Drugs targeting atrial-specific ion channels, Kv1.5 or Kir3.1/3.4, are being developed as new therapeutic strategies for atrial fibrillation. However, current preclinical studies carried out in non-cardiac cell lines or animal models may not accurately represent the physiology of a human cardiomyocyte (CM). In the current study, we tested whether human embryonic stem cell (hESC)-derived atrial CMs could predict atrial selectivity of pharmacological compounds. By modulating retinoic acid signaling during hESC differentiation, we generated atrial-like (hESC-atrial) and ventricular-like (hESC-ventricular) CMs. We found the expression of atrial-specific ion channel genes, KCNA5 (encoding Kv1.5) and KCNJ3 (encoding Kir 3.1), in hESC-atrial CMs and further demonstrated that these ion channel genes are regulated by COUP-TF transcription factors. Moreover, in response to multiple ion channel blocker, vernakalant, and Kv1.5 blocker, XEN-D0101, hESC-atrial but not hESC-ventricular CMs showed action potential (AP) prolongation due to a reduction in early repolarization. In hESC-atrial CMs, XEN-R0703, a novel Kir3.1/3.4 blocker restored the AP shortening caused by CCh. Neither CCh nor XEN-R0703 had an effect on hESC-ventricular CMs. In summary, we demonstrate that hESC-atrial CMs are a robust model for pre-clinical testing to assess atrial selectivity of novel antiarrhythmic drugs.

SummaryMaximizing baseline function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is essential for their effective application in models of cardiac toxicity and disease. Here, we aimed to identify factors that would promote an adequate level of function to permit robust single-cell contractility measurements in a human induced pluripotent stem cell (hiPSC) model of hypertrophic cardiomyopathy (HCM). A simple screen revealed the collaborative effects of thyroid hormone, IGF-1 and the glucocorticoid analog dexamethasone on the electrophysiology, bioenergetics, and contractile force generation of hPSC-CMs. In this optimized condition, hiPSC-CMs with mutations in MYBPC3, a gene encoding myosin-binding protein C, which, when mutated, causes HCM, showed significantly lower contractile force generation than controls. This was recapitulated by direct knockdown of MYBPC3 in control hPSC-CMs, supporting a mechanism of haploinsufficiency. Modeling this disease in vitro using human cells is an important step toward identifying therapeutic interventions for HCM.

The inability of multipotent cardiovascular progenitor cells (CPCs) to undergo multiple divisions in culture has precluded stable expansion of precursors of cardiomyocytes and vascular cells. This contrasts with neural progenitors, which can be expanded robustly and are a renewable source of their derivatives. Here we use human pluripotent stem cells bearing a cardiac lineage reporter to show that regulated MYC expression enables robust expansion of CPCs with insulin-like growth factor-1 (IGF-1) and a hedgehog pathway agonist. The CPCs can be patterned with morphogens, recreating features of heart field assignment, and controllably differentiated to relatively pure populations of pacemaker-like or ventricular-like cardiomyocytes. The cells are clonogenic and can be expanded for >40 population doublings while retaining the ability to differentiate into cardiomyocytes and vascular cells. Access to CPCs will allow precise recreation of elements of heart development in vitro and facilitate investigation of the molecular basis of cardiac fate determination. This technology is applicable for cardiac disease modeling, toxicology studies and tissue engineering.

SummaryDifferentiated derivatives of human pluripotent stem cells in culture are generally phenotypically immature compared to their adult counterparts. Their identity is often difficult to determine with certainty because little is known about their human fetal equivalents in vivo. Cellular identity and signaling pathways directing differentiation are usually determined by extrapolating information from either human adult tissue or model organisms, assuming conservation with humans. To resolve this, we generated a collection of human fetal transcriptional profiles at different developmental stages. Moreover, we developed an algorithm, KeyGenes, which uses this dataset to quantify the extent to which next-generation sequencing or microarray data resemble specific cell or tissue types in the human fetus. Using KeyGenes combined with the human fetal atlas, we identified multiple cell and tissue samples unambiguously on a limited set of features. We thus provide a flexible and expandable platform to monitor and evaluate the efficiency of differentiation in vitro.

Genetic causes of many familial arrhythmia syndromes remain elusive. In this study, whole‐exome sequencing (WES) was carried out on patients from three different families that presented with life‐threatening arrhythmias and high risk of sudden cardiac death (SCD). Two French Canadian probands carried identical homozygous rare variant in TECRL gene (p.Arg196Gln), which encodes the trans‐2,3‐enoyl‐CoA reductase‐like protein. Both patients had cardiac arrest, stress‐induced atrial and ventricular tachycardia, and QT prolongation on adrenergic stimulation. A third patient from a consanguineous Sudanese family diagnosed with catecholaminergic polymorphic ventricular tachycardia (CPVT) had a homozygous splice site mutation (c.331+1G>A) in TECRL. Analysis of intracellular calcium ([Ca2+]i) dynamics in human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) generated from this individual (TECRLH om‐hiPSCs), his heterozygous but clinically asymptomatic father (TECRLH et‐hiPSCs), and a healthy individual (CTRL‐hiPSCs) from the same Sudanese family, revealed smaller [Ca2+]i transient amplitudes as well as elevated diastolic [Ca2+]i in TECRLH om‐hiPSC‐CMs compared with CTRL‐hiPSC‐CMs. The [Ca2+]i transient also rose markedly slower and contained lower sarcoplasmic reticulum (SR) calcium stores, evidenced by the decreased magnitude of caffeine‐induced [Ca2+]i transients. In addition, the decay phase of the [Ca2+]i transient was slower in TECRLH om‐hiPSC‐CMs due to decreased SERCA and NCX activities. Furthermore, TECRLH om‐hiPSC‐CMs showed prolonged action potentials (APs) compared with CTRL‐hiPSC‐CMs. TECRL knockdown in control human embryonic stem cell‐derived CMs (hESC‐CMs) also resulted in significantly longer APs. Moreover, stimulation by noradrenaline (NA) significantly increased the propensity for triggered activity based on delayed afterdepolarizations (DADs) in TECRLH om‐hiPSC‐CMs and treatment with flecainide, a class Ic antiarrhythmic drug, significantly reduced the triggered activity in these cells. In summary, we report that mutations in TECRL are associated with inherited arrhythmias characterized by clinical features of both LQTS and CPVT. Patient‐specific hiPSC‐CMs recapitulated salient features of the clinical phenotype and provide a platform for drug screening evidenced by initial identification of flecainide as a potential therapeutic. These findings have implications for diagnosis and treatment of inherited cardiac arrhythmias.