Genetically Engineered Excitable Cardiac Myofibroblasts Coupled to Cardiomyocytes Rescue Normal Propagation and Reduce Arrhythmia Complexity in Heterocellular Monolayers

Rationale and ObjectiveThe use of genetic engineering of unexcitable cells to enable expression of gap junctions and inward rectifier potassium channels has suggested that cell therapies aimed at establishing electrical coupling of unexcitable donor cells to host cardiomyocytes may be arrhythmogenic. Whether similar considerations apply when the donor cells are electrically excitable has not been investigated. Here we tested the hypothesis that adenoviral transfer of genes coding Kir2.1 (IK1), NaV1.5 (INa) and connexin-43 (Cx43) proteins into neonatal rat ventricular myofibroblasts (NRVF) will convert them into fully excitable cells, rescue rapid conduction velocity (CV) and reduce the incidence of complex reentry arrhythmias in an in vitro model. Methods and ResultsWe used adenoviral (Ad-) constructs encoding Kir2.1, NaV1.5 and Cx43 in NRVF. In single NRVF, Ad-Kir2.1 or Ad-NaV1.5 infection enabled us to regulate the densities of IK1 and INa, respectively. At varying MOI ratios of 10/10, 5/10 and 5/20, NRVF co-infected with Ad-Kir2.1+ NaV1.5 were hyperpolarized and generated action potentials (APs) with upstroke velocities >100 V/s. However, when forming monolayers only the addition of Ad-Cx43 made the excitable NRVF capable of conducting electrical impulses (CV = 20.71±0.79 cm/s). When genetically engineered excitable NRVF overexpressing Kir2.1, NaV1.5 and Cx43 were used to replace normal NRVF in heterocellular monolayers that included neonatal rat ventricular myocytes (NRVM), CV was significantly increased (27.59±0.76 cm/s vs. 21.18±0.65 cm/s, p ConclusionViral transfer of genes coding Kir2.1, NaV1.5 and Cx43 to cardiac myofibroblasts endows them with the ability to generate and propagate APs. The results provide proof of concept that cell therapies with excitable donor cells increase safety and reduce arrhythmogenic potential.研究背景与目的 通过对非兴奋性细胞实施基因工程改造以使其表达间隙连接（gap junctions）和内向整流钾通道（inward rectifier potassium channels）的前期研究提示，旨在将非兴奋性供体细胞与宿主心肌细胞（cardiomyocytes）实现电耦合的细胞疗法或存在致心律失常性（arrhythmogenic）风险。目前尚未明确，当供体细胞本身具备电兴奋性时，是否仍需遵循同类考量。本研究验证了如下假说：将编码Kir2.1（IK1）、NaV1.5（INa）及连接蛋白-43（Cx43）的基因通过腺病毒转染（adenoviral transfer）至新生大鼠心室成纤维细胞（NRVF），可将其转化为完全可兴奋细胞，在体外模型中恢复快速传导速度（CV）并降低复杂折返性心律失常（reentry arrhythmias）的发生率。 研究方法与结果 本研究采用携带Kir2.1、NaV1.5及Cx43编码序列的腺病毒（Ad-）载体转染NRVF。在单个NRVF中，Ad-Kir2.1或Ad-NaV1.5转染可分别实现对IK1与INa通道密度的精准调控。当采用10/10、5/10及5/20等不同感染复数（MOI）比例共转染Ad-Kir2.1与Ad-NaV1.5时，NRVF发生超极化，并可产生动作电位（APs），其动作电位上升支速度超过100 V/s。但在构建细胞单层时，仅共转染Ad-Cx43才可使可兴奋NRVF实现电冲动传导（传导速度CV=20.71±0.79 cm/s）。当将过表达Kir2.1、NaV1.5及Cx43的基因工程改造可兴奋NRVF，用于替换包含新生大鼠心室肌细胞（NRVM）的异细胞单层培养体系中的正常NRVF时，传导速度显著提升（27.59±0.76 cm/s 对比 21.18±0.65 cm/s，p < 0.05）。 研究结论 将编码Kir2.1、NaV1.5及Cx43的基因通过腺病毒转染至心脏成纤维细胞，可赋予其产生并传播动作电位的能力。本研究结果证实了如下概念：采用可兴奋性供体细胞的细胞疗法可提升治疗安全性并降低致心律失常风险。