Optogenetic silencing to achieve antiarrhythmic effect of renal denervation in chronic heart failure

项目摘要 室性心律失常是慢性心力衰竭(CHF)患者的主要死亡原因。尽管 去肾神经支配(RDN)治疗室性心律失常的潜力已被广泛报道。 引起的不良反应严重限制了其在临床上的应用。我们最近的研究表明，巨噬细胞 星状神经节(SG)的扩张和神经炎症导致CHF细胞兴奋性增加 心脏交感神经节后(CSP)神经元，从而促进心脏交感 充血性心力衰竭大鼠过度兴奋与室性心律失常的发生。粒细胞-巨噬细胞集落刺激因子 (GM-CSF)是巨噬细胞活化的重要介质。我们的试验数据显示，RDN可以减弱心脏 交感神经过度兴奋和室性心律失常，并伴有GM- 充血性心力衰竭大鼠海马区脑脊液水平及巨噬细胞活化。然而，目前尚不清楚该药是否具有抗心律失常作用。 慢性肾小球肾炎的发生是通过减弱GM-CSF介导的炎症通路而实现的。紧随其后的是 RDN抗心律失常机制的发现，这一建议旨在开发一种新的临床干预措施 以实现RDN的治疗作用，避免其局限性。由于肾脏的交感神经支配 主要起源于主肾神经节(ARG)中的神经元，靶向ARG神经元可能是一种合乎逻辑的 实现RDN抗心律失常作用的治疗策略。考虑到光遗传学的优势， 包括通过光敏视蛋白、腺相关病毒- 古紫质(ARCHT，一种抑制性光敏视蛋白)基因将被导入CHF大鼠ARG神经元 老鼠。ARCHT在ARG神经元中表达的特异性将通过将神经元特异性的 ARCHT基因的启动子。在ARG神经元中持续的光遗传沉默将通过照射一种 LED探头，在自由活动的动物中无线控制和供电。我们假设光遗传 抑制ARG神经元将降低CHF升高的SGS中GM-CSF水平，这随后会减轻 SGS中巨噬细胞的激活和神经炎症，从而减轻CSP神经元的兴奋性，心脏 交感神经过度激活与充血性心力衰竭的室性心律失常。使用多方面的技术方法 从整个动物到细胞分子水平，我们将设计体内和体外研究来评估这些 问题。具体目标1，我们将测试GM-CSF信号轴是否有助于巨噬细胞的激活和 慢性心力衰竭动物SGS中的神经炎症。具体目标2，我们将解决如果GM-CSF信号通路 导致充血性心力衰竭CSP神经元细胞兴奋性增加，心脏交感神经过度激活，以及 室性心律失常。具体目标3，我们将确定ARG中的光遗传沉默是否可以实现 雷公藤多苷通过减轻GM-CSF诱导的巨噬细胞活化和神经炎症而抗心律失常 在瑞士瑞士的SGS。这些研究将为治疗致死性室性心律失常开辟新的途径。 提供一种新的临床干预措施，以降低CHF患者的死亡率，改善预后和生活质量。

Project Summary Ventricular arrhythmia is the leading cause of death for chronic heart failure (CHF) patients. Although the therapeutic potential of renal denervation (RDN) for ventricular arrhythmias has been reported extensively, RDN- induced adverse complications severely limit its use in the clinic. Our recent study revealed that macrophage expansion and neuroinflammation in the stellate ganglion (SG) contribute to CHF-increased cell excitability of cardiac sympathetic postganglionic (CSP) neurons, which subsequently promotes cardiac sympathetic overactivation and ventricular arrhythmogenesis in CHF rats. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a crucial mediator in macrophage activation. Our pilot data showed that RDN attenuates cardiac sympathetic overactivation and ventricular arrhythmias, which are accompanied by the marked reduction of GM- CSF level and macrophage activation in SGs in CHF rats. However, it remains unclear if the antiarrhythmic effect of RDN is achieved via attenuating GM-CSF-mediated inflammatory pathways in SGs in CHF. Following the discovery of the antiarrhythmic mechanisms of RDN, this proposal aims to develop a novel clinical intervention to achieve the therapeutic role of RDN and avoid its limitations. Since sympathetic innervation of the kidney originates primarily from neurons in the aorticorenal ganglion (ARG), targeting ARG neurons could be a logical therapeutic strategy for achieving the antiarrhythmic role of RDN. Considering the advantages of optogenetics, including rapid, specific control of neuronal activities by light-sensitive opsins, adeno-associated-virus- Archaerhodopsin (ArchT, an inhibitory light-sensitive opsin) gene will be transfected into ARG neurons in CHF rats. Specificity of neuronal expression of ArchT in ARG neurons will be achieved by linking a neuron-specific promoter to the ArchT gene. Continual optogenetic silencing in ARG neurons will be achieved by illuminating a LED probe that is controlled and powered wirelessly in freely moving animals. We hypothesize that optogenetic inhibition of ARG neurons would reduce CHF-elevated GM-CSF level in SGs, which subsequently alleviates macrophage activation and neuroinflammation in SGs, thereby attenuating CSP neuronal excitability, cardiac sympathetic overactivation, and ventricular arrhythmogenesis in CHF. Using multi-faceted technical approaches ranging from whole-animals to cellular-molecular levels, we will design in vivo and in vitro studies to assess these questions. Specific Aim 1, we will test if GM-CSF signaling axis contributes to macrophage activation and neuroinflammation in SGs from CHF animals. Specific Aim 2, we will address if GM-CSF signaling pathway contributes to CHF-increased cell excitability of CSP neurons, cardiac sympathetic overactivation, and ventricular arrhythmogenesis. Specific Aim 3, we will determine if optogenetic silencing in ARGs can achieve the antiarrhythmic effect of RDN by attenuating GM-CSF-induced macrophage activation and neuroinflammation in SGs in CHF. These studies will open a new avenue in therapeutics against lethal ventricular arrhythmia and provide a novel clinical intervention to reduce mortality and improve outcomes and quality of life in CHF patients.