Induced Pluripotent Stem Cell-derived Cardiomyocytes Research Articles

The hERG PAS domain: A major regulator of IKr and a promising anti-arrhythmic drug target.

The hERG1 potassium channel conducts the critical cardiac repolarizing current IKr. Given its role in mediating electrical excitability, several groups have investigated the antiarrhythmic capacity of hERG1 activators in cardiac diseases marked by prolonged action potential duration (APD), such as long QT Syndrome. hERG1 activators have had limited success due to their off-target effects. We recently generated a single chain variable fragment (scFv) antibody, scFv2.10, that selectively binds the hERG1 Per-Arnt-Sim (PAS) domain. The PAS domain binds to the C-terminal cyclic nucleotide binding homology domain (CNBHD) and slows channel activation and promotes inactivation. scFv2.10 disrupts the PAS-CNBHD interaction, which in turn causes a two-fold increase in the time course of hERG1 activation and inactivation recovery, thereby increasing hERG1 current in HEK293 cells. In human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), disrupting the PAS-CNBHD interaction with scFv2.10 increases IKr and shortens APD. We hypothesized that the hERG1 PAS domain could represent a novel therapeutic target for diseases of impaired cardiac repolarization. To test this, we measured the antiarrhythmic capacity of scFv2.10 on a novel hiPSC-CM line derived from a patient with Jervell and Lange Nielsen syndrome (JLN). JLN syndrome is a severe form of long QT Syndrome Type 1 characterized by increased APD and incidence of sudden cardiac death. Compared to hiPSC-CMs derived from a healthy genetic background, JLN hiPSC-CMs display hallmarks of pro-arrhythmia including prolonged APD, increased action potential variability, and early afterdepolarizations. scFv2.10 reduced APD, action potential variability, and the incidence of early afterdepolarizations in JLN hiPSC-CMs, compared to GFP controls. These data demonstrate that the hERG1 PAS domain is a potential therapeutic target to treat disorders of electrical excitability.

SIRT3 promotes metabolic maturation of human iPSC-derived cardiomyocytes via OPA1-controlled mitochondrial dynamics.

The metabolic patterns and energetics of human induced pluripotent stem cell-derived cardiomyocytes (HiPSC-CMs) are much less than those of normal adult cardiomyocytes, which has limited their application in disease therapy and regenerative medicine. It has been demonstrated that SIRT3, a mitochondria-target deacetylase, controls mitochondrial metabolism in physiological and pathological conditions. In this research, We investigated the role and regulatory mechanism of SIRT3 in energy metabolism in HiPSC-CMs. We found that the expression of SIRT3 was increased during the differentiation and maturation of HiPSC-CMs. Knocking down SIRT3 impaired mitochondrial structure, mitochondrial respiration capacity, and fatty acid oxidation but enhanced glycolysis. However, honokiol, a pharmacological activator of SIRT3, improved the mitochondrial ultrastructure and energetics, and promoted oxidative phosphorylation in HiPSC-CMs. Furthermore, SIRT3 regulated the acetylation of OPA1, and the knockdown of OPA1 blocked the promotion of energy metabolism by honokiol, meanwhile, knocking down OPA1 impaired mitochondrial fusion, mitochondrial respiration capacity, and fatty acid oxidation which were reversed by M1 (a mitochondrial fusion promoter) in HiPSC-CMs. In summary, SIRT3 regulated energetics and promoted metabolism remodeling by targeting the OPA1-controlled mitochondrial dynamics in HiPSC-CMs, and targeting SIRT3 may have revelatory implications in the treatment of cardiovascular diseases and the application of HiPSC-CMs to regenerative medicine.

Leveraging optogenetics for CRISPRi screens in human iPSC-cardiomyocytes.

New gene modulation methods, e.g., interference or activation CRISPR (CRISPRi/a), are deployable in a highly parallel format and allow selective perturbation of genes of interest in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). When combined with high-throughput (HT) methods for quantitative assessment of cellular responses, they provide new means for functional genomics in human heart cells and engineered tissues for translational studies. Here we demonstrate how all-optical electrophysiology, in part fueled by optogenetics for actuation and/or sensing of functional responses, can be used in conjunction with CRISPRi modulation to yield a HT platform. Our recent studies explore the possibility to use CRISPRi in post-differentiated hiPSC-CMs to selectively perturb key genes underlying cardiac electrophysiology. We validated the use of our all-optical technology for HT analysis as part of a comprehensive characterization pipeline to link electromechanical functional outputs to molecular correlates, identified by CRISPRi perturbation in hiPSC-CMs. The approach entails the short-term genetic engineering of already differentiated cells and the validation of a pipeline for mRNA or protein analysis post-functional assessment, along with correlative and dimension-reduction studies. Our results demonstrate proof of principle utility of optical methods as partner technology to CRISPRi perturbations that yield mild but specific functional changes relevant to cardiac electromechanics and remodeling during disease. This technology can be useful for multiparametric assessment of cardiotoxicity and drug testing in human cells.

An amphiphilic plasma membrane-targeted photo-transducer mediates optical pacing in cardiomyocytes derived from human induced pluripotent stem cells.

The use of light to control the activity of different cell-types has recently come at the forefront of the scientific community thanks to a series of key-enabling characteristics (i.e. spatial/temporal selectivity, and lower invasiveness). Notably, non-genetic photostimulation is a novel and rapidly growing multidisciplinary field of research that aims to induce light sensitivity in living systems by exploiting exogeneous phototransducers. Here we propose a recently synthetized actuator, based on an azobenzene derivative, for cardiac applications. The light-mediated photostimulation process has been studied by applying several characterization techniques to detect the effect on the cellular properties of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). In particular, we recorded changes in membrane capacitance, in membrane potential (Vm), intracellular Ca2+ dynamics and cell contractility. Light stimulation of the photo-transducers causes a transient Vm hyperpolarization followed by a delayed depolarization and action potential (AP) generation. The observed increase in AP frequency nicely correlates with changes in Ca2+ dynamics and contraction rate. Altogether, these preliminrary evidences provide the proof of concept that our amphiphilic plasma membrane-targeted photo-transducer is a viable tool to modulate electrical activity and contractility in hiPSC-CMs, opening up a broader field of applications in cardiac electrophysiology.

Functional characterization and identification of a therapeutic for a novel SCN5A-F1760C variant causing type 3 long QT syndrome refractory to all guideline-directed therapies

Pathogenic variants in the SCN5A-encoded Nav1.5 sodium channel cause type 3 long QT syndrome (LQT3). We present the case of an infant with severe LQT3 who was refractory to multiple pharmacologic therapies as well as bilateral stellate ganglionectomy. The patient's novel variant, p.F1760C-SCN5A, involves a critical residue of the Nav1.5's local anesthetic binding domain. The purpose of this study was to characterize functionally the p.F1760C-SCN5A variant using TSA-201 and patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Whole-cell patch clamp was used to assess p.F1760C-SCN5A associated sodium currents with/without lidocaine (Lido), flecainide, and phenytoin (PHT) in TSA-201 cells. p.F1760C-SCN5A and CRISPR-Cas9 variant-corrected isogenic control (IC) iPSC-CMs were generated. FluoVolt voltage dye was used to measure the action potential duration (APD) with/without mexiletine or PHT. V1/2 of inactivation was right-shifted significantly in F1760C cells (-72.2 ± 0.7 mV) compared to wild-type (WT) cells (-86.3 ± 0.9 mV; P <.0001) resulting in a marked increase in windowcurrent. F1760C increased sodium late current 2-fold from 0.18% ± 0.04% of peak in WT to 0.49% ± 0.07% of peak in F1760C (P = .0005). Baseline APD to 90% repolarization (APD90) was increased markedly in F1760C iPSC-CMs (601 ± 4 ms) compared to IC iPSC-CMs (423 ± 15 ms; P <.0001). However, 4-hour treatment with 10 μM mexiletine failed to shorten APD90, and treatment with 5μM PHT significantly decreased APD90 of F1760C iPSC-CMs (453 ± 6 ms; P <.0001). PHT rescued electrophysiological phenotype and APD of a novel p.F1760C-SCN5A variant. The antiepileptic drug PHT may be an effective alternative therapeutic for the treatment of LQT3, especially for variants that disrupt the Lido/mexiletine binding site.

PTX3 from vascular endothelial cells contributes to trastuzumab-induced cardiac complications.

Trastuzumab, the first humanized monoclonal antibody that targets human epidermal growth factor receptor 2 (ERBB2/HER2), is currently used as a first-line treatment for HER2 (+) tumours. However, trastuzumab increases the risk of cardiac complications without affecting myocardial structure, suggesting a distinct mechanism of cardiotoxicity. We used medium from trastuzumab-treated human umbilical vein endothelial cells (HUVECs) to treat CCC-HEH-2 cells, the human embryonic cardiac tissue-derived cell lines, and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to assess the crosstalk between vascular endothelial cells (VECs) and cardiomyocytes. Protein mass spectrometry analysis was used to identify the key factors from VECs that regulate the function of cardiomyocytes. We applied RNA-sequencing to clarify the mechanism, by which PTX3 causes cardiac dysfunction. We used an anti-human/rat HER2 (neu) monoclonal antibody to generate a rat model that was used to evaluate the effects of trastuzumab on cardiac structure and function and the rescue effects of lapatinib on trastuzumab-induced cardiac side effects. Medium from trastuzumab-treated HUVECs apparently impaired the contractility of CCC-HEH-2 cells and iPSC-CMs. PTX3 from VECs caused defective cardiomyocyte contractility and cardiac dysfunction in mice, phenocopying trastuzumab treatment. PTX3 affected calcium homoeostasis in cardiomyocytes, which led to defective contractile properties. EGFR/STAT3 signalling in VECs contributed to the increased expression and release of PTX3. Notably, lapatinib, a dual inhibitor of EGFR/HER2, could rescue the cardiac complications caused by trastuzumab by blocking the release of PTX3. We identified a distinct mode of cardiotoxicity, wherein the activation of EGFR/STAT3 signalling by trastuzumab in VECs promotes PTX3 excretion, which contributes to the impaired contractility of cardiomyocytes by inhibiting cellular calcium signalling. We confirmed that lapatinib could be a feasible preventive agent against trastuzumab-induced cardiac complications and provided the rationale for the combined application of lapatinib and trastuzumab in cancer therapy.

Harshening stem cell research and precision medicine: The states of human pluripotent cells stem cell repository diversity, and racial and sex differences in transcriptomes.

In vitro investigation on human development, disease modeling, and drug discovery has been empowered by human induced pluripotent stem cell (hiPSC) technologies that form the foundation of precision medicine. Race and sex genetic backgrounds have become a major focus of many diseases modeling and drug response evaluation in the pharmaceutical industry. Here, we gathered data from major stem cell repositories to analyze the diversity with respect to ethnicity, sex, and disease types; and we also analyzed public datasets to unravel transcriptomics differences between samples of different ethnicities and sexes. We found a lack of diversity despite the large sample size of human induced pluripotent stem cells. In the ethnic comparison, the White group made up the majority of the banked hiPSCs. Similarly, for the organ/disease type and sex comparisons, the neural and male hiPSCs accounted for the majority of currently available hiPSCs. Bulk RNA-seq and single-cell transcriptomic analysis coupled with Machine Learning and Network Analysis revealed panels of gene features differently expressed in healthy hiPSCs and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) of different races and sexes. The data highlights the current ethnic and sex inequality in stem cell research and demonstrates the molecular biological diversity of hiPSCs and cardiomyocytes from different races and genders. We postulate that future efforts in stem cell biology, regenerative and precision medicine should be guided towards an inclusive, diverse repository reflecting the prevalence of diseases across racial and ethnic groups and the sexes, important for both common and rare disease modeling, drug screening, and cell therapeutics.

Cardiac Piezo1 Exacerbates Lethal Ventricular Arrhythmogenesis by Linking Mechanical Stress with Ca2+ Handling After Myocardial Infarction.

Ventricular arrhythmogenesis is a key cause of sudden cardiac death following myocardial infarction (MI). Accumulating data show that ischemia, sympathetic activation, and inflammation contribute to arrhythmogenesis. However, the role and mechanisms of abnormal mechanical stress in ventricular arrhythmia following MI remain undefined. We aimed to examine the impact of increased mechanical stress and identify the role of the key sensor Piezo1 in ventricular arrhythmogenesis in MI. Concomitant with increased ventricular pressure, Piezo1, as a newly recognized mechano-sensitive cation channel, was the most up-regulated mechanosensor in the myocardium of patients with advanced heart failure. Piezo1 was mainly located at the intercalated discs and T-tubules of cardiomyocytes, which are responsible for intracellular calcium homeostasis and intercellular communication. Cardiomyocyte-conditional Piezo1 knockout mice (Piezo1Cko) exhibited preserved cardiac function after MI. Piezo1Cko mice also displayed a dramatically decreased mortality in response to the programmed electrical stimulation after MI with a markedly reduced incidence of ventricular tachycardia. In contrast, activation of Piezo1 in mouse myocardium increased the electrical instability as indicated by prolonged QT interval and sagging ST segment. Mechanistically, Piezo1 impaired intracellular calcium cycling dynamics by mediating the intracellular Ca2+ overload and increasing the activation of Ca2+-modulated signaling, CaMKII, and calpain, which led to the enhancement of phosphorylation of RyR2 and further increment of Ca2+ leaking, finally provoking cardiac arrhythmias. Furthermore, in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), Piezo1 activation remarkably triggered cellular arrhythmogenic remodeling by significantly shortening the duration of the action potential, inducing early afterdepolarization, and enhancing triggered activity.This study uncovered a proarrhythmic role of Piezo1 during cardiac remodeling, which is achieved by regulating Ca2+ handling, implying a promising therapeutic target in sudden cardiac death and heart failure.

Titin-truncating variants in hiPSC cardiomyocytes induce pathogenic proteinopathy and sarcomere defects with preserved core contractile machinery.

Titin-truncating variants (TTNtv) are the single largest genetic cause of dilated cardiomyopathy (DCM). In this study we modeled disease phenotypes of A-band TTNtv-induced DCM in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) using genome editing and tissue engineering technologies. Transcriptomic, cellular, and micro-tissue studies revealed that A-band TTNtv hiPSC-CMs exhibit pathogenic proteinopathy, sarcomere defects, aberrant Na+ channel activities, and contractile dysfunction. These phenotypes establish a dual mechanism of poison peptide effect and haploinsufficiency that collectively contribute to DCM pathogenesis. However, TTNtv cellular defects did not interfere with the function of the core contractile machinery, the actin-myosin-troponin-Ca2+ complex, and preserved the therapeutic mechanism of sarcomere modulators. Treatment of TTNtv cardiac micro-tissues with investigational sarcomere modulators augmented contractility and resulted in sustained transcriptomic changes that promote reversal of DCM disease signatures. Together, our findings elucidate the underlying pathogenic mechanisms of A-band TTNtv-induced DCM and demonstrate the validity of sarcomere modulators as potential therapeutics.

Assessing proarrhythmic potential of environmental chemicals using a high throughput in vitro-in silico model with human induced pluripotent stem cell-derived cardiomyocytes_suppl1

Assessing proarrhythmic potential of environmental chemicals using a high throughput in vitro-in silico model with human induced pluripotent stem cell-derived cardiomyocytes_suppl1

Assessing proarrhythmic potential of environmental chemicals using a high throughput in vitro-in silico model with human induced pluripotent stem cell-derived cardiomyocytes_suppl2

Assessing proarrhythmic potential of environmental chemicals using a high throughput in vitro-in silico model with human induced pluripotent stem cell-derived cardiomyocytes_suppl2

Assessing proarrhythmic potential of environmental chemicals using a high throughput in vitro-in silico model with human induced pluripotent stem cell-derived cardiomyocytes.

QT prolongation and the potentially fatal arrhythmia Torsades de Pointes are common causes for withdrawing or restricting drugs; however, little is known about similar liabilities of environmental chemicals. Current in vitro-in silico models for testing proarrhythmic liabilities, using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM), provide an opportunity to address this data gap. These methods are still low- to medium-throughput and not suitable for testing the tens of thousands of chemicals in commerce. We hypothesized that combining high-throughput population- based in vitro testing in hiPSC-CMs with a fully in silico data analysis workflow can offer sensitive and specific predictions of proarrhythmic potential. We calibrated the model with a published hiPSC-CM dataset of drugs known to be positive or negative for proarrhythmia and tested its performance using internal cross-validation and external validation. Additionally, we used computational down-sampling to examine three study designs for hiPSC-CM data: one replicate of one donor, five replicates of one donor, and one replicate of a population of five donors. We found that the population of five donors had the best performance for predicting proarrhythmic potential. The resulting model was then applied to predict the proarrhythmic potential of environmental chemicals, additionally characterizing risk through margin of exposure (MOE) calculations. Out of over 900 environmental chemicals tested, over 150 were predicted to have proarrhythmic potential, but only seven chemicals had a MOE < 1. We conclude that a high-throughput in vitro-in silico approach using population-based hiPSC-CM testing provides a reasonable strategy to screen environmental chemicals for proarrhythmic potential.

Small molecule-mediated rapid maturation of human induced pluripotent stem cell-derived cardiomyocytes

BackgroundHuman induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) do not display all hallmarks of mature primary cardiomyocytes, especially the ability to use fatty acids (FA) as an energy source, containing high mitochondrial mass, presenting binucleation and increased DNA content per nuclei (polyploidism), and synchronized electrical conduction. This immaturity represents a bottleneck to their application in (1) disease modelling—as most cardiac (genetic) diseases have a middle-age onset—and (2) clinically relevant models, where integration and functional coupling are key. So far, several methods have been reported to enhance iPSC-CM maturation; however, these protocols are laborious, costly, and not easily scalable. Therefore, we developed a simple, low-cost, and rapid protocol to promote cardiomyocyte maturation using two small molecule activators of the peroxisome proliferator-activated receptor β/δ and gamma coactivator 1-alpha (PPAR/PGC-1α) pathway: asiatic acid (AA) and GW501516 (GW).Methods and ResultsMonolayers of iPSC-CMs were incubated with AA or GW every other day for ten days resulting in increased expression of FA metabolism-related genes and markers for mitochondrial activity. AA-treated iPSC-CMs responsiveness to the mitochondrial respiratory chain inhibitors increased and exhibited higher flexibility in substrate utilization. Additionally, structural maturity improved after treatment as demonstrated by an increase in mRNA expression of sarcomeric-related genes and higher nuclear polyploidy in AA-treated samples. Furthermore, treatment led to increased ion channel gene expression and protein levels.ConclusionsCollectively, we developed a fast, easy, and economical method to induce iPSC-CMs maturation via PPAR/PGC-1α activation. Treatment with AA or GW led to increased metabolic, structural, functional, and electrophysiological maturation, evaluated using a multiparametric quality assessment.

Prediction of inotropic effect based on calcium transients in human iPSC-derived cardiomyocytes and machine learning

Functional changes to cardiomyocytes are undesirable during drug discovery and identifying the inotropic effects of compounds is hence necessary to decrease the risk of cardiovascular adverse effects in the clinic. Recently, approaches leveraging calcium transients in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been developed to detect contractility changes, induced by a variety of mechanisms early during drug discovery projects. Although these approaches have been able to provide some predictive ability, we hypothesised that using additional waveform parameters could offer improved insights, as well as predictivity. In this study, we derived 25 parameters from each calcium transient waveform and developed a modified Random Forest method to predict the inotropic effects of the compounds. In total annotated data for 48 compounds were available for modelling, out of which 31 were inotropes. The results show that the Random Forest model with a modified purity criterion performed slightly better than an unmodified algorithm in terms of the Area Under the Curve, giving values of 0.84 vs 0.81 in a cross-validation, and outperformed the ToxCast Pipeline model, for which the highest value was 0.76 when using the best-performing parameter, PW10. Our study hence demonstrates that more advanced parameters derived from waveforms, in combination with additional machine learning methods, provide improved predictivity of cardiovascular risk associated with inotropic effects.

Metabolic perturbations in hypertrophic cardiomyopathy – Insights from human induced pluripotent stem cell-derived cardiomyocytes

Hypertrophic cardiomyopathy (HCM) is one of the most commonly inherited cardiac disorders, and is defined as left ventricular hypertrophy without chamber dilation in the absence of an identifiable cause. It is characterised by cardiomyocyte hypertrophy and disarray, interstitial fibrosis, impaired left ventricular relaxation, and diastolic dysfunction, which can manifest in exercise intolerance, angina, dyspnoea, dizziness, syncope, or sudden cardiac death.

Investigating the utilisation of human induced pluripotent stem cell-derived cardiomyocytes to study metabolic alterations in the heart of Duchenne muscular dystrophy patients

Investigating the utilisation of human induced pluripotent stem cell-derived cardiomyocytes to study metabolic alterations in the heart of Duchenne muscular dystrophy patients

Investigating the molecular disease mechanisms of the human p.G592R PRKD1 mutation in human induced pluripotent stem cell-derived cardiomyocytes

Investigating the molecular disease mechanisms of the human p.G592R PRKD1 mutation in human induced pluripotent stem cell-derived cardiomyocytes

Transcriptomics reveal stretched human pluripotent stem cell-derived cardiomyocytes as an advantageous hypertrophy model

Left ventricular hypertrophy, characterized by hypertrophy of individual cardiomyocytes, is an adaptive response to an increased cardiac workload that eventually leads to heart failure. Previous studies using neonatal rat ventricular myocytes (NRVMs) and animal models have revealed several genes and signaling pathways associated with hypertrophy and mechanical load. However, these models are not directly applicable to humans. Here, we studied the effect of cyclic mechanical stretch on gene expression of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) using RNA sequencing. hiPSC-CMs showed distinct hypertrophic changes in gene expression at the level of individual genes and in biological processes. We also identified several differentially expressed genes that have not been previously associated with cardiomyocyte hypertrophy and thus serve as attractive targets for future studies. When compared to previously published data attained from stretched NRVMs and human embryonic stem cell-derived cardiomyocytes, hiPSC-CMs displayed a smaller number of changes in gene expression, but the differentially expressed genes revealed more pronounced enrichment of hypertrophy-related biological processes and pathways. Overall, these results establish hiPSC-CMs as a valuable in vitro model for studying human cardiomyocyte hypertrophy. • Mechanical load-associated genes in human cardiomyocytes are not fully understood. • Changes in response to mechanical stretch in hiPSC-CMs were investigated by RNAseq. • hiPSC-CMs showed distinct gene expression changes compared to NRVMs or hESC-CMs. • Differentially expressed genes were restricted to hypertrophy-related processes. • GAL, LINC00648 and SLC16A9 identified as new cardiac stretch-responsive genes.

Generation of human induced pluripotent stem cell line encoding for a genetically encoded voltage indicator Arclight A242

Genetically encoded voltage indicators (GEVIs) allow for monitoring membrane potential changes in neurons and cardiomyocytes (CMs) as an alternative to patch-clamp techniques. GEVIs facilitate non-invasive, high throughput screening of electrophysiological properties of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). A dual transgenic hiPSC line with Arclight A242 (GEVI) and an antibiotic resistance cardiac selection cassette was successfully generated from an earlier established hiPSC line MHHi001-A. After cardiac differentiation and selection, purified populations of CMs with constitutive GEVI expression can be utilized for studying cardiac development, disease modeling, and drug testing.

Acetylcholine Reduces L-Type Calcium Current without Major Changes in Repolarization of Canine and Human Purkinje and Ventricular Tissue.

Vagal nerve stimulation (VNS) holds a strong basis as a potentially effective treatment modality for chronic heart failure, which explains why a multicenter VNS study in heart failure with reduced ejection fraction is ongoing. However, more detailed information is required on the effect of acetylcholine (ACh) on repolarization in Purkinje and ventricular cardiac preparations to identify the advantages, risks, and underlying cellular mechanisms of VNS. Here, we studied the effect of ACh on the action potential (AP) of canine Purkinje fibers (PFs) and several human ventricular preparations. In addition, we characterized the effects of ACh on the L-type Ca2+ current (ICaL) and AP of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and performed computer simulations to explain the observed effects. Using microelectrode recordings, we found a small but significant AP prolongation in canine PFs. In the human myocardium, ACh slightly prolonged the AP in the midmyocardium but resulted in minor AP shortening in subepicardial tissue. Perforated patch-clamp experiments on hiPSC-CMs demonstrated that 5 µM ACh caused an ≈15% decrease in ICaL density without changes in gating properties. Using dynamic clamp, we found that under blocked K+ currents, 5 µM ACh resulted in an ≈23% decrease in AP duration at 90% of repolarization in hiPSC-CMs. Computer simulations using the O'Hara-Rudy human ventricular cell model revealed that the overall effect of ACh on AP duration is a tight interplay between the ACh-induced reduction in ICaL and ACh-induced changes in K+ currents. In conclusion, ACh results in minor changes in AP repolarization and duration of canine PFs and human ventricular myocardium due to the concomitant inhibition of inward ICaL and outward K+ currents, which limits changes in net repolarizing current and thus prevents major changes in AP repolarization.