Optical control of cardiac myocyte activity with ChR2 and NpHR

描述（由申请人提供）：心律失常是美国死亡率和发病率的主要原因之一，给公共卫生带来了巨大负担。虽然目前的疗法已经证明了症状和死亡率的好处，有许多缺点，每一个导致显着的发病率。药理学方法通常缺乏细胞和时间精度，虽然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。在过去的几年中，斯坦福大学细胞光学界面实验室（COIL）已经成功地开发了一种系统，该系统利用两个单组分离子通道对细胞放电进行遗传靶向的、时间上精确的光学控制，所述单组分离子通道具有响应于光刺激的亚毫秒开放动力学，特别是视紫红质-2（ChR 2）和盐视紫红质（NpHR）。该技术允许在体内对特定神经元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。关于心律失常，局部激活心脏内的特定区域或细胞类型的能力将产生治疗选择的新视野，从抑制心律失常的传播到更生理性的起搏，到改变对心脏的交感神经和副交感神经输入。到目前为止，这种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。本研究的具体目的如下：1）证明ChR 2对心肌细胞放电的遗传靶向、时间精确的光学激活。2)证明NpHR对肌细胞活性的遗传靶向、时间上精确的光学抑制。此外，通过ChR 2和NpHR的共表达，我们预测可以在同一细胞内实现对肌细胞膜电位的双向光学控制。3)证明ChR 2和NpHR的表达对培养的心肌细胞的基本电特性和存活率的影响最小。慢病毒基因递送将用于实现ChR 2和NpHR的肌细胞表达。将通过全细胞膜片钳技术收集电生理数据，以证明脉冲光引起的单个尖峰以及尖峰串的激活和抑制。随着老年人口的增长，心律失常的发病率预计将在未来50年内增加三倍，从而带来重大的经济、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，可能性开放到下一个发展阶段，使这项技术更接近临床应用，具有开发心律失常上级治疗选择的巨大潜力。

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.