The extracellular matrix protein agrin is essential for epicardial epithelial-to-mesenchymal transition during heart development.

During heart development, epicardial cells residing within the outer layer undergo epithelial-mesenchymal transition (EMT) and migrate into the underlying myocardium to support organ growth and morphogenesis. Disruption of epicardial EMT results in embryonic lethality, yet its regulation is poorly understood. Here, we report epicardial EMT within the mesothelial layer of the mouse embryonic heart at ultra-high resolution using scanning electron microscopy combined with immunofluorescence analyses. We identified morphologically active EMT regions that associated with key components of the extracellular matrix, including the basement membrane-associated proteoglycan agrin. Deletion of agrin resulted in impaired EMT and compromised development of the epicardium, accompanied by downregulation of Wilms' tumor 1. Agrin enhanced EMT in human embryonic stem cell-derived epicardial-like cells by decreasing ß-catenin and promoting pFAK localization at focal adhesions, and promoted the aggregation of dystroglycan within the Golgi apparatus in murine epicardial cells. Loss of agrin resulted in dispersal of dystroglycan in vivo, disrupting basement membrane integrity and impairing EMT. Our results provide new insights into the role of the extracellular matrix in heart development and implicate agrin as a crucial regulator of epicardial EMT.

BGP-15 Improves Aspects of the Dystrophic Pathology in mdx and dko Mice with Differing Efficacies in Heart and Skeletal Muscle.

Duchenne muscular dystrophy is a severe and progressive striated muscle wasting disorder that leads to premature death from respiratory and/or cardiac failure. We have previously shown that treatment of young dystrophic mdx and dystrophin/utrophin null (dko) mice with BGP-15, a coinducer of heat shock protein 72, ameliorated the dystrophic pathology. We therefore tested the hypothesis that later-stage BGP-15 treatment would similarly benefit older mdx and dko mice when the dystrophic pathology was already well established. Later stage treatment of mdx or dko mice with BGP-15 did not improve maximal force of tibialis anterior (TA) muscles (in situ) or diaphragm muscle strips (in vitro). However, collagen deposition (fibrosis) was reduced in TA muscles of BGP-15-treated dko mice but unchanged in TA muscles of treated mdx mice and diaphragm of treated mdx and dko mice. We also examined whether BGP-15 treatment could ameliorate aspects of the cardiac pathology, and in young dko mice it reduced collagen deposition and improved both membrane integrity and systolic function. These results confirm BGP-15's ability to improve aspects of the dystrophic pathology but with differing efficacies in heart and skeletal muscles at different stages of the disease progression. These findings support a role for BGP-15 among a suite of pharmacological therapies for Duchenne muscular dystrophy and related disorders.

Citrulline does not prevent skeletal muscle wasting or weakness in limb-casted mice.

BACKGROUND: Increasing arginine (Arg) availability reduces atrophy in cultured skeletal muscle cells. Supplementation with its metabolic precursor citrulline (Cit) is more effective at improving skeletal muscle Arg concentrations. OBJECTIVE: We tested the hypothesis that Cit supplementation would attenuate skeletal muscle atrophy and loss of function during hindlimb immobilization in mice. METHODS: Male C57BL/6JArc mice underwent 14 d of unilateral hindlimb immobilization/plaster casting and were supplemented with ~0.81 g Cit · kg⁻¹ · d⁻¹ (CIT group) or Ala (ALA group) mixed into their food. The uncasted contralateral limb (internal control) and an uncasted group (CON) served as controls. Muscle atrophy was evaluated with mass, fiber area, and in situ muscle function. RESULTS: Tibialis anterior (TA) muscle mass [ALA: 37.6 ± 0.92 mg; CIT: 38.3 ± 1.25 mg] and peak tetanic force (ALA: 1150 ± 38.5 mN; CIT: 1150 ± 52.0 mN) were lower (P < 0.001) in the ALA (53.9 ± 0.42 mg) and CIT (1760 ± 28.5 mN) groups than in the CON group. No difference was found between ALA and CIT groups for TA mass, fiber area, or peak force. The mRNA expression of the nitric oxide synthase 2, inducible (Nos2; ~15-fold) and B-cell chronic lymphoid leukemia/lymphoma 2/adenovirus E1B 19 kDa interacting protein 3 (Bnip3; ~17-fold) genes and the ratio of microtubule-associated protein light chain 3BII to 3BI (LC3BII:LC3BI) (50.5% ± 17.7%) were higher (P < 0.05) in the ALA group than in the CON group, suggesting increased autophagy. In the CIT group, Bnip3 mRNA was lower (-70%; P < 0.05) and Nos2 mRNA tended to be lower (-45%; P = 0.05) than in the ALA group, whereas LC3BII:LC3BI was not different from the CON group. CONCLUSIONS: Cit treatment of male mice did not affect therapeutically relevant outcome measures such as skeletal muscle mass and peak muscle force after 14 d of hindlimb immobilization.

Cardiac and Skeletal Muscle Pathology in the D2/mdx Mouse Model and Caveats Associated with the Quantification of Utrophin.

Duchenne muscular dystrophy (DMD) (the most common form of muscular dystrophy) is caused by a lack of dystrophin protein. Currently, although many therapeutic strategies are under investigation, there is no cure for DMD and unfortunately, patients succumb to respiratory and/or cardiac failure in their second or third decade of life. Preclinical work has focused on the mouse model C57BL/10ScSn-Dmdmdx/J (BL10/mdx), which does not exhibit a robust pathophenotype. More recently, the D2.B10-Dmdmdx/J (D2/mdx) mouse has been utilized, which presents a more severe pathology and therefore more closely mimics the human pathophenotype, particularly in the heart. Here, we outline important considerations when utilizing the D2/mdx model by highlighting the differences between these models in addition to describing histological and immunohistochemical methods utilized in Kennedy et al. (Mol Ther Methods Clin Dev 11:92-105, 2018) for both cardiac and skeletal muscle, which can quantify these differences. These considerations are particularly important when investigating treatment strategies that may be affected by regeneration; such is the case for upregulation of the dystrophin paralogue, utrophin.

Regenerative potential of epicardium-derived extracellular vesicles mediated by conserved miRNA transfer.

AIMS: After a myocardial infarction, the adult human heart lacks sufficient regenerative capacity to restore lost tissue, leading to heart failure progression. Finding novel ways to reprogram adult cardiomyocytes into a regenerative state is a major therapeutic goal. The epicardium, the outermost layer of the heart, contributes cardiovascular cell types to the forming heart and is a source of trophic signals to promote heart muscle growth during embryonic development. The epicardium is also essential for heart regeneration in zebrafish and neonatal mice and can be reactivated after injury in adult hearts to improve outcome. A recently identified mechanism of cell-cell communication and signalling is that mediated by extracellular vesicles (EVs). Here, we aimed to investigate epicardial signalling via EV release in response to cardiac injury and as a means to optimize cardiac repair and regeneration. METHODS AND RESULTS: We isolated epicardial EVs from mouse and human sources and targeted the cardiomyocyte population. Epicardial EVs enhanced proliferation in H9C2 cells and in primary neonatal murine cardiomyocytes in vitro and promoted cell cycle re-entry when injected into the injured area of infarcted neonatal hearts. These EVs also enhanced regeneration in cryoinjured engineered human myocardium (EHM) as a novel model of human myocardial injury. Deep RNA-sequencing of epicardial EV cargo revealed conserved microRNAs (miRs) between human and mouse epicardial-derived exosomes, and the effects on cell cycle re-entry were recapitulated by administration of cargo miR-30a, miR-100, miR-27a, and miR-30e to human stem cell-derived cardiomyocytes and cryoinjured EHM constructs. CONCLUSION: Here, we describe the first characterization of epicardial EV secretion, which can signal to promote proliferation of cardiomyocytes in infarcted mouse hearts and in a human model of myocardial injury, resulting in enhanced contractile function. Analysis of exosome cargo in mouse and human identified conserved pro-regenerative miRs, which in combination recapitulated the therapeutic effects of promoting cardiomyocyte proliferation.

Experimental limitations of extracellular vesicle-based therapies for the treatment of myocardial infarction.

Extracellular vesicles (EVs) are particles secreted by a vast variety of cells and are often recognised to mimic the properties of their parent cell, as such those derived from developmental sources hold promise for the treatment of various diseases including myocardial infarction (MI). Here we review the experimental approaches taken for assessing the therapeutic efficacy of EVs for MI and find overt shortcomings regarding purity of isolated EVs, quantitation, dosing, EV labelling/uptake, route of administration and use of appropriate controls that renders much of the data uninterpretable. Overall, the EV/MI field has suffered from experimental approaches that are not fully standardised or validated. Fundamental improvements in EV study design are required to improve interpretation of efficacy and to ensure reproducibility and comparability across preclinical MI studies.

Glycine administration attenuates progression of dystrophic pathology in prednisolone-treated dystrophin/utrophin null mice.

Duchenne muscular dystrophy (DMD) is an X-linked genetic disease characterized by progressive muscle wasting and weakness and premature death. Glucocorticoids (e.g. prednisolone) remain the only drugs with a favorable impact on DMD patients, but not without side effects. We have demonstrated that glycine preserves muscle in various wasting models. Since glycine effectively suppresses the activity of pro-inflammatory macrophages, we investigated the potential of glycine treatment to ameliorate the dystrophic pathology. Dystrophic mdx and dystrophin-utrophin null (dko) mice were treated with glycine or L-alanine (amino acid control) for up to 15 weeks and voluntary running distance (a quality of life marker and strong correlate of lifespan in dko mice) and muscle morphology were assessed. Glycine increased voluntary running distance in mdx mice by 90% (P < 0.05) after 2 weeks and by 60% (P < 0.01) in dko mice co-treated with prednisolone over an 8 week treatment period. Glycine treatment attenuated fibrotic deposition in the diaphragm by 28% (P < 0.05) after 10 weeks in mdx mice and by 22% (P < 0.02) after 14 weeks in dko mice. Glycine treatment augmented the prednisolone-induced reduction in fibrosis in diaphragm muscles of dko mice (23%, P < 0.05) after 8 weeks. Our findings provide strong evidence that glycine supplementation may be a safe, simple and effective adjuvant for improving the efficacy of prednisolone treatment and improving the quality of life for DMD patients.

Micro-utrophin Improves Cardiac and Skeletal Muscle Function of Severely Affected D2/mdx Mice.

Duchenne muscular dystrophy (DMD) is an X-linked muscle-wasting disease caused by mutations in the dystrophin gene. DMD boys are wheelchair-bound around 12 years and generally survive into their twenties. There is currently no effective treatment except palliative care, although personalized treatments such as exon skipping, stop codon read-through, and viral-based gene therapies are making progress. Patients present with skeletal muscle pathology, but most also show cardiomyopathy by the age of 10. A systemic therapeutic approach is needed that treats the heart and skeletal muscle defects in all patients. The dystrophin-related protein utrophin has been shown to compensate for the lack of dystrophin in the mildly affected BL10/mdx mouse. The purpose of this investigation was to demonstrate that AAV9-mediated micro-utrophin transgene delivery can not only functionally replace dystrophin in the heart, but also attenuate the skeletal muscle phenotype in severely affected D2/mdx mice. The data presented here show that utrophin can indeed alleviate the pathology in skeletal and cardiac muscle in D2/mdx mice. These results endorse the view that utrophin modulation has the potential to increase the quality life of all DMD patients whatever their mutation.

Utrophin influences mitochondrial pathology and oxidative stress in dystrophic muscle.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a lethal X-linked muscle wasting disorder caused by the absence of dystrophin, a large cytoskeletal muscle protein. Increasing the levels of the dystrophin-related-protein utrophin is a highly promising therapy for DMD and has been shown to improve pathology in dystrophin-deficient mice. One contributing factor to muscle wasting in DMD is mitochondrial pathology that contributes to oxidative stress and propagates muscle damage. The purpose of this study was to assess whether utrophin could attenuate mitochondria pathology and oxidative stress. METHODS: Skeletal muscles from wildtype C57BL/10, dystrophin-deficient mdx, dystrophin/utrophin double knockout (dko) and dystrophin-deficient mdx/utrophin over-expressing mdx-Fiona transgenic mice were assessed for markers of mitochondrial damage. RESULTS: Using transmission electron microscopy, we show that high levels of utrophin ameliorate the aberrant structure and localisation of mitochondria in mdx mice whereas absence of utrophin worsened these features in dko mice. Elevated utrophin also reverts markers of protein oxidation and oxidative stress, elevated in mdx and dko mice, to wildtype levels. These changes were observed independently of a shift in oxidative phenotype. CONCLUSION: These findings show that utrophin levels influence mitochondrial pathology and oxidative stress. While utrophin deficiency worsens the pathology, utrophin over-expression in dystrophic muscle benefits mitochondria and attenuates the downstream pathology associated with aberrant mitochondrial function.