TY - JOUR
AU - Sasse,, Philipp
AB - Abstract Aims The primary goal in the treatment of symptomatic atrial fibrillation/flutter (AF) is to restore sinus rhythm by cardioversion. Electrical shocks are highly effective, but have to be applied under analgo-sedation and can further harm the heart. In order to develop a novel pain-free and less harmful approach, we explored herein the optogenetic cardioversion by light-induced depolarization. Methods and results Hearts from mice expressing Channelrhodopsin-2 (ChR2) and the AF-promoting loss-of-function Connexin 40 Ala96Ser mutation were explanted and perfused with low K+ Tyrode’s solution and an atrial KATP-channel activator. This new protocol shortened atrial refractoriness as well as slowed atrial conduction and thereby enabled the induction of sustained AF. AF episodes could be terminated by epicardial illumination of the atria with focussed blue light (470 nm, 0.4 mW/mm2) with an efficacy of ∼97% (n = 17 hearts). In > 80% of cases, light directly terminated the AF episode with onset of illumination. Because similar illumination intensity was able to locally inhibit atrial activity, we propose that a light-induced block of electrical activity is responsible for reliable AF termination. The success rate was strongly depending on the illuminated area, applied light intensity and duration of illumination. Importantly, we were also able to demonstrate optogenetic termination of AF in vivo, using epicardial illumination through the open chest (n = 3 hearts). To point towards a translational potential, we systemically injected an adeno-associated virus to express ChR2 in wild type hearts. After 6–8 months, we found robust ChR2 expression in the atria, enabling light-mediated AF termination in six of seven mice tested. Conclusion We provide the first evidence for optogenetic termination of atrial tachyarrhythmia in intact hearts from transgenic as well as wild type mice ex and in vivo. Thus, this report could lay the foundation for the development of implantable devices for pain-free termination of AF. Defibrillation, Cardioversion, Gene therapy, Arrhythmia, Supraventricular arrhythmia, Atrial fibrillation 1. Introduction Atrial fibrillation/flutter (AF) is the most common cardiac arrhythmia with substantial impact on morbidity and mortality as well as increasing incidence and prevalence.1 In clinics, restoring sinus rhythm is the first goal to relieve the patients from their symptoms and electrical cardioversion has the highest efficacy in acutely terminating AF. However, electrical shocks require high electrical currents inducing substantial pain and patient discomfort and can damage the heart.2 Therefore analgo-sedation is indispensable and miniaturized implantable ‘atrioverter’ are generally not accepted by the patients.2 Thus, electrical overdrive pacing with well-tolerable electrical currents, so called anti-tachycardia pacing was developed,3 but turned out to be ineffective in terminating AF episodes with short and irregular cycle lengths.4 Hence, an effective and pain-free therapeutic approach for AF termination on demand is currently lacking. In clear contrast to electrical field stimulation, optogenetics allows selective stimulation of defined cell types, which were genetically engineered to express light-sensitive proteins.5 The light-gated non-selective cation channel Channelrhodopsin-2 (ChR2) has been applied before for ventricular and atrial pacing of intact mouse hearts by short light pulses.6,7 Importantly, ChR2 expression could be restricted to cardiomyocytes, allowing light-induced pain-free stimulation without side effects. Furthermore, optogenetics provides the advantage of uniform and sustained depolarization which results in refractoriness of cardiomyocytes6 and this was recently used to terminate re-entrant electrical wavefronts in a monolayer of neonatal, atrial rat cardiomyocytes in vitro.8 To investigate if optogenetic methods could be used to terminate AF in the complex 3D architecture of intact atria, we sought to establish optogenetic cardioversion in the murine heart. Here we demonstrate for the first time, that epicardial illumination is a highly reliable approach to terminate AF in ChR2 expressing transgenic mice and in wild type mice after adeno-associated virus (AAV)-mediated gene delivery. 2. Methods 2.1 Animals All animal experiments were carried out according to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and were approved by the National Office for Nature, Environment and Consumer Protection in Recklinghausen, Nordrhein-Westfalen (Permit Numbers: 84-02.04.2015.A267 and 84.02.04.2011.A292). A new optogenetic AF-susceptible mouse line was generated by crossing mice expressing ChR2 with the H134R mutation9 under control of the chicken β-actin (CAG) promoter6 with mice carrying a targeted heterozygous Ala96Ser mutation of the mouse Connexin 40 gene (Cx40 Ala96Ser).10 ChR2 mice had CD1 genetic background (backcrossed at least 10 generations) and Cx40 Ala96Ser mice had C57/Bl6 background (backcrossed at least 10 generations). For ex vivo Langendorff perfusion of explanted hearts, we used ChR2 positive/Cx40 Ala96Ser (+/−) mice (n = 17, 47% female, 31 ± 4 weeks), ChR2 negative/Cx40 Ala96Ser (+/−) mice (n = 6, 17% female, 51 ± 1 weeks), CD1 wild type mice (n = 9, 78% female, 58 ± 5 weeks) and CD1 wild type mice after AAV-mediated gene transfer (n = 7, all female, 45 ± 1 weeks). For in vivo stimulation we used ChR2 positive/Cx40 Ala96Ser (+/−) mice (n = 3, 1 female, 18 ± 3 weeks). 2.2 AAV-based gene transfer The AAV9-CAG-hChR2(H134R)-mCherry virus (AAV9 capsid and AAV2 DNA) was obtained from Penn Vector Core (University of Pennsylvania, PA, USA). 2 × 1011 genome copies diluted in 100 µl phosphate buffered saline (PBS) were injected systemically via the internal jugular vein of 10-week-old female CD1 wild type mice (Charles River Laboratories, Wilmington, MA, USA) as described earlier.7,11 Mice were analyzed ex vivo 29 (n = 2), 36 (n = 3), and 37 (n = 2) weeks after virus injection at a mean age of 45 ± 1 weeks. For quantification of viral transduction, the right atria were dissected after the experiments and incubated in collagenase B (1 mg/ml; Roche, Basel, Switzerland) for 30 min at 37°C and shaken at 500 rpm. Subsequently cells were dissociated by pipetting, centrifuged (10 min, 5000 rpm), re-suspended in Tyrode’s solution and plated on laminin coated glass cover slips, for counting by fluorescence microscopy (10× objective, numerical aperture (NA): 0.3, Zeiss, Jena, Germany). At least five fields of view per heart were analyzed for the percentage of mCherry positive cells as earlier reported.7 The remaining heart with the intact left atrium was used for histological analysis (see below). 2.3 Ex vivo Langendorff-perfused hearts The experimental setup is summarized in Figure 2A. Hearts of heparinized mice were explanted after cervical dislocation and immediately placed in ice-cold PBS. Surrounding tissue was dissected and hearts were retrogradely perfused via the aorta with Tyrode’s solution containing (in mM): NaCl 140, KCl 5.4, MgCl2 2, CaCl2 1.8, glucose 10 and HEPES 10; pH 7.35–7.45 at 37°C. Bipolar cardiac electrograms were recorded between right atrium and a metal spoon placed under the heart’s apex with a bio-amplifier system (PowerLab 8/30, Animal Bio Amp ML 136, LabChart 7.1 software, AD Instruments, Dunedin, New Zealand). Additionally, either a bipolar epicardial atrial electrogram was recorded between electrodes at the left and right atrium or bipolar atrial and ventricular electrograms were recorded with a 2-French octapolar mouse electrophysiological catheter (Ciber Mouse, NuMed Inc., NY, USA) inserted via the orifice of the superior vena cava into the right heart chambers (Figure 2A). Epicardial illumination of the atria was performed with a macroscope (MVX10, Olympus, Tokyo, Japan) and a 1× objective (MVLPAPO1x, NA: 0.25) using a light-guide (Ø 3 mm) coupled 470 nm LED and a constant current driver (GCS-0470-50-A0510 and BLS-13000-1, Mightex, Pleasanton, CA, USA) controlled by the PowerLab system and the LabChart software. The area of illumination was set with the zoom function of the macroscope to circles of 100, 26, or 10 mm2 in order to cover both atria, the whole right atrium and parts of the right atrium, respectively (Figure 2A). A powermeter (PM100, Thorlabs, Newton, NJ, USA) was used to calculate the light intensity (light power divided by illuminated area in mW/mm2). After establishment of a regular sinus rhythm, we performed in some experiments an illumination protocol with light pulses of 3 s duration and stepwise increasing light intensity (∼0.02 mW/mm2 increase per step, 2 s delay in-between). Thereby we analyzed the individual lowest light intensities for induction of a premature supraventricular contraction (PSVC) and for complete local block of atrial electrical activity, which was defined as absence of atrial signals in the local atrial electrogram. To determine the atrial refractory period (ARP), programmed atrial electrical stimulation with shortening of one extra S2 stimulus (6× S1 with 150 ms cycle length, 1× S1S2 decreased by 2 or 3 ms each step) was performed with constant current applied slightly above the pacing threshold (stimulator 2100, AM Systems, Sequim, WA, USA) through bipolar silver chloride electrodes placed epicardially on the right atrium. ARP was defined as shortest S1S2 with effective atrial response in the electrograms. To increase the susceptibility to sustained episodes of atrial tachyarrhythmia (>15 s), perfusion solution was changed to a modified Tyrode’s solution with low potassium concentration (2 mM) constantly supplemented with 300 µM Diazoxide. To detect the effect of this perfusion solution, we waited for at least 7 min and reanalyzed P-wave duration as well as ARP. In this condition, electrical atrial burst stimulation (duration: 5 s, cycle length: 10–50 ms, current: 1–10 mA) reliably induced atrial flutter or fibrillation (AF) episodes characterized by high-frequency atrial electrical activity and a regular (atrial flutter) or irregular (atrial fibrillation) ventricular activation. The dominant cycle length of the respective arrhythmia was calculated from extracardiac or intracardiac electrograms. To determine success rates of optogenetic cardioversion and to compare different illumination parameters, only AF episodes lasting for at least 3 s after electrical burst stimulation were analyzed in order to exclude shorter self-terminating AF episodes (Figure 2C). In each attempt 4 light pulses of identical illuminated area (10, 26, and 100 mm2), light intensity (0.1, 0.2, 0.4, and 1 mW/mm2) and pulse duration (1, 3, 10, 30, 100, 300, and 1000 ms) were applied with 1 s delay in between. AF termination between the first light pulse and 3 s after the last light pulse (Figure 2C; highlighted as green box) was defined as successful optogenetic cardioversion. If AF persisted, the attempt was classified as non-successful and high intensity illumination (up to 5 mW/mm2) was applied to terminate AF, allowing subsequent cardioversion attempts in the same heart. If AF still persisted, perfusion was temporally changed to normal Tyrode’s solution until AF ceased. For statistical analysis of success rates, each illumination setting was tested for at least five attempts and the average success rate of each individual heart was used for comparison and is displayed as one data point in the figures. For mechanistic insights, we classified AF termination by light into three categories: (i) direct block, defined as cessation of high frequent atrial electrical activity exactly with onset of illumination or within one AF cycle thereafter, (ii) termination during, or (iii) after the illumination. To allow comparison to spontaneous AF termination, we analyzed and classified AF terminations likewise in ChR2 positive and ChR2 negative hearts using the identical illumination protocol (1 s, 0.4 mW/mm2, 100 mm2). Hearts of CD1 wild type mice with or without AAV-injection had shorter episodes of AF, most likely due to the lack of the Cx40 Ala96Ser mutation. Therefore, a one light pulse (1 s, 5 mW/mm2, 100 mm2) protocol was used for statistical analysis of cardioversion success rates (effective AF termination between onset and 1 s after the first light pulse). For some noisy electrogram recordings, a low pass filter of 70–90 Hz was applied. 2.4 In vivo electrophysiological investigation and optogenetic cardioversion Thirty minutes prior to the procedure, buprenorphine (0.1 mg/kg) was subcutaneously injected for intraoperative analgesia. Anaesthesia was induced by 1.5–2.5 vol.% isoflurane in 50% N2O/50% O2. Mice were intubated and ventilated (MINIVENT Mouse Ventilator Type 845, Harvard Apparatus Inc., Holliston, MA, USA) and placed on a warming plate (37°C). The right jugular vein was dissected from surrounding tissue and the 2-French octapolar mouse electrophysiological catheter (see above) was inserted and advanced into the right heart chambers as previously reported.10 Bipolar electrograms between neighboring electrodes showed atrial (proximal electrodes) or ventricular signals (distal electrodes). Subsequently the rib cage was carefully surgically removed and the heart exposed as described before.6,7 A spoon was placed below the heart’s apex for stabilization and to expose the atria for illumination. A ‘surface’ ECG was recorded between the right front leg and the spoon using the PowerLab system (see above). Illumination of the anterior portion of both atria (∼100 mm2) was performed with a macroscope (see above). AF was induced by intra-atrial burst stimulation using the proximal electrode pairs of the intracardiac catheter showing clear atrial signals (duration: 5 s, cycle length: 10–50 ms, current: 1–3 mA). After the experiment mice were sacrificed by cervical dislocation. 2.5 Histology and immunofluorescence Epicardial mCherry fluorescence was determined with a MVX10 macroscope (1× objective, NA: 0.25) using a mCherry filter set (F36-504, AHF Analysentechnik, Tübingen, Germany), a calibrated light source system (X-Cite 120 PC, Lumen Dynamics, Mississauga, Ontario, Canada), and an EMCCD camera (iXon 885+ with Solis software, Andor Technology, Belfast, United Kingdom). Explanted hearts with intact left atria were perfused with and stored in 4% paraformaldehyde for 24 h, subsequently dehydrated in 20% sucrose and frozen. Sections were cut with a cryotome (10 µm; Cryostat CM3050 S, Leica, Wetzlar, Germany). Staining and image acquisition were performed as reported earlier.7,11 Heart slices were permeabilized with 0.2% TritonX (Sigma-Aldrich, St Louis, MO, USA) for 20 min and subsequently stained for 2 h at room temperature with primary antibodies against CD45 (1:400; Merck Millipore, Billerica, MA, USA) and Connexin 43 (1:200; MyBiosource, San Diego, CA, USA) diluted in 5% donkey serum (Jackson ImmunoResearch, West Grove, PA, USA). Cy5 conjugated secondary antibodies (Jackson ImmunoResearch) diluted in 0.1% Hoechst 33342 (Sigma-Aldrich) were applied for 1 h at room temperature. To determine cell viability, we performed a TUNEL assay (In Situ Cell Death Detection Kit Fluorescein, Roche, Basel, Switzerland) according to the manufacturer’s instructions. Images were taken with an AxioZoom.V16 macroscope equipped with a PlanApoZ 1× objective lens (NA: 0.25), a 43 HE filter set, and an AxioCam MRm camera with ZEN 2012 software (all Zeiss) or with an inverted fluorescence microscope (Axiovert 200 M) equipped with the Apotome section module, a 40× Oil-Plan-Apochromat objective (NA: 1.4), Immersol 518 F, AxioCam MRm camera, AxioVision software (all Zeiss) and mCherry (F46-008), Tritc HC (F36-503) or Cy5 (F46-006) filter sets (all AHF Analysentechnik). 2.6 Statistical analysis Data are shown as mean ± standard error of the mean. Statistical analyses were performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA). P-wave duration and ARP before and after changing to Tyrode’s solution with 2 mM potassium and 300 µM Diazoxide as well as between ChR2 positive and ChR2 negative hearts were compared using the two-way repeated measures ANOVA with Bonferroni post-test (Figure 1B and C). The arrhythmia cycle lengths in ChR2 expressing and non-expressing hearts were compared using the Chi-square test for trend. Success rates with and without illumination in ChR2 expressing versus ChR2 non-expressing hearts were compared using the one-way ANOVA Kruskal-Wallis with Dunn’s multiple comparison post-test because of the non-normal distribution of each parameter tested and unequal variances of average cardioversion success rates. The differently classified AF terminations were compared between ChR2 positive and ChR2 negative hearts using the Chi-square test. The effect of different illumination parameters (illuminated area, light intensity and light pulse duration) was tested using the Friedman with Dunn’s multiple comparison post-test because of the non-normal distribution, unequal variances and the repeated measures in identical hearts. A P-value < 0.05 was considered statistically significant. Asterisks in the figures indicate significant levels (*P < 0.05; **P < 0.01; ***P < 0.001). Figure 1 Open in new tabDownload slide Ex vivo model for sustained atrial tachyarrhythmia. (A) Representative examples of P-waves from whole heart electrograms from hearts of Cx40 Ala96Ser (+/−) mice without (left) and with (right) expression of ChR2 during perfusion with normal Tyrode’s solution (black) and modified Tyrode’s solution with lower K+ concentration (2 mM) and 300 µM Diazoxide (red). (B, C) Statistical analysis of the influence of ChR2 expression and modified Tyrode’s solution (normal solution in black, modified Tyrode’s solution in red) on P-wave duration (B; n = 6–7; P = 0.6507 for ChR2 genotype, P = 0.0014 for solution) and ARP (C; n = 6; P = 0.1468 for ChR2 genotype, P < 0.0001 for solution). Statistical analysis by two-way repeated measures ANOVA, asterisks indicate Bonferroni post-test significances. Note that also unpaired, two tailed student’s t-tests did not reveal any significant differences for ChR2 genotype (normal Tyrode’s solution: P = 0.7129 for P-wave duration and P = 0.0954 for ARP; modified Tyrode’s solution: P = 0.6118 for P-wave duration and P = 0.7470 for ARP). (D) Whole heart electrograms of AF episodes with atrial flutter (top) and atrial fibrillation (bottom) induced by atrial electrical burst stimulation (5 s, 50 Hz; red boxes). Note the regular or the irregular ventricular activation during atrial flutter or atrial fibrillation, respectively. (E) Incidence of AF episodes with the indicated cycle lengths in ChR2 negative (grey, n = 223) and positive (blue, n = 236) hearts (P = 0.8902, Chi-square test for trend). Figure 1 Open in new tabDownload slide Ex vivo model for sustained atrial tachyarrhythmia. (A) Representative examples of P-waves from whole heart electrograms from hearts of Cx40 Ala96Ser (+/−) mice without (left) and with (right) expression of ChR2 during perfusion with normal Tyrode’s solution (black) and modified Tyrode’s solution with lower K+ concentration (2 mM) and 300 µM Diazoxide (red). (B, C) Statistical analysis of the influence of ChR2 expression and modified Tyrode’s solution (normal solution in black, modified Tyrode’s solution in red) on P-wave duration (B; n = 6–7; P = 0.6507 for ChR2 genotype, P = 0.0014 for solution) and ARP (C; n = 6; P = 0.1468 for ChR2 genotype, P < 0.0001 for solution). Statistical analysis by two-way repeated measures ANOVA, asterisks indicate Bonferroni post-test significances. Note that also unpaired, two tailed student’s t-tests did not reveal any significant differences for ChR2 genotype (normal Tyrode’s solution: P = 0.7129 for P-wave duration and P = 0.0954 for ARP; modified Tyrode’s solution: P = 0.6118 for P-wave duration and P = 0.7470 for ARP). (D) Whole heart electrograms of AF episodes with atrial flutter (top) and atrial fibrillation (bottom) induced by atrial electrical burst stimulation (5 s, 50 Hz; red boxes). Note the regular or the irregular ventricular activation during atrial flutter or atrial fibrillation, respectively. (E) Incidence of AF episodes with the indicated cycle lengths in ChR2 negative (grey, n = 223) and positive (blue, n = 236) hearts (P = 0.8902, Chi-square test for trend). 3. Results 3.1 Establishing AF induction and optogenetic cardioversion in the isolated perfused mouse heart Because analysis of optogenetic cardioversion requires sustained episodes of AF, we took advantage of a mouse model carrying the loss-of-function Cx40 Ala96Ser mutation. These mice were crossed with transgenic mice expressing the ChR2 H134R variant9 under control of the ubiquitous chicken β-actin promoter with a CMV enhancer element resulting in particularly strong transgene expression in all cardiomyocytes.6,12 Hearts from ChR2 positive/Cx40 Ala96Ser (+/−) or ChR2 negative/Cx40 Ala96Ser (+/−) offspring were explanted and retrogradely perfused in Langendorff configuration. After regular sinus rhythm was observed the ARP and P-wave duration was determined. Subsequently the external solution was changed to a modified Tyrode’s solution with low K+ (2 mM) concentration supplemented with the KATP-channel activator Diazoxide (300 µM). While basic electrophysiological properties of the atria were not different between ChR2 negative or ChR2 positive hearts, the modified Tyrode’s solution caused a prolongation of the P-wave duration (Figure 1A and 1B) indicating reduction of conduction velocity in the atria, and a shortening of the ARP (Figure 1C) indicating reduction of action potential duration. Both effects would lead to a decreased cardiac wavelength (conduction velocity * refractoriness), which is the most important factor for micro-re-entrant waves in cardiac tissue.13 In consequence, bipolar epicardial electrical burst stimulation (5 s, cycle length 10–50 ms, 1–10 mA) of the right atrium was able to reliably induce sustained episodes (> 15 s) of atrial flutter (Figure 1D top) or atrial fibrillation (Figure 1D bottom) characterized in accordance to clinical decision criteria by high-frequency atrial electrical activity and regular or irregular ventricular activation, respectively. Since atrial flutter was only observed in 17.2% of ChR2 positive (n = 238 episodes) and in 17.9% of ChR2 negative hearts (n = 223 episodes), we merged atrial flutter and fibrillation in one AF group for statistical comparisons. The majority of AF episodes had a predominant cycle length between 30 and 70 ms which were equally distributed and not different between ChR2 positive and negative hearts (Figure 1E). 3.2 Efficacy of optogenetic cardioversion For optogenetic cardioversion, blue light (470 nm, 1 s, 0.4 mW/mm2, 100 mm2) was focused onto the epicardial surface of both atria (Figure 2A), which terminated AF in all ChR2 expressing hearts (n = 17; Figure 2B). To determine cardioversion efficacy, we developed an illumination protocol using four consecutive light pulses (1 s, 0.4 mW/mm2, 100 mm2) and 1 s delay in-between (Figure 2C) with the rationale that, in contrast to electrical shocks, multiple light stimulations are technically feasible. Analysing all AF episodes (n = 101) from ten ChR2 positive hearts, the AF termination efficacy after the first and second light pulse was already 91 and 98%, respectively whereas these values were only 13 and 25% in all AF episodes (n = 114) of six ChR2 negative hearts (Figure 2D). Importantly the overall average success rate per heart after the four light pulse protocol was 96.5 ± 3.5% for optogenetic cardioversion in ChR2 positive hearts (n = 17), whereas control experiments revealed significantly lower spontaneous conversion rates with an average of 43.1 ± 4.8% in ChR2 positive hearts within the identical timeframe but without illumination (n = 10) and of 54.9 ± 9.2% in ChR2 negative hearts with the same illumination protocol (n = 6) (Figure 2E and Supplementary material online, Figure S1A–C). This indicates that ChR2 expression and illumination are required for optogenetic cardioversion. In addition, the identical spontaneous conversion rates in both control groups exclude altered electrophysiological behaviour and influence on spontaneous AF termination by ChR2 expression alone. Figure 2 Open in new tabDownload slide Optogenetic cardioversion in Cx40 Ala96Ser (+/−) hearts ex vivo. (A) Hearts were explanted and retrogradely perfused in Langendorff configuration. An electrogram was recorded with electrodes (grey) placed at the right atrium and at the apex of the heart. In some experiments an octapolar electrophysiology catheter was additionally introduced via the superior vena cava into the right heart chamber (dark grey). AF was induced by electrical burst stimulation with an atrial electrode pair (red). Circular illumination was performed with a blue LED (470 nm) focused on the epicardial atrial surface at three different sizes covering both atria (100 mm2, light blue), the whole right atrium (26 mm2, blue) or parts of the right atrium (10 mm2, dark blue). (B) Example of optogenetic cardioversion with whole heart electrogram (surface) and local recordings from the right atrium (atrial) and the right ventricle (ventricular). AF was terminated by 1 s long illumination (470 nm, 0.4 mW/mm2, 100 mm2; blue box). Insert (black box) highlights the high-frequency electrical activity (cycle length ∼55 ms) in the atria during AF and the irregular conduction to the ventricles. (C) Schematic of the protocol used for efficacy testing of optogenetic cardioversion in explanted hearts (for details see Section 2). Optogenetic termination was classified successful when AF terminated between onset of illumination and 3 s after the last illumination (time period indicated by green box). (D) Cumulative AF termination rate by each of the four light pulses applied (1 s, 0.4 mW/mm2, 100 mm2) in all episodes (n = 101) of ten ChR2 positive (blue circles) and six ChR2 negative (grey triangles, n = 114 episodes) Cx40 Ala96Ser (+/−) hearts. (E) Average AF termination rates per heart by four consecutive light pulses (1 s, 0.4 mW/mm2, 100 mm2) in ChR2 positive hearts with illumination (blue circles; n = 17) or during the same time period without illumination (grey squares, n = 10) and in ChR2 negative hearts with the same illumination protocol (grey triangles, n = 6). Kruskal-Wallis test: P < 0.0001; asterisks indicate the significances of Dunn’s multiple comparison post-test. Figure 2 Open in new tabDownload slide Optogenetic cardioversion in Cx40 Ala96Ser (+/−) hearts ex vivo. (A) Hearts were explanted and retrogradely perfused in Langendorff configuration. An electrogram was recorded with electrodes (grey) placed at the right atrium and at the apex of the heart. In some experiments an octapolar electrophysiology catheter was additionally introduced via the superior vena cava into the right heart chamber (dark grey). AF was induced by electrical burst stimulation with an atrial electrode pair (red). Circular illumination was performed with a blue LED (470 nm) focused on the epicardial atrial surface at three different sizes covering both atria (100 mm2, light blue), the whole right atrium (26 mm2, blue) or parts of the right atrium (10 mm2, dark blue). (B) Example of optogenetic cardioversion with whole heart electrogram (surface) and local recordings from the right atrium (atrial) and the right ventricle (ventricular). AF was terminated by 1 s long illumination (470 nm, 0.4 mW/mm2, 100 mm2; blue box). Insert (black box) highlights the high-frequency electrical activity (cycle length ∼55 ms) in the atria during AF and the irregular conduction to the ventricles. (C) Schematic of the protocol used for efficacy testing of optogenetic cardioversion in explanted hearts (for details see Section 2). Optogenetic termination was classified successful when AF terminated between onset of illumination and 3 s after the last illumination (time period indicated by green box). (D) Cumulative AF termination rate by each of the four light pulses applied (1 s, 0.4 mW/mm2, 100 mm2) in all episodes (n = 101) of ten ChR2 positive (blue circles) and six ChR2 negative (grey triangles, n = 114 episodes) Cx40 Ala96Ser (+/−) hearts. (E) Average AF termination rates per heart by four consecutive light pulses (1 s, 0.4 mW/mm2, 100 mm2) in ChR2 positive hearts with illumination (blue circles; n = 17) or during the same time period without illumination (grey squares, n = 10) and in ChR2 negative hearts with the same illumination protocol (grey triangles, n = 6). Kruskal-Wallis test: P < 0.0001; asterisks indicate the significances of Dunn’s multiple comparison post-test. 3.3 Mechanism of optogenetic cardioversion To investigate which illumination parameters affect optogenetic cardioversion, we performed the same protocol but altered the size of illumination, the light intensities and the pulse durations. We found a significant influence of the size of the illuminated area (Figure 3A) and the applied light intensity (Figure 3B) on efficacy, indicating that affecting a critical area of the atria and also deeper myocardial cell layers is important for successful optogenetic cardioversion. Furthermore, efficacy was lower when reducing the pulse duration to 10 ms and close to spontaneous termination rate when using 3 or 1 ms pulses (Figure 3C) highlighting that illuminations longer than the AF cycle length (30–70 ms, Figure 1E) are important. To further delineate the differences between spontaneous conversion and optogenetic cardioversion, we classified AF termination in ChR2 positive (Figure 3D) and ChR2 negative mice (Supplementary material online, Figure S1B and C) into AF episodes (i) blocked directly with onset of illumination (Figure 3D top), (ii) terminating during the light pulse (Figure 3D middle andSupplementary material online, Figure S1B), or (iii) terminating after illumination (Figure 3D bottom andSupplementary material online, Figure S1C). In ChR2 expressing hearts over 80% of AF episodes were terminated directly with onset of illumination (direct block) whereas in ChR2 non-expressing hearts, AF episodes terminated predominantly (>90%) during or after the illumination (Figure 3E). Figure 3 Open in new tabDownload slide Mechanistic insights into optogenetic cardioversion. AF termination rates using different illumination areas (A; 1 s, 0.1 mW/mm2; n = 5, P = 0.0394, Friedman test), light intensities (B; 1 s, 10 mm2; n = 7, P = 0.0021, Friedman test) or durations of light pulses (C; 0.2 mW/mm2, 26 mm2; n = 7, P < 0.0001, Friedman test). (D) Representative ECG traces for light (470 nm, 1 s, 0.4 mW/mm2, 100 mm2; blue bar) terminating AF with onset of illumination (direct block; top, example from Figure 2B), during the light pulse (middle) or after the light pulse (bottom). (E) Comparison of the relative occurrence of AF termination timings in relation to the light pulse (in ChR2 positive (examples in D) and ChR2 negative hearts (examples in Supplementary material online, Figure S1B and C) (P < 0.0001, Chi-square test). Figure 3 Open in new tabDownload slide Mechanistic insights into optogenetic cardioversion. AF termination rates using different illumination areas (A; 1 s, 0.1 mW/mm2; n = 5, P = 0.0394, Friedman test), light intensities (B; 1 s, 10 mm2; n = 7, P = 0.0021, Friedman test) or durations of light pulses (C; 0.2 mW/mm2, 26 mm2; n = 7, P < 0.0001, Friedman test). (D) Representative ECG traces for light (470 nm, 1 s, 0.4 mW/mm2, 100 mm2; blue bar) terminating AF with onset of illumination (direct block; top, example from Figure 2B), during the light pulse (middle) or after the light pulse (bottom). (E) Comparison of the relative occurrence of AF termination timings in relation to the light pulse (in ChR2 positive (examples in D) and ChR2 negative hearts (examples in Supplementary material online, Figure S1B and C) (P < 0.0001, Chi-square test). To determine possible cardioversion mechanisms, we applied light pulses of increasing intensities to both atria during sinus rhythm and analysed local atrial electrograms. In all hearts tested, light intensities as low as 0.05 mW/mm2 were sufficient to evoke atrial excitation indicated by light-induced PSVC (Figure 4A and C). In principle, such local excitation of the atria could terminate AF by a mechanism termed ‘filling of the excitable gap’ (see discussion). In contrast, higher light intensities were able to induce a total block of atrial electrical activity indicated by lack of electrical signals within atrial recordings (Figure 4B and C). During AF, this would result in a block of electrical activity within re-entrant waves. Figure 4 Open in new tabDownload slide Influence of light on atrial electrical activity. Representative surface (top) and local atrial (lower) electrograms during epicardial illumination. (A) Light-induced PSVC during low light intensity illumination (3 s, 0.05 mW/mm2, 100 mm2) with changes in P-wave and atrial signal morphology (red arrows in the boxed insert). (B) Example of complete block of atrial electrical activity during higher light intensity illumination (3 s, 0.13 mW/mm2, 100 mm2); PSVC indicated by red arrow, remaining signals in the atrial recording are indicating ventricular activity. (C) Cumulative percentage of hearts with light-induced PSVC or complete block of atrial activity in dependence of the applied light intensity (n = 7). Figure 4 Open in new tabDownload slide Influence of light on atrial electrical activity. Representative surface (top) and local atrial (lower) electrograms during epicardial illumination. (A) Light-induced PSVC during low light intensity illumination (3 s, 0.05 mW/mm2, 100 mm2) with changes in P-wave and atrial signal morphology (red arrows in the boxed insert). (B) Example of complete block of atrial electrical activity during higher light intensity illumination (3 s, 0.13 mW/mm2, 100 mm2); PSVC indicated by red arrow, remaining signals in the atrial recording are indicating ventricular activity. (C) Cumulative percentage of hearts with light-induced PSVC or complete block of atrial activity in dependence of the applied light intensity (n = 7). 3.4 Optogenetic cardioversion in vivo To demonstrate the feasibility of optogenetic AF termination in vivo, we intubated and ventilated mice and performed an ‘open chest’ preparation6,7 for illumination of the atria. AF was induced by endocardial electrical burst stimulation with a transvenous stimulation catheter placed in the right atrium. Illumination of the atrial region (1 s, 0.4 mW/mm2, 100 mm2) terminated AF in all mice tested (n = 3 mice; Figure 5). These results prove the feasibility of optogenetic cardioversion by epicardial illumination in vivo, despite the fact that hemoglobin in the atrial wall absorbs light and impairs light penetration.14 In addition, hearts were illuminated through the pericardial sack which adds another tissue layer the light has to penetrate.11 Figure 5 Open in new tabDownload slide Optogenetic cardioversion in vivo. Representative (n = 3 hearts) ECG (top) and endocardial atrial electrogram (lower). AF was initiated in vivo by endocardial electrical burst stimulation (5 s, 100 Hz; red) with a transvenous catheter and terminated by 1 s long epicardial illumination (0.4 mW/mm2, 100 mm2; blue) of the atria through the open chest. Figure 5 Open in new tabDownload slide Optogenetic cardioversion in vivo. Representative (n = 3 hearts) ECG (top) and endocardial atrial electrogram (lower). AF was initiated in vivo by endocardial electrical burst stimulation (5 s, 100 Hz; red) with a transvenous catheter and terminated by 1 s long epicardial illumination (0.4 mW/mm2, 100 mm2; blue) of the atria through the open chest. 3.5 Optogenetic cardioversion in wild type hearts Because of the tantalizing possibility of optogenetic cardioversion in patients, we assessed the feasibility in non-transgenic hearts after viral gene transfer for ChR2 expression, which is a prerequisite for clinical applicability. We previously reported long-lasting ventricular ChR2 expression in mouse hearts after one systemic injection of AAV, which was sufficient for optogenetic pacing7 and defibrillation of ventricular tachycardia and fibrillation.11 To show long-lasting expression of ChR2 also in the atria, we analyzed hearts 6–8 months after injecting AAV expressing ChR2 in fusion to mCherry. We found bright mCherry signals in epicardial images of the atria (Figure 6A) and no obvious adverse side effects, such as wall thinning (Figure 6B). Detailed histological analysis revealed the presence of CD45-positive immune cells within the atrial myocardium of AAV transduced mice compared with only sparsely distributed immune cells in uninfected control mice (Supplementary material online, Figure S2A and B). However this had no influence on cardiomyocyte survival because we could not detect any TUNEL-positive apoptotic cardiomyocytes and gap junctions were similar distributed in the atria of AAV-transfected and control hearts (Supplementary material online, Figure S2C–F). Expression of mCherry was equally distributed throughout the whole atrial wall (Figure 6C) and localized within the membrane of the expressing cardiomyocytes (Figure 6D). AF episodes were induced in explanted hearts as reported above and were shorter than in Cx40 Ala96Ser (+/−) mice but lasted for at least 5 s. Thus we compared the efficacy of a single light pulse to the spontaneous conversion rate in the same timeframe in AAV transduced hearts without illumination and in CD1 wild type hearts with the same illumination protocol. We found that illuminating (1 s, 5 mW/mm2, 100 mm2) the epicardium of both atria (Figure 7A) terminated AF with an average efficacy of 74.7 ± 9.1% (n = 7) in AAV transduced hearts. This was significantly higher than the spontaneous conversion rate in these hearts (9.1 ± 3.8%; n = 7) and the termination rate in hearts from non-transduced wild type mice using the same illumination protocol (24.7 ± 5.9%; n = 9) (Figure 7B). After the experiments, we dissociated the right atrium and determined the percentage of ChR2 expressing atrial myocytes (Figure 7B). This value correlated well with the success rates of cardioversion and importantly suggested that one heart could not be cardioverted because of the low expression rate (8%). Figure 6 Open in new tabDownload slide ChR2 expression in wild type hearts after systemic AAV injection. (A) Epicardial mCherry fluorescence from the right atrium 8 months after systemic AAV injection. (B, C) Sections through the left atrium showing normal atrial morphology in haematoxylin eosin staining (B) and transmural mCherry fluorescence (C). (D) Membrane-bound ChR2-mCherry signals (red) in α-actinin positive atrial cardiomyocytes (white). All pictures are taken from the heart with 38% mCherry positive cardiomyocytes (see Figure 7B). Scale bars: 1 mm (A), 500 µm (B, C), 25 μm (D); nuclear staining in blue. Figure 6 Open in new tabDownload slide ChR2 expression in wild type hearts after systemic AAV injection. (A) Epicardial mCherry fluorescence from the right atrium 8 months after systemic AAV injection. (B, C) Sections through the left atrium showing normal atrial morphology in haematoxylin eosin staining (B) and transmural mCherry fluorescence (C). (D) Membrane-bound ChR2-mCherry signals (red) in α-actinin positive atrial cardiomyocytes (white). All pictures are taken from the heart with 38% mCherry positive cardiomyocytes (see Figure 7B). Scale bars: 1 mm (A), 500 µm (B, C), 25 μm (D); nuclear staining in blue. Figure 7 Open in new tabDownload slide Optogenetic cardioversion ex vivo in wild type hearts after AAV-mediated ChR2 gene transfer. (A) Representative example of an AF episode induced by electrical burst stimulation (5 s, 50 Hz; red) and terminated by epicardial illumination (470 nm, 1 s, 5 mW/mm2, 100 mm2; blue). Inserts highlight the high-frequency electrical activity in the atrium during AF, (red box; cycle length 35–40 ms) and the regular P-waves during sinus rhythm before (black box) and after (blue box) the AF episode. (B) AF termination rate 6–8 months after AAV injection by one light pulse (1 s, 5 mW/mm2, 100 mm2; blue circle; n = 7) compared with spontaneous termination during the same time window without illumination (grey squares; n = 7) and to hearts from untreated wild type mice with the same illumination protocol (grey triangles; n = 9). Kruskal-Wallis test: P = 0.0010; asterisks indicate the significances of Dunn’s multiple comparison post-test. The percentages of atrial cardiomyocytes expressing ChR2 are indicated next to the AF termination rate of the corresponding heart. Due to a technical problem during the dissociation of one heart, percentage values could only be obtained for six hearts. Figure 7 Open in new tabDownload slide Optogenetic cardioversion ex vivo in wild type hearts after AAV-mediated ChR2 gene transfer. (A) Representative example of an AF episode induced by electrical burst stimulation (5 s, 50 Hz; red) and terminated by epicardial illumination (470 nm, 1 s, 5 mW/mm2, 100 mm2; blue). Inserts highlight the high-frequency electrical activity in the atrium during AF, (red box; cycle length 35–40 ms) and the regular P-waves during sinus rhythm before (black box) and after (blue box) the AF episode. (B) AF termination rate 6–8 months after AAV injection by one light pulse (1 s, 5 mW/mm2, 100 mm2; blue circle; n = 7) compared with spontaneous termination during the same time window without illumination (grey squares; n = 7) and to hearts from untreated wild type mice with the same illumination protocol (grey triangles; n = 9). Kruskal-Wallis test: P = 0.0010; asterisks indicate the significances of Dunn’s multiple comparison post-test. The percentages of atrial cardiomyocytes expressing ChR2 are indicated next to the AF termination rate of the corresponding heart. Due to a technical problem during the dissociation of one heart, percentage values could only be obtained for six hearts. 4. Discussion We herein present a novel protocol to induce sustained episodes of AF in mouse hearts using three perturbations of atrial electrophysiology: (i) Cx40 is the predominant atrial connexin isoform and the human Ala96Ser mutation was demonstrated to increase susceptibility for AF in patients.15 In the mouse model this mutation led to a decreased P-wave amplitude, prolonged P-wave duration and decreased atrial conduction velocities compared with wild type mice.10 (ii) Opening of KATP-channels with Diazoxide shortens action potential duration and refractoriness,16,17 which is one key component of electrical remodelling during AF.18,19 (iii) Low K+ is known to increase the susceptibility for AF in patients.20 Interestingly, we were able to detect that this combined approach led to prolonged P-waves and thus further slowed atrial conduction velocity. Altogether, these measures will reduce the cardiac wavelength enabling the accommodation of re-entrant waves in the small mouse atrium. In accordance, this allowed not only to provide the first evidence for optogenetic cardioversion of AF but also to determine the AF termination efficacy with various illumination parameters. Importantly, ChR2 overexpression per se did not affect atrial electrophysiology or AF stability, which is suggested by unaltered P-wave duration, ARP and similar spontaneous AF termination rate in ChR2 expressing and non-expressing hearts. Three possible mechanisms could underlie the observed optogenetic AF termination: (i) Similar to pharmacological approaches, constant optogenetic depolarization could modulate atrial conduction, excitability or refractoriness and thereby modify AF activity within the atrium, eventually leading to colliding and self-extinction of re-entrant waves. Such a ‘modulatory mechanism’ was proposed in one of the recent reports on optogenetic defibrillation, which suggested light-induced prolongation of action potentials as the underlying mechanism.21 However, because AF terminated in most (>80%) of cases directly at onset of illumination (Figure 3E), we consider this mechanism of minor importance. (ii) Light pulses could induce action potentials in the excitable parts of the atrium on the trailing edge of the re-entrant wave and thereby create new excitation waves that collide with the re-entrant AF waves. Such a mechanism is termed ‘filling of the excitable gap’ and is considered to be the underlying mechanism of anti-tachycardia pacing by electrical stimulation, which has a low efficacy in patients.4 This is most likely due to the fact that the excitable gaps are small in AF and thus difficult to be filled all at once with non-triggered random pacing. In line with this, we observed low AF termination efficacy using brief light pulses (1–10 ms, Figure 3C) applied to one atrium although the intensity used is above the pacing threshold (Figure 4A and C). Thus reliable filling of the excitable gaps by optogenetic stimulation would require local illumination triggered after the trailing edge of re-entrant waves or global illumination affecting all fibrillating regions,22 which is both technically challenging. (iii) Sustained and higher intensity illumination can constantly depolarize cardiomyocytes,6,11 which would not only fill the excitable gap but also keep the illuminated tissue in refractory state and thus could block electrical conduction.6,8,11 Such a ‘block of electrical activity’ mechanism is suggested by our data showing the ability to block atrial electrical activity by epicardial illumination (Figure 4B and C), which indicates sufficient light penetration throughout the atrial wall. Although this mechanism requires higher light intensity compared with pacing and filling of excitable gaps (Figure 4C), it does not require to analyse the underlying re-entry wavefront which would simplify devices and makes AF termination more robust. In line with these considerations, we found robust optogenetic AF termination with success rates of up to 97% when using illumination durations longer than one AF cycle length (Figure 3C) and when using the high light intensities required to block atrial activity (Figure 4B and C). This ‘block of electrical activity’ mechanism is also well in line with our recent findings in simulations of optogenetic defibrillation in a human heart by sustained depolarization of all myocardial layers.11 Although we cannot discriminate between the ‘filling of excitable gap’ and the ‘block of electrical activity’ mechanism in each AF termination event, our data strongly suggest that the latter mechanism will add to the efficacy of AF termination. Importantly, this effect can only be achieved by an optogenetic method allowing constant and homogenous depolarization and tissue refractoriness by illumination. In the future, local brief illumination triggered by local electrograms will be useful to identify novel illumination patterns to fill the excitable gaps for efficient cardioversion and thereby might provide also insights to optimize low energy electrical cardioversion strategies.23–25 Furthermore, the ability of optogenetic methods to induce uniform de- and hyperpolarization5 as well as the use of spatial and temporal defined illumination patterns opens up new avenues to understand the mechanisms underlying AF induction, maintenance and cardioversion. Importantly light stimulation can be feed-back controlled by electrical or contractile activity and this would allow an in-depth interrogation with the biological system, a technology which was shown before by spatially dynamic precise optogenetic modulation of re-entrant wavefronts and rotors in a two dimensional cardiac tissue.26 Excitingly, we were also able to prove optogenetic termination of AF in wild type hearts up to 8 months after one single AAV injection. In light of recent successful optogenetic defibrillation with blue light in mouse11,27 and rat hearts21 whose ventricles have comparable myocardial wall thicknesses to human atria,28 especially in patients with AF,29 our results raise hope for a possible clinical alternative to electrical shocks. Optogenetic cardioversion could offer pain-free AF termination, since illumination does not activate sensory or pain neurons when ChR2 is selectively expressed in cardiomyocytes. Importantly light itself does not induce considerable heat production during continuous illumination even when using light intensities much higher than the ones used in our study.30,31 However, cardiac specific expression of ChR2 is pivotal, which can be achieved by selective tropism of the AAV towards cardiomyocytes and by expression driven by myocardial specific promotors. Such cardiomyocyte specific overexpression has been recently optimized using the cardiac Troponin T promoter and AAV with the serotype 9 capsid.32,33 For optogenetic termination of AF only, without requirement for ventricular defibrillation or pacing, atrial-specific ChR2 expression could be sufficient using the sarcolipin promoter.34 Alternatively, also the promotors of atrial natriuretic peptide35 or Cx4036 could be used, with the draw back that the former is upregulated in failing ventricular myocardium37 and the latter is also expressed in the cardiac conduction system and endothelial cells. However, despite the safety of AAV cardiac gene delivery,38 long-term expression and potential activation of the immune system by the non-mammalian ChR2 have to be carefully evaluated and strategies must be optimized to circumvent immune reactions towards ChR2 or AAV. This is of special concern as we found a low level of immune cell infiltration in the AAV transduced hearts. However it is known that the immune response can vary dramatically between species.39 Furthermore, mouse models are suited for prove-of-concept studies but provide limited translational evidence considering the complex pathophysiology of AF and atrial anatomy in humans.40 These include differences in atrial ionic currents and Ca2+ handling, ectopic electrical activity from the pulmonary veins as well as electrical and structural remodeling in AF patients.41,42 Importantly, limiting ChR2 expression to cardiomyocytes could likely impede the efficacy of optogenetic cardioversion in patients with a high level of atrial fibrosis. This could be addressed by overexpressing ChR2 in fibroblasts and cardiomyocytes given an electrical coupling between both cell types.43 Thus, the promising results of our proof-of-concept study have to be verified in pre-clinical large animal models with human-like anatomy and comparable atrial arrhythmia. Such animal models will not only allow predicting the therapeutic value of optogenetic cardioversion but will also be of great value to solve open questions on progressive AF disease mechanisms such as AF begetting AF18 by performing repetitive cardioversions on demand with implanted light sources. Cardiac illumination could be achieved by the recent developments in implantable optoelectronics, such as cell-scale injectable LEDs,44–46 LEDs integrated in stretchable integumentary membranes47 and µLED arrays.48 Taken together, the herein presented first prove of shock-less cardioversion by light in explanted hearts and mice in vivo could lay the foundation for future developments towards implantable optical cardioverters as a novel therapeutic option for patients with AF. Supplementary material Supplementary material is available at Cardiovascular Research online. Acknowledgements We thank F. Holst and H. Panatzek for technical assistance and Penn Vector Core, Gene Therapy Programme (University of Pennsylvania), for providing the AAV9 CAG-hChR2(H134R)-mCherry.WPRE.SV40 virus vector. Conflict of interest: none declared. Funding This work was supported by the German Research Foundation (SA-1785/7 1, SA-1785/9 1 and Research Training Group 1873 to P.S.); the BONFOR Programme, Medical Faculty, University of Bonn (O-162.0011 to P.S., O-109.0052 to T.Be.); and by the German Federal Ministry of Education and Research, funding programme Photonics Research Germany, project BioPACE (13N14087 to P.S.). References 1 Lip GY , Tse HF , Lane DA. Atrial fibrillation . Lancet 2012 ; 379 : 648 – 661 . Google Scholar Crossref Search ADS PubMed WorldCat 2 Geller JC , Reek S , Timmermans C , Kayser T , Tse HF , Wolpert C , Jung W , Camm AJ , Lau CP , Wellens HJ , Klein HU. Treatment of atrial fibrillation with an implantable atrial defibrillator–long term results . Eur Heart J 2003 ; 24 : 2083 – 2089 . 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WorldCat 48 Gossler C , Bierbrauer C , Moser R , Kunzer M , Holc K , Pletschen W , Kohler K , Wagner J , Schwaerzle M , Ruther P , Paul O , Neef J , Keppeler D , Hoch G , Moser T , Schwarz UT. GaN-based micro-LED arrays on flexible substrates for optical cochlear implants . J Phys D Appl Phys 2014 ; 47 :205401. WorldCat Author notes Tobias Bruegmann and Thomas Beiert authors contributed equally to the study. Time for primary review: 44 days Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2017. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)
TI - Optogenetic termination of atrial fibrillation in mice
JO - Cardiovascular Research
DO - 10.1093/cvr/cvx250
DA - 2018-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/optogenetic-termination-of-atrial-fibrillation-in-mice-ft4jJUfqC8
SP - 713
VL - 114
IS - 5
DP - DeepDyve
ER -