Various protocols have been proven effective in the directed differentiation of mouse and human pluripotent stem cells into skeletal muscles and used to study myogenesis. Current 2D myogenic differentiation protocols can mimic muscle development and its alteration under pathological conditions such as muscular dystrophies. 3D skeletal muscle differentiation approaches can, in addition, model the interaction between the various cell types within the developing organoid. Our protocol ensures the differentiation of human embryonic/induced pluripotent stem cells (hESC/hiPSC) into skeletal muscle organoids (SMO) via cells with paraxial mesoderm and neuromesodermal progenitors\' identity and further production of organized structures of the neural plate margin and the dermomyotome. Continuous culturing omits neural lineage differentiation and promotes fetal myogenesis, including the maturation of fibroadipogenic progenitors and PAX7-positive myogenic progenitors. The PAX7 progenitors resemble the late fetal stages of human development and, based on single-cell transcriptomic profiling, cluster close to adult satellite cells of primary muscles. To overcome the limited availability of muscle biopsies from patients with muscular dystrophy during disease progression, we propose to use the SMO system, which delivers a stable population of skeletal muscle progenitors from patient-specific iPSCs to investigate human myogenesis in healthy and diseased conditions. Key features • Development of skeletal muscle organoid differentiation from human pluripotent stem cells, which recapitulates myogenesis. • Analysis of early embryonic and fetal myogenesis. • Provision of skeletal muscle progenitors for in vitro and in vivo analysis for up to 14 weeks of organoid culture. • In vitro myogenesis from patient-specific iPSCs allows to overcome the bottleneck of muscle biopsies of patients with pathological conditions.

Duchenne Muscular Dystrophy (DMD) is marked by genetic mutations occurring in the DMD gene, which is widely expressed in the cardiovascular system. In addition to developing cardiomyopathy, patients with DMD have been reported to be susceptible to the development of symptomatic hypotension, although the mechanisms are unclear. Analysis of single-cell RNA sequencing data has identified potassium voltage-gated channel subfamily Q member 5 (KCNQ5) and possibly ryanodine receptor 2 (RyR2) as potential candidate hypotension genes whose expression is significantly upregulated in the vascular smooth muscle cells of DMD mutant mice. We hypothesize that heightened KCNQ5 and RyR2 expression contributes to decreased arterial blood pressure in patients with DMD. Exploring pharmacological approaches to inhibit the KCNQ5 and RyR2 channels holds promise in managing the systemic hypotension observed in individuals with DMD. This avenue of investigation presents new prospects for improving clinical outcomes for these patients.

The dystrophin-glycoprotein complex (DGC) plays a crucial role in maintaining the structural integrity of the plasma membrane and the neuromuscular junction. In this study, we investigated the impact of the deficiency of α-dystrobrevin (αdbn), a component of the DGC, on the homeostasis of intracellular organelles, specifically mitochondria and the sarcoplasmic reticulum (SR). In αdbn deficient muscles, we observed a significant increase in the membrane-bound ATP synthase complex levels, a marker for mitochondria in oxidative muscle fiber types compared to wild-type. Furthermore, examination of muscle fibers deficient in αdbn using electron microscopy revealed profound alterations in the organization of mitochondria and the SR within certain myofibrils of muscle fibers. This included the formation of hyper-branched intermyofibrillar mitochondria with extended connections, an extensive network spanning several myofibrils, and a substantial increase in the number/density of subsarcolemmal mitochondria. Concurrently, in some cases, we observed significant structural alterations in mitochondria, such as cristae loss, fragmentation, swelling, and the formation of vacuoles and inclusions within the mitochondrial matrix cristae. Muscles deficient in αdbn also displayed notable alterations in the morphology of the SR, along with the formation of distinct anomalous concentric SR structures known as whorls. These whorls were prevalent in αdbn-deficient mice but were absent in wild-type muscles. These results suggest a crucial role of the DGC αdbn in regulating intracellular organelles, particularly mitochondria and the SR, within muscle cells. The remodeling of the SR and the formation of whorls may represent a novel mechanism of the unfolded protein response (UPR) in muscle cells.

The muscle is the principal tissue that is capable to transform potential energy into kinetic energy. This process is due to the transformation of chemical energy into mechanical energy to enhance the movements and all the daily activities. However, muscular tissues can be affected by some pathologies associated with genetic alterations that affect the expression of proteins. As the muscle is a highly organized structure in which most of the signaling pathways and proteins are related to one another, pathologies may overlap. Duchenne muscular dystrophy (DMD) is one of the most severe muscle pathologies triggering degeneration and muscle necrosis. Several mathematical models have been developed to predict muscle response to different scenarios and pathologies. The aim of this review is to describe DMD and Becker muscular dystrophy in terms of cellular behavior and molecular disorders and to present an overview of the computational models implemented to understand muscle behavior with the aim of improving regenerative therapy.

Myopathies are a common manifestation of endocrine disorders. Endocrine myopathies are often overlooked while considering differential diagnoses in patients with musculoskeletal symptoms. The hindrance to mobility and the musculoskeletal discomfort owing to these myopathies are important causes of disability and depreciated quality of life in these patients. Endocrine myopathies occur due to the effects of endogenous or iatrogenic hormonal imbalance on skeletal muscle protein and glucose metabolism, disrupting the excitation-contraction coupling. Abnormalities of the pituitary, thyroid, parathyroid, adrenal, and gonadal hormones have all been associated with myopathies and musculoskeletal symptoms. Endocrine myopathies can either be the complication of a secondary endocrine disorder or a presenting symptom of a missed underlying disorder. Therefore, an underlying endocrine abnormality must always be excluded in all patients with musculoskeletal symptoms. This review presents a compilation of various endocrine myopathies, their etiopathogenesis, clinical presentation, diagnostic modalities, and treatment protocols.

The vegetal polyphenol curcumin displays beneficial effects against skeletal muscle derangement induced by oxidative stress, disuse or aging. Since oxidative stress and inflammation are involved in the progression of muscle dystrophy, the effects of curcumin administration were investigated in the diaphragm of mdx mice injected intraperitoneally or subcutaneously with curcumin for 4-12-24 weeks. Curcumin treatment independently of the way and duration of administration (i) ameliorated myofiber maturation index without affecting myofiber necrosis, inflammation and degree of fibrosis; (ii) counteracted the decrease in type 2X and 2B fiber percentage; (iii) increased about 30% both twitch and tetanic tensions of diaphragm strips; (iv) reduced myosin nitrotyrosination and tropomyosin oxidation; (v) acted on two opposite nNOS regulators by decreasing active AMP-Kinase and increasing SERCA1 protein levels, the latter effect being detectable also in myotube cultures from mdx satellite cells. Interestingly, increased contractility, decreased myosin nitrotyrosination and SERCA1 upregulation were also detectable in the mdx diaphragm after a 4-week administration of the NOS inhibitor 7-Nitroindazole, and were not improved further by a combined treatment. In conclusion, curcumin has beneficial effects on the dystrophic muscle, mechanistically acting for the containment of a deregulated nNOS activity.

299In Duchenne muscular dystrophy, dystrophic muscle phenotypes are closely associated with the exhaustion of muscle stem cells. Transplantation of muscle stem cells has been widely studied for improving muscle regeneration, but poor cell survival and self-renewal, rapid loss of stemness, and limited dispersion of grafted cells following transplantation have collectively hindered the overall success of this strategy. Optimized mechanisms for maintaining and improving stem cell function are naturally present in the microenvironment of the stem cell niche in healthy muscles. Therefore, one logical strategy toward improving stem cell function and efficiency of stem cell transplantation in diseased muscles would be the establishment of a microenvironment mimicking some key aspects of healthy native stem cell niches. Here, we applied inkjet-based bioprinting technology to engineer a mimicked artificial stem cell niche in dystrophic muscle, comprising stem cell niche regulating factors (Notch activator DLL1) bioprinted onto 3D DermaMatrix construct. The recombinant DLL1 protein, DLL1 (mouse): Fc (human) (rec), was applied here as the Notch activator. Bioprinted DermaMatrix construct was seeded with muscle stem cells in vitro, and increased stem cell maintenance and repressed myogenic differentiation process was observed. DLL1 bioprinted DermaMatrix construct was then engrafted into dystrophic muscle of mdx/scid mice, and the improved cell engraftment and progression of muscle regeneration was observed 10 days after engraftment. Our results demonstrated that bioprinting of Notch activator within 3D construct can be applied to serve as muscle stem cell niche and improve the efficacy of muscle stem cell transplantation in diseased muscle.

MRI\'s T2 relaxation time is a valuable biomarker for neuromuscular disorders and muscle dystrophies. One of the hallmarks of these pathologies is the infiltration of adipose tissue and a loss of muscle volume. This leads to a mixture of two signal components, from fat and from water, to appear in each imaged voxel, each having a specific T2 relaxation time. In this proof-of-concept work, we present a technique that can separate the signals from water and from fat within each voxel, measure their separate T2 values, and calculate their relative fractions. The echo modulation curve (EMC) algorithm is a dictionary-based technique that offers accurate and reproducible mapping of T2 relaxation times. We present an extension of the EMC algorithm for estimating subvoxel fat and water fractions, alongside the T2 and proton-density values of each component. To facilitate data processing, calf and thigh anatomy were automatically segmented using a fully convolutional neural network and FSLeyes software. The preprocessing included creating two signal dictionaries, for water and for fat, using Bloch simulations of the prospective protocol. Postprocessing included voxelwise fitting for two components, by matching the experimental decay curve to a linear combination of the two simulated dictionaries. Subvoxel fat and water fractions and relaxation times were generated and used to calculate a new quantitative biomarker, termed viable muscle index, and reflecting disease severity. This biomarker indicates the fraction of remaining muscle out of the entire muscle region. The results were compared with those using the conventional Dixon technique, showing high agreement (R = 0.98, p < 0.001). It was concluded that the new extension of the EMC algorithm can be used to quantify abnormal fat infiltration as well as identify early inflammatory processes corresponding to elevation in the T2 value of the water (muscle) component. This new ability may improve the diagnostic accuracy of neuromuscular diseases, help stratification of patients according to disease severity, and offer an efficient tool for tracking disease progression.

OBJECTIVE: In muscular dystrophies (MD) patients with end-stage heart failure (HF), continuous flow left ventricular assist device (cf-LVAD) therapy is still controversial due to a progressive nature of MD-associated muscle weakness.
METHODS: All the MD patients who had cf- VAD implants between March 2013 and August 2019 in our hospital were retrospectively studied. Study end points were death, major LVAD-associated complications or respiratory dysfunction caused by muscular weakness.
RESULTS: A total of 11 MD patients (Becker type: n = 6; Emery-Dreifuss Myodystrophy: n = 2; Fukuyama subtype: n = 1; Limb-girdle 1B: n = 2) were enrolled.
METHODS: median age 41 years (IQR; 29-47); median Japanese Registry for Mechanically Assisted Circulatory Support: level 3 (2-3); a median interval between MD diagnosis and LVAD implantation 9 years (6-18). The pulmonary function test at LVAD implantation showed a median of %VC; 62% (45-82), FEV1%, 82% (81-88). Survival to discharge was 100% without pulmonary complication and early VAD-related complications. During a median follow-up of 38 months (27-53), re-admissions were needed due to device infection (n = 2), cerebrovascular accidents (disabling, n = 2 and non-disabling, n = 2), ventricular tachycardia (n = 4), and right HF (n = 3), respectively. 7 patients received successful heart transplant after a median waiting time of 44 months (34-61); 3 patients are still on the waiting list (waiting time: 21, 38, and 39 months). One patient died of right HF 15 months after VAD implantation. No one had overt pulmonary dysfunction during LVAD support.
CONCLUSIONS: In selected MD patients with end-stage HF, cf-LVAD therapy is a viable therapeutic option as bridge to heart transplant.

The clinical epidemiology of inherited myopathies in sub-Saharan Africa (SSA) is unknown but likely underdiagnosed due to problems of scientific research and social issues. We report a case series of patients born in SSA, evacuated to Portugal through an international health protocol and seen at a single neuromuscular disorders centre, between January/2004 and August/2021. We identified 9 patients (5 males), 35.6 ± 19.3 years-old (10-64), from Cape Verde (n = 4), Angola (n = 2), Sao Tome and Principe (n = 2) and Guinea-Bissau (n = 1), with a delay in diagnosis of 19.7 ± 14.3 years. Seven patients (77.8 %) had positive family history. Most patients had significant morbidity, requiring wheelchair (55.6 %), and nocturnal non-invasive ventilation (55.6 %). The diagnosis included Bethlem myopathy (n = 2), Duchenne muscular dystrophy (n = 2), Emery-Dreifuss muscular dystrophy (n = 1), LGMDR1 (n = 2), LGMDR2 (n = 1), and type-1 myotonic dystrophy (n = 1). Genetic testing was remarkable for 3 mutations previously not described. Despite the small sample, the spectrum of hereditary myopathies in our cohort is like western studies. Further studies are needed to better understand the epidemiology of muscle diseases in SSA.