Human Engineered Heart Tissue Patches Remuscularize the Injured Heart in a Dose-Dependent Manner.

BACKGROUND: Human engineered heart tissue (EHT) transplantation represents a potential regenerative strategy for patients with heart failure and has been successful in preclinical models. Clinical application requires upscaling, adaptation to good manufacturing practices, and determination of the effective dose. METHODS: Cardiomyocytes were differentiated from 3 different human induced pluripotent stem cell lines including one reprogrammed under good manufacturing practice conditions. Protocols for human induced pluripotent stem cell expansion, cardiomyocyte differentiation, and EHT generation were adapted to substances available in good manufacturing practice quality. EHT geometry was modified to generate patches suitable for transplantation in a small-animal model and perspectively humans. Repair efficacy was evaluated at 3 doses in a cryo-injury guinea pig model. Human-scale patches were epicardially transplanted onto healthy hearts in pigs to assess technical feasibility. RESULTS: We created mesh-structured tissue patches for transplantation in guinea pigs (1.5×2.5 cm, 9-15×106 cardiomyocytes) and pigs (5×7 cm, 450×106 cardiomyocytes). EHT patches coherently beat in culture and developed high force (mean 4.6 mN). Cardiomyocytes matured, aligned along the force lines, and demonstrated advanced sarcomeric structure and action potential characteristics closely resembling human ventricular tissue. EHT patches containing ≈4.5, 8.5, 12×106, or no cells were transplanted 7 days after cryo-injury (n=18-19 per group). EHT transplantation resulted in a dose-dependent remuscularization (graft size: 0%-12% of the scar). Only high-dose patches improved left ventricular function (+8% absolute, +24% relative increase). The grafts showed time-dependent cardiomyocyte proliferation. Although standard EHT patches did not withstand transplantation in pigs, the human-scale patch enabled successful patch transplantation. CONCLUSIONS: EHT patch transplantation resulted in a partial remuscularization of the injured heart and improved left ventricular function in a dose-dependent manner in a guinea pig injury model. Human-scale patches were successfully transplanted in pigs in a proof-of-principle study.

Intermittent Optogenetic Tachypacing of Atrial Engineered Heart Tissue Induces Only Limited Electrical Remodelling.

ABSTRACT: Atrial tachypacing is an accepted model for atrial fibrillation (AF) in large animals and in cellular models. Human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CM) provide a novel human source to model cardiovascular diseases. Here, we investigated whether optogenetic tachypacing of atrial-like hiPSC-CMs grown into engineered heart tissue (aEHT) can induce AF-remodeling. After differentiation of atrial-like cardiomyocytes from hiPSCs using retinoic acid, aEHTs were generated from ∼1 million atrial-like hiPSC-CMs per aEHT. AEHTs were transduced with lentivirus expressing channelrhodopsin-2 to enable optogenetic stimulation by blue light pulses. AEHTs underwent optical tachypacing at 5 Hz for 15 seconds twice a minute over 3 weeks and compared with transduced spontaneously beating isogenic aEHTs (1.95 ± 0.07 Hz). Force and action potential duration did not differ between spontaneously beating and tachypaced aEHTs. Action potentials in tachypaced aEHTs showed higher upstroke velocity (138 ± 15 vs. 87 ± 11 V/s, n = 15-13/3; P = 0.018), possibly corresponding to a tendency for more negative diastolic potentials (73.0 ± 1.8 vs. 68.0 ± 1.9 mV; P = 0.07). Tachypaced aEHTs exhibited a more irregular spontaneous beating pattern (beat-to-beat scatter: 0.07 ± 0.01 vs. 0.03 ± 0.004 seconds, n = 15-13/3; P = 0.008). Targeted expression analysis showed higher RNA levels of KCNJ12 [Kir2.2, inward rectifier (IK1); 69 ± 7 vs. 44 ± 4, P = 0.014] and NPPB (NT-proBNP; 39,690 ± 4834 vs. 23,671 ± 3691; P = 0.024). Intermittent tachypacing in aEHTs induces some electrical alterations found in AF and induces an arrhythmic spontaneous beating pattern, but does not affect resting force. Further studies using longer, continuous, or more aggressive stimulation may clarify the contribution of different rate patterns on the changes in aEHT mimicking the remodeling process from paroxysmal to persistent atrial fibrillation.

Differential regulation of node formation, nodal ciliogenesis and cilia positioning by Noto and Foxj1.

The mouse transcription factor Noto is expressed in the node and controls node morphogenesis, formation of nodal cilia and left-right asymmetry. Noto acts upstream of Foxj1, which regulates ciliogenesis in other mouse tissues. However, the significance of Foxj1 for the formation of cilia in the mouse node is unclear; in non-amniote species Foxj1 is required for ciliogenesis in the structures equivalent to the node. Here, we analyzed nodes, nodal cilia and nodal flow in mouse embryos in which we replaced the Noto-coding sequence with that of Foxj1, or in embryos that were deficient for Foxj1. We show that Foxj1 expressed from the Noto locus is functional and restores the formation of structurally normal motile cilia in the absence of Noto. However, Foxj1 is not sufficient for the correct positioning of cilia on the cell surface within the plane of the nodal epithelium, and cannot restore normal node morphology. We also show that Foxj1 is essential for ciliogenesis upstream of Rfx3 in the node. Thus, the function of Foxj1 in vertebrate organs of asymmetry is conserved, and Noto regulates node morphogenesis and the posterior localization of cilia on node cells independently of Foxj1.

Embryonic exposure to propylthiouracil disrupts left-right patterning in Xenopus embryos.

Antithyroid medications are the preferred therapy for the treatment of Graves' disease during pregnancy. Propylthiouracil (PTU) is favored over methimazole (MMI) due to potential teratogenic concerns with MMI. This study was to determine the teratogenic potential of MMI and PTU using a validated Xenopus tropicalis embryo model. Embryos were exposed to 1 mM PTU (EC(50)=0.88 mM), 1 mM MMI, or vehicle control (water) from stages 2 to 45. Treated embryos were examined for gross morphological defects, ciliary function, and gene expression by in situ hybridization. Exposure to PTU, but not MMI, led to cardiac and gut looping defects and shortening along the anterior-posterior axis. PTU exposure during gastrulation (stage 8-12.5) was identified as the critical period of exposure leading to left-right (LR) patterning defects. Abnormal cilia polarization, abnormal cilia-driven leftward flow at the gastrocoel roof plate (GRP), and aberrant expression of both Coco and Pitx2c were associated with abnormal LR symmetry observed following PTU exposure. PTU is teratogenic during late blastula, gastrulation, and neurulation; whereas MMI is not. PTU alters ciliary-driven flow and disrupts the normal genetic program involved in LR axis determination. These studies have important implications for women taking PTU during early pregnancy.

Physioxia rewires mitochondrial complex composition to protect stem cell viability.

Human induced pluripotent stem cells (hiPSCs) are an invaluable tool to study molecular mechanisms on a human background. Culturing stem cells at an oxygen level different from their microenvironmental niche impacts their viability. To understand this mechanistically, dermal skin fibroblasts of 52 probands were reprogrammed into hiPSCs, followed by either hyperoxic (20 % O2) or physioxic (5 % O2) culture and proteomic profiling. Analysis of chromosomal stability by Giemsa-banding revealed that physioxic -cultured hiPSC clones exhibited less pathological karyotypes than hyperoxic (e.g. 6 % vs. 32 % mosaicism), higher pluripotency as evidenced by higher Stage-Specific Embryonic Antigen 3 positivity, higher glucose consumption and lactate production. Global proteomic analysis demonstrated lower abundance of several subunits of NADH:ubiquinone oxidoreductase (complex I) and an underrepresentation of pathways linked to oxidative phosphorylation and cellular senescence. Accordingly, release of the pro-senescent factor IGFBP3 and ß-galactosidase staining were lower in physioxic hiPSCs. RNA- and ATAC-seq profiling revealed a distinct hypoxic transcription factor-binding footprint, amongst others higher expression of the HIF1α-regulated target NDUFA4L2 along with increased chromatin accessibility of the NDUFA4L2 gene locus. While mitochondrial DNA content did not differ between groups, physioxic hiPSCs revealed lower polarized mitochondrial membrane potential, altered mitochondrial network appearance and reduced basal respiration and electron transfer capacity. Blue-native polyacrylamide gel electrophoresis coupled to mass spectrometry of the mitochondrial complexes detected higher abundance of NDUFA4L2 and ATP5IF1 and loss of incorporation into complex IV or V, respectively. Taken together, physioxic culture of hiPSCs improved chromosomal stability, which was associated with downregulation of oxidative phosphorylation and senescence and extensive re-wiring of mitochondrial complex composition.

Human model of primary carnitine deficiency cardiomyopathy reveals ferroptosis as a novel mechanism.

Primary carnitine deficiency (PCD) is an autosomal recessive monogenic disorder caused by mutations in SLC22A5. This gene encodes for OCTN2, which transports the essential metabolite carnitine into the cell. PCD patients suffer from muscular weakness and dilated cardiomyopathy. Two OCTN2-defective human induced pluripotent stem cell lines were generated, carrying a full OCTN2 knockout and a homozygous OCTN2 (N32S) loss-of-function mutation. OCTN2-defective genotypes showed lower force development and resting length in engineered heart tissue format compared with isogenic control. Force was sensitive to fatty acid-based media and associated with lipid accumulation, mitochondrial alteration, higher glucose uptake, and metabolic remodeling, replicating findings in animal models. The concordant results of OCTN2 (N32S) and -knockout emphasizes the relevance of OCTN2 for these findings. Importantly, genome-wide analysis and pharmacological inhibitor experiments identified ferroptosis, an iron- and lipid-dependent cell death pathway associated with fibroblast activation as a novel PCD cardiomyopathy disease mechanism.

Human-Engineered Atrial Tissue for Studying Atrial Fibrillation.

This chapter details the generation of atrial fibrin-based engineered heart tissue (EHT) in standard 24-well format as a 3D model for the human atrium. Compared to 2D cultivation, human-induced pluripotent stem cells (hiPSCs)-derived atrial cardiomyocytes demonstrated a higher degree of maturation in 3D format. Furthermore, we have demonstrated in previous work that the model displayed atrial characteristics in terms of contraction and gene expression patterns, electrophysiology, and pharmacological response. Here, we describe how to embed atrial cardiomyocytes differentiated from hiPSCs in a fibrin hydrogel to form atrial EHT attached to elastic silicone posts, allowing auxotonic contraction. In addition, we describe how force and other contractility parameters can be derived from these beating atrial EHTs by video-optical monitoring. The presented atrial EHT model is suitable to study chamber-specific mechanisms, drug effects and to serve for disease modeling.

Sulforaphane exposure impairs contractility and mitochondrial function in three-dimensional engineered heart tissue.

Sulforaphane (SFN) is a phytochemical compound extracted from cruciferous plants, like broccoli or cauliflower. Its isothiocyanate group renders SFN reactive, thus allowing post-translational modification of cellular proteins to regulate their function with the potential for biological and therapeutic actions. SFN and stabilized variants recently received regulatory approval for clinical studies in humans for the treatment of neurological disorders and cancer. Potential unwanted side effects of SFN on heart function have not been investigated yet. The present study characterizes the impact of SFN on cardiomyocyte contractile function in cardiac preparations from neonatal rat, adult mouse and human induced-pluripotent stem cell-derived cardiomyocytes. This revealed a SFN-mediated negative inotropic effect, when administered either acutely or chronically, with an impairment of the Frank-Starling response to stretch activation. A direct effect of SFN on myofilament function was excluded in chemically permeabilized mouse trabeculae. However, SFN pretreatment increased lactate formation and enhanced the mitochondrial production of reactive oxygen species accompanied by a significant reduction in the mitochondrial membrane potential. Transmission electron microscopy revealed disturbed sarcomeric organization and inflated mitochondria with whorled membrane shape in response to SFN exposure. Interestingly, administration of the alternative energy source l-glutamine to the medium that bypasses the uptake route of pyruvate into the mitochondrial tricarboxylic acid cycle improved force development in SFN-treated EHTs, suggesting indeed mitochondrial dysfunction as a contributor of SFN-mediated contractile dysfunction. Taken together, the data from the present study suggest that SFN might impact negatively on cardiac contractility in patients with cardiovascular co-morbidities undergoing SFN supplementation therapy. Therefore, cardiac function should be monitored regularly to avoid the onset of cardiotoxic side effects.

Generation of bi-allelic MYBPC3 truncating mutant and isogenic control from an iPSC line of a patient with hypertrophic cardiomyopathy.

MYBPC3 is the most frequently affected gene in hypertrophic cardiomyopathy (HCM), which is an autosomal-dominant cardiac disease caused by mutations in sarcomeric proteins. Bi-allelic truncating MYBPC3 mutations are associated with severe forms of neonatal cardiomyopathy. We reprogrammed skin fibroblasts from a HCM patient carrying a heterozygous MYBPC3 truncating mutation into human induced pluripotent stem cells (iPSC) and used CRISPR/Cas9 to generate bi-allelic MYBPC3 truncating mutation and isogenic control hiPSC lines. All lines expressed pluripotency markers, had normal karyotype and differentiated into endoderm, ectoderm and cardiomyocytes in vitro. This set of three lines provides a useful tool to study HCM pathomechanisms.

Impairment of the ER/mitochondria compartment in human cardiomyocytes with PLN p.Arg14del mutation.

The phospholamban (PLN) p.Arg14del mutation causes dilated cardiomyopathy, with the molecular disease mechanisms incompletely understood. Patient dermal fibroblasts were reprogrammed to hiPSC, isogenic controls were established by CRISPR/Cas9, and cardiomyocytes were differentiated. Mutant cardiomyocytes revealed significantly prolonged Ca2+ transient decay time, Ca2+ -load dependent irregular beating pattern, and lower force. Proteomic analysis revealed less endoplasmic reticulum (ER) and ribosomal and mitochondrial proteins. Electron microscopy showed dilation of the ER and large lipid droplets in close association with mitochondria. Follow-up experiments confirmed impairment of the ER/mitochondria compartment. PLN p.Arg14del end-stage heart failure samples revealed perinuclear aggregates positive for ER marker proteins and oxidative stress in comparison with ischemic heart failure and non-failing donor heart samples. Transduction of PLN p.Arg14del EHTs with the Ca2+ -binding proteins GCaMP6f or parvalbumin improved the disease phenotype. This study identified impairment of the ER/mitochondria compartment without SR dysfunction as a novel disease mechanism underlying PLN p.Arg14del cardiomyopathy. The pathology was improved by Ca2+ -scavenging, suggesting impaired local Ca2+ cycling as an important disease culprit.

Human pluripotent stem cell-derived cardiomyocytes for studying energy metabolism.

Cardiomyocyte energy metabolism is altered in heart failure, and primary defects of metabolic pathways can cause heart failure. Studying cardiac energetics in rodent models has principal shortcomings, raising the question to which extent human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) can provide an alternative. As metabolic maturation of CM occurs mostly after birth during developmental hypertrophy, the immaturity of hiPSC-CM is an important limitation. Here we shortly review the physiological drivers of metabolic maturation and concentrate on methods to mature hiPSC-CM with the goal to benchmark the metabolic state of hiPSC-CM against in vivo data and to see how far known abnormalities in inherited metabolic disorders can be modeled in hiPSC-CM. The current data indicate that hiPSC-CM, despite their immature, approximately mid-fetal state of energy metabolism, faithfully recapitulate some basic metabolic disease mechanisms. Efforts to improve their metabolic maturity are underway and shall improve the validity of this model.

Effects of the Delta Opioid Receptor Agonist DADLE in a Novel Hypoxia-Reoxygenation Model on Human and Rat-Engineered Heart Tissue: A Pilot Study.

Intermittent hypoxia and various pharmacological compounds protect the heart from ischemia reperfusion injury in experimental approaches, but the translation into clinical trials has largely failed. One reason may lie in species differences and the lack of suitable human in vitro models to test for ischemia/reperfusion. We aimed to develop a novel hypoxia-reoxygenation model based on three-dimensional, spontaneously beating and work performing engineered heart tissue (EHT) from rat and human cardiomyocytes. Contractile force, the most important cardiac performance parameter, served as an integrated outcome measure. EHTs from neonatal rat cardiomyocytes were subjected to 90 min of hypoxia which led to cardiomyocyte apoptosis as revealed by caspase 3-staining, increased troponin I release (time control vs. 24 h after hypoxia: cTnI 2.7 vs. 6.3 ng/mL, ** p = 0.002) and decreased contractile force (64 ± 6% of baseline) in the long-term follow-up. The detrimental effects were attenuated by preceding the long-term hypoxia with three cycles of 10 min hypoxia (i.e., hypoxic preconditioning). Similarly, [d-Ala2, d-Leu5]-enkephalin (DADLE) reduced the effect of hypoxia on force (recovery to 78 ± 5% of baseline with DADLE preconditioning vs. 57 ± 5% without, p = 0.012), apoptosis and cardiomyocyte stress. Human EHTs presented a comparable hypoxia-induced reduction in force (55 ± 5% of baseline), but DADLE failed to precondition them, likely due to the absence of δ-opioid receptors. In summary, this hypoxia-reoxygenation in vitro model displays cellular damage and the decline of contractile function after hypoxia allows the investigation of preconditioning strategies and will therefore help us to understand the discrepancy between successful conditioning in vitro experiments and its failure in clinical trials.

Chronic intermittent tachypacing by an optogenetic approach induces arrhythmia vulnerability in human engineered heart tissue.

AIMS: Chronic tachypacing is commonly used in animals to induce cardiac dysfunction and to study mechanisms of heart failure and arrhythmogenesis. Human induced pluripotent stem cells (hiPSC) may replace animal models to overcome species differences and ethical problems. Here, 3D engineered heart tissue (EHT) was used to investigate the effect of chronic tachypacing on hiPSC-cardiomyocytes (hiPSC-CMs). METHODS AND RESULTS: To avoid cell toxicity by electrical pacing, we developed an optogenetic approach. EHTs were transduced with lentivirus expressing channelrhodopsin-2 (H134R) and stimulated by 15 s bursts of blue light pulses (0.3 mW/mm2, 30 ms, 3 Hz) separated by 15 s without pacing for 3 weeks. Chronic optical tachypacing did not affect contractile peak force, but induced faster contraction kinetics, shorter action potentials, and shorter effective refractory periods. This electrical remodelling increased vulnerability to tachycardia episodes upon electrical burst pacing. Lower calsequestrin 2 protein levels, faster diastolic depolarization (DD) and efficacy of JTV-519 (46% at 1 µmol/L) to terminate tachycardia indicate alterations of Ca2+ handling being part of the underlying mechanism. However, other antiarrhythmic compounds like flecainide (69% at 1 µmol/L) and E-4031 (100% at 1 µmol/L) were also effective, but not ivabradine (1 µmol/L) or SEA0400 (10 µmol/L). CONCLUSION: We demonstrated a high vulnerability to tachycardia of optically tachypaced hiPSC-CMs in EHT and the effective termination by ryanodine receptor stabilization, sodium or hERG potassium channel inhibition. This new model might serve as a preclinical tool to test antiarrhythmic drugs increasing the insight in treating ventricular tachycardia.

Case Report on: Very Early Afterdepolarizations in HiPSC-Cardiomyocytes-An Artifact by Big Conductance Calcium Activated Potassium Current (Ibk,Ca).

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent an unlimited source of human CMs that could be a standard tool in drug research. However, there is concern whether hiPSC-CMs express all cardiac ion channels at physiological level and whether they might express non-cardiac ion channels. In a control hiPSC line, we found large, "noisy" outward K+ currents, when we measured outward potassium currents in isolated hiPSC-CMs. Currents were sensitive to iberiotoxin, the selective blocker of big conductance Ca2+-activated K+ current (IBK,Ca). Seven of 16 individual differentiation batches showed a strong initial repolarization in the action potentials (AP) recorded from engineered heart tissue (EHT) followed by very early afterdepolarizations, sometimes even with consecutive oscillations. Iberiotoxin stopped oscillations and normalized AP shape, but had no effect in other EHTs without oscillations or in human left ventricular tissue (LV). Expression levels of the alpha-subunit (KCa1.1) of the BKCa correlated with the presence of oscillations in hiPSC-CMs and was not detectable in LV. Taken together, individual batches of hiPSC-CMs can express sarcolemmal ion channels that are otherwise not found in the human heart, resulting in oscillating afterdepolarizations in the AP. HiPSC-CMs should be screened for expression of non-cardiac ion channels before being applied to drug research.

Comparison of 10 Control hPSC Lines for Drug Screening in an Engineered Heart Tissue Format.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are commercially available, and cardiac differentiation established routine. Systematic evaluation of several control hiPSC-CM is lacking. We investigated 10 different control hiPSC-CM lines and analyzed function and suitability for drug screening. Five commercial and 5 academic hPSC-CM lines were casted in engineered heart tissue (EHT) format. Spontaneous and stimulated EHT contractions were analyzed, and 7 inotropic indicator compounds investigated on 8 cell lines. Baseline contractile force, kinetics, and rate varied widely among the different lines (e.g., relaxation time range: 118-471 ms). In contrast, the qualitative correctness of responses to BayK-8644, nifedipine, EMD-57033, isoprenaline, and digoxin in terms of force and kinetics varied only between 80% and 93%. Large baseline differences between control cell lines support the request for isogenic controls in disease modeling. Variability appears less relevant for drug screening but needs to be considered, arguing for studies with more than one line.

Cell Banking of hiPSCs: A Practical Guide to Cryopreservation and Quality Control in Basic Research.

The reproducibility of stem cell research relies on the constant availability of quality-controlled cells. As the quality of human induced pluripotent stem cells (hiPSCs) can deteriorate in the course of a few passages, cell banking is key to achieve consistent results and low batch-to-batch variation. Here, we provide a cost-efficient route to generate master and working cell banks for basic research projects. In addition, we describe minimal protocols for quality assurance including tests for sterility, viability, pluripotency, and genetic integrity. © 2020 The Authors. Basic Protocol 1: Expansion of hiPSCs Basic Protocol 2: Cell banking of hiPSCs Support Protocol 1: Pluripotency assessment by flow cytometry Support Protocol 2: Thawing control: Viability and sterility Support Protocol 3: Potency, viral clearance, and pluripotency: Spontaneous differentiation and qRT-PCR Support Protocol 4: Identity: Short tandem repeat analysis.

Metabolic Maturation Media Improve Physiological Function of Human iPSC-Derived Cardiomyocytes.

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have enormous potential for the study of human cardiac disorders. However, their physiological immaturity severely limits their utility as a model system and their adoption for drug discovery. Here, we describe maturation media designed to provide oxidative substrates adapted to the metabolic needs of human iPSC (hiPSC)-CMs. Compared with conventionally cultured hiPSC-CMs, metabolically matured hiPSC-CMs contract with greater force and show an increased reliance on cardiac sodium (Na+) channels and sarcoplasmic reticulum calcium (Ca2+) cycling. The media enhance the function, long-term survival, and sarcomere structures in engineered heart tissues. Use of the maturation media made it possible to reliably model two genetic cardiac diseases: long QT syndrome type 3 due to a mutation in the cardiac Na+ channel SCN5A and dilated cardiomyopathy due to a mutation in the RNA splicing factor RBM20. The maturation media should increase the fidelity of hiPSC-CMs as disease models.

Dissecting hiPSC-CM pacemaker function in a cardiac organoid model.

Biological pacemakers could be a promising alternative to electronic pacemakers and human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) may represent a suitable source for implantable cells. To further unravel this potential a thorough understanding of pacemaker function with regard to coupling processes both in the physiological and in the graft-host context is required. Here we developed a 2-component cardiac organoid model with a hiPSC-CM embryoid body (EB) as trigger casted into a rat engineered heart tissue (EHT) as arrhythmic beating substrate. Contractility recordings revealed that the EB controlled the beating activity of the EHT, leading to a regular hiPSC-CM-like beating pattern instead of the irregular beating typically seen in rat EHT. Connectivity was observed with action potential (AP) measurements and calcium transients transmitting from the EB directly into the rat EHT. Immunohistochemistry and genetically labeled hiPSC-CMs demonstrated that EB-derived and rat cells intermingled and formed a transitional zone. Connexin 43 expression followed the same pattern as histological and computer models have indicated for the human sinoatrial node. In conclusion, hiPSC-CM EBs function as a biological pacemaker in a 2-component cardiac organoid model, which provides the possibility to study electrophysiological and structural coupling mechanisms underlying propagation of pacemaker activity.

Author Correction: Differentiation of cardiomyocytes and generation of human engineered heart tissue.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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

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.