Publications by authors named "Marina Bouchè"

31 Publications

Muscle Diversity, Heterogeneity, and Gradients: Learning from Sarcoglycanopathies.

Int J Mol Sci 2021 Mar 2;22(5). Epub 2021 Mar 2.

Department of AHFMO, University of Rome "la Sapienza", Via A. Scarpa 14, 00161 Rome, Italy.

Skeletal muscle, the most abundant tissue in the body, is heterogeneous. This heterogeneity forms the basis of muscle diversity, which is reflected in the specialized functions of muscles in different parts of the body. However, these different parts are not always clearly delimitated, and this often gives rise to gradients within the same muscle and even across the body. During the last decade, several studies on muscular disorders both in mice and in humans have observed particular distribution patterns of muscle weakness during disease, indicating that the same mutation can affect muscles differently. Moreover, these phenotypical differences reveal gradients of severity, existing alongside other architectural gradients. These two factors are especially prominent in sarcoglycanopathies. Nevertheless, very little is known about the mechanism(s) driving the phenotypic diversity of the muscles affected by these diseases. Here, we will review the available literature on sarcoglycanopathies, focusing on phenotypic differences among affected muscles and gradients, characterization techniques, molecular signatures, and cell population heterogeneity, highlighting the possibilities opened up by new technologies. This review aims to revive research interest in the diverse disease phenotype affecting different muscles, in order to pave the way for new therapeutic interventions.
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http://dx.doi.org/10.3390/ijms22052502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7958856PMC
March 2021

A novel approach for the isolation and long-term expansion of pure satellite cells based on ice-cold treatment.

Skelet Muscle 2021 Mar 17;11(1). Epub 2021 Mar 17.

Department of Anatomical, Histological, Forensic and Orthopedic Sciences, Section of Histology and Embryology, Sapienza University of Rome, Rome, Italy.

Satellite cells (SCs) are muscle stem cells capable of regenerating injured muscle. The study of their functional potential depends on the availability of methods for the isolation and expansion of pure SCs with preserved myogenic properties after serial passages in vitro. Here, we describe the ice-cold treatment (ICT) method, which is a simple, economical, and efficient method for the isolation and in vitro expansion of highly pure mouse and human SCs. It involves a brief (15-30 min) incubation on ice (0 °C) of a dish containing a heterogeneous mix of adherent muscle mononuclear cells, which leads to the detachment of only the SCs, and gives rise to cultures of superior purity compared to other commonly used isolation methods. The ICT method doubles up as a gentle passaging technique, allowing SC expansion over extended periods of time without compromising their proliferation and differentiation potential. Moreover, SCs isolated and expanded using the ICT method are capable of regenerating injured muscle in vivo. The ICT method involves minimal cell manipulation, does not require any expertise or expensive reagents, it is fast, and highly reproducible, and greatly reduces the number of animals or human biopsies required in order to obtain sufficient number of SCs. The cost-effectiveness, accessibility, and technical simplicity of this method, as well as its remarkable efficiency, will no doubt accelerate SC basic and translational research bringing their therapeutic use closer to the clinic.
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http://dx.doi.org/10.1186/s13395-021-00261-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7968259PMC
March 2021

Activation of skeletal muscle-resident glial cells upon nerve injury.

JCI Insight 2021 Apr 8;6(7). Epub 2021 Apr 8.

IRCCS Fondazione Santa Lucia, Rome, Italy.

Here, we report on the identification of Itga7-expressing muscle-resident glial cells activated by loss of neuromuscular junction (NMJ) integrity. Gene expression analysis at the bulk and single-cell level revealed that these cells are distinct from Itga7-expressing muscle satellite cells. We show that a selective activation and expansion of Itga7+ glial cells occur in response to muscle nerve lesion. Upon activation, muscle glial-derived progenies expressed neurotrophic genes, including nerve growth factor receptor, which enables their isolation by FACS. We show that activated muscle glial cells also expressed genes potentially implicated in extracellular matrix remodeling at NMJs. We found that tenascin C, which was highly expressed by muscle glial cells, activated upon nerve injury and preferentially localized to NMJ. Interestingly, we observed that the activation of muscle glial cells by acute nerve injury was reversible upon NMJ repair. By contrast, in a mouse model of ALS, in which NMJ degeneration is progressive, muscle glial cells steadily increased over the course of the disease. However, they exhibited an impaired neurotrophic activity, suggesting that pathogenic activation of glial cells may be implicated in ALS progression.
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http://dx.doi.org/10.1172/jci.insight.143469DOI Listing
April 2021

A Pound of Flesh: What Cachexia Is and What It Is Not.

Diagnostics (Basel) 2021 Jan 12;11(1). Epub 2021 Jan 12.

DAHFMO Unit of Histology and Medical Embryology, Sapienza University of Rome, 00161 Rome, Italy.

Body weight loss, mostly due to the wasting of skeletal muscle and adipose tissue, is the hallmark of the so-called cachexia syndrome. Cachexia is associated with several acute and chronic disease states such as cancer, chronic obstructive pulmonary disease (COPD), heart and kidney failure, and acquired and autoimmune diseases and also pharmacological treatments such as chemotherapy. The clinical relevance of cachexia and its impact on patients' quality of life has been neglected for decades. Only recently did the international community agree upon a definition of the term cachexia, and we are still awaiting the standardization of markers and tests for the diagnosis and staging of cancer-related cachexia. In this review, we discuss cachexia, considering the evolving use of the term for diagnostic purposes and the implications it has for clinical biomarkers, to provide a comprehensive overview of its biology and clinical management. Advances and tools developed so far for the in vitro testing of cachexia and drug screening will be described. We will also evaluate the nomenclature of different forms of muscle wasting and degeneration and discuss features that distinguish cachexia from other forms of muscle wasting in the context of different conditions.
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http://dx.doi.org/10.3390/diagnostics11010116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7828214PMC
January 2021

HDAC inhibitors tune miRNAs in extracellular vesicles of dystrophic muscle-resident mesenchymal cells.

EMBO Rep 2020 09 5;21(9):e50863. Epub 2020 Aug 5.

Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Fondazione Santa Lucia, Rome, Italy.

We show that extracellular vesicles (EVs) released by mesenchymal cells (i.e., fibro-adipogenic progenitors-FAPs) mediate microRNA (miR) transfer to muscle stem cells (MuSCs) and that exposure of dystrophic FAPs to HDAC inhibitors (HDACis) increases the intra-EV levels of a subset of miRs, which cooperatively target biological processes of therapeutic interest, including regeneration, fibrosis, and inflammation. Increased levels of miR-206 in EVs released by FAPs of muscles from Duchenne muscular dystrophy (DMD) patients or mdx mice exposed to HDACi are associated with enhanced regeneration and decreased fibrosis. Consistently, EVs from HDACi-treated dystrophic FAPs can stimulate MuSC activation and expansion ex vivo, and promote regeneration, while inhibiting fibrosis and inflammation of dystrophic muscles, upon intramuscular transplantation in mdx mice, in vivo. AntagomiR-mediated blockade of individual miRs reveals a specific requirement of miR-206 for EV-induced expansion of MuSCs and regeneration of dystrophic muscles, and indicates that cooperative activity of HDACi-induced miRs accounts for the net biological effect of these EVs. These data point to pharmacological modulation of EV content as novel strategy for therapeutic interventions in muscular dystrophies.
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http://dx.doi.org/10.15252/embr.202050863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7507515PMC
September 2020

Targeting PKCθ Promotes Satellite Cell Self-Renewal.

Int J Mol Sci 2020 Mar 31;21(7). Epub 2020 Mar 31.

Dept AHFMO, University of Rome "La Sapienza", Via A. Scarpa 14, 00161 Rome, Italy.

Skeletal muscle regeneration following injury depends on the ability of satellite cells (SCs) to proliferate, self-renew, and eventually differentiate. The factors that regulate the process of self-renewal are poorly understood. In this study we examined the role of PKCθ in SC self-renewal and differentiation. We show that PKCθ is expressed in SCs, and its active form is localized to the chromosomes, centrosomes, and midbody during mitosis. Lack of PKCθ promotes SC symmetric self-renewal division by regulating Pard3 polarity protein localization, without affecting the overall proliferation rate. Genetic ablation of PKCθ or its pharmacological inhibition in vivo did not affect SC number in healthy muscle. By contrast, after induction of muscle injury, lack or inhibition of PKCθ resulted in a significant expansion of the quiescent SC pool. Finally, we show that lack of PKCθ does not alter the inflammatory milieu after acute injury in muscle, suggesting that the enhanced self-renewal ability of SCs in PKCθ-/- mice is not due to an alteration in the inflammatory milieu. Together, these results suggest that PKCθ plays an important role in SC self-renewal by stimulating their expansion through symmetric division, and it may represent a promising target to manipulate satellite cell self-renewal in pathological conditions.
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http://dx.doi.org/10.3390/ijms21072419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7177808PMC
March 2020

Lack of PKCθ Promotes Regenerative Ability of Muscle Stem Cells in Chronic Muscle Injury.

Int J Mol Sci 2020 Jan 31;21(3). Epub 2020 Jan 31.

Department of AHFMO, University of Rome "la Sapienza", Via A. Scarpa 14, 00161 Rome, Italy.

Duchenne muscular dystrophy (DMD) is a genetic disease characterized by muscle wasting and chronic inflammation, leading to impaired satellite cells (SCs) function and exhaustion of their regenerative capacity. We previously showed that lack of PKCθ in mice, a mouse model of DMD, reduces muscle wasting and inflammation, and improves muscle regeneration and performance at early stages of the disease. In this study, we show that muscle regeneration is boosted, and fibrosis reduced in θ mice, even at advanced stages of the disease. This phenotype was associated with a higher number of Pax7 positive cells in θ muscle compared with muscle, during the progression of the disease. Moreover, the expression level of Pax7 and Notch1, the pivotal regulators of SCs self-renewal, were upregulated in SCs isolated from θ muscle compared with derived SCs. Likewise, the expression of the Notch ligands Delta1 and Jagged1 was higher in θ muscle compared with . The expression level of Delta1 and Jagged1 was also higher in PKCθ muscle compared with WT muscle following acute injury. In addition, lack of PKCθ prolonged the survival and sustained the differentiation of transplanted myogenic progenitors. Overall, our results suggest that lack of PKCθ promotes muscle repair in dystrophic mice, supporting stem cells survival and maintenance through increased Delta-Notch signaling.
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http://dx.doi.org/10.3390/ijms21030932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037041PMC
January 2020

Splenic Ly6Chi monocytes are critical players in dystrophic muscle injury and repair.

JCI Insight 2020 01 30;5(2). Epub 2020 Jan 30.

Dystrophic muscle is characterized by chronic injury and a steady recruitment of inflammatory Ly6Chi monocytes. Recent studies have identified the spleen as the dominant reservoir of these cells during chronic inflammation. Here, we investigated the contribution of splenic Ly6Chi monocytes to dystrophic muscle pathology. Using the mdx mouse model of muscular dystrophy, we show that Ly6Chi monocytes accumulate in great numbers in the spleen over the course of the disease. The chemokine receptor CCR2 was upregulated on Ly6Chi monocytes in mdx spleen before disease onset, thereby enabling their recruitment to dystrophic muscle. Splenectomy performed before disease onset significantly reduced the number of Ly6Chi monocytes infiltrating dystrophic limb muscle. Moreover, in the absence of splenic Ly6Chi monocytes there was a significant reduction in dystrophic muscle inflammation and necrosis, along with improved regeneration during early disease. However, during late disease, a lack of splenic Ly6Chi monocytes adversely affected muscle fiber repair, due to a delay in the phenotypic shift of proinflammatory F4/80+Ly6ChiCD206lo to antiinflammatory F4/80+Ly6CloCD206+ macrophages. Overall, we show that the spleen is an indispensable source of Ly6Chi monocytes in muscular dystrophy and that splenic monocytes are critical players in both muscle fiber injury and repair.
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http://dx.doi.org/10.1172/jci.insight.130807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7098719PMC
January 2020

Macrophages fine tune satellite cell fate in dystrophic skeletal muscle of mdx mice.

PLoS Genet 2019 10 18;15(10):e1008408. Epub 2019 Oct 18.

Institute of Biochemistry and Cell Biology (IBBC), National Research Council (CNR), Rome, Italy.

Satellite cells (SCs) are muscle stem cells that remain quiescent during homeostasis and are activated in response to acute muscle damage or in chronic degenerative conditions such as Duchenne Muscular Dystrophy. The activity of SCs is supported by specialized cells which either reside in the muscle or are recruited in regenerating skeletal muscles, such as for instance macrophages (MΦs). By using a dystrophic mouse model of transient MΦ depletion, we describe a shift in identity of muscle stem cells dependent on the crosstalk between MΦs and SCs. Indeed MΦ depletion determines adipogenic conversion of SCs and exhaustion of the SC pool leading to an exacerbated dystrophic phenotype. The reported data could also provide new insights into therapeutic approaches targeting inflammation in dystrophic muscles.
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http://dx.doi.org/10.1371/journal.pgen.1008408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821135PMC
October 2019

Skeletal Muscle: A Significant Novel Neurohypophyseal Hormone-Secreting Organ.

Front Physiol 2018 8;9:1885. Epub 2019 Jan 8.

Section of Histology & Medical Embryology, Department of Anatomical, Histological, Forensic and Orthopedic Sciences, Interuniversity Institute of Myology, Sapienza University of Rome, Rome, Italy.

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http://dx.doi.org/10.3389/fphys.2018.01885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331439PMC
January 2019

Do neurogenic and cancer-induced muscle atrophy follow common or divergent paths?

Eur J Transl Myol 2018 Nov 13;28(4):7931. Epub 2018 Dec 13.

IRCCS, Fondazione Santa Lucia, Rome, Italy.

Skeletal muscle is a dynamic tissue capable of responding to a large variety of physiological stimuli by adjusting muscle fiber size, metabolism and function. However, in pathological conditions such as cancer and neural disorders, this finely regulated homeostasis is impaired leading to severe muscle wasting, reduced muscle fiber size (atrophy), and impaired function. These disease features develop due to enhanced protein breakdown, which relies on two major degradation systems: the ubiquitin-proteasome and the autophagy-lysosome. These systems are independently regulated by different signalling pathways, which in physiological conditions, determine protein and organelle turnover. However, alterations in one or both systems, as it happens in several disorders, leads to enhanced protein breakdown and muscle atrophy. Although this is a common feature in the different types of muscle atrophy, the relative contribution of each of these systems is still under debate. Here, we will briefly describe the regulation and the activity of the ubiquitin-proteasome and the autophagy-lysosome systems during muscle wasting. We will then discuss what we know regarding how these pathways are involved in cancer induced and in neurogenic muscle atrophy, highlighting common and divergent paths. It is now clear that there is no one unifying common mechanism that can be applied to all models of muscle loss. Detailed understanding of the pathways and proteolysis mechanisms involved in each model will hopefully help the development of drugs to counteract muscle wasting in specific conditions.
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http://dx.doi.org/10.4081/ejtm.2018.7931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6317130PMC
November 2018

Culture conditions influence satellite cell activation and survival of single myofibers.

Eur J Transl Myol 2018 Apr 29;28(2):7567. Epub 2018 May 29.

DAHFMO Unit of Histology and Medical Embryology, InterUniversity Institute of Myology, Sapienza University of Rome, Italy.

Single myofiber isolation protocols allow to obtain an in vitro system in which the physical association between the myofiber and its stem cells, the satellite cells, is adequately preserved. This technique is an indispensable tool by which the muscle regeneration process can be recapitulated and studied in each specific phase, from satellite cell activation to proliferation, from differentiation to fusion. This study aims to clarify the effect of different culture conditions on single myofibers, their associated satellite cells, and the physiological behavior of the satellite cells upon long term culture. By direct observations of the cultures, we compared different experimental conditions and their effect on both satellite cell behavior and myofiber viability.
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http://dx.doi.org/10.4081/ejtm.2018.7567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6036316PMC
April 2018

Disruption of MEK/ERK/c-Myc signaling radiosensitizes prostate cancer cells in vitro and in vivo.

J Cancer Res Clin Oncol 2018 Sep 29;144(9):1685-1699. Epub 2018 Jun 29.

Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, Via Vetoio, Coppito 2, 67100, L'Aquila, Italy.

Purpose: Prostate cancer (PCa) cell radioresistance causes the failure of radiation therapy (RT) in localized or locally advanced disease. The aberrant accumulation of c-Myc oncoprotein, known to promote PCa onset and progression, may be due to the control of gene transcription and/or MEK/ERK-regulated protein stabilization. Here, we investigated the role of MEK/ERK signaling in PCa.

Methods: LnCAP, 22Rv1, DU145, and PC3 PCa cell lines were used in in vitro and in vivo experiments. U0126, trametinib MEK/ERK inhibitors, and c-Myc shRNAs were used. Radiation was delivered using an x-6 MV photon linear accelerator. U0126 in vivo activity alone or in combination with irradiation was determined in murine xenografts.

Results: Inhibition of MEK/ERK signaling down-regulated c-Myc protein in PCa cell lines to varying extents by affecting expression of RNA and protein, which in turn determined radiosensitization in in vitro and in vivo xenograft models of PCa cells. The crucial role played by c-Myc in the MEK/ERK pathways was demonstrated in 22Rv1 cells by the silencing of c-Myc by means of short hairpin mRNA, which yielded effects resembling the targeting of MEK/ERK signaling. The clinically approved compound trametinib used in vitro yielded the same effects as U0126 on growth and C-Myc expression. Notably, U0126 and trametinib induced a drastic down-regulation of BMX, which is known to prevent apoptosis in cancer cells.

Conclusions: The results of our study suggest that signal transduction-based therapy can, by disrupting the MEK/ERK/c-Myc axis, reduce human PCa radioresistance caused by increased c-Myc expression in vivo and in vitro and restores apoptosis signals.
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http://dx.doi.org/10.1007/s00432-018-2696-3DOI Listing
September 2018

Targeting early PKCθ-dependent T-cell infiltration of dystrophic muscle reduces disease severity in a mouse model of muscular dystrophy.

J Pathol 2018 03 9;244(3):323-333. Epub 2018 Jan 9.

Department of Anatomical, Histological, Forensic Medicine and Orthopedic Sciences (DAHFMO), Sapienza University of Rome, Rome, Italy.

Chronic muscle inflammation is a critical feature of Duchenne muscular dystrophy and contributes to muscle fibre injury and disease progression. Although previous studies have implicated T cells in the development of muscle fibrosis, little is known about their role during the early stages of muscular dystrophy. Here, we show that T cells are among the first cells to infiltrate mdx mouse dystrophic muscle, prior to the onset of necrosis, suggesting an important role in early disease pathogenesis. Based on our comprehensive analysis of the kinetics of the immune response, we further identify the early pre-necrotic stage of muscular dystrophy as the relevant time frame for T-cell-based interventions. We focused on protein kinase C θ (PKCθ, encoded by Prkcq), a critical regulator of effector T-cell activation, as a potential target to inhibit T-cell activity in dystrophic muscle. Lack of PKCθ not only reduced the frequency and number of infiltrating T cells but also led to quantitative and qualitative changes in the innate immune cell infiltrate in mdx/Prkcq muscle. These changes were due to the inhibition of T cells, since PKCθ was necessary for T-cell but not for myeloid cell infiltration of acutely injured muscle. Targeting T cells with a PKCθ inhibitor early in the disease process markedly diminished the size of the inflammatory cell infiltrate and resulted in reduced muscle damage. Moreover, diaphragm necrosis and fibrosis were also reduced following treatment. Overall, our findings identify the early T-cell infiltrate as a therapeutic target and highlight the potential of PKCθ inhibition as a therapeutic approach to muscular dystrophy. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.5016DOI Listing
March 2018

Muscle Expression of SOD1 Triggers the Dismantlement of Neuromuscular Junction via PKC-Theta.

Antioxid Redox Signal 2018 04 30;28(12):1105-1119. Epub 2017 Oct 30.

1 Center for Life Nano Science at Sapienza , Istituto Italiano di Tecnologia, Rome, Italy .

Aim: Neuromuscular junction (NMJ) represents the morphofunctional interface between muscle and nerve. Several chronic pathologies such as aging and neurodegenerative diseases, including muscular dystrophy and amyotrophic lateral sclerosis, display altered NMJ and functional denervation. However, the triggers and the molecular mechanisms underlying the dismantlement of NMJ remain unclear.

Results: Here we provide evidence that perturbation in redox signaling cascades, induced by muscle-specific accumulation of mutant SOD1 in transgenic MLC/SOD1 mice, is causally linked to morphological alterations of the neuromuscular presynaptic terminals, high turnover rate of acetylcholine receptor, and NMJ dismantlement. The analysis of potential molecular mechanisms that mediate the toxic activity of SOD1 revealed a causal link between protein kinase Cθ (PKCθ) activation and NMJ disintegration.

Innovation: The study discloses the molecular mechanism that triggers functional denervation associated with the toxic activity of muscle SOD1 expression and suggests the possibility of developing a new strategy to counteract age- and pathology-associated denervation based on pharmacological inhibition of PKCθ activity.

Conclusions: Collectively, these data indicate that muscle-specific accumulation of oxidative damage can affect neuromuscular communication and induce NMJ dismantlement through a PKCθ-dependent mechanism. Antioxid. Redox Signal. 28, 1105-1119.
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http://dx.doi.org/10.1089/ars.2017.7054DOI Listing
April 2018

Phosphotyrosine phosphatase inhibitor bisperoxovanadium endows myogenic cells with enhanced muscle stem cell functions via epigenetic modulation of Sca-1 and Pw1 promoters.

FASEB J 2016 Apr 15;30(4):1404-15. Epub 2015 Dec 15.

*Stem Cells and Regenerative Medicine, Institute of Cardiometabolism and Nutrition Unité Mixte de Recherche en Santé 1166 INSERM/Sorbonne University (Pierre and Marie Curie University, Paris VI), Paris, France; Department of Anatomy, Histology, Forensic Medicine, and Orthopedics, Unit of Histology, Sapienza University of Rome, Rome, Italy; INSERM Unité 955 Institut Mondor de Recherche Biomédicale, Creteil, France; Université Paris-Est Créteil, Faculty of Medicine, Creteil, France; Sorbonne Universités, Pierre and Marie Curie University, Paris VI, INSERM Unité Mixte de Recherche en Santé 974, Centre National de la Recherche Scientifique FRE3617, Center for Research in Myology, Paris, France; Etablissement Français du Sang, Creteil, France; and Université Paris Est, Ecole Nationale Veterinaire d'Alfort, Maison Alfort, France

Understanding the regulation of the stem cell fate is fundamental for designing novel regenerative medicine strategies. Previous studies have suggested that pharmacological treatments with small molecules provide a robust and reversible regulation of the stem cell program. Previously, we showed that treatment with a vanadium compound influences muscle cell fatein vitro In this study, we demonstrate that treatment with the phosphotyrosine phosphatase inhibitor bisperoxovanadium (BpV) drives primary muscle cells to a poised stem cell stage, with enhanced function in muscle regenerationin vivofollowing transplantation into injured muscles. Importantly, BpV-treated cells displayed increased self-renewal potentialin vivoand replenished the niche in both satellite and interstitial cell compartments. Moreover, we found that BpV treatment induces specific activating chromatin modifications at the promoter regions of genes associated with stem cell fate, includingSca-1andPw1 Thus, our findings indicate that BpV resets the cell fate program by specific epigenetic regulations, such that the committed myogenic cell fate is redirected to an earlier progenitor cell fate stage, which leads to an enhanced regenerative stem cell potential.-Smeriglio, P., Alonso-Martin, S., Masciarelli, S., Madaro, L., Iosue, I., Marrocco, V., Relaix, F., Fazi, F., Marazzi, G., Sassoon, D. A., Bouché, M. Phosphotyrosine phosphatase inhibitor bisperoxovanadium endows myogenic cells with enhanced muscle stem cell functionsviaepigenetic modulation of Sca-1 and Pw1 promoters.
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http://dx.doi.org/10.1096/fj.15-275420DOI Listing
April 2016

Cavin-1 and Caveolin-1 are both required to support cell proliferation, migration and anchorage-independent cell growth in rhabdomyosarcoma.

Lab Invest 2015 Jun 30;95(6):585-602. Epub 2015 Mar 30.

1] Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy [2] Interuniversity Institute of Myology (IIM), Rome, Italy.

Rhabdomyosarcoma (RMS) is a childhood soft tissue tumor with broad expression of markers that are typically found in skeletal muscle. Cavin-1 is a recently discovered protein actively cooperating with Caveolin-1 (Cav-1) in the morphogenesis of caveolae and whose role in cancer is drawing increasing attention. Using a combined in silico and in vitro analysis here we show that Cavin-1 is expressed in myogenic RMS tumors as well as in human and primary mouse RMS cultures, exhibiting a broad subcellular localization, ranging from nuclei and cytosol to plasma membrane. In particular, the coexpression and plasma membrane interaction between Cavin-1 and Cav-1 characterized the proliferation of human and mouse RMS cell cultures, while a downregulation of their expression levels was observed during the myogenic differentiation. Knockdown of Cavin-1 or Cav-1 in the human RD and RH30 cells led to impairment of cell proliferation and migration. Moreover, loss of Cavin-1 in RD cells impaired the anchorage-independent cell growth in soft agar. While the loss of Cavin-1 did not affect the Cav-1 protein levels in RMS cells, Cav-1 overexpression and knockdown triggered a rise or depletion of Cavin-1 protein levels in RD cells, respectively, in turn reflecting on increased or decreased cell proliferation, migration and anchorage-independent cell growth. Collectively, these data indicate that the interaction between Cavin-1 and Cav-1 underlies the cell growth and migration in myogenic tumors.
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http://dx.doi.org/10.1038/labinvest.2015.45DOI Listing
June 2015

Targeting PKCθ in skeletal muscle and muscle diseases: good or bad?

Biochem Soc Trans 2014 Dec;42(6):1550-5

*DAHFMO, Unit of Histology and Medical Embryology, Sapienza University of Rome, Via A. Scarpa 14, 00161 Rome, Italy.

Protein kinase Cθ (PKCθ) is a member of the novel calcium-independent PKC family, with a relatively selective tissue distribution. Most studies have focused on its unique role in T-lymphocyte activation and suggest that inhibition of PKCθ could represent a novel therapeutic approach in the treatment of chronic inflammation, autoimmunity and allograft rejection. However, considering that PKCθ is also expressed in other cell types, including skeletal muscle cells, it is important to understand its function in different tissues before proposing it as a molecular target for the treatment of immune-mediated diseases. A number of studies have highlighted the role of PKCθ in mediating several intracellular pathways, regulating muscle cell development, homoeostasis and remodelling, although a comprehensive picture is still lacking. Moreover, we recently showed that lack of PKCθ in a mouse model of Duchenne muscular dystrophy (DMD) ameliorates the progression of the disease. In the present article, we review new developments in our understanding of the involvement of PKCθ in intracellular mechanisms regulating skeletal muscle development, growth and maintenance under physiological conditions and recent advances showing a hitherto unrecognized role of PKCθ in promoting muscular dystrophy.
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http://dx.doi.org/10.1042/BST20140207DOI Listing
December 2014

Inflammation in muscle repair, aging, and myopathies.

Biomed Res Int 2014 4;2014:821950. Epub 2014 Aug 4.

B2A Biology of Adaptation and Aging, Pierre and Marie Curie University (Paris 6), 7 quai St Bernard, 75252 Paris Cedex 5, France.

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http://dx.doi.org/10.1155/2014/821950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4137693PMC
May 2015

From innate to adaptive immune response in muscular dystrophies and skeletal muscle regeneration: the role of lymphocytes.

Biomed Res Int 2014 16;2014:438675. Epub 2014 Jun 16.

DAHFMO, Unit of Histology and Medical Embryology, Sapienza University of Rome, Via Antonio Scarpa 14, 00161 Rome, Italy.

Skeletal muscle is able to restore contractile functionality after injury thanks to its ability to regenerate. Following muscle necrosis, debris is removed by macrophages, and muscle satellite cells (MuSCs), the muscle stem cells, are activated and subsequently proliferate, migrate, and form muscle fibers restoring muscle functionality. In most muscle dystrophies (MDs), MuSCs fail to properly proliferate, differentiate, or replenish the stem cell compartment, leading to fibrotic deposition. However, besides MuSCs, interstitial nonmyogenic cells and inflammatory cells also play a key role in orchestrating muscle repair. A complete understanding of the complexity of these mechanisms should allow the design of interventions to attenuate MDs pathology without disrupting regenerative processes. In this review we will focus on the contribution of immune cells in the onset and progression of MDs, with particular emphasis on Duchenne muscular dystrophy (DMD). We will briefly summarize the current knowledge and recent advances made in our understanding of the involvement of different innate immune cells in MDs and will move on to critically evaluate the possible role of cell populations within the acquired immune response. Revisiting previous observations in the light of recent evidence will likely change our current view of the onset and progression of the disease.
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http://dx.doi.org/10.1155/2014/438675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4083765PMC
March 2015

Protein kinase C theta (PKCθ) modulates the ClC-1 chloride channel activity and skeletal muscle phenotype: a biophysical and gene expression study in mouse models lacking the PKCθ.

Pflugers Arch 2014 Dec 20;466(12):2215-28. Epub 2014 Mar 20.

Section of Pharmacology, Department of Pharmacy & Drug Sciences, University of Bari - Aldo Moro, 70125, Bari, Italy.

In skeletal muscle, the resting chloride conductance (gCl), due to the ClC-1 chloride channel, controls the sarcolemma electrical stability. Indeed, loss-of-function mutations in ClC-1 gene are responsible of myotonia congenita. The ClC-1 channel can be phosphorylated and inactivated by protein kinases C (PKC), but the relative contribution of each PKC isoforms is unknown. Here, we investigated on the role of PKCθ in the regulation of ClC-1 channel expression and activity in fast- and slow-twitch muscles of mouse models lacking PKCθ. Electrophysiological studies showed an increase of gCl in the PKCθ-null mice with respect to wild type. Muscle excitability was reduced accordingly. However, the expression of the ClC-1 channel, evaluated by qRT-PCR, was not modified in PKCθ-null muscles suggesting that PKCθ affects the ClC-1 activity. Pharmacological studies demonstrated that although PKCθ appreciably modulates gCl, other isoforms are still active and concur to this role. The modification of gCl in PKCθ-null muscles has caused adaptation of the expression of phenotype-specific genes, such as calcineurin and myocyte enhancer factor-2, supporting the role of PKCθ also in the settings of muscle phenotype. Importantly, the lack of PKCθ has prevented the aging-related reduction of gCl, suggesting that its modulation may represent a new strategy to contrast the aging process.
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http://dx.doi.org/10.1007/s00424-014-1495-1DOI Listing
December 2014

Knock down of caveolin-1 affects morphological and functional hallmarks of human endothelial cells.

J Cell Biochem 2013 Aug;114(8):1843-51

Department of Anatomy, Histology, Forensic Medicine and Orthopedics, Section of Histology and Medical Embryology, Sapienza University of Rome, Rome, Italy.

Caveolin-1 (CAV1) is the principal structural component of caveolae which functions as scaffolding protein for the integration of a variety of signaling pathways. In this study, we investigated the involvement of CAV1 in endothelial cell (EC) functions and show that siRNA-induced CAV1 silencing in the human EC line EA.hy926 induces distinctive morphological changes, such as a marked increase in cell size and formation of stress fibers. Design-based stereology was employed in this work to make unbiased quantification of morphometric properties such as volume, length, and surface of CAV1 silenced versus control cells. In addition, we showed that downregulation of CAV1 affects cell cycle progression at G1/S phase transition most likely by perturbation of AKT signaling. With the aim to assess the contribution of CAV1 to typical biological processes of EC, we report here that CAV1 targeting affects cell migration and matrix metalloproteinases (MMPs) activity, and reduces angiogenesis in response to VEGF, in vitro. Taken together our data suggest that the proper expression of CAV1 is important not only for maintaining the appropriate morphology and size of ECs but it might represent a prospective molecular target for studying key biological mechanisms such as senescence and tumorigenesis.
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http://dx.doi.org/10.1002/jcb.24526DOI Listing
August 2013

Intracellular signaling in ER stress-induced autophagy in skeletal muscle cells.

FASEB J 2013 May 6;27(5):1990-2000. Epub 2013 Feb 6.

DAHFMO, Unit of Histology, Sapienza University of Rome, Via A. Scarpa 14, 00161 Rome, Italy.

Skeletal muscle remodeling in response to muscle disuse and unloading is known to be associated with so-called ER stress, which, in turn, activates autophagy and contributes to muscle atrophy. Different molecules are involved in ER stress-induced autophagy, among which PKCθ has recently been described. In this study, we dissected both in vitro and in vivo ER stress-induced autophagy pathways in muscle. Using C2C12 muscle cells in culture, we demonstrated that PKC activation induced autophagy in the absence of ER stress. We further demonstrated that PKCθ was strongly activated in cultured myoblasts and myotubes during ER stress induced by different stimuli, such as TG or TN treatment, and that it localized into Lc3-positive autophagic dots upon TG treatment. Neither Akt dephosphorylation nor Foxo or GSK3β activation was observed in these conditions. Moreover, PKCθ inhibition in myoblasts and myotubes prevented ER stress-induced Lc3 activation and autophagic dot formation, but not ER stress. In vivo, lack of PKCθ prevented both food deprivation- and immobilization-induced autophagy and muscle atrophy, irrespective of Akt pathway inhibition. Taken together, these results demonstrate that PKCθ functions as an ER stress sensor in skeletal muscle, required for ER-stress-dependent autophagy activation, and can be proposed as a novel molecular target to maintain muscle homeostasis in response to external stimuli, such as disuse and unloading, still allowing intracellular clearance.
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http://dx.doi.org/10.1096/fj.12-215475DOI Listing
May 2013

PKC theta ablation improves healing in a mouse model of muscular dystrophy.

PLoS One 2012 14;7(2):e31515. Epub 2012 Feb 14.

Unit of Histology, and IIM, Sapienza University, DAHFMO, Rome, Italy.

Inflammation is a key pathological characteristic of dystrophic muscle lesion formation, limiting muscle regeneration and resulting in fibrotic and fatty tissue replacement of muscle, which exacerbates the wasting process in dystrophic muscles. Limiting immune response is thus one of the therapeutic options to improve healing, as well as to improve the efficacy of gene- or cell-mediated strategies to restore dystrophin expression. Protein kinase C θ (PKCθ) is a member of the PKCs family highly expressed in both immune cells and skeletal muscle; given its crucial role in adaptive, but also innate, immunity, it is being proposed as a valuable pharmacological target for immune disorders. In our study we asked whether targeting PKCθ could represent a valuable approach to efficiently prevent inflammatory response and disease progression in a mouse model of muscular dystrophy. We generated the bi-genetic mouse model mdx/θ(-/-), where PKCθ expression is lacking in mdx mice, the mouse model of Duchenne muscular dystrophy. We found that muscle wasting in mdx/θ(-/-) mice was greatly prevented, while muscle regeneration, maintenance and performance was significantly improved, as compared to mdx mice. This phenotype was associated to reduction in inflammatory infiltrate, pro-inflammatory gene expression and pro-fibrotic markers activity, as compared to mdx mice. Moreover, BM transplantation experiments demonstrated that the phenotype observed was primarily dependent on lack of PKCθ expression in hematopoietic cells.These results demonstrate a hitherto unrecognized role of immune-cell intrinsic PKCθ activity in the development of DMD. Although the immune cell population(s) involved remain unidentified, our findings reveal that PKCθ can be proposed as a new pharmacological target to counteract the disease, as well as to improve the efficacy of gene- or cell- therapy approaches.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0031515PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3279361PMC
July 2012

Thyroid hormone T3 counteracts STZ induced diabetes in mouse.

PLoS One 2011 27;6(5):e19839. Epub 2011 May 27.

Dipartimento di Medicina Sperimentale, Sapienza Università di Roma, Roma, Italy.

This study intended to demonstrate that the thyroid hormone T3 counteracts the onset of a Streptozotocin (STZ) induced diabetes in wild type mice. To test our hypothesis diabetes has been induced in Balb/c male mice by multiple low dose Streptozotocin injection; and a group of mice was contemporaneously injected with T3. After 48 h mice were tested for glucose tolerance test, insulin serum levels and then sacrificed. Whole pancreata were utilized for morphological and biochemical analyses, while protein extracts and RNA were utilized for expression analyses of specific molecules. The results showed that islets from T3 treated mice were comparable to age- and sex-matched control, untreated mice in number, shape, dimension, consistency, ultrastructure, insulin and glucagon levels, Tunel positivity and caspases activation, while all the cited parameters and molecules were altered by STZ alone. The T3-induced pro survival effect was associated with a strong increase in phosphorylated Akt. Moreover, T3 administration prevented the STZ-dependent alterations in glucose blood level, both during fasting and after glucose challenge, as well as in insulin serum level. In conclusion we demonstrated that T3 could act as a protective factor against STZ induced diabetes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0019839PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3103518PMC
September 2011

Synthetic sulfonyl-hydrazone-1 positively regulates cardiomyogenic microRNA expression and cardiomyocyte differentiation of induced pluripotent stem cells.

J Cell Biochem 2011 Aug;112(8):2006-14

Translational Cardiomyology, Stem Cell Institute, K.U.Leuven, Leuven, Belgium.

Induced pluripotent stem cells (iPSCs) are obtained from adult cells through overexpression of pluripotency factors. iPSCs share many features with embryonic stem cells (ESCs), circumventing ethical issues, and, noteworthy, match donor's genotype. iPSCs represent therefore a valuable tool for regenerative medicine. Cardiac differentiation of ESCs can be enhanced via microRNAs (miRNAs) and small chemical compounds, which probably act as chromatin remodelers. Cardiomyogenic potential of iPSCs is currently intensely investigated for cell therapy or in vitro drug screening and disease modeling. However, influences of small compounds on iPSC-related cardiomyogenesis have not yet been investigated in details. Here, we compared the effects of two small molecules, bis-peroxo-vanadium (bpV) and sulfonyl-hydrazone-1 (SHZ) at varying concentrations, during cardiac differentiation of murine iPSCs. SHZ (5 µM) enhanced specific marker expression and cardiomyocyte yield, without loss of cell viability. In contrast, bpV showed negligible effects on cardiac differentiation rate and appeared to induce Casp3-dependent apoptosis in differentiating iPSCs. Furthermore, SHZ-treated iPSCs were able to increase beating foci rate and upregulate early and late cardiomyogenic miRNA expression (miR-1, miR-133a, and miR-208a). Thus, our results demonstrate that small chemical compounds, such as SHZ, can constitute a novel and clinically feasible strategy to improve iPSC-derived cardiac differentiation.
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http://dx.doi.org/10.1002/jcb.23118DOI Listing
August 2011

PKCθ signaling is required for myoblast fusion by regulating the expression of caveolin-3 and β1D integrin upstream focal adhesion kinase.

Mol Biol Cell 2011 Apr 23;22(8):1409-19. Epub 2011 Feb 23.

Department of Anatomy, Sapienza University of Rome, Rome, Italy.

Fusion of mononucleated myoblasts to form multinucleated myofibers is an essential phase of skeletal myogenesis, which occurs during muscle development as well as during postnatal life for muscle growth, turnover, and regeneration. Many cell adhesion proteins, including integrins, have been shown to be important for myoblast fusion in vertebrates, and recently focal adhesion kinase (FAK), has been proposed as a key mediator of myoblast fusion. Here we focused on the possible role of PKC, the PKC isoform predominantly expressed in skeletal muscle, in myoblast fusion. We found that the expression of PKC is strongly up-regulated following freeze injury-induced muscle regeneration, as well as during in vitro differentiation of satellite cells (SCs; the muscle stem cells). Using both PKC knockout and muscle-specific PKC dominant-negative mutant mouse models, we observed delayed body and muscle fiber growth during the first weeks of postnatal life, when compared with wild-type (WT) mice. We also found that myofiber formation, during muscle regeneration after freeze injury, was markedly impaired in PKC mutant mice, as compared with WT. This phenotype was associated with reduced expression of the myogenic differentiation program executor, myogenin, but not with that of the SC marker Pax7. Indeed in vitro differentiation of primary muscle-derived SCs from PKC mutants resulted in the formation of thinner myotubes with reduced numbers of myonuclei and reduced fusion rate, when compared with WT cells. These effects were associated to reduced expression of the profusion genes caveolin-3 and β1D integrin and to reduced activation/phosphorylation of their up-stream regulator FAK. Indeed the exogenous expression of a constitutively active mutant form of PKC in muscle cells induced FAK phosphorylation. Moreover pharmacologically mediated full inhibition of FAK activity led to similar fusion defects in both WT and PKC-null myoblasts. We thus propose that PKC signaling regulates myoblast fusion by regulating, at least in part, FAK activity, essential for profusion gene expression.
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http://dx.doi.org/10.1091/mbc.E10-10-0821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3078083PMC
April 2011

Transgenic mice with dominant negative PKC-theta in skeletal muscle: a new model of insulin resistance and obesity.

J Cell Physiol 2003 Jul;196(1):89-97

Department of Histology and Medical Embryology, University of Rome La Sapienza, Rome, Italy.

Protein kinase C theta (PKC-theta) is the PKC isoform predominantly expressed in skeletal muscle, and it is supposed to mediate many signals necessary for muscle histogenesis and homeostasis, such as TGFbeta, nerve-dependent signals and insulin. To study the role of PKC-theta in these mechanisms we generated transgenic mice expressing a "kinase dead" mutant form of PKC-theta (PKC-thetaK/R), working as "dominant negative," specifically in skeletal muscle. These mice are viable and fertile, however, by the 6-7 months of age, they gain weight, mainly due to visceral fat deposition. Before the onset of obesity (4 months of age), they already show increased fasting and fed insulin levels and reduced insulin-sensitivity, as measured by ipITT, but normal glucose tolerance, as measured by ipGTT. After the 6-7 months of age, transgenic mice develop hyperinsulinemia in the fasting and fed state. The ipGTT revealed in the transgenic mice both hyperglycemia and hyperinsulinemia. At the molecular level, impaired activation of the IR/IRS/PI3K pathway and a significant decrease both in the levels and in insulin-stimulated activation of the serine/threonine kinase Akt were observed. Taken together these data demonstrate that over-expression of dominant negative PKC-theta in skeletal muscle causes obesity associated to insulin resistance, as demonstrated by defective receptor and post-receptorial activation of signaling cascade.
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http://dx.doi.org/10.1002/jcp.10278DOI Listing
July 2003

The block of ryanodine receptors selectively inhibits fetal myoblast differentiation.

J Cell Sci 2003 Apr;116(Pt 8):1589-97

Department of Histology and Medical Embryology, University of Rome 'La Sapienza', 00161 Rome, Italy.

Differentiation and morphogenesis of skeletal muscle are complex and asynchronous events that involve various myogenic cell populations and extracellular signals. Embryonic and fetal skeletal myoblasts are responsible for the formation of primary and secondary fibers, respectively, although the mechanism that diversifies their fate is not fully understood. Calcium transients appear to be a signaling mechanism that is widely utilized in differentiation and embryogenesis. In mature skeletal muscle, calcium transients are generated mainly by ryanodine receptors (type 1 and type 3), which are involved in excitation-contraction coupling. However, it is not clear whether the activity of these receptors is important for contractile activity alone or whether it may also play a role in regulating the differentiation/developmental processes. To clarify this point, we first examined the expression of the receptors during development. The results show that the expression of both receptors appears as early as E13 during limb muscle development and parallels the expression of skeletal myosin. The expression and the activity of both receptors is maintained in vitro by all myogenic cell populations isolated from different stages of development, including somitic, embryonic and fetal myoblasts and satellite cells. Blocking ryanodine receptor activity by using ryanodine inhibits in vitro differentiation of fetal myoblasts (judged by the expression of sarcomeric myosin and formation of multinucleated myotubes) but not of somitic or embryonic and satellite muscle cells. This block is caused by the transcriptional inhibition of markers characteristic of terminal differentiation, rather than commitment, as the expression of muscle regulatory factors is not impaired by ryanodine treatment. Taken together, the data reported in this paper demonstrate that, although calcium transients represent a general mechanism for the control of differentiation and development, multiple calcium-dependent pathways may be relevant in different myogenic populations during development. Moreover, since fetal myoblasts are responsible for the formation of secondary fibers during development, and therefore for the building of the bulk of muscular mass, these results suggest that calcium release from ryanodine receptors plays a role in the histogenesis of mammalian skeletal muscle.
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http://dx.doi.org/10.1242/jcs.00358DOI Listing
April 2003