MUSCARINIC SIGNLING: REGULATION OF VENTRICULAR FUNCTION

描述（由申请人提供）：心脏的机械性能是由其内在的收缩特性决定的，并受自主神经系统的平衡调节。当a-肾上腺素能刺激增加泵功能时，胆碱能刺激通过降低先前由cAMP升高的l型Ca2+通道（ICa-L）的电导而降低泵功能（“强化拮抗”）。两种不同的G蛋白，Go和Gi-2，已经被认为是这种效应所必需的，但它们的确切作用和相互作用以及所涉及的信号机制仍然不清楚。我们有初步的数据表明，Go蛋白也可能通过改变兴奋收缩（E-C）偶联和肌丝对Ca2+的反应性来调节Ca2+循环和力的产生。本研究的总体目标是确定将Go和/或Gi2蛋白激活与心室Ca2+通量和收缩功能的毒蕈碱调节联系起来的信号机制，并验证对该途径的选择性干扰可以用来增强体内收缩功能的假设。具体目标是：(1)验证激活的Gao表达通过增加（而不是减少）E-C偶联增益和肌丝对Ca2+的反应，以及其对ICa-L的钝化作用，调节心室心肌Ca2+循环和细胞缩短的假设；(2)测试细胞渗透性肽导入是否适用于将显性负抑制肽引入成人心室心肌细胞，作为腺病毒基因转移的一种替代和可能的改进；(3)测试Go和Gi2蛋白作为m2受体对ICa-L作用的介质协同作用的假设；Ca2+循环和收缩性，以及Galpha和Gbetagamma都有助于这种效果(4)，以验证在心室心肌中干扰Go和/或gi介导的信号转导可以用来增强心室收缩功能的假设。毒蕈碱“强化拮抗”在正常和病理生理条件下都具有重要的生理意义。更好地了解毒蕈碱受体介导的β -肾上腺素能刺激的对抗和收缩功能的钝化过程，可能会通过迄今尚未探索的机制形成针对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.