Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 18 de 18
Filtrar
Mais filtros

Base de dados
Tipo de documento
País de afiliação
Intervalo de ano de publicação
1.
J Biol Chem ; 298(9): 102362, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35963431

RESUMO

The activity of protein phosphatase 2A (PP2A) is determined by the expression and localization of the regulatory B-subunits. PP2A-B56α is the dominant isoform of the B'-family in the heart. Its role in regulating the cardiac response to ß-adrenergic stimulation is not yet fully understood. We therefore generated mice deficient in B56α to test the functional cardiac effects in response to catecholamine administration versus corresponding WT mice. We found the decrease in basal PP2A activity in hearts of KO mice was accompanied by a counter-regulatory increase in the expression of B' subunits (ß and γ) and higher phosphorylation of sarcoplasmic reticulum Ca2+ regulatory and myofilament proteins. The higher phosphorylation levels were associated with enhanced intraventricular pressure and relaxation in catheterized KO mice. In contrast, at the cellular level, we detected depressed Ca2+ transient and sarcomere shortening parameters in KO mice at basal conditions. Consistently, the peak amplitude of the L-type Ca2+ current was reduced and the inactivation kinetics of ICaL were prolonged in KO cardiomyocytes. However, we show ß-adrenergic stimulation resulted in a comparable peak amplitude of Ca2+ transients and myocellular contraction between KO and WT cardiomyocytes. Therefore, we propose higher isoprenaline-induced Ca2+ spark frequencies might facilitate the normalized Ca2+ signaling in KO cardiomyocytes. In addition, the application of isoprenaline was associated with unchanged L-type Ca2+ current parameters between both groups. Our data suggest an important influence of PP2A-B56α on the regulation of Ca2+ signaling and contractility in response to ß-adrenergic stimulation in the myocardium.


Assuntos
Adrenérgicos , Proteína Fosfatase 2 , Adrenérgicos/metabolismo , Adrenérgicos/farmacologia , Animais , Cálcio/metabolismo , Isoproterenol/farmacologia , Camundongos , Camundongos Knockout , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Fosforilação , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Retículo Sarcoplasmático/metabolismo
2.
FASEB J ; 34(8): 11272-11291, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32602979

RESUMO

ICER (inducible cAMP early repressor) isoforms are transcriptional repressors encoded by the Crem (cAMP responsive element modulator) gene. They were linked to the regulation of a multitude of cellular processes and pathophysiological mechanisms. Here, we show for the first time that two independent induction patterns for CREM repressor isoforms exist in the heart, namely for ICER and smICER (small ICER), which are induced in response to ß-adrenergic stimulation in a transient- and saturation-like manner, respectively. This time-shifted induction pattern, driven by two internal promoters in the Crem gene, leads to the predominant transcription of smIcer after prolonged ß-adrenergic stimulation. Using an ICER knockout mouse model with preserved smICER induction, we show that the transient-like induction of Icer itself has minor effects on gene regulation, cardiac hypertrophy or contractile function in the heart. We conclude that the functions previously linked to ICER may be rather attributed to smICER, also beyond the cardiac background.


Assuntos
Agonistas Adrenérgicos beta/farmacologia , Modulador de Elemento de Resposta do AMP Cíclico/genética , Receptores Adrenérgicos beta/genética , Animais , Cardiomegalia/tratamento farmacológico , Linhagem Celular , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Células HEK293 , Coração/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética
3.
J Mol Cell Cardiol ; 125: 195-204, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30389400

RESUMO

RATIONALE: A higher expression/activity of type 1 serine/threonine protein phosphatase 1 (PP1) may contribute to dephosphorylation of cardiac regulatory proteins triggering the development of heart failure. OBJECTIVE: Here, we tested the putatively protective effects of PP1 inhibitor-2 (I2) overexpression using a heart failure model induced by chronic ß-adrenergic stimulation. METHODS AND RESULTS: Transgenic (TG) and wild-type (WT) mice were subjected to isoprenaline (ISO) or isotonic NaCl solution supplied via osmotic minipumps for 7 days. I2 overexpression was associated with a depressed PP1 activity. Basal contractility was unchanged in catheterized mice and isolated cardiomyocytes between TGNaCl and WTNaCl. TGISO mice exhibited more fibrosis and a higher expression of hypertrophy marker proteins as compared to WTISO. After acute administration of ISO, the contractile response was accompanied by a higher sensitivity in TGISO as compared to WTISO. In contrast to basal contractility, the peak amplitude of [Ca]i and SR Ca load were reduced in TGNaCl as compared to WTNaCl. These effects were normalized to WT levels after chronic ISO stimulation. Cardiomyocyte relaxation and [Ca]i decay kinetics were hastened in TGISO as compared to WTISO, which can be explained by a higher phospholamban phosphorylation at Ser16. Chronic catecholamine stimulation was followed by an enhanced expression of GSK3ß, whereas the phosphorylation at Ser9 was lower in TG as compared to the corresponding WT group. This resulted in a higher I2 phosphorylation that may reactivate PP1. CONCLUSION: Our findings suggest that the basal desensitization of ß-adrenergic signaling and the depressed Ca handling in TG by inhibition of PP1 is restored by a GSK3ß-dependent phosphorylation of I2.


Assuntos
Cálcio/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Oncogênicas/metabolismo , Proteína Fosfatase 1/metabolismo , Animais , Células Cultivadas , Proteínas de Ligação a DNA , Insuficiência Cardíaca/metabolismo , Chaperonas de Histonas , Humanos , Isoproterenol/farmacologia , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/efeitos dos fármacos , Sarcômeros/efeitos dos fármacos , Sarcômeros/genética , Cloreto de Sódio/farmacologia
4.
Basic Res Cardiol ; 113(4): 27, 2018 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-29881975

RESUMO

Understanding molecular mechanisms involved in atrial tissue remodeling and arrhythmogenesis in atrial fibrillation (AF) is essential for developing specific therapeutic approaches. Two-pore-domain potassium (K2P) channels modulate cellular excitability, and TASK-1 (K2P3.1) currents were recently shown to alter atrial action potential duration in AF and heart failure (HF). Finding animal models of AF that closely resemble pathophysiological alterations in human is a challenging task. This study aimed to analyze murine cardiac expression patterns of K2P channels and to assess modulation of K2P channel expression in murine models of AF and HF. Expression of cardiac K2P channels was quantified by real-time qPCR and immunoblot in mouse models of AF [cAMP-response element modulator (CREM)-IbΔC-X transgenic animals] or HF (cardiac dysfunction induced by transverse aortic constriction, TAC). Cloned murine, human, and porcine TASK-1 channels were heterologously expressed in Xenopus laevis oocytes. Two-electrode voltage clamp experiments were used for functional characterization. In murine models, among members of the K2P channel family, TASK-1 expression displayed highest levels in both atrial and ventricular tissue samples. Furthermore, K2P2.1, K2P5.1, and K2P6.1 showed significant expression levels. In CREM-transgenic mice, atrial expression of TASK-1 was significantly reduced in comparison with wild-type animals. In a murine model of TAC-induced pressure overload, ventricular TASK-1 expression remained unchanged, while atrial TASK-1 levels were significantly downregulated. When heterologously expressed in Xenopus oocytes, currents of murine, porcine, and human TASK-1 displayed similar characteristics. TASK-1 channels display robust cardiac expression in mice. Murine, porcine, and human TASK-1 channels share functional similarities. Dysregulation of atrial TASK-1 expression in murine AF and HF models suggests a mechanistic contribution to arrhythmogenesis.


Assuntos
Fibrilação Atrial/metabolismo , Remodelamento Atrial , Átrios do Coração/metabolismo , Insuficiência Cardíaca/metabolismo , Ventrículos do Coração/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Remodelação Ventricular , Potenciais de Ação , Animais , Fibrilação Atrial/genética , Fibrilação Atrial/patologia , Fibrilação Atrial/fisiopatologia , Clonagem Molecular , Modelos Animais de Doenças , Feminino , Átrios do Coração/patologia , Átrios do Coração/fisiopatologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Frequência Cardíaca , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Oócitos , Canais de Potássio de Domínios Poros em Tandem/genética , Transdução de Sinais , Sus scrofa , Remodelação Ventricular/efeitos dos fármacos , Xenopus laevis
5.
FASEB J ; 29(9): 3773-87, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26023182

RESUMO

Annexin A4 (AnxA4), a Ca(2+)- and phospholipid-binding protein, is up-regulated in the human failing heart. In this study, we examined the impact of AnxA4 on ß-adrenoceptor (ß-AR)/cAMP-dependent signal transduction. Expression of murine AnxA4 in human embryonic kidney (HEK)293 cells dose-dependently inhibited cAMP levels after direct stimulation of adenylyl cyclases (ACs) with forskolin (FSK), as determined with an exchange protein activated by cAMP-Förster resonance energy transfer (EPAC-FRET) sensor and an ELISA (control vs. +AnxA4: 1956 ± 162 vs. 1304 ± 185 fmol/µg protein; n = 8). Disruption of the anxA4 gene led to a consistent increase in intracellular cAMP levels in isolated adult mouse cardiomyocytes, with heart-directed expression of the EPAC-FRET sensor, stimulated with FSK, and as determined by ELISA, also in mouse cardiomyocytes stimulated with the ß-AR agonist isoproterenol (ISO) (anxA4a(+/+) vs. anxA4a(-/-): 5.1 ± 0.3 vs. 6.7 ± 0.6 fmol/µg protein) or FSK (anxA4a(+/+) vs. anxA4a(-/-): 1891 ± 238 vs. 2796 ± 343 fmol/µg protein; n = 9-10). Coimmunoprecipitation experiments in HEK293 cells revealed a direct interaction of murine AnxA4 with human membrane-bound AC type 5 (AC5). As a functional consequence of AnxA4-mediated AC inhibition, AnxA4 inhibited the FSK-induced transcriptional activation mediated by the cAMP response element (CRE) in reporter gene studies (10-fold vs. control; n = 4 transfections) and reduced the FSK-induced phosphorylation of the CRE-binding protein (CREB) measured on Western blots (control vs. +AnxA4: 150 ± 17% vs. 105 ± 10%; n = 6) and by the use of the indicator of CREB activation caused by phosphorylation (ICAP)-FRET sensor, indicating CREB phosphorylation. Inactivation of AnxA4 in anxA4a(-/-) mice was associated with an increased cardiac response to ß-AR stimulation. Together, these results suggest that AnxA4 is a novel direct negative regulator of AC5, adding a new facet to the functions of annexins.


Assuntos
Adenilil Ciclases/metabolismo , Anexina A4/metabolismo , Membrana Celular/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Adenilil Ciclases/genética , Animais , Anexina A4/genética , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Membrana Celular/genética , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Fosforilação/fisiologia
7.
J Biol Chem ; 289(49): 33862-73, 2014 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-25320082

RESUMO

Dephosphorylation of important myocardial proteins is regulated by protein phosphatase 2A (PP2A), representing a heterotrimer that is comprised of catalytic, scaffolding, and regulatory (B) subunits. There is a multitude of B subunit family members directing the PP2A holoenzyme to different myocellular compartments. To gain a better understanding of how these B subunits contribute to the regulation of cardiac performance, we generated transgenic (TG) mice with cardiomyocyte-directed overexpression of B56α, a phosphoprotein of the PP2A-B56 family. The 2-fold overexpression of B56α was associated with an enhanced PP2A activity that was localized mainly in the cytoplasm and myofilament fraction. Contractility was enhanced both at the whole heart level and in isolated cardiomyocytes of TG compared with WT mice. However, peak amplitude of [Ca]i did not differ between TG and WT cardiomyocytes. The basal phosphorylation of cardiac troponin inhibitor (cTnI) and the myosin-binding protein C was reduced by 26 and 35%, respectively, in TG compared with WT hearts. The stimulation of ß-adrenergic receptors by isoproterenol (ISO) resulted in an impaired contractile response of TG hearts. At a depolarizing potential of -5 mV, the ICa,L current density was decreased by 28% after administration of ISO in TG cardiomyocytes. In addition, the ISO-stimulated phosphorylation of phospholamban at Ser(16) was reduced by 27% in TG hearts. Thus, the increased PP2A-B56α activity in TG hearts is localized to specific subcellular sites leading to the dephosphorylation of important contractile proteins. This may result in higher myofilament Ca(2+) sensitivity and increased basal contractility in TG hearts. These effects were reversed by ß-adrenergic stimulation.


Assuntos
Coração/fisiologia , Contração Miocárdica/fisiologia , Miócitos Cardíacos/metabolismo , Proteína Fosfatase 2/metabolismo , Troponina I/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Regulação da Expressão Gênica , Coração/efeitos dos fármacos , Isoproterenol/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miofibrilas/genética , Miofibrilas/metabolismo , Fosforilação , Ligação Proteica , Multimerização Proteica , Proteína Fosfatase 2/genética , Transdução de Sinais , Troponina I/genética
8.
Arthritis Rheum ; 65(3): 792-804, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23440693

RESUMO

OBJECTIVE: There is increasing evidence that serotonin (5-hydroxytryptamine [5-HT]) and distinct 5-HT receptors are involved in the pathogenesis of systemic sclerosis. The aim of this study was to test the hypothesis that tropisetron, a routinely used antiemetic agent previously characterized as a 5-HT(3/4) receptor-modulating agent, can directly affect collagen synthesis in vitro and attenuate experimentally induced fibrosis in vivo. METHODS: Functional in vitro studies were performed using human dermal fibroblasts (HDFs). Signal transduction studies included immunofluorescence analysis, Western immunoblotting, promoter reporter assays, cAMP/Ca(2+) measurements, and use of pharmacologic activators and inhibitors. Gene silencing was performed using small interfering RNA. Putative receptors of tropisetron were detected by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. The murine model of bleomycin-induced scleroderma was used to assess the antifibrogenic and antifibrotic effects of tropisetron in vivo. Collagen expression in vitro, ex vivo, and in situ was determined by real-time RT-PCR analysis, Western immunoblotting, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunohistochemical analysis. RESULTS: Tropisetron suppressed collagen synthesis induced by transforming growth factor ß1 (TGFß1). This effect was independent of 5-HT(3/4) receptor but was mediated via α7 nicotinic acetylcholine receptor (α7nAChR). Suppression of TGFß1-induced collagen synthesis occurred via an unknown molecular mechanism not involving modulation of the Smad, cAMP, Akt, c-Jun, or MAPK pathway. In vivo, tropisetron not only prevented skin fibrosis but also reduced the collagen content in established dermal fibrosis induced by bleomycin. CONCLUSION: Tropisetron directly reduces collagen synthesis in HDFs via an α7nAChR-dependent mechanism. The antifibrogenic and antifibrotic effects of this agent observed in a mouse model of bleomycin- induced scleroderma indicate the future potential of tropisetron in the treatment of fibrotic diseases such as scleroderma.


Assuntos
Colágeno/biossíntese , Indóis/farmacologia , Receptores Nicotínicos/metabolismo , Escleroderma Sistêmico/tratamento farmacológico , Células 3T3 , Adulto , Idoso , Animais , Antibióticos Antineoplásicos/toxicidade , Bleomicina/toxicidade , Derme/efeitos dos fármacos , Derme/metabolismo , Derme/patologia , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/tratamento farmacológico , Fibrose/metabolismo , Fibrose/patologia , Humanos , Camundongos , Pessoa de Meia-Idade , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/patologia , Antagonistas da Serotonina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo , Tropizetrona , Receptor Nicotínico de Acetilcolina alfa7
9.
Front Cardiovasc Med ; 11: 1419597, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38863902

RESUMO

Background: Protein phosphatase 2A (PP2A) is a serine/threonine-selective holoenzyme that controls Ca2+ homeostasis and contractility of the heart via dephosphorylation of regulatory proteins. In some genetically modified mouse models with increased arrhythmogenicity, a reduced expression of the regulatory subunit B56α of PP2A was found as a concomitant effect. Whether there is a general correlation between the abundance of B56α and the promotion of cardiac arrhythmogenesis remains unclear. Methods: The aim of this study was therefore to investigate the role of PP2A-B56α in the propensity for arrhythmic activity in the heart. The experimental analysis of this question has been addressed by using a mouse model with deletion of the PP2A-B56α gene, PPP2R5A (KO), in comparison to wild-type animals (WT). Evidence for arrhythmogenicity was investigated in whole animal, isolated heart and cardiomyocytes by ECG, recording of monophasic action potential (MAP) induced by programmed electrical stimulation (PES), measurement of Ca2+ transients under increased pacing frequencies and determination of total K+ channel currents (I K). Results: ECG measurements showed a prolongation of QT time in KO vs. WT. KO mice exhibited a higher rate of premature ventricular contractions in the ECG. MAP measurements in Langendorff-perfused KO hearts showed increased episodes of ventricular tachyarrhythmia induced by PES. However, the KO hearts showed values for MAP duration that were similar to those in WT hearts. In contrast, KO showed more myocardial cells with spontaneous arrhythmogenic Ca2+ transient events compared to WT. The whole-cell patch-clamp technique applied to ventricular cardiomyocytes revealed comparable peak potassium channel current densities between KO and WT. Conclusion: These findings support the assumption that a decrease or even the loss of PP2A-B56α leads to an increased propensity of triggered arrhythmias. This could be based on the increased spontaneous Ca2+ tansients observed.

10.
Int J Cardiol Heart Vasc ; 44: 101168, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36620202

RESUMO

Aims: Na+/Ca2+ exchanger (NCX) upregulation in cardiac diseases like heart failure promotes as an independent proarrhythmic factor early and delayed afterdepolarizations (EADs/DADs) on the single cell level. Consequently, NCX inhibition protects against EADs and DADs in isolated cardiomyocytes. We here investigate, whether these promising cellular in vitro findings likewise apply to an in vivo setup. Methods/Results: Programmed ventricular stimulation (PVS) and isoproterenol were applied to a murine heterozygous NCX-knockout model (KO) to investigate ventricular arrhythmia initiation and perpetuation compared to wild-type (WT). KO displayed a reduced susceptibility towards isoproterenol-induced premature ventricular complexes. During PVS, initiation of single or double ectopic beats was similar between KO and WT. But strikingly, perpetuation of ventricular tachycardia (VT) was significantly increased in KO (animals with VT - KO: 82 %; WT: 47 %; p = 0.0122 / median number of VTs - KO: 4.5 (1.0, 6.25); WT: 0.0 (0.0, 4.0); p = 0.0039). The median VT duration was prolonged in KO (in s; KO: 0.38 (0.19, 0.96); WT: 0.0 (0.0, 0.60); p = 0.0239). The ventricular refractory period (VRP) was shortened in KO (in ms; KO: 15.1 ± 0.7; WT: 18.7 ± 0.7; p = 0.0013). Conclusions: Not the initiation, but the perpetuation of provoked whole-heart in vivo ventricular arrhythmia was increased in KO. As a potential mechanism, we found a significantly reduced VRP, which may promote perpetuation of reentrant ventricular arrhythmia. On a translational perspective, the antiarrhythmic concept of therapeutic NCX inhibition seems to be ambivalent by protecting from initiating afterdepolarizations but favoring arrhythmia perpetuation in vivo at least in a murine model.

11.
Basic Res Cardiol ; 107(2): 247, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22327339

RESUMO

The cardiac Na(+)/Ca(2+) exchanger (NCX) generates an inward electrical current during SR-Ca(2+) release, thus possibly promoting afterdepolarizations of the action potential (AP). We used transgenic mice 12.5 weeks or younger with cardiomyocyte-directed overexpression of NCX (NCX-Tg) to study the proarrhythmic potential and mechanisms of enhanced NCX activity. NCX-Tg exhibited normal echocardiographic left ventricular function and heart/body weight ratio, while the QT interval was prolonged in surface ECG recordings. Langendorff-perfused NCX-Tg, but not wild-type (WT) hearts, developed ventricular tachycardia. APs and ionic currents were measured in isolated cardiomyocytes. Cell capacitance was unaltered between groups. APs were prolonged in NCX-Tg versus WT myocytes along with voltage-activated K(+) currents (K(v)) not being reduced but even increased in amplitude. During abrupt changes in pacing cycle length, early afterdepolarizations (EADs) were frequently recorded in NCX-Tg but not in WT myocytes. Next to EADs, delayed afterdepolarizations (DAD) triggering spontaneous APs (sAPs) occurred in NCX-Tg but not in WT myocytes. To test whether sAPs were associated with spontaneous Ca(2+) release (sCR), Ca(2+) transients were recorded. Despite the absence of sAPs in WT, sCR was observed in myocytes of both genotypes suggesting a facilitated translation of sCR into DADs in NCX-Tg. Moreover, sCR was more frequent in NCX-Tg as compared to WT. Myocardial protein levels of Ca(2+)-handling proteins were not different between groups except the ryanodine receptor (RyR), which was increased in NCX-Tg versus WT. We conclude that NCX overexpression is proarrhythmic in a non-failing environment even in the absence of reduced K(V). The underlying mechanisms are: (1) occurrence of EADs due to delayed repolarization; (2) facilitated translation from sCR into DADs; (3) proneness to sCR possibly caused by altered Ca(2+) handling and/or increased RyR expression.


Assuntos
Potenciais de Ação/fisiologia , Arritmias Cardíacas/metabolismo , Coração/fisiologia , Proteínas de Homeodomínio/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Arritmias Cardíacas/genética , Western Blotting , Modelos Animais de Doenças , Eletrocardiografia , Proteínas de Homeodomínio/genética , Camundongos , Técnicas de Cultura de Órgãos
12.
Fundam Clin Pharmacol ; 33(1): 43-51, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30092622

RESUMO

Administration of digitalis in heart failure (HF) increases quality of life but does not carry a prognostic benefit. Digitalis is an indirect inhibitor of the Na+ /Ca2+ exchanger (NCX), which is overexpressed in HF. We therefore used the cardiac glycoside ouabain in Ca2+ imaging experiments and patch-clamp experiments in isolated ventricular myocytes from nonfailing transgenic NCX overexpressor mice (OE). In field-stimulated myocytes, ouabain (1-100 µm) increased the amplitude of the Ca2+ transient in OE and wild-type (WT) similarly. Ouabain-mediated spontaneous Ca2+ -activity was significantly more pronounced in OE compared to WT myocytes at higher concentrations (100 µm). Also, at very high concentrations (1000 µm) of ouabain, the number of cells with hypercontraction leading to cell death was higher in OE. Ouabain (10 µm) shortened the action potential duration in both genotypes. Our findings suggest that the proarrhythmic but not the inotropic effects of cardiac glycosides are enhanced by increased NCX expression. This may offer an explanation for the observed lack of prognostic benefit but increased quality of life in HF, which is accompanied by NCX upregulation.


Assuntos
Cálcio/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Ouabaína/administração & dosagem , Trocador de Sódio e Cálcio/genética , Potenciais de Ação/efeitos dos fármacos , Animais , Cardiotônicos/administração & dosagem , Cardiotônicos/farmacologia , Relação Dose-Resposta a Droga , Feminino , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/citologia , Ventrículos do Coração/efeitos dos fármacos , Masculino , Camundongos , Camundongos Transgênicos , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ouabaína/farmacologia , Técnicas de Patch-Clamp , Qualidade de Vida
13.
Front Pharmacol ; 9: 933, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30186171

RESUMO

Background: Principal mechanisms of arrhythmia have been derived from ventricular but not atrial cardiomyocytes of animal models despite higher prevalence of atrial arrhythmia (e.g., atrial fibrillation). Due to significant ultrastructural and functional differences, a simple transfer of ventricular proneness toward arrhythmia to atrial arrhythmia is critical. The use of murine models in arrhythmia research is widespread, despite known translational limitations. We here directly compare atrial and ventricular mechanisms of arrhythmia to identify critical differences that should be considered in murine models for development of antiarrhythmic strategies for atrial arrhythmia. Methods and Results: Isolated murine atrial and ventricular myocytes were analyzed by wide field microscopy and subjected to a proarrhythmic protocol during patch-clamp experiments. As expected, the spindle shaped atrial myocytes showed decreased cell area and membrane capacitance compared to the rectangular shaped ventricular myocytes. Though delayed afterdepolarizations (DADs) could be evoked in a similar fraction of both cell types (80% of cells each), these led significantly more often to the occurrence of spontaneous action potentials (sAPs) in ventricular myocytes. Interestingly, numerous early afterdepolarizations (EADs) were observed in the majority of ventricular myocytes, but there was no EAD in any atrial myocyte (EADs per cell; atrial myocytes: 0 ± 0; n = 25/12 animals; ventricular myocytes: 1.5 [0-43]; n = 20/12 animals; p < 0.05). At the same time, the action potential duration to 90% decay (APD90) was unaltered and the APD50 even increased in atrial versus ventricular myocytes. However, the depolarizing L-type Ca2+ current (ICa) and Na+/Ca2+-exchanger inward current (INCX) were significantly smaller in atrial versus ventricular myocytes. Conclusion: In mice, atrial myocytes exhibit a substantially distinct occurrence of proarrhythmic afterdepolarizations compared to ventricular myocytes, since they are in a similar manner susceptible to DADs but interestingly seem to be protected against EADs and show less sAPs. Key factors in the generation of EADs like ICa and INCX were significantly reduced in atrial versus ventricular myocytes, which may offer a mechanistic explanation for the observed protection against EADs. These findings may be of relevance for current studies on atrial level in murine models to develop targeted strategies for the treatment of atrial arrhythmia.

14.
Front Pharmacol ; 8: 649, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28983248

RESUMO

Background/Objective: The cardiac Na+/Ca2+ exchanger (NCX) has been identified as a promising target to counter arrhythmia in previous studies investigating the benefit of NCX inhibition. However, the consequences of NCX inhibition have not been investigated in the setting of altered NCX expression and function, which is essential, since major cardiac diseases (heart failure/atrial fibrillation) exhibit NCX upregulation. Thus, we here investigated the effects of the NCX inhibitor SEA0400 on the Ca2+ transient amplitude and on proarrhythmia in homozygous NCX overexpressor (OE) and heterozygous NCX knockout (hetKO) mice compared to corresponding wild-types (WTOE/WThetKO). Methods/Results: Ca2+ transients of field-stimulated isolated ventricular cardiomyocytes were recorded with fluo-4-AM or indo-1-AM. SEA0400 (1 µM) significantly reduced NCX forward mode function in all mouse lines. SEA0400 (1 µM) significantly increased the amplitude of field-stimulated Ca2+ transients in WTOE, WThetKO, and hetKO, but not in OE (% of basal; OE = 98.7 ± 5.0; WTOE = 137.8 ± 5.2*; WThetKO = 126.3 ± 6.0*; hetKO = 140.6 ± 12.8*; *p < 0.05 vs. basal). SEA0400 (1 µM) significantly reduced the number of proarrhythmic spontaneous Ca2+ transients (sCR) in OE, but increased it in WTOE, WThetKO and hetKO (sCR per cell; basal/+SEA0400; OE = 12.5/3.7; WTOE = 0.2/2.4; WThetKO = 1.3/8.8; hetKO = 0.2/5.5) and induced Ca2+ overload with subsequent cell death in hetKO. Conclusion: The effects of SEA0400 on Ca2+ transient amplitude and the occurrence of spontaneous Ca2+ transients as a proxy measure for inotropy and cellular proarrhythmia depend on the NCX expression level. The antiarrhythmic effect of SEA0400 in conditions of increased NCX expression promotes the therapeutic concept of NCX inhibition in heart failure/atrial fibrillation. Conversely, in conditions of reduced NCX expression, SEA0400 suppressed the NCX function below a critical level leading to adverse Ca2+ accumulation as reflected by an increase in Ca2+ transient amplitude, proarrhythmia and cell death. Thus, the remaining NCX function under inhibition may be a critical factor determining the inotropic and antiarrhythmic efficacy of SEA0400.

15.
Prog Biophys Mol Biol ; 130(Pt B): 233-243, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28526353

RESUMO

Two-pore-domain potassium (K2P) channels modulate cellular excitability. The significance of stretch-activated cardiac K2P channels (K2P2.1, TREK-1, KCNK2; K2P4.1, TRAAK, KCNK4; K2P10.1, TREK-2, KCNK10) in heart disease has not been elucidated in detail. The aim of this work was to assess expression and remodeling of mechanosensitive K2P channels in atrial fibrillation (AF) and heart failure (HF) patients in comparison to murine models. Cardiac K2P channel levels were quantified in atrial (A) and ventricular (V) tissue obtained from patients undergoing open heart surgery. In addition, control mice and mouse models of AF (cAMP-response element modulator (CREM)-IbΔC-X transgenic animals) or HF (cardiac dysfunction induced by transverse aortic constriction, TAC) were employed. Human and murine KCNK2 displayed highest mRNA abundance among mechanosensitive members of the K2P channel family (V > A). Disease-associated K2P2.1 remodeling was studied in detail. In patients with impaired left ventricular function, atrial KCNK2 (K2P2.1) mRNA and protein expression was significantly reduced. In AF subjects, downregulation of atrial and ventricular KCNK2 (K2P2.1) mRNA and protein levels was observed. AF-associated suppression of atrial Kcnk2 (K2P2.1) mRNA and protein was recapitulated in CREM-transgenic mice. Ventricular Kcnk2 expression was not significantly altered in mouse models of disease. In conclusion, mechanosensitive K2P2.1 and K2P10.1 K+ channels are expressed throughout the heart. HF- and AF-associated downregulation of KCNK2 (K2P2.1) mRNA and protein levels suggest a mechanistic contribution to cardiac arrhythmogenesis.


Assuntos
Fibrilação Atrial/metabolismo , Insuficiência Cardíaca/metabolismo , Fenômenos Mecânicos , Miocárdio/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Idoso , Fibrilação Atrial/genética , Fenômenos Biomecânicos , Regulação para Baixo , Feminino , Insuficiência Cardíaca/genética , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Canais de Potássio de Domínios Poros em Tandem/química , Canais de Potássio de Domínios Poros em Tandem/genética , Conformação Proteica , Transporte Proteico , Regulação para Cima
16.
Circ Arrhythm Electrophysiol ; 8(5): 1210-8, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26338832

RESUMO

BACKGROUND: The Na(+)/Ca(2+) exchanger (NCX) has been implied to cause arrhythmias. To date, information on the role of NCX in arrhythmogenesis derived from models with increased NCX expression, hypertrophy, and heart failure. Furthermore, the exact mechanism by which NCX exerts its potentially proarrhythmic effect, ie, by promoting early afterdepolarization (EAD) or delayed afterdepolarization (DAD) or both, is unknown. METHODS AND RESULTS: We investigated isolated cardiomyocytes from a murine model with heterozygous knockout of NCX (hetKO) using the patch clamp and Ca(2+) imaging techniques. Action potential duration was shorter in hetKO with IKtot not being increased. The rate of spontaneous Ca(2+) release events and the rate of DADs were unaltered; however, DADs had lower amplitude in hetKO. A DAD triggered a spontaneous action potential significantly less often in hetKO when compared with wild-type. The occurrence of EADs was also drastically reduced in hetKO. ICa activity was reduced in hetKO, an effect that was abolished in the presence of the Ca(2+) buffer BAPTA. CONCLUSIONS: Genetic suppression of NCX reduces both EADs and DADs. The following molecular mechanisms apply: (1) Although the absolute number of DADs is unaffected, an impaired translation of DADs into spontaneous action potentials results from a reduced DAD amplitude. (2) EADs are reduced in absolute number of occurrence, which is presumably a consequence of shortened action potential duration because of reduced NCX activity but also reduced ICa the latter possibly being caused by a direct modulation of Ca(2+)-dependent ICa inhibition by reduced NCX activity. This is the first study to demonstrate that genetic inhibition of NCX protects against afterdepolarizations and to investigate the underlying mechanisms.


Assuntos
Arritmias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Potenciais de Ação/fisiologia , Animais , Arritmias Cardíacas/genética , Modelos Animais de Doenças , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Heterozigoto , Immunoblotting , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp , Trocador de Sódio e Cálcio/genética
17.
Circ Heart Fail ; 8(1): 79-88, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25424392

RESUMO

BACKGROUND: New therapeutic approaches to improve cardiac contractility without severe risk would improve the management of acute heart failure. Increasing systolic sodium influx can increase cardiac contractility, but most sodium channel activators have proarrhythmic effects that limit their clinical use. Here, we report the cardiac effects of a novel positive inotropic peptide isolated from the toxin of the Black Judean scorpion that activates neuronal tetrodotoxin-sensitive sodium channels. METHODS AND RESULTS: All venoms and peptides were isolated from Black Judean Scorpions (Buthotus Hottentotta) caught in the Judean Desert. The full scorpion venom increased left ventricular function in sedated mice in vivo, prolonged ventricular repolarization, and provoked ventricular arrhythmias. An inotropic peptide (BjIP) isolated from the full venom by chromatography increased cardiac contractility but did neither provoke ventricular arrhythmias nor prolong cardiac repolarization. BjIP increased intracellular calcium in ventricular cardiomyocytes and prolonged inactivation of the cardiac sodium current. Low concentrations of tetrodotoxin (200 nmol/L) abolished the effect of BjIP on calcium transients and sodium current. BjIP did not alter the function of Nav1.5, but selectively activated the brain-type sodium channels Nav1.6 or Nav1.3 in cellular electrophysiological recordings obtained from rodent thalamic slices. Nav1.3 (SCN3A) mRNA was detected in human and mouse heart tissue. CONCLUSIONS: Our pilot experiments suggest that selective activation of tetrodotoxin-sensitive neuronal sodium channels can safely increase cardiac contractility. As such, the peptide described here may become a lead compound for a new class of positive inotropic agents.


Assuntos
Insuficiência Cardíaca/tratamento farmacológico , Ventrículos do Coração/efeitos dos fármacos , Coração/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Sódio/metabolismo , Tetrodotoxina/farmacologia , Animais , Modelos Animais de Doenças , Coração/fisiologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/metabolismo , Camundongos , Contração Miocárdica/efeitos dos fármacos , Projetos Piloto , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/metabolismo
18.
Int J Cardiol ; 166(2): 366-74, 2013 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-22093963

RESUMO

BACKGROUND AND METHODS: Atrial fibrillation (AF) is the most common cardiac arrhythmia in clinical practice. The substrate of AF is composed of a complex interplay between structural and functional changes of the atrial myocardium often preceding the occurrence of persistent AF. However, there are only few animal models reproducing the slow progression of the AF substrate to the spontaneous occurrence of the arrhythmia. Transgenic mice (TG) with cardiomyocyte-directed expression of CREM-IbΔC-X, an isoform of transcription factor CREM, develop atrial dilatation and spontaneous-onset AF. Here we tested the hypothesis that TG mice develop an arrhythmogenic substrate preceding AF using physiological and biochemical techniques. RESULTS: Overexpression of CREM-IbΔC-X in young TG mice (<8weeks) led to atrial dilatation combined with distension of myocardium, elongated myocytes, little fibrosis, down-regulation of connexin 40, loss of excitability with a number of depolarized myocytes, atrial ectopies and inducibility of AF. These abnormalities continuously progressed with age resulting in interatrial conduction block, increased atrial conduction heterogeneity, leaky sarcoplasmic reticulum calcium stores and the spontaneous occurrence of paroxysmal and later persistent AF. This distinct atrial remodelling was associated with a pattern of non-regulated and up-regulated marker genes of myocardial hypertrophy and fibrosis. CONCLUSIONS: Expression of CREM-IbΔC-X in TG hearts evokes abnormal growth and development of the atria preceding conduction abnormalities and altered calcium homeostasis and the development of spontaneous and persistent AF. We conclude that transcription factor CREM is an important regulator of atrial growth implicated in the development of an arrhythmogenic substrate in TG mice.


Assuntos
Fibrilação Atrial/metabolismo , Modulador de Elemento de Resposta do AMP Cíclico/biossíntese , Regulação da Expressão Gênica , Átrios do Coração/metabolismo , Miocárdio/metabolismo , Animais , Fibrilação Atrial/patologia , Fibrilação Atrial/fisiopatologia , Átrios do Coração/patologia , Átrios do Coração/fisiopatologia , Camundongos , Camundongos Transgênicos , Miocárdio/patologia , Técnicas de Cultura de Órgãos , Fatores de Tempo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA