Research in the inhibitory effect of sarcoplasmic reticulum Ca release channel stabilizer on the development of heart failure

在心力衰竭中，肌浆网功能异常是心力衰竭的主要致病机制之一。在这里，我们评估了1,4-苯并噻唑类衍生物JTV519（细胞内Ca2+调节剂）和心得安对心脏和SR功能的影响。从狗LV肌肉中分离SR囊泡{正常(N), N =11；联合或不联合JTV519 （JT:14.4 mg/Kg/d）或普萘洛尔（PL:0.05mg/ Kg/d） 4周RV快速起搏[未治疗组：n=10， JT(+): n=10， PL(+): n=10]}。1)无论是JT（+）组还是PL（+）组，心功能均较未治疗组改善。2)未治疗的衰竭SR存在异常的SR Ca2+泄漏，苯并噻唑类Ca^<2+>拮抗剂地尔硫卓和JTV519可明显抑制衰竭SR Ca^<2+>泄漏（IC50分别为0.3μM和0.03μM），硝苯地平和维拉帕米对Ca^<2+>泄漏均无影响。在JT（+）和PL（+）中均未发现异常的SR Ca^<2+>渗漏。3) JTV519和普萘洛尔均能阻止RyR相对于FKBP12.6的化学计量下降，[^3H]赖亚定和[^3H] fk506结合测定[正常组1:6 .6，未治疗组1:1.3，JT（+）组1:6 .6，PL（+）组1:4 .4]。4)未治疗组RyR发生PKA-过度磷酸化，而在JT（+）和PL（+）中RyR均发生逆转。5)未处理组RyR结合FKBP12.6的数量明显少于正常RyR，而在JT（+）和PL（+）组则相反。6)用荧光构象探针甲基香豆素醋酸酯（MCA）对RyR进行了位点定向标记。在J（+）和P（+）中，未处理组较高的MCA荧光水平均下降至正常水平，表明两种处理均改善了RyR构象状态。JTV519和普萘洛尔通过恢复RyR的pka -超磷酸化和RyR随后的构象改变，改善FKBP12.6与RyR的缺陷相互作用，从而改善心功能和减轻左室重构。

In heart failure, abnormal function of sarcoplasmic reticulum (SR) is one of the major pathogenic mechanisms in heart failure. Here, we assessed the effects of 1,4-benzothiazepine derivative JTV519 (intracellular Ca2+ modulator) and propranolol on cardiac and SR functions. SR vesicles were isolated from dog LV muscles {normal (N), n=11; 4-weeks rapid RV pacing with or without JTV519 (JT:14.4 mg/Kg/day) or propranolol (PL:0.05mg/kg/day) [untreated: n=10, JT(+): n=10, PL(+): n=10, respectively]}. 1) In either JT(+) or PL(+), cardiac function was improved compared with untreated group. 2) Abnormal SR Ca2+ leak was found in untreated failing SR. A benzothiazepine Ca^<2+> antagonist diltiazem as well as JTV519 acutely inhibited this Ca^<2+> leak in failing SR (IC50= 0.3μM, 0.03μM, respectively), and neither nifedipine nor verapamil affected the Ca^<2+> leak. There was no abnormal SR Ca^<2+> leak either in JT(+) and PL(+). 3) Both JTV519 and propranolol prevented the decrease in the stoichiometry of RyR vs FKBP12.6 assessed by [^3H]ryanodine and [^3H]FK 506-bindng assays [1:3.6 in normal, 1:1.3 in untreated, 1:3.6 in JT(+), 1:2.4 in PL(+)]. 4) In untreated group, RyR was PKA- hyperphosphorylated, whereas it was reversed both in JT(+) and in PL(+). 5) The amount of RyR-bound FKBP12.6 was tremendously less in untreated group than normal RyR, whereas it was reversed both in JT(+) and PL(+). 6) RyR was labeled in a site-directed fashion with the fluorescent conformational probe methylcoumarin acetate (MCA). In both J(+) and P(+), the level of MCA fluorescence, which was higher in untreated group, was decreased back towards normal, suggesting the improvement of RyR conformational state by both treatments. Both JTV519 and proplanolol improved cardiac function and attenuated LV remodeling by ameliorating the defective interaction of FKBP12.6 with RyR through restoration of PKA-hyperphosphorylation of RyR and the following conformational change of RyR.