Myopathy and exercise intolerance are prominent clinical features in carriers of a point-mutation or large-scale deletion in the mitochondrial DNA (mtDNA). In the majority of patients, the mtDNA mutation is heteroplasmic with varying mutation loads between tissues of an individual. Exercise-induced muscle regeneration has been shown to be beneficial in some mtDNA mutation carriers, but is often not feasible for this patient group. In this study, we performed in vitro analysis of mesoangioblasts from mtDNA mutation carriers to assess their potential to be used as source for autologous myogenic cell therapy.

Duchenne muscular dystrophy, like other muscular dystrophies, is a progressive disorder hallmarked by muscle degeneration, inflammation, and fibrosis. Latent transforming growth factor β (TGFβ) binding protein 4 (LTBP4) is an extracellular matrix protein found in muscle. LTBP4 sequesters and inhibits a precursor form of TGFβ. LTBP4 was originally identified from a genome-wide search for genetic modifiers of muscular dystrophy in mice, where there are two different alleles. The protective form of LTBP4, which contains an insertion of 12 amino acids in the protein’s hinge region, was linked to increased sequestration of latent TGFβ, enhanced muscle membrane stability, and reduced muscle fibrosis. The deleterious form of LTBP4 protein, lacking 12 amino acids, was more susceptible to proteolysis and promoted release of latent TGF-β, and together, these data underscored the functional role of LTBP4’s hinge. Here, we generated a monoclonal human anti-LTBP4 antibody directed toward LTBP4’s hinge region. In vitro, anti-LTBP4 bound LTBP4 protein and reduced LTBP4 proteolytic cleavage. In isolated myofibers, the LTBP4 antibody stabilized the sarcolemma from injury. In vivo, anti-LTBP4 treatment of dystrophic mice protected muscle against force loss induced by eccentric contraction. Anti-LTBP4 treatment also reduced muscle fibrosis and enhanced muscle force production, including in the diaphragm muscle, where respiratory function was improved. Moreover, the anti-LTBP4 in combination with prednisone, a standard of care for Duchenne muscular dystrophy, further enhanced muscle function and protected against injury in mdx mice. These data demonstrate the potential of anti-LTBP4 antibodies to treat muscular dystrophy.

Exogenous glucocorticoids interact with the circadian clock, but little attention is paid to the timing of intake. We recently found that intermittent once-weekly prednisone improved nutrient oxidation in dystrophic muscle. Here, we investigated whether dosage time affected prednisone effects on muscle bioenergetics. In mice treated with once-weekly prednisone, drug dosing in the light-phase promoted nicotinamide adenine dinucleotide (NAD+) levels and mitochondrial function in wild-type muscle, while this response was lost with dark-phase dosing. These effects depended on a normal circadian clock since they were disrupted in muscle from [Brain and muscle Arnt-like protein-1 (Bmal1)]-knockout mice. The light-phase prednisone pulse promoted BMAL1-dependent glucocorticoid receptor recruitment on noncanonical targets, including Nampt and Ppargc1a [peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α)]. In mice with muscle-restricted inducible PGC1α ablation, bioenergetic stimulation by light-phase prednisone required PGC1α. These results demonstrate that glucocorticoid "chronopharmacology" for muscle bioenergetics requires an intact clock and muscle PGC1α activity.

Disruption of the plasma membrane often accompanies cellular injury, and in muscle, plasma membrane resealing is essential for efficient recovery from injury. Muscle contraction, especially of lengthened muscle, disrupts the sarcolemma. To define the molecular machinery that directs repair, we applied laser wounding to live mammalian myofibers and assessed translocation of fluorescently tagged proteins using high-resolution microscopy. Within seconds of membrane disruption, annexins A1, A2, A5, and A6 formed a tight repair "cap." Actin was recruited to the site of damage, and annexin A6 cap formation was both actin dependent and Ca(2+) regulated. Repair proteins, including dysferlin, EHD1, EHD2, MG53, and BIN1, localized adjacent to the repair cap in a "shoulder" region enriched with phosphatidlyserine. Dye influx into muscle fibers lacking both dysferlin and the related protein myoferlin was substantially greater than control or individual null muscle fibers, underscoring the importance of shoulder-localized proteins. These data define the cap and shoulder as subdomains within the repair complex accumulating distinct and nonoverlapping components.

Genetic disruption of the dystrophin complex produces muscular dystrophy characterized by a fragile muscle plasma membrane leading to excessive muscle degeneration. Two genetic modifiers of Duchenne Muscular Dystrophy implicate the transforming growth factor β (TGFβ) pathway, osteopontin encoded by the SPP1 gene and latent TGFβ binding protein 4 (LTBP4). We now evaluated the functional effect of these modifiers in the context of muscle injury and repair to elucidate their mechanisms of action. We found that excess osteopontin exacerbated sarcolemmal injury, and correspondingly, that loss of osteopontin reduced injury extent both in isolated myofibers and in muscle in vivo. We found that ablation of osteopontin was associated with reduced expression of TGFβ and TGFβ-associated pathways. We identified that increased TGFβ resulted in reduced expression of Anxa1 and Anxa6, genes encoding key components of the muscle sarcolemma resealing process. Genetic manipulation of Ltbp4 in dystrophic muscle also directly modulated sarcolemmal resealing, and Ltbp4 alleles acted in concert with Anxa6, a distinct modifier of muscular dystrophy. These data provide a model in which a feed forward loop of TGFβ and osteopontin directly impacts the capacity of muscle to recover from injury, and identifies an intersection of genetic modifiers on muscular dystrophy.

Glucocorticoid steroids are commonly prescribed for many inflammatory conditions, but chronic daily use produces adverse effects, including muscle wasting and weakness. In contrast, shorter glucocorticoid pulses may improve athletic performance, although the mechanisms remain unclear. Muscle is sexually dimorphic and comparatively little is known about how male and female muscles respond to glucocorticoids. We investigated the impact of once-weekly glucocorticoid exposure on skeletal muscle performance comparing male and female mice. One month of once-weekly glucocorticoid dosing improved muscle specific force in both males and females. Transcriptomic profiling of isolated myofibers identified a striking sexually dimorphic response to weekly glucocorticoids. Male myofibers had increased expression of genes in the IGF1/PI3K pathway and calcium handling, while female myofibers had profound upregulation of lipid metabolism genes. Muscles from weekly prednisone-treated males had improved calcium handling, while comparably treated female muscles had reduced intramuscular triglycerides. Consistent with altered lipid metabolism, weekly prednisone-treated female mice had greater endurance relative to controls. Using chromatin immunoprecipitation, we defined a sexually dimorphic chromatin landscape after weekly prednisone. These results demonstrate that weekly glucocorticoid exposure elicits distinct pathways in males versus females, resulting in enhanced performance.

Skeletal muscle holds an intrinsic capability of growth and regeneration both in physiological conditions and in case of injury. Chronic muscle illnesses, generally caused by genetic and acquired factors, lead to deconditioning of the skeletal muscle structure and function, and are associated with a significant loss in muscle mass. At the same time, progressive muscle wasting is a hallmark of aging. Given the paracrine properties of myogenic stem cells, extracellular vesicle-derived signals have been studied for their potential implication in both the pathogenesis of degenerative neuromuscular diseases and as a possible therapeutic target. In this study, we screened the content of extracellular vesicles from animal models of muscle hypertrophy and muscle wasting associated with chronic disease and aging. Analysis of the transcriptome, protein cargo, and microRNAs (miRNAs) allowed us to identify a hypertrophic miRNA signature amenable for targeting muscle wasting, consisting of miR-1 and miR-208a. We tested this signature among others in vitro on mesoangioblasts (MABs), vessel-associated adult stem cells, and we observed an increase in the efficiency of myogenic differentiation. Furthermore, injections of miRNA-treated MABs in aged mice resulted in an improvement in skeletal muscle features, such as muscle weight, strength, cross-sectional area, and fibrosis compared to controls. Overall, we provide evidence that the extracellular vesicle-derived miRNA signature we identified enhances the myogenic potential of myogenic stem cells.

Duchenne muscular dystrophy (DMD) is a progressive severe muscle-wasting disease caused by mutations in DMD, encoding dystrophin, that leads to loss of muscle function with cardiac/respiratory failure and premature death. Since dystrophic muscles are sensed by infiltrating inflammatory cells and gut microbial communities can cause immune dysregulation and metabolic syndrome, we sought to investigate whether intestinal bacteria support the muscle immune response in mdx dystrophic murine model. We highlighted a strong correlation between DMD disease features and the relative abundance of Prevotella. Furthermore, the absence of gut microbes through the generation of mdx germ-free animal model, as well as modulation of the microbial community structure by antibiotic treatment, influenced muscle immunity and fibrosis. Intestinal colonization of mdx mice with eubiotic microbiota was sufficient to reduce inflammation and improve muscle pathology and function. This work identifies a potential role for the gut microbiota in the pathogenesis of DMD.

Sarcopenia burdens the elderly population through loss of muscle energy and mass, yet treatments to functionally rescue both parameters are missing. The glucocorticoid prednisone remodels muscle metabolism based on frequency of intake, but its mechanisms in sarcopenia are unknown. We found that once-weekly intermittent prednisone rescued muscle quality in aged 24-month-old mice to levels comparable to young 4-month-old mice. We discovered an age- and sex-independent glucocorticoid receptor transactivation program in muscle encompassing PGC1alpha and its co-factor Lipin1. Treatment coordinately improved mitochondrial abundance through isoform 1 and muscle mass through isoform 4 of the myocyte-specific PGC1alpha, which was required for the treatment-driven increase in carbon shuttling from glucose oxidation to amino acid biogenesis. We also probed the myocyte-specific Lipin1 as non-redundant factor coaxing PGC1alpha upregulation to the stimulation of both oxidative and anabolic capacities. Our study unveils an aging-resistant druggable program in myocytes to coordinately rescue energy and mass in sarcopenia.

Satellite cells that reside on the myofibre surface are crucial for the muscle homeostasis and regeneration. Aging goes along with a less effective regeneration of skeletal muscle tissue mainly due to the decreased myogenic capability of satellite cells. This phenomenon impedes proper maintenance and contributes to the age-associated decline in muscle mass, known as sarcopenia. The myogenic potential impairment does not depend on a reduced myogenic cell number, but mainly on their difficulty to complete a differentiation program. The unbalanced production of reactive oxygen species in elderly people could be responsible for skeletal muscle impairments. microRNAs are conserved post-transcriptional regulators implicated in numerous biological processes including adult myogenesis. Here, we measure the ROS level and analyze myomiR (miR-1, miR-133b and miR-206) expression in human myogenic precursors obtained from Vastus lateralis of elderly and young subjects to provide the molecular signature responsible for the differentiation impairment of elderly activated satellite cells.

The Duchenne and Becker muscular dystrophies are caused by mutation of dystrophin gene and primarily affect skeletal and cardiac muscles. Cardiac involvement in dystrophic GRMD dogs has been demonstrated by electrocardiographic studies with the onset of a progressive cardiomyopathy similar to the cardiac disease in DMD patients. In this respect, GRMD is a useful model to explore cardiac and skeletal muscle pathogenesis and for developing new therapeutic protocols. Here we describe a protocol to convert GRMD canine fibroblasts isolated from heart and skin into induced cardiac-like myocytes (ciCLMs). We used a mix of transcription factors (GATA4, HAND2, TBX5, and MEF2C), known to be able to differentiate mouse and human somatic cells into ciCLMs. Exogenous gene expression was obtained using four lentiviral vectors carrying transcription factor genes and different resistance genes. Our data demonstrate a direct switch from fibroblast into ciCLMs with no activation of early cardiac genes. ciCLMs were unable to contract spontaneously, suggesting, differently from mouse and human cells, an incomplete differentiation process. However, when transplanted in neonatal hearts of SCID/Beige mice, ciCLMs participate in cardiac myogenesis.

The effectiveness of therapeutic treatment based on regenerative medicine for degenerative diseases (i.e., neurodegenerative or cardiac diseases) requires tools allowing the visualization and analysis of the three-dimensional (3D) distribution of target drugs within the tissue. Here, we present a new computational procedure able to overcome the limitations of visual analysis emerging by the examination of a molecular signal within images of serial tissue/organ sections by using the conventional techniques. Together with the 3D anatomical reconstitution of the tissue/organ, our framework allows the detection of signals of different origins (e.g., marked generic molecules, colorimetric, or fluorimetric substrates for enzymes; microRNA; recombinant protein). Remarkably, the application does not require the employment of specific tracking reagents for the imaging analysis. We report two different representative applications: the first shows the reconstruction of a 3D model of mouse brain with the analysis of the distribution of the β-Galactosidase, the second shows the reconstruction of a 3D mouse heart with the measurement of the cardiac volume.

The skeletal fibers have different embryological origin; the extraocular and jaw-closer muscles develop from prechordal mesoderm while the limb and trunk muscles from somites. These different origins characterize also the adult muscle stem cells, known as satellite cells (SCs) and responsible for the fiber growth and regeneration. The physiological properties of presomitic SCs and their epigenetics are poorly studied despite their peculiar characteristics to preserve muscle integrity during chronic muscle degeneration. Here, we isolated SCs from canine somitic [somite-derived muscle (SDM): vastus lateralis, rectus abdominis, gluteus superficialis, biceps femoris, psoas] and presomitic [pre-somite-derived muscle (PSDM): lateral rectus, temporalis, and retractor bulbi] muscles as myogenic progenitor cells from young and old animals. In addition, SDM and PSDM-SCs were obtained also from golden retrievers affected by muscular dystrophy (GRMD). We characterized the lifespan, the myogenic potential and functions, and oxidative stress of both somitic and presomitic SCs with the aim to reveal differences with aging and between healthy and dystrophic animals. The different proliferation rate was consistent with higher telomerase activity in PSDM-SCs compared to SDM-SCs, although restricted at early passages. SDM-SCs express early (Pax7, MyoD) and late (myosin heavy chain, myogenin) myogenic markers differently from PSDM-SCs resulting in a more efficient and faster cell differentiation. Taken together, our results showed that PSDM-SCs elicit a stronger stem cell phenotype compared to SDM ones. Finally, myomiR expression profile reveals a unique epigenetic signature in GRMD SCs and miR-206, highly expressed in dystrophic SCs, seems to play a critical role in muscle degeneration. Thus, miR-206 could represent a potential target for novel therapeutic approaches.

The fat-muscle communication regulates metabolism and involves circulating signals like adiponectin. Modulation of this cross-talk could benefit muscle bioenergetics and exercise tolerance in conditions like obesity. Chronic daily intake of exogenous glucocorticoids produces or exacerbates metabolic stress, often leading to obesity. In stark contrast to the daily intake, we discovered that intermittent pulses of glucocorticoids improve dystrophic muscle metabolism. However, the underlying mechanisms, particularly in the context of obesity, are still largely unknown. Here we report that in mice with diet-induced obesity, intermittent once-weekly prednisone increased total and high-molecular weight adiponectin levels and improved exercise tolerance and energy expenditure. These effects were dependent upon adiponectin, as shown by genetic ablation of the adipokine. Upregulation of Adipoq occurred through the glucocorticoid receptor (GR), as this effect was blocked by inducible GR ablation in adipocytes. The treatment increased the muscle metabolic response of adiponectin through the CAMKK2-AMPK cascade. Our study demonstrates that intermittent glucocorticoids produce healthful metabolic remodeling in diet-induced obesity.

Muscular regeneration is a complex biological process that occurs during acute injury and chronic degeneration, implicating several cell types. One of the earliest events of muscle regeneration is the inflammatory response, followed by the activation and differentiation of muscle progenitor cells. However, the process of novel neuromuscular junction formation during muscle regeneration is still largely unexplored. Here, we identify by single-cell RNA sequencing and isolate a subset of vessel-associated cells able to improve myogenic differentiation. We termed them 'guide' cells because of their remarkable ability to improve myogenesis without fusing with the newly formed fibers. In vitro, these cells showed a marked mobility and ability to contact the forming myotubes. We found that these cells are characterized by CD44 and CD34 surface markers and the expression of Ng2 and Ncam2. In addition, in a murine model of acute muscle injury and regeneration, injection of guide cells correlated with increased numbers of newly formed neuromuscular junctions. Thus, we propose that guide cells modulate de novo generation of neuromuscular junctions in regenerating myofibers. Further studies are necessary to investigate the origin of those cells and the extent to which they are required for terminal specification of regenerating myofibers.

Mitochondrial capacity is critical to adapt the high energy demand of the heart to circadian oscillations and diseased states. Glucocorticoids regulate the circadian cycle of energy metabolism, but little is known about how circadian timing of exogenous glucocorticoid dosing directly regulates heart metabolism through cardiomyocyte-autonomous mechanisms. While chronic once-daily intake of glucocorticoids promotes metabolic stress and heart failure, we recently discovered that intermittent once-weekly dosing of exogenous glucocorticoids promoted muscle metabolism in normal and obese skeletal muscle. However, the effects of glucocorticoid intermittence on heart metabolism and heart failure remain unknown. Here we investigated the extent to which circadian time of dosing regulates the effects of the glucocorticoid prednisone in heart metabolism and function in conditions of single pulse or chronic intermittent dosing.

Circadian time of intake determines the cardioprotective outcome of glucocorticoids in normal and infarcted hearts. The cardiomyocyte-specific glucocorticoid receptor (GR) is genetically required to preserve normal heart function in the long-term. The GR co-factor KLF15 is a pleiotropic regulator of cardiac metabolism. However, the cardiomyocyte-autonomous metabolic targets of the GR-KLF15 concerted epigenetic action remain undefined. Here we report that circadian time of intake determines the activation of a transcriptional and functional glucose oxidation program in heart by the glucocorticoid prednisone with comparable magnitude between sexes. We overlayed transcriptomics, epigenomics and cardiomyocyte-specific inducible ablation of either GR or KLF15. Downstream of a light-phase prednisone stimulation in mice, we found that both factors are non-redundantly required in heart to transactivate the adiponectin receptor expression (Adipor1) and promote insulin-stimulated glucose uptake, as well as transactivate the mitochondrial pyruvate complex expression (Mpc1/2) and promote pyruvate oxidation. We then challenged this time-specific drug effect in obese diabetic db/db mice, where the heart shows insulin resistance and defective glucose oxidation. Opposite to dark-phase dosing, light-phase prednisone rescued glucose oxidation in db/db cardiomyocytes and diastolic function in db/db hearts towards control-like levels with sex-independent magnitude of effect. In summary, our study identifies novel cardiomyocyte-autonomous metabolic targets of the GR-KLF15 concerted program mediating the time-specific cardioprotective effects of glucocorticoids on cardiomyocyte glucose utilization.

Muscular dystrophies are a heterogeneous group of myopathies, characterized by muscle weakness and degeneration, without curative treatment. Mesoangioblasts (MABs) have been proposed as a potential regenerative therapy. To improve our understanding of the in vivo behavior of MABs and the effect of different immunosuppressive therapies, like cyclosporine A or co-stimulation-adhesion blockade therapy, on cell survival noninvasive cell monitoring is required. Therefore, cells were transduced with a lentiviral vector encoding firefly luciferase (Fluc) and the human sodium iodide transporter (hNIS) to allow cell monitoring via bioluminescence imaging (BLI) and small-animal positron emission tomography (PET). Non-H2 matched mMABs were injected in the femoral artery of dystrophic mice and were clearly visible via small-animal PET and BLI. Based on noninvasive imaging data, we were able to show that co-stim was clearly superior to CsA in reducing cell rejection and this was mediated via a reduction in cytotoxic T cells and upregulation of regulatory T cells.

Induced pluripotent stem cells (iPSCs) hold great potential not only for human but also for veterinary purposes. The equine industry must often deal with health issues concerning muscle and cartilage, where comprehensive regenerative strategies are still missing. In this regard, a still open question is whether equine iPSCs differentiate toward muscle and cartilage, and whether donor cell type influences their differentiation potential. We addressed these questions through an isogenic system of equine iPSCs obtained from myogenic mesoangioblasts (MAB-iPSCs) and chondrogenic mesenchymal stem cells (MSC-iPSCs). Despite similar levels of pluripotency characteristics, the myogenic differentiation appeared enhanced in MAB-iPSCs. Conversely, the chondrogenic differentiation was augmented in MSC-iPSCs through both teratoma and in vitro differentiation assays. Thus, our data suggest that equine iPSCs can differentiate toward the myogenic and chondrogenic lineages, and can present a skewed differentiation potential in favor of the source cell lineage.

The muscular dystrophies are genetically diverse. Shared pathological features among muscular dystrophies include breakdown, or loss of muscle, and accompanying fibrotic replacement. Novel strategies are needed to enhance muscle repair and function and to slow this pathological remodeling. Glucocorticoid steroids, like prednisone, are known to delay loss of ambulation in patients with Duchenne muscular dystrophy but are accompanied by prominent adverse effects. However, less is known about the effects of steroid administration in other types of muscular dystrophies, including limb-girdle muscular dystrophies (LGMDs). LGMD 2B is caused by loss of dysferlin, a membrane repair protein, and LGMD 2C is caused by loss of the dystrophin-associated protein, γ-sarcoglycan. Herein, we assessed the efficacy of steroid dosing on sarcolemmal repair, muscle function, histopathology, and the regenerative capacity of primary muscle cells. We found that in murine models of LGMD 2B and 2C, daily prednisone dosing reduced muscle damage and fibroinflammatory infiltration. However, daily prednisone dosing also correlated with increased muscle adipogenesis and atrophic remodeling. Conversely, intermittent dosing of prednisone, provided once weekly, enhanced muscle repair and did not induce atrophy or adipogenesis, and was associated with improved muscle function. These data indicate that dosing frequency of glucocorticoid steroids affects muscle remodeling in non-Duchenne muscular dystrophies, suggesting a positive outcome associated with intermittent steroid dosing in LGMD 2B and 2C muscle.