Optical control of cardiac myocyte activity with ChR2 and NpHR

ChR2 和 NpHR 对心肌细胞活性的光学控制

• 批准号: 7749610
• 负责人: Shirley Park
• 金额: $ 5.72万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-11 至 2011-08-10
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7749610
• 关键词: Action Potentials; Adrenergic Agents; Anti-Arrhythmia Agents; Arrhythmia; Automobile Driving; Cardiac; Cardiac Myocytes; Cardiac ablation; Cell Survival; Cells; Data; Development; Devices; Economics; Elderly; Electrodes; Gene Delivery; Halorhodopsins; Heart; In Vitro; Incidence; Intervention; Ion Channel; Ion Pumps; Kinetics; Laboratories; Light; Membrane Potentials; Morbidity - disease rate; Movement; Muscle Cells; Myocardial; Neurons; Optics; Patch-Clamp Techniques; Physiologic pulse; Population; Public Health; Resolution; Specificity; Staging; Stimulus; System; Techniques; Technology; Therapeutic; Tissues; Training; adrenergic; base; cell type; clinical application; electrical property; in vivo; in vivo Model; millisecond; mortality; mouse model; prevent; response; sleep regulation; social

DESCRIPTION (provided by applicant): Cardiac arrhythmias constitute one of the major causes of mortality and morbidity in the U.S. and present an enormous burden to public health. While current therapies have proven symptomatic and mortality benefit, there are a number of drawbacks with each leading to significant morbidity. Pharmacological approaches generally lack cellular and temporal precision, and while electrode-based approaches have better temporal resolution, they still lack cell specificity. Within the last several years, the Stanford Cellular-Optical Interface Laboratory (COIL) has successfully developed a system of genetically targeted, temporally precise optical control of cell firing utilizing two single-component ion channels with submillisecond opening kinetics in response to light stimuli, specifically Channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR). This technique has allowed for functional control of specific neuronal cell types in vivo, with mouse models successfully demonstrating optical control of movement and sleep regulation. With regard to cardiac arrhythmias, the ability to locally activate specific regions or cell types within the heart would give rise to a new horizon of therapeutic options, from inhibiting propagation of arrhythmias, to pacing more physiologically, to altering sympathetic and parasympathetic input to the heart. Thus far, this technique has been demonstrated reliably in neurons, however has not been applied to other populations of excitable cells. The specific aims of this project are as follows: 1) To demonstrate genetically targeted, temporally precise optical activation of myocyte firing with ChR2. 2) To demonstrate genetically targeted, temporally precise optical inhibition of myocyte activity with NpHR. Furthermore, with co-expression of ChR2 and NpHR, we predict that bidirectional optical control of myocyte membrane potential can be achieved within the same cell. 3) To demonstrate that expression of ChR2 and NpHR will have minimal effects on the basic electrical properties and survival of cultured myocytes. Lentiviral gene delivery will be used to achieve myocyte expression of ChR2 and NpHR. Electrophysiological data will be gathered by whole cell patch clamp technique to demonstrate both activation and inhibition of single spikes as well as spike trains elicited by pulsed light. As the elderly population grows, the incidence of arrhythmias is projected to increase by three fold in the next fifty years, presenting a significant economic, social, and public health burden. By successfully demonstrating the fundamental principle of optogenetics at the cellular level, the possibilities are open to the next stage of development in bringing this technology closer to clinical application, with enormous potential to develop superior therapeutic options for cardiac arrhythmias.

描述（由申请人提供）：心律失常是美国死亡和发病的主要原因之一，对公共卫生构成了巨大的负担。虽然目前的治疗方法已被证明对症状和死亡率有利，但仍有许多缺点，每种缺点都会导致严重的发病率。药理学方法通常缺乏细胞和时间精度，而基于电极的方法具有更好的时间分辨率，但它们仍然缺乏细胞特异性。在过去的几年里，斯坦福细胞光学界面实验室（COIL）已经成功地开发了一种基因靶向系统，利用两个单组分离子通道，在响应光刺激时具有亚毫秒的开放动力学，特别是通道视紫红质-2 （ChR2）和盐视紫红质（NpHR），对细胞放电进行暂时精确的光学控制。这项技术允许在体内对特定神经细胞类型进行功能控制，小鼠模型成功地展示了运动和睡眠调节的光学控制。对于心律失常，局部激活心脏内特定区域或细胞类型的能力将带来新的治疗选择，从抑制心律失常的传播，到更生理的起搏，到改变对心脏的交感和副交感输入。到目前为止，这项技术已经在神经元中得到了可靠的证明，但尚未应用于其他可兴奋细胞群。该项目的具体目的如下：1)证明ChR2对肌细胞放电的基因靶向、时间精确的光学激活。2)证明NpHR对肌细胞活性的基因靶向、时间精确的光学抑制。此外，通过ChR2和NpHR的共表达，我们预测可以在同一细胞内实现肌细胞膜电位的双向光学控制。3)证明ChR2和NpHR的表达对培养肌细胞的基本电学特性和存活的影响很小。慢病毒基因传递将用于实现心肌细胞ChR2和NpHR的表达。电生理数据将通过全细胞膜片钳技术收集，以证明单尖峰和脉冲光引起的尖峰序列的激活和抑制。随着老年人口的增长，心律失常的发病率预计将在未来五十年增加三倍，带来重大的经济、社会和公共卫生负担。通过在细胞水平上成功展示光遗传学的基本原理，使这项技术更接近临床应用的可能性进入了下一阶段的发展，具有开发心律失常优越治疗方案的巨大潜力。