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

Abstract

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: arrhythmia; cardiac regeneration; cardiomyocyte; cell therapy; optogenetics; stem cell.

Figure 1

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.

Figure 2

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).

Figure 3

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.