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1.
J Physiol Sci ; 70(1): 39, 2020 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-32895058

RESUMO

Signal regulators during early cardiogenetic differentiation for the cellular automaticity are largely unknown. Our investigations were designed to clarify the role of transcription factors and their modulators in P19-derived cardiomyocytes to the expression of cardiac pacemaker ion channels. Transcription factors Csx/Nkx2.5 and GATA4 but not MEF2C were markedly inhibited by p38 MAP kinase inhibition in a distinct manner; expression but not phosphorylation of GATA4 was reduced by inhibition of p38 MAP kinase actions. In the presence of an ERK1/2,5 inhibitor PD98059 or a JNK MAP kinase inhibitor SP600125, P19 cells successfully differentiated into cardiomyocytes displaying spontaneous beatings with expression of three types of pacemaker ion channels. We demonstrate that acquisition of cellular automaticity and the expression of pacemaker ion channels are regulated by the transcription factors, Csx/Nkx2.5 and GATA4, through intracellular signals including p38 MAP kinase in the process of P19-derived pluripotent cells differentiation into cardiomyocytes.


Assuntos
Relógios Biológicos , Diferenciação Celular , Frequência Cardíaca , Células-Tronco Pluripotentes Induzidas/enzimologia , Canais Iônicos/metabolismo , Miócitos Cardíacos/enzimologia , Fatores de Transcrição/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Potenciais de Ação , Animais , Linhagem Celular Tumoral , Fator de Transcrição GATA4/metabolismo , Proteína Homeobox Nkx-2.5/metabolismo , Fatores de Transcrição MEF2/metabolismo , Camundongos , Fenótipo , Fosforilação , Transdução de Sinais , Fatores de Tempo , Fatores de Transcrição/genética
2.
J Physiol Sci ; 70(1): 20, 2020 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-32213161

RESUMO

The homeobox-containing gene Csx/Nkx2.5 codes several cardiac transcription factors and plays a critical role in early cardiogenesis. We investigated the effect of Csx/Nkx2.5 on the expression of cardiac ion channels using P19-derived cardiomyocytes. P19CL6 cells and P19CL6 cells with Csx/Nkx2.5 overexpression (P19CL6-Csx cells) were induced to differentiate into cardiomyocytes by treatment with dimethyl sulfoxide. Action potentials and membrane currents were measured by whole cell patch clamp at different differentiation stage: the early stage (1-5 days after beating had begun) and the late stage (10-15 days after beating). Expression of Csx/Nkx2.5 mRNA was increased as the differentiation stages advanced in both P19CL6 and P19CL6-Csx cells. In action potential configuration, maximal diastolic potentials in P19CL6-Csx cells exhibited more hyperpolarized potential (‒ 64.2 mV) than those in P19CL6 cells (‒ 54.8 mV, p < 0.01) in the early stage. In P19CL6 cells, among 6 different voltage-gated and ligand-operated K+ channels expressed during the early stage, the transient-outward K+ channel was most predominant. By overexpression of Csx/Nkx2.5, developmental decrease in the transient-outward K+ channel was suppressed. Homeobox-containing gene Csx/Nkx2.5 modifies the amount of distinct ionic channels, during differentiation periods, predominantly changing the expression of the transient-outward K+ channel.


Assuntos
Proteína Homeobox Nkx-2.5/metabolismo , Miócitos Cardíacos/metabolismo , Canais de Potássio/metabolismo , Fatores de Transcrição/metabolismo , Potenciais de Ação , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Camundongos , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo
3.
Heart Vessels ; 28(5): 658-66, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23329163

RESUMO

We hypothesized that Ca(2+) entry through the window T-type Ca(2+) current causes apoptosis. To test this hypothesis, we transfected human embryonic kidney (HEK) 293 cells to express recombinant Cav3.2 T-type Ca(2+) channels (hereafter called HEK-Cav3.2 cells). After incubation in media containing a high concentration (7.2 mM) of Ca(2+), intracellular Ca(2+) levels increased in HEK-Cav3.2 cells without electrical stimulation but not in untransfected HEK293 cells. In quiescent HEK-Cav3.2 cells exposed to high Ca(2+) media, apoptosis, as indicated by the appearance of hypodiploid cells, loss of mitochondrial transmembrane potential, and activation of caspases-3 and -9 was observed, while caspase-8 was not activated. These apoptosis-associated changes were blunted by pretreatment with the R(-)-isomer of efonidipine, a selective blocker of T-type Ca(2+) channels. High Ca(2+) did not induce apoptosis in untransfected HEK293 cells. Our findings show that Ca(2+) entry through the steady-state window current of T-type Ca(2+) channels causes apoptosis via mitochondrial pathways, and suggests that T-type Ca(2+) channels may be novel therapeutic targets for several diseases associated with abnormal apoptosis.


Assuntos
Apoptose , Canais de Cálcio Tipo T/metabolismo , Sinalização do Cálcio , Mitocôndrias/metabolismo , Apoptose/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/efeitos dos fármacos , Canais de Cálcio Tipo T/genética , Caspase 3/metabolismo , Caspase 9/metabolismo , Ativação Enzimática , Células HEK293 , Humanos , Cinética , Potencial da Membrana Mitocondrial , Potenciais da Membrana , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Transfecção
4.
Endocrinology ; 150(2): 879-88, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18832095

RESUMO

T-type Ca(2+) channel current (I(Ca,T)) plays an important role for spontaneous pacemaker activity and is involved in the progression of structural heart diseases. Estrogens are of importance for the regulation of growth and differentiation and function in a wide array of target tissues, including those in the cardiovascular system. The aim of this study was to elucidate the short-term and long-term effects of 17beta-estradiol (E(2)) on I(Ca,T) in cardiomyocytes. We employed in vivo and in vitro techniques to clarify E(2)-mediated modulation of heart rate (HR) in ovariectomized rats and I(Ca,T) in cardiomyocytes. Ovariectomy increased HR and E(2) supplement reduced HR in ovariectomized rats. Slowing of E(2)-induced HR was consistent with the deceleration of automaticity in E(2)-treated neonatal cardiomyocytes. Short-term application of E(2) did not have significant effects on I(Ca,T), whereas in cardiomyocytes treated with 10 nm E(2) for 24 h, estrogen receptor-independent down-regulation of peak I(Ca,T) and declination of Ca(V)3.2 mRNA were observed. Expression of a cardiac-specific transcription factor Csx/Nkx2.5 was also suppressed by E(2) treatment for 24 h. On the other hand, expression of Ca(V)3.1 mRNA was unaltered by E(2) treatment in this study. An ERK-1/2, 5 inhibitor, PD-98059, abolished the effects of E(2) on I(Ca,T) and Ca(V)3.2 mRNA as well as Csx/Nkx2.5 mRNA. These findings indicate that E(2) decreases Ca(V)3.2 I(Ca,T) through activation of ERK-1/2, 5, which is mediated by the suppression of Csx/Nkx2.5-dependent transcription, suggesting a genomic effect of E(2) as a negative chronotropic factor in the heart.


Assuntos
Canais de Cálcio Tipo T/genética , Estradiol/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Miócitos Cardíacos/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Canais de Cálcio Tipo T/metabolismo , Canais de Cálcio Tipo T/fisiologia , Células Cultivadas , Eletrofisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Humanos , Miócitos Cardíacos/metabolismo , Ovariectomia/veterinária , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo
5.
Hypertens Res ; 31(9): 1781-90, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18971557

RESUMO

Pulmonary hypertension (PH) is a disease of unknown etiology that ultimately causes right ventricle heart failure with a lethal outcome. An increase in circulating endothelin (ET)-1 levels may contribute to disease progression. This study aimed to examine the possible effects of an orally active ET receptor antagonist, sulfisoxazole (SFX), for the rescue of PH, right ventricular hypertrophy, and eventual right ventricular failure. PH rats (single injection of monocrotaline [MCT]) were treated with an ET antagonist, SFX, an orally active sulfonamide antibody. Effects of SFX on PH rats were assessed in terms of survival rate, pulmonary artery blood pressure (PABP), autonomic nerve activity, and atrial natriuretic peptide (ANP) concentration in right ventricular myocytes and plasma. SFX did not change systemic blood pressure, however, it significantly suppressed the elevation of PABP. SFX maintained the derangement of autonomic nerve control, blunted an increase in ANP in myocytes and plasma, and significantly improved survival in right heart failure and/or related organs dysfunction in PH rats. The ET antagonistic action of the antimicrobial agent, SFX, was experimentally confirmed for treatment of PH in rats.


Assuntos
Anti-Infecciosos/farmacologia , Antagonistas dos Receptores de Endotelina , Hipertensão Pulmonar/tratamento farmacológico , Hipertrofia Ventricular Direita/tratamento farmacológico , Sulfisoxazol/farmacologia , Administração Oral , Animais , Fator Natriurético Atrial/sangue , Fator Natriurético Atrial/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Modelos Animais de Doenças , Eletrocardiografia , Endotelina-1/metabolismo , Frequência Cardíaca , Hipertensão Pulmonar/mortalidade , Hipertrofia Ventricular Direita/mortalidade , Hipertrofia Ventricular Direita/patologia , Masculino , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Artéria Pulmonar/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores de Endotelina/metabolismo , Sistema Nervoso Simpático/fisiologia
6.
Circ J ; 72(6): 1012-7, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18503231

RESUMO

BACKGROUND: Sepsis can be exacerbated by an inappropriate immune response and the severe impact of this disease on the cardiovascular system is well documented. High mobility group box 1 (HMGB1) protein is an important mediator in the pathogenesis of sepsis and its role in cardiovascular system dysfunction was investigated in an lipopolysaccharide (LPS)-induced rat model of sepsis. METHODS AND RESULTS: Twelve hours after intravenous bolus injections of LPS (5 mg/kg), rats were killed and heart samples were harvested. Immunoblot analysis was performed to assess expression levels of HMGB1 in cardiac myocytes. Left ventricular developed pressure (LVDP) served as a measure of systolic function. LPS administration was associated with an increase in the expression of HMGB1 in cardiac myocytes and a decrease in cardiac function. Hearts from the LPS-treated rats were also perfused with recombinant HMGB1 and cardiac function measured. The dose-dependent effects observed with elevated HMGB1 included decreased LVDP, decreased left ventricular (LV) + dP/dt(max), decreased absolute value of LV- dP/dt(min), and increased LV end-diastolic pressure. CONCLUSIONS: HMGB1 stimulation produces a negative inotropic effect during septic shock, suggesting an important role for this molecule in cardiovascular system dysfunction during sepsis.


Assuntos
Proteína HMGB1/metabolismo , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Choque Séptico/metabolismo , Choque Séptico/fisiopatologia , Animais , Modelos Animais de Doenças , Frequência Cardíaca , Lipopolissacarídeos/toxicidade , Masculino , Miocárdio/patologia , Projetos Piloto , Ratos , Ratos Wistar , Choque Séptico/induzido quimicamente , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/fisiopatologia , Pressão Ventricular
7.
J Mol Cell Cardiol ; 42(6): 1045-53, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17498735

RESUMO

The cardiac transcription factors Csx/Nkx2.5 and GATA4 play important roles in vertebrate heart development. Although mutations of Csx/Nkx2.5 or GATA4 are associated with various congenital heart diseases, their mechanism of action on cardiomyocyte function is not completely elucidated. In this study, we therefore investigated the actions of these transcription factors on the electrophysiological features and expression of ion channels in cardiomyocytes. Genes for transcription factors Csx/Nkx2.5 and GATA4 were transfected into rat neonatal cardiomyocytes by adenoviral infection. Action potentials, L-, T-type Ca(2+) channels and hyperpolarization-activated cation current (I(h)) of rat neonatal myocytes were recorded by patch clamp technique after adenoviral infection. Expression of ion channels was confirmed by real-time PCR. In Csx/Nkx2.5 overexpression myocytes, the spontaneous beating rate was markedly increased with an up-regulation of the Ca(v)3.2 T-type Ca(2+) channel, while in GATA4 overexpression myocytes, the T-type Ca(2+) channel was unchanged. On the other hand, the L-type Ca(2+) channel was down-regulated by both Csx/Nkx2.5 and GATA4 overexpression; the level of Ca(v)1.3 mRNA was dramatically decreased by Csx/Nkx2.5 overexpression. These results indicate that Csx/Nkx2.5 and GATA4 play important roles on the generation of pacemaker potentials modulating voltage-dependent Ca(2+) channels in the neonatal cardiomyocyte.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Fator de Transcrição GATA4/fisiologia , Regulação da Expressão Gênica/fisiologia , Miocárdio/metabolismo , Fatores de Transcrição/fisiologia , Potenciais de Ação , Adenoviridae/genética , Animais , Animais Recém-Nascidos , Canais de Cálcio Tipo L/genética , Células Cultivadas , Fator de Transcrição GATA4/genética , Ventrículos do Coração/citologia , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fatores de Transcrição/genética , Transfecção
8.
Pharmacology ; 78(1): 11-20, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16899990

RESUMO

We compared detailed efficacy of efonidipine and nifedipine, dihydropyridine analogues, and mibefradil using recombinant T- and L-type Ca2+ channels expressed separately in mammalian cells. All these Ca2+ channel antagonists blocked T-type Ca2+ channel currents (I(Ca(T))) with distinct blocking manners: I(Ca(T)) was blocked mainly by a tonic manner by nifedipine, by a use-dependent manner by mibefradil, and by a combination of both manners by efonidipine. IC50s of these Ca2+ channel antagonists to I(Ca(T)) and L-type Ca2+ channel current (I(Ca(L))) were 1.2 micromol/l and 0.14 nmol/l for nifedipine; 0.87 and 1.4 micromol/l for mibefradil, and 0.35 micromol/l and 1.8 nmol/l for efonidipine, respectively. Efonidipine, a dihydropyridine analogue, showed high affinity to T-type Ca2+ channel.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Di-Hidropiridinas/farmacologia , Mibefradil/farmacologia , Nifedipino/farmacologia , Nitrofenóis/farmacologia , Linhagem Celular , Humanos , Compostos Organofosforados/farmacologia , Proteínas Recombinantes/antagonistas & inibidores
9.
Pharmacology ; 76(4): 192-200, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16543777

RESUMO

Lysophosphatidylcholine (LPC) has been shown to induce electrophysiological disturbances to arrhythmogenesis. However, the effects of LPC on the low-voltage-activated T-type Ca(2+) channels in the heart are not understood yet. We found that LPC increases the T-type Ca(2+) channel current (I(Ca.T)) in neonatal rat cardiomyocytes. To further investigate the underlying modulatory mechanism of LPC on T-type Ca(2+) channels, we utilized HEK-293 cells stably expressing alpha1G and alpha1H subunits (HEK-293/alpha1G and HEK-293/alpha1H), by use of patch-clamp techniques. A low concentration of LPC (10 micromol/l) significantly increased Ca(v)3.2 I(Ca.T) (alpha1H) that were similar to those observed in neonatal rat cardiomyocytes. Activation and steady-state inactivation curves were shifted in the hyperpolarized direction by 5.1 +/- 0.2 and 4.6 +/- 0.4 mV, respectively, by application of 10 micromol/l LPC. The pretreatment of cells with a protein kinase C inhibitor (chelerythrine) attenuated the effects of LPC on I(Ca.T) (alpha1H). However, the application of LPC failed to modify Ca(v)3.1 (alpha1G) I(Ca.T) at concentrations of 10-50 micromol/l. In conclusion, these data demonstrate that extracellularly applied LPC augments Ca(v)3.2 I(Ca.T) (alpha1H) but not Ca(v)3.1 I(Ca.T) (alpha1G) in a heterologous expression system, possibly by modulating protein kinase C signaling.


Assuntos
Canais de Cálcio Tipo T/fisiologia , Lisofosfatidilcolinas/farmacologia , Alcaloides , Animais , Animais Recém-Nascidos , Benzofenantridinas , Canais de Cálcio Tipo T/genética , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Técnicas In Vitro , Miócitos Cardíacos/fisiologia , Técnicas de Patch-Clamp , Fenantridinas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Ratos , Ratos Wistar
10.
Mol Pharmacol ; 69(5): 1684-91, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16443692

RESUMO

Low-voltage-activated T-type Ca2+ channels have been recognized recently in the mechanisms underlying atrial arrhythmias. However, the pharmacological effects of amiodarone on the T-type Ca2+ channel remain unclear. We investigated short- and long-term effects of amiodarone on the T-type (Cav 3.2) Ca2+ channel. The Cav3.2 alpha1H subunit derived from human heart was stably transfected into cells [human embryonic kidney (HEK)-Cav3.2] cultured with or without 5 muM amiodarone. Patch-clamp recordings in the conventional whole-cell configuration were used to evaluate the actions of amiodarone on the T-type Ca2+ channel current (ICa.T). Amiodarone blockade of ICa.T occurred in a dose- and holding potential-dependent manner, shifting the activation and the steady-state inactivation curves in the hyperpolarization direction, when amiodarone was applied immediately to the bath solution. However, when the HEK-Cav3.2 cells were incubated with 5 microM amiodarone for 72 h, ICa.T density was significantly decreased by 31.7+/-2.3% for control,-93.1+/-4.3 pA/pF (n=8), versus amiodarone,-56.5+/-3.2 pA/pF (n=13), P<0.001. After the prolonged administration of amiodarone, the activation and the steady-state inactivation curves were shifted in the depolarization direction by -7.1 (n=41) and -5.5 mV (n=37), respectively, and current inactivation was significantly delayed [time constant (tau): control, 13.3+/-1.1 ms (n=6) versus amiodarone, 39.6+/-5.5 ms (n=6) at -30 mV, P<0.001)]. Nevertheless, short-term inhibitory effects of amiodarone on the modified T-type Cav3.2 Ca2+ channel created by long-term amiodarone treatment were functionally maintained. We conclude that amiodarone exerts its short- and long-term inhibitory actions on ICa.T via distinct blocking mechanisms.


Assuntos
Amiodarona/farmacologia , Canais de Cálcio Tipo T/fisiologia , Canais de Cálcio Tipo T/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Coração/efeitos dos fármacos , Coração/fisiologia , Cinética , Técnicas de Patch-Clamp , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Hormônios Tireóideos/fisiologia , Transfecção
11.
Pharmacology ; 74(4): 174-81, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15855830

RESUMO

Effects of bepridil on the low voltage-activated T-type Ca2+ channel (CaV3.2) current stably expressed in human embryonic kidney (HEK)-293 cells were examined using patch-clamp techniques. Bepridil potently inhibited ICa,T with a markedly voltage-dependent manner; the IC50 of bepridil was 0.4 micromol/l at the holding potential of -70 mV, which was 26 times as potent as that at -100 mV (10.6 micromol/l). Steady-state inactivation curve (8.4 +/- 1.7 mV) and conductance curve (5.9 +/- 1.9 mV) were shifted to the hyperpolarized potential by 10 micromol/l bepridil. Bepridil exerted the tonic blocking action but not the use-dependent block. Bepridil had no effect on the recovery from inactivation of T-type Ca2+ channels. Thus, high efficacy of bepridil for terminating atrial fibrillation and atrial flutter may be considered to be attributed, at least in a part, to the T-type Ca2+ channel-blocking actions.


Assuntos
Bepridil/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/fisiologia , Canais de Cálcio Tipo L/genética , Linhagem Celular , Relação Dose-Resposta a Droga , Estimulação Elétrica , Vetores Genéticos/genética , Humanos , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Fatores de Tempo , Transfecção
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