Apelin stimulation of the vascular skeletal muscle stem cell niche enhances endogenous repair in dystrophic mice.

Impaired skeletal muscle stem cell (MuSC) function has long been suspected to contribute to the pathogenesis of muscular dystrophy (MD). Here, we showed that defects in the endothelial cell (EC) compartment of the vascular stem cell niche in mouse models of Duchenne MD, laminin α2-related MD, and collagen VI-related myopathy were associated with inefficient mobilization of MuSCs after tissue damage. Using chemoinformatic analysis, we identified the 13-amino acid form of the peptide hormone apelin (AP-13) as a candidate for systemic stimulation of skeletal muscle ECs. Systemic administration of AP-13 using osmotic pumps generated a pro-proliferative EC-rich niche that supported MuSC function through angiocrine factors and markedly improved tissue regeneration and muscle strength in all three dystrophic mouse models. Moreover, EC-specific knockout of the apelin receptor led to regenerative defects that phenocopied key pathological features of MD, including vascular defects, fibrosis, muscle fiber necrosis, impaired MuSC function, and reduced force generation. Together, these studies provide in vivo proof of concept that enhancing endogenous skeletal muscle repair by targeting the vascular niche is a viable therapeutic avenue for MD and characterized AP-13 as a candidate for further study for the systemic treatment of MuSC dysfunction.

Proof of concept of a two-stage GMR sensor-based lab-on-a-chip for early diagnostic tests.

The development of rapid, sensitive, portable and inexpensive early diagnostic techniques is a real challenge in the fields of health, defense and in the environment. The current global pandemic has also shown the need for such tests. The World Health Organization has defined ASSURED criteria (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable to end-users) that field diagnostic tests must fulfill, which proves the real need in terms of public health. Giant magnetoresistance (GMR) sensors, which have flourished in a wide variety of spintronic applications (automobile industry, Information Technology, etc.), also have real potential in the field of health, particularly for the development of early diagnostic point-of-care devices. This work presents a new type of innovative biochip, consisting of GMR sensors arranged on both sides of a microfluidic channel which allow on the one hand to count magnetic objects one by one but also to better distinguish false positives (aggregates of beads, etc.) from labelled biological targets of interest by determining their magnetic moment. We present the operating principle of this new tool and its great potential as a versatile diagnostic test.

An engineered multicellular stem cell niche for the 3D derivation of human myogenic progenitors from iPSCs.

Fate decisions in the embryo are controlled by a plethora of microenvironmental interactions in a three-dimensional niche. To investigate whether aspects of this microenvironmental complexity can be engineered to direct myogenic human-induced pluripotent stem cell (hiPSC) differentiation, we here screened murine cell types present in the developmental or adult stem cell niche in heterotypic suspension embryoids. We identified embryonic endothelial cells and fibroblasts as highly permissive for myogenic specification of hiPSCs. After two weeks of sequential Wnt and FGF pathway induction, these three-component embryoids are enriched in Pax7-positive embryonic-like myogenic progenitors that can be isolated by flow cytometry. Myogenic differentiation of hiPSCs in heterotypic embryoids relies on a specialized structural microenvironment and depends on MAPK, PI3K/AKT, and Notch signaling. After transplantation in a mouse model of Duchenne muscular dystrophy, embryonic-like myogenic progenitors repopulate the stem cell niche, reactivate after repeated injury, and, compared to adult human myoblasts, display enhanced fusion and lead to increased muscle function. Altogether, we provide a two-week protocol for efficient and scalable suspension-based 3D derivation of Pax7-positive myogenic progenitors from hiPSCs.

Assessment of Muscle Function Following hiPSC-Derived Myoblast Transplantation in Dystrophic Mice.

Muscular dystrophies are caused by genetic variants in genes encoding for proteins important for muscle structure or function, leading to a loss of muscle integrity and muscle wasting. To this day, no cure has been found for these diseases. Different therapeutic approaches are under intensive investigation. Cellular therapy has been extensively studied for diseases such as Duchenne Muscular Dystrophy, a debilitating disease caused by a mutation in the DMD gene, encoding for the dystrophin protein. Healthy myogenic cells transplanted into dystrophic muscles have the potential to engraft at long-term and fuse to donate their nuclei to the dystrophin-deficient myofibers, thereby restoring normal gene expression. Despite promising preclinical studies, the clinical trials had limited success so far due to many technical limitations. The recent technological advances in induced-pluripotent stem cells and genome editing opened new opportunities in this field. One of the keys to efficiently translate these new technologies into clinical benefits is to use relevant endpoints for preclinical studies. Considering that dystrophic muscles are susceptible to contraction-induced injury, the assessment of their resistance to repeated eccentric contractions is an optimal outcome to evaluate their functional recovery following cell transplantation. This protocol describes the procedure to generate induced-pluripotent stem cell-derived myoblasts, transplant these cells into skeletal muscle of immunosuppressed dystrophic mice, and assess muscle function in situ by measuring the resistance of the transplanted muscle to repeated eccentric contractions. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Generation of hiPSC-derived myoblasts. Basic Protocol 2: Transplantation of hiPSC-derived myoblasts in skeletal muscle of dystrophic mice. Basic Protocol 3: Assessment of muscle function in situ.

Fibro-adipogenic progenitors in skeletal muscle homeostasis, regeneration and diseases.

Skeletal muscle possesses a remarkable regenerative capacity that relies on the activity of muscle stem cells, also known as satellite cells. The presence of non-myogenic cells also plays a key role in the coordination of skeletal muscle regeneration. Particularly, fibro-adipogenic progenitors (FAPs) emerged as master regulators of muscle stem cell function and skeletal muscle regeneration. This population of muscle resident mesenchymal stromal cells has been initially characterized based on its bi-potent ability to differentiate into fibroblasts or adipocytes. New technologies such as single-cell RNAseq revealed the cellular heterogeneity of FAPs and their complex regulatory network during muscle regeneration. In acute injury, FAPs rapidly enter the cell cycle and secrete trophic factors that support the myogenic activity of muscle stem cells. Conversely, deregulation of FAP cell activity is associated with the accumulation of fibrofatty tissue in pathological conditions such as muscular dystrophies and ageing. Considering their central role in skeletal muscle pathophysiology, the regulatory mechanisms of FAPs and their cellular and molecular crosstalk with muscle stem cells are highly investigated in the field. In this review, we summarize the current knowledge on FAP cell characteristics, heterogeneity and the cellular crosstalk during skeletal muscle homeostasis and regeneration. We further describe their role in muscular disorders, as well as different therapeutic strategies targeting these cells to restore muscle regeneration.

Resolvin-D2 targets myogenic cells and improves muscle regeneration in Duchenne muscular dystrophy.

Lack of dystrophin causes muscle degeneration, which is exacerbated by chronic inflammation and reduced regenerative capacity of muscle stem cells in Duchenne Muscular Dystrophy (DMD). To date, glucocorticoids remain the gold standard for the treatment of DMD. These drugs are able to slow down the progression of the disease and increase lifespan by dampening the chronic and excessive inflammatory process; however, they also have numerous harmful side effects that hamper their therapeutic potential. Here, we investigated Resolvin-D2 as a new therapeutic alternative having the potential to target multiple key features contributing to the disease progression. Our in vitro findings showed that Resolvin-D2 promotes the switch of macrophages toward their anti-inflammatory phenotype and increases their secretion of pro-myogenic factors. Moreover, Resolvin-D2 directly targets myogenic cells and promotes their differentiation and the expansion of the pool of myogenic progenitor cells leading to increased myogenesis. These effects are ablated when the receptor Gpr18 is knocked-out, knocked-down, or blocked by the pharmacological antagonist O-1918. Using different mouse models of DMD, we showed that Resolvin-D2 targets both inflammation and myogenesis leading to enhanced muscle function compared to glucocorticoids. Overall, this preclinical study has identified a new therapeutic approach that is more potent than the gold-standard treatment for DMD.

Para-Substituted α-Phenyl-N-tert-butyl Nitrones: Spin-Trapping, Redox and Neuroprotective Properties.

In this work, a series of para-substituted α-phenyl-N-tert-butyl nitrones (PBN) were studied. Their radical-trapping properties were evaluated by electron paramagnetic resonance, with 4-CF3-PBN being the fastest derivative to trap the hydroxymethyl radical (•CH2OH). The redox properties of the nitrones were further investigated by cyclic voltammetry, and 4-CF3-PBN was the easiest to reduce and the hardest to oxidize. This is due to the presence of the electron-withdrawing CF3 group. Very good correlations between the Hammett constants (σp) of the substituents and both spin-trapping rates and redox potentials were observed. These correlations were further supported by computationally determined ionization potentials and atom charge densities. Finally, the neuroprotective effect of these derivatives was studied using two different in vitro models of cell death on primary cortical neurons injured by glutamate exposure or on glial cells exposed to t BuOOH. Trends between the protection afforded by the nitrones and their lipophilicity were observed. 4-CF3-PBN was the most potent agent against t BuOOH-induced oxidative stress on glial cells, while 4-Me2N-PBN showed potency in both models.

Macrophages Are Key Regulators of Stem Cells during Skeletal Muscle Regeneration and Diseases.

Muscle regeneration is a closely regulated process that involves a variety of cell types such as satellite cells, myofibers, fibroadipogenic progenitors, endothelial cells, and inflammatory cells. Among these different cell types, macrophages emerged as a central actor coordinating the different cellular interactions and biological processes. Particularly, the transition of macrophages from their proinflammatory to their anti-inflammatory phenotype was shown to regulate inflammation, myogenesis, fibrosis, vascularization, and return to homeostasis. On the other hand, deregulation of macrophage accumulation or polarization in chronic degenerative muscle disorders was shown to impair muscle regeneration. Considering the key roles of macrophages in skeletal muscle, they represent an attractive target for new therapeutic approaches aiming at mitigating various muscle disorders. This review aims at summarizing the novel insights into macrophage heterogeneity, plasticity, and functions in skeletal muscle homeostasis, regeneration, and disease.

Biallelic variants in the transcription factor PAX7 are a new genetic cause of myopathy.

PURPOSE: Skeletal muscle growth and regeneration rely on muscle stem cells, called satellite cells. Specific transcription factors, particularly PAX7, are key regulators of the function of these cells. Knockout of this factor in mice leads to poor postnatal survival; however, the consequences of a lack of PAX7 in humans have not been established. METHODS: Here, we study five individuals with myopathy of variable severity from four unrelated consanguineous couples. Exome sequencing identified pathogenic variants in the PAX7 gene. Clinical examination, laboratory tests, and muscle biopsies were performed to characterize the disease. RESULTS: The disease was characterized by hypotonia, ptosis, muscular atrophy, scoliosis, and mildly dysmorphic facial features. The disease spectrum ranged from mild to severe and appears to be progressive. Muscle biopsies showed the presence of atrophic fibers and fibroadipose tissue replacement, with the absence of myofiber necrosis. A lack of PAX7 expression was associated with satellite cell pool exhaustion; however, the presence of residual myoblasts together with regenerating myofibers suggest that a population of PAX7-independent myogenic cells partially contributes to muscle regeneration. CONCLUSION: These findings show that biallelic variants in the master transcription factor PAX7 cause a new type of myopathy that specifically affects satellite cell survival.

2-Aryl-3H-indol-3-ones: synthesis, electrochemical behaviour and antiplasmodial activities.

The synthesis of indolone derivatives and their antiplasmodial activity in vitro against Plasmodium falciparum at the blood stage are described. The 2-aryl-3H-indol-3-ones were synthesized via deoxygenation of indolone-N-oxides. Electrochemical behaviour, antiplasmodial activity and cytotoxicity on human tumor cell lines were compared to those of indolone-N-oxides. The antiplasmodial IC50 (concentrations at 50% inhibition) of these compounds ranged between 49 and 1327 nM. Among them, the 2-(4-dimethylaminophenyl)-5-methoxy-indol-3-one, 7, had the best antiplasmodial activity in vitro (IC50 = 49 nM; FcB1 strain) and selectivity index (SI (CC50 MCF7/IC50 FcB1) = 423.4). Thus, the hits identified in this deoxygenated series correspond to their structural homologs in the N-oxide series with comparable electrochemical behaviour at the nitrogen-carbon double bond.

EPR spectroelectrochemical investigation of guanine radical formation and environment effects.

Guanine radical detection was carried out by a new convenient and efficient method coupling electron paramagnetic resonance spectroscopy and indirect electrooxidation of guanine in different biological environments, from the free nucleotide to several types of DNA substrates. Compared to the widely used photoirradiation method, this method appeared more selective in the choice of the electrochemical mediator. Carried out in presence of a ruthenium mediator and PBN as spin trap, this method revealed two types of EPR spectra depending of the environment of the guanine radical. Both EPR spectra show the trapping of the neutral guanine radical G(-H)(•) obtained after fast deprotonation of the radical cation G(•+). However, they differ by the atom where the trapped radical is centered. This difference highlights the structural dependency of the environment on the nature of the radical formed. This work gave the evidence of an innovative method to detect in situ the guanine radical.

Pro-oxidant properties of indolone-N-oxides in relation to their antimalarial properties.

Indolone-N-oxides (INODs) are bioreducible and possess remarkable anti-malarial activities in the low nanomolar range in vitro against different Plasmodium falciparum (P. falciparum) strains and in vivo. INODs have an original mechanism of action: they damage the host cell membrane without affecting non-parasitized erythrocytes. These molecules produce a redox signal which activates SYK tyrosine kinases and induces a hyperphosphorylation of AE1 (band 3, erythrocyte membrane protein). The present work aimed to understand the early stages of the biochemical interactions of these compounds with some erythrocyte components from which the redox signal could originate. The interactions were studied in a biomimetic model and compared with those of chloroquine and artemisinin. The results showed that INODs i) do not enter the coordination sphere of the metal in the heme iron complex as does chloroquine; ii) do not generate iron-dependent radicals as does artemisinin; iii) generate stable free radical adducts after reduction at one electron; iv) cannot trap free radicals after reduction. These results confirm that the bioactivity of INODs does not lie in their spin-trapping properties but rather in their pro-oxidant character. This property may be the initiator of the redox signal which activates SYK tyrosine kinases.

Unexpected effect of copper ions on electrochemical impedance behaviour of self-assembled alkylaminethiol monolayer.

Effect of copper ions on the electrochemical behaviour of an alkylaminethiol monolayer has been studied by electrochemical impedance spectrosocpy. RAMAN experiment shows the effective adsorption of receptor onto the gold surfaces. The study of Nyquist plot shows that the gold/monolayer/electrolyte interface can be described by a serial combination of two R, CPE electrical circuits. In the presence of increasing amounts of copper, the Nyquist plots at low frequencies were modified showing an increase of the resistance of the second R, CPE electrical circuit. Moreover, this increase of resistance varies linearly with the amounts of copper ions added in solution from 10(-8) mol·L(-1) to 10(-5) mol·L(-1).

Electrochemical behavior of indolone-N-oxides: relationship to structure and antiplasmodial activity.

Indolone-N-oxides exert high parasiticidal activity at the nanomolar level in vitro against Plasmodium falciparum, the parasite responsible for malaria. The bioreductive character of these molecules was investigated using cyclic voltammetry and EPR spectroelectrochemistry to examine the relationship between electrochemical behavior and antimalarial activity and to understand their mechanisms of action. For all the compounds (37 compounds) studied, the voltammograms recorded in acetonitrile showed a well-defined and reversible redox couple followed by a second complicated electron transfer. The first reduction (-0.88V

Correlations between electrochemical behaviors and DNA photooxidative properties of non-steroïdal anti-inflammatory drugs and their photoproducts.

Alkali-labile lesion to DNA photosensitized, via an electron transfer mechanism, by three non-steroidal anti-inflammatory drugs (NSAIDs), ketoprofen, tiaprofenic acid and naproxen and their photoproducts during drug photolysis, was investigated using (32)P-end labelled synthetic oligonucleotide. These photooxidative damages were correlated with the photophysical and electrochemical properties of drugs, appearing as the photosensitizer PS. Photophysical studies provided the excited state energies of the photosensitizer while their redox potentials and the relative stabilities of the PS(-) radical-anions were determined by cyclic voltammetry. On the basis of these data, we have calculated the Gibbs energy of photoinduced electron-transfer and evaluated the exergonicity of the oxidative photodamage. Moreover, kinetic control may be invoked according to the stabilities of PS(-). Applied to this NSAIDs family, the photoxidative damages through electron transfer mechanism were analyzed and a good correlation with photoredox and photobiological properties was established.

Characterization of oxidative stress in Leishmaniasis-infected or LPS-stimulated macrophages using electrochemical impedance spectroscopy.

The physiological changes caused by external stimuli can be employed as parameters to study pathogen infection in cells and the effect of drugs. Among analytical methods, impedance is potentially useful to give insight into cellular behavior by studying morphological changes, alterations in the physiological state, production of charged or redox species without interfering with in vitro cellular metabolism and labeling. The present work describes the use of electrochemical impedance spectroscopy to simply monitor by modeling impedance plots (Nyquist diagram) in appropriate equivalent circuit, the changes affecting murine macrophage cell line (RAW 264.7) in response to parasite infection by Leishmania amazonensis or to lipopolysaccharide (LPS) treatment. These results demonstrate the ability of electrochemical impedance spectroscopy to discriminate between two opposite cell responses associated to two different stimuli, one caused by the internalization of a parasite, and the other by activation by a bacterium component. Indeed, the study has allowed the characterization, from an electrical point of view, of the extra-cellular NO radical produced endogenously and in great quantities by the inducible form of NO-synthase in the case of LPS-stimulated macrophages. This production was not observed in the case of Leishmania-infected macrophages for which to survive and multiply, the parasite itself possesses mechanisms which may interfere with NO production. In this latest case, only the intracellular production of ROS was observed. To confirm these interpretations confocal microscopy analysis using the ROS (reactive oxygen species) fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate and electron paramagnetic resonance experiments using Fe(DETC)(2) as NO radical spin trap were carried out.

Electrochemical impedance spectroscopy to study physiological changes affecting the red blood cell after invasion by malaria parasites.

The malaria parasite, Plasmodium falciparum, invades human erythrocytes and induces dramatic changes in the host cell. The idea of this work was to use RBC modified electrode to perform electrochemical impedance spectroscopy (EIS) with the aim of monitoring physiological changes affecting the erythrocyte after invasion by the malaria parasite. Impedance cell-based devices are potentially useful to give insight into cellular behavior and to detect morphological changes. The modelling of impedance plots (Nyquist diagram) in equivalent circuit taking into account the presence of the cellular layer, allowed us pointing out specific events associated with the development of the parasite such as (i) strong changes in the host cell cytoplasm illustrated by changes in the film capacity, (ii) perturbation of the ionic composition of the host cell illustrated by changes in the film resistance, (iii) releasing of reducer (lactic acid or heme) and an enhanced oxygen consumption characterized by changes in the charge transfer resistance and in the Warburg coefficient characteristic of the redox species diffusion. These results show that the RBC-based device may help to analyze strategic events in the malaria parasite development constituting a new tool in antimalarial research.

Comparative study of the photophysical properties of indoprofen photoproducts in relation with their DNA photosensitizing properties.

The photophysical properties of indoprofen photoproducts have been examined in various solvents by absorbance and emission spectroscopies in relation with their photosensitizing properties. The photophysical properties of 2-[4-(1-hydroxy)ethylphenyl]isoindolin-1-one (HOINP) and 2-(4-ethylphenyl)isoindolin-1-one (ETINP) are typical of a singlet excited state when the ones of 2-(4-acetylphenyl)isoindolin-1-one (KINP) are based on its triplet excited state according to previous work. The effect of solvent polarity on the absorption and fluorescence properties of HOINP and ETINP has been investigated as a function of Delta f, the Lippert solvent polarity parameter. A solvatochromic effect, function of the polarity region, has been observed for both photoproducts due to a change in the dipole moment of the compound upon excitation. In low-polarity regions, the excited state dipole moment of HOINP undergoes only a moderate increase (11.5 D) as compared to the dipole moment of the ground state (4.5 D) suggesting that the fluorescence arises from the locally excited state while in high-polarity regions it is strongly increased (42.9 D), which can imply that the emission takes place from a charge transfer state. In the case of ETINP, it would seem that the emitting state is rather a charge transfer state whatever the region is (16.9 and 31.8 D for the calculated excited-state dipole moments in the low and high-polarity regions, respectively). HOINP and ETINP do not produce thymine dimers by photosensitization but induce photooxidative damage via an electron transfer mechanism.