Modulation of cAMP/cGMP signaling as prevention of congenital heart defects in Pde2A deficient embryos: a matter of oxidative stress.

Phosphodiesterase 2A (Pde2A) is a dual-specific PDE that breaks down both cAMP and cGMP cyclic nucleotides. We recently highlighted a direct relationship between Pde2A impairment, a consequent increase of cAMP, and the appearance of mouse congenital heart defects (CHDs). Here we aimed to characterize the pathways involved in the development of CHDs and in their prevention by pharmacological approaches targeting cAMP and cGMP signaling. Transcriptome analysis revealed a modulation of more than 500 genes affecting biological processes involved in the immune system, cardiomyocyte development and contractility, angiogenesis, transcription, and oxidative stress in hearts from Pde2A-/- embryos. Metoprolol and H89 pharmacological administration prevented heart dilatation and hypertabeculation in Pde2A-/- embryos. Metoprolol was also able to partially impede heart septum defect and oxidative stress at tissue and molecular levels. Amelioration of cardiac defects was also observed by using the antioxidant NAC, indicating oxidative stress as one of the molecular mechanisms underpinning the CHDs. In addition, Sildenafil treatment recovered cardiac defects suggesting the requirement of cAMP/cGMP nucleotides balance for the correct heart development.

Physiological determinants of mechanical efficiency during advanced ageing and disuse.

This study aimed to determine which physiological factors impact net efficiency (ηnet) in oldest-old individuals at different stages of skeletal muscle disuse. To this aim, we examined ηnet, central haemodynamics, peripheral circulation, and peripheral factors (skeletal muscle fibre type, capillarization and concentration of mitochondrial DNA [mtDNA]). Twelve young (YG; 25 ± 2 years), 12 oldest-old mobile (OM; 87 ± 3 years), and 12 oldest-old immobile (OI; 88 ± 4 years) subjects performed dynamic knee extensor (KE) and elbow flexors (EF) exercise. Pulmonary oxygen uptake, photoplethysmography, Doppler ultrasound and muscle biopsies of the vastus lateralis and biceps brachii were used to assess central and peripheral adaptations to advanced ageing and disuse. Compared to the YG (12.1 ± 2.4%), the ηnet of lower-limb muscle was higher in the OM (17.6 ± 3.5%, P < 0.001), and lower in the OI (8.9 ± 1.9%, P < 0.001). These changes in ηnet during KE were coupled with significant peripheral adaptations, revealing strong correlations between ηnet and the proportion of type I muscle fibres (r = 0.82), as well as [mtDNA] (r = 0.77). No differences in ηnet were evident in the upper-limb muscles between YG, OM and OI. In view of the differences in limb-specific activity across the lifespan, these findings suggest that ηnet is reduced by skeletal muscle inactivity and not by chronological age, per se. Likewise, this study revealed that the age-related changes in ηnet are not a consequence of central or peripheral haemodynamic adaptations, but are likely a product of peripheral changes related to skeletal muscle fibre type and mitochondrial density. KEY POINTS: Although the effects of ageing and muscle disuse deeply impact the cardiovascular and skeletal muscle function, the combination of these factors on the mechanical efficiency are still a matter of debate. By measuring both upper- and lower-limb muscle function, which experience differing levels of disuse, we examined the influence of central and peripheral haemodynamics, and skeletal muscle factors linked to mechanical efficiency. Across the ages and degree of disuse, upper-limb muscles exhibited a preserved work economy. In the legs the oldest-old without mobility limitations exhibited an augmented mechanical efficiency, which was reduced in those with an impairment in ambulation. These changes in mechanical efficiency were associated with the proportion of type I muscle fibres. Recognition that the mechanical efficiency is not simply age-dependent, but the consequence of inactivity and subsequent skeletal muscle changes, highlights the importance of maintaining physical activity across the lifespan.

The phosphodiesterase 2A controls lymphatic junctional maturation via cGMP-dependent notch signaling.

The molecular mechanisms by which lymphatic vessels induce cell contact inhibition are not understood. Here, we identify the cGMP-dependent phosphodiesterase 2A (PDE2A) as a selective regulator of lymphatic but not of blood endothelial contact inhibition. Conditional deletion of Pde2a in mouse embryos reveals severe lymphatic dysplasia, whereas blood vessel architecture remains unaltered. In the absence of PDE2A, human lymphatic endothelial cells fail to induce mature junctions and cell cycle arrest, whereas cGMP levels, but not cAMP levels, are increased. Loss of PDE2A-mediated cGMP hydrolysis leads to the activation of p38 signaling and downregulation of NOTCH signaling. However, DLL4-induced NOTCH activation restores junctional maturation and contact inhibition in PDE2A-deficient human lymphatic endothelial cells. In postnatal mouse mesenteries, PDE2A is specifically enriched in collecting lymphatic valves, and loss of Pde2a results in the formation of abnormal valves. Our data demonstrate that PDE2A selectively finetunes a crosstalk of cGMP, p38, and NOTCH signaling during lymphatic vessel maturation.

Cellular Redox Metabolism Is Modulated by the Distinct Localization of Cyclic Nucleotide Phosphodiesterase 5A Isoforms.

3'-5' cyclic nucleotide phosphodiesterases (PDEs) are a family of evolutionarily conserved cAMP and/or cGMP hydrolyzing enzymes, components of transduction pathways regulating crucial aspects of cell life. Among them, cGMP-specific PDE5-being a regulator of vascular smooth muscle contraction-is the molecular target of several drugs used to treat erectile dysfunction and pulmonary hypertension. Production of full-length murine PDE5A isoforms in the milk-yeast Kluyveromyces lactis showed that the quaternary assembly of MmPDE5A1 is a mixture of dimers and tetramers, while MmPDE5A2 and MmPDE5A3 only assembled as dimers. We showed that the N-terminal peptide is responsible for the tetramer assembly of MmPDE5A1, while that of the MmPDE5A2 is responsible for its mitochondrial localization. Overexpression of the three isoforms alters at different levels the cAMP/cGMP equilibrium as well as the NAD(P)+/NAD(P)H balance and induces a metabolic switch from oxidative to fermentative. In particular, the mitochondrial localization of MmPDE5A2 unveiled the existence of a cAMP-cGMP signaling cascade in this organelle, for which we propose a metabolic model that could explain the role of PDE5 in some cardiomyopathies and some of the side effects of its inhibitors.

An Electroconductive, Thermosensitive, and Injectable Chitosan/Pluronic/Gold-Decorated Cellulose Nanofiber Hydrogel as an Efficient Carrier for Regeneration of Cardiac Tissue.

Myocardial infarction is a major cause of death worldwide and remains a social and healthcare burden. Injectable hydrogels with the ability to locally deliver drugs or cells to the damaged area can revolutionize the treatment of heart diseases. Herein, we formulate a thermo-responsive and injectable hydrogel based on conjugated chitosan/poloxamers for cardiac repair. To tailor the mechanical properties and electrical signal transmission, gold nanoparticles (AuNPs) with an average diameter of 50 nm were physically bonded to oxidized bacterial nanocellulose fibers (OBC) and added to the thermosensitive hydrogel at the ratio of 1% w/v. The prepared hydrogels have a porous structure with open pore channels in the range of 50−200 µm. Shear rate sweep measurements demonstrate a reversible phase transition from sol to gel with increasing temperature and a gelation time of 5 min. The hydrogels show a shear-thinning behavior with a shear modulus ranging from 1 to 12 kPa dependent on gold concentration. Electrical conductivity studies reveal that the conductance of the polymer matrix is 6 × 10−2 S/m at 75 mM Au. In vitro cytocompatibility assays by H9C2 cells show high biocompatibility (cell viability of >90% after 72 h incubation) with good cell adhesion. In conclusion, the developed nanocomposite hydrogel has great potential for use as an injectable biomaterial for cardiac tissue regeneration.

MANP Activation Of The cGMP Inhibits Aldosterone Via PDE2 And CYP11B2 In H295R Cells And In Mice.

BACKGROUND: Aldosterone is a critical pathological driver for cardiac and renal diseases. We recently discovered that mutant atrial natriuretic peptide (MANP), a novel atrial natriuretic peptide (ANP) analog, possessed more potent aldosterone inhibitory action than ANP in vivo. MANP and natriuretic peptide (NP)-augmenting therapy sacubitril/valsartan are under investigations for human hypertension treatment. Understanding the elusive mechanism of aldosterone inhibition by NPs remains to be a priority. Conflicting results were reported on the roles of the pGC-A (particulate guanylyl cyclase A receptor) and NP clearance receptor in aldosterone inhibition. Furthermore, the function of PKG (protein kinase G) and PDEs (phosphodiesterases) on aldosterone regulation are not clear. METHODS: In the present study, we investigated the molecular mechanism of aldosterone regulation in a human adrenocortical cell line H295R and in mice. RESULTS: We first provided evidence to show that pGC-A, not NP clearance receptor, mediates aldosterone inhibition. Next, we confirmed that MANP inhibits aldosterone via PDE2 (phosphodiesterase 2) not PKG, with specific agonists, antagonists, siRNA silencing, and fluorescence resonance energy transfer experiments. Further, the inhibitory effect is mediated by a reduction of intracellular Ca2+ levels. We then illustrated that MANP directly reduces aldosterone synthase CYP11B2 (cytochrome p450 family 11 subfamily b member 2) expression via PDE2. Last, in PDE2 knockout mice, consistent with in vitro findings, embryonic adrenal CYP11B2 is markedly increased. CONCLUSIONS: Our results innovatively explore and expand the NP/pGC-A/3',5', cyclic guanosine monophosphate (cGMP)/PDE2 pathway for aldosterone inhibition by MANP in vitro and in vivo. In addition, our data also support the development of MANP as a novel ANP analog drug for aldosterone excess treatment.

Cell Shortening and Calcium Homeostasis Analysis in Adult Cardiomyocytes via a New Software Tool.

Intracellular calcium (Ca2+) is the central regulator of heart contractility. Indeed, it couples the electrical signal, which pervades the myocardium, with cardiomyocytes contraction. Moreover, alterations in calcium management are the main factors contributing to the mechanical and electrical dysfunction observed in failing hearts. So, simultaneous analysis of the contractile function and intracellular Ca2+ is indispensable to evaluate cardiomyocytes activity. Intracellular Ca2+ variations and fraction shortening are commonly studied with fluorescent Ca2+ indicator dyes associated with microscopy techniques. However, tracking and dealing with multiple files manually is time-consuming and error-prone and often requires expensive apparatus and software. Here, we announce a new, user-friendly image processing and analysis tool, based on ImageJ-Fiji/MATLAB® software, to evaluate the major cardiomyocyte functional parameters. We succeeded in analyzing fractional cell shortening, Ca2+ transient amplitude, and the kinematics/dynamics parameters of mouse isolated adult cardiomyocytes. The proposed method can be applied to evaluate changes in the Ca2+ cycle and contractile behavior in genetically or pharmacologically induced disease models, in drug screening and other common applications to assess mammalian cardiomyocyte functions.

Phosphodiesterase 5a Signalling in Skeletal Muscle Pathophysiology.

Phosphodiesterase 5A (PDE5A) is involved in cGMP hydrolysis, regulating many physiological processes. Increased activity of PDE5A has been found in several pathological conditions, and the pharmacological inhibition of PDE5 has been demonstrated to have several therapeutic applications. We have identified the presence of three different Pde5a isoforms in cardiomyocytes, and we have found that the expression of specific Pde5a isoforms may have a causal role in the onset of pathological responses in these cells. In our previous study, we demonstrated that PDE5A inhibition could ameliorate muscular dystrophy by acting at different levels, as assessed by the altered genomic response of muscular cells following treatment with the PDE5A inhibitor tadalafil. Thus, considering the importance of PDE5A in various pathophysiological conditions, we further investigated the regulation of this enzyme. Here, we analysed the expression of Pde5a isoforms in the pathophysiology of skeletal muscle. We found that skeletal muscle tissues and myogenic cells express Pde5a1 and Pde5a2 isoforms, and we observed an increased expression of Pde5a1 in damaged skeletal muscles, while Pde5a2 levels remained unchanged. We also cloned and characterized the promoters that control the transcription of Pde5a isoforms, investigating which of the transcription factors predicted by bioinformatics analysis could be involved in their modulation. In conclusion, we found an overexpression of Pde5a1 in compromised muscle and identified an involvement of MyoD and Runx1 in Pde5a1 transcriptional activity.

Preclinical Evidence of Intra-Articular Autologous Cartilage Micrograft for Osteochondral Repair: Evaluation in a Rat Model.

OBJECTIVE: The search for an effective and long-lasting strategy to treat osteochondral defects (OCD) is a great challenge. Regenerative medicine launched a new era of research in orthopaedics for restoring normal tissue functions. The aim of this study was to test the healing potential of Rigenera micrografting technology in a rat model of OCD by investigating 2 cartilage donor sites. METHODS: Full-thickness OCD was bilaterally created in the knee joints of rats. Animals were randomly divided into 2 groups based on the anatomical site used for micrograft collection: articular (TO) and xiphoid (XA). Micrograft was injected into the knee via an intra-articular approach. The contralateral joint served as the control. Euthanasia was performed 2 months after the set-up of OCD. Histological evaluations foresaw hematoxylin/eosin and safranin-O/fast green staining, the modified O'Driscoll score, and collagen 1A1 and 2A1 immunostaining. Kruskal-Wallis and the post hoc Dunn test were performed to evaluate differences among groups. RESULTS: Histological results showed defect filling in both autologous micrografts. The TO group displayed tissue repair with more hyaline-like characteristics than its control (P < 0.01). A fibrocartilaginous aspect was instead noticed in the XA group. Immunohistochemical assessments on type 2A1 and type 1 collagens confirmed the best histological results in the TO group. CONCLUSIONS: TO and XA groups contributed to a different extent to fill the OCD lesions. TO group provided the best histological and immunohistochemical results; therefore, it could be a promising method to treat OCD after the validation in a larger animal model.

Phosphodiesterases Expression during Murine Cardiac Development.

3'-5' cyclic nucleotide phosphodiesterases (PDEs) are a large family of enzymes playing a fundamental role in the control of intracellular levels of cAMP and cGMP. Emerging evidence suggested an important role of phosphodiesterases in heart formation, but little is known about the expression of phosphodiesterases during cardiac development. In the present study, the pattern of expression and enzymatic activity of phosphodiesterases was investigated at different stages of heart formation. C57BL/6 mice were mated and embryos were collected from 14.5 to 18.5 days of development. Data obtained by qRT-PCR and Western blot analysis showed that seven different isoforms are expressed during heart development, and PDE1C, PDE2A, PDE4D, PDE5A and PDE8A are modulated from E14.5 to E18.5. In heart homogenates, the total cAMP and cGMP hydrolytic activity is constant at the evaluated times, and PDE4 accounts for the majority of the cAMP hydrolyzing ability and PDE2A accounts for cGMP hydrolysis. This study showed that a subset of PDEs is expressed in developing mice heart and some of them are modulated to maintain constant nucleotide phosphodiesterase activity in embryonic and fetal heart.

TLQP-21 changes in response to a glucose load.

BACKGROUND: The TLQP-21 peptide potentiates glucose-stimulated insulin secretion, hence we investigated its endogenous response to glucose. METHODS: Fasted mice received intraperitoneal glucose (3 g/kg), or saline (controls), and were sacrificed 30 and 120 min later (4 groups, n = 6/group). We investigated TLQP-21 in pancreas and plasma using immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and high performance liquid chromatography (HPLC), as well as TLQP-21 receptors (gC1q-R and C3a-R1) expression in pancreas by immunohistochemistry. RESULTS: In pancreas, TLQP-immunoreactivity (TLQP-ir.) was shown in insulin-, glucagon- and somatostatin-containing cells. Upon glucose, TLQP-ir. decreased at 30 min (∼40 % vs. controls), while returning to basal values at 120 min. In all groups, C3a-R1 was localized in ∼50 % of TLQP labelled islet cells (mostly central), while gC1q-R was detected in ∼25 % of TLQP cells (mainly peripheral). HPLC fractions of control pancreas extracts, assessed by ELISA, confirmed the presence of a TLQP-21 compatible-form (∼2.5 kDa MW). In plasma, TLQP-ir. increased at 30 min (∼30 %), with highest concentrations at 120 min (both: p<0.05 vs. controls), while HPLC fractions showed an increase in the TLQP-21 compatible form. CONCLUSIONS: Upon hyperglycaemia, TLQP-21 would be released from islets, to enhance insulin secretion but we cannot exclude an autocrine activity which may regulate insulin storage/secretion.

Bone Marrow Transplantation as Therapy for Ataxia-Telangiectasia: A Systematic Review.

Ataxia-Telangiectasia (A-T) is a rare autosomal recessive disorder, first reported in 1926, caused by a deficiency of ATM (Ataxia-Telangiectasia Mutated) protein. The disease is characterized by progressive cerebellar neurodegeneration, immunodeficiency, leukemia, and lymphoma cancer predisposition. Immunoglobulin replacement, antioxidants, neuroprotective factors, growth, and anti-inflammatory hormones are commonly used for A-T treatment, but, to date, there is no known cure. Bone marrow transplantation (BMT) is a successful therapy for several forms of diseases and it is a valid approach for tumors, hemoglobinopathies, autoimmune diseases, inherited disorders of metabolism, and other pathologies. Some case reports of A-T patients have shown that BMT is becoming a good option, as a correct engraftment of healthy cells can restore some aspects of immunologic capacity. However, due to a high risk of mortality as a result of a clinical and cellular hypersensitivity to ionizing radiation and radiomimetic drugs, a specific non-myeloablative conditioning is required before BMT. Although BMT might be considered as one promising therapy for the treatment of immunological defects and cancer prevention in selected A-T patients, the therapy is currently not recommended or recognized and the eligibility of A-T patients for BMT is a point to deepen and deliberate.

Exercise training improves vascular function in patients with Alzheimer's disease.

PURPOSE: Vascular dysfunction has been demonstrated in patients with Alzheimer's disease (AD). Exercise is known to positively affect vascular function. Thus, the aim of our study was to investigate exercise-induced effects on vascular function in AD. METHODS: Thirty-nine patients with AD (79 ± 8 years) were recruited and randomly assigned to exercise training (EX, n = 20) or control group (CTRL, n = 19). All subjects performed 72 treatment sessions (90 min, 3 t/w). EX included moderate-high-intensity aerobic and strength training. CTRL included cognitive stimuli (visual, verbal, auditive). Before and after the 6-month treatment, the vascular function was measured by passive-leg movement test (PLM, calculating the variation in blood flow: ∆peak; and area under the curve: AUC) tests, and flow-mediated dilation (FMD, %). A blood sample was analyzed for vascular endothelial growth factor (VEGF). Arterial blood flow (BF) and shear rate (SR) were measured during EX and CTRL during a typical treatment session. RESULTS: EX group has increased FMD% (+ 3.725%, p < 0.001), PLM ∆peak (+ 99.056 ml/min, p = 0.004), AUC (+ 37.359AU, p = 0.037) and VEGF (+ 8.825 pg/ml, p = 0.004). In the CTRL group, no difference between pre- and post-treatment was found for any variable. Increase in BF and SR was demonstrated during EX (BF + 123%, p < 0.05; SR + 134%, p < 0.05), but not during CTRL treatment. CONCLUSION: Exercise training improves peripheral vascular function in AD. These ameliorations may be due to the repetitive increase in SR during exercise which triggers NO and VEGF upregulation. This approach might be included in standard AD clinical practice as an effective strategy to treat vascular dysfunction in this population.

Phosphodiesterase Inhibitors: Could They Be Beneficial for the Treatment of COVID-19?

In March 2020, the World Health Organization declared the severe acute respiratory syndrome corona virus 2 (SARS-CoV2) infection to be a pandemic disease. SARS-CoV2 was first identified in China and, despite the restrictive measures adopted, the epidemic has spread globally, becoming a pandemic in a very short time. Though there is growing knowledge of the SARS-CoV2 infection and its clinical manifestations, an effective cure to limit its acute symptoms and its severe complications has not yet been found. Given the worldwide health and economic emergency issues accompanying this pandemic, there is an absolute urgency to identify effective treatments and reduce the post infection outcomes. In this context, phosphodiesterases (PDEs), evolutionarily conserved cyclic nucleotide (cAMP/cGMP) hydrolyzing enzymes, could emerge as new potential targets. Given their extended distribution and modulating role in nearly all organs and cellular environments, a large number of drugs (PDE inhibitors) have been developed to control the specific functions of each PDE family. These PDE inhibitors have already been used in the treatment of pathologies that show clinical signs and symptoms completely or partially overlapping with post-COVID-19 conditions (e.g., thrombosis, inflammation, fibrosis), while new PDE-selective or pan-selective inhibitors are currently under study. This review discusses the state of the art of the different pathologies currently treated with phosphodiesterase inhibitors, highlighting the numerous similarities with the disorders linked to SARS-CoV2 infection, to support the hypothesis that PDE inhibitors, alone or in combination with other drugs, could be beneficial for the treatment of COVID-19.

PDE2A Is Indispensable for Mouse Liver Development and Hematopoiesis.

Phosphodiesterase 2A (PDE2A) is a cAMP-cGMP hydrolyzing enzyme essential for mouse development and the PDE2A knockout model (PDE2A-/-) is embryonic lethal. Notably, livers of PDE2A-/- embryos at embryonic day 14.5 (E14.5) have extremely reduced size. Morphological, cellular and molecular analyses revealed loss of integrity in the PDE2A-/- liver niche that compromises the hematopoietic function and maturation. Hematopoietic cells isolated from PDE2A-/- livers are instead able to differentiate in in vitro assays, suggesting the absence of blood cell-autonomous defects. Apoptosis was revealed in hepatoblasts and at the endothelial and stromal compartments in livers of PDE2A-/- embryos. The increase of the intracellular cAMP level and of the inducible cAMP early repressor (ICER) in liver of PDE2A-/- embryos might explain the impairment of liver development by downregulating the expression of the anti-apoptotic gene Bcl2. In summary, we propose PDE2A as an essential gene for integrity maintenance of liver niche and the accomplishment of hematopoiesis.

Corrigendum: Skeletal Muscle Fiber Size and Gene Expression in the Oldest-Old With Differing Degrees of Mobility.

[This corrects the article DOI: 10.3389/fphys.2019.00313.].

Therapeutic use of pulsed electromagnetic field therapy reduces prostate volume and lower urinary tract symptoms in benign prostatic hyperplasia.

BACKGROUND: Benign prostatic hyperplasia (BPH) etiology remains poorly understood, but chronic low-grade inflammation plays a role. Pulsed electromagnetic field therapy (PEMF) (1-50 Hz) is effective in reducing tissue inflammation. OBJECTIVES: We designed a pilot study to evaluate the effects of PEMF on prostate volume (PV) in BPH. MATERIALS AND METHODS: This is a prospective interventional trial on 27 naive patients with BPH and lower urinary tract symptoms (LUTS). At baseline (V0 ), all patients had blood tests, transrectal ultrasound, and questionnaires (IPSS, IIEF-15) and received a perineal PEMF device (Magcell® Microcirc, Physiomed Elektromedizin). PEMF was delivered on perineal area 5 minutes twice daily for 28 days, then (V1 ) all baseline evaluations were repeated. Afterward, nine patients continued therapy for 3 more months (PT group) and 15 discontinued (FU group). A 4-month evaluation (V2 ) was performed in both groups. RESULTS: A reduction was observed both at V1 and at V2 in PV: PVV0 44.5 mL (38.0;61.6) vs PVV1 42.1 mL (33.7;61.5, P = .039) vs PVV2 41.7mL (32.7;62.8, P = .045). IPSS was reduced both at V1 and at V2 : IPSSV0 11 (5.7;23.2) vs IPSSV1 10 (6;16, P = .045) vs IPSSV2 9 (6;14, P = .015). Baseline IPSS was related to IPSS reduction both at V1 (rs  = 0.313;P = .003) and at V2 (rs  = 0.664;P < .001). PV reduction in patients without metabolic syndrome (ΔPVV1nMetS -4.7 mL, 95%CI -7.3;-2.0) was greater than in affected patients (ΔPVV1MetS 1.7 mL, 95%CI -2.69;6.1)(P = .017, Relative RiskMetS  = 6). No changes were found in gonadal hormones or sexual function. DISCUSSION: PEMF was able to reduce PV after 28 days of therapy. Symptoms improved in a short time, with high compliance and no effects on hormonal and sexual function or any side effects. Patients with moderate-severe LUTS and without MetS seem to benefit more from this treatment. CONCLUSION: PEMF reduces PV and improves LUTS in a relative short time, in BPH patients. These benefits seem greater in those patients with moderate-severe LUTS but without MetS.

A Three-Dimensional Culture Model of Reversibly Quiescent Myogenic Cells.

Satellite cells (SC) are the stem cells of skeletal muscles. They are quiescent in adult animals but resume proliferation to allow muscle hypertrophy or regeneration after injury. The mechanisms balancing quiescence, self-renewal, and differentiation of SC are difficult to analyze in vivo owing to their complexity and in vitro because the staminal character of SC is lost when they are removed from the niche and is not adequately reproduced in the culture models currently available. To overcome these difficulties, we set up a culture model of the myogenic C2C12 cell line in suspension. When C2C12 cells are cultured in suspension, they enter a state of quiescence and form three-dimensional aggregates (myospheres) that produce the extracellular matrix and express markers of quiescent SC. In the initial phase of culture, a portion of the cells fuses in syncytia and abandons the myospheres. The remaining cells are mononucleated and quiescent but resume proliferation and differentiation when plated in a monolayer. The notch pathway controls the quiescent state of the cells as shown by the fact that its inhibition leads to the resumption of differentiation. Within this context, notch3 appears to play a central role in the activity of this pathway since the expression of notch1 declines soon after aggregation. In summary, the culture model of C2C12 in suspension may be used to study the cellular interactions of muscle stem cells and the pathways controlling SC quiescence entrance and maintenance.