Intrafamilial phenotypic heterogeneity related to a new DMD splice site variant.

Dystrophinopathies are a group of X-linked neuromuscular disorders that result from pathogenic variants in the DMD gene. Their pathophysiological substrate is the defective expression of dystrophin in many tissues. While patients from the same pedigree usually present similar dystrophin expression and clinical course, the extent of cardiac and skeletal muscle involvement may not correlate in the same individual. We identified a new splice site variant c.2803+5G>C (NM_004006) ClinVar VCV000803902, located in intron 22 of DMD in a Brazilian family that present a broad phenotypic and histological heterogeneity. One of the subjects had a typical Duchenne muscular dystrophy (DMD) phenotype, whereas the others had Becker muscular dystrophy (BMD). Cardiac involvement was remarkable in some of the BMD patients, but not in the DMD patient. Western blot analysis of skeletal muscle revealed much lower levels of calsequestrin in the most severely affected patient compared to his brother, whose phenotype is BMD, highlighting the potential role of proteins involved in skeletal muscle calcium homeostasis in differential degrees of dystrophinopathies.

Heterologous fibrin biopolymer associated to a single suture stitch enables the return of neuromuscular junction to its mature pattern after peripheral nerve injury.

Denervation leads to severe atrophy of neuromuscular junction (NMJ) structure including decrease of the expression of fundamental proteins. Up to now, conventional suture has been the gold standard method used to correct this injury. Fibrin sealant is one of the alternatives proposed to optimize this method. This study verified if the association of fibrin sealant - Heterologous Fibrin Biopolymer (HFB) and a single suture stitch promotes return of morphology and NMJ structure to mature pattern after peripheral nerve injury. Forty Wistar rats were distributed into 4 groups: Sham-Control (SC), Denervated-Control (DC), Suture-Lesion (SL) and Suture-Lesion + HFB (SFS). In SC group only the right sciatic nerve identification was done. In DC, SL and SFS groups fixation of nerve stumps on musculature immediately after neurotmesis was performed. After seven days, stump reconnection with 3 stitches in SL and a single stitch associated with HFB in SFS were done. After sixty days right soleus muscles were prepared for nicotinic acetylcholine receptors (nAChRs) and nerve terminal confocal analyses, and for nAChRs (α1, ε e γ), S100, Agrin, LRP-4, MMP-3, Rapsyn western blotting analyses. SC group presented normal morphology. In DC group it was observed flattening of NMJ, fragmentation of nAChRs and tangled nerve terminals. The majority of the parameters of SL and SFS groups presented values in between SC and DC groups. There was an increase of relative planar area in these groups (SL and SFS) highlighting that there was less nAChRs fragmentation and the values of protein expression showed return of nAChRs to mature pattern. Use of HFB associated with a single suture stitch decreased surgical time, minimized suture injuries, did not alter nerve regeneration and presented potential to reestablish the NMJ apparatus. These consolidated results encourage surgeons to develop future clinical trials to install definitively this new approach both for reconstructive surgery and neurosurgery.

Identification of plasma interleukins as biomarkers for deflazacort and omega-3 based Duchenne muscular dystrophy therapy.

Duchenne muscular dystrophy (DMD) is a progressive and fatal disease, characterized by the absence of dystrophin, muscle degeneration and cardiorespiratory failure. Creatine kinase is the classic marker to screen for DMD. However, other markers are needed to follow disease progression and to evaluate the response to therapy over longer periods. In the present study, we aim to identify interleukins in the plasma of the mdx mice model of DMD that could serve as biomarkers to monitor dystrophy progression, at distinct stages of the disease (1, 3 and 8 months of age). We used deflazacort and omega-3 therapies to validate the biomarkers studied. Plasma levels of TNF-α and TGF-ß were increased in mdx mice in relation to control, at all times studied. Differences in IFN-γ and IL-10 contents, comparing mdx x CTRL, were detected only at the early stage (1 month). IL-6 decreased at 3 and 8 months and IL-13 increased at 8 months in the mdx compared to control. Deflazacort and omega-3 reduced the plasma levels of the pro-inflammatory (TNF-α, INF-γ, IL-6) and pro-fibrotic (IL-13 and TGF-ß) interleukins and increased the plasma levels of IL-10. It is suggested that TNF-α and TGF-ß in plasma would be the best markers to follow disease progression. IL-6, INF-γ and IL-10 would be suitable markers to the earlier stages of dystrophy and IL-13 a suitable marker to the later stages of dystrophy.

Effects of dietary omega-3 on dystrophic cardiac and diaphragm muscles as evaluated by 1H magnetic resonance spectroscopy: Metabolic profile and calcium-related proteins.

BACKGROUND & AIMS: Duchenne muscular dystrophy (DMD) is characterized by the absence of dystrophin and muscle degeneration. Calcium dysregulation and oxidative stress also contribute to the disease progression. We evaluated the potential therapeutic benefits of supplementation with omega-3 on the metabolic profile, calcium-related proteins and oxidative stress response in the heart and diaphragm (DIA) of the mdx mouse model of DMD at later stages of the disease (13 months). METHODS: Mdx mice (8 months old) received omega-3 via a dietary supplement for 5 months. Metabolites were analyzed by 1H magnetic resonance spectroscopy. Muscle total calcium was evaluated by inductively coupled plasma-optical emission spectrometry. Calsequestrin, TRPC1 and 4-HNE were determined via Western blot. RESULTS: Omega-3 decreased the metabolites taurine (related to calcium regulation and oxidative stress), aspartate (related to inflammation) and oxypurinol (related to oxidative stress) in the heart (aspartate) and DIA (taurine, aspartate and oxypurinol). Omega-3 also significantly decreased total calcium and TRPC1 levels in cardiac and DIA muscles and increased the levels of calsequestrin (cardiac and skeletal) and decreased the oxidative stress marker 4-HNE. CONCLUSIONS: The current study suggests that supplementation with omega-3 may generate therapeutic benefits on dystrophy progression, at later stages of the disease, with changes in the metabolic profile that may be correlated to omega-3 therapy.

Effects of omega-3 on matrix metalloproteinase-9, myoblast transplantation and satellite cell activation in dystrophin-deficient muscle fibers.

In Duchenne muscular dystrophy (DMD), lack of dystrophin leads to progressive muscle degeneration, with DMD patients suffering from cardiorespiratory failure. Cell therapy is an alternative to life-long corticoid therapy. Satellite cells, the stem cells of skeletal muscles, do not completely compensate for the muscle damage in dystrophic muscles. Elevated levels of proinflammatory and profibrotic factors, such as metalloproteinase 9 (MMP-9), impair muscle regeneration, leading to extensive fibrosis and poor results with myoblast transplantation therapies. Omega-3 is an anti-inflammatory drug that protects against muscle degeneration in the mdx mouse model of DMD. In the present study, we test our hypothesis that omega-3 affects MMP-9 and thereby benefits muscle regeneration and myoblast transplantation in the mdx mouse. We observe that omega-3 reduces MMP-9 gene expression and improves myoblast engraftment, satellite cell activation, and muscle regeneration by mechanisms involving, at least in part, the regulation of macrophages, as shown here with the fluorescence-activated cell sorting technique. The present study demonstrates the benefits of omega-3 on satellite cell survival and muscle regeneration, further supporting its use in clinical trials and cell therapies in DMD.

Changes in calsequestrin, TNF-α, TGF-ß and MyoD levels during the progression of skeletal muscle dystrophy in mdx mice: a comparative analysis of the quadriceps, diaphragm and intrinsic laryngeal muscles.

In Duchenne muscular dystrophy (DMD), the search for new biomarkers to follow the evolution of the disease is of fundamental importance in the light of the evolving gene and pharmacological therapies. In addition to the lack of dystrophin, secondary events including changes in calcium levels, inflammation and fibrosis greatly contribute to DMD progression and the molecules involved in these events may represent potential biomarkers. In this study, we performed a comparative evaluation of the progression of dystrophy within muscles that are differently affected by dystrophy (diaphragm; DIA and quadriceps; QDR) or spared (intrinsic laryngeal muscles) using the mdx mice model of DMD. We assessed muscle levels of calsequestrin (calcium-related protein), tumour necrosis factor (TNF-α; pro-inflammatory cytokine), tumour growth factor (TGF-ß; pro-fibrotic factor) and MyoD (muscle proliferation) vs. histopathology at early (1 and 4 months of age) and late (9 months of age) stages of dystrophy. Fibrosis was the primary feature in the DIA of mdx mice (9 months: 32% fibrosis), which was greater than in the QDR (9 months: 0.6% fibrosis). Muscle regeneration was the primary feature in the QDR (9 months: 90% of centrally nucleated fibres areas vs. 33% in the DIA). The QDR expressed higher levels of calsequestrin than the DIA. Laryngeal muscles showed normal levels of TNF-α, TGF-ß and MyoD. A positive correlation between histopathology and cytokine levels was observed only in the diaphragm, suggesting that TNF-α and TGF-ß serve as markers of dystrophy primarily for the diaphragm.

Long-Term Therapy With Omega-3 Ameliorates Myonecrosis and Benefits Skeletal Muscle Regeneration in Mdx Mice.

In Duchenne muscle dystrophy (DMD) and in the mdx mouse model of DMD, a lack of dystrophin leads to myonecrosis and cardiorespiratory failure. Several lines of evidence suggest a detrimental role of the inflammatory process in the dystrophic process. Previously, we demonstrated that short-term therapy with eicosapentaenoic acid (EPA), at early stages of disease, ameliorated dystrophy progression in the mdx mouse. In the present study, we evaluated the effects of a long-term therapy with omega-3 later in dystrophy progression. Three-month-old mdx mice received omega-3 (300 mg/kg) or vehicle by gavage for 5 months. The quadriceps and diaphragm muscles were removed and processed for histopathology and Western blot. Long-term therapy with omega-3 increased the regulatory protein MyoD and muscle regeneration and reduced markers of inflammation (TNF-α and NF-kB) in both muscles studied. The present study supports the long-term use of omega-3 at later stages of dystrophy as a promising option to be investigated in DMD clinical trials.