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

我们的目标是首次在心脏中开发用于心脏刺激的声遗传学， 超声波敏感的离子通道在大鼠心脏和刺激心脏功能使用超声波。心律失常 是死亡率和发病率的主要来源。药物治疗未达到可接受的结局 in a large大fraction部分of patients患者.导管消融和手术消融可能有效，但它们需要侵入性 手术在过去的十年中，光遗传学被研究作为电子起搏器的替代品， 提供非电的、低能量的起搏，其可以是细胞类型特异性的和无痛的。然而，有限的光线 穿透胸腔并进入心肌， 心脏应用为了解决心律失常管理方面尚未满足的需求，我们建议开发一种新的 - 将机械敏感离子通道引入心脏的策略，即心脏声遗传学， 使用低强度聚焦超声（LIFU）激活这些通道进行抗肿瘤治疗。 目的1：筛选和优化适合心脏声遗传学研究的离子通道。 机械敏感离子通道需要满足以下标准才能适用于心脏声遗传学： 它们可以被LIFU激活，并且对起搏大鼠心脏足够快地响应; 2）它们可以是病毒性的， 在心脏中表达，并且当被超声激活以激发心肌细胞时， 3）它们被模仿心脏收缩过程中的肌肉收缩的机械刺激最低限度地激活。 正常的心脏跳动，这样它们就不会严重改变正常的心脏功能。根据这些标准，我们 将评估两个候选通道，MscL-G22 S（具有突变G22 S的MscL通道）和TRPV 4。我们 可以使这些通道突变以增强超声灵敏度。为此，我们将使用体外模型， 细胞和电生理学和Ca 2+成像与Aim 2中的离体模型并行进行验证。 目标二。证明声遗传学在大鼠心脏中的可行性和安全性。 我们将在大鼠心脏中表达MscL-G22 S和TRPV 4通道，并测试LIFU起搏心率的能力 使用Langendorff制剂，以各种能量、持续时间、频率和波形离体进行。我们还将 评估外源性机械敏感离子通道对心脏生理的影响。评估 声刺激的安全性，我们将监测动物的存活率、体重、摄食量和心电图 与表达外源性离子通道的对照动物相比， 载体和无外源表达的动物。我们还将比较动作电位波形、传导 速度和激活模式的声遗传修饰和控制动物使用光学映射。 这些目标的成功完成将为心血管界提供一个变革性的工具， 能够在体内非侵入性地刺激大型动物和人类的心脏。这个工具有潜力 成为抗肿瘤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.