Atrial-like Engineered Heart Tissue: An In Vitro Model of the Human Atrium

Abstract

Cardiomyocytes (CMs) generated from human induced pluripotent stem cells (hiPSCs) are under investigation for their suitability as human models in preclinical drug development. Antiarrhythmic drug development focuses on atrial biology for the treatment of atrial fibrillation. Here we used recent retinoic acid-based protocols to generate atrial CMs from hiPSCs and establish right atrial engineered heart tissue (RA-EHT) as a 3D model of human atrium. EHT from standard protocol-derived hiPSC-CMs (Ctrl-EHT) and intact human muscle strips served as comparators. RA-EHT exhibited higher mRNA and protein concentrations of atrial-selective markers, faster contraction kinetics, lower force generation, shorter action potential duration, and higher repolarization fraction than Ctrl-EHTs. In addition, RA-EHTs but not Ctrl-EHTs responded to pharmacological manipulation of atrial-selective potassium currents. RA- and Ctrl-EHTs' behavior reflected differences between human atrial and ventricular muscle preparations. Taken together, RA-EHT is a model of human atrium that may be useful in preclinical drug screening.

Keywords: atrial differentiation; atrial fibrillation; atrial myocytes; atrial-like cells; cardiac tissue engineering; engineered heart tissue; hiPSC-CMs; pluripotent stem cells; retinoic acid.

Graphical abstract

Figure 1

Effect of RA on Cardiac Differentiation Protocol (A) EB-based cardiac differentiation protocol. RA (1 μmol/L) was added from day 4 to day 7 to induce differentiation toward an atrial-like phenotype. (B) Flow cytometry analysis of RA-treated and Ctrl cells. (C) Analysis of cell area using α-actinin 2 staining. RA-treated cells show smaller area than Ctrl cells (1,736.2 ± 64.4 μm² versus 2,468.7 ± 192 μm², n = 209 and 88 from three batches each; p < 0.05, unpaired t test). (D) Box plots showing median, first, and third quartile of the volume of Ctrl versus RA-treated CMs. (Left) Volume of RA-treated cells after differentiation was smaller than Ctrl cells (560 ± 1.3 μm³ versus 588 ± 1.6 μm³, n = 26,508 and 21,577 from three batches each; p < 0.05, unpaired t test). (Right) Volume of cells dissociated from RA-EHTs was smaller than from Ctrl-EHTs (569 ± 3 μm³ versus 854 ± 2.8 μm³, n = 15,474 and 20,931 from 4 EHTs each; p < 0.05, unpaired t test). Error bars show means ± SEM.

Figure 2

RA Treatment of hiPSCs Promotes Expression of Atrial-Specific Genes (A) RT-qPCR of selected genes at day 14 to validate upregulation of atrial and downregulation of ventricular markers in RA-MLs/EHTs (n = 9 from three batches each) compared with Ctrl-MLs/EHTs (n = 9 from three batches each). Cycle treshold (CT) values were normalized with CT values for human GUSB. Transcript concentrations are shown in the graph as folds of their respective Ctrl. (B) Western blotting of 14-day-old MLs with antibodies against α-actinin 2, COUPTFII, PITX2, MLC2A, and ANP (n = 3 batches). Error bars show means ± SEM.

Figure 3

RA Treatment Shifts Expression of MLC from MLC2V to MLC2A (A) Immunofluorescence labeling of 14-day-old MLs. 2.5× (left) and 40× (right) magnification of Ctrl and RA-MLs. Merged staining of MLC2V (green), MLC2A (red), and DAPI (blue). (B) Immunohistochemistry of MLC2V and MLC2A expression in EHTs and human tissue (first two columns). Immunofluorescence of MLC2V (green), MLC2A (red), and DAPI (blue) (last two columns). Scale bar for all images represents 100 μm. Please note that the images show similar areas of evenly distributed CM network throughout the diameter of EHTs while generally the majority of the cells are located at the outer layer of the EHT (Figure S6C; Hirt et al., 2014, Vollert et al., 2013).

Figure 4

Effect of RA Treatment on Beating Rate and Contraction Kinetics (A) Effects in MLs: averaged contractions obtained from six different single wells recorded by CellOPTIQ. RA-MLs (n = 13 wells from two batches) showed faster kinetic parameters (TTP and RT) and faster spontaneous beating compared with Ctrl-MLs (n = 12 wells from two batches). (B) Effects on EHTs: average contraction peaks were calculated from six different EHTs. RA-EHTs (n = 17 from three batches) showed faster contraction kinetics, faster spontaneous beating, and smaller force compared with Ctrl-EHTs (n = 10 from three batches). All average contraction peaks were normalized. Y axes differ between MLs and EHTs to better visualize the effect of RA. Error bars show means ± SEM.

Figure 5

Effect of RA Treatment on APD₉₀ and Repolarization Fraction The scatterplot for APD₉₀ versus repolarization fraction includes AP recordings of MLs and EHTs measured by cellOPTIQ and sharp microelectrodes, respectively. For each group a representative original AP trace is shown. The bar graphs (right) show APD₉₀ and repolarization fraction (APD₉₀ − APD₅₀)/APD₉₀ in Ctrl-ML (n = 70 wells from two batches), RA-ML (n = 140 wells from two batches), Ctrl-EHT (n = 90/6, number of impalements/EHTs, three batches), RA-EHT (n = 157/6, number of impalements/EHTs, three batches), LV (n = 20 patients), and RAA (n = 38 patients). Error bars show means ± SEM.

Figure 6

CCh Effect on AP Recordings Original traces of APs recorded by sharp microelectrodes before and after 2 min exposure to CCh (10 μmol/L) in Ctrl- and RA-EHTs (n = 5 from three batches) (A) and in RAA (n = 10 patients) and LV (n = 3 patients) (B). On the right, mean values for TOP/RMP and APD₉₀ are given before and 2 min after CCh exposure. AP traces were recorded at 37°C with 2 Hz pacing for EHTs obtained from ERC18 cell line. LV and RAA APs were field stimulated at 1 Hz. Y axes differ between EHT and human adult cardiac tissues to better visualize the effect of CCh. Error bars show means ± SEM.

Figure 7

4-AP Effect on AP Recordings Original traces of APs recorded by sharp microelectrodes before and after 15 min exposure to 4-AP (50 μmol/L) in Ctrl- and RA-EHTs (n = 6 from three batches) (A) and in RAA (n = 8 patients) and LV (n = 8 patients) (B). On the right, mean values for APD₂₀ and APA are given before and 15 min after 4-AP exposure. AP recordings were obtained at 37°C with 2 Hz pacing for Ctrl-EHTs and 4 Hz pacing for RA-EHTs. LV and RAA APs were field stimulated at 1 Hz. Y axes differ between EHT and human adult cardiac tissues to better visualize the effect of 4-AP. Error bars show means ± SEM.