Identification of murine phosphodiesterase 5A isoforms and their functional characterization in HL-1 cardiac cell line

Phosphodiesterase 5A (PDE5A) specifically degrades the ubiquitous second messenger cGMP and experimental and clinical data highlight its important role in cardiac diseases. To address PDE5A role in cardiac physiology, three splice variants of the PDE5A were cloned for the first time from mouse cDNA library (mPde5a1, mPde5a2 and mPde5a3). The predicted amino acidic sequences of the three murine isoforms are different in the N-terminal regulatory domain. mPDE5A isoforms were transfected in HEK293T cells and they showed high affinity for cGMP and similar sensitivity to sildenafil inhibition. RT-PCR analysis showed that mPde5a1, mPde5a2 and mPde5a3 had differential tissue distribution. In the adult heart, mPde5a1 and mPde5a2 were expressed at different levels whereas mPde5a3 was undetectable. Overexpression of mPDE5As induced an increase of HL-1 number cells which progress into cell cycle. mPDE5A1 and mPDE5A3 overexpression increased the number of polyploid and binucleated cells, mPDE5A3 widened HL-1 areas and modulated hypertrophic markers more efficiently respect to the other mPDE5A isoforms. Moreover, mPDE5A isoforms had differential subcellular localization: mPDE5A1 was mainly localized in the cytoplasm, mPDE5A2 and mPDE5A3 were also nuclear localized. These results demonstrate for the first time the existence of three PDE5A isoforms in mouse and highlight their potential role in the induction of hypertrophy. This article is protected by copyright. All rights reserved

Identification of Drosophila Mitotic Genes by Combining Co-Expression Analysis and RNA Interference

RNAi screens have, to date, identified many genes required for mitotic divisions of Drosophila tissue culture cells. However, the inventory of such genes remains incomplete. We have combined the powers of bioinformatics and RNAi technology to detect novel mitotic genes. We found that Drosophila genes involved in mitosis tend to be transcriptionally co-expressed. We thus constructed a co-expression–based list of 1,000 genes that are highly enriched in mitotic functions, and we performed RNAi for each of these genes. By limiting the number of genes to be examined, we were able to perform a very detailed phenotypic analysis of RNAi cells. We examined dsRNA-treated cells for possible abnormalities in both chromosome structure and spindle organization. This analysis allowed the identification of 142 mitotic genes, which were subdivided into 18 phenoclusters. Seventy of these genes have not previously been associated with mitotic defects; 30 of them are required for spindle assembly and/or chromosome segregation, and 40 are required to prevent spontaneous chromosome breakage. We note that the latter type of genes has never been detected in previous RNAi screens in any system. Finally, we found that RNAi against genes encoding kinetochore components or highly conserved splicing factors results in identical defects in chromosome segregation, highlighting an unanticipated role of splicing factors in centromere function. These findings indicate that our co-expression–based method for the detection of mitotic functions works remarkably well. We can foresee that elaboration of co-expression lists using genes in the same phenocluster will provide many candidate genes for small-scale RNAi screens aimed at completing the inventory of mitotic proteins

Disease Reactivation after Fingolimod Discontinuation in Pregnant Multiple Sclerosis Patients

Recent studies estimated an incidence of 4-25% of disease rebound after withdrawal of fingolimod (FTY) for any reason, but specific data on disease reactivation after FTY withdrawal due to pregnancy are limited. The aim of the study was to evaluate the frequency and predictors of disease reactivation in patients who stopped FTY for pregnancy. A multicentre retrospective cohort study was conducted in four Italian MS centres in 2013-2019. Both planned and unplanned pregnancies were included. The annualized relapse rate (ARR) was calculated before FTY treatment, during FTY treatment, during pregnancy and during the year after delivery. In total, 27 patients (mean age 29 years) were included. The ARR 1 year before FTY treatment was 1.3. Patients were exposed to FTY for a median of 2.9 years. The ARR was 0.04 during the last year before conception (p < 0.001 compared with the ARR before FTY treatment). Eleven patients became pregnant after a mean of 88 days following FTY discontinuation, whereas 16 patients stopped FTY after pregnancy confirmation. Relapses were observed in 22% of patients during pregnancy and in 44% in the postpartum period. ARR increased both during pregnancy (0.49; p = 0.027) and in the first year after delivery (0.67; p < 0.001) compared to the last year before pregnancy. Compared with radiological assessment before pregnancy, more patients showed new or enlarging T2 lesions (63% vs 30%; p = 0.02) and gadolinium-enhancing lesions (44% vs 0; p = 0.0001) on brain Magnetic Resonance Imaging. Relapses during pregnancy were the only significant predictor for postpartum relapses (OR 1.9, 95% CI 1.11-3.1). One case of spontaneous abortion and no cases of abnormal foetal development were observed. Despite adequate and prolonged control of disease activity, women who discontinue FTY because of pregnancy are at risk for disease reactivation. In patients who relapsed during pregnancy, the initiation of high-efficacy disease modifying drugs (DMDs) soon after delivery is advisable to prevent postpartum relapses

β1-Syntrophin Modulation by miR-222 in mdx Mice

Background: In mdx mice, the absence of dystrophin leads to the deficiency of other components of the dystrophin-glycoprotein complex (DAPC), making skeletal muscle fibers more susceptible to necrosis. The mechanisms involved in the disappearance of the DAPC are not completely understood. The muscles of mdx mice express normal amounts of mRNA for the DAPC components, thus suggesting post-transcriptional regulation. Methodology/Principal Findings: We investigated the hypothesis that DAPC reduction could be associated with the microRNA system. Among the possible microRNAs (miRs) found to be upregulated in the skeletal muscle tissue of mdx compared to wt mice, we demonstrated that miR-222 specifically binds to the 3′-UTR of β1-syntrophin and participates in the downregulation of β1-syntrophin. In addition, we documented an altered regulation of the 3′-UTR of β1-syntrophin in muscle tissue from dystrophic mice. Conclusion/Significance: These results show the importance of the microRNA system in the regulation of DAPC components in dystrophic muscle, and suggest a potential role of miRs in the pathophysiology of dystrophy. © 2010 De Arcangelis et al

V1a vasopressin receptor expression is modulated during myogenic differentiation.

International audienceNeurohypophyseal peptides potently stimulate myogenic differentiation by acting through different receptors of the same family. Here, we show that L6C5 myogenic cells express, at a high density, a single class of V1a Arg8-vasopressin (AVP) receptor. The expression of the vasopressin receptor of type 1a (V1aR) is significantly higher in proliferating myoblasts than in differentiated myotubes. The differentiation-related decrease of V1aR expression was evident both at the mRNA and at the protein level as shown by the reduction of [(3)H]-AVP binding. However, in L6C5 cells transfected with a synthetic construct containing the luciferase gene driven by the 2 kb upstream region of V1aR, we observed a stimulation of the activity of the promoter when the cells were cultured in differentiative medium. The down-regulation of the V1aR correlated with a decreased half-life of its mRNA (half-life 5.86+/-0.74 hr in 10% fetal bovine serum [FBS] versus 3.53+/-0.72 hr in 1% FBS). Cyclosporine A and dexamethasone, but not 5'-azacytidine, treatments of cells in differentiation medium restored the V1aR level to that measured in proliferating L6C5 cells, thus confirming the role of post-transcriptional mechanisms in the modulation of V1aR expression. Taken together, these data show that mRNA stability plays a role in modulating protein expression during the myogenic differentiation process

V1a vasopressin receptor expression is modulated during myogenic differentiation

Neurohypophyseal peptides potently stimulate myogenic differentiation by acting through different receptors of the same family. Here, we show that L6C5 myogenic cells express, at a high density, a single class of V1a Arg8-vasopressin (AVP) receptor. The expression of the vasopressin receptor of type 1a (V1aR) is significantly higher in proliferating myoblasts than in differentiated myotubes. The differentiation-related decrease of V1aR expression was evident both at the mRNA and at the protein level as shown by the reduction of [H-3]-AVP binding. However, in L6C5 cells transfected with a synthetic construct containing the luciferase gene driven by the 2 kb upstream region of V1aR, we observed a stimulation of the activity of the promoter when the cells were cultured in differentiative medium. The down-regulation of the V1aR correlated with a decreased half-life of its mRNA (half-life 5.86 +/- 0.74 hr in 10% fetal bovine serum [FBS] versus 3.53 +/- 0.72 hr in 1% FBS). Cyclosporine A and dexamethasone, but not 5'-azacytidine, treatments of cells in differentiation medium restored the V1aR level to that measured in proliferating L6C5 cells, thus confirming the role of post-transcriptional mechanisms in the modulation of V1aR expression. Taken together, these data show that mRNA stability plays a role in modulating protein expression during the myogenic differentiation process

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

Inflammation in muscular dystrophy and the beneficial effects of non-steroidal anti-inflammatory drugs

Introduction: Glucocorticoids are the only drugs available for the treatment of Duchenne muscular dystrophy (DMD), but it is unclear whether their efficacy is dependent on their anti-inflammatory activity. Methods: To address this issue, mdx mice were treated daily with methylprednisolone and non-steroidal anti-inflammatory drugs (NSAIDs: aspirin, ibuprofen, parecoxib). Results: NSAID treatment was effective in ameliorating muscle morphology and reducing macrophage infiltration and necrosis. The percentage of regenerating myofibers was not modified by the treatments. The drugs were effective in reducing COX-2 expression and inflammatory cytokines, but they did not affect utrophin levels. The effects of the treatments on contractile performance were analyzed. Isometric tension did not differ in treated and untreated muscle, but the resistance to fatigue was decreased by treatment with methylprednisolone and aspirin. Conclusions: NSAIDs have a beneficial effect on mdx muscle morphology, pointing to a crucial role of inflammation in the progression of DMD. Muscle Nerve, 201

PDE5 Inhibition Counteracts beta-Adrenergic Induction Of Cardiac Hypertrophy

The beta-adrenoreceptors play important roles in cardiovascular function regulation mediated by the sympathetic nervous system. It is known that sustained beta-adrenergic stimulations promotes cardiac hypertrophy. Recently, an anti-hypertrophic role of sildenafil, that acts as a specific phosphodiesterase 5 (PDE5) inhibitor, has been demonstrated in mice where hypertrophy was mechanically induced. We report the results obtained on an in vitro model of cardiac hypertrophy. By using three-dimensional cultures of mouse ventricular cardiomyocytes we show that: 1) these cells express levels of PDE5 comparable with the ones in normal heart, 2) treatment of the cultures with the beta-adrenoreceptors agonist isoproterenol induces cell hypertrophy accompanied by an increment of the level of PDE5 expression and 3) sildenafil prevents the development of such hypertrophy. In summary, we present a test system that may contribute to clarify intracellular signaling pathways leading to cardiac hypertrophy and to identify molecular targets, like the ones involved in PDE5 activity, on which to steer the development of new drugs and to design new clinical therapies