Reversal of isoproterenol-induced downregulation of phospholamban and FKBP12.6 by CPU0213-mediated antagonism of endothelin receptors

Aim： The downregulation of phospholamban （PLB） and FKBP12.6 as a result of β- receptor activation is involved in the pathway（s） of congestive heart failure. We hypothesized that the endothelin （ET）-1 system may link to downregulated PLB and FKBP12.6. Methods： Rats were subjected to ischemia/reperfusion （I/R） to cause heart failure （HF）. 1 mg/kg isoproterenol （ISO） was injected subcutaneously （sc） for 10 d to worsen HF. 30 mg/kg CPU0213 （sc）, a dual ET receptor （ETAR/ETBR） antagonist was given from d 6 to d 10. On d 11, cardiac function was assessed together with the determination of mRNA levels of ryanodine receptor 2, calstabin-2 （FKBP12.6）, PLB, and sarcoplasmic reticulum Ca^2＋-ATPase. Isolated adult rat ventricular myocytes were incubated with ISO at 1 × 10^-6 mol/L to set up an in vitro model of HF. Propranolol （PRO）, CPU0213, and darusentan （DAR, an ETAR antagonist） were incubated with cardiomyocytes at 1× 10^-5 mol/L or 1× 10^-6 mol/L in the presence of ISO （1×10^-6 mol/L）. Immunocytochemistry and Western blotting were applied for measuring the protein levels of PLB and FKBP12.6. Results： The worsened hemodynamics produced by I/R were exacerbated by ISO pretreatment. The significant downregulation of the gene expression of PLB and FKBP12.6 and worsened cardiac function by ISO were reversed by CPU0213. In vitro ISO 1× 10^-6 mol/L produced a sharp decline of PLB and FKBP12.6 proteins relative to the control. The downregulation of the protein expression was significantly reversed by the ET receptor antagonist CPU0213 or DAR, comparable to that achieved by PRO. Conclusion： This study demonstrates a role of ET in mediating the downregulation of the cardiac Ca^2＋-handling protein by ISO.     

内皮素受体拮抗剂CPU0213对异丙肾上腺素心肌病SERCA2a表达的改善作用

目的：研究异丙肾上腺素心肌病模型心肌细胞肌浆网钙ATP酶（sarco-endoplasmic reticulum ATPase 2a,SERCA2a）表达的改变,以及新型内皮素受体拮抗剂CPU0213的治疗作用。方法：雄性SD大鼠皮下给予异丙肾上腺素（2mg·kg^-1·d^-1）10d,治疗组动物在第6到第10天皮下给予CPU0213（30mg·kg^-1·d^-1）。各组动物颈动脉插管记录心功能指标：左心室收缩压（LVSP）,左心室舒张末期压（LVEDP）,和左心室压力变化最大速率（±dp/dtmax）。左心室心肌组织SERCA2a的mRNA及蛋白表达分别用逆转录聚合酶链反应（RT-PCR）和蛋白免疫印迹法（Western blotting）测定。结果：异丙肾上腺素引起左心室收缩和舒张功能明显下降,同时SERCA2a的mRNA及蛋白表达均显著下调（P〈0.05）。CPU0213显著提高SERCA2a表达（P〈0.05）,明显改善心功能（P〈0.05）。结论：CPU0213可通过逆转肌浆网钙调控蛋白SERCA2a的表达下调,使异丙肾上腺素引起的心功能下降得以恢复。本实验证明SERCA2a是β受体过度激活引发心衰过程中的重要靶点。内皮素受体介导了β受体过度激活状态下SER-CA2a的表达下调,是内皮素受体拮抗剂治疗心衰的重要依据之一。     

Sildenafil improves diabetic vascular activity through suppressing endothelin receptor A, iNOS and NADPH oxidase which is comparable with the endothelin receptor antagonist CPU0213 in STZ-injected rats

Objectives Abnormal vascular activity in diabetes is related not only to impaired nitric oxide bioavailability but also to inflammatory cytokines, endothelin A receptor (ET_A) activation and NADPH oxidase in the vasculature. The potential role of sildenafil in improving vascular function was investigated. Its action was likely blocking upregulated ET_A and NADPH oxidase, and was compared with the endothelin receptor antagonist CPU0213. Methods Diabetes was induced by single‐dose administration of streptozotocin (65 mg/kg, i.p.) to rats and the vascular activity of the thoracic aorta was measured. Key findings An increase in contractile tone to phenylephrine and a decrease in relaxant tone to acetylcholine was found in the thoracic aorta. Oxidative stress was evident by increased malondialdehyde and reduced glutathione peroxidase levels in serum and upregulation of ET_A, MMP‐9 (matrix metalloproteinase‐9), inducible nitric oxide synthase and NADPH oxidase p67^phox were found in the vascular wall. The vascular abnormalities and abnormal biomarkers were attenuated significantly by either sildenafil or CPU0213 along with an improvement of nitric oxide bioavailability and vascular activity. Conclusions Improvement of diabetic vascular abnormal activity by sildenafil results from its suppression of activation of ET_A and NADPH oxidase in the vasculature, and these actions are comparable with those of the endothelin receptor antagonist CPU0213.     

CPU0123, a novel endothelin receptor antagonist,relieves hypoxic pulmonary hypertension in rats by suppressing excessive ET‐ROS pathway

Pulmonary artery hypertension (PAH, PH) is a chronic and progressive disease with high morbidity and mortality and is resistant to vasodilative drugs in the clinic due to remodeling of pulmonary arterioles involved in PAH. The endothelin (ET) receptor antagonist bosentan is an effective oral medication in relieving PAH due to its antiproliferative effects. The dual ET_A/ET_B antagonist receptor antagonist, CPU0213, was compared with nifedipine in treating hypoxic PAH in SD rats that were exposed to 28 days of hypoxia (O₂ 10±0.5%). In untreated animals, elevated right ventricular systolic pressure (RVSP), central vein pressure (CVP), and remodeling of pulmonary arterioles were predominant. In the pulmonary tissue of PAH rats, the ET‐ROS pathway was over‐activated in association with a reduced NO level, increased iNOS activity and hydroxyproline contents. Following oral treatment with CPU0213, (50 mg/mg, 100 mg/kg, and 200 mg/kg, p.o.), the hemodynamic indices and pulmonary arteriole remodeling were significantly improved in a dose‐dependent manner. Elevated ET‐1 levels, iNOS activity, and maladjustment of pulmonary redox system were reversed, accompanied by a reduction of hydroxyproline in pulmonary tissue. An antiproliferative effect of CPU0213 was superior to that of nifedipine. The efficacy of 50 mg/kg CPU0213 was reduced. It is concluded that the low‐selective ET_A/ET_B receptor antagonist, CPU0213, is effective in relieving hypoxia‐induced pulmonary hypertension by suppressing an over‐activated ET‐ROS pathway in pulmonary tissue. Drug Dev Res 68:42–50, 2007. © 2007 Wiley‐Liss, Inc.     

Endothelin receptor antagonist CPU0213 and vitamin E reverse downregulation of FKBP12.6 and SERCA2a: a role of hyperphosphorylation of PKCepsilon

Downregulation of FKBP12.6 and sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) contributes to sudden cardiac death and heart failure. We aimed to test the hypothesis that (i) downregulation of FKBP12.6 and SERCA2a can be taken as molecular markers for drug interventions and (ii) such downregulation is produced by crosstalk between endothelin-reactive oxygen species and beta-adrenoceptors stimulation, mediated by hyperphosphorylation of protein kinase Cvarepsilon (PKCvarepsilon). Rat cardiomyocytes were incubated with isoproterenol (1 microM), endothelin-1 (0.1 microM) or hydrogen peroxide (10 microM) for 18 h, resulting in downregulation of mRNA and protein of FKBP12.6 and SERCA2a, as well as upregulation of PKCvarepsilon mRNA and phosphorylated PKCvarepsilon protein. These changes were reversed by an application of either propranolol (1 microM), endothelin receptor antagonist CPU0213 (1 microM) or vitamin E (1 microM). As indicated by the fluorescent dye Fluo3, diastolic [Ca(2+)](i) in rat ventricular myocytes was increased after incubation with isoproterenol (0.1 microM). The increased [Ca(2+)](i) in diastole was dramatically decreased by CPU0213. Thus, the downregulation of FKBP12.6 and SERCA2a, and hyperphosphorylation of PKCvarepsilon, appear to be related to crosstalk between over-activated endothelin-reactive oxygen species and a beta-adrenoceptor pathway. CPU0213 is beneficial in treating cardiac insufficiency and preventing cardiac arrhythmias possibly by normalizing hyperphosphorylation of PKCvarepsilon and abnormal FKBP12.6 and SERCA2a. The antioxidant activity of vitamin E was sufficient to normalize the levels of FKBP12.6 and SERCA2a and phosphorylation of PKCvarepsilon. Thus by testing with biomarkers FKBP12.6 and SERCA2a, we have shown that the endothelin receptor antagonist CPU0213 and the antioxidant vitamin E may relieve risk of lethal arrhythmias and heart failure by suppressing PKCvarepsilon.     

Effect of the endothelin receptor antagonist CPU0213, and its modulation by rifampin, on cardiac and vascular tissue following chronic isoproterenol treatment

1. The aim of the present study was to investigate the effects of the endothelin (ET) receptor antagonist CPU0213 on cardiac and vascular tissues after impairment by chronic isoproterenol treatment. Because rifampin reduces plasma concentrations of CPU0213, the modulation of the effects of CPU0213 by rifampin was also investigated. 2. Thirty rats were randomly divided into five groups as follows: (i) control; (ii) isoproterenol treated (1 mg/kg, s.c., for 10 days); (iii) isoproterenol treated with a single injection of CPU0213 (30 mg/kg, s.c., on Day 11); (iv) isoproterenol treated with a single injection of rifampin (50 mg/kg, p.o., on Day 11); and (v) isoproterenol treated with rifampin gvien 3 h before CPU0213 on Day 11. Serum concentrations of CPU0213, haemodynamic and biochemical parameters, mRNA and protein expression levels of the ET(A) receptor (ET(A)R), calstabin 2 (FKBP12.6) and sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a), and vasoactivity of the thoracic aorta were determined. 3. Haemodynamic parameters, serum creatine phosphokinase, lactate dehydrogenase and malondialdehyde levels, mRNA and protein expression of FKBP12.6, SERCA2a and vascular responses were altered following isoproterenol treatment for 10 days. These effects were significantly reversed by CPU0213. Rifampin caused a reduction in serum concentrations of CPU0213 to 36% of control values. However, this reduction in the serum concentrations of CPU0213 did not affect its effects on the heart, but did eliminate its beneficial action on vascular responses. Rifampin alone had no effect these paramters. 4. The data suggest that isoproterenol acts on the myocardium to cause cardiac insufficiency by upregulating ET(A)R and downregulating FKBP12.6 and SERCA2a. These effects were ameliorated by CPU0213, but were resistant to rifampin-induced decreases in plasma CPU0213 concentrations. In vascular tissue, the pathological effects of isoproterenol were ameliorated by CPU0213; however, lowering plasma CPU0213 concentrations with rifampin did partly eliminate the amelioration in vascular activity in respones to CPU0213.     

内皮素受体拮抗剂CPU0213对博莱霉素及油酸的小鼠肺损伤及纤维化的改善作用

目的：本实验采用两种小鼠急性肺损伤与肺纤维化相关的模型,探讨新内皮素受体拮抗剂CPU0213对肺纤维化病变明显的肺动脉高压是否具有改善作用。方法：实验分正常组、博莱霉素或油酸模型组、阳性药硝苯地平（10mg/kg）组和CPU0213（50、100、200mg/kg）治疗组（n=10）。博莱霉素模型中,观察肺重系数、支气管肺泡灌洗液（BALF）回收率、BALF白细胞数量及分类、丙二醛（MDA）含量以及肺组织羟脯氨酸含量。油酸模型中,小鼠尾静脉注射伊文氏兰（50mg/kg）1h后,接着静注油酸100mg/kg,4h后分离左肺,烘干后测定小鼠左肺中伊文氏兰的含量。结果：CPU021350、100、200mg/kg可以降低肺重系数,减少肺组织和血清MDA含量,减少肺组织羟脯氨酸含量,降低油酸所致肺纤维化模型中肺组织通透性的增加。其中,CPU0213200mg/kg的治疗效果优于阳性药硝苯地平（10mg/kg）。结论：内皮素受体拮抗剂CPU0213能减轻小鼠两种急性肺损伤与纤维化病变,对临床肺动脉高压有较好疗效。     

CPU228, a derivative of dofetilide,relieves cardiac dysfunction by normalizing FKBP12.6, NADPH oxidase and protein kinase C ε in the myocardium

Objectives The aim of this study was to determine if CPU228, a derivative of dofetilide, is more effective than dofetilide in attenuating isoproterenol‐induced heart failure by recovering downregulated FK506 binding protein (FKBP12.6), and suppressing oxidative stress, upregulated NADPH oxidase and protein kinase C ε (PKCε) hyperphosphorylation in the myocardium. Methods Heart failure was induced by isoproterenol (1 mg/kg s.c. for 5 days) in male Sprague‐Dawley rats. Intervention with either CPU228 or dofetilide (2 mg/kg on Days 3–5) was then conducted in vivo and in vitro. Key findings Isoproterenol produced compromised left ventricular systolic pressure, left ventricular pressure rise (dp/dt_max) and fall (dp/dt_min), and left ventricular end‐diastolic pressure, associated with oxidative stress, abnormal FKBP12.6, NADPH oxidase p67phox and PKCε in the myocardium. CPU228 was more effective in attenuating these changes than dofetilide in vivo. Dofetilide produced a prolonged QTc to replace a shortened one. In primary neonatal cardiomyocytes, cultured with isoproterenol and treated with either CPU228 or dofetilide at 10^?8, 10^?7 and 10^?6 mol/l, isoproterenol produced a hyperadrenergic state characterized by downregulated FKBP12.6, upregulated NADPH oxidase p67phox and PKCε in vitro. CPU228 was more effective than dofetilide in recovering these changes in a dose‐dependent manner without a prolonged QTc. Conclusions CPU228 was more effective than dofetilide in attenuating heart failure by normalizing isoproterenol‐induced changes, including downregulation of FKBP12.6, upregulation of NADPH oxidase and PKCε hyperphosphorylation in vivo and in vitro.