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. 2024 Jul 25;19(8):1053–1060. doi: 10.1016/j.stemcr.2024.06.012

Impulse initiation in engrafted pluripotent stem cell-derived cardiomyocytes can stimulate the recipient heart

Tim Stüdemann 1,2,6, Barbora Schwarzová 1,2, Till Schneidewind 1,2, Birgit Geertz 1, Constantin von Bibra 1,2,7, Marie Nehring 1,2, Judith Rössinger 1,2, J Simon Wiegert 3,4, Thomas Eschenhagen 1,2, Florian Weinberger 1,2,5,8,∗
PMCID: PMC11368679  PMID: 39059379

Summary

Transplantation of pluripotent stem cell-derived cardiomyocytes is a novel promising cell-based therapeutic approach for patients with heart failure. However, engraftment arrhythmias are a predictable life-threatening complication and represent a major hurdle for clinical translation. Thus, we wanted to experimentally study whether impulse generation by transplanted cardiomyocytes can propagate to the host myocardium and overdrive the recipient rhythm. We transplanted human induced pluripotent stem cell-derived cardiomyocytes expressing the optogenetic actuator Bidirectional Pair of Opsins for Light-induced Excitation and Silencing (BiPOLES) in a guinea pig injury model. Eight weeks after transplantation ex vivo, Langendorff perfusion was used to assess electrical coupling. Pulsed photostimulation was applied to specifically activate the engrafted cardiomyocytes. Photostimulation resulted in ectopic pacemaking that propagated to the host myocardium, caused non-sustained arrhythmia, and stimulated the recipient heart with higher pacing frequency (4/9 hearts). Our study demonstrates that transplanted cardiomyocytes can (1) electrically couple to the host myocardium and (2) stimulate the recipient heart. Thus, our results provide experimental evidence for an important aspect of engraftment-induced arrhythmia induction and thereby support the current hypothesis that cardiomyocyte automaticity can serve as a trigger for ventricular arrhythmias.

Keywords: cell therapy, cardiac regeneration, stem cell, cardiomyocyte, optogenetics, arrhythmia

Highlights

  • •

    hiPSC-cardiomyocyte expressing the optogenetic actuator BiPOLES can be paced with light

  • •

    BiPOLES cardiomyocytes engraft in the injured heart

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    Photostimulation results in ectopic pacemaking that stimulates the host myocardium

Weinberger and colleagues show that hiPSC-cardiomyocytes expressing the optogenetic actuator BiPOLES engrafted in the injured heart after transplantation. Pulsed photostimulation resulted in ectopic pacemaking that propagated to the host myocardium, caused non-sustained arrhythmia, and stimulated the recipient heart. Their results provide evidence for the hypothesis that cardiomyocyte automaticity can serve as a trigger for ventricular arrhythmias.

Introduction

Transplantation of pluripotent stem cell-derived cardiomyocytes is a regenerative therapeutic strategy that holds great promise for patients with heart failure (Weinberger and Eschenhagen 2021; Querdel et al., 2021; Selvakumar et al., 2024; Kobayashi et al., 2024). First clinical trials have started, and several others are currently in the planning stage. Ventricular arrhythmias after cardiomyocyte transplantation (so-called engraftment arrhythmia) are the main hurdle for clinical translation. Engraftment arrhythmia is a common, potentially life-threatening complication that mainly occurs within the first three weeks after the transplantation (Chong et al., 2014; Shiba et al., 2016; Romagnuolo et al., 2019). Catheter-based electrophysiological studies in primates and pigs have provided evidence that it is caused by automaticity of the immature transplanted cells (Liu et al., 2018; Romagnuolo et al., 2019). Here, we wanted to investigate whether impulse generation in the engrafted cells can propagate to the injured heart and overdrive the recipient heart rhythm. We used optogenetics to specifically activate engrafted cardiomyocytes and show that impulse generation in the engrafted cardiomyocytes can stimulate the recipient injured heart, providing evidence that cardiomyocyte automaticity can serve as a trigger for ventricular arrhythmias.

Results

To assess whether impulse generation in the engrafted cardiomyocytes can propagate to the host myocardium, we used induced pluripotent stem cell (iPSC)-derived cardiomyocytes which expressed the optogenetic actuator Bidirectional Pair of Opsins for Light-induced Excitation and Silencing (BiPOLES) consisting of the anion channel GtACR2 (blue excitation spectrum) and the cation channel Chrimson (red excitation spectrum). BiPOLES iPSC were generated by the targeted integration of BiPOLES in the adeno-associated virus integration site 1 (AAVS1) locus (Vierock et al., 2021). BiPOLES iPSC displayed high pluripotency (stage specific embryonic antigen 3 [SSEA3] positivity 99%, Figures 1A and 1B) and could be differentiated into cardiomyocytes with high purity (average troponin T positivity: 89%; Figures 1C and 1D, and Table S1). BiPOLES iPSCs and cardiomyocytes showed a (mainly) membranous transgene localization (Figures 1B and 1D). A detailed (electro)physiological characterization of the BiPOLES cell line has been published previously (Schwarzová et al., 2023). Engineered heart tissue (EHT) derived from BiPOLES cardiomyocytes could be activated by pulsed photostimulation with blue and red light (Video S1). BiPOLES-EHTs developed less force compared to EHTs derived from unedited cardiomyocytes but otherwise showed no major physiological alterations. Beating frequency did not differ from EHT derived from unedited cardiomyocytes (∼50 beats per minute [bpm]) (Schwarzová et al., 2023).

Figure 1.

Figure 1

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Differentiation of BiPOLES pluripotent stem cell-derived cardiomyocytes

(A) Immunohistology of BiPOLES hiPSC.

(B) Flow cytometry of BiPOLES iPSC stained for stage-specific embryonic antigen 3 (SSEA3, red).

(C) BiPOLES cardiomyocytes stained for Cerulean and ACTN2.

(D) Flow cytometry of BiPOLES cardiomyocytes stained for cardiac troponin T (cTnT, red). Isotype controls are shown in gray in (B) and (D). Scale bars: 10 μm.

Video S1. Optogenetically paced BiPOLES expressing engineered heart tissue
Download video file (2.6MB, mp4)

In this study we transplanted BiPOLES cardiomyocytes in a guinea pig injury model. This experimental setup allowed us to specifically activate the engrafted cardiomyocytes with pulsed photostimulation. BiPOLES cardiomyocytes were injected transepicardially (30 × 106 cardiomyocytes per animal, n = 9). 56-day post-transplantation, the hearts showed transmural scarring (scar area was 28% ± 5% of the left ventricle; Figures 2A and 2B, and Table S1). Engrafted human cardiomyocytes were visualized by staining for human Ku80. Partial remuscularization by islands of human myocardium was identified in 5/9 animals (graft size: 15% ± 7% of the scar; Figures 2A and 2B). While some grafts were entirely surrounded by scar tissue (Figures 2A, 2B, and S1A) or separated from the host myocardium by a thin fibrotic layer (Figures S1A and S1B), there was also histological evidence for direct graft-host interactions (Figures 2C–2F, S1A, and S1B). We studied the expression pattern of the cell-cell contacts connexin 43 and N-cadherin (which are both mainly localized at intercalated discs in mature myocardium) as well as cell-cycle activity and the troponin I and myosin light-chain isoform expression to assess the maturation state of the engrafted cardiomyocytes. Connexin 43 was circumferentially localized, indicating immaturity, but N-cadherin showed signs of polarization and formed intercalated-disc-like structures (Figures 2G and 2H). Cell-cycle activity was still ongoing (8% ± 2% Ki67-positive cardiomyocyte nuclei, Figure 2I). Cardiomyocytes demonstrated advanced sarcomere structure and mainly expressed the ventricular (mature) isoform of the myosin light chain (93% ± 2%, Figure 2J). A beginning switch from the fetal slow skeletal to the cardiac troponin I isoform (cardiac troponin I expression: 28% ± 2%; Figure 2K) further indicated maturation.

Figure 2.

Figure 2

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BiPOLES cardiomyocyte transplantation partially remuscularized the injured heart

(A and B) Short-axis sections of a heart eight weeks after cardiomyocyte transplantation stained for dystrophin (A) and human Ku80 (B). Inset in B is shown in higher magnification on the right.

(C–F) Graft-host interaction in lower and higher magnification. Grafts are visualized with mCerulean staining (C and D). Asterisks mark structural graft-host coupling (E and F).

(G and H) High-magnification images showing (G) connexin 43 and (H) N-cadherin expression in the human grafts. Higher magnification depicts the inset to visualize an intercalated-disc-like structure.

(I) Analysis of cell-cycle activity. Each data point represents one heart. Four images per heart were used for quantification. Data is represented as mean ± SEM. Asterisks mark human nuclei in the cell cycle.

(J) Staining for myosin light-chain isoform expression.

(K) Staining for troponin I isoform expression. Quantification for (E) and (F) was based on pixel area per image. Four images per heart from three different hearts were used for quantification. Scale bars: 2 mm in (A) and (B). 100 μm in (B) (high magnification) and 10 μm in (C–K).

Ex vivo Langendorff perfusion was used to assess electrical coupling. The aorta was cannulated, a balloon catheter was used to measure left-ventricular isovolumetric pressure, and two electrocardiogram (ECG) electrodes were placed on the surface of the heart (Figure 3A). Pulsed photostimulation (blue [470 nm] or red [635 nm] light, pulse duration 40–100 ms) was applied to the site of injury at the anterior wall of spontaneously beating hearts (average beating frequency 181 ± 15 bpm, Video S2) after a run-in period of 10–15 min. Individual premature ectopic beats occurred during the run-in phase, but we did not detect sustained arrhythmias. Photostimulation with frequencies similar to the host frequency caused non-sustained arrhythmias (stimulation frequency 180–210 bpm/host heart frequency ∼205 bpm) that terminated with the end of photostimulation (Figures 3B, S2A, and S2B). We then adjusted the photostimulation frequency to be higher than the spontaneous beating frequency (spontaneous beating frequency +0.25 to 1 Hz). Pulsed photostimulation resulted in ectopic pacemaking in 4 out of 9 recipient hearts (Figures 3C and 3D). This was followed by a stepwise increase in stimulation frequency. All hearts that showed electrical coupling could be paced up to 5 Hz (300 bpm), and the maximum pacing frequency was 10 Hz (600 bpm, Figure 3E). There was no difference between red and blue light photostimulation with respect to capture or maximum pacing frequency (which is in line with the in vitro characterization of BiPOLES in cardiomyocytes [Schwarzová et al., 2023]). Adenosine (0.3 mg) was applied to inhibit atrio-ventricular conduction and to induce transient ventricular asystole. Photostimulation was initiated after the onset of ventricular asystole. Again, pulsed photostimulation resulted in optogenetic impulse generation that initiated a ventricular rhythm in 4 out of 9 hearts (Figures 3F and S2C). Comparing electrophysiological results to histological analysis revealed that cardiomyocytes were not engrafted in 4 of the 5 hearts that failed to respond to photostimulation. Consequently, 4 of the 5 hearts with engrafted cardiomyocytes exhibited ectopic pacemaking with pulsed photostimulation (Figure 3G and Table S1).

Figure 3.

Figure 3

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Impulse generation in engrafted cardiomyocytes stimulates the recipient heart

(A) Photography of the experimental setup.

(B) Original recording of an ECG (middle row) and left-ventricular pressure (lower row) under baseline condition and upon photostimulation with blue light in low frequency (470 nm, 3 Hz, upper row).

(C and D) Original recording of an ECG (middle row) and left-ventricular pressure (lower row) under baseline condition and upon photostimulation with high-frequency (C) blue light (470 nm, 6 Hz, upper row) and (D) red light (635 nm, 6 Hz, upper row).

(E) Quantification of pacing capabilities with red light. Nine individual hearts are shown.

(F) Original ECG and left-ventricular pressure recording while adenosine was applied via the coronaries. Pulsed photostimulation with blue light (470 nm, 4 Hz). Photostimulation was initiated after ventricular asystole was established.

(G) Comparison between coupling and cardiomyocyte engraftment. Each data point represents one heart.

Video S2. Optogenetic pacing of a BiPOLES cardiomyocyte transplanted heart with red light (635 nm, 5 Hz)
Download video file (1.7MB, mp4)

Discussion

Transplantation of pluripotent stem cell-derived cardiomyocytes represents a promising novel regenerative therapy for heart failure patients. First clinical trials are ongoing. Yet, engraftment-induced ventricular arrhythmias occurred as a potentially life-threatening complication after stem cell-derived cardiomyocyte transplantation in clinically relevant large animal models. Stem cell derivatives are immature, and automaticity is one hallmark of immature cardiomyocytes (Guo and Pu 2020). Ventricular arrhythmias occurred within the first week after transplantation and ceased after three to four weeks (Chong et al., 2014; Shiba et al., 2016; Romagnuolo et al., 2019). The time frame of the occurrence of ventricular arrhythmias mirrored the maturation process indicating that the cardiomyocytes’ immaturity caused the engraftment-induced arrhythmias. Accordingly, catheter-based electrophysiological studies provided evidence that ectopic pacemaking at the site of cell engraftment initiated the ventricular arrhythmias (Liu et al., 2018; Romagnuolo et al., 2019).

Previous studies with genetically encoded calcium and voltage indicators showed that engrafted cardiomyocytes can be stimulated by the recipient myocardium (Shiba et al., 2012; Dhahri et al., 2022). We used a contrary approach and stimulated the engrafted cardiomyocytes to experimentally assess whether impulse initiation in the engrafted cardiomyocytes can propagate to the host myocardium and stimulate the recipient heart. By applying optogenetics, our study provides evidence that (1) transplanted human cardiomyocytes electrically couple to the host myocardium and that (2) impulse initiation in the engrafted cardiomyocytes can stimulate the injured recipient heart. Small animal models have inherent limitations when studying arrhythmogenicity: (1) the high heart rate and (2) the small volume of the heart. Transplantation of pluripotent stem cell-derived cardiomyocytes suppressed arrhythmias in a small animal model (Shiba et al., 2012), and only in later studies large animal models revealed engraftment-induced arrhythmias as a major risk for successful clinical translation (Chong et al., 2014). We detected a temporary rhythm disturbance when stimulating the heart with frequencies similar to the host rhythm but no sustained arrhythmias. Photostimulation with higher frequencies resulted in ectopic pacemaking. Thereby our study provides evidence for one important aspect of the hypothesis that automatism triggers the engraftment-induced ventricular arrhythmias: the impulse propagation from the engrafted cardiomyocytes to the injured host myocardium. The exact mechanisms of impulse propagation to the host tissue remain not fully understood. While we have seen direct structural coupling between engrafted and host myocytes (even though to a lesser degree than in a former study [Stüdemann et al., 2022]), most grafts were structurally separated from the host myocardium (at least by a thin fibrotic layer). There is evidence that impulse propagation can also occur via fibroblasts (Quinn et al., 2016; Wang et al., 2023), but the exact mechanism of electrical coupling remains unknown at present.

Besides the replacement of working myocardium, transplantation of pluripotent stem cell-derived cardiomyocytes has also been suggested as a biological pacemaker (Kehat et al., 2004) potentially in combination with optogenetics (Nussinovitch and Gepstein 2015). It was not our intention to study this application and we therefore (1) did not transplant cardiomyocytes in the uninjured heart and (2) did not assess whether a smaller number of engrafted cardiomyocytes can be sufficient to pace the heart. Yet, our study also provides evidence that engrafted cardiomyocytes could serve as biological pacemakers.

Experimental procedures

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Florian Weinberger (floriananton.weinberger@cnic.es).

Materials availability

The BiPOLES human iPSC (hiPSC) line used in this study will be made available on request with a completed Materials Transfer Agreement.

Data and code availability

This study did not generate new datasets.

hiPSC culture and cardiac differentiation

Generation of the BiPOLES iPSC line was recently described (Schwarzová et al., 2023). In brief, the BiPOLES transgene was integrated in the AAVS1 locus of the UKEi001-A hiPSC line (hPSCreg: RRID:CVCL_A8PR). A master cell bank was generated at passage 40, and a working cell bank at passage 41. Karyotyping by NanoString analysis was performed on the master cell bank and revealed a regular karyotype. hiPSCs were cultured in FTDA medium on Geltrex-coated cell culture vessels. SSEA analysis by fluorescence-activated cell sorting (FACS) was performed prior to cardiac differentiation. Formation of embryoid bodies was performed in spinner flasks. Embryoid body formation was followed by differentiation with the sequential administration of growth factor- and small-molecule-based cocktails to induce mesodermal progenitors, cardiac progenitors, and cardiomyocytes. Cardiomyocytes were dissociated with collagenase. The protocol in detail can be found in the supplemental methods. Cardiomyocytes underwent heat shock 24 h before dissociation as previously described (Laflamme et al., 2005) and were either used directly after differentiation or cryopreserved and thawed on the day of transplantation. Cardiomyocytes from four differentiation runs were used in this study. Control for mycoplasma contamination was performed bi-weekly.

Flow cytometry

Single-cell suspensions of iPSC-derived cardiomyocytes were fixed in Histofix (Roth A146.3) for 20 min at 4°C and stained in a buffer, containing 5% fetal bovine serum, 0.5% Saponin, and 0.05% sodium azide in PBS. Antibodies are listed in Table S2. Samples were analyzed with a BD FACSCanto II flow cytometer and the BD FACSDiva software 6.0 or BD FlowJo V10.

Animal care and experimental protocol approval

The investigation conforms to the guide for the care and use of laboratory animals published by the NIH (publication no. 85-23, revised 1985) and was approved by the local authorities (Behörde für Gesundheit und Verbraucherschutz, Freie und Hansestadt Hamburg: N123/021).

Injury model, cardiomyocyte transplantation, and histology

Cryoinjury of the left-ventricular wall was induced in female guinea pigs (∼300 g, ∼8 weeks of age, Envigo) as previously described by our group (von Bibra et al., 2022, 2023). Animals were anesthetized with fentanyl (0.05 mg/kg body weight), midazolam (3.3 mg/kg body weight), and medetomidine (0.33 mg/kg body weight). Buprenorphine (0.05 mg/kg body weight 3x per day) and carprofen (5 mg/kg body weight once daily) were administered postoperative for 7 days. Cardiomyocyte transplantation was performed seven days after the injury. During this procedure, 30 × 106 cardiomyocytes (directly after differentiation [5 animals] or after cryopreservation [4 animals]), resuspended in pro-survival cocktail (Laflamme et al., 2007; Romagnuolo et al., 2019) (Matrigel [∼50% v/v], cyclosporine A [200 nM], pinacidil [50 μM], insulin growth factor 1 [100 ng/mL], and B-cell lymphoma-extra large Bcl-2 homology 4 [Bcl-XL BH4] [50nM], total volume: 150 μL), were injected into three separate injection sites targeting central lesion and the flanking lateral border zones. Immunosuppression was performed with cyclosporine A beginning three days prior to transplantation (7.5 mg/kg body weight/day for the first three postoperative days and 5 mg/body weight/day for the following 53 days) and methylprednisolone (2 mg/kg body weight/day). Animals were euthanized by heart explantation under anesthesia (fentanyl, midazolam, and medetomidine).

Hearts were sectioned into four to five slices after fixation. Serial paraffin sections were acquired from each slice and used for histological analysis. Antigen retrieval and antibody dilution combinations used are summarized in Table S2. The primary antibody was either visualized with the multimer-technology-based ultraView Universal 3,3′-Diaminobenzidin (DAB) detection kit (Ventana BenchMark XT; Roche) or a fluorochrome-labeled secondary antibody (Alexa-conjugated, Thermo Fisher Scientific). Confocal images were acquired with an LSM 800 (Zeiss). For morphometry, images of dystrophin-stained sections were acquired with a Hamamatsu NanoZoomer whole slide scanner and viewed with NDP software (NDP.view 2.6.13). Graft size was measured in dystrophin-stained short-axis sections and expressed as a percentage of the scar area measured in the same section with the NPD2.view software. A detailed summary is provided in Table S1.

Langendorff perfusion

Guinea pigs (body weight 520–700 g) were injected with heparin (1,000 U/kg, subcutaneous) and anesthetized with fentanyl (0.05 mg/kg body weight), midazolam (3.3 mg/kg body weight), and medetomidine (0.33 mg/kg body weight). Hearts were excised and immersed in ice-cold modified Krebs-Henseleit solution containing (in mM) NaCl 120, KCl 4.7, MgSO4 1.2, NaHCO3 25.0, KH2PO4 1.2, glucose 11.1, Na-pyruvate 2.0, and CaCl2 1.8. The aorta was cannulated, and the heart was connected to a custom-made Langendorff apparatus. The heart was perfused using hydrostatic pressure (∼55 mmHg) with warm (36.5°C ± 1°C) modified Krebs-Henseleit solution equilibrated with a mixture of 95% O2 and 5% CO2 (pH 7.35–7.40). Left-ventricular pressure was recorded with a self-assembled balloon catheter, and data were acquired using Chart5 (ADInstruments). After a run-in period of >10 min, the hearts were illuminated with pulsed photo stimuli (40 ms pulse duration). Light was applied on the anterior wall of the left ventricle with blue (470 nm, irradiance 1–2 mW/mm2) or red light (635 nm, irradiance 1–2 mW/mm2) with a pE-4000 CoolLED for 30–60 s. Stepwise increase (0.5 Hz per step) was performed over a time period of 30–45 min.

Acknowledgments

We thank Kristin Hartmann (UKE, Mouse Pathology Core Facility) for technical assistance in immunohistochemistry. We would like to acknowledge Jutta Starbatty, Thomas Schulze, and Birgit Klampe for technical assistance. We would like to appreciate the contribution of Dr. Sandra Laufer during reprogramming the UKEi001-A line. Flow cytometry was conducted in the FACS Core Facility, UKE. We would like to particularly thank the laboratory animal facility staff (UKE) for their support. This work was supported by a Translational Research Grant from the German Centre for Cardiovascular Research (DZHK; 81X2710153 to T.E.), the European Research Council (ERC-AG IndivuHeart to T.E.), the German Research Foundation (DFG; WE5620/3-1 to F.W.), the European Union’s Horizon 2020 FetOpen RIA (964800; to F. W.), and the Werner Otto Foundation (to T.S. and F.W.).

Author contributions

Conceptualization: T. Stüdemann, T.E., and F.W. Funding acquisition: T. Stüdemann, T.E., and F.W. Investigation: T. Stüdemann, B.S., T. Schneidewind, B.G., C.v.B., M.N., and J.R. Methodology: J.S.W. Project administration: T. Stüdemann and F.W. Resources: T. Stüdemann, T.E., and F.W. Supervision: F.W. Visualization: T. Stüdemann, B.S., T. Schneidewind, and F.W. Writing – original draft: T. Stüdemann and F.W. Writing – review and editing: B.S., J.S.W., and T.E.

Declaration of interests

T.E. and F.W. participate in a structured partnership between Evotec AG and the University Medical Center Hamburg-Eppendorf (UKE) on the development of a cell-based therapy for patients with heart failure.

Published: July 25, 2024

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.stemcr.2024.06.012.

Supplemental information

Document S1. Figures S1 and S2, Tables S1 and S2, and supplemental methods
mmc1.pdf (837.4KB, pdf)
Document S2. Article plus supplemental information
mmc4.pdf (5.4MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Video S1. Optogenetically paced BiPOLES expressing engineered heart tissue
Download video file (2.6MB, mp4)
Video S2. Optogenetic pacing of a BiPOLES cardiomyocyte transplanted heart with red light (635 nm, 5 Hz)
Download video file (1.7MB, mp4)
Document S1. Figures S1 and S2, Tables S1 and S2, and supplemental methods
mmc1.pdf (837.4KB, pdf)
Document S2. Article plus supplemental information
mmc4.pdf (5.4MB, pdf)

Data Availability Statement

This study did not generate new datasets.

Articles from Stem Cell Reports are provided here courtesy of Elsevier

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