Cardiac Sonogenetics: Noninvasive Stimulation of the Heart With Low-Intensity Focused Ultrasound

我们的目标是通过表达外源基因，首次在心脏中开发用于心脏刺激的声遗传学。 超声敏感的大鼠心脏离子通道，并利用超声刺激心脏功能。心律不齐 是死亡率和发病率的主要来源。药物治疗不能取得可接受的结果 在很大一部分患者中。导管消融和外科消融是有效的，但它们需要侵入性。 做手术。在过去的十年里，光遗传学作为电子起搏器的替代品进行了研究， 提供非电的、低能量的起搏，可以是特定细胞类型的无痛起搏。然而，有限的光线 通过肋骨和进入心肌的穿透减少了光遗传学在临床上的翻译 心脏应用。为了解决心律失常管理中未得到满足的需求，我们建议开发一种新的 策略，即心脏声学，将机械敏感的离子通道引入心脏和 使用低强度聚焦超声(LIFU)激活这些通道以进行抗心律失常治疗。 目的1：筛选和优化适合心脏声学的离子通道。 机械敏感离子通道需要满足以下标准才能适用于心脏声学：1) 它们可以被利福激活，并对大鼠心脏起搏做出足够快的反应；2)它们可以是病毒 在心脏中表达，当被超声波激活时，可以去极化膜电位，从而兴奋 心脏；以及3)它们被模拟肌肉收缩的机械刺激最小程度地激活 正常的心跳使它们不会严重改变正常的心脏功能。基于这些标准，我们 将评估两个候选通道，MSCL-G22S(带有突变G22S的MSCL通道)和TRPV4。我们 可能会使这些通道发生突变，以提高超声敏感性。为了达到这个目的，我们将使用体外模型 细胞与电生理学和钙离子成像的并行体外模型在AIM 2中进行验证。 目的2.论证声遗传学在大鼠心脏中应用的可行性和安全性。 我们将在大鼠心脏中表达MSCL-G22S和TRPV4通道，并检测利福对心率起搏的能力 具有不同的能量、持续时间、频率和波形，体外使用朗宁多夫制剂。我们还将 评价外源性机械敏感离子通道对心脏生理的影响。要评估 声学刺激的安全性，我们将监测动物的存活率、体质量、摄食量和心电图 外源离子通道的表达，与病毒表达的对照动物进行比较 载体，以及没有外源表达的动物。我们还将比较动作电位波形、传导 声学修饰动物和对照动物使用光学映射的速度和激活模式。 这些目标的成功完成将为心血管学界提供一种变革性的工具， 能够在活体内无创地刺激大型动物和人类的心脏。这个工具有可能 成为抗心律失常研究的下一个前沿和人类治疗应用的未来。

Our goal is to develop for the first time sonogenetics in the heart for cardiac stimulation, by expressing exogenous ultrasound-sensitive ion channels in rat hearts and stimulating cardiac function using ultrasound. Arrhythmias are a major source of mortality and morbidity. Pharmacological treatment does not achieve acceptable outcomes in a large fraction of patients. Catheter ablation and surgical ablation can be effective, but they require invasive surgery. Optogenetics has been investigated in the past decade as an alternative to electronic pacemakers, offering non-electrical, low-energy pacing that can be cell-type specific and painless. However, the limited light penetration through the rib cage and into the myocardium curtails the clinical translation of optogenetics in cardiac applications. To address the unmet need in arrhythmia management, we propose to develop a new strategy, namely cardiac sonogenetics, to introduce mechanically sensitive ion channels into the heart and activate these channels using low-intensity focused ultrasound (LIFU) for antiarrhythmic therapy. Aim 1: Select and optimize the ion channels suitable for cardiac sonogenetics. Mechanosensitive ion channels need to meet the following criteria to be suitable for cardiac sonogenetics: 1) they can be activated by LIFU and respond sufficiently quickly for pacing rat hearts; 2) they can be virally expressed in the heart and can depolarize the membrane potential when activated by ultrasound to excite the heart; and 3) they are minimally activated by mechanical stimulation that mimics the muscle contraction during the normal heart beat such that they do not severely alter normal cardiac function. Based on these criteria, we will evaluate two candidate channels, MscL-G22S (a MscL channel with the mutation G22S) and TRPV4. We may make mutations of these channels to enhance ultrasound sensitivity. In this aim, we will use in vitro model cells and electrophysiology and Ca2+ imaging in parallel with the ex vivo model in Aim 2 for the validation. Aim 2. Demonstrate the feasibility and safety of sonogenetics in rat hearts. We will express MscL-G22S and TRPV4 channels in rat hearts and test the ability of LIFU to pace the heart rate with various energy, duration, frequency, and waveforms ex vivo using a Langendorff preparation. We will also evaluate the influence of the exogenous mechanosensitive ion channels on heart physiology. To assess the safety of sonogenetic stimulation, we will monitor survival rate, body mass, food intake, and ECG of the animals with expression of the exogenous ion channels, compare them with control animals with expression of viral vectors, and animals with no exogenous expression. We will also compare action potential waveform, conduction velocity, and activation patterns for sonogenetically modified and control animals using optical mapping. Successful completion of these aims will provide the cardiovascular community with a transformative tool, capable of noninvasively stimulating the hearts of large animals and humans in vivo. This tool has the potential to become the next frontier in antiarrhythmic research and future of therapeutic applications in humans.