Optogenetic termination of atrial tachyarrhythmias by brief pulsed light stimulation

https://doi.org/10.1016/j.yjmcc.2023.03.006Get rights and content

Highlights

  • Optogenetics can be used to terminate reentrant arrhythmias, which may provide a painless method of cardioversion.

  • Mechanisms of optogenetic arrhythmia termination include (i) transient conduction block or (ii) filling of the excitable gap.

  • We demonstrated light-induced APD and ERP prolongation even within the absolute refractory period in ChR2-epressing miced.

  • APD and ERP prolongation may function as a previously unrecognized mechanism of optogenetic atrial arrhythmia termination.

  • For clinical application of optogenetic termination, our findings could help to establish an efficient stimulation protocol.

Abstract

Aims

The most efficient way to acutely restore sinus rhythm from atrial fibrillation (AF) is electrical cardioversion, which is painful without adequate sedation. Recent studies in various experimental models have indicated that optogenetic termination of AF using light-gated ion channels may provide a myocardium-specific and potentially painless alternative future therapy. However, its underlying mechanism(s) remain(s) incompletely understood. As brief pulsed light stimulation, even without global illumination, can achieve optogenetic AF termination, besides direct conduction block also modulation of action potential (AP) properties may be involved in the termination mechanism. We studied the relationship between optogenetic AP duration (APD) and effective refractory period (ERP) prolongation by brief pulsed light stimulation and termination of atrial tachyarrhythmia (AT).

Methods and results

Hearts from transgenic mice expressing the H134R variant of channelrhodopsin-2 in atrial myocytes were explanted and perfused retrogradely. AT induced by electrical stimulation was terminated by brief pulsed blue light stimulation (470 nm, 10 ms, 16 mW/mm2) with 68% efficacy. The termination rate was dependent on pulse duration and light intensity. Optogenetically imposed APD and ERP changes were systematically examined and optically monitored. Brief pulsed light stimulation (10 ms, 6 mW/mm2) consistently prolonged APD and ERP when light was applied at different phases of the cardiac action potential. Optical tracing showed light-induced APD prolongation during the termination of AT.

Conclusion

Our results directly demonstrate that cationic channelrhodopsin activation by brief pulsed light stimulation prolongs the atrial refractory period suggesting that this is one of the key mechanisms of optogenetic termination of AT.

Graphical abstract

Prolongation of action potential (AP) duration and effective refractory period by brief pulsed light stimulation of optogenetically modified hearts during an AP leads to cardioversion.

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Introduction

Atrial fibrillation (AF) is the most common tachyarrhythmia and has a large socioeconomic impact due to its associated morbidity, mortality, reduction in quality of life and health care costs [1]. The prevalence of AF is rapidly growing globally due to aging of the human population and adherence to unhealthy lifestyles. Although clinical trials have confirmed the efficacy of treating AF by pulmonary vein isolation [2], many patients show refractory symptomatic AF despite multiple ablation procedures and drug therapies [[3], [4], [5]]. A considerable number of these patients require electrical cardioversion to terminate AF [6]. However, electrical cardioversion is distressing and requires adequate sedation [7]. To improve quality of life and simplify the care of patients with recurrent AF, we require a novel therapeutic approach that enables painless cardioversion [8].

Optogenetics is an innovative technology that includes the delivery of microbial light-sensitive proteins called opsins to excitable cells, which enables either light-based depolarization or hyperpolarization [9,10]. Channelrhodopsin-2 (ChR2) is a light-gated cation channel activated by blue light. Early studies have shown that expressing ChR2 in cardiomyocytes and applying blue pulsed light to the myocardium resulted in depolarization of the sarcolemma, which enabled optogenetic pacing [[11], [12], [13], [14], [15]]. More recent studies have shown that long light illumination can terminate reentrant tachyarrhythmias [[16], [17], [18], [19], [20]] and could provide a method for painless cardioversion.

The mechanism(s) of optogenetic termination by depolarization with optogenetic tools is/are not well understood. Possible mechanisms include (i) establishment of a transient conduction block [17,21,22] and (ii) filling of the excitable gap [8]. Filling of the excitable gap involves only brief light exposure (of a few milliseconds [ms]) but requires global illumination of the excitable myocardium for optimal termination efficacy [23], which is difficult to achieve especially in anatomically complicated structures such as the atria [8]. In in situ experiments in transverse ventricular slices, the photostimulation of the depolarizing light-activated ion channel Ca2+-translocating channelrhodopsin caused a complete conduction block and terminated reentrant arrhythmias [22]. It was proposed that the conduction block was achieved by the inactivation of voltage-dependent Na+ channels via resting membrane potential elevation during light stimulation [8,13]. Based on this hypothesis, illumination should be conducted for the duration of the tachycardia cycle to terminate reentrant arrythmias. However, local brief (10–30 ms) pulsed light stimulation without global illumination can also terminate reentrant arrhythmias with termination rates of ∼80% [23,24]. Therefore, we presumed that an additional mechanism is involved in the optogenetic termination of arrhythmias.

ChR2 shows an inwardly rectifying current-voltage relationship with a reversal potential of approximately 0 mV [10]. As a consequence, light stimulation of ChR2 leads to influx of cations, even when the cell membrane is (partially) depolarized. It is hypothesized that brief pulsed light stimulation, even within the absolute and relative refractory period, may prolong the action potential (AP) duration (APD) and, therefore, the effective refractory period (ERP), effectively increasing the arrhythmia termination rate beyond that derived from the instant conduction block imposed by the elevated resting membrane potential. This phenomenon has been observed in in silico and in vitro experiments and has been hypothesized to modulate AP shape [[25], [26], [27]]. However, the effect of brief pulsed light stimulation on APD and ERP duration and the influence of refractory period on optogenetic termination rate have not yet been studied in whole hearts.

The aims of this study were to systematically investigate optogenetic APD and ERP prolongation by brief pulsed light stimulation and to describe the relationship between refractory period extension and arrhythmia termination rate in an ex vivo whole heart model of atrial tachyarrhythmia (AT).

Section snippets

Animals

Cx40-CreERT2 mice (Institut de Biologie du Développement de Marseille, Aix-Marseille Université) [28] were bred to B6.Cg-Gt(ROSA)26Sortm27.1(CAG-COP4⁎H134R/tdTomato)Hze/J15 mice (The Jackson Laboratory, Bar Harbor, ME) to generate transgenic mice harboring a taxomifen-inducible Cre recombinase (CreERT2) gene under the control of the Cx40 promoter and a ChR2(H134R) ∼ tdTomato fusion protein gene driven by the strong ubiquitously active CAG promoter [15]. The Cx40 promoter mediates CreERT2

Histological and electrophysiological characterization of ChR2-expressing atrial tissue

ChR2 expression was histologically assessed by tdTomato fluorescence, which confirmed that ChR2 was expressed in the atria of mice after tamoxifen treatment and was mainly located at the plasma membrane (Fig. 1C). To confirm the function of ChR2, the epicardium of the right atria was irradiated with blue light (470 nm, 10 mW/mm2) during sinus rhythm to examine photostimulation (Fig. 1B). As expected, pulsed light stimulation onto the right atrial surface resulted in consistent atrial pacing.

Discussion

In the present study, we systematically investigated the influence of brief pulsed light stimulation on atrial APs in intact mouse hearts expressing the H134R variant of ChR2. We clearly demonstrated that brief pulsed light stimulation applied during an AP could prolong the APD and ERP. We also observed that optogenetic termination of ATs was achieved with the prolongation of APD. Based on the results of our study, we propose that ERP prolongation by light stimulation is one of the key

Conclusion

Our results directly demonstrated that brief pulsed light stimulation prolonged the atrial refractory period depending on pulse duration in perfused mouse hearts and suggested that prolongation of the refractory period is one of the key mechanisms of optogenetic termination of AT.

The following are the supplementary data related to this article.

Funding

This work was supported, in part, by Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research 18 K08058 (M.W.) and 22_K16093 (M.N.), and research grants from the Fukuda Foundation for Medical Technology (M.W.) and SENSHIN Medical Research Foundation (M.W.).

Author contribution statement

M.N., M.W. conceptualized the project. M.N., M.W. and L.M. performed experimentsa. M.N. and M.W. analyzed data. H.N., T.K., T.K., T.K., H.H., R.K., and T.T. cinterpreted the data. M.N. and M.W. wrote the manuscript. A.A.F.d.V. critically reviewed and edited the article. T.A. supervised and validatedthe revised article. All authors approved the final version of the article and agree to be accountable for all aspects of the work.

Declaration of Competing Interest

None declared.

Acknowledgments

We thank Noriko Tosa, Norio Takei, Yuki Kimura, and Miwako Yamane for their technical assistance in the experiments, and thank Prof. Katja Zeppefeld and Prof. Daniël A. Pijnappels for  insightful discussion.

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