Basic Research for Gene Therapy of Heart Failure by Transcoronay Gene Transfer

我们进行了基础实验，以研究心力衰竭时通过GTP结合蛋白相关通路的心肌收缩力调节机制的变化。该项目的主要目的是确定可能的靶基因，以防止正常心脏发生心力衰竭的过程。我们获得了以下发现。(A)在犬衰竭心脏中，1）GTP结合蛋白Gq-RhoA-Rho激酶信号转导上调与肌球蛋白轻链磷酸化水平的增加相关，这导致肌原纤维Ca^2+敏感性增加; 2）β-肾上腺素能刺激诱导的肌原纤维Ca^2+敏感性降低减弱。这些肌原纤维Ca^2+敏感性的异常调节可能与衰竭心脏收缩功能下降有关。(B)与平滑肌一样，心肌肌球蛋白轻链的磷酸化也受到钙调素和环磷酸鸟苷的调节，这两种物质都能调节心肌肌原纤维的Ca^<2+>敏感性。(C)使用选择性Rho激酶抑制剂Y-27632，我们测试了Rho激酶的慢性抑制可能能够防止导致心力衰竭的心肌肥大过程的假设。我们发现，慢性抑制Rho激酶可防止高血压引起的心肌肥大，从而保护心肌收缩功能。Rho激酶可能是治疗心力衰竭的重要靶点。(D)我们正在进行另一项实验，以研究瞬时受体电位（TRP）蛋白在心肌细胞中的功能。TRP蛋白超家族由一组由Gq依赖性机制激活的受体操纵的Ca^2+通道组成。假设培养的心肌细胞转染TRP基因，纳入腺病毒载体，发展肥大时，血管紧张素II刺激。

We performed basic experiments to investigate changes in the regulatory mechanisms of cardiac contractility through the GTP binding protein-related pathways in heart failure. The main purpose of this project was to determine possible target genes to prevent the process by which normal hearts develop heart failure. We obtained the following findings.(A) In canine failing hearts, 1) GTP-binding protein Gq-RhoA-Rho kinase signaling was upregulated in association with an increase in the phosphorylation level of myosin light chain, which caused an increase in myofibrillar Ca^<2+> sensitivity, 2) β-adrenergicstimulation-induced decreasein the myofibrillar Ca^<2+> sensitivity was attenuated. These abnormal regulation of the myofibrillar Ca^<2+> sensitivity may be related to decreased contractile function in the failing hearts.(B) In cardiacmuscle, as in smooth muscle, the phosphorylation of myosin light chain was modulatedby calmodulin and cyclic GMP, both of which were able to regulate the cardiacmyofibrillar Ca^<2+> sensitivity.(C) Using a selective Rho kinase inhibitor Y-27632, we tested the hypothesis that chronic inhibition of Rho kinase might be able to prevent the process of myocardial hypertrophy resulting in heart failure. We found that chronic inhibition of Rho kinase prevented myocardial hypertrophy induced by hypertension and thereby preserved myocardial contractile function. Rho kinase may be an important target in treatment of heart failure.(D) We are performing another experiment to investigate the function of the transient receptor potential (TRP) proteins in cardiac cells. The TRP protein superfamily consists of a diverse group of receptor-operated Ca^<2+> channels activated by Gq-dependent mechanisms. The hypothesis is that cultured cardiomyocytes transfected with the TRP gene, incorporated into adenovirus vector, develop hypertrophy when stimulated by angiotensin II.