Degenerative Muscle Disease Research Articles

3D printing a biocompatible elastomer for modeling muscle regeneration after volumetric muscle loss

Volumetric muscle loss (VML) injuries due to trauma, tumor ablation, or other degenerative muscle diseases are debilitating and currently have limited options for self-repair. Advancements in 3D printing allow for the rapid fabrication of biocompatible scaffolds with designer patterns. However, the materials chosen are often stiff or brittle, which is not optimal for muscle tissue engineering. This study utilized a photopolymerizable biocompatible elastomer – poly (glycerol sebacate) acrylate (PGSA) – to develop an in vitro model of muscle regeneration and proliferation into an acellular scaffold after VML injury. Mechanical properties of the scaffold were tuned by controlling light intensity during the 3D printing process to match the specific tension of skeletal muscle. The effect of both geometric (channel sizes between 300 and 600 μm) and biologic (decellularized muscle extracellular matrix (dECM)) cues on muscle progenitor cell infiltration, proliferation, organization, and maturation was evaluated in vitro using a near-infrared fluorescent protein (iRFP) transfected cell line to assess cells in the 3D scaffold. Larger channel sizes and dECM coating were found to enhance cell proliferation and maturation, while no discernable effect on cell alignment was observed. In addition, a pilot experiment was carried out to evaluate the regenerative capacity of this scaffold in vivo after a VML injury. Overall, this platform demonstrates a simple model to study muscle progenitor recruitment and differentiation into acellular scaffolds after VML repair.

Interleukins 4 and 13 Induce Exon Skipping of Mutant Dystrophin Pre-mRNA to Restore Dystrophin Production

Duchenne Muscular Dystrophy is (DMD) a lethal muscle degenerative disease caused by nonsense or out of frame deletion mutations in the DMD gene [1], which encodes Dystrophin [2,3]. While multiple therapeutic strategies to ameliorate the disease symptoms are under development, there is currently no cure

Mineralocorticoid Receptor Signaling in the Inflammatory Skeletal Muscle Microenvironments of Muscular Dystrophy and Acute Injury

Duchenne muscular dystrophy (DMD) is a striated muscle degenerative disease due to loss of functional dystrophin protein. Loss of dystrophin results in susceptibility of muscle membranes to damage, leading to muscle degeneration and continuous inflammation and fibrosis that further exacerbate pathology. Long-term glucocorticoid receptor (GR) agonist treatment, the current standard-of-care for DMD, modestly improves prognosis but has serious side effects. The mineralocorticoid receptor (MR), a ligand-activated transcription factor present in many cell types, has been implicated as a therapeutic target for DMD. MR antagonists (MRAs) have fewer side effects than GR agonists and are used clinically for heart failure. MRA efficacy has recently been demonstrated for DMD cardiomyopathy and in preclinical studies, MRAs also alleviate dystrophic skeletal muscle pathology. MRAs lead to improvements in muscle force and membrane stability and reductions in degeneration, inflammation, and fibrosis in dystrophic muscles. Myofiber-specific MR knockout leads to most of these improvements, supporting an MR-dependent mechanism of action, but MRAs additionally stabilize myofiber membranes in an MR-independent manner. Immune cell MR signaling in dystrophic and acutely injured normal muscle contributes to wound healing, and myeloid-specific MR knockout is detrimental. More research is needed to fully elucidate MR signaling in striated muscle microenvironments. Direct comparisons of genomic and non-genomic effects of glucocorticoids and MRAs on skeletal muscles and heart will contribute to optimal temporal use of these drugs, since they compete for binding conserved receptors. Despite the advent of genetic medicines, therapies targeting inflammation and fibrosis will be necessary to achieve optimal patient outcomes.

EP017: GNE gene variants associated with thrombocytopenia with or without GNE myopathy

GNE Myopathy is a rare, degenerative skeletal muscle disease, first presenting with anterior tibialis weakness then progressing proximally, relatively sparing the quadriceps. Symptoms usually develop in early adulthood (between 20-40 years) and include bilateral foot drop and rimmed vacuoles on muscle biopsy. Most patients become wheelchair dependent within a decade after onset. Upper limbs are usually involved around a decade later than the lower limbs. GNE myopathy is caused by biallelic variants in GNE, which encodes the rate limiting bifunctional enzyme in sialic acid synthesis, UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE), leading to decreased sialic acid production. Sialic acid is a negatively charged sugar, that serves as the terminal sugar of many N- and O-linked glycoproteins; it serves many functions in cell interactions and signaling. Recently, GNE variants have been associated with thrombocytopenia, with or without manifestations of myopathy. To investigate this phenomenon, we performed a literature review and analyzed data of patients in our GNE myopathy prospective natural history (NH) study. Data were collected through a prospective NH study of individuals with GNE myopathy (NCT1417533). Patients are admitted to the NIH Clinical Center for investigations, including physical function tests, questionnaires, blood analysis (including a complete blood count at each visit), and cardiac and pulmonary function tests. Patient platelet data were compared to normal values. For the literature review, two authors performed a search of all reports including GNE and thrombocytopenia. Clinical manifestations as well as variants identified were compared to those of our cohort. We analyzed a total of 126 platelet counts obtained from 51 GNE myopathy patients (61% female), between 21 and 65 years of age at their baseline to our NH study, with at least one platelet count obtained during subsequent visits. The average platelet count for females was 251 X 109 cells/L (range 144–390) and 205 X 109 cells/L for males (range 103-300), which are within the normal range for our laboratory (173–369 and 161-347, respectively). However, these values are lower than the expected means of 271 for females (p < 0.001) or 254 for males (p < 0.0001), based upon our lab’s normal ranges. A total of 8 individuals were below the normal range at some point, but above the pathologic thrombocytopenia cutoff (100). None presented signs of thrombocytopenia. The literature review yielded 25 patients with thrombocytopenia (17 without myopathy and 8 with myopathy) and biallelic GNE variants. The GNE variants in the 25 patients included 7 novel variants not reported in individuals with GNE myopathy and 11 variants previously associated with GNE myopathy. The novel variants were not localized to specific functional GNE domains but were scattered across the entire GNE gene and found in Chinese, European, Pakistani, Thai, and Palestinian Arab populations. None of our 51 NH study patients had any of the 18 GNE variants associated with thrombocytopenia. Although there was no evident thrombocytopenia in our NH study cohort, the average platelet count was slightly lower than expected, suggesting a potential role of GNE in platelet survival. Previous studies indicate that sialic acid has a significant role in platelet function. Mice deficient in a sialyltransferase had decreased sialic acid and platelet survival times, leading to thrombocytopenia. Patients with GNE myopathy and thrombocytopenia had increased reticulated platelets, suggesting that pathogenic GNE variants may contribute to platelet surface hyposialylation, resulting in increased platelet clearance by the Ashwell-Morell receptor.

Paeonia lactiflora extract improves the muscle function of mdx mice, an animal model of Duchenne muscular dystrophy, via downregulating the high mobility group box 1 protein

Ethnopharmacological relevancePaeonia lactiflora Pall. is an ethnopharmacological medicine with a long history of human use for treating various inflammatory diseases in many Asian countries.Aim of the study: Duchenne muscular dystrophy (DMD) is an X-linked degenerative muscle disease affecting 1 in 3500 males and is characterized by severe muscle inflammation and a progressive decline in muscle function. This study aimed to elucidate the effects of an ethanol extract of the root of Paeonia lactiflora Pall. (PL) on the muscle function in the muscular dystrophy X-linked (mdx) mouse, the most commonly used animal model of DMD. Materials and methodsMale mdx mice and wild-type controls aged 5 weeks were orally treated with PL for 4 weeks. The corticosteroid prednisolone was used as a comparator drug. Muscle strength and motor coordination were assessed via the grip-strength and rotarod tests, respectively. Muscle damage was evaluated via histological examination and assessment of plasma creatine-kinase activity. Proteomic analyses were conducted to identify the muscle proteins whose levels were significantly affected by PL (ProteomeXchange identifier: PXD028886). Muscle and plasma levels of these proteins, and their corresponding mRNAs were measured using western blotting and ELISA, and quantitative reverse transcription–polymerase chain reaction, respectively. ResultsThe muscle strength and motor coordination of mdx mice were significantly increased by the oral treatment of PL. PL significantly reduced the histological muscle damage and plasma creatine-kinase activity. Proteomic analyses of the muscle showed that PL significantly downregulated the high mobility group box 1 (HMGB1) protein and Toll-like receptor (TLR) 4, thus suppressing the HMGB1–TLR4–NF-κB signaling, in the muscle of mdx mice. Consequently, the muscle levels of proinflammatory cytokines/chemokines, which play crucial roles in inflammation, were downregulated. ConclusionPL improves the muscle function and reduces the muscle damage in mdx mice via suppressing the HMGB1-TLR4-NF-κB signaling and downregulating proinflammatory cytokines/chemokines.

Development of a functional antibody against Ca&lt;sup&gt;2+&lt;/sup&gt;permeable channel TRPV2 for treatment of human muscle degenerative diseases

Development of a functional antibody against Ca&lt;sup&gt;2+&lt;/sup&gt;permeable channel TRPV2 for treatment of human muscle degenerative diseases

A new therapeutic effect of fenofibrate in Duchenne muscular dystrophy: The promotion of myostatin degradation.

Duchenne muscular dystrophy (DMD) is a degenerative muscle disease with no effective drug treatment. This study investigated the positive effects of fenofibrate on dystrophic muscles. Myostatin expression in serum and muscle tissue from patients with Duchenne muscular dystrophy and mdx mice were tested. Primary myoblasts isolated from mdx mice were challenged with an inflammatory stimulus and treated with fenofibrate. In animal experiments, 6-week-old male mdx mice were treated with fenofibrate (100 mg kg-1 ) administered orally once per day for 6weeks. Effects of fenofibrate were evaluated by tests of muscle function plus histology and biochemical analyses of serum. Expression of myostatin, MuRF1, and atrogin-1 in skeletal muscle was evaluated by western blotting and real-time PCR. Total and oxidative myosin heavy chain (MHC) were assessed via immunofluorescence. Expression of myostatin protein was increased in dystrophic muscle of patients with Duchenne muscular dystrophy and mdx mice. Fenofibrate enhanced myofibre differentiation by down-regulating the expression of myostatin protein but not mRNA in primary myoblasts of mdx mice. Fenofibrate significantly improved muscle function while ameliorating muscle damage in mdx mice. These benefits were accompanied by an anti-inflammatory effect. Fenofibrate treatment returned myofibre function by inhibiting the expressions of myostatin, MuRF1, and atrogin-1 protein in the gastrocnemius muscle and diaphragm, while leaving the mRNA level of myostatin unaffected. Fenofibrate substantially slows muscle dystrophy by promoting the degradation of myostatin protein, which may indicate a new therapeutic focus for patients with Duchenne muscular dystrophy.

Female Outperformance in Voluntary Running Persists in Dystrophin-Null and Klotho-Overexpressing Mice.

Duchenne muscular dystrophy is a degenerative muscle disease that results from impairment of the dystrophin gene. The disease causes progressive loss in muscle mass and function. The anti-aging protein, α-klotho, has been implicated in the regulation of muscle regeneration. We previously discovered that mice harboring reduced α-klotho levels exhibited a decline in muscle strength and running endurance. To investigate the ability of α-klotho to improve overall endurance in a dystrophin null murine model, we examined the voluntary wheel running performance of dystrophin-null, mdx4cv mice overexpressing an α-klotho transgene. As expected, compared to wild type, both male and female dystrophic mice exhibited reduced running ability that was characterized by shorter running duration and longer periods of rest between cycles of activity. While our results did not detect an improvement in running performance with α-klotho overexpression, we identified distinct differences in the running patterns between females and males from all mouse strains analyzed (i.e., mdx4cv, mdx4cv overexpressing α-klotho, α-klotho overexpressing, α-klotho hypomorph, and wild type). For all strains, male mice displayed significantly reduced voluntary running ability compared to females. Further analysis of the mdx4cv strains demonstrated that male mice ran for shorter lengths of time and took longer breaks. However, we did not identify gender-associated differences in the actual speed at which mdx4cv mice ran. Our data suggest key differences in the running capabilities of female and male mice, which are of particular relevance to studies of dystrophin-null mice.

Rheological properties of skeletal muscles in a Duchenne muscular dystrophy murine model before and after autologous cell therapy

Duchenne muscular dystrophy (DMD) is still an incurable muscle degenerative disease; thus, numerous studies focused on novel therapeutic approaches. However, a simple assay of muscle function restoration remains needed. Herein, we used an oscillatory shear rheometer to evaluate changes in rheological properties of mouse muscles (tibialis anterior, TA) and their restoration upon autologous cell therapy by comparing the viscoelastic properties of normal, diseased and treated muscles. Amplitude sweep tests of muscle samples were performed under 20% compression over a range of shear strain between 0.01 and 2% and frequency of 1 rad/s. The samples were tested in plane-plane geometry and horizontal myofiber alignment. Typical linear viscoelastic region (LVER) patterns were found for each muscle type. For healthy muscles, a broad LVER between shear deformations (γ) of 0.013–0.62% was observed. The LVER of DMD mdx/SCID muscles was found at 0.14% to 0.46% shear deformation, and no shear dependence of storage (G') and loss (G“) moduli at γ range changing from 0.034% to 0.26% was found for transplanted tissues. G'LVER and G”LVER moduli of healthy muscles were significantly higher than G'LVER and G“LVER of dystrophic tissues. Additionally, muscle resistance assessment by rheometer indicated that muscles transplanted with stem cells restored elastic properties to levels close to those of healthy muscles. Interestingly, histological staining and rheological data indicate that the loss factor is strongly related to structural changes of examined muscles.

Role of DYSF Genetic Variant in Limb Girdle Muscular Dystrophy: A Case Report

Introduction: Muscular dystrophy is a hereditary degenerative muscle disease which progressively reduces the strength of the muscles that control movement. In this study, we tried to investigate genetic variants in muscular dystrophy using sequencing of whole exons. Case Presentation: A family with two affected patients with muscular dystrophy was referred for genetic counseling followed by exome sequencing testing on the proband. After filling out informed consent, blood samples were obtained from each available family member. Candidate genetic variant was confirmed using Sanger sequencing. Conclusions: Exome data analysis revealed a variant of c.2864 + 1G &gt; A in the proband, which altered the exon-intron 26 splice site within the DYSF gene. Genetic changes in this gene are known to be associated with muscular disorders, such as limb-girdle muscular dystrophy and other dysferlinopathies. Assessment of this genetic variant in the patient's sister also showed homozygous variant. Since the patient's sister was married to her cousin, the same variant was tested in her husband, which was normal homozygous. NGS-based techniques, including whole-exome sequencing, can identify the molecular genetic basis of the disease in families with limb-girdle muscular dystrophy. The results can be helpful in identifying potential carriers in the family and in prenatal diagnosis to the families involved.

MRTF-A regulates myoblast commitment to differentiation by targeting PAX7 during muscle regeneration.

Myocardin‐related transcription factor‐A/serum response factor (MRTF‐A/SRF), a well‐known transcriptional programme, has been proposed to play crucial roles in skeletal muscle development and function. However, whether MRTF‐A participates in muscle regeneration and the molecular mechanisms are not completely understood. Here, we show that MRTF‐A levels are highly correlated with myogenic genes using a RNA‐seq assay, which reveal that MRTF‐A knockdown in C2C12 cells significantly reduces PAX7 expression. Subsequent in vitro and in vivo data show that MRTF‐A and PAX7 present identical expression patterns during myoblast differentiation and CTX‐induced muscle injury and repair. Remarkably, MRTF‐A overexpression promotes myoblast proliferation, while inhibiting cell differentiation and the expression of MyoD and MyoG. MRTF‐A loss of function produces the opposite effect. Moreover, mice with lentivirus (MRTF‐A) injection possesses more PAX7+ satellite cells, but less differentiating MyoD+ and MyoG+ cells, leading subsequently to diminished muscle regeneration. Our mechanistic results reveal that MRTF‐A contributes to PAX7‐mediated myoblast self‐renewal, proliferation, and differentiation by binding to its distal CArG box. Overall, we propose that MRTF‐A functions as a novel PAX7 regulator upon myoblast commitment to differentiation, which could provide pathways for dictating muscle stem cell fate and open new avenues to explore stem cell‐based therapy for muscle degenerative diseases.

CircRNA FUT10 regulates the regenerative potential of aged skeletal muscle stem cells by targeting HOXA9.

Skeletal muscle is capable of repairing itself after injury to maintain the stability of its own tissue, but this ability declines with aging. Circular RNAs (circRNAs) are involved in cell aging. However, there is little research into their role and underlying mechanisms, especially in skeletal muscle stem cells (SkMSCs). In this study, we assessed circRNA FUT10 expression in aged and adult SkMSCs. We observed that circRNA FUT10 was upregulated in aged SkMSCs compared with that in adult SkMSCs. Furthermore, we identified putative miR-365-3p binding sites on circRNA FUT10, suggesting that this circRNA sponges miR-365a-3p. We also found that HOXA9 is a downstream target of miR-365a-3p and confirmed that miR-365a-3p can bind to circRNA FUT10 and the 3′-untranslated region of HOXA9 mRNA. This finding indicated that miR-365a-3p might serve as a “bridge” between circRNA FUT10 and HOXA9. Finally, we found that the circRNA FUT10/miR365a-3p/HOXA9 axis is involved in SkMSC aging. Collectively, our results show that the circRNA FUT10/miR365a-3p/HOXA9 axis is a promising therapeutic target and are expected to facilitate the development of therapeutic strategies to improve the prognosis of degenerative muscle disease.

Nrf2 activation induces mitophagy and reverses Parkin/Pink1 knock down-mediated neuronal and muscle degeneration phenotypes

The balanced functionality of cellular proteostatic modules is central to both proteome stability and mitochondrial physiology; thus, the age-related decline of proteostasis also triggers mitochondrial dysfunction, which marks multiple degenerative disorders. Non-functional mitochondria are removed by mitophagy, including Parkin/Pink1-mediated mitophagy. A common feature of neuronal or muscle degenerative diseases, is the accumulation of damaged mitochondria due to disrupted mitophagy rates. Here, we exploit Drosophila as a model organism to investigate the functional role of Parkin/Pink1 in regulating mitophagy and proteostatic responses, as well as in suppressing degenerative phenotypes at the whole organism level. We found that Parkin or Pink1 knock down in young flies modulated proteostatic components in a tissue-dependent manner, increased cell oxidative load, and suppressed mitophagy in neuronal and muscle tissues, causing mitochondrial aggregation and neuromuscular degeneration. Concomitant to Parkin or Pink1 knock down cncC/Nrf2 overexpression, induced the proteostasis network, suppressed oxidative stress, restored mitochondrial function, and elevated mitophagy rates in flies’ tissues; it also, largely rescued Parkin or Pink1 knock down-mediated neuromuscular degenerative phenotypes. Our in vivo findings highlight the critical role of the Parkin/Pink1 pathway in mitophagy, and support the therapeutic potency of Nrf2 (a druggable pathway) activation in age-related degenerative diseases.

A novel passive neck orthosis for patients with degenerative muscle diseases: Development & evaluation

The current study evaluated the effect of a passive neck orthosis, developed for patients suffering from progressive muscular diseases, on neck muscle activity in 10 adult healthy participants.The participants performed discrete head movements involving pure neck flexion (−10 to 30°), pure neck rotation (up to 30° left and right) and combined neck flexion-rotation (−10 to 30°) in steps of 10° by moving a cursor on a screen to reach predefined targets and staying on target for 10 s. Surface electromyography (EMG) was recorded from upper trapezius and sternocleidomastoid muscles and amplitudes were averaged over the static phases in trials with and without the orthosis. Moreover, the variability in head position and time required to perform the tasks were compared between conditions.Wearing the orthosis caused significant reductions (p = 0.027) in upper trapezius activity (a change of 0.2–1.5% EMGmax) while working against gravity. The activity level of the sternocleidomastoid muscle increased (p ≤ 0.025) by 0.3–1.0% EMGmax during pure and combined rotations without any pain reported.The orthosis showed potential to reduce the activity level of the upper trapezius muscle, the main load bearing muscle of the neck. Further study will be carried out to evaluate the effect in different patient groups.

Lgr5 Marks Adult Progenitor Cells Contributing to Skeletal Muscle Regeneration and Sarcoma Formation

Regeneration of adult skeletal muscle is driven largely by resident satellite cells, a stem cell population increasingly considered to display a high degree of molecular heterogeneity. In this study, we find that Lgr5, a receptor for Rspo and a potent mediator of Wnt/β-catenin signaling, marks a subset of activated satellite cells that contribute to muscle regeneration. Lgr5 is found to be rapidly upregulated in purified myogenic progenitors following acute cardiotoxin-induced injury. Invivo lineage tracing using our Lgr5-2ACreERT2R26tdTomatoLSL reporter mouse model shows that Lgr5+ cells can reconstitute damaged muscle fibers following muscle injury, as well as replenish the quiescent satellite cell pool. Moreover, conditional mutation in Lgr52ACreERT2;KrasG12D;Trp53flox/flox mice drives undifferentiated pleomorphic sarcoma formation in adult mice, thereby substantiating Lgr5+ cells as a cell of origin of sarcomas. Our findings provide the groundwork for developing Rspo/Wnt-signaling-based therapeutics to potentially enhance regenerative outcomes of skeletal muscles in degenerative muscle diseases.

Effect of myofibril architecture on the active contraction of dystrophic muscle. A mathematical model

Duchenne muscular dystrophy (DMD) is a muscle degenerative disease caused by a mutation in the dystrophin gene. The lack of dystrophin leads to persistent inflammation, degeneration/regeneration cycles of muscle fibers, Ca2＋ dysregulation, incompletely regenerated fibers, necrosis, fibrotic tissue replacement, and alterations in the fiber ultrastructure i.e., myofibril misalignment and branched fibers. This work aims to develop a comprehensive chemo-mechanical model of muscle-skeletal tissue accounting for dispersion in myofibrillar orientations, in addition to the disorders in sarcomere pattern and the fiber branching. The model results confirm a significant correlation between the myofibrillar dispersion and the reduction of isometric force in the dystrophic muscle and indicate that the reduction of contraction velocity in the dystrophic muscle seems to be associated with the local disorders in the sarcomere patterns of the myofibrils. Also, the implemented model can predict the force–velocity response to both concentric and eccentric loading. The resulting model represents an original approach to account for defects in the muscle ultrastructure caused by pathologies as DMD.

Abstract 14072: Bioenergetic and Metabolic Impairments in Duchenne Muscular Dystrophy (DMD) Patients' Induced Pluripotent Stem Cell-derived Cardiomyocytes (iPSC-CMs)

Introduction: DMD, an X-linked muscle degenerative fatal disease, is caused by mutations in the dystrophin gene. Dilated cardiomyopathy (DCM) is a major cause of morbidity and mortality in DMD patients. Since the DMD DCM treatment is limited, novel therapeutic modalities are urgently needed. Hypothesis: We hypothesized that dystrophin mutations in DMD lead to cardiomyopathy-causing bioenergetic/metabolic impairments, which can be therapeutically targeted for improving cardiac function. Methods: The study included iPSC-CMs from a healthy volunteer and 3 DMD patients (young male, YM; adult male, AM; adult female, AF) and mdx mice. We investigated the bioenergetics, electrophysiology, mitochondrial and metabolic features of iPSC-CMs and heart tissues using the SeaHorse Flux Analyzer, patch clamp, confocal fluorescence microscopy and Liquid Chromatography Mass Spectrometry (LC-MS) technologies, respectively. Results: To test the hypothesis, we measured the iPSC-CM respiration rate using the SeaHorse. Compared to healthy, in both AM and AF DMD, but not in YM DMD cardiomyocytes, ATP production decreased by 75%, and basal respiration decreased by 80% and 45%, respectively. In agreement with impaired oxidative phosphorylation capacity, mitochondrial activity measured by 3D life confocal microscopy was attenuated by 35% in AM, but not in YM. Concurring with the healthy-like bioenergetic status, YM iPSC-CMs fired regularly with no arrhythmias, in contrast to the prominent arrhythmias in AM and AF cardiomyocytes. Further, the LC-MS analysis demonstrated that the levels of 13%, 6% and 7% of the 120 metabolite measured, were lower in AM, AF and YM, while 10%, 18% and 43% were higher, respectively, compared to healthy iPSC-CMs ( p&lt;0.05 ). For example, in AM and AF (but not in YM) the phosphocreatine (PCr) levels were diminished by &gt;80%, compared to healthy iPSC-CMs, indicating a dysfunctional PCr energy system. Finally, the LC-MS analysis of mdx ventricular tissue demonstrated a statistically-significant different metabolic profile in 8 mdx mice vs 8 healthy mice. Conclusion: DMD iPSC-CMs and mdx mice exhibit bioenergetic/metabolic impairments, which constitute novel targets for alleviating the cardiomyopathy in DMD patients.

Bioenergetic and metabolic impairments in Duchenne Muscular Dystrophy (DMD) patients' iPSC-derived cardiomyocytes

Abstract Introduction DMD, an X-linked muscle degenerative fatal disease, is caused by mutations in the dystrophin gene. Dilated cardiomyopathy (DCM) is a major cause of morbidity and mortality in DMD patients. Treatments for DCM in DMD are limited to steroids and standard heart failure medications such as β-blockers and ACE-inhibitors, and therefore novel therapeutic modalities are urgently needed. Purpose We hypothesized that dystrophin mutations in DMD lead to cardiomyopathy-causing bioenergetic/metabolic impairments, which can be therapeutically targeted for improving cardiac function. Methods Induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs) were generated from healthy volunteer and 3 DMD patients: young male (YM), adult male (AM) and adult female (AF). We investigated the bioenergetics, electrophysiology, mitochondrial and metabolic features of healthy and DMD iPSC-CMs using the Seahorse Flux analyzer, patch clamp, confocal fluorescence microscopy and Liquid chromatography mass spectrometry (LC-MS) technologies, respectively. Results To test the hypothesis, we measured respiration and glycolytic rates of healthy and DMD iPSC-CMs. Compared to healthy iPSC-CMs, in both AM and AF DMD, but not in YM DMD cardiomyocytes, there was a 75% decrease in ATP production, and 80% and 45% decrease in basal respiration, respectively. In agreement with the healthy-like bioenergetic status of YM, the iPSC-CMs showed no arrhythmias, in contrast to the prominent arrhythmias in AM and AF cardiomyocytes. To determine whether the impairment in the phosphorylation pathway (OXPHOS) affects glycolysis, we measured the cardiomyocytes' response to glycolytic stress test. These experiments showed that the glycolytic rates were similar in healthy and DMD iPSC-CMs. In agreement with impaired OXPHOS, mitochondrial activity measured by 3D life confocal microscopy was attenuated in the DMD male by 35%, compared to healthy cardiomyocytes. Furthermore, the metabolomic LC-MS analyses demonstrated significant differences in metabolite levels in YM, AM and AF DMD iPSC-CMs relative to healthy iPSC-CMs. For example, compared to healthy iPSC-CMs, there was a dramatic fall to undetected levels in phosphocreatine in both AM and AF, but not in YM DMD, indicating a dysfunctional phosphocreatine energy system. Conclusions DMD iPSC-CMs exhibit bioenergetic/metabolic impairments, which constitute novel targets for alleviating the cardiomyopathy in DMD patients. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): ISF - Israel Science Foundation

The clock regulator Bmal1 protects against muscular dystrophy

The muscle-intrinsic clock machinery is required for the maintenance of muscle growth, remodeling and function. Our previous studies demonstrated that the essential transcription activator of the molecular clock feed-back loop, Brain and Muscle Arnt-Like 1(Bmal1), plays a critical role in myogenic progenitor behavior to promote and regenerative myogenesis. Using genetic approaches targeting Bmal1 in the DMDmdx (mdx) dystrophic mouse model, here we report that the loss of Bmal1 function significantly accelerated dystrophic disease progression. In contrast to the mild dystrophic changes in mdx mice, the genetic loss-of-function of Bmal1 aggravated muscle damage in this dystrophic disease background, as indicated by persistently elevated creatine kinase levels, increased injury area and reduced muscle grip strength. Mechanistic studies revealed that markedly impaired myogenic progenitor proliferation and myogenic response underlie the defective new myofiber formation in the chronic dystrophic milieu. Taken together, our study identified the function of pro-myogenic clock gene Bmal1 in protecting against dystrophic damage, suggesting the potential for augmenting Bmal1 function to ameliorate dystrophic or degenerative muscle diseases.

Tissue specific expression of sialic acid metabolic pathway: role in GNE myopathy.

GNE myopathy is an adult-onset degenerative muscle disease that leads to extreme disability in patients. Biallelic mutations in the rate-limiting enzyme UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine-kinase (GNE) of sialic acid (SA) biosynthetic pathway, was shown to be the cause of this disease. Other genetic disorders with muscle pathology where defects in glycosylation are known. It is yet not clear why a defect in SA biosynthesis and glycosylation affect muscle cells selectively even though they are ubiquitously present in all tissues. Here we have comprehensively examined the complete SA metabolic pathway involving biosynthesis, sialylation, salvage, and catabolism. To understand the reason for tissue-specific phenotype caused by mutations in genes of this pathway, we analysed the expression of different SA pathway genes in various tissues, during the muscle tissue development and in muscle tissues from GNE myopathy patients (p.Met743Thr) using publicly available databases. We have also analysed gene co-expression networks with GNE in different tissues as well as gene interactions that are unique to muscle tissues only. The results do show a few muscle specific interactions involving ANLN, MYO16 and PRAMEF25 that could be involved in specific phenotype. Overall, our results suggest that SA biosynthetic and catabolic genes are expressed at a very low level in skeletal muscles that also display a unique gene interaction network.