MEK-inhibitors decrease Nfix in muscular dystrophy but induce unexpected calcifications, partially rescued with Cyanidin diet.

Muscular dystrophies (MDs) are incurable genetic myopathies characterized by progressive degeneration of skeletal muscles. Dystrophic mice lacking the transcription factor Nfix display morphological and functional improvements of the disease. Recently, we demonstrated that MAPK signaling pathway positively regulates Nfix in muscle development and that Cyanidin, a natural antioxidant molecule, strongly ameliorates the pathology. To explore a synergistic approach aimed at treating MDs, we administered Trametinib, a clinically approved MEK inhibitor, alone or combined with Cyanidin to adult Sgca null mice. We observed that chronic treatment with Trametinib and Cyanidin reduced Nfix in myogenic cells but, unexpectedly, caused ectopic calcifications exclusively in dystrophic muscles. The combined treatment with Cyanidin resulted in histological improvements by preventing Trametinib-induced calcifications in Diaphragm and Soleus. Collectively, this first pilot study revealed that Nfix is modulated by the MAPK pathway in MDs, and that Cyanidin partly rescued the unexpected ectopic calcifications caused by MEK inhibition.

To be born twin: effects on long-term neurodevelopment of very preterm infants-a cohort study.

Objective: To examine the effect of twin birth on long-term neurodevelopmental outcomes in a cohort of Italian preterm infants with very low birth weight. Study design: We performed a retrospective cohort study on children born in a tertiary care centre. We included children born between 1 January 2007 and 31 December 2013 with a gestational age (GA) of ≤32 weeks and birth weight of <1,500 g. The infants born from twin pregnancies complicated by twin-to-twin transfusion syndrome and from higher-order multiple pregnancies were excluded. The children were evaluated both at 2 years corrected age and 5 years chronological age with Griffiths mental development scales revised (GMDS-R). The linear mixed effects models were used to study the effect of being a twin vs. being a singleton on GMDS-R scores, adjusting for GA, being born small for gestational age, sex, length of NICU stay, socio-economic status, and comorbidity score (CS) calculated as the sum of the weights associated with each of the major morbidities of the infants. Results: A total of 301 children were included in the study, of which 189 (62.8%) were singletons and 112 (37.2%) were twins; 23 out of 112 twins were monochorionic (MC). No statistically significant differences were observed between twins and singletons in terms of mean general quotient and subscales at both 2 and 5 years. No effect of chorionicity was found when comparing scores of MC and dichorionic twins vs. singletons; however, after adjusting for the CS, the MC twins showed lower scores in the hearing and language and performance subscales at 5 years. Conclusion: Overall, in our cohort of children born very preterm, twin infants were not at higher risk of neurodevelopmental impairment compared with singletons at pre-school age.

Lack of the transcription factor Nfix causes tachycardia in mice sinus node and rats neonatal cardiomyocytes.

AIMS: Nfix is a transcription factor belonging to the Nuclear Factor I (NFI) family comprising four members (Nfia, b, c, x). Nfix plays important roles in the development and function of several organs. In muscle development, Nfix controls the switch from embryonic to fetal myogenesis by promoting fast twitching fibres. In the adult muscle, following injury, lack of Nfix impairs regeneration, inducing higher content of slow-twitching fibres. Nfix is expressed also in the heart, but its function has been never investigated before. We studied Nfix role in this organ. METHODS: Using Nfix-null and wild type (WT) mice we analyzed: (1) the expression pattern of Nfix during development by qPCR and (2) the functional alterations caused by its absence, by in vivo telemetry and in vitro patch clamp analysis. RESULTS AND CONCLUSIONS: Nfix expression start in the heart from E12.5. Adult hearts of Nfix-null mice show a hearts morphology and sarcomeric proteins expression similar to WT. However, Nfix-null animals show tachycardia that derives form an intrinsic higher beating rate of the sinus node (SAN). Molecular and functional analysis revealed that sinoatrial cells of Nfix-null mice express a significantly larger L-type calcium current (Cacna1d + Cacna1c). Interestingly, downregulation of Nfix by sh-RNA in primary cultures of neonatal rat ventricular cardiomyocytes induced a similar increase in their spontaneous beating rate and in ICaL current. In conclusion, our data provide the first demonstration of a role of Nfix that, increasing the L-type calcium current, modulates heart rate.

Selective ablation of Nfix in macrophages attenuates muscular dystrophy by inhibiting fibro-adipogenic progenitor-dependent fibrosis.

Muscular dystrophies are genetic diseases characterized by chronic inflammation and fibrosis. Macrophages are immune cells that sustain muscle regeneration upon acute injury but seem deleterious in the context of chronic muscle injury such as in muscular dystrophies. Here, we observed that the number of macrophages expressing the transcription factor Nfix increases in two distinct mouse models of muscular dystrophies. We showed that the deletion of Nfix in macrophages in dystrophic mice delays the establishment of fibrosis and muscle wasting, and increases grasp force. Macrophages lacking Nfix expressed more TNFα and less TGFß1, thus promoting apoptosis of fibro-adipogenic progenitors. Moreover, pharmacological treatment of dystrophic mice with a ROCK inhibitor accelerated fibrosis through the increase of Nfix expression by macrophages. Thus, we have identified Nfix as a macrophage profibrotic factor in muscular dystrophies, whose inhibition could be a therapeutic route to reduce severity of the dystrophic disease. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Mesoangioblasts at 20: From the embryonic aorta to the patient bed.

In 2002 we published an article describing a population of vessel-associated progenitors that we termed mesoangioblasts (MABs). During the past decade evidence had accumulated that during muscle development and regeneration things may be more complex than a simple sequence of binary choices (e.g., dorsal vs. ventral somite). LacZ expressing fibroblasts could fuse with unlabelled myoblasts but not among themselves or with other cell types. Bone marrow derived, circulating progenitors were able to participate in muscle regeneration, though in very small percentage. Searching for the embryonic origin of these progenitors, we identified them as originating at least in part from the embryonic aorta and, at later stages, from the microvasculature of skeletal muscle. While continuing to investigate origin and fate of MABs, the fact that they could be expanded in vitro (also from human muscle) and cross the vessel wall, suggested a protocol for the cell therapy of muscular dystrophies. We tested this protocol in mice and dogs before proceeding to the first clinical trial on Duchenne Muscular Dystrophy patients that showed safety but minimal efficacy. In the last years, we have worked to overcome the problem of low engraftment and tried to understand their role as auxiliary myogenic progenitors during development and regeneration.

Synthesis and characterization of 13C labeled carnosine derivatives for isotope dilution mass spectrometry measurements in biological matrices.

Due to the physiological properties of l-carnosine (l-1), supplementation of this dipeptide has both a nutritional ergogenic application and a therapeutic potential for the treatment of numerous diseases in which ischemic or oxidative stress are involved. Quantitation of carnosine and its analogs in biological matrices results to be crucial for these applications and HPLC-MS procedures with isotope-labeled internal standards are the state-of-the-art approach for this analytical need. The use of these standards allows to account for variations during the sample preparation process, between-sample matrix effects, and variations in instrument performance over analysis time. Although literature reports a number of studies involving carnosine, isotope-labeled derivatives of the dipeptide are not commercially available. In this work we present a fast, flexible, and convenient strategy for the synthesis of the 13C-labeled carnosine analogs and their application as internal standards for the quantitation of carnosine and anserine in a biological matrix.

The Transcription Factor Nfix Requires RhoA-ROCK1 Dependent Phagocytosis to Mediate Macrophage Skewing during Skeletal Muscle Regeneration.

Macrophages (MPs) are immune cells which are crucial for tissue repair. In skeletal muscle regeneration, pro-inflammatory cells first infiltrate to promote myogenic cell proliferation, then they switch into an anti-inflammatory phenotype to sustain myogenic cells differentiation and myofiber formation. This phenotypical switch is induced by dead cell phagocytosis. We previously demonstrated that the transcription factor Nfix, a member of the nuclear factor I (Nfi) family, plays a pivotal role during muscle development, regeneration and in the progression of muscular dystrophies. Here, we show that Nfix is mainly expressed by anti-inflammatory macrophages. Upon acute injury, mice deleted for Nfix in myeloid line displayed a significant defect in the process of muscle regeneration. Indeed, Nfix is involved in the macrophage phenotypical switch and macrophages lacking Nfix failed to adopt an anti-inflammatory phenotype and interact with myogenic cells. Moreover, we demonstrated that phagocytosis induced by the inhibition of the RhoA-ROCK1 pathway leads to Nfix expression and, consequently, to acquisition of the anti-inflammatory phenotype. Our study identified Nfix as a link between RhoA-ROCK1-dependent phagocytosis and the MP phenotypical switch, thus establishing a new role for Nfix in macrophage biology for the resolution of inflammation and tissue repair.

Nutritional intervention with cyanidin hinders the progression of muscular dystrophy.

Muscular Dystrophies are severe genetic diseases due to mutations in structural genes, characterized by progressive muscle wasting that compromises patients' mobility and respiratory functions. Literature underlined oxidative stress and inflammation as key drivers of these pathologies. Interestingly among different myofiber classes, type I fibers display a milder dystrophic phenotype showing increased oxidative metabolism. This work shows the benefits of a cyanidin-enriched diet, that promotes muscle fiber-type switch and reduced inflammation in dystrophic alpha-sarcoglyan (Sgca) null mice having, as a net outcome, morphological and functional rescue. Notably, this benefit is achieved also when the diet is administered in dystrophic animals when the signs of the disease are seriously evident. Our work provides compelling evidence that a cyanidin-rich diet strongly delays the progression of muscular dystrophies, paving the way for a combinatorial approach where nutritional-based reduction of muscle inflammation and oxidative stress facilitate the successful perspectives of definitive treatments.

RhoA and ERK signalling regulate the expression of the transcription factor Nfix in myogenic cells.

The transcription factor Nfix belongs to the nuclear factor one family and has an essential role in prenatal skeletal muscle development, where it is a master regulator of the transition from embryonic to foetal myogenesis. Recently, Nfix was shown to be involved in adult muscle regeneration and in muscular dystrophies. Here, we have investigated the signalling that regulates Nfix expression, and show that JunB, a member of the AP-1 family, is an activator of Nfix, which then leads to foetal myogenesis. Moreover, we demonstrate that their expression is regulated through the RhoA/ROCK axis, which maintains embryonic myogenesis. Specifically, RhoA and ROCK repress ERK kinase activity, which promotes JunB and Nfix expression. Notably, the role of ERK in the activation of Nfix is conserved postnatally in satellite cells, which represent the canonical myogenic stem cells of adult muscle. As lack of Nfix in muscular dystrophies rescues the dystrophic phenotype, the identification of this pathway provides an opportunity to pharmacologically target Nfix in muscular dystrophies.

Silencing Nfix rescues muscular dystrophy by delaying muscle regeneration.

Muscular dystrophies are severe disorders due to mutations in structural genes, and are characterized by skeletal muscle wasting, compromised patient mobility, and respiratory functions. Although previous works suggested enhancing regeneration and muscle mass as therapeutic strategies, these led to no long-term benefits in humans. Mice lacking the transcription factor Nfix have delayed regeneration and a shift toward an oxidative fiber type. Here, we show that ablating or silencing the transcription factor Nfix ameliorates pathology in several forms of muscular dystrophy. Silencing Nfix in postnatal dystrophic mice, when the first signs of the disease already occurred, rescues the pathology and, conversely, Nfix overexpression in dystrophic muscles increases regeneration and markedly exacerbates the pathology. We therefore offer a proof of principle for a novel therapeutic approach for muscular dystrophies based on delaying muscle regeneration.

Nfix Regulates Temporal Progression of Muscle Regeneration through Modulation of Myostatin Expression.

Nfix belongs to a family of four highly conserved proteins that act as transcriptional activators and/or repressors of cellular and viral genes. We previously showed a pivotal role for Nfix in regulating the transcriptional switch from embryonic to fetal myogenesis. Here, we show that Nfix directly represses the Myostatin promoter, thus controlling the proper timing of satellite cell differentiation and muscle regeneration. Nfix-null mice display delayed regeneration after injury, and this deficit is reversed upon in vivo Myostatin silencing. Conditional deletion of Nfix in satellite cells results in a similar delay in regeneration, confirming the functional requirement for Nfix in satellite cells. Moreover, mice lacking Nfix show reduced myofiber cross sectional area and a predominant slow twitching phenotype. These data define a role for Nfix in postnatal skeletal muscle and unveil a mechanism for Myostatin regulation, thus providing insights into the modulation of its complex signaling pathway.

PW1/Peg3 expression regulates key properties that determine mesoangioblast stem cell competence.

Mesoangioblasts are vessel-associated progenitor cells that show therapeutic promise for the treatment of muscular dystrophy. Mesoangioblasts have the ability to undergo skeletal muscle differentiation and cross the blood vessel wall regardless of the developmental stage at which they are isolated. Here we show that PW1/Peg3 is expressed at high levels in mesoangioblasts obtained from mouse, dog and human tissues and its level of expression correlates with their myogenic competence. Silencing PW1/Peg3 markedly inhibits myogenic potential of mesoangioblasts in vitro through MyoD degradation. Moreover, lack of PW1/Peg3 abrogates mesoangioblast ability to cross the vessel wall and to engraft into damaged myofibres through the modulation of the junctional adhesion molecule-A. We conclude that PW1/Peg3 function is essential for conferring proper mesoangioblast competence and that the determination of PW1/Peg3 levels in human mesoangioblasts may serve as a biomarker to identify the best donor populations for therapeutic application in muscular dystrophies.