Engineered BacNav and BacCav for Improved Excitability and Contraction

心肌兴奋性和收缩功能受损是预防心肌梗死的重要靶点 发生心脏性猝死和心力衰竭进展。不断增长的机械论理解 心脏病理和日益安全有效的病毒携带方法使基因进入人体 治疗是对抗各种心脏病的一种有吸引力的策略。具体地说，从基因上讲，在一个 稳定的方式，直接增强心肌细胞的钠或L型钙电流可直接增强细胞 兴奋性和收缩性，并抵消各种心脏疾病中电异常的发生。 然而，心肌钠或L型钙通道基因太大，不能通过治疗有效地传递 病毒包括腺相关病毒(AAV)载体。为了应对这一挑战，我们建议开发一种 利用小得多的原核电压门控钠的工程的基于AAV的新疗法 (BacNav)和钙(BacCav)通道基因。我们的初步结果表明，基因工程杆菌Nav 通道可改善体外和矽肺模型心肌细胞兴奋性和动作电位传导 大鼠和人的纤维化心脏组织。此外，我们还展示了成功的心肌细胞特异性AAV9 在健康小鼠心脏内传递BacNav通道不会对心脏电生理产生任何不良影响 或收缩功能。在这些有希望的结果的基础上，我们建议：1)识别基因工程的BacNav变体 具有特定的突变和运输基序，使心肌细胞的兴奋性和动作电位速度最大化 通过利用体外细胞培养、体外心脏切片制备和计算机模拟以及2)工程新技术 BacCav的变种，单独或与BacNav联合使用不仅可以增强心肌细胞的兴奋性 而且还有收缩强度，这将在体外使用工程3D心脏组织模型进行研究。最后，我们 将利用心肌组织兴奋性受损的小鼠模型(遗传性心肌钠电流丢失(SCN5A/- )或收缩功能障碍(心肌梗死)，以探索已识别的BacNav和BacCav基因中的哪一个 以AAV为载体的载体在体内可诱导最佳的长期治疗效果。如果成功，这些研究将 为今后哺乳动物原核细胞通道调控的机制研究奠定基础 并将指导工程BacNav和BacCav通道疗法在大动物身上的测试 心脏病的模型。

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.