The PKA-p38MAPK-NFAT5-organic osmolytes pathway in Duchenne muscular dystrophy : from essential player in osmotic homeostasis, inflammation and skeletal muscle regeneration to therapeutic target

In Duchenne muscular dystrophy (DMD), the absence of dystrophin from the dystrophin-associated protein complex (DAPC) causes muscle membrane instability, which leads to myofiber necrosis, hampered regeneration, and chronic inflammation. The resulting disabled DAPC-associated cellular pathways have been described both at the molecular and the therapeutical level, with the Toll-like receptor nuclear factor kappa-light-chain-enhancer of activated B cells pathway (NF-kappa B), Janus kinase/signal transducer and activator of transcription proteins, and the transforming growth factor-beta pathways receiving the most attention. In this review, we specifically focus on the protein kinase A/ mitogen-activated protein kinase/nuclear factor of activated T-cells 5/organic osmolytes (PKA-p38MAPK-NFAT5-organic osmolytes) pathway. This pathway plays an important role in osmotic homeostasis essential to normal cell physiology via its regulation of the influx/efflux of organic osmolytes. Besides, NFAT5 plays an essential role in cell survival under hyperosmolar conditions, in skeletal muscle regeneration, and in tissue inflammation, closely interacting with the master regulator of inflammation NF-kappa B. We describe the involvement of the PKA-p38MAPK-NFAT5-organic osmolytes pathway in DMD pathophysiology and provide a clear overview of which therapeutic molecules could be of potential benefit to DMD patients. We conclude that modulation of the PKA-p38MAPK-NFAT5-organic osmolytes pathway could be developed as supportive treatment for DMD in conjunction with genetic therapy

Abnormal NFAT5 physiology in Duchenne muscular dystrophy fibroblasts as a putative explanation for the permanent fibrosis formation in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is characterized by chronic inflammation and fibrotic tissue production by fibroblasts. The promyogenic factor nuclear factor of activated T-cells 5 (NFAT5) is virtually present in all cells, responding to hyperosmolar or pro-inflammatory stress. In embryogenic fibroblasts, absence of NFAT5 results in cell cycle arrest. Here, unaffected skeletal muscle fibroblasts from one healthy donor showed NFAT5 nuclear translocation upon hyperosmolar stress and normal cell viability. Absence of NFAT5 translocation under pro-inflammatory conditions resulted in decreased cell growth (Incucyte ZOOM). In DMD skeletal muscle fibroblasts from one DMD patient, NFAT5 was merely located in the nucleus. Exposure to hyperosmolar conditions or pro-inflammatory cytokines IFN-γ, IL-1β and TNF-α had no influence on NFAT5 physiology (immunofluorescence, western blotting, RT-qPCR). Hyperosmolarity resulted in decreased cell viability and pro-inflammatory stress in unaltered cell growth. These findings suggest that NFAT5 is vital to DMD fibroblast survival. Exposure to pro-inflammatory or hyperosmolar stress in DMD fibroblasts results in an unexpected NFAT5 response, where fibroblasts are not triggered by inflammatory cytokines and do not withstand hyperosmolarity. Chronic inflammation could be viewed as a non-restrictive factor in the formation of fibrosis in DMD. Abnormal NFAT5 physiology could provide a molecular explanation for permanent fibrotic matrix production by DMD fibroblasts

Description of a novel mechanism possibly explaining the antiproliferative properties of glucocorticoids in Duchenne muscular dystrophy fibroblasts based on glucocorticoid receptor GR and NFAT5

Glucocorticoids are drugs of choice in Duchenne muscular dystrophy (DMD), prolonging patients’ ambulation. Their mode of action at the protein level is not completely understood. In DMD, muscle tissue is replaced by fibrotic tissue produced by fibroblasts, reducing mobility. Nuclear factor of activated T-cells 5 (NFAT5) is involved in fibroblast proliferation. By treating one DMD fibroblast cell culture and one of unaffected skeletal muscle fibroblasts with methylprednisolone (MP) or hydrocortisone (HC) for 24 h or 12 d, the antiproliferative properties of glucocorticoids could be unraveled. NFAT5 localization and expression was explored by immunocytochemistry (ICC), Western blotting (WB) and RT-qPCR. NFAT5 and glucocorticoid receptor (GR) colocalization was measured by ImageJ. GR siRNA was used, evaluating GR’s influence on NFAT5 expression during MP and HC treatment. Cell proliferation was monitored by IncuCyte ZOOM. In DMD fibroblasts, treatment with MP for 24 h induced dots (ICC) positive for NFAT5 and colocalizing with GR. After 12 d of MP or HC in DMD fibroblasts, NFAT5 expression was decreased (RT-qPCR and WB) and growth arrest was observed (Incucyte ZOOM), whereas NFAT5 expression and cell growth remained unchanged in unaffected skeletal muscle fibroblasts. This study may help understand the antiproliferative properties of glucocorticoids in DMD fibroblasts

Syncope in dental practices:a systematic review on aetiology and management

Introduction This systematic review aimed to give an overview of the current evidence sur-rounding the aetiology and management in terms of treatment and prevention of syncope in dental practices. Alongside the occurrence, the practitioner's com-petence, and the association between syncope and local anaesthetics were dis-cussed. Methods An electronic search in EMBASE, Web of Science, PubMed, Cochrane databases and a hand search were performed by 2 independent reviewers to identify stud-ies up to November 2019. Eligibility criteria were applied and relevant data was extracted. Inclusion criteria covered all types of dental treatment under local anaesthesia or conscious sedation performed by a wide range of oral health care workers in their practices. Risk of bias of the included studies was as-sessed using the methodological tools recommend by Zeng et al.1 No restric-tions were made to exclude papers from qualitive analysis based on risk of bias assessment. Results The search yielded a total of 18 studies for qualitative analysis. With the ex-ception of one prospective cohort study, all articles were considered having a high risk of bias. Meta-analysis showed that dentists encountered on average 1.2 cases of syncope per year. The male gender (RR=2.69 [1.03, 7.02]), dental fear (RR=3.55 [2.22, 5.70]), refusal of local anaesthesia in non-acute situations (OR = 12.9) and the use of premedication (RR = 4.70, [1.30, 16.90]) increased the risk for syncope. Treatment and prevention were underreported as both were solely discussed in one study. The supine recovery position with raised legs and oxygen administration (15l/min) was presented as an effective treatment. The Medical Risk-Related History (MRRH) system was proposed as prevention pro-tocol, yet this protocol was ineffective in reducing incidence rates (p = 0.27). The majority of dentists (79.2%) were able to diagnose syncope, yet most (86%) lacked the skills for appropriate treatment. Only 57,6% of dental practices were equipped with an oxygen cylinder. Conclusions Syncope is the most common emergency in dental practices. Nonetheless, the vast majority of dentists do not seem competent nor prepared to manage this emergency. Psychogenic factors seem to play an important role in provoking syncope. Placing the patient in a supine reclined position with raised legs in combination with the administration of oxygen seems effective for regaining consciousness. Although valuable in many aspects, risk assessment by medical history taking is not proven to result in fewer episodes. The strength of these conclusions is low based on GRADE guidelines.(2

Anti-inflammatory and general glucocorticoid physiology in skeletal muscles affected by Duchenne muscular dystrophy : exploration of steroid-sparing agents

In Duchenne muscular dystrophy (DMD), the activation of proinflammatory and metabolic cellular pathways in skeletal muscle cells is an inherent characteristic. Synthetic glucocorticoid intake counteracts the majority of these mechanisms. However, glucocorticoids induce burdensome secondary effects, including hypertension, arrhythmias, hyperglycemia, osteoporosis, weight gain, growth delay, skin thinning, cushingoid appearance, and tissue-specific glucocorticoid resistance. Hence, lowering the glucocorticoid dosage could be beneficial for DMD patients. A more profound insight into the major cellular pathways that are stabilized after synthetic glucocorticoid administration in DMD is needed when searching for the molecules able to achieve similar pathway stabilization. This review provides a concise overview of the major anti-inflammatory pathways, as well as the metabolic effects of glucocorticoids in the skeletal muscle affected in DMD. The known drugs able to stabilize these pathways, and which could potentially be combined with glucocorticoid therapy as steroid-sparing agents, are described. This could create new opportunities for testing in DMD animal models and/or clinical trials, possibly leading to smaller glucocorticoids dosage regimens for DMD patients

Hyperadrenocorticisme bij de fret: een overzicht van de huidige kennis aan de hand van twee klinische cases

Hyperadrenocorticism is a frequently occurring disease in middle-aged ferrets. It is caused by a neoplasm of the adrenal cortex. Otherwise than in dogs and cats, hyperadrenocorticism in the ferret is always independent of adrenocorticotropic hormone (ACTH). The tumor of the adrenal cortex can be removed surgically, mostly with satisfying results. After surgery, a regression of symptoms occurs after two to eight weeks, and the animals are without symptoms after five to eight months. In recent years, there has been a growing interest in using a chemical therapy aimed at decreasing hormone production through the administration of GnRH agonists. These agonists initially cause a temporary increase of the sex steroids, and then a long-term decrease of them through desensitization of the GnRH receptors. A deslorelin implant seems to be a promising alternative for the surgical treatment of hyperadrenocorticism in ferrets. Some authors even recommend it as a standard preventive treatment after neutering in both genders and for every ferret over four years of age. Moreover, it is an excellent alternative for surgical castration

Localization and expression of nuclear factor of activated T-cells 5 in myoblasts exposed to pro-inflammatory cytokines or hyperosmolar stress and in biopsies from myositis patients

Aims: Regeneration in skeletal muscle relies on regulated myoblast migration and differentiation, in which the transcription factor nuclear factor of activated T-cells 5 (NFAT5) participates. Impaired muscle regeneration and chronic inflammation are prevalent in myositis. Little is known about the impact of inflammation on NFAT5 localization and expression in this group of diseases. The goal of this study was to investigate NFAT5 physiology in unaffected myoblasts exposed to cytokine or hyperosmolar stress and in myositis. Methods: NFAT5 intracellular localization and expression were studied in vitro using a cell culture model of myositis. Myoblasts were exposed to DMEM solutions enriched with pro-inflammatory cytokines IFN-gamma with IL-1 beta) or hyperosmolar DMEM obtained by NaCI supplementation. NFAT5 localization was visualized using immunohistochemistry (IHC) and Western blotting (WB) in fractionated cell lysates. NFAT5 expression was assessed by WB and RT-qPCR. In vivo localization and expression of NFAT5 were studied in muscle biopsies of patients diagnosed with polymyositis (n = 6), dermatomyositis (n = 10), inclusion body myositis (n = 11) and were compared to NFAT5 localization and expression in non-myopathic controls (n = 13). Muscle biopsies were studied by means of quantitative IHC and WB of total protein extracts. Results: In unaffected myoblasts, hyperosmolar stress ensues in NFAT5 nuclear translocation and increased NFAT5 mRNA and protein expression. In contrast, pro-inflammatory cytokines did not lead to NFAT5 nuclear translocation nor increased expression. Cytokines IL-1 beta with IFN-gamma induced colocalization of NFAT5 with histone deacetylase 6 (HDAC6), involved in cell motility. In muscle biopsies from dermatomyositis and polymyositis patients, NFAT5 colocalized with HDAC6, while in IBM, this was often absent. Conclusions: Our data suggest impaired NFAT5 localization and expression in unaffected myoblasts in response to inflammation. This disturbed myogenic NFAT5 physiology could possibly explain deleterious effects on muscle regeneration in myositis