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.

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.

Regulation of Satellite Cells Functions during Skeletal Muscle Regeneration: A Critical Step in Physiological and Pathological Conditions.

Skeletal muscle regeneration is a complex process involving the generation of new myofibers after trauma, competitive physical activity, or disease. In this context, adult skeletal muscle stem cells, also known as satellite cells (SCs), play a crucial role in regulating muscle tissue homeostasis and activating regeneration. Alterations in their number or function have been associated with various pathological conditions. The main factors involved in the dysregulation of SCs' activity are inflammation, oxidative stress, and fibrosis. This review critically summarizes the current knowledge on the role of SCs in skeletal muscle regeneration. It examines the changes in the activity of SCs in three of the most common and severe muscle disorders: sarcopenia, muscular dystrophy, and cancer cachexia. Understanding the molecular mechanisms involved in their dysregulations is essential for improving current treatments, such as exercise, and developing personalized approaches to reactivate SCs.

Rebalancing expression of HMGB1 redox isoforms to counteract muscular dystrophy.

Muscular dystrophies (MDs) are a group of genetic diseases characterized by progressive muscle wasting associated to oxidative stress and persistent inflammation. It is essential to deepen our knowledge on the mechanism connecting these two processes because current treatments for MDs have limited efficacy and/or are associated with side effects. Here, we identified the alarmin high-mobility group box 1 (HMGB1) as a functional link between oxidative stress and inflammation in MDs. The oxidation of HMGB1 cysteines switches its extracellular activities from the orchestration of tissue regeneration to the exacerbation of inflammation. Extracellular HMGB1 is present at high amount and undergoes oxidation in patients with MDs and in mouse models of Duchenne muscular dystrophy (DMD) and limb-girdle muscular dystrophy 3 (LGMDR3) compared to controls. Genetic ablation of HMGB1 in muscles of DMD mice leads to an amelioration of the dystrophic phenotype as evidenced by the reduced inflammation and muscle degeneration, indicating that HMGB1 oxidation is a detrimental process in MDs. Pharmacological treatment with an engineered nonoxidizable variant of HMGB1, called 3S, improves functional performance, muscle regeneration, and satellite cell engraftment in dystrophic mice while reducing inflammation and fibrosis. Overall, our data demonstrate that the balance between HMGB1 redox isoforms dictates whether skeletal muscle is in an inflamed or regenerating state, and that the nonoxidizable form of HMGB1 is a possible therapeutic approach to counteract the progression of the dystrophic phenotype. Rebalancing the HMGB1 redox isoforms may also be a therapeutic strategy for other disorders characterized by chronic oxidative stress and inflammation.

Trabectedin and Lurbinectedin Extend Survival of Mice Bearing C26 Colon Adenocarcinoma, without Affecting Tumor Growth or Cachexia.

Trabectedin (ET743) and lurbinectedin (PM01183) limit the production of inflammatory cytokines that are elevated during cancer cachexia. Mice carrying C26 colon adenocarcinoma display cachexia (i.e., premature death and body wasting with muscle, fat and cardiac tissue depletion), high levels of inflammatory cytokines and subsequent splenomegaly. We tested whether such drugs protected these mice from cachexia. Ten-week-old mice were inoculated with C26 cells and three days later randomized to receive intravenously vehicle or 0.05 mg/kg ET743 or 0.07 mg/kg PM01183, three times a week for three weeks. ET743 or PM01183 extended the lifespan of C26-mice by 30% or 85%, respectively, without affecting tumor growth or food intake. Within 13 days from C26 implant, both drugs did not protect fat, muscle and heart from cachexia. Since PM01183 extended the animal survival more than ET743, we analyzed PM01183 further. In tibialis anterior of C26-mice, but not in atrophying myotubes, PM01183 restrained the NF-κB/PAX7/myogenin axis, possibly reducing the pro-inflammatory milieu, and failed to limit the C/EBPß/atrogin-1 axis. Inflammation-mediated splenomegaly of C26-mice was inhibited by PM01183 for as long as the treatment lasted, without reducing IL-6, M-CSF or IL-1ß in plasma. ET743 and PM01183 extend the survival of C26-bearing mice unchanging tumor growth or cachexia but possibly restrain muscle-related inflammation and C26-induced splenomegaly.

Oxidation of HMGB1 Is a Dynamically Regulated Process in Physiological and Pathological Conditions.

Acute inflammation is a complex biological response of tissues to harmful stimuli, such as pathogens or cell damage, and is essential for immune defense and proper healing. However, unresolved inflammation can lead to chronic disorders, including cancer and fibrosis. The High Mobility Group Box 1 (HMGB1) protein is a Damage-Associated Molecular Pattern (DAMP) molecule that orchestrates key events in inflammation by switching among mutually exclusive redox states. Fully reduced HMGB1 (frHMGB1) supports immune cell recruitment and tissue regeneration, while the isoform containing a disulphide bond (dsHMGB1) promotes secretion of inflammatory mediators by immune cells. Although it has been suggested that the tissue itself determines the redox state of the extracellular space and of released HMGB1, the dynamics of HMGB1 oxidation in health and disease are unknown. In the present work, we analyzed the expression of HMGB1 redox isoforms in different inflammatory conditions in skeletal muscle, from acute injury to muscle wasting, in tumor microenvironment, in spleen, and in liver after drug intoxication. Our results reveal that the redox modulation of HMGB1 is tissue-specific, with high expression of dsHMGB1 in normal spleen and liver and very low in muscle, where it appears after acute damage. Similarly, dsHMGB1 is highly expressed in the tumor microenvironment while it is absent in cachectic muscles from the same tumor-bearing mice. These findings emphasize the accurate and dynamic regulation of HMGB1 redox state, with the presence of dsHMGB1 tightly associated with leukocyte infiltration. Accordingly, we identified circulating, infiltrating, and resident leukocytes as reservoirs and transporters of dsHMGB1 in tissue and tumor microenvironment, demonstrating that the redox state of HMGB1 is controlled at both tissue and cell levels. Overall, our data point out that HMGB1 oxidation is a timely and spatially regulated process in physiological and pathological conditions. This precise modulation might play key roles to finetune inflammatory and regenerative processes.

Exploiting Live Imaging to Track Nuclei During Myoblast Differentiation and Fusion.

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear positioning occurs during skeletal muscle differentiation. Muscle fibers (myofibers) are multinucleated cells formed by the fusion of muscle precursor cells (myoblasts) derived from muscle stem cells (satellite cells) that undergo proliferation and differentiation. Correct nuclear positioning within myofibers is required for the proper muscle regeneration and function. The common procedure to assess myoblast differentiation and myofiber formation relies on fixed cells analyzed by immunofluorescence, which impedes the study of nuclear movement and cell behavior over time. Here, we describe a method for the analysis of myoblast differentiation and myofiber formation by live cell imaging. We provide a software for automated nuclear tracking to obtain a high-throughput quantitative characterization of nuclear dynamics and myoblast behavior (i.e., the trajectory) during differentiation and fusion.

High mobility group box 1 orchestrates tissue regeneration via CXCR4.

Inflammation and tissue regeneration follow tissue damage, but little is known about how these processes are coordinated. High Mobility Group Box 1 (HMGB1) is a nuclear protein that, when released on injury, triggers inflammation. We previously showed that HMGB1 with reduced cysteines is a chemoattractant, whereas a disulfide bond makes it a proinflammatory cytokine. Here we report that fully reduced HMGB1 orchestrates muscle and liver regeneration via CXCR4, whereas disulfide HMGB1 and its receptors TLR4/MD-2 and RAGE (receptor for advanced glycation end products) are not involved. Injection of HMGB1 accelerates tissue repair by acting on resident muscle stem cells, hepatocytes, and infiltrating cells. The nonoxidizable HMGB1 mutant 3S, in which serines replace cysteines, promotes muscle and liver regeneration more efficiently than the wild-type protein and without exacerbating inflammation by selectively interacting with CXCR4. Overall, our results show that the reduced form of HMGB1 coordinates tissue regeneration and suggest that 3S may be used to safely accelerate healing after injury in diverse clinical contexts.

HMGB1 as biomarker and drug target.

High Mobility Group Box 1 protein was discovered as a nuclear protein, but it has a "second life" outside the cell where it acts as a damage-associated molecular pattern. HMGB1 is passively released or actively secreted in a number of diseases, including trauma, chronic inflammatory disorders, autoimmune diseases and cancer. Extracellular HMGB1 triggers and sustains the inflammatory response by inducing cytokine release and by recruiting leucocytes. These characteristics make extracellular HMGB1 a key molecular target in multiple diseases. A number of strategies have been used to prevent HMGB1 release or to inhibit its activities. Current pharmacological strategies include antibodies, peptides, decoy receptors and small molecules. Noteworthy, salicylic acid, a metabolite of aspirin, has been recently found to inhibit HMGB1. HMGB1 undergoes extensive post-translational modifications, in particular acetylation and oxidation, which modulate its functions. Notably, high levels of serum HMGB1, in particular of the hyper-acetylated and disulfide isoforms, are sensitive disease biomarkers and are associated with different disease stages. In the future, the development of isoform-specific HMGB1 inhibitors may potentiate and fine-tune the pharmacological control of inflammation. We review here the current therapeutic strategies targeting HMGB1, in particular the emerging and relatively unexplored small molecules-based approach.