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Background: Becker muscular dystrophy (BMD) is a dystrophinopathy caused by a pathological variant of the DMD gene. Urinary titin, a degradation product of the giant protein titin present in muscle sarcomeres, has been used as a biomarker to reflect muscle degradation in Duchenne muscular dystrophy, a more severe dystrophinopathy. However, the clinical significance of urinary titin levels in BMD remains unclear. This study aimed to investigate the relationship between urinary titin levels and the clinical data in patients with BMD., Methods: Urine samples were collected from 123 patients with BMD, and urinary titin levels were measured. The association of urinary titin with clinical data, including age, physical measurements, physical activity, blood tests, and cardiopulmonary test results, was examined., Results: A total of 257 urine samples were obtained from patients of 5-79 years of age. The median urinary titin level was 72.6 pmol/mg Cr (range 0.2-4325.0 pmol/mg Cr). No strong correlation was found between urinary titin levels and age, physical measurements, physical function, blood test results, or cardiopulmonary function. However, on comparing clinical data between the age-matched high urinary titin (N = 94) and normal (N = 29) groups, the high urinary titin group had a significantly greater number of non-ambulatory cases (23.9 % vs. 3.6 %), weaker grip strength (16.3 vs. 32.0 kg), and higher serum creatine kinase (1072 vs. 398 U/L) and cardiac troponin I (10.6 vs. 2.5 pg/mL) levels., Conclusion: Urinary titin was identified as a biomarker reflecting walking ability, muscle strength, and skeletal and cardiac damage in patients with BMD., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations of the dystrophin gene, which spans 2.4 Mb on the X chromosome. Creatine kinase (CK) activity in blood and titin fragment levels in urine have been identified as biomarkers in DMD to monitor disease progression and evaluate therapeutic intervention. However, the difference in the sensitivity of these biomarkers in DMD remains unclear. Previously, we generated transchromosomic mice carrying the full-length human dystrophin gene on a human artificial chromosome (DYS-HAC1) vector. The human dystrophin derived from DYS-HAC1 improved pathological phenotypes observed in DMD-null mice, which lack the entire 2.4 Mb of the dystrophin gene. In this study, we compared the values of plasma CK activity and urine/plasma titin fragment levels in wild-type (WT), DYS-HAC1, DMD-null, and DYS-HAC1; DMD-null mice. Plasma CK activity and urine/plasma titin fragment levels in DMD-null mice were significantly higher than those in WT mice. Although plasma CK activity showed no significant difference between WT and DYS-HAC1; DMD-null mice, urine/plasma titin fragment levels in DYS-HAC1; DMD-null mice were higher than those in WT mice. Human dystrophin in DYS-HAC1; DMD-null mice drastically improved muscular dystrophy phenotypes seen in DMD-null mice; however, the proportion of myofibers with central nuclei in DYS-HAC1; DMD-null mice had a tendency to be slightly higher than that in WT mice. These results suggest that urine/plasma titin fragment levels could be a more sensitive biomarker than plasma CK activity., Competing Interests: Declarations. Ethics approval and consent to participate: This study was approved by the Animal Care and Use Committee of Tottori University (Permit Numbers: 16-Y-20, 17-Y-28, 19-Y-22, 20-Y-31, 21-Y-26, and 22-Y-36). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Duchenne muscular dystrophy (DMD) results in a progressive loss of functional skeletal muscle mass (MM) and replacement with fibrofatty tissue. Accurate evaluation of MM in DMD patients has not previously been available. Our objective was to measure MM using the D 3 creatine (D 3 Cr) dilution method and determine its relationship with strength and functional capacity in patients with DMD over a wide range of ages. Subjects were recruited for participation in a 12 month, longitudinal, observational study. Here, we report the baseline data. A 20 mg dose of D 3 Cr dissolved in water was ingested by 92 patients with DMD (ages 4-25 years) followed later with a fasting urine sample. Enrichment of D 3 creatinine was determined by liquid chromatography-mass spectrometry analysis. The North Star Ambulatory Assessment (NSAA) total score was determined for ambulatory participants, and the Performance of Upper Limb (PUL 2.0) total score and grip strength for all participants. We observed a significant age-associated increase in body weight along with a substantial decrease in MM/body weight (%MM). MM and %MM were associated with PUL score (r = 0.517, P < 0.0001 and r = 0.764, P < 0.0001 respectively). The age-associated decrease in MM and %MM was strongly associated with ambulatory status. We observed very little overlap in %MM between ambulant and non-ambulant subjects, suggesting a threshold of 18-22% associated with loss of ambulation. MM is substantially diminished with advancing age and is highly related to clinically meaningful functional status. The D 3 Cr dilution method may provide a biomarker of disease progression and therapeutic efficacy in patients with DMD or other neuromuscular disorders. KEY POINTS: The non-invasive D 3 creatine dilution method provides novel data on whole body functional muscle mass (MM) in a wide range of ages in patients with DMD and reveals profoundly low functional MM in older non-ambulant patients. The difference in %MM between ambulant and non-ambulant subjects suggests a threshold for loss of ambulatory ability between 18 and 22% MM. The data suggest that as functional MM declines with age, maintaining a lower body weight may help to conserve ambulatory ability., (© 2024 The Author(s). The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Duchenne muscular dystrophy (DMD), the most common lethal muscular disorder, affects 1 in 5000 male births. It is caused by mutations in the X-linked dystrophin gene (DMD), and there is no effective treatment currently. Gene addition is a promising strategy owing to its universality for patients with all gene mutations types. In this study, we describe a site-specific gene addition strategy in induced pluripotent stem cells (iPSCs) derived from a DMD patient with exon 50 deletion. By using transcription activator-like effector nickases (TALENickases), the mini-dystrophin cassette was precisely targeted at the ribosomal RNA gene (rDNA) locus via homologous recombination with high targeting efficiency. The targeted clone retained the main pluripotent properties and was differentiated into cardiomyocytes. Significantly, the dystrophin expression and membrane localization were restored in the genetic corrected iPSCs and their derived cardiomyocytes. More importantly, the enhanced spontaneous contraction was observed in modified cardiomyocytes. These results provide a proof of principle for an efficient targeted gene addition for DMD gene therapy and represents a significant step toward precisely therapeutic for DMD., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Since the protein constituents of urine present a dynamic proteome that can reflect a variety of disease-related alterations in the body, the mass spectrometric survey of proteome-wide changes in urine promises new insights into pathogenic mechanisms. Urine can be investigated in a completely non-invasive way and provides valuable biomedical information on body-wide changes. In this report, we have focused on the urine proteome in X-linked muscular dystrophy using the established mdx-4cv mouse model of dystrophinopathy. In order to avoid potential artefacts due to the manipulation of the biofluid proteome prior to mass spectrometry, crude urine specimens were analyzed without the prior usage of centrifugation steps or concentration procedures. Comparative proteomics revealed 21 increased and 8 decreased proteins out of 870 identified urinary proteoforms using 50 μl of biofluid per investigated sample, i.e. 14 wild type versus 14 mdx-4cv specimens. Promising marker proteins that were almost exclusively found in mdx-4cv urine included nidogen, parvalbumin and titin. Interestingly, the mass spectrometric identification of urine-associated titin revealed a wide spread of peptides over the sequence of this giant muscle protein. The newly established urinomic signature of dystrophinopathy might be helpful for the design of non-invasive assays to improve diagnosis, prognosis, therapy-monitoring and evaluation of potential harmful side effects of novel treatments in the field of muscular dystrophy research.

Urine is increasingly being considered as a source of biomarker development in Duchenne Muscular Dystrophy (DMD), a severe, life-limiting disorder that affects approximately 1 in 4500 boys. In this study, we considered the mdx mice-a murine model of DMD-to discover biomarkers of disease, as well as pharmacodynamic biomarkers responsive to prednisolone, a corticosteroid commonly used to treat DMD. Longitudinal urine samples were analyzed from male age-matched mdx and wild-type mice randomized to prednisolone or vehicle control via liquid chromatography tandem mass spectrometry. A large number of metabolites (869 out of 6,334) were found to be significantly different between mdx and wild-type mice at baseline (Bonferroni-adjusted p-value < 0.05), thus being associated with disease status. These included a metabolite with m/z = 357 and creatine, which were also reported in a previous human study looking at serum. Novel observations in this study included peaks identified as biliverdin and hypusine. These four metabolites were significantly higher at baseline in the urine of mdx mice compared to wild-type, and significantly changed their levels over time after baseline. Creatine and biliverdin levels were also different between treated and control groups, but for creatine this may have been driven by an imbalance at baseline. In conclusion, our study reports a number of biomarkers, both known and novel, which may be related to either the mechanisms of muscle injury in DMD or prednisolone treatment., Competing Interests: The authors have declared that no competing interests exist.

Background: Edasalonexent is an orally administered small molecule designed to inhibit NF-κB, which is activated from infancy in Duchenne muscular dystrophy and is central to causing muscle damage and preventing muscle regeneration., Objective: Evaluate the safety, tolerability, pharmacokinetics and exploratory pharmacodynamics of three doses of edasalonexent in ambulatory males ≥4 to <8 years of age with genetically confirmed Duchenne muscular dystrophy., Methods: This was a 1-week, open-label, multiple-dose study with 3 sequential ascending doses (33, 67 and 100 mg/kg/day) of edasalonexent administered under different dietary conditions to 17 males with a mean age of 5.5 years., Results: All doses of edasalonexent were well tolerated, with no serious adverse events, no drug discontinuations and no dose reductions. The majority of adverse events were mild, and the most common adverse events were gastrointestinal (primarily diarrhea). Edasalonexent was rapidly absorbed with peak levels observed 2-6 hours after dosing and exposures appeared to increase nearly proportionally to dose for the 2 lower and all 3 doses under low-fat and high-fat meal conditions, respectively. Only minor plasma accumulation of edasalonexent was observed with 7 days of dosing. After treatment with edasalonexent for 7 days, levels of NF-κB-regulated genes and serum proteins were decreased., Conclusions: This first report of edasalonexent oral administration for one week in male pediatric patients with Duchenne muscular dystrophy showed that treatment was well tolerated and inhibited NF-kB pathways.

Background: Duchenne muscular dystrophy (DMD) is a progressive, fatal muscle wasting disease. Early detection of DMD by mass screening may enable the early treatment of these patients. We have reported that urinary titin concentration, an indicator of severe muscle wasting, is a diagnostic biomarker for DMD., Methods: Urinary titin concentrations were measured in healthy 3-y-old children and, by comparison with concentrations in 4 DMD patients, and validated as a screening biomarker for DMD. Urine samples were obtained from 100 healthy Japanese children, 52 boys and 48 girls, and their urinary titin concentrations measured by ELISA., Results: The mean ± SD urinary titin concentration was 1.5 ± 2.5 nmol/l, and the mean urinary titin concentration normalized to creatinine was 2.2 ± 4.1 pmol/mg creatinine, with no differences between boys and girls. Histograms and box-and-whisker plots showed that almost all titin and normalized titin concentrations were in narrow ranges, with one outlier in common. Receiver operating characteristic curve analysis showed that titin and normalized-titin concentrations from healthy 3-y-olds were completely separate from those of 3-y-old DMD patients., Conclusions: These findings indicate that urinary titin may be an excellent non-invasive biomarker to screen for DMD., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Patients with Duchenne muscular dystrophy (DMD) exhibit increased prostaglandin D 2 (PGD 2 ) expression in necrotic muscle and increased PGD 2 metabolites in their urine. In mouse models, inhibiting PGD 2 production suppresses muscle necrosis, suggesting a possible intervention through PGD 2 -mediated activities., Objective: We investigated the involvement of PGD 2 and its potential use as a marker of pathological progression in DMD., Methods: Sixty-one male children with DMD and thirty-five age-matched controls were enrolled in the study. DMD patients were divided into "ambulant" and "non-ambulant" groups, which were further subdivided into "steroid" and "non-steroid" therapy groups. Levels of the PGD 2 metabolite tetranor-PGDM (t-PGDM) and creatinine were measured in both spot and 24-hour urine samples, with comparisons between groups made according to geometric mean values., Results: DMD patients had significantly higher levels of creatinine-corrected t-PGDM in spot urine samples as compared with the control group. Additionally, both ambulant and non-ambulant DMD groups had significantly higher levels of t-PGDM as compared with controls, with no significant difference in t-PGDM levels observed between steroid and non-steroid groups. Moreover, total creatinine excretion in 24-hour urine samples was significantly lower in DMD patients as compared with controls, and although DMD patients had lower muscle mass than controls, their overall levels of t-PGDM did not differ significantly from those in the non-ambulant and control groups., Conclusion: PGD 2 might help explain the progression and symptomatic presentations (e.g., ambulatory difficulty) associated with DMD, suggesting it as a useful pathological marker and use of a selective PGD 2 inhibitor as a potential treatment modality., (Copyright © 2018 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

Urine contains extracellular RNA (exRNA) markers of urogenital cancers. However, the capacity of genetic material in urine to identify systemic diseases is unknown. Here we describe exRNA splice products in human urine as a source of biomarkers for the two most common forms of muscular dystrophies, myotonic dystrophy (DM) and Duchenne muscular dystrophy (DMD). Using a training set, RT-PCR, droplet digital PCR, and principal component regression, we identify ten transcripts that are spliced differently in urine exRNA from patients with DM type 1 (DM1) as compared to unaffected or disease controls, form a composite biomarker, and develop a predictive model that is 100% accurate in our independent validation set. Urine also contains mutation-specific DMD mRNAs that confirm exon-skipping activity of the antisense oligonucleotide drug eteplirsen. Our results establish that urine mRNA splice variants can be used to monitor systemic diseases with minimal or no clinical effect on the urinary tract.

We recently found that renal carbonic anhydrase (CA) is involved in the reabsorption of inorganic nitrite (NO 2 - ), an abundant reservoir of nitric oxide (NO) in tissues and cells. Impaired NO synthesis in the endothelium and decreased NO bioavailability in the circulation are considered major contributors to the development and progression of renal and cardiovascular diseases in different conditions including diabetes. Isolated human and bovine erythrocytic CAII and CAIV can convert nitrite to nitrous acid (HONO) and its anhydride N 2 O 3 which, in the presence of thiols (RSH), are further converted to S-nitrosothiols (RSNO) and NO. Thus, CA may be responsible both for the homeostasis of nitrite and for its bioactivation to RSNO/NO. We hypothesized that enhanced excretion of nitrite in the urine may contribute to NO-related dysfunctions in the renal and cardiovascular systems, and proposed the urinary nitrate-to-nitrite molar ratio, i.e., U NOx R, as a measure of renal CA-dependent excretion of nitrite. Based on results from clinical and experimental animal studies, here, we report on a first evaluation of U NOx R. We determined U NOx R values in preterm neonates, healthy children, and adults, in children suffering from type 1 diabetes mellitus (T1DM) or Duchenne muscular dystrophy (DMD), in elderly subjects suffering from chronic rheumatic diseases, type 2 diabetes mellitus (T2DM), coronary artery disease (CAD), or peripheral arterial occlusive disease (PAOD). We also determined U NOx R values in healthy young men who ingested isosorbide dinitrate (ISDN), pentaerythrityl tetranitrate (PETN), or inorganic nitrate. In addition, we tested the utility of U NOx R in two animal models, i.e., the LEW.1AR1-iddm rat, an animal model of human T1DM, and the APOE*3-Leiden.CETP mice, a model of human dyslipidemia. Mean U NOx R values were lower in adult patients with rheumatic diseases (187) and in T2DM patients of the DALI study (74) as compared to healthy elderly adults (660) and healthy young men (1500). The intra- and inter-variabilities of U NOx R were of the order of 50% in young and elderly healthy subjects. U NOx R values were lower in black compared to white boys (314 vs. 483, P = 0.007), which is in line with reported lower NO bioavailability in black ethnicity. Mean U NOx R values were lower in DMD (424) compared to healthy (730) children, but they were higher in T1DM children (1192). ISDN (3 × 30 mg) decreased stronger U NOx R compared to PETN (3 × 80 mg) after 1 day (P = 0.046) and after 5 days (P = 0.0016) of oral administration of therapeutically equivalent doses. In healthy young men who ingested NaNO 3 (0.1 mmol/kg/d), U NOx R was higher than in those who ingested the same dose of NaCl (1709 vs. 369). In LEW.1AR1-iddm rats, mean U NOx R values were lower than in healthy rats (198 vs. 308) and comparable to those in APOE*3-Leiden.CETP mice (151).

New Findings: What is the central question of this study? We examined whether the macrophage-synthesized antioxidant 7,8-dihydroneopterin was elevated in Duchenne muscular dystrophy (DMD) patients. We then examined whether 7,8-dihydroneopterin could protect dystrophic skeletal mouse muscle from eccentric contraction-induced force loss and improve recovery. What is the main finding and its importance? Urinary neopterin/creatinine and 7,8-dihydroneopterin/creatinine were elevated in DMD patients. 7,8-Dihydroneopterin attenuated eccentric contraction-induced force loss of dystrophic skeletal mouse muscle and accelerated recovery of force. These results suggest that eccentric contraction-induced force loss is mediated, in part, by an oxidative component and provides a potential protective role for 7,8-dihydroneopterin in DMD., Abstract: Macrophage infiltration is a hallmark of dystrophin-deficient muscle. We tested the hypothesis that Duchenne muscular dystrophy (DMD) patients would have elevated levels of the macrophage-synthesized pterins, neopterin and 7,8-dihydroneopterin, compared with unaffected age-matched control subjects. Urinary neopterin/creatinine and 7,8-dihydroneopterin/creatinine were elevated in DMD patients, and 7,8-dihydroneopterin/creatinine was associated with patient age and ambulation. Urinary 7,8-dihydroneopterin corrected for specific gravity was also elevated in DMD patients. Given that 7,8-dihydroneopterin is an antioxidant, we then identified a potential role for 7,8-dihydroneopterin in disease pathology. We assessed whether 7,8-dihydroneopterin could: (i) protect against isometric force loss in wild-type skeletal muscle exposed to various pro-oxidants; and (ii) protect wild-type and mdx muscle from eccentric contraction-induced force loss, which has an oxidative component. Force loss was elicited in isolated extensor digitorum longus (EDL) muscles by 10 eccentric contractions, and recovery of force after the contractions was measured in the presence of exogenous 7,8-dihydroneopterin. 7,8-Dihydroneopterin attenuated isometric force loss by wild-type EDL muscles when challenged by H 2 O 2 and HOCl, but exacerbated force loss when challenged by SIN-1 (NO • , O 2 • , ONOO - ). 7,8-Dihydroneopterin attenuated eccentric contraction-induced force loss in mdx muscle. Isometric force production by EDL muscles of mdx mice also recovered to a greater degree after eccentric contractions in the presence of 7,8-dihydroneopterin. The results corroborate macrophage activation in DMD patients, provide a potential protective role for 7,8-dihydroneopterin in the susceptibility of dystrophic muscle to eccentric contractions and indicate that oxidative stress contributes to eccentric contraction-induced force loss in mdx skeletal muscle., (© 2018 The Authors. Experimental Physiology © 2018 The Physiological Society.)

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by mutations in the dystrophin gene leading to the absence of the normal dystrophin protein. The efforts of many laboratories brought new treatments of DMD to the reality, but ongoing and forthcoming clinical trials suffer from absence of valuable biomarkers permitting to follow the outcome of the treatment day by day and to adjust the treatment if needed. In the present study the levels of 128 urinary proteins including growth factors, cytokines and chemokines were compared in urine of DMD patients and age related control subjects by antibody array approach. Surprisingly, statistically significant difference was observed only for urinary ferritin whose level was 50 times higher in young DMD patients. To explain the observed high urinary ferritin content we analysed the levels of iron, iron containing proteins and proteins involved in regulation of iron metabolism in serum and urine of DMD patients and their age-matched healthy controls. Obtained data strongly suggest that elevated level of urinary ferritin is functionally linked to the renal management of myoglobin iron derived from leaky muscles of DMD patients. This first observation of the high level of ferritin in urine of DMD patients permits to consider this protein as a new urinary biomarker in muscular dystrophies and sheds light on the mechanisms of iron metabolism and kidney functioning in DMD., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Aim: To study the signature of 87 urinary miRNAs in Duchenne muscular dystrophy (DMD) patients, select the most dysregulated and determine statistically significant differences in their expression between controls, ambulant (A) and nonambulant (NA) DMD patients, and patients on different corticosteroid regimens. Patients/materials & methods: Urine was collected from control (n = 20), A (n = 31) and NA (n = 23) DMD patients. miRNA expression was measured by reverse transcription-quantitative PCR., Results: miR-29c-3p was significantly downregulated in A DMD patients while miR-23b-3p and miR-21-5p were significantly downregulated in NA DMD patients compared with age-matched controls., Conclusion: miR-29c-3p, miR-23b-3p and miR-21-5p are promising novel noninvasive biomarkers for DMD, and miR-29c-3p levels are differentially affected by different steroid regimens, supporting the antifibrotic effect of steroid therapy.

Duchenne muscular dystrophy (DMD) is a fatal progressive muscle wasting disease of childhood. Titin in sarcomere is digested by calcium dependent protease. To explore muscle damage in DMD, the urinary concentrations of the N-terminal fragment of titin were determined using a newly developed enzyme linked immune sorbent assay kit. The urinary titin concentrations were normalized to creatinine (Cr). A total of 145 urine samples were obtained at a single Japanese hospital from 113 DMD patients aged 3-29years. Normalized urinary titin concentration was 965.8±1011.9 (Mean±SD) pmol/mg Cr in patients with DMD. This was nearly 700-fold higher than healthy children (1.4±0.8pmol/mg Cr). The concentration was significantly higher in DMD than in BMD patients who had significantly higher urinary titin than normal. Urinary titin in DMD patients tended to decrease with age. The median concentration of urinary titin in the youngest (aged 3-7years) and oldest (aged ≥16years) groups was 1468.3 and 411.3pmol/mg Cr, respectively, with significant difference. Urinary concentration of titin correlated significantly with serum creatine kinase concentration, the best-known biomarker of DMD. The N-terminal fragment of titin in urine has potential as a diagnostic and clinical biomarker for DMD., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Enzyme-linked and electrochemiluminescence immunoassays were developed for quantification of amino (N-) terminal fragments of the skeletal muscle protein titin (N-ter titin) and qualified for use in detection of urinary N-ter titin excretion. Urine from normal subjects contained a small but measurable level of N-ter titin (1.0 ± 0.4 ng/ml). A 365-fold increase (365.4 ± 65.0, P = 0.0001) in urinary N-ter titin excretion was seen in Duchene muscular dystrophy (DMD) patients. Urinary N-ter titin was also evaluated in dystrophin deficient rodent models. Mdx mice exhibited low urinary N-ter titin levels at 2 weeks of age followed by a robust and sustained elevation starting at 3 weeks of age, coincident with the development of systemic skeletal muscle damage in this model; fold elevation could not be determined because urinary N-ter titin was not detected in age-matched wild type mice. Levels of serum creatine kinase and serum skeletal muscle troponin I (TnI) were also low at 2 weeks, elevated at later time points and were significantly correlated with urinary N-ter titin excretion in mdx mice. Corticosteroid treatment of mdx mice resulted in improved exercise performance and lowering of both urinary N-ter titin and serum skeletal muscle TnI concentrations. Low urinary N-ter titin levels were detected in wild type rats (3.0 ± 0.6 ng/ml), while Dmd mdx rats exhibited a 556-fold increase (1652.5 ± 405.7 ng/ml, P = 0.002) (both at 5 months of age). These results suggest that urinary N-ter titin is present at low basal concentrations in normal urine and increases dramatically coincident with muscle damage produced by dystrophin deficiency. Urinary N-ter titin has potential as a facile, non-invasive and translational biomarker for DMD., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Muscle damage and loss of muscle mass are triggered by immobilization, loss of appetite, dystrophies and chronic wasting diseases. In addition, physical exercise causes muscle damage. In damaged muscle, the N-terminal and C-terminal regions of titin, a giant sarcomere protein, are cleaved by calpain-3, and the resulting fragments are excreted into the urine via glomerular filtration. Therefore, we considered titin fragments as promising candidates for reliable and non-invasive biomarkers of muscle injury. Here, we established a sandwich ELISA that can measure the titin N-terminal fragment over a biologically relevant range of concentrations, including those in urine samples from older, non-ambulatory Duchenne muscular dystrophy patients and from healthy donors under everyday life conditions and after exercise. Our results indicate that the established ELISA could be a useful tool for the screening of muscular dystrophies and also for monitoring the progression of muscle disease, evaluating the efficacy of therapeutic approaches, and investigating exercise-related sarcomeric disruption and repair processes., Competing Interests: Regarding potential conflicts of interest, please note that three of the authors (N.M., K.M. and M.M.) are employed by a company that plans to make the ELISA kit commercially available in the near future.

The L-arginine/nitric oxide (L-Arg/NO) pathway regulates endothelial function and may play an important role in the pathogenesis of Duchenne muscular dystrophy (DMD). Yet, this pathway is poorly investigated in children suffering from DMD. Endothelial dysfunction can affect the perfusion of contracting muscles, thus leading to ischemia and hypoxia. In the present study, we tested the hypothesis that reduced NO production due to elevated synthesis of N (G),N (G)-dimethyl-L-arginine (asymmetric dimethylarginine, ADMA), an endogenous inhibitor of NO synthesis, is a possible pathophysiological mechanism for progressive intramuscular muscle ischemia and disturbed endothelial function in children with DMD. Given the possible antagonistic action of homoarginine (hArg) on ADMA, we also analyzed this amino acid. We investigated 55 male patients with DMD and 54 healthy male controls (HC; aged 11.9 ± 4.8 vs. 11.1 ± 4.9 years, mean ± SD). Urinary creatinine and metabolites of the L-Arg/NO pathway were measured in plasma and urine by GC-MS or GC-MS/MS. Urine levels of ADMA and its major urinary metabolite dimethylamine (DMA), nitrite and nitrate (P < 0.001 for all) and hArg (P = 0.002) were significantly higher in DMD patients compared to HC, while the urinary DMA/ADMA molar ratio was lower (P = 0.002). In plasma, nitrate (P < 0.001), hArg (P = 0.002) and the hArg/ADMA ratio (P < 0.001) were lower in DMD than in HC. In plasma, ADMA (631 ± 119 vs. 595 ± 129 nM, P = 0.149), arginine and nitrite did not differ between DMD and HC. In DMD, positive correlations between ADMA, DMA or nitrate excretion and the stage of disease (according to Vignos and Thompson) were found. In DMD patients on steroid medication, lower concentrations of ADMA in plasma, and of DMA, ADMA, nitrate and hArg in urine were observed compared to non-treated patients. The L-Arg/NO pathway is impaired in DMD patients, with the disease progression being clinically negatively correlated with the extent of impairment. One of the underlying mechanisms in DMD may involve insufficient antagonism of ADMA by hArg. Steroids, but not creatine supplementation, seems to improve the L-Arg/NO pathway in DMD.

Improved life expectancy and the need for robust tools to monitor renal safety of emerging new therapies have fueled the interest in renal function in Duchenne muscular dystrophy (DMD) patients. We aimed to establish a methodology to accurately assess their renal function. Twenty DMD patients (5-22 years) were included in this prospective study. After obtaining medical history, all patients underwent a clinical examination, 24-hour ambulatory blood pressure monitoring, ultrasound of the kidneys, direct GFR measurement ((51)Cr-EDTA, mGFR), complete blood and urine analysis. Seventeen of 20 patients were treated with corticosteroids and 5/20 with angiotensin converting enzyme inhibitor (lisinopril). No patient suffered from urinary tract infections or other renal diseases. Hypertension (systolic or diastolic blood pressure >P95) was found in 9/20 patients (8/9 patients were on steroid treatment) and a non-dipping blood pressure profile in 13/20 subjects (10/13 patients were on steroid treatment). Urinary protein to creatinine ratio was elevated in 17/18 patients, whereas 24-hour urine protein excretion was normal in all subjects. Median interquartile range (IQR) mGFR was 130.4 (29.1) mL/min/1.73 m(2). Hyperfiltration (mGFR >150 mL/min/1.73 m(2)) was found in 5/20 patients. Inverse correlation between mGFR and age was observed (R(2) = 0.45, p = 0.001). Serum creatinine based estimated GFR (eGFR) equations overestimated mGFR up to 300%. eGFR based on cystatin C Filler equation was closest to the mGFR (median eGFR (IQR) of 129.5 (39.7) mL/min/1.73 m(2)). Our study demonstrates a high prevalence of hyperfiltration and hypertension in children and adolescents with DMD. Because the majority of hypertensive patients were under corticosteroid treatment, the iatrogenic cause of hypertension cannot be excluded. Serum or urine creatinine measurements are of no value to evaluate renal function in DMD patients due to the reduced skeletal muscle mass., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Diagnosis of muscular dystrophies is currently based on invasive methods requiring muscle biopsies or blood tests. The aim of the present study was to identify urinary biomarkers as a diagnostic tool for muscular dystrophies. Here, the urinary proteomes of Duchenne muscular dystrophy (DMD) patients and healthy donors were compared with a bottom-up proteomic approach. Label-free analysis of more than 1100 identified proteins revealed that 32 of them were differentially expressed between healthy controls and DMD patients. Among these 32 proteins, titin showed the highest fold change between healthy subjects and DMD patients. Interestingly, most of the sequenced peptides belong to the N-terminal and C-terminal parts of titin, and the presence of the corresponding fragments in the urine of DMD patients was confirmed by Western blot analysis. Analysis of a large cohort of DMD patients and age-matched controls (a total of 104 individuals aged from 3 to 20 years) confirmed presence of the N-ter fragment in all but two patients. In two DMD patients aged 16 and 20 years this fragment was undetectable and two healthy controls of 16 and 19 years with serum CK >800 IU/L demonstrated a low level of the fragment. N- and C-terminal titin fragments were also detected in urine from patients with other muscular dystrophies such as Becker muscular dystrophy and Limb-girdle muscular dystrophy (type 1D, 2D and 2J) but not in neurogenic spinal muscular atrophy. They were also present in urine of dystrophin-deficient animal models (GRMD dogs and mdx mice). Titin is the first urinary biomarker that offers the possibility to develop a simple, non-invasive and easy-to-use test for pre-screening of muscular dystrophies, and may also prove to be useful for the non-invasive follow up of DMD patients under treatment., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The ability to extract somatic cells from a patient and reprogram them to pluripotency opens up new possibilities for personalized medicine. Induced pluripotent stem cells (iPSCs) have been employed to generate beating cardiomyocytes from a patient's skin or blood cells. Here, iPSC methods were used to generate cardiomyocytes starting from the urine of a patient with Duchenne muscular dystrophy (DMD). Urine was chosen as a starting material because it contains adult stem cells called urine-derived stem cells (USCs). USCs express the canonical reprogramming factors c-myc and klf4, and possess high telomerase activity. Pluripotency of urine-derived iPSC clones was confirmed by immunocytochemistry, RT-PCR and teratoma formation. Urine-derived iPSC clones generated from healthy volunteers and a DMD patient were differentiated into beating cardiomyocytes using a series of small molecules in monolayer culture. Results indicate that cardiomyocytes retain the DMD patient's dystrophin mutation. Physiological assays suggest that dystrophin-deficient cardiomyocytes possess phenotypic differences from normal cardiomyocytes. These results demonstrate the feasibility of generating cardiomyocytes from a urine sample and that urine-derived cardiomyocytes retain characteristic features that might be further exploited for mechanistic studies and drug discovery., (Copyright © 2013. Published by Elsevier B.V.)

Background: Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by muscle dystrophin deficiency. Downstream of the primary dystrophin deficiency is not well elucidated. Here, the hypothesis that prostaglandin D2 (PGD2)-mediated inflammation is involved in the pathology of DMD was examined by measuring tetranor PGDM, a major PGD2 metabolite, in urine of DMD patients., Methods: We measured tetranor PGDM in urine using LC-MS/MS. First morning urine samples were collected from genetically confirmed DMD patients and age-matched healthy controls aged 4 to 15y., Results: The urinary tetranor PGDM concentration was 3.08±0.15 and 6.90±0.35ng/mg creatinine (mean±SE) in 79 control and 191 DMD samples, respectively. The mean concentration was approximately 2.2-times higher in DMD patients than in controls (p<0.05). Remarkably, urinary tetranor PGDM concentrations in DMD patients showed chronological changes: it stayed nearly 1.5 times higher than in controls until 7y but surged at the age of 8y to a significantly higher concentration., Conclusion: Urinary tetranor PGDM concentrations were shown to be increased in DMD patients and became higher with advancing age. It was indicated that PGD2-mediated inflammation plays a role in the pathology of DMD., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We describe two cases of Duchenne's cardiomyopathy with severe cardiac dysfunction, sporadic episodes of myoglobinuria induced by effort and increased levels of serum creatine kinase. Very mild signs of skeletal myopathy were clinically evident. Left ventriculography showed diffuse severe hypokinesia. Skeletal muscle biopsy demonstrated a dystrophic process. The patients had no familial background of the disease. These 2 patients might have a sporadic inheritance pattern with severe cardiac involvement.

Duchenne muscular dystrophy (DMD) is associated with an increase in oxidative stress. We measured 24 h 8-hydroxy-2'-deoxyguanosine (8-OHdG) excretion in 24 patients with MD (DMD + Becker's MD), 23 with myotonic dystrophy, and 34 healthy controls. The 8-OHdG/creatinine ratio was higher in patients with dystrophinopathy ( upward arrow 48%, p <.01) but not myotonic dystrophy, as compared to healthy controls. These results indicate that 8-OHdG excretion can be used as a marker of oxidative stress in clinical trials with dystrophinopathy.

We examined the energy expenditure in patients with Duchenne muscular dystrophy(DMD) to evaluate the cause of the paradoxical weight loss observed in large numbers of adolescent patients before any obvious impairment of their swallowing function. In the morning, resting energy expenditure (REE)/m(2) was almost the same as that in normal controls despite a reduction in fat-free mass (FFM); thus, REE/m(2)/FFM was significantly increased in patients (median, 21.2 kcal/m(2)/FFM kg; range, 17.7 to 44.2, P =.012). A thermographic examination in the morning showed an obvious elevation of the body surface temperature on the back. This phenomenon was consistent with a paradoxical fall in the low frequency (LF)/high frequency (HF) ratio at night analyzed using the inter-RR spectrum by 24-hour electrocardiogram, which indicated relative activation of the sympathetic nervous system. The urinary secretion of norepinephrine at night was also significantly greater in patients (median, 0.119 microg/kg/h; range, 0.061 to 0.219, P =.011). These results suggest that paradoxical activation of the sympathetic nervous system may accelerate the production of heat in brown adipose tissue (BAT) and increase the level of energy consumption in patients, and that adolescent DMD patients may require greater caloric intake than expected to maintain body weight, which is important to improve the prognosis of their respiratory function., (Copyright 2001 by W.B. Saunders Company)