Generation of cardiomyocytes from human induced pluripotent stem cells resembling atrial cells with ability to respond to adrenoceptor agonists

Abstract

Cardiovascular disease is the leading cause of global mortality and morbidity. Cardiac dysrhythmias contribute significantly to this disease burden. Atrial fibrillation (AF) is the most common chronic dysrhythmia. Human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-AMs) present an exciting new model for AF but currently fail to reach maturity and so are limited in translational potential currently. We report a new approach using a combination of Gremlin 2 and retinoic acid treatment of human iPSCs for generating cardiomyocytes resembling atrial cells. More than 40% of myocytes generated by this approach showed rod-shaped morphology, expression of cardiomyocyte proteins (including RyR2 receptors, a-actinin-2, F-actin) and typically a striated appearance, all of which were broadly similar to the characteristics of adult atrial myocytes. Isolated myocytes were electrically quiescent until stimulated to fire action potentials with an atrial myocyte profile and an amplitude of approximately 100 mV, arising from a resting potential of approximately -70 mV. Single-cell RNA sequence (scRNASeq) analysis showed a high level of expression of several atrial specific transcripts including NPPA, MYL7, HOXA3, SLN, KCNJ4, KCNJ5 and KCNA5. Amplitudes of calcium transients recorded from spontaneously beating cultures were increased by the stimulation of α-adrenoceptors (activated by phenylephrine and blocked by prazosin) or β-adrenoceptors (activated by isoproterenol and blocked by CGP20712A). Thus, our new method provides an efficient approach for differentiating human atrial myocytes with mature characteristics from hiPSCs. This preparation will be very useful for studying signalling pathways in human atrial myocytes, and provides a valuable model for investigating atrial fibrillation and drug discovery.

Cadherin‐5 facilitated the differentiation of human induced pluripotent stem cells into sinoatrial node‐like pacemaker cells by regulating β‐catenin

“…Pioneering studies have successfully recapitulated cardiac development in a dish, resulting in the generation of functional ventricular, atrial, and even specialized conduction system cells including SANPCs (Devalla et al, 2015; Hausburg et al, 2017; Lee et al, 2017; Li et al, 2021; Protze et al, 2017, 2019; Ren et al, 2019; Zhang et al, 2011; Zhao, Shao, et al, 2020). Despite significant strides in differentiating human pluripotent stem cells into ventricular or atrial myocytes (Ahmad et al, 2023; Cyganek et al, 2018; Devalla et al, 2015; Lee et al, 2017; Zhang et al, 2011), the directed differentiation toward SANPCs has relatively lagged behind. The distinct molecular and functional characteristics of cardiac pacemaker cells, which confer their cardiac pacemaking properties, present unique challenges in recreating these cells in vitro (Barbuti & Robinson, 2015; Christoffels et al, 2010; Kapoor et al, 2013; Liang et al, 2017).…”

hiPSC-derived cardiomyocytes as a model to study the role of small-conductance Ca2+-activated K+ (SK) ion channel variants associated with atrial fibrillation

“…In hiPSC-aCMs derived from RA-guided differentiation, I Kur is relatively smaller than adult atrial cells but was sufficient to produce a shorter APD compared to hiPSC-vCMs (Hilderink et al, 2020). Otherwise, I Ca,L , I Kr , and I Ks current densities and the expression of their encoding channels are considered comparable to adult cardiomyocytes (Itzhaki et al, 2011;Ma et al, 2011;Lahti et al, 2012), and I KAch was recently shown to be comparable in hiPSC-aCMs (Ahmad et al, 2023).…”

Cardiomyocyte electrophysiology and its modulation: current views and future prospects

“…They showed low resting membrane potentials [33], low/ absent I K1 [34], low membrane capacitances [35], immature AP profiles and slow electric impulse propagation velocities [32], and their generation primarily focused on ventricular rather than atrial phenotypes. However, Ahmad et al [36] describe hiPSC-CMs with AP properties and acetylcholine (ACh)-activated I K expression characteristic of atrial cells. iPSCs have also been explored as possible models for normal and disease-related changes in ion channel expression, Ca 2+ homeostatic phenotypes, neurocardiac interactions and cardiac hypertrophic change (see: Chen et al [37]; Zhou et al [38]; Li et al [39] and Langa et al [40], respectively).…”

“…Closer characterization of such signalling pathways in iPSC cells is a relatively new area of study. Ahmad et al [36] describe differentiated human iPSCs resembling an atrial phenotype, with the expected electrophysiological and Ca 2+ signalling properties, and specific transcripts, responsive to adrenergic stimulation, therefore permitting studies of such effects.…”

“…However, Ahmad et al . [36] describe hiPSC-CMs with AP properties and acetylcholine (ACh)-activated I K expression characteristic of atrial cells. iPSCs have also been explored as possible models for normal and disease-related changes in ion channel expression, Ca 2+ homeostatic phenotypes, neurocardiac interactions and cardiac hypertrophic change (see: Chen et al .…”