The muscle regeneration marker FOXP3 is associated with muscle injury in Duchenne muscular dystrophy.

BACKGROUND: In Duchenne muscular dystrophy (DMD), the immune system cells (ISC) synthesize molecules to regulate inflammation, a process needed to regenerate muscle. The relationship between those molecules and the muscle injury is unknown. Monocytes belonging to ISC are regulated by omega-3 fatty acids (ω-3 LCPUFAs) in DMD, but whether those fatty acids influence other ISC like T-cells is unknown. OBJECTIVE: We analyzed the expression of the muscle regeneration markers (FOXP3 and AREG) in circulating leukocytes of DMD patients with different lower limb muscle functions and whether ω-3 LCPUFAs regulate the expression of those markers, and the populations of circulating T-cells, their intracellular cytokines, and disease progression (CD69 and CD49d) markers. METHODS: This placebo-controlled, double-blind, randomized study was conducted in DMD boys supplemented with ω-3 LCPUFAs (n = 18) or placebo (sunflower oil, n = 13) for six months. FOXP3 and AREG mRNA expression in leukocytes, immunophenotyping of T-cell populations, CD49d and CD69 markers, and intracellular cytokines in blood samples were analyzed at baseline and months 1, 2, 3, and 6 of supplementation. RESULTS: Patients with assisted ambulation expressed higher (P = 0.015) FOXP3 mRNA levels than ambulatory patients. The FOXP3 mRNA expression correlated (Rho = -0.526, P = 0.03) with the Vignos scale score at month six of supplementation with ω-3 LCPUFAs. CD49d + CD8 + T-cells population was lower (P = 0.037) in the ω -3 LCPUFAs group than placebo at month six of supplementation. CONCLUSION: FOXP3 is highly expressed in circulating leukocytes of DMD patients with the worst muscle function. Omega-3 LCPUFAs might modulate the synthesis of the adhesion marker CD49d + CD8 + T-cells, but their plausible impact on FOXP3 needs more research.

Circulating Nrf2, Glutathione, and Malondialdehyde Correlate with Disease Severity in Duchenne Muscular Dystrophy.

Oxidative stress (OS) plays an essential role in the pathophysiology of Duchenne muscular dystrophy (DMD). However, the actors that regulate OS need to be better studied. We aimed to evaluate whether NFE2-like bZIP transcription factor 2 (Nrf2), glutathione, malondialdehyde (MDA), and protein carbonyl concentrations change according to the disease severity in DMD patients. Moreover, we assessed whether OS correlated with muscle injury, clinical characteristics, physical activity, and antioxidant food consumption (AFC). A total of 28 DMD patients participated in this study. OS markers, metabolic indicators, and enzymatic markers of muscle injury were measured in circulation. Muscle injury was measured with clinical scales, and physical activity and AFC were evaluated with questionnaires. Nrf2 concentration was lower (p ≤ 0.01), and malondialdehyde concentration was higher (p < 0.05) in non-ambulatory patients than in ambulatory patients. Nrf2 correlated with age (rho = -0.387), Vignos scale (rho = -0.328), GMFCS scale (rho = -0.399), and Brooke scale scores (rho = -0.371) (p < 0.05). MDA correlated with Vignos (rho = 0.317) and Brooke scale scores (rho = 0.414) (p ≤ 0.05). In conclusion, DMD patients with the worst muscle function had more significant oxidative damage and lower antioxidant function than DMD patients with better muscle function.

Natural history of circulating miRNAs in Duchenne disease: Association with muscle injury and metabolic parameters.

OBJECTIVES: This study aimed to evaluate whether the expression of circulating dystromiRs and a group of oxidative stress-related (OS-R) miRNAs is associated with muscle injury and circulating metabolic parameters in Duchenne muscular dystrophy (DMD) patients. METHODS: Twenty-four DMD patients were included in this cross-sectional study. Clinical scales to evaluate muscle injury (Vignos, GMFCS, Brooke, and Medical Research Council), enzymatic muscle injury parameters (CPK, ALT, and AST), anthropometry, metabolic indicators, physical activity, serum dystromiRs (miR-1-3p, miR-133a-3p, and miR-206), and OS-R miRNAs (miR-21-5p, miR-31-5p, miR-128-3p, and miR-144-3p) levels were measured in ambulatory and non-ambulatory DMD patients. RESULTS: DystromiRs (except miR-1-3p) and miRNAs OS-R levels were lower (p-value <.05) in the non-ambulatory group than the ambulatory group. The expression of those miRNAs correlated with Vignos scale score (For instance, rho = -0.567, p-value <0.05 for miR-21-5p) and with other scales scores of muscle function and strength. CPK, AST, and ALT concentration correlated with expression of all miRNAs (For instance, rho = 0.741, p-value <.05 between miR-206 level and AST concentration). MiR-21-5p level correlated with glucose concentration (rho = -0.369, p-value = .038), and the miR-1-3p level correlated with insulin concentration (rho = 0.343, p-value = .05). CONCLUSIONS: Non-ambulatory DMD patients have lower circulating dystromiRs and OS-R miRNAs levels than ambulatory DMD patients. The progressive muscle injury is associated with a decrease in the expression of those miRNAs, evidencing DMD progress. These findings add new information about the natural history of DMD.

Pro-inflammatory monocytes are increased in Duchenne muscular dystrophy and suppressed with omega-3 fatty acids: A double-blind, randomized, placebo-controlled pilot study.

BACKGROUND: Omega-3 long chain polyunsaturated fatty acids (LCPUFA) reduce circulating cytokines produced by monocytes. Nevertheless, whether the omega-3 LCPUFA regulate the monocytes and their cytokines in Duchenne muscular dystrophy (DMD) is unknown. The aim of this study was to evaluate whether circulating pro-inflammatory monocytes are increased and whether omega-3 LCPUFA selectively suppress these monocytes and their cytokines in patients with DMD. METHODS: This was a double-blind, randomized, placebo-controlled pilot study carried out in patients with DMD supplemented with omega-3 LCPUFA (n = 6) or sunflower oils (placebo, n = 6) for 6 months. Monocytes and their cytokines were measured at baseline and after 1, 2, 3, and 6 months of supplementation. RESULTS: The anti-inflammatory monocytes (median, [95% CI]) are increased at month 3 (-0.46 [-13.5-9.5] vs. 8.4 [5.5-12.5], p = 0.05) in the omega-3 LCPUFA group compared with the placebo group. The pro-inflammatory monocytes (-5.7 [-63.8-114.1] vs. -51.9 [-91.2 to -25.4], p = 0.026 and -16.4 [-50.8-50.6] vs. -57.9 [-86.9 to -18.5], p = 0.045 at months 3 and 6, respectively) and their cytokine interleukin 6 (-11.9 [-93.5-148.9] vs. -64.7 [-77.8 to -42.6], p = 0.019 at month 6) decreased in the omega-3 LCPUFA group compared with the placebo group. Pro-inflammatory monocytes decreased and anti-inflammatory monocytes were augmented (p < 0.05) during the 6 months of supplementation with omega-3 LCPUFA. CONCLUSIONS: This pilot study suggests that supplementation with omega-3 LCPUFA could have a selective reductive effect on pro-inflammatory monocytes and their cytokines in patients with DMD. These findings also support the performance of studies in a significant population to explore the role of omega-3 LCPUFA on monocyte populations and their cytokines in patients with DMD. This research was registered at clinicaltrials.gov (NCT018264229).

Circulating markers of oxidative stress are associated with a muscle injury in patients with muscular dystrophy Duchenne.

BACKGROUND: A small number of studies have confirmed the presence of oxidative damage in patients with Duchenne muscular dystrophy (DMD). Nevertheless, it is unknown if there a relationship of circulating markers of oxidative stress with a muscle injury. OBJECTIVE: We evaluated if oxidative damage and anti-oxidant markers are associated with muscle damage in DMD. METHODS: This cross-sectional study included 24 patients with DMD classified in ambulatory and non-ambulatory. Markers of muscle damage (creatine kinase [CK]), oxidative damage (malondialdehyde [MDA], and 8-isoprostane), anti-oxidant function (Thiol and mRNA of NRF2 and NF-κB) and nitric oxide (NO) were quantified in circulation. RESULTS: Total NO, MDA, and 8-isoprostane concentrations were significantly (p < 0.05) higher, and thiol concentration was lower in non-ambulatory than ambulatory patients. A significant correlation (p < 0.05) between muscle injury (evaluated by Vignos scale) with CK (r = -0.382), NO (r = 0.444), MDA (r = 0.503), 8-isoprostanes (r = 0.435) and thiol (r = -0.430) was observed. CONCLUSION: These findings suggest that non-ambulatory have high oxidative damage and low anti-oxidant function than ambulatory patients with DMD. Total nitric oxide and oxidative damage plasma markers increase, but the anti-oxidant marker thiol decreases with a muscle injury in boys with DMD. The findings of this study suggest that these markers could be considered as goods indicators of oxidative damage in longitudinal studies to evaluate the muscle injury during DMD progression. Additionally, these findings add new information about the pathophysiology of DMD.

Natural History of Serum Enzyme Levels in Duchenne Muscular Dystrophy and Implications for Clinical Practice.

OBJECTIVE: In Duchenne muscular dystrophy, creatine kinase and transaminases are released into the circulation, indicating muscle injury. Their usefulness in monitoring muscle injury or disease progression has not yet been fully evaluated. Thus, this study examined serum creatine kinase and transaminase concentrations at different ages in patients with Duchenne muscular dystrophy and evaluated their association with muscle injury. DESIGN: This is a prospective cohort study that included 110 patients with Duchenne muscular dystrophy categorized by age groups. Creatine kinase and transaminases were quantified in the serum; the Vignos scale evaluated the muscle function. RESULTS: Creatine kinase and transaminase levels were higher in ambulatory than that in nonambulatory patients, which significantly decreased as age increased. Serum creatine kinase and transaminase concentrations were elevated in all ages, and those aged 3-4 yrs had the highest concentrations. Age and Vignos Scale were significantly correlated with creatine kinase and transaminase concentrations. Age, creatine kinase, and transaminases explained the 42.5% of loss of muscle function. CONCLUSIONS: This study added the knowledge on the natural history of Duchenne muscular dystrophy at different ages and confirmed that creatine kinase and transaminases decrease with age and loss of muscle function, making them generally inappropriate for monitoring response to therapy, although they are useful for the clinical diagnosis.

Muscle function and age are associated with loss of bone mineral density in Duchenne muscular dystrophy.

INTRODUCTION: Patients with Duchenne muscular dystrophy (DMD) demonstrate decreased bone mineral density (BD). It is not clear which factors exert the greatest impact on BD loss in these patients. METHODS: In 63 patients with DMD, serum cytokines (interleukin [IL]-1, IL-6, and tumor necrosis factor-beta [TNF-ß]), C-reactive protein (CRP), creatine kinase (CK), muscle function (by Vignos scale), body composition, and total BD (the latter 2 measured by dual-energy X-ray absorptiometry, or DEXA) were determined. RESULTS: The main factors associated with BD loss were muscle function (34.0%; ß = -0.139; P < 0.023) and age (36.7%; ß = -0.151; P = 0.004). Cytokines, CRP, body fat mass, and CK did not contribute to BD loss. DISCUSSION: Muscle function and age contribute to BD loss in DMD. We propose that a cut-off of at least 6 points for the Vignos scale and at least 10.5 years of age predict a Z-score of less than or equal to -2.0. Muscle Nerve 59:417-421, 2019.

Body composition and body mass index in Duchenne muscular dystrophy: Role of dietary intake.

INTRODUCTION: In Duchenne muscular dystrophy (DMD) muscle is replaced by adipose tissue. The role of dietary intake (DI) in DMD has not been evaluated. In this study we examined body composition, body mass index (BMI), and adequacy of DI in patients with DMD and evaluated the influence of DI on body composition. METHODS: Patients (n = 101; age 3-18 years; BMI 11.8-29.5 kg/m2 ) completed a dietary recall to determine DI and then underwent dual-energy X-ray absorptiometry to determine body composition. RESULTS: Preschool-age and school-age boys with DMD had high total energy intake. Protein intake per kilogram exceeded recommendations. As age increased, the percentage of boys with abnormal BMI and fat mass increased, while lean mass decreased. Dietary intake did not predict body fat or lean mass. DISCUSSION: Age-dependent changes in BD in boys with DMD may be due to endogenous metabolic factors related to the underlying disease process and to disease-related mobility impairments. Muscle Nerve 59:295-302, 2019.

Evidence of muscle loss delay and improvement of hyperinsulinemia and insulin resistance in Duchenne muscular dystrophy supplemented with omega-3 fatty acids: A randomized study.

BACKGROUND & AIMS: Duchenne Muscular Dystrophy (DMD) is the most prevalent dystrophy of childhood and is characterized by generalized motor delays due to progressive muscular weakness, leading to loss of muscle mass. Additionally, patients with DMD develop obesity, hyperinsulinemia, and Insulin Resistance (IR). Omega-3 Long-Chain PolyUnsaturated Fatty Acids (Ω-3LCPUFA) increase fat mass, decrease lean mass, and decrease hyperinsulinemia and IR. The aim of this study was to analyze the impact of Ω-3LCPUFA consumption on lean mass, fat mass, hyperinsulinemia, and IR in children with DMD. METHODS: This placebo-controlled, double-blind, randomized study was carried out in 28 patients with DMD supplemented with 2.9 g/d of Ω-3LCPUFA (n = 14) or sunflower oil (placebo, n = 14) during 6 months. Serum glucose and insulin were measured at baseline and thereafter at months 3 and 6 of the intervention to estimate IR by HOmeostasis Model Assessment. Body composition was assessed by Dual Energy X-ray Absorptiometry. RESULTS: The percentage of change in EicosaPentaenoic Acid (EPA) and DocosaHexaenoic Acid (DHA) in erythrocytes was significantly (p < 0.05) higher in boys who consumed Ω-3LCPUFA than in the placebo group. Lean mass and fat mass (both in g/kg of Body Weight [BW]) had a trend toward being higher (p = 0.07 at month 3 and p = 0.085 at month 6) and lower (p = 0.05 at month 3 and p = 0.085 at month 6) respectively, in boys with DMD supplemented with Ω-3LCPUFA compared with the placebo group. The loss of lean mass was delayed in the Ω-3LCPUFA group; it started at month 6 but, in placebo, it started at month 3 of supplementation in comparison with the baseline of each group. Fasting insulin, percentage of boys with hyperinsulinemia, and IR were similar between the placebo and Ω-3LCPUFA groups during the 6 months of supplementation. The percentage of boys with IR was significantly (p = 0.045) lower at month 6 of supplementation in the Ω-3LCPUFA group than in the placebo group. CONCLUSION: This study suggests that Ω-3LCPUFA (2.9 g/day) intake during 6 months likely slows the progression of muscle loss, decreases the fat mass, and reduces IR in boys with DMD. The findings of this study provide scientific background for conducting a randomized trial focused of confirming the possible beneficial role of Ω-3LCPUFA on the previously mentioned alterations mentioned in boys with early muscle damage (without fibrosis) DMD. This research was registered at clinicaltrials.gov (NCT018264229).

Potential therapeutic impact of omega-3 long chain-polyunsaturated fatty acids on inflammation markers in Duchenne muscular dystrophy: A double-blind, controlled randomized trial.

BACKGROUND & AIMS: Duchenne Muscular Dystrophy (DMD) is the most frequent dystrophy in childhood generated by a deficiency in dystrophin. DMD is a neuromuscular disease and its clinical course comprises chronic inflammation and gradual muscle weakness. Supplementation of omega-3 long chain-Polyunsaturated Fatty Acids (ω-3 long chain-PUFA) reduces inflammatory markers in various disorders. The goal of this research was to analyze the influence of ω-3 long chain-PUFA intake on gene expression and blood inflammatory markers in boys with DMD. METHODS: In a placebo-controlled, double. Blind, randomized trial, boys with DMD (n = 36) consumed 2.9 g/day of ω-3 long chain-PUFA or sunflower oil as control, in capsules, for a period of 6 months. Blood was analyzed at baseline and at months 1, 2, 3, and 6 of supplementation for expression of inflammatory markers in leukocytes and serum. RESULTS: There was high adherence to capsule intake (control: 95.3% ± 7.2%, and ω-3 long chain-PUFA: 97.4% ± 3.7% at month 6). Enrichment of EicosaPentaenoic Acid (EPA) and DocosaHexaenoic Acid (DHA) in erythrocytes increased significantly in patients supplemented with ω-3 long chain-PUFA compared with the placebo group during the 6 months of supplementation. Messenger RNA (mRNA) of the Nuclear Factor kappa beta (NF-κB) and its target genes InterLeukin 1 beta (IL-1ß) and IL-6 was downregulated significantly (p < 0.05) in leukocytes from DMD boys supplemented with ω-3 long chain-PUFA for 6 months, compared to the placebo group. Omega-3 long chain-PUFA intake decreased the serum IL-1ß (-59.5%; p = 0.011) and IL-6 (-54.8%; p = 0.041), and increased the serum IL-10 (99.9%, p < 0.005), in relation to those with placebo treatment. CONCLUSION: Supplementation with ω-3 long chain-PUFA 2.9 g/day is well-tolerated, has a beneficial reductive effect on proinflammatory markers, and increases an anti-inflammatory marker, indicating that ω-3 long chain-PUFA could have a potential therapeutic impact on chronic inflammation in DMD. This research is registered at clinicaltrials.gov (NCT018264229).