Impaired cardiomyocyte excitability and contractile function represent important targets for preventing the occurrence of sudden cardiac death and progression of heart failure. Growing mechanistic understanding of cardiac pathologies and increasingly safe and effective methods to deliver viruses to human body make gene therapies an attractive strategy for combatting various heart diseases. Specifically, the ability to genetically, in a stable fashion, directly augment sodium or L-type calcium current in cardiomyocytes could directly enhance cell excitability and contractility and counteract occurrence of electrical abnormalities in a variety of heart diseases. However, cardiac Na+ or L-type Ca2+ channel genes are too large to be effectively delivered by therapeutic viruses including adeno-associated viral (AAV) vectors. To address this challenge, we propose to develop a novel AAV-based therapy that leverages engineering of much smaller prokaryotic voltage-gated sodium (BacNav) and calcium (BacCav) channel genes. Our preliminary results show that genetically engineered BacNav channels can improve cardiomyocyte excitability and action potential conduction in in vitro and in silico models of rat and human fibrotic heart tissues. Furthermore, we demonstrate successful cardiomyocyte-specific AAV9 delivery of BacNav channels in healthy murine hearts without any adverse effects on cardiac electrophysiology or contractile function. Building on these promising results, we propose to: 1) identify engineered BacNav variants with specific mutations and trafficking motifs that maximize cardiomyocyte excitability and action potential speed by utilizing in vitro cell culture, ex vivo heart slice preparations, and computer simulations and 2) engineer new variants of BacCav, which alone or in combination with BacNav can augment not only cardiomyocyte excitability but also contractile strength, which will be studied using engineered 3D heart tissue models in vitro. Finally, we will exploit murine models of impaired cardiac tissue excitability (genetic loss of cardiac Na+ current (SCN5A+/- )) or contractile dysfunction (myocardial infarction) to explore which of the identified BacNav and BacCav genes delivered by AAV vector will induce optimal long-term therapeutic effects in vivo. If successful, these studies will create a foundation for the future mechanistic studies of prokaryotic channel regulation in mammalian cardiomyocytes and will guide testing of the engineered BacNav and BacCav channel therapies in large animal models of heart disease.

Heart diseases remain leading causes of death in industrialized countries including the US. The overall goal of this project is to develop new gene therapies that employ engineered prokaryotic sodium and calcium channels. The ability of these therapies to improve electrical and mechanical function of the heart will be tested in mouse models of disease.