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...