Abstract
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<0.05), reaching values similar to those of pure myocytes monolayers (27.27±0.72 cm/s). Moreover, during reentry, propagation was faster and more organized, with a significantly lower number of wavebreaks in heterocellular monolayers formed by excitable compared with unexcitable NRVF.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.
Highlights
Cell therapy, which involves the introduction of new cells into host tissue, is emerging as a novel therapeutic approach in heart disease [1]
Viral transfer of genes coding Kir2.1, NaV1.5 and Cx43 to cardiac myofibroblasts endows them with the ability to generate and propagate action potentials (APs)
The results provide proof of concept that cell therapies with excitable donor cells increase safety and reduce arrhythmogenic potential
Summary
Cell therapy, which involves the introduction of new cells into host tissue, is emerging as a novel therapeutic approach in heart disease [1]. Recent studies suggested that coupling of unexcitable cells, which are commonly used in cell therapy, to cardiomyocytes might yield different functional outcomes that can be arrhythmogenic [6]. Yankelson et al showed that when fibroblasts transfected to express the voltage-sensitive potassium channel KV1.3 were co-cultured with neonatal rat ventricular myocytes (NRVM) they reduced significantly (68%) the spontaneous beating frequency of the cultures [5]. Our previous studies using co-cultures of randomly distributed NRVM and neonatal rat ventricular myofibroblasts (NRVF), which express low levels of Cx43 and repolarizing potassium currents, demonstrated correlations between the proportion of myofibroblasts and the amount of conduction and rotor frequency slowing, the number of wavebreaks, and the degree of fibrillatory conduction [8]. The above studies brought attention to the potential arrhythmogenic consequences of using unexcitable cells in an attempt to regenerate normal cardiac function. No one has studied the electrophysiological consequences when the donor cells are of cardiac origin and engineered to be fully excitable and capable of generating action potentials (APs)
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