Ascorbic Acid Significantly Decreases Creatine Kinase Plasma Levels in an Animal Model of Statin/Fibrate-Induced Myopathy

Background. Myopathy is one of the side effects of lipid-lowering drugs, especially statins and particularly when combined with a fibrate. To diagnose myopathy and determine its severity, the plasma levels of three enzymes, creatine kinase (CK), aldolase, and lactate dehydrogenase (LDH), are routinely measured. Physical exercise can aggravate the statin-associated muscular disease. The question is whether antioxidants like ascorbic acid (Vit. C) can prevent such myopathy. Methods. In this experiment, a combination of atorvastatin (ATV, 80 mg/kg/day) and gemfibrozil (GMF, 1000 mg/kg/day) orally for 10 days as well as exercise as forced swimming on days 8, 9, and 10 were used to induce myopathy. Ascorbic acid (50 mg/kg/day, orally) was added to ATV/GMF plus exercise regimen throughout the 10 days in the treatment group. Mean blood levels of CK, aldolase, and LDH were measured in addition to swimming tolerance times. Results. There was a significantly higher swimming tolerance time P < 0.05 and lower CK levels P < 0.01 in rats receiving ATV/GMF/Vit. C plus exercise compared with rats not taking Vit. C. LDH and aldolase did not decrease significantly. Conclusion. The results of this study showed that Vit. C can be effective in preventing myopathy caused by fat-lowering drugs.

Simple and efficient differentiation of human iPSCs into contractible skeletal muscles for muscular disease modeling

Pathophysiological analysis and drug discovery targeting human diseases require disease models that suitably recapitulate patients pathology. Disease-specific human induced pluripotent stem cells (hiPSCs) can potentially recapitulate disease pathology more accurately than existing disease models when differentiated into affected cell types. Thus, successful modeling of muscular diseases requires efficient differentiation of hiPSCs into skeletal muscles. hiPSCs transduced with doxycycline-inducible MYOD1 (MYOD1-hiPSCs) have been widely used; however, they require time- and labor-consuming clonal selection procedures, and clonal variations must be overcome. Moreover, their functionality to exhibit muscular contraction has never been reported. Here, we demonstrated that bulk MYOD1-hiPSCs established with puromycin selection, but not with G418 selection, showed high differentiation efficiency, generating more than 80% Myogenin (MyoG)+ and Myosin heavy chain (MHC)+ muscle cells within seven days. Interestingly, bulk MYOD1-hiPSCs exhibited average differentiation properties compared with those of clonally established MYOD1-hiPSCs, suggesting that the bulk method may minimize the effects of clonal variations. Finally, three-dimensional muscle tissues were fabricated from bulk MYOD1-hiPSCs, which exhibited contractile force upon electrical pulse stimulation, indicating their functionality. Together, the findings indicate that our bulk differentiation requires less time and labor than existing methods, efficiently generates contractible skeletal muscles, and facilitates the generation of muscular disease models.

DMD/BMD prenatal diagnosis and treatment expectation in a single centre in China for 15 years

Objective DMD/BMD prenatal diagnosis for 931 foetuses. Background DMD is the most common fatal X-linked recessive muscular disease. There is no effective clinical treatment method at present. Accurate gene diagnosis and prenatal diagnosis technology are important ways for early detection, early prevention and early treatment. Methods A total of 931 prenatal diagnoses were performed for pregnant women with a definite family history of DMD or a history of DMD childbirth between 2005 and 2019. This report may be considered the largest DMD prenatal diagnosis report in a single centre worldwide. Multiple ligation-dependent probe amplification (MLPA) and next-generation sequencing were used in combination. Techniques and short tandem repeat (STR) linkage analysis were used to determine the location of the DMD gene mutation in the pregnant woman and then to detect the DMD gene in the foetuses. Results There were 872 families in our study. Among all 931 foetuses, 20.73% (193/931) were males expected to develop DMD and 16.33% (152/931) were female carriers. In addition, gonadal mosaicism was observed in 5 mothers, and gene recombination was identified in three foetuses. The results of the prenatal diagnosis were consistent with the results of the CPK analysis, and the results of the prenatal diagnosis were 100% accurate. Conclusions MLPA and Sanger sequencing, when combined with STR linkage analyses, can provide an accurate and rapid prenatal diagnosis. Due to the high de novo rate, prenatal diagnosis and genetic counselling should be given great attention.

Establishment of a Robust Platform for Induced Pluripotent Stem Cell Research Using Maholo LabDroid

Induced pluripotent stem cells (iPSCs) are attractive for use in early drug discovery because they can differentiate into any cell type. Maintenance cultures and differentiation processes for iPSCs, however, require a high level of technical expertise. To overcome this problem, technological developments such as enhanced automation are necessary to replace manual operation. In addition, a robot system with the flexibility and expandability to carry out maintenance culture and each of the required differentiation processes would also be important. In this study, we established a platform to enable the multiple processes required for iPSC experiments using the Maholo LabDroid, which is a humanoid robotic system with excellent reproducibility and flexibility. The accuracy and robustness of Maholo LabDroid enabled us to cultivate undifferentiated iPSCs for 63 days while maintaining their ability to differentiate into the three embryonic germ layers. Maholo LabDroid maintained and harvested iPSCs in six-well plates, then seeded them into 96-well plates, induced differentiation, and implemented immunocytochemistry. As a result, Maholo LabDroid was confirmed to be able to perform the processes required for myogenic differentiation of iPSCs isolated from a patient with muscular disease and achieved a high differentiation rate with a coefficient of variation (CV) <10% in the first trial. Furthermore, the expandability and flexibility of Maholo LabDroid allowed us to experiment with multiple cell lines simultaneously.

Ascorbic Acid Greatly Decreases Creatine Kinase Levels in An Animal Model of Statin/Fibrate-Induced Myopathy

BackgroundDrug-induced myopathy is one of the frequent forms of muscle disease, and drugs used for hyperlipidemia, especially the statins are a common culprit, and particularly when combined with a fibrate. Clinicians usually measure plasma levels of three enzymes, creatine kinase (CK), aldolase and lactate dehydrogenase (LDH) for diagnosis of myopathy and determination of its severity. Physical exercise can aggravate statin-associated muscular disease. The question is whether antioxidants like ascorbic acid (vit. C) can prevent such myopathy.MethodsIn this experiment a combination of oral atorvastatin (ATV, 80 mg/kg/day, orally) and gemfibrozil (GMF, 1000 mg/kg/day, orally) was used for ten days plus exercise in days 8, 9 and 10 to induce myopathy in rats. To add physical exercise, the forced swimming test was applied in the last three days. Ascorbic acid (50 mg/kg/day, orally) was added to ATV/GMF plus exercise regimen throughout the 10 days in the treatment group. The mean blood levels of CK, aldolase and LDH were measured in addition to swimming tolerance times. ResultsThere was a significantly lower swimming tolerance time (P < 0.05) and higher CK levels (P < 0.01) in rats receiving ATV/GMF/Vit.C plus exercise compared with rats not taking Vit.C. LDH and aldolase didn’t decrease significantly.ConclusionsA protective role of vit.C against drug-induced myopathy is suggested by the findings of this study.

Long-Term Exercise Reduces Formation of Tubular Aggregates and Promotes Maintenance of Ca2+ Entry Units in Aged Muscle

Tubular aggregates (TAs) in skeletal muscle fibers are unusual accumulation of sarcoplasmic reticulum (SR) tubes that are found in different disorders including TA myopathy (TAM). TAM is a muscular disease characterized by muscle pain, cramping, and weakness that has been recently linked to mutations in STIM1 and ORAI1. STIM1 and ORAI1 are the two main proteins mediating store-operated Ca2+ entry (SOCE), a mechanism activated by depletion of intracellular Ca2+ stores (e.g., SR) that allows recovery of Ca2+ from the extracellular space during repetitive muscle activity. We have recently shown that exercise triggers the formation of unique intracellular junctions between SR and transverse tubules named Ca2+entry units (CEUs). CEUs promote colocalization of STIM1 with ORAI1 and improve muscle function in presence of external Ca2+. TAs virtually identical to those of TAM patients are also found in fast-twitch fibers of aging male mice. Here, we used a combination of electron and confocal microscopy, Western blotting, and ex vivo stimulation protocols (in presence or absence of external Ca2+) to evaluate the presence of TAs, STIM1-ORAI1 localization and expression and fatigue resistance of intact extensor digitorum longus (EDL) muscles in wild-type male adult (4-month-old) and aged (24-month-old) mice and in mice trained in wheel cages for 15 months (from 9 to 24 months of age). The results collected indicate that (i) aging causes STIM1 and ORAI1 to accumulate in TAs and (ii) long-term exercise significantly reduced formation of TAs. In addition, (iii) EDL muscles from aged mice exhibited a faster decay of contractile force than adult muscles, likely caused by their inability to refill intracellular Ca2+ stores, and (iv) exercise in wheel cages restored the capability of aged EDL muscles to use external Ca2+ by promoting maintenance of CEUs. In conclusion, exercise prevented improper accumulation of STIM1 and ORAI1 in TAs during aging, maintaining the capability of aged muscle to refill intracellular Ca2+ stores via SOCE.

Phenotype and clinical outcomes of dystrophin associated dilated cardiomyopathy

Background Mutations in dystrophin gene (DMD) can cause skeletal myopathy and dilated cardiomyopathy (DCM) independently or in combination. Natural history of DMD mutation carriers and dystrophin-associated DCM is poorly understood. Objectives This study sought to describe phenotype and prognosis of DMD mutations in a large multicenter cohort of Non-Duchenne DMD mutations carriers. Methods The study cohort comprised 223 individuals with a DMD mutation (83% males, 33±15 years at first evaluation) followed at 26 European centers. Major adverse cardiac events (MACE) were defined as a composite of cardiac death, heart transplant, LVAD implantation, aborted SCD or appropriate ICD shock. Results At initial evaluation, 85 patients (38%) had DCM (52 in combination with muscular disease) and 92 (41%) had isolated muscular disease. After a median follow-up of 96 months, 112 individuals (53%) had DCM and 20% of the individuals who had normal cardiac function at baseline developed DCM. DCM penetrance by age 30 was 56%. DCM onset was associated with male sex and was independent of the type of mutation, the presence of skeletal myopathy or serum creatine kinase levels. MACE occurred in 11% and 22% individuals from the entire cohort and with DCM respectively, and were more frequent in DCM patients without muscular disease than in those with skeletal myopathy (35.5% vs 17.7%; p=0.04). Among patients with DCM, 18% developed end-stage heart failure and 9% a major arrhythmic event (SCD/aborted SCD/ICD shock/VT). There were not differences in survival between patients with isolated DCM and those with DCM and muscular phenotype. Decreased LVEF and increased left ventricular end-diastolic diameter at baseline were associated with MACE. Atrial fibrillation and neurological events were also frequent. Prognosis of individuals who did not develop DCM was good with 96% survival during follow-up. Conclusions DCM caused by mutations in DMD is characterized by moderate penetrance but a high risk of MACE, progression to end-stage heart failure and ventricular arrythmias. DCM onset is the major determinant of prognosis in DMD mutation carriers with similar survival irrespectively of the presence of concomitant muscular disease. Survival free of MACE analysis Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Instituto de Salud Carlos III. Contratos i-PFIS: Doctorados IIS-empresa en Ciencias y Tecnologías de la Salud

DUX4 Expression in FSHD Muscles: Focus on Its mRNA Regulation

Facioscapulohumeral dystrophy (FSHD) is the most frequent muscular disease in adults. FSHD is characterized by a weakness and atrophy of a specific set of muscles located in the face, the shoulder, and the upper arms. FSHD patients may present different genetic defects, but they all present epigenetic alterations of the D4Z4 array located on the subtelomeric part of chromosome 4, leading to chromatin relaxation and, ultimately, to the aberrant expression of one gene called DUX4. Once expressed, DUX4 triggers a cascade of deleterious events, eventually leading to muscle dysfunction and cell death. Here, we review studies on DUX4 expression in skeletal muscle to determine the genetic/epigenetic factors and regulatory proteins governing DUX4 expression, with particular attention to the different transcripts and their very low expression in muscle.

Urinary Dimethylamine (DMA) and Its Precursor Asymmetric Dimethylarginine (ADMA) in Clinical Medicine, in the Context of Nitric Oxide (NO) and Beyond

Asymmetric protein-arginine dimethylation is a major post-translational modification (PTM) catalyzed by protein-arginine methyltransferase (PRMT). Regular proteolysis releases asymmetric dimethylarginine (ADMA). Of the daily produced ADMA, about 10% are excreted unchanged in the urine. The remaining 90% are hydrolyzed by dimethylarginine dimethylaminohydrolase (DDAH) to L-citrulline and dimethylamine (DMA), which is readily excreted in the urine. The PRMT/DDAH pathway is almost the exclusive origin of urinary ADMA and the major source of urinary DMA. Dietary fish and seafood represent additional abundant sources of urinary DMA. The present article provides an overview of urinary ADMA and DMA reported thus far in epidemiological, clinical and pharmacological studies, in connection with the L-arginine/nitric oxide (NO) pathway and beyond, in neonates, children and adolescents, young and elderly subjects, males and females. Discussed diseases mainly include those relating to the renal and cardiovascular systems such as peripheral arterial occlusive disease, coronary artery disease, chronic kidney disease, rheumatoid arthritis, Becker muscular disease, Duchenne muscular disease (DMD), attention deficit hyperactivity disorder (ADHD), and type I diabetes. Under standardized conditions involving the abstinence of DMA-rich fresh and canned fish and seafood, urinary DMA and ADMA are useful as measures of whole-body asymmetric arginine-dimethylation in health and disease. The creatinine-corrected excretion rates of DMA range from 10 to 80 µmol/mmol in adults and up to 400 µmol/mmol in children and adolescents. The creatinine-corrected excretion rates of ADMA are on average 10 times lower. In general, diseases are associated with higher urinary DMA and ADMA excretion rates, and pharmacological treatment, such as with steroids and creatine (in DMD), decreases their excretion rates, which may be accompanied by a decreased urinary excretion of nitrate, the major metabolite of NO. In healthy subjects and in rheumatoid arthritis patients, the urinary excretion rate of DMA correlates positively with the excretion rate of dihydroxyphenylglycol (DHPG), the major urinary catecholamines metabolite, suggesting a potential interplay in the PRMT/DDAH/NO pathway.