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Pharmacologic Therapy for Engraftment Arrhythmia Induced by Transplantation of Human Cardiomyocytes Brief title: Drug therapy for engraftment arrhythmia following human cardiomyocyte transplantation

1. Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle 2. Center for Cardiovascular Biology, University of Washington, Seattle 3. Division of Cardiology, Department of Medicine, University of Washington, Seattle 4. Department of Comparative Medicine, University of Washington, Seattle 5. Department of Laboratory Medicine & Pathology, University of Washington, Seattle 6. Sana Biotechnology, Seattle, Washington 7. Department of Biostatics, University of Washington, Seattle 8. Department of Medicinal Chemistry, University of Washington, Seattle 9. Division of Clinical Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 10. Department of Bioengineering, University of Washington, Seattle


Introduction
Ischemic heart disease including myocardial infarction (MI) and heart failure remains the leading cause of death in the United States and around the world (1). Approximately one billion cardiomyocytes are permanently lost during MI (2) and an increasing proportion of MI survivors-an estimated 20 to 40% (3,4)-later develop heart failure. Current treatments can slow the initiation and progression of heart failure, but none replaces lost myocardium, short of orthotopic heart transplantation, which remains restricted in availability and indication (5). Human pluripotent stem cells (hPSCs, comprising embryonic stem cells [ESCs] and their reprogrammed cousins, induced pluripotent stem cells [iPSCs]) are a renewable source of cardiomyocytes (CMs). Transplantation of hPSC-derived cardiomyocytes (hPSC-CMs) into infarcted myocardium of small animals-mice, rats, and guinea pigs-has shown stable engraftment (6)(7)(8)(9)(10). More recently, our group and others have shown remuscularization and functional benefit in infarcted non-human primates (NHP) following transplantation of pluripotent stem cellderived cardiomyocytes (11)(12)(13). In addition to functional remuscularization, the human graft vascularizes and electromechanically couples with the host myocardium within one-month posttransplant and remains durable up to three months, the longest time tested.
Although no arrhythmias were observed in smaller animals, we and others consistently observe ventricular arrhythmias following hPSC-CM transplantation in NHPs (11)(12)(13) and pigs (14) which we have called "engraftment arrhythmias" (EAs). EAs are generally transient, occurring within a week of transplantation and typically resolve spontaneously after approximately one-month. Based on electrical mapping, overdrive pacing, and cardioversion studies, EAs appear to originate focally in the graft or peri-graft myocardium and function as automatous foci rather than reentrant pathways (12,14). Although EA is reasonably well-tolerated in NHPs, the Laflamme group (14) reported that EA can be lethal in (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
We hypothesized that the risk of EA may be mitigated by treatment with clinically available antiarrhythmic drugs. Because the pig shows heightened sensitivity to EAs, is a well-established model in cardiovascular research (16) and cell therapy (17), whose larger heart permits the use of percutaneous delivery catheters, we chose to test the hypothesis in this large animal model. In the first phase of our study, we screened a panel of seven anti-arrhythmic agents. The broad-acting (class III) antiarrhythmic amiodarone and the pacemaker inhibitor (class 0) ivabradine emerged independently as the most promising agents for control of rhythm and rate, respectively. We therefore performed a second phase to test the effect of combined amiodarone and ivabradine treatment. We found that this regimen reduced sudden cardiac death, as well as suppressed tachycardia and arrhythmia.

hESC-CM production
These studies were approved by the University of Washington Stem Cell Research Oversight Committee. Two lines of hESCs were used in this study. Initial subjects received H7 (WiCell)-derived cardiomyocytes that were cultured, expanded, and differentiated in suspension-culture format by collaborators at the Center for Applied Technology Development at the City of Hope in California, all as previously described (11,12,18). Most subjects received RUES2 (Rockefeller University)-derived cardiomyocytes produced in our laboratory in stirred suspension culture format. Briefly, RUES2 hESC were cultured to form aggregates and were expanded in commercially available media (Essential 8, Gibco). For cardiac differentiation, suspension adapted pluripotent aggregates were induced to differentiate in RPMI-1640, MCDB-131, or M199 supplemented with B-27 (all from Gibco) or serum (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Subsequent wash steps were done using RPMI-1640 basal media in progressively smaller volumes to concentrate the cell suspension. For the last centrifugation step, the cell pellet was resuspended in a sufficient volume of RPMI-1640 to achieve a target cell density for injection of ~3 x10 9 cells/mL in 1.6 mL. The final volume of the cell suspension was determined by the results of a count sampled before the final centrifugation step. Cell counts were performed as described previously to achieve a final total dose of 500x10 6 live cells per transplant (12).

Study design
The objective of this study was to identify a pharmacological regimen to attenuate arrhythmias following cardiac remuscularization therapy. This study was designed in two phases: the first to observe the natural history of EA in the minipig model and screen various antiarrhythmic agents for possible efficacy, and the second to rigorously test for efficacy of selected candidates (Fig. 1). All subjects were 30-40 kg castrated male Yucatan minipigs between 6-13 months of age (Premier BioSource). In Phase 1, nine subjects underwent cardiac remuscularization therapy with 500 x 10 6 hESC-CMs delivered by direct surgical trans-epicardial injections or, later by percutaneous trans-endocardial injections (Table 1).
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The first four subjects (one non-infarcted and three infarcted) were followed to learn the natural history of EA and establish clinical endpoints and parameters for the Phase 2 drug trial. The subsequent five subjects underwent systematic dosing with antiarrhythmic agents with continuous electrocardiography (ECG) monitoring to determine effect on rhythm and rate (Online Table 1, Online Methods). Among the nine subjects in Phase 1, we observed high mortality, with six out of nine experiencing ventricular fibrillation (VF) or tachycardia-induced heart failure requiring euthanasia. VF typically followed frequent episodes of unstable EA >350 beats per minute (bpm), and tachycardia-induced heart failure requiring euthanasia was characterized by chronically elevated heart rates >150 bpm.
In Phase 2, we conducted a two-drug antiarrhythmic study with amiodarone and ivabradine, enrolling an additional 17 subjects (9 treated, 8 untreated) that underwent MI and percutaneous transplantation with hESC-CMs at two weeks post-MI. Two additional subjects underwent MI with sham vehicle injection to serve as sham transplant controls (Fig. 1, Online Fig. 1). The primary endpoint was prespecified as combined cardiac death (either spontaneous death from arrhythmia or heart failure, or clinically directed euthanasia necessitated by sustained tachycardia >350 bpm or signs of heart failure). Prespecified secondary endpoints were suppression of tachycardia, percent time in arrhythmia (arrhythmia burden) and resolution of arrhythmia, termed "electrical maturation" and defined as arrhythmia burden <25% for 48 consecutive hours. Antiarrhythmic therapy was discontinued after electrical maturation or posttransplantation day 30, whichever was earlier. To prevent tachycardia-induced cardiomyopathy, we titrated ivabradine treatment to maintain target heart rate <150 bpm. Based on early experience that tachycardia >350 bpm often degenerated to VF, subjects were euthanized humanely if heart rates >350 bpm were reached. Continuous telemetric ECG was monitored for eight weeks total (two weeks post-MI and six weeks post-transplantation). Of note, subjects 1 and 2 (untreated) and 3 and 4 (treated) received H7 hESC-CM and subjects 1, 2 and 3 were transplanted surgically prior to adopting percutaneous (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted February 16, 2021. ; https://doi.org/10.1101/2021.02.15.431108 doi: bioRxiv preprint delivery. Subject 5 was euthanized on day 37 as a prespecified endpoint following electrical maturation, prior to extending the study duration to 6 weeks post-transplantation for extended treatment washout and monitoring.

Cardiac remuscularization therapy
Cell transplantation for our three initial subjects (1-3) was performed by direct transepicardial injection into the peri-infarction region as previously described for NHP with minor modification (12). Briefly, a partial median sternotomy was performed to expose the infarcted anterior left ventricle. Purse-string sutures were preplaced at five discrete locations subtended by the LAD, targeting the central ischemic region and lateral border zones. After cinching the purse-string tightly around the needle, three injections of 100 µL each were performed by partial withdrawal and lateral repositioning, for a total of

Antiarrhythmic treatment
The treated cohort was loaded with oral amiodarone 1000-1200 mg orally twice daily starting seven days prior to cell transplantation followed by maintenance dose of 400-1000 mg orally twice daily to maintain a steady-state plasma level of 1.5-4.0 µg/mL (Online Fig. 2). Ivabradine was started at 2.5 mg orally twice daily when sustained tachycardia reached ≥ 150 bpm and titrated every 3 days up to 15 mg twice daily for goal heart rate < 125 bpm. All but one subject (1) required adjunctive ivabradine for additional heart rate control. Antiarrhythmics were discontinued after electrical maturation was achieved or post-transplantation day 30, whichever was earlier, to allow for treatment washout and assess for recrudescence of arrhythmia. All subjects tolerated the antiarrhythmic regimen without complication.
Untreated and sham transplant control subjects did not receive antiarrhythmic agents following the MI procedure, but otherwise received all immunosuppression and standard care.

Statistical analysis
Statistical analyses and graphing were performed using Prism 8.4.2 software (GraphPad) and Stata 15 (StataCorp, College Station, Texas). Data are presented as mean ± standard error of the mean (SEM).
Comparisons were performed using Mann-Whitney test with significance threshold of P < 0.05 unless otherwise specified. The sample size to demonstrate a difference in mortality rate of 67% (untreated group) versus 0% (treatment group), with alpha 0.05 and 90% power, was estimated to be 8 per cohort.
Kaplan-Meier plots show survival curves for the primary endpoint of cardiac death, unstable EA or heart failure, and for all-cause mortality. Cox proportional regression models are used to estimate the hazard ratio (HR) between the two treatment groups, for the primary outcome and for mortality. Significance is based on the likelihood ratio test and confidence intervals on HR are computed by inverting the likelihood test, based on varying the offset term in the stcox procedure in Stata.
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Clinical history of engraftment arrhythmia
A flow chart for all subjects in the study is shown in Fig. 1 and clinical summaries are provided in Table   1. No significant arrhythmias were noted in the two untreated sham transplant control subjects (9 and 10) that underwent myocardial infarction and percutaneous intracardiac injection of vehicle. All subjects that received human cardiomyocyte grafts developed EA between 2-6 days following cell transplantation. Initiation of EA was characterized by salvos of non-sustained VT, and this typically progressed to periods of sustained VT with rates ranging from 110 to 250 bpm (Fig. 2). The VT was often polymorphic, with the same subject showing different electrical axes and both wide-and narrowcomplex tachycardia at different times. In four of the eight untreated subjects, EA was either fatal or necessitated euthanasia due to a prespecified endpoint of unstable tachycardia (defined as sustained heart rate > 350 bpm). In one additional untreated case (subject 12), acute heart failure was noted clinically shortly after initiation of EA at a rate of 300 bpm, and based on recommendations from veterinary staff, the subject was euthanized. Signs of heart failure were subsequently confirmed on necropsy. In all other cases, EA was noted with a rapid acceleration to > 350 bpm (subjects 11 and 12) and, in two cases, deterioration to VF prior to euthanasia (subjects 1 and 2) ( Table 1). Three out of four arrhythmic endpoints occurred within the first three days of developing EA, and they occurred when tachyarrhythmia was nearly constant. Mean heart rate peaked at day 8 post-transplantation and began to decline after that, whereas the arrhythmia burden plateaued from days 8-16 and began to normalize thereafter. Of the three survivors in the untreated cohort, two did not normalize rhythm and experienced on average 42% arrhythmia burden at the end of study (subjects 15 and 17). The single subject in the untreated cohort that normalized heart rate and rhythm did so on day post-transplant day 26 (subject 11).
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Screening drugs for anti-arrhythmic effects
In Phase 1 of the study, we screened six canonical antiarrhythmic agents broadly targeting sodium channels, potassium channels, and beta-adrenergic receptors: lidocaine (class Ib Vaughan-Williams-Singh antiarrhythmic, sodium channel inhibitor), flecainide (Ic, sodium channel inhibitor), propafenone (Ic, sodium channel inhibitor), amiodarone (III, potassium channel inhibitor), sotalol (III, potassium channel inhibitor) and metoprolol (β1-adrenergic receptor inhibitor) for effect on EA heart rate and rhythm. In addition, the funny current/HCN4 channel antagonist, ivabradine, was tested (Online Table   1). This series was not meant to be comprehensive but rather to rapidly identify candidate agents.
Animals were brought into the laboratory while in EA, anesthetized, and the effects of short-term intravenous infusion or oral treatment of anti-arrhythmic agents were studied. In three instances, intravenous amiodarone successfully cardioverted unstable EA from >350 bpm to a lower heart rate, typically including brief episodes of sinus rhythm (Fig. 3A). Oral ivabradine demonstrated robust dosedependent effects on heart rate but did not restore sinus rhythm (Fig. 3B). Four of the other drugs had no significant effect in this screen (lidocaine, flecainide, sotalol, and metoprolol). Propafenone briefly reduced heart rate and restored sinus rhythm in two drug challenges, but this drug was associated with substantial gastrointestinal toxicity and not studied further (data not shown).

Amiodarone-ivabradine enhance survival
Given their distinct mechanisms of action and complementary effects on heart rate and rhythm, we formally tested the hypothesis that amiodarone along with ivabradine would reduce a combined primary endpoint of cardiac death, unstable EA >350 bpm and heart failure in Phase 2 of the study. A total of nine treated, eight untreated, and two sham transplant subjects were enrolled in the study with similar baseline and cell transplantation characteristics ( Table 1). As detailed in the Methods, treated animals received bolus and maintenance doses of amiodarone, and ivabradine was given as needed to keep heart (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Suppression of tachycardia and arrhythmia burden
Pooled and individual subject-level data of heart rate and arrhythmia burden are provided in Figs 5A/5B and 5C/5D, respectively. The average heart rate was significantly lower with antiarrhythmic treatment compared to no treatment. Mean heart rates peaked at post-transplantation day 7 in untreated animals at 163 ± 35 bpm, versus average heart rates of 90 ± 10 bpm in the treated cohort (p = 0.03) ( Table 1 and Fig. 5). Heart rate in the treated animals was not significantly different than the normal resting heart rate prior to MI and transplant (84 ± 1 bpm, p = 0.21). Following transplantation, peak daily heart rate for the study duration averaged 305 ± 29 bpm in untreated animals, whereas treatment significantly attenuated peak heart rate to 185 ± 9 bpm (p = 0.005) (Fig. 5E). We defined arrhythmia burden as the percentage of the day spent in arrhythmia. Treatment reduced peak arrhythmia burden from 96.8 ± 2.9% to 76.5 ± 7.9% (p = 0.003) (Fig. 5F). No differences in heart rate or arrhythmia burden were noted at posttransplant day 30, as the majority of arrhythmia had resolved irrespective of treatment (Fig. 5A/B) (p = 0.09 and p = 0.52, respectively).
Antiarrhythmic treatment was safely discontinued between day 24-34 in all treated subjects that achieved electrical maturation without recrudescence of arrhythmia (Fig. 5). Two treated and two untreated subjects (3, 4 and 15, 17, respectively) failed to mature electrically and exhibited significant (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. arrhythmia at the end of study. In these four animals, heart rates were well controlled irrespective of treatment, and they survived until the study's completion. Average serum amiodarone was subtherapeutic at 0.42 ± 0.12 µg/mL within 1 week of discontinuation (Online Fig. 2).

Percutaneous delivery of hESC-CMs in infarcted porcine model
Catheter-based endocardial delivery of hESC-CMs was safe and effective in remuscularizing the infarcted porcine heart (Online Fig. 3). No significant differences in myocardial infarct or cardiomyocyte graft sizes were observed between the treatment groups. The average infarct size for the treated and untreated cohorts were comparable at 11.7±1.1% and 10.5±2.0% of the left ventricle, respectively (p=0.59). Graft size relative to infarct size was also comparable at 2.3±0.7% and 2.8±1.3% for treated and untreated, respectively (p= 0.74). Delivery of hESC-CM successfully targeted the periinfarct border zone and central ischemic regions as intended and resulted in discrete hPSC-CM grafts transplanted into host myocardium, as previously reported (11)(12)(13)(14). All grafts localized to the anterior, antero-septal and antero-lateral walls and as previously reported in pig (14), appeared structurally immature at early time points before two weeks post-transplantation with increasing maturity up to the end of study.

Graft interaction with host Purkinje conduction system
The narrow-complex tachycardia that resembles accelerated junctional rhythm (Fig. 2) was not observed in our previous NHP studies (11,12) but was common in the minipig. Pigs are known to have an extensive Purkinje fiber network that extends transmurally throughout the ventricular myocardium, whereas in macaques and humans the Purkinje network is subendocardial (19,20). We hypothesized that the narrow-complex VT resulted from graft automaticity conducting through intramural Purkinje fibers and propagating to the rest of the ventricle. Histology confirmed the mesh-like network of intramural Purkinje fibers (PFs) throughout the minipig left ventricle (Online Fig. 4A, Online Video 1). There were (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. multiple examples of hESC-CM grafts in direct contact with these intramural branches of the Purkinje system. (Fig. 6, Online Video 2). We used connexin 40 (Cx40) immunostaining to specifically stain Purkinje fiber gap junctions (19,21), and confirmed their identity by their reduced myofibril content and the absence of T tubules (Online Fig. 4B). This supports the hypothesis that the pig's unique Purkinje network anatomy contributes to narrow-complex engraftment arrhythmia.

Discussion
Intramyocardial transplantation of hPSC-CM is a promising strategy to remuscularize the infarcted heart and restore function (5). Such a therapy to prevent and treat heart failure would be a seminal advance in addressing a large unmet clinical need. Studies in large animals have demonstrated long-term efficacy but also defined a significant safety signal of generally transient but potentially fatal arrhythmias. As demonstrated in earlier studies (12)(13)(14), EA is a predictable complication of cardiac remuscularization therapy for myocardial infarction (22). In the NHP, EA typically presents as a wide-complex tachycardia with a variable electrical axis (11,12), and this was reproduced in the minipig recently by Laflamme laboratory (14). Here, we further describe EA as polymorphic and interpret the changes in electrical axis as ectopy originating from different graft foci. Interestingly, in the pig we also observed a narrowcomplex VT that alternated with a wide-complex tachycardia, a pattern not seen in the NHP. Histology of native and grafted porcine myocardium support the hypothesis that the wide-complex beats originate from grafts interacting with the working cardiac myocytes with slow conduction, and that the narrow complex beats originate when grafts interact with the intramural Purkinje fibers that diffusely permeate the porcine heart (19,20).
All 17 subjects transplanted with 500 x 10 6 hESC-CMs demonstrated significant burden of arrhythmia that, while typically transient, was associated with high mortality in pigs. We observed higher morbidity (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. and mortality related to EA than the recent study by Laflamme and colleagues (14), perhaps reflecting differences in our animal model including use of Yucatan minipigs, percutaneous cell delivery, or our cell product. Our experience with this model suggests two primary mechanisms of cardiac morbidity.
Firstly, rapid EA > 350 bpm often degenerates to fatal ventricular fibrillation, and secondly, heart failure commonly ensues in pigs with chronic tachycardia > 230 bpm (23). Consequently, our primary endpoint included these parameters to limit excessive mortality in our antiarrhythmic trial.
Combined antiarrhythmic treatment with baseline amiodarone and adjunctive ivabradine safely prevented the combined primary endpoint of cardiac death, unstable EA and heart failure in all treated subjects, indicating that the risk of EA may be mitigated through pharmacology. Treatment was associated with significantly decreased peak tachycardia and arrhythmia. Once subjects experienced sustained improvement in arrhythmia burden, termed electrical maturation, antiarrhythmic therapy was successfully withdrawn in all subjects. Thus, short-term amiodarone and ivabradine treatment promoted electrical stability until the grafts became less arrhythmogenic.
The mechanism of benefit for our antiarrhythmic treatment may be related to suppression of automaticity, reducing both heart rate and arrhythmia burden. The drugs were particularly beneficial during the early phase of EA, which carries the greatest risk of deterioration to VF. Electrophysiological studies performed by our group and the Laflamme laboratory in NHP (12) and pig (14), respectively, suggests that the etiology of EA is increased focal automaticity, rather than macro-reentry typically observed with clinical ventricular tachycardia (24). As EA became unstable in the untreated animals, heart rates rapidly accelerate to >350 bpm, and we cannot exclude the possibility that this escalation could have a distinct mechanism, e.g., automaticity leading to reentry. This may explain why treatment successfully suppressed unstable and fatal arrhythmias but was unable to prevent EA altogether.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The efficacy of ivabradine to rate-control EA suggests that its pharmacologic target, the I f current carried by the HCN4 channel, which is highly expressed in immature cardiomyocytes and hPSC-CMs (25), may be an important mediator. Ivabradine, by itself, never abrogated EA, suggesting that while the I f current can accelerate the rate of EAs, I f is not the current responsible for generating the ventricular automaticity triggered by engraftment. In contrast, amiodarone reduced the burden of EA chronically and clearly restored sinus rhythm in some acute infusion experiments (Fig. 3). Although classified principally as a K + channel blocker (class III), amiodarone is well-known also to antagonize Na + channels, Ca 2+ channels, and β -adrenergic receptors (26). Thus, it is difficult to gain insights into the mechanism of EA from amiodarone's efficacy. The disappearance of EA coincides with maturation of the stem cell-derived graft (11,12,27), and we with others have hypothesized that the window of arrhythmogenicity may reflect a period of in vivo graft maturation prior to reaching a state more similar to host myocardium (25,(28)(29)(30)(31)(32). Additional strategies such as promoting maturation prior to Engraftment arrhythmia is the most significant barrier to clinical translation of cardiac remuscularization therapy. The natural history of EA emerging from the NHP and more recent porcine data suggests that, once EA resolves, there is low risk for further arrhythmia. This study provides a proof-of-concept that clinically relevant antiarrhythmic drug treatment can successfully suppress fatal arrhythmias and control (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted February 16, 2021. ; https://doi.org/10.1101/2021.02.15.431108 doi: bioRxiv preprint tachycardia to achieve electrical quiescence. This could be an important tool toward reaching an acceptable safety profile for clinical development.
While this study demonstrates that EA is responsive to pharmacologic suppression, there are several limitations. It would be useful to perform a longer follow-up to establish the long-term effectiveness of EA mitigation as well as dosing studies to optimize the treatment regimen. We did not randomize enrollment of animals or assess whether sex is a biological variable. Although we took pains to administer clinically relevant doses of amiodarone and ivabradine, we cannot exclude the possibility that EA in itself is dependent on the dose of cells transplanted. The dose utilized is comparable to that utilized to demonstrate long-term function benefit in NHP (500 x 10 6 versus 750 x 10 6 hESC-CMs, respectively),(12) but dosing studies have not been reported. Future studies will also ideally include functional endpoints to determine mechanical efficacy with background guideline-directed medical therapy such as inhibitors of the renin-angiotensin-aldosterone and β -adrenergic systems.

Conclusions
In this study utilizing a porcine infarction model of cardiac remuscularization therapy, EA was universally observed and associated with significant mortality. Chronic amiodarone treatment combined with adjunctive ivabradine successfully prevented the combined primary endpoint of cardiac death, unstable EA and heart failure. Overall survival was significantly improved with antiarrhythmic treatment and associated with heart rate and rhythm control. The mechanisms of engraftment arrhythmia remain incompletely understood and merit concerted scientific inquiry.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted February 16, 2021. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Clinical Perspectives
Heart failure remains a significant cause of morbidity and mortality following myocardial infarction (MI). Cardiac remuscularization with transplantation of pluripotent stem cell-derived cardiomyocytes is a promising preclinical therapy to restore function. Recent large animal data, however, have revealed a significant risk of engraftment arrhythmia (EA). The present study provides proof-of-concept evidence that a combination of amiodarone and ivabradine can effectively prevent EA-related mortality and suppresses tachycardia and arrhythmia burden. Thus, pharmacologic suppression of EA may be a viable strategy to improve safety and allow further clinical development of cardiac remuscularization therapy.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.  Fig. 1 Study Design Flowchart of the study design. Phase 1 consisted of nine total subjects, four untreated with any antiarrhythmic to study the natural history of engraftment arrythmia (EA) and five used to screen seven candidate antiarrhythmic agents. Amiodarone and ivabradine were found to have promising signals of effect and advanced for further study. Phase 2 consisted of 19 total subjects: nine treated with amiodarone and ivabradine and eight untreated following hESC-CM transplantation, and two untreated following sham transplantation.  as an intravenous bolus to cardiovert engraftment arrythmia to normal sinus or a lower heart rate transiently in three separate instances (A, red line). Ivabradine administered orally significantly slowed EA but did not cardiovert two days following initiation (B). These data supported a combined (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Fig. 5 Heart Rate and Engraftment Arrhythmia Burden
Effect of antiarrhythmic treatment on heart rate and arrhythmia burden. Pooled daily average heart rate (A) and pooled daily average arrhythmia burden (B) in treated (blue) compared to untreated (red) cohort. The difference in heart rate or arrhythmia burden between treated and untreated cohorts was not significant (NS) by day 30 post-transplantation. Sham transplant (grey) did not induce tachycardia or arrythmia. Subject-level averaged daily heart rate (C) and arrhythmia burden (D) for antiarrhythmic treated (blue), untreated (red) and sham transplant (grey). Unexpected death or euthanasia denoted by black symbol. Overall peak and mean daily heart rate (E) and overall peak and mean daily arrythmia burden (F) were significantly reduced in treated (blue) compared to untreated (red) cohorts. No tachycardia or arrhythmias were noted in the sham transplant control cohort. ** p ≤ 0.01.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.  Myocardial infarction (MI) was induced by 90-minute balloon occlusion of the mid-left anterior descending artery two weeks prior to human embryonic stem cell-derived cardiomyocyte transplantation (day 0). All subjects received multi-drug immunosuppression. Treated cohort received rate and rhythm control with combined oral amiodarone and adjunctive oral ivabradine.

Online Fig. 2 Amiodarone Levels
Plasma amiodarone levels in pigs. Amiodarone levels were measured in plasma by a custom liquid chromatography-mass spectrometry assay. Chronic oral amiodarone in six pigs was discontinued after achieving electrical maturation and stabilization of engraftment arrythmia. Serum through concentrations of amiodarone were assayed weekly including 3-4 weeks after discontinuation.
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