RESUMEN
BACKGROUND: The purpose of this study was to investigate the inhibitory effects of long-term electroacupuncture at BaiHui (DU20) and ZuSanLi (ST36) on cardiovascular remodeling in spontaneously hypertensive rats (SHR) and underlying mechanisms. METHODS: 6-weeks-old SHR or Wistar male rats were randomly, divided into 6 groups: the control group (SHR/Wistar), the non-acupoint electroacupuncture stimulation group (SHR-NAP/Wistar-NAP) and the electroacupuncture stimulation at DU20 and ST36 group (SHR-AP/Wistar-AP), 24 rats in each group. Rats were treated with or without electroacupuncture at DU20 and ST36, once every other day for a period of 8 weeks. The mean arterial pressure (MAP) was measured once every 2 weeks. By the end of the 8th week, the left ventricular structure and function were assessed by echocardiography. The content of angiotensin II (Ang II), endothelin-1 (ET-1) and nitric oxide (NO) in the plasma was determined using enzyme-linked immunosorbent assay. Histological studies on the heart and the ascending aorta were performed. The expression of angiotensin II type 1 receptor (AT1R), endothelin-1 type A receptor (ETAR), eNOS and iNOS in rat myocardium and ascending aorta was investigated by Western blotting. RESULTS: The MAP in SHR increased linearly over the observation period and significantly reduced following electroacupuncture as compared with sham control SHR rats, while no difference in MAP was observed in Wistar rats between electroacupuncture and sham control. The aortic wall thickness, cardiac hypertrophy and increased collagen level in SHR were attenuated by long term electroacupuncture. The content of Ang II, ET-1 in the plasma decreased, but the content of NO increased after electroacupuncture stimulation in SHR. Long term electroacupuncture significantly inhibited the expression of AT1R, ETAR and iNOS, whereas increased eNOS expression, in myocardium and ascending aorta of SHR. CONCLUSIONS: The long term electroacupuncture stimulation at DU20 and ST36 relieves the increased MAP and cardiovascular abnormality in both structure and function in SHR, this beneficial action is most likely mediated via modulation of AT1R-AT1R-ET-1-ETAR and NOS/NO pathway.
Asunto(s)
Aorta/patología , Presión Sanguínea , Cardiomegalia/terapia , Electroacupuntura , Hipertensión/terapia , Miocardio/patología , Remodelación Vascular , Puntos de Acupuntura , Angiotensina II/sangre , Angiotensina II/metabolismo , Animales , Aorta/metabolismo , Colágeno/metabolismo , Endotelina-1/sangre , Endotelina-1/metabolismo , Ensayo de Inmunoadsorción Enzimática , Hipertensión/sangre , Masculino , Miocardio/metabolismo , Óxido Nítrico/sangre , Óxido Nítrico Sintasa/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Ratas Wistar , Receptor de Angiotensina Tipo 1/sangre , Receptor de Angiotensina Tipo 1/metabolismoRESUMEN
RATIONALE: During the transition from compensated hypertrophy to heart failure, the signaling between L-type Ca(2+) channels in the cell membrane/T-tubules and ryanodine receptors in the sarcoplasmic reticulum becomes defective, partially because of the decreased expression of a T-tubule-sarcoplasmic reticulum anchoring protein, junctophilin-2. MicroRNA (miR)-24, a junctophilin-2 suppressing miR, is upregulated in hypertrophied and failing cardiomyocytes. OBJECTIVE: To test whether miR-24 suppression can protect the structural and functional integrity of L-type Ca(2+) channel-ryanodine receptor signaling in hypertrophied cardiomyocytes. METHODS AND RESULTS: In vivo silencing of miR-24 by a specific antagomir in an aorta-constricted mouse model effectively prevented the degradation of heart contraction, but not ventricular hypertrophy. Electrophysiology and confocal imaging studies showed that antagomir treatment prevented the decreases in L-type Ca(2+) channel-ryanodine receptor signaling fidelity/efficiency and whole-cell Ca(2+) transients. Further studies showed that antagomir treatment stabilized junctophilin-2 expression and protected the ultrastructure of T-tubule-sarcoplasmic reticulum junctions from disruption. CONCLUSIONS: MiR-24 suppression prevented the transition from compensated hypertrophy to decompensated hypertrophy, providing a potential strategy for early treatment against heart failure.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Acoplamiento Excitación-Contracción/efectos de los fármacos , Insuficiencia Cardíaca/prevención & control , Hipertrofia Ventricular Izquierda/tratamiento farmacológico , MicroARNs/antagonistas & inhibidores , Miocitos Cardíacos/efectos de los fármacos , Oligonucleótidos Antisentido/uso terapéutico , Animales , Estenosis Aórtica Subvalvular/complicaciones , Canales de Calcio Tipo L/fisiología , Señalización del Calcio/fisiología , Progresión de la Enfermedad , Evaluación Preclínica de Medicamentos , Regulación de la Expresión Génica , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/metabolismo , Hipertrofia Ventricular Izquierda/complicaciones , Hipertrofia Ventricular Izquierda/fisiopatología , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , MicroARNs/fisiología , Modelos Cardiovasculares , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/ultraestructura , Oligonucleótidos Antisentido/farmacología , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/fisiología , Retículo Sarcoplasmático/ultraestructuraRESUMEN
Myocardial fibrosis is a key pathological change in a variety of heart diseases contributing to the development of heart failure, arrhythmias, and sudden death. Recent studies have shown that relaxin prevents and reverses cardiac fibrosis. Endogenous expression of relaxin was elevated in the setting of heart disease; the extent of such up-regulation, however, is insufficient to exert compensatory actions, and the mechanism regulating relaxin expression is poorly defined. In the rat relaxin-1 (RLN1, Chr1) gene promoter region we found presence of repeated guanine (G)-rich sequences, which allowed formation and stabilization of G-quadruplexes with the addition of a G-quadruplex interactive ligand berberine. The G-rich sequences and the G-quadruplexes were localized adjacent to the binding motif of signal transducer and activator of transcription (STAT)3, which negatively regulates relaxin expression. Thus, we hypothesized that the formation and stabilization of G-quadruplexes by berberine could influence relaxin expression. We found that berberine-induced formation of G-quadruplexes did increase relaxin gene expression measured at mRNA and protein levels. Formation of G-quadruplexes significantly reduced STAT3 binding to the promoter of relaxin gene. This was associated with consequent increase in the binding of RNA polymerase II and STAT5a to relaxin gene promoter. In cardiac fibroblasts and rats treated with angiotensin II, berberine was found to suppress fibroblast activation, collagen synthesis, and extent of cardiac fibrosis through up-regulating relaxin. The antifibrotic action of berberine in vitro and in vivo was similar to that by exogenous relaxin. Our findings document a novel therapeutic strategy for fibrosis through up-regulating expression of endogenous relaxin.
Asunto(s)
Berberina/farmacología , Berberina/uso terapéutico , Fibrosis Endomiocárdica/metabolismo , Fibrosis Endomiocárdica/terapia , G-Cuádruplex/efectos de los fármacos , Animales , Western Blotting , Células Cultivadas , Inmunoprecipitación de Cromatina , Dicroismo Circular , Ensayo de Inmunoadsorción Enzimática , Humanos , Masculino , Microscopía Confocal , Regiones Promotoras Genéticas/genética , Ratas , Ratas Sprague-Dawley , RelaxinaRESUMEN
1. Ketamine is widely used for the induction of anaesthesia in high-risk patients with cardiovascular instability or severe hypovolaemia. However, the ionic mechanisms involved in the effects of ketamine at therapeutically relevant concentrations in human cardiac myocytes are unclear. The present study was designed to investigate the effects of ketamine on L-type Ca2+ (I(Ca)), transient outward K+ (I(to)), ultra-rapid delayed rectifier K+ (I(Kur)) and inward rectifier potassium (I(K1)) currents, as well as on action potentials, in human isolated atrial myocytes. 2. Atrial myocytes were isolated enzymatically from specimens of human atrial appendage obtained from patients undergoing coronary artery bypass grafting. The action potential and membrane currents were recorded in both current- and voltage-clamp modes using the patch-clamp technique. 3. Ketamine inhibited I(Ca) with an IC(50) of 1.8 micromol/L. In addition, 10 micromol/L ketamine decreased the I(Ca) peak current at +10 mV from 5.1 +/- 0.3 to 2.1 +/- 0.4 pA/pF (P < 0.01), but did not change the threshold potential, peak current potential and reverse potential. 4. Ketamine had no effect on I(to), I(Kur) or I(K1), but it reversibly shortened the duration of the action potential in human atrial myocytes. 5. In conclusion, ketamine, at a clinically relevant concentration, shortens the action potential duration of the human atrial myocytes, probably by inhibiting I(Ca).
Asunto(s)
Ketamina/farmacología , Ketamina/uso terapéutico , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Adulto , Anciano , Relación Dosis-Respuesta a Droga , Técnicas Electrofisiológicas Cardíacas , Atrios Cardíacos/citología , Atrios Cardíacos/efectos de los fármacos , Humanos , Persona de Mediana Edad , Miocitos Cardíacos/citología , Factores de Tiempo , Adulto JovenRESUMEN
AIM: To investigate the alpha1B-adrenoceptor (alpha1B-AR)-mediated cAMP response and underlying mechanisms in HEK293 cells. METHODS: Full-length cDNA encoding alpha1B-AR was transfected into HEK293 cells using the calcium phosphate precipitation method, and alpha1B-AR expression and cAMP accumulation were determined by using the saturation radioligand binding assay and ion-exchange chromatography, respectively. RESULTS: Under agonist stimulation, alpha1B-AR mediated cAMP synthesis in HEK293 cells, and blockade by PLC-PKC or tyrosine kinase did not reduce cAMP accumulation induced by NE. Pretreatment with pertussis toxin (PTX) had little effect on basal cAMP accumulation as well as norepinephrine (NE)-stimulated cAMP accumulation. In addition, pretreatment with cholera toxin (CTX) neither mimicked nor blocked the effect induced by NE. The extracellular Ca2+ chelator egtazic acid (EGTA), nonselective Ca2+ channel blocker CdCl2 and calmodulin (CaM) inhibitor W-7 significantly reduced NE-induced cAMP accumulation from 1.59%+/-0.47% to 1.00%+/-0.31%, 0.78%+/-0.23%, and 0.90%+/-0.40%, respectively. CONCLUSION: By coupling with a PTX-insensitive G protein, alpha1BAR promotes Ca2+ influx via receptor-dependent Ca2+ channels, then Ca2+ is linked to CaM to form a Ca2+-CaM complex, which stimulates adenylyl cyclase (AC), thereby increasing the cAMP production in HEK293 cell lines.
Asunto(s)
Calcio/metabolismo , AMP Cíclico/metabolismo , Riñón/citología , Receptores Adrenérgicos alfa 1/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Calmodulina/antagonistas & inhibidores , Línea Celular , ADN Complementario/genética , Expresión Génica , Humanos , Norepinefrina/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Receptores Adrenérgicos alfa 1/genética , Transducción de Señal , Transfección , Fosfolipasas de Tipo C/antagonistas & inhibidoresRESUMEN
AIM: To investigate the characterization of cAMP response mediated by alpha1-adrenoceptor (alpha1-AR) subtypes in HEK293 cells. METHODS: (1) Full-length cDNA encoding three alpha1-AR subtypes were transfected into HEK293 cells by the calcium phosphate precipitation method, respectively. (2) The densities of alpha1-AR subtypes expressed in HEK293 cells were measured by radioligand binding assay. (3) cAMP accumulation was measured by [3H]adenine prelabeling method. RESULTS: (1) Activation of each of three subtypes resulted in an increase of cAMP accumulation in HEK293 cells in a dose-dependent manner, which was inhibited by selective alpha1-AR antagonist prazosin. (2) Comparing the pharmacological property, the maximal responses of alpha1A-AR to agonists were the most potent, while the sensitivity of alpha1-AR subtypes to norepinephrine (NE) was the highest. CONCLUSION: Each of three alpha1-AR subtypes can mediate cAMP accumulation in HEK293 cell line, and there are differences in pharmacological property.