MUSCARINIC SIGNLING: REGULATION OF VENTRICULAR FUNCTION

说明(申请人提供)：心脏的机械性能由其固有的收缩特性决定，并受自主神经系统的平衡调节。刺激α-肾上腺素能增强泵功能，而胆碱能刺激则通过降低先前被cAMP升高的L钙通道(ICa-L)的电导而降低泵功能(“增强的拮抗作用”)。两种不同的G蛋白Go和Gi-2被认为是这一效应所必需的，但它们的确切作用和相互作用以及涉及的信号机制仍不清楚。我们已有的初步数据表明，GO蛋白还可能通过改变兴奋收缩(E-C)偶联和肌丝对钙的反应性来调节钙循环和力的产生。本研究的总体目标是明确GO和/或GI2蛋白激活与毒鼠碱调节心肌细胞钙离子流量和收缩功能之间的联系的信号机制，并验证选择性干扰这一途径可用于增强体内收缩功能的假设。其具体目的是：(1)验证激活的GaO的表达通过增加(而不是降低)E-C偶联增益和肌丝对钙的反应性来调节心肌细胞内钙循环和细胞缩短的假说，与其对ICa-L的钝化作用相一致；(2)测试细胞通透性多肽导入是否能够适应将优势的负性抑制肽引入成年心室肌细胞作为腺病毒基因转移的替代和可能的改进；(3)检验GO和GI2蛋白协同作用于ICa-L上M2受体作用的假设。(4)验证干扰GO和/或Gi介导的心肌信号转导可被用来增强心室收缩功能的假说。无论是在正常生理条件下还是在病理生理条件下，毒扁豆碱的“强化拮抗”都具有重要的生理意义。更好地理解毒扁豆碱受体介导的对抗β-肾上腺素能刺激和钝化收缩功能的过程，可能成为旨在通过迄今未被探索的机制向心脏长期自主神经输入的治疗干预的基础。

DESCRIPTION (provided by applicant): The mechanical performance of the heart is determined by its intrinsic contractile properties and is subject to counterbalancing regulation by the autonomic nervous system. While a-adrenergic stimulation increases pump function, cholinergic stimulation decreases it by reducing the conductance of the L-type Ca2+ channel (ICa-L) previously elevated by cAMP ("accentuated antagonism"). Two different G proteins, Go and Gi-2, have been implicated to be absolutely required for this effect, but their exact role and interplay as well as the signaling mechanisms involved are still poorly defined. We have preliminary data suggesting that Go protein may also regulate Ca2+ cycling and force generation by altering excitation contraction (E-C) coupling and the responsiveness of the myofilaments to Ca2+. The overall goal of this investigation is to define the signaling mechanisms that link Go and/or Gi2 protein activation to the muscarinic regulation of ventricular Ca2+ fluxes and contractile function and to test the hypothesis that selective interference with this pathway can be utilized to enhance contractile function in vivo. The Specific Aims are: (1) to test the hypothesis that expression of activated Gao regulates Ca2+ cycling and cell shortening in the ventricular myocardium by increasing (rather than decreasing) E-C coupling gain and myofilament responsiveness to Ca2+ in concert to its blunting effect on ICa-L, (2) to test whether cell-permeable peptide import can be adapted for introduction of dominant negative inhibitory peptides into adult ventricular cardiocytes as an alternative and possible improvement to adenoviral gene transfer, (3) to test the hypotheses that Go and Gi2 proteins act in concert as mediators of M2-receptor effects on ICa-L, Ca2+ cycling and contractility and that both Galpha and Gbetagamma contribute to this effect (4) to test the hypothesis that interference with Go and/or Gi-mediated signal transduction in the ventricular myocardium can be utilized to enhance ventricular contractile function. Muscarinic "accentuated antagonism" is of physiological importance both under normal and pathophysiological conditions. A better understanding of the processes involved in muscarinic receptor-mediated opposition of beta-adrenergic stimulation and blunting of contractile function may form the basis for therapeutic interventions aimed at the long-term autonomic input to the heart through a so far underexplored mechanism.