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1.
Biochem Biophys Res Commun ; 514(4): 1198-1203, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31104767

RESUMO

Inflammation is a major driver of cardiac remodeling. Cardiac fibroblasts play an integral role in cardiac inflammation, fibrosis and remodeling. The orphan G-protein-coupled-receptor 5B of family C (GPRC5B) has recently been shown to have pro-inflammatory effects in adipocytes via the NFκB-signaling-pathway. Here, we investigated whether GPRC5B is involved in myocardial inflammation and fibrosis. Using neonatal rat cardiac fibroblasts (NRCF) we show that the transcription and the expression of endogenous GPRC5B is induced by stimulation with TNFα and LPS as well as through cyclic mechanical stretch, while the principle pro-fibrotic factor TGFß has no effect on the GPRC5B expression. Furthermore, we demonstrate that adenoviral overexpression and siRNA-mediated knockdown of GPRC5B in NRCF significantly alters the transcription level of the pro-inflammatory and pro-fibrotic cytokines TNFα, IL-1ß, IL-6 and MCP-1, and extracellular matrix-degrading MMP-9 in vitro. Additionally, in adult GPRC5B-transgenic mice the protein expression of collagen-1A1 is decreased and the production of MMP-9 is increased, indicating remodeling of the extracellular matrix in vivo. Our data show that GPRC5B is up-regulated by inflammatory signals and mechanical stress in NRCF, while GPRC5B modulates the inflammatory response of cardiac fibroblasts and the degradation of extracellular matrix-proteins in the mice heart. Thus, our findings are the first to report a novel role of the orphan receptor GPRC5B in fibroblast-driven myocardial inflammation and cardiac remodeling.

2.
Naunyn Schmiedebergs Arch Pharmacol ; 392(8): 887-911, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31101932

RESUMO

Karl H. Jakobs, former editor-in-chief of Naunyn-Schmiedeberg's Archives of Pharmacology and renowned molecular pharmacologist, passed away in April 2018. In this article, his scientific achievements regarding G protein-mediated signal transduction and regulation of canonical pathways are summarized. Particularly, the discovery of inhibitory G proteins for adenylyl cyclase, methods for the analysis of receptor-G protein interactions, GTP supply by nucleoside diphosphate kinases, mechanisms in phospholipase C and phospholipase D activity regulation, as well as the development of the concept of sphingosine-1-phosphate as extra- and intracellular messenger will presented. His seminal scientific and methodological contributions are put in a general and timely perspective to display and honor his outstanding input to the current knowledge in molecular pharmacology.

3.
Europace ; 21(9): 1410-1421, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31106349

RESUMO

AIMS: Brugada syndrome (BrS) is associated with a pronounced risk to develop sudden cardiac death (SCD). Up to 21% of patients are related to mutations in SCN5A. Studies identified SCN10A as a contributor of BrS. However, the investigation of the human cellular phenotype of BrS in the presence of SCN10A mutations remains lacking. The objective of this study was to establish a cellular model of BrS in presence of SCN10A mutations using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). METHODS AND RESULTS: Dermal fibroblasts obtained from a BrS patient suffering from SCD harbouring the SCN10A double variants (c.3803G>A and c.3749G>A) and three independent healthy control subjects were reprogrammed to hiPSCs. Human-induced pluripotent stem cells were differentiated into cardiomyocytes (hiPSC-CMs).The hiPSC-CMs from the BrS patient showed a significantly reduced peak sodium channel current (INa) and a significantly reduced ATX II (sea anemone toxin, an enhancer of late INa) sensitive as well as A-887826 (a blocker of SCN10A channel) sensitive late sodium channel current (INa) when compared with the healthy control hiPSC-CMs, indicating loss-of-function of sodium channels. Consistent with reduced INa the action potential amplitude and upstroke velocity (Vmax) were significantly reduced, which may contribute to arrhythmogenesis of BrS. Moreover, Ajmaline effects on action potentials were stronger in BrS-hiPSC-CMs than in healthy control cells. This is in agreement with the higher susceptibility of patients to sodium channel blocking drugs in unmasking BrS. CONCLUSION: Patient-specific hiPSC-CMs are able to recapitulate single-cell phenotype features of BrS with SCN10A mutations and may provide novel opportunities to further elucidate the cellular disease mechanism.

4.
Sci Rep ; 9(1): 5651, 2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30948775

RESUMO

Acute myocardial infarction (MI) evokes a systemic inflammatory response and locally the degradation of the necrotic tissue, followed by scar formation. The mechanisms for containment of the infarct zone are not studied well. The study aimed to examine the response of healthy cardiomyocytes to serum of patients with myocardial infarction. Human iPSC-cardiomyocytes (iPSC-CM) generated from two healthy donors were incubated with serum of patients with MI with and without ventricular fibrillation (VF) or of healthy controls. Different cell adhesion molecules were studied by flow cytometry and immunostaining. Cellular electrophysiology was studied by patch clamp. The cell adhesion molecules CD54/ICAM-1, CD58/LFA-3 and CD321/JAM-A were expressed on iPSC-CM within the plasma membrane. Incubation with serum of MI patients reduced the levels of expression of CD54/ICAM-1 and CD321/JAM-A by 15-20%. VF serum was less effective than serum of MI patients without VF. MI serum or VF serum did not affect resting potential, action potential duration or maximum depolarization velocity. Myocardial infarction serum exerts anti-inflammatory effects on healthy cardiomyocytes without affecting their electrical activity, thus helping to contain the infarct zone and to protect healthy tissue. Ventricular fibrillation during MI drives healthy cardiomyocytes towards a pro-inflammatory phenotype.

5.
Clin Pharmacol Ther ; 106(3): 642-651, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30947366

RESUMO

Short QT syndrome (SQTS) predisposes afflicted patients to sudden cardiac death. Until now, only one drug-quinidine-has been shown to be effective in patients with SQTS type 1(SQTS1). The objective of this study was to use human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from a patient with SQTS1 to search for potentially effective drugs for the treatment of SQTS1 patients. Patch clamp and single-cell contraction measurements were employed to assess drug effects. Ivabradine, mexiletine, and ajmaline but not flecainide, ranolazine, or amiodarone prolonged the action potential duration (APD) in hiPSC-CMs from an SQTS1 patient. Ivabradine, ajmaline, and mexiletine inhibited KCNH2 channel currents significantly, which may underlie their APD-prolonging effects. Under proarrhythmic epinephrine stimulation in spontaneously beating SQTS1 hiPSC-CMs, ivabradine, mexiletine, and ajmaline but not flecainide reduced the epinephrine-induced arrhythmic events. The results demonstrate that ivabradine, ajmaline, and mexiletine may be candidate drugs for preventing tachyarrhythmias in SQTS1 patients.

6.
Mol Syst Biol ; 15(2): e8503, 2019 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-30777892

RESUMO

Genome-, transcriptome- and proteome-wide measurements provide insights into how biological systems are regulated. However, fundamental aspects relating to which human proteins exist, where they are expressed and in which quantities are not fully understood. Therefore, we generated a quantitative proteome and transcriptome abundance atlas of 29 paired healthy human tissues from the Human Protein Atlas project representing human genes by 18,072 transcripts and 13,640 proteins including 37 without prior protein-level evidence. The analysis revealed that hundreds of proteins, particularly in testis, could not be detected even for highly expressed mRNAs, that few proteins show tissue-specific expression, that strong differences between mRNA and protein quantities within and across tissues exist and that protein expression is often more stable across tissues than that of transcripts. Only 238 of 9,848 amino acid variants found by exome sequencing could be confidently detected at the protein level showing that proteogenomics remains challenging, needs better computational methods and requires rigorous validation. Many uses of this resource can be envisaged including the study of gene/protein expression regulation and biomarker specificity evaluation.


Assuntos
Genoma Humano/genética , Proteoma/genética , Distribuição Tecidual/genética , Transcriptoma/genética , Regulação da Expressão Gênica/genética , Humanos , Espectrometria de Massas/métodos , Proteômica/métodos , RNA Mensageiro/genética , Análise de Sequência de RNA/métodos
7.
Mol Syst Biol ; 15(2): e8513, 2019 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-30777893

RESUMO

Despite their importance in determining protein abundance, a comprehensive catalogue of sequence features controlling protein-to-mRNA (PTR) ratios and a quantification of their effects are still lacking. Here, we quantified PTR ratios for 11,575 proteins across 29 human tissues using matched transcriptomes and proteomes. We estimated by regression the contribution of known sequence determinants of protein synthesis and degradation in addition to 45 mRNA and 3 protein sequence motifs that we found by association testing. While PTR ratios span more than 2 orders of magnitude, our integrative model predicts PTR ratios at a median precision of 3.2-fold. A reporter assay provided functional support for two novel UTR motifs, and an immobilized mRNA affinity competition-binding assay identified motif-specific bound proteins for one motif. Moreover, our integrative model led to a new metric of codon optimality that captures the effects of codon frequency on protein synthesis and degradation. Altogether, this study shows that a large fraction of PTR ratio variation in human tissues can be predicted from sequence, and it identifies many new candidate post-transcriptional regulatory elements.


Assuntos
Proteínas/genética , Proteoma/genética , Distribuição Tecidual/genética , Transcriptoma/genética , Regulação da Expressão Gênica/genética , Genoma Humano/genética , Humanos , Espectrometria de Massas/métodos , Proteômica/métodos , RNA Mensageiro/genética , Análise de Sequência de RNA/métodos
8.
Hum Genet ; 137(9): 753-768, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30167850

RESUMO

NALCN is a conserved cation channel, which conducts a permanent sodium leak current and regulates resting membrane potential and neuronal excitability. It is part of a large ion channel complex, the "NALCN channelosome", consisting of multiple proteins including UNC80 and UNC79. The predominant neuronal expression pattern and its function suggest an important role in neuronal function and disease. So far, biallelic NALCN and UNC80 variants have been described in a small number of individuals leading to infantile hypotonia, psychomotor retardation, and characteristic facies 1 (IHPRF1, OMIM 615419) and 2 (IHPRF2, OMIM 616801), respectively. Heterozygous de novo NALCN missense variants in the S5/S6 pore-forming segments lead to congenital contractures of the limbs and face, hypotonia, and developmental delay (CLIFAHDD, OMIM 616266) with some clinical overlap. In this study, we present detailed clinical information of 16 novel individuals with biallelic NALCN variants, 1 individual with a heterozygous de novo NALCN missense variant and an interesting clinical phenotype without contractures, and 12 individuals with biallelic UNC80 variants. We report for the first time a missense NALCN variant located in the predicted S6 pore-forming unit inherited in an autosomal-recessive manner leading to mild IHPRF1. We show evidence of clinical variability, especially among IHPRF1-affected individuals, and discuss differences between the IHPRF1- and IHPRF2 phenotypes. In summary, we provide a comprehensive overview of IHPRF1 and IHPRF2 phenotypes based on the largest cohort of individuals reported so far and provide additional insights into the clinical phenotypes of these neurodevelopmental diseases to help improve counseling of affected families.


Assuntos
Proteínas de Transporte/genética , Canalopatias/genética , Deficiências do Desenvolvimento/genética , Marcadores Genéticos , Variação Genética , Proteínas de Membrana/genética , Canais de Sódio/genética , Adolescente , Adulto , Canalopatias/patologia , Criança , Pré-Escolar , Deficiências do Desenvolvimento/patologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Fenótipo , Adulto Jovem
9.
Sci Rep ; 8(1): 10581, 2018 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-30002415

RESUMO

Nucleoside diphosphate kinase B (NDPK-B) acts as a protective factor in the retinal vasculature. NDPK-B deficiency leads to retinal vasoregression mimicking diabetic retinopathy (DR). Angiopoetin 2 (Ang-2), an initiator of retinal vasoregression in DR, is upregulated in NDPK-B deficient retinas and in NDPK-B depleted endothelial cells (ECs) in vitro. We therefore investigated the importance of Ang-2 in NDPK-B deficient retinas and characterized the mechanisms of Ang-2 upregulation upon NDPK-B depletion in cultured ECs. The crucial role of retinal Ang-2 in the initiation of vasoregression was verified by crossing NDPK-B deficient with Ang-2 haplodeficient mice. On the molecular level, FoxO1, a transcription factor regulating Ang-2, was upregulated in NDPK-B depleted ECs. Knockdown of FoxO1 abolished the elevation of Ang-2 induced by NDPK-B depletion. Furthermore O-GlcNAcylated FoxO1 was found preferentially in the nucleus. An increased O-GlcNAcylation of FoxO1 was revealed upon NDPK-B depletion. In accordance, the inhibition of protein O-GlcNAcylation normalized NDPK-B depletion induced Ang-2 upregulation. In summary, we demonstrated that the upregulation of Ang-2 upon NDPK-B deficiency is driven by O-GlcNAcylation of FoxO1. Our data provide evidence for a central role of protein O-GlcNAcylation in NDPK-B associated vascular damage and point to the hexosamine pathway as an important target in retinal vasoregression.

10.
Orphanet J Rare Dis ; 13(1): 120, 2018 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-30025539

RESUMO

BACKGROUND: Mitochondrial acyl-CoA dehydrogenase family member 9 (ACAD9) is essential for the assembly of mitochondrial respiratory chain complex I. Disease causing biallelic variants in ACAD9 have been reported in individuals presenting with lactic acidosis and cardiomyopathy. RESULTS: We describe the genetic, clinical and biochemical findings in a cohort of 70 patients, of whom 29 previously unpublished. We found 34 known and 18 previously unreported variants in ACAD9. No patients harbored biallelic loss of function mutations, indicating that this combination is unlikely to be compatible with life. Causal pathogenic variants were distributed throughout the entire gene, and there was no obvious genotype-phenotype correlation. Most of the patients presented in the first year of life. For this subgroup the survival was poor (50% not surviving the first 2 years) comparing to patients with a later presentation (more than 90% surviving 10 years). The most common clinical findings were cardiomyopathy (85%), muscular weakness (75%) and exercise intolerance (72%). Interestingly, severe intellectual deficits were only reported in one patient and severe developmental delays in four patients. More than 70% of the patients were able to perform the same activities of daily living when compared to peers. CONCLUSIONS: Our data show that riboflavin treatment improves complex I activity in the majority of patient-derived fibroblasts tested. This effect was also reported for most of the treated patients and is mirrored in the survival data. In the patient group with disease-onset below 1 year of age, we observed a statistically-significant better survival for patients treated with riboflavin.

11.
EMBO Mol Med ; 10(7)2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29907596

RESUMO

The myocyte enhancer factor 2 (MEF2) regulates transcription in cardiac myocytes and adverse remodeling of adult hearts. Activators of G protein-coupled receptors (GPCRs) have been reported to activate MEF2, but a comprehensive analysis of GPCR activators that regulate MEF2 has to our knowledge not been performed. Here, we tested several GPCR agonists regarding their ability to activate a MEF2 reporter in neonatal rat ventricular myocytes. The inflammatory mediator prostaglandin E2 (PGE2) strongly activated MEF2. Using pharmacological and protein-based inhibitors, we demonstrated that PGE2 regulates MEF2 via the EP3 receptor, the ßγ subunit of Gi/o protein and two concomitantly activated downstream pathways. The first consists of Tiam1, Rac1, and its effector p21-activated kinase 2, the second of protein kinase D. Both pathways converge on and inactivate histone deacetylase 5 (HDAC5) and thereby de-repress MEF2. In vivo, endotoxemia in MEF2-reporter mice induced upregulation of PGE2 and MEF2 activation. Our findings provide an unexpected new link between inflammation and cardiac remodeling by de-repression of MEF2 through HDAC5 inactivation, which has potential implications for new strategies to treat inflammatory cardiomyopathies.

12.
Sci Rep ; 8(1): 5975, 2018 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-29654304

RESUMO

By N-ethyl-N-nitrosourea (ENU) mutagenesis, we generated the mutant mouse line TUB6 that is characterised by severe combined immunodeficiency (SCID) and systemic sterile autoinflammation in homozygotes, and a selective T cell defect in heterozygotes. The causative missense point mutation results in the single amino acid exchange G170W in multicatalytic endopeptidase complex subunit-1 (MECL-1), the ß2i-subunit of the immuno- and thymoproteasome. Yeast mutagenesis and crystallographic data suggest that the severe TUB6-phenotype compared to the MECL-1 knockout mouse is caused by structural changes in the C-terminal appendage of ß2i that prevent the biogenesis of immuno- and thymoproteasomes. Proteasomes are essential for cell survival, and defective proteasome assembly causes selective death of cells expressing the mutant MECL-1, leading to the severe immunological phenotype. In contrast to the immunosubunits ß1i (LMP2) and ß5i (LMP7), mutations in the gene encoding MECL-1 have not yet been assigned to human disorders. The TUB6 mutant mouse line exemplifies the involvement of MECL-1 in immunopathogenesis and provides the first mouse model for primary immuno- and thymoproteasome-associated immunodeficiency that may also be relevant in humans.

13.
Circ Genom Precis Med ; 11(3): e001893, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29545480

RESUMO

BACKGROUND: Limb-Girdle muscular dystrophies (LGMD) are a heritable group of genetically determined disorders with a primary involvement of the pelvic or shoulder girdle musculature with partially cardiac manifestation, such as dilated cardiomyopathy (DCM) and life-threatening tachyarrhythmia. We report here that human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes from a patient with LGMD2I and DCM associated with recurrent ventricular tachycardia displayed ion channel dysfunction and abnormality of calcium homeostasis. METHODS: Dermal fibroblasts obtained from a patient with LGMD2I harboring a fukutin-related protein gene mutation (826C>A; Leu276Ile) and 3 healthy donors were reprogrammed to hiPSCs. The hiPSCs were differentiated into cardiomyocytes and used for biological and electrophysiological studies. RESULTS: Compared with hiPSC cardiomyocytes from the healthy donors, the hiPSC cardiomyocytes from the patient exhibited abnormal action potentials characterized by reduced amplitude and upstroke velocity. The peak and late Na channel currents (INa) as well as the peak L-type calcium channel currents were significantly reduced. The expression of SCN5A and CACNA1C was reduced in DCM cardiomyocytes, consistent with reduction of INa and L-type calcium channel currents. In addition, the rapidly activating delayed rectifier potassium current (IKr) was reduced, whereas the transient outward current (Ito) and slowly activating delayed rectifier potassium current (IKs) were similar in DCM and control cardiomyocytes. Finally, a significant reduction of systolic and diastolic intracellular Ca2+ concentrations was detected in DCM cardiomyocytes. CONCLUSIONS: This study demonstrates that patient-specific hiPSC cardiomyocytes can recapitulate some phenotypic properties of LGMD2I with DCM and provide a platform for studies on the cardiac events in LGMD.

14.
Stem Cells Int ; 2018: 6067096, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29535773

RESUMO

Background: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are providing new possibilities for the biological study, cell therapies, and drug discovery. However, the ion channel expression and functions as well as regulations in hiPSC-CMs still need to be fully characterized. Methods: Cardiomyocytes were derived from hiPS cells that were generated from two healthy donors. qPCR and patch clamp techniques were used for the study. Results: In addition to the reported ion channels, INa, ICa-L, ICa-T, If, INCX, IK1, Ito, IKr, IKs IKATP, IK-pH, ISK1-3, and ISK4, we detected both the expression and currents of ACh-activated (KACh) and Na+-activated (KNa) K+, volume-regulated and calcium-activated (Cl-Ca) Cl-, and TRPV channels. All the detected ion currents except IK1, IKACh, ISK, IKNa, and TRPV1 currents contribute to AP duration. Isoprenaline increased ICa-L, If, and IKs but reduced INa and INCX, without an effect on Ito, IK1, ISK1-3, IKATP, IKr, ISK4, IKNa, ICl-Ca, and ITRPV1. Carbachol alone showed no effect on the tested ion channel currents. Conclusion: Our data demonstrate that most ion channels, which are present in healthy or diseased cardiomyocytes, exist in hiPSC-CMs. Some of them contribute to action potential performance and are regulated by adrenergic stimulation.

15.
J Am Heart Assoc ; 7(7)2018 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-29574456

RESUMO

BACKGROUND: Short QT syndrome (SQTS), a disorder associated with characteristic ECG QT-segment abbreviation, predisposes affected patients to sudden cardiac death. Despite some progress in assessing the organ-level pathophysiology and genetic changes of the disorder, the understanding of the human cellular phenotype and discovering of an optimal therapy has lagged because of a lack of appropriate human cellular models of the disorder. The objective of this study was to establish a cellular model of SQTS using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). METHODS AND RESULTS: This study recruited 1 patient with short QT syndrome type 1 carrying a mutation (N588K) in KCNH2 as well as 2 healthy control subjects. We generated hiPSCs from their skin fibroblasts, and differentiated hiPSCs into cardiomyocytes (hiPSC-CMs) for physiological and pharmacological studies. The hiPSC-CMs from the patient showed increased rapidly activating delayed rectifier potassium channel current (IKr) density and shortened action potential duration compared with healthy control hiPSC-CMs. Furthermore, they demonstrated abnormal calcium transients and rhythmic activities. Carbachol increased the arrhythmic events in SQTS but not in control cells. Gene and protein expression profiling showed increased KCNH2 expression in SQTS cells. Quinidine but not sotalol or metoprolol prolonged the action potential duration and abolished arrhythmic activity induced by carbachol. CONCLUSIONS: Patient-specific hiPSC-CMs are able to recapitulate single-cell phenotype features of SQTS and provide novel opportunities to further elucidate the cellular disease mechanism and test drug effects.

16.
Europace ; 20(FI1): f46-f56, 2018 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-29566126

RESUMO

Aims: Our aim is to investigate the arrhythmogenic mechanism in arrhythmogenic right ventricular cardiomyopathy (ARVC)-patients by using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Methods and results: Human-induced pluripotent stem cell-derived cardiomyocytes were generated from human skin fibroblasts of two healthy donors and an ARVC-patient with a desmoglein-2 (DSG2) mutation. Patch clamp, quantitative polymerase chain reaction, and calcium imaging techniques were employed for the study. The amplitude and maximal upstroke velocity (Vmax) of action potential (AP) in ARVC-cells were smaller than that in healthy donor cells, whereas the resting potential and AP duration (APD) was not changed. The reduced Vmax resulted from decreased peak sodium current. The reason for undetected changes in APD may be the counter-action of reduced transient outward, small conductance Ca2+-activated, adenosine triphosphate-sensitive, Na/Ca exchanger (INCX) currents, and enhanced rapidly delayed rectifier currents. Isoprenaline (Iso) reduced INCX and shortened APD in both donor and ARVC-hiPSC-CMs. However, the effects of Iso in ARVC-cells are significantly larger than that in donor cells. In addition, ARVC-hiPSC-CMs showed more frequently than donor cells arrhythmogenic events induced by adrenergic stimulation. Conclusion: Cardiomyocytes derived from the ARVC patient with a DSG2 mutation displayed multiple ion channel dysfunctions and abnormal cellular electrophysiology as well as enhanced sensitivity to adrenergic stimulation. These may underlie the arrhythmogenesis in ARVC patients.

17.
Int J Cardiol ; 254: 195-202, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29407091

RESUMO

BACKGROUND AND PURPOSE: Previous studies revealed that Takotsubo cardiomyopathy (TTC), a transient disorder of ventricular dysfunction affecting predominantly postmenopausal women, is associated with acquired long QT syndrome and arrhythmias, but the exact pathophysiologic mechanism is unknown. Our aim is to investigate the electrophysiological mechanism for QT-prolongation in TTC-patients by using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). METHODS: hiPSC-CMs, which were generated from human skin fibroblasts of three healthy donors, were treated by estradiol (10µM for one week) and a toxic concentration of isoprenaline (Iso, 1mM for 2h). Patch clamp techniques, qPCR and fluorescence-activated cell sorting (FACS) were employed for the study. KEY RESULTS: Iso enhanced late INa and suppressed Ito and thus prolonged the action potential duration (APD), suggesting possible reasons for arrhythmias in TTC. Iso elevated the production of reactive oxygen species (ROS). N-acetylcystein (1mM), a ROS-blocker, abolished the effects of Iso on late INa and Ito. H2O2 (100µM) mimicked Iso effects on late INa and Ito. These data indicate that the effects of Iso were mediated by ROS. Metoprolol (1mM), a beta-blocker, prevented the effects of Iso on late INa and APD, confirming the adrenoceptor-dependent effects of Iso. Estradiol treatment prevented the APD-prolongation, attenuated the enhancement of INa, diminished the reduction of Ito, suppressed ROS-production induced by Iso and reduced the expression levels of adrenoceptors, suggesting protective effects of estragon against toxic effects of catecholamine. CONCLUSIONS: Estradiol has protective effects against catecholamine excess and hence reduction in estrogen level may increase the risk of acquired long QT syndrome in TTC.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Catecolaminas/toxicidade , Citoproteção/efeitos dos fármacos , Estradiol/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Potenciais de Ação/fisiologia , Células Cultivadas , Citoproteção/fisiologia , Estradiol/uso terapêutico , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Síndrome do QT Longo/tratamento farmacológico , Síndrome do QT Longo/fisiopatologia , Miócitos Cardíacos/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Cardiomiopatia de Takotsubo/tratamento farmacológico , Cardiomiopatia de Takotsubo/fisiopatologia
18.
Oncotarget ; 9(2): 1492-1493, 2018 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-29416708
19.
PLoS One ; 13(1): e0191423, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29373579

RESUMO

Stimulation of renal collecting duct principal cells with antidiuretic hormone (arginine-vasopressin, AVP) results in inhibition of the small GTPase RhoA and the enrichment of the water channel aquaporin-2 (AQP2) in the plasma membrane. The membrane insertion facilitates water reabsorption from primary urine and fine-tuning of body water homeostasis. Rho guanine nucleotide exchange factors (GEFs) interact with RhoA, catalyze the exchange of GDP for GTP and thereby activate the GTPase. However, GEFs involved in the control of AQP2 in renal principal cells are unknown. The A-kinase anchoring protein, AKAP-Lbc, possesses GEF activity, specifically activates RhoA, and is expressed in primary renal inner medullary collecting duct principal (IMCD) cells. Through screening of 18,431 small molecules and synthesis of a focused library around one of the hits, we identified an inhibitor of the interaction of AKAP-Lbc and RhoA. This molecule, Scaff10-8, bound to RhoA, inhibited the AKAP-Lbc-mediated RhoA activation but did not interfere with RhoA activation through other GEFs or activities of other members of the Rho family of small GTPases, Rac1 and Cdc42. Scaff10-8 promoted the redistribution of AQP2 from intracellular vesicles to the periphery of IMCD cells. Thus, our data demonstrate an involvement of AKAP-Lbc-mediated RhoA activation in the control of AQP2 trafficking.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Aquaporina 2/metabolismo , Membrana Celular/metabolismo , Túbulos Renais Coletores/citologia , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína rhoA de Ligação ao GTP/metabolismo , Membrana Celular/efeitos dos fármacos , Células HEK293 , Humanos , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade
20.
FASEB J ; 32(4): 2021-2035, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29208700

RESUMO

G protein-mediated signaling plays a decisive role in blood pressure regulation and the phenotype of vascular smooth muscle cells (VSMCs); however, the relevance of proteins that restrict G protein activity is not well characterized in this context. Here, we investigated the influence of regulator of G protein signaling 5 (RGS5), an inhibitor of Gαq/11 and Gαi/o activity, on blood pressure and the VSMC phenotype during experimental hypertension. In mice, loss of RGS5 did not affect baseline blood pressure, but prevented hypertension-induced structural remodeling. RGS5-deficient arterial VSMCs did not acquire a synthetic phenotype as evidenced by their inability to decrease the abundance of contractile markers-α-smooth muscle actin and smooth muscle-myosin heavy chain-or to proliferate under these conditions. Mechanistically, hypertensive pressure levels or biomechanical stretch are sufficient to increase the expression of RGS5. Loss of RGS5 severely impairs the activation of RhoA and stress fiber formation. In stretch-exposed VSMCs, RhoA activity was amplified upon inhibition of PKC, which mimics the downstream effects evoked by RGS5-mediated inhibition of Gαq/11 signaling. Collectively, our findings underline that RhoA activation may depend on the restriction of G protein activity and identify RGS5 as a mechanosensitive regulatory protein that is required to promote the synthetic VSMC phenotype as a prerequisite for structural renovation of the arterial wall during hypertension.-Arnold, C., Demirel, E., Feldner, A., Genové, G., Zhang, H., Sticht, C., Wieland, T., Hecker, M., Heximer, S., Korff, T. Hypertension-evoked RhoA activity in vascular smooth muscle cells requires RGS5.

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