background Multiple types of fast and slow skeletal muscle fibers form during early embryogenesis in vertebrates. In zebrafish, formation of the earliest slow myofibers in fin muscles requires expression of the zinc-finger transcriptional repressor Prdm1 (also known as Blimp1). To further understand how the role of Prdm1 in early myogenesis may vary through evolution and during development, we have now analyzed Prdm1 expression in the diverse types of myotubes that form in culture from somitic, embryonic, and fetal chicken myoblasts. Principal Findings In cultures of somitic, embryonic limb, and fetal limb chicken cells, we found that Prdm1 was expressed in all of the differentiated muscle cells that formed, including those that expressed only fast myosin heavy chain isoforms, as well as those that co-expressed both fast and slow myosin heavy chain isoforms. Prdm1 was also expressed in Pax7-positive myoblasts, as well as in non-myogenic cells in the cultures. Furthermore, though all differentiated cells in control somite cultures co-expressed fast and slow myosin heavy chains, antisense knockdown of Prdm1 expression inhibited the formation of these co-expressing cells in somite cultures. Conclusions In chicken myogenic cell cultures, Prdm1 was expressed in most Pax7-positive myoblasts and in all differentiated muscle cells, irrespective of the developmental stage of cell donor or the pattern of fast and slow myosin heavy chains expressed in the differentiated cells that were formed. Thus, Prdm1 was expressed in myogenic cells prior to terminal differentiation; and, after differentiation, Prdm1 expression was not limited to cells that expressed slow myosin heavy chain isoforms. In addition, Prdm1 appeared to be required for differentiation of the somitic myocytes, which are the earliest myocytes to form in the avian embryo.

miR-410 and miR-495 Are Dynamically Regulated in Diverse Cardiomyopathies and Their Inhibition Attenuates Pathological Hypertrophy

Noncoding RNAs have emerged as important modulators in cardiac development and pathological remodeling. Recently, we demonstrated that regulation of the Gtl2-Dio3 noncoding RNA locus is dependent on the MEF2 transcription factor in cardiac muscle, and that two of its encoded miRNAs, miR-410 and miR-495, induce robust cardiomyocyte proliferation. Given the possibility of manipulating the expression of these miRNAs to repair the damaged heart by stimulating cardiomyocyte proliferation, it is important to determine whether the Gtl2-Dio3 noncoding RNAs are regulated in cardiac disease and whether they function downstream of pathological cardiac stress signaling. Therefore, we examined expression of the above miRNAs processed from the Gtl2-Dio3 locus in various cardiomyopathies. These noncoding RNAs were upregulated in all cardiac disease models examined including myocardial infarction (MI) and chronic angiotensin II (Ang II) stimulation, and in the cardiomyopathies associated with muscular dystrophies. Consistent with these observations, we show that the Gtl2-Dio3 proximal promoter is activated by stress stimuli in cardiomyocytes and requires MEF2 for its induction. Furthermore, inhibiting miR-410 or miR-495 in stressed cardiomyocytes attenuated the hypertrophic response. Thus, the Gtl2-Dio3 noncoding RNA locus is a novel marker of cardiac disease and modulating the activity of its encoded miRNAs may mitigate pathological cardiac remodeling in these diseases.

IGF-1/GH axis enhances losartan treatment in Lama2-related muscular dystrophy

As the complexities of dystrophic pathology have been elucidated over the last few years, it has become increasingly clear that primary monogenetic defects result in multiple secondary pathologies capable of autonomously driving disease progression. Consequently, single-mode therapies fail to comprehensively ameliorate all aspects of pathology. Lama2-related muscular dystrophy (MDC1A) is a devastating congenital muscular dystrophy caused by mutations in the LAMA2 gene that results in multi-faceted secondary pathologies that include inflammation, fibrosis, apoptosis, and necrosis leading to severe muscle weakness and minimal postnatal growth. This study sought to implement a novel combinatorial treatment utilizing losartan, previously shown to ameliorate fibrosis and inflammation in conjunction with transgenic IGF-1 overexpression to improve postnatal growth. We found that dual-therapy rescued inflammation and fibrosis, improved weight gain, and led to remarkable restoration of muscle architecture and locomotory function in DyW mice (mouse model of MDC1A). We further showed using murine growth hormone that postnatal intervention with both therapies also yielded impressive amelioration of dystrophic pathology. Our results suggest for the first time that a combinatorial anti-fibrotic and pro-myogenic therapy could be the foundation of future therapies to a population of afflicted children in serious need.