RESUMO
AIMS: In atrial fibrillation, increased function of the Na+/Ca2+-exchanger (NCX) is one among several electrical remodeling mechanisms. METHODS/RESULTS: Using the patch-clamp- and Ca2+ imaging-methods, we investigated atrial myocytes from NCX-homozygous-overexpressor (OE)- and heterozygous-knockout (KO)-mice and their corresponding wildtypes (WTOE; WTKO). NCX mediated Ca2+ extrusion capacity was reduced in KO and increased in OE. There was no evidence for structural or molecular remodeling. During a proarrhythmic pacing-protocol, the number of low amplitude delayed afterdepolarizations (DADs) was unaltered in OE vs. WTOE and KO vs. WTKO. However, DADs triggered full spontaneous action potentials (sAP) significantly more often in OE vs. WTOE (ratio sAP/DAD: OE:0.18±0.05; WTOE:0.02±0.02; p<0.001). Using the same protocol, a DAD triggered an sAP by tendency less often in KO vs. WTKO (p=0.06) and significantly less often under a more aggressive proarrhythmic protocol (ratio sAP/DAD: KO:0.01±0.003; WT KO: 0.12±0.05; p=0.007). The DAD amplitude was increased in OE vs. WTOE and decreased in KO vs. WTKO. There were no differences in SR-Ca2+-load, the number of spontaneous Ca2+-release-events or IKACh/IK1. CONCLUSIONS: Atrial myocytes with increased NCX expression exhibited increased vulnerability towards sAPs while atriomyocytes with reduced NCX expression were protected. The underlying mechanism consists of a modification of the DAD-amplitude by the level of NCX-activity. Thus, although the number of spontaneous Ca2+-releases and therefore DADs is unaltered, the higher DAD-amplitude in OE made a transgression of the voltage-threshold of an sAP more likely. These findings indicate that the level of NCX activity could influence triggered activity in atrial myocytes independent of possible remodeling processes.
Assuntos
Átrios do Coração/metabolismo , Miócitos Cardíacos/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Potenciais de Ação/genética , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Feminino , Expressão Gênica , Masculino , Potenciais da Membrana/genética , Camundongos , Camundongos Transgênicos , Contração Miocárdica/genética , Miocárdio/metabolismo , Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/genéticaRESUMO
Chronic ß-adrenergic stimulation is regarded as a pivotal step in the progression of heart failure which is associated with a high risk for arrhythmia. The cAMP-dependent transcription factors cAMP-responsive element binding protein (CREB) and cAMP-responsive element modulator (CREM) mediate transcriptional regulation in response to ß-adrenergic stimulation and CREM repressor isoforms are induced after stimulation of the ß-adrenoceptor. Here, we investigate whether CREM repressors contribute to the arrhythmogenic remodeling in the heart by analyzing arrhythmogenic alterations in ventricular cardiomyocytes (VCMs) from mice with transgenic expression of the CREM repressor isoform CREM-IbΔC-X (TG). Patch clamp analyses, calcium imaging, immunoblotting and real-time quantitative RT-PCR were conducted to study proarrhythmic alterations in TG VCMs vs. wild-type controls. The percentage of VCMs displaying spontaneous supra-threshold transient-like Ca(2+) releases was increased in TG accompanied by an enhanced transduction rate of sub-threshold Ca(2+) waves into these supra-threshold events. As a likely cause we discovered enhanced NCX-mediated Ca(2+) transport and NCX1 protein level in TG. An increase in I NCX and decrease in I to and its accessory channel subunit KChIP2 was associated with action potential prolongation and an increased proportion of TG VCMs showing early afterdepolarizations. Finally, ventricular extrasystoles were augmented in TG mice underlining the in vivo relevance of our findings. Transgenic expression of CREM-IbΔC-X in mouse VCMs leads to distinct arrhythmogenic alterations. Since CREM repressors are inducible by chronic ß-adrenergic stimulation our results suggest that the inhibition of CRE-dependent transcription contributes to the formation of an arrhythmogenic substrate in chronic heart disease.
Assuntos
Arritmias Cardíacas/metabolismo , Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Potenciais de Ação , Animais , Arritmias Cardíacas/fisiopatologia , Cálcio/metabolismo , Células Cultivadas , Modulador de Elemento de Resposta do AMP Cíclico/antagonistas & inibidores , Modulador de Elemento de Resposta do AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ventrículos do Coração/fisiopatologia , Isoproterenol , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Potássio/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/metabolismoRESUMO
The cAMP response element binding protein (CREB) belongs to the CREB/cAMP response element binding modulator/activating transcription factor 1 family of cAMP-dependent transcription factors mediating a regulation of gene transcription in response to cAMP. Chronic stimulation of ß-adrenergic receptors and the cAMP-dependent signal transduction pathway by elevated plasma catecholamines play a central role in the pathogenesis of heart failure. Ion channel remodeling, particularly a decreased transient outward current (I(to)), and subsequent action potential (AP) prolongation are hallmarks of the failing heart. Here, we studied the role of CREB for ion channel regulation in mice with a cardiomyocyte-specific knockout of CREB (CREB KO). APs of CREB KO cardiomyocytes were prolonged with increased AP duration at 50 and 70% repolarization and accompanied by a by 51% reduction of I(to) peak amplitude as detected in voltage-clamp measurements. We observed a 29% reduction of Kcnd2/Kv4.2 mRNA in CREB KO cardiomyocytes mice while the other I(to)-related channel subunits Kv4.3 and KChIP2 were not different between groups. Accordingly, Kv4.2 protein was reduced by 37% in CREB KO. However, we were not able to detect a direct regulation of Kv4.2 by CREB. The I(to)-dependent AP prolongation went along with an increase of I(Na) and a decrease of I(Ca,L) associated with an upregulation of Scn8a/Nav1.6 and downregulation of Cacna1c/Cav1.2 mRNA in CREB KO cardiomyocytes. Our results from mice with cardiomyocyte-specific inactivation of CREB definitively indicate that CREB critically regulates the AP shape and duration in the mouse ventricle, which might have an impact on ion channel remodeling in situations of altered cAMP-dependent signaling like heart failure.
Assuntos
Potenciais de Ação/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Ventrículos do Coração/citologia , Canais Iônicos/fisiologia , Miócitos Cardíacos/fisiologia , Função Ventricular/fisiologia , Animais , Canais de Cálcio Tipo L/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/deficiência , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Regulação para Baixo/fisiologia , Camundongos , Camundongos Knockout , Modelos Animais , Miócitos Cardíacos/citologia , Canal de Sódio Disparado por Voltagem NAV1.6 , Proteínas do Tecido Nervoso/fisiologia , Técnicas de Patch-Clamp , Transdução de Sinais/fisiologia , Canais de Sódio/fisiologia , Regulação para Cima/fisiologiaRESUMO
We wanted to elucidate whether extracellular calcium may regulate the expression of the cardiac gap-junction proteins connexin 40 and connexin43. In the free wall of the left atria of 126 cardiac surgery patients with either sinus rhythm (SR) or chronic atrial fibrillation (AF), we determined the expression of the cardiac gap-junction proteins Cx43 and Cx40 by Western blot and immunohistology. For deeper investigation, we incubated cultured neonatal rat cardiomyocytes at 2 or 4 mM Ca(++) for 24 h and determined intercellular coupling, Cx40, Cx43 protein and mRNA expression, protein trafficking and sensitivity to verapamil (10-100 nM), cyclosporin A (1 microM),and BMS605401 (100 nM), a specific inhibitor of Ca(2+)-sensing receptor (CaSR). We found in patients that both Cx are up-regulated in AF in the left atrium (by 100-200%). Interestingly, Cx40 was mainly up-regulated, if total serum calcium was >or=2.2 mM, while Cx43 was independent from extracellular [Ca(++)]. In cultured cells, 4 mM Ca(++)-exposure lead to up-regulation of Cx40, but not Cx43. We found enhanced Cx40 in the plasma membrane and reduced Cx40 in the Golgi apparatus. The membrane Cx40 up-regulation resulted in enhanced gap-junction intercellular coupling with a shift in the Boltzmann fit of voltage-dependent inactivation indicating a higher contribution of Cx40 as revealed by dual whole cell voltage clamp experiments. BMS605401 could prevent all Ca(2+)-induced changes. Moreover, cyclosporin A completely abolished the Ca(2+)-induced changes, while verapamil was ineffective. We conclude that extracellular calcium (24 h exposure) seems to up-regulate Cx40 but not Cx43.
Assuntos
Cálcio/fisiologia , Junções Comunicantes/fisiologia , Animais , Fibrilação Atrial/metabolismo , Células Cultivadas , Conexina 43/análise , Conexina 43/fisiologia , Conexinas/análise , Conexinas/fisiologia , Ciclosporina/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ratos , Ratos Wistar , Transdução de Sinais , Verapamil/farmacologia , Proteína alfa-5 de Junções ComunicantesRESUMO
BACKGROUND: The molecular pathomechanisms underlying atrial thrombogenesis are multifactorial and still require detailed investigations. Transgenic mice with cardiomyocyte-directed expression of the transcriptional repressor CREM-IbΔC-X (CREM-TG) represent an experimental model of atrial fibrillation (AF) that shows a gradual, age-dependent progression from atrial ectopy to persistent AF. Importantly, this model develops biatrial thrombi. The molecular characteristics related to the thrombogenesis in CREM-TG mice have not been studied, yet. METHODS: The inflammatory and prothrombotic state was evaluated at the transcriptional (qRT-PCR) and protein level in the left (LA) and right atria (RA) from CREM-TG mice at the age of 20weeks and compared to wild-type controls. Moreover, histological analyses of atrial thrombi were performed. RESULTS: The endocardial dysfunction was mirrored by diminished levels of eNOS-mRNA in both atria (RA: 0.79±0.04, LA: 0.72±0.06; each P<0.05). Moreover, the PAI-1/t-PA mRNA ratio was significantly increased in both atria (RA: 3.6±0.6; P<0.01, LA: 4.0±1.0; P<0.05) indicating a high risk of thrombus formation. However, the inflammatory phenotype was more pronounced in the RA and was reflected by a significant increase in the mRNA levels encoding adhesion molecules ICAM-1 (2.1±0.2; P<0.01), VCAM-1 (2.3±0.5; P<0.05), and selectin P (3.6±0.5: P<0.05). CONCLUSIONS: CREM-TG mice represent a valuable model for studying atrial thrombogenesis and assessing therapeutic approaches preventing embolic events in the systemic and pulmonary circulation.
Assuntos
Fibrilação Atrial/genética , Trombose/genética , Animais , Fibrilação Atrial/metabolismo , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Trombose/metabolismoRESUMO
Human congestive heart failure is accompanied by structural and electrical alterations leading to the development of an arrhythmogenic substrate. This substrate is associated with the "sudden cardiac death" due to ventricular tachycardia or ventricular fibrillation. Multiple studies link distinct transcription factors to the transcriptional regulation of genes related to the formation of an arrhythmogenic substrate. In addition to cardiac hypertrophy the up- or downregulation of ion channels, calcium-handling proteins, and proteins forming gap junctions play a pivotal role in the progression of heart failure. This review summarizes the transcriptional regulation of selected genes implicated in the formation of an arrhythmogenic substrate. In this context we provide an overview of relevant transcription factors, activating stimuli and pathways, the evidence of binding to respective elements in the promoter of target genes and the associated mRNA regulation in animal models. Finally, possible therapeutic consequences are discussed.