Pluripotent Stem Cell-derived Ventricular Cardiomyocytes Research Articles

Abstract P3140: Extracellular Vesicle MicroRNA Cargo Drives Ventricular Arrhythmia In Heart Failure Patients By Recapitulating Developmental Genes

Heart Failure (HF) is the leading cause of death worldwide, of which arrhythmias account for 50%. The relationship between HF and arrhythmias is closely intertwined. Since novel biomarkers associated with HF that lead to these arrhythmogenic events are an area of unmet need, we aimed to identify the specific plasma Extracellular vesicles (EV)-driven microRNA cargo that could predispose ventricular cardiomyocytes to arrhythmia during HF as well as delineate targetable pathways to rescue the arrhythmic phenotype. We treated human pluripotent stem cell-derived ventricular cardiomyocytes (hPSC-VCM) with plasma-derived EVs from HF and control samples and observed significantly prolonged action potential duration in HF-treated hPSC-VCM (94.41 ± 16.13 msec) using optical voltage mapping. Small RNA cargo of the plasma EVs were sequenced to reveal 26 downregulated and 70 upregulated differentially expressed miRNAs between HF and control. Upon deconvolution analysis, many of these plasma miRNA transcripts were found to be enriched in the brain, RBCs, monocytes and other tissues. We also sequenced the long-RNA transcriptome of the recipient cardiomyocytes. We prioritized 33 targets for qRT-PCR validation based on predicted protein-coding targets of miRNA, and 10 of these mRNAs were different between HF and control EV-treated hPSC-VCMs. Interestingly, some mRNAs (EIF4E, NEDD4, ID3, ID1, CDKN1A, HSPD1) are known to be expressed during development. Pathway enrichment analysis of the validated targets converged in TGF beta signaling and Hippo pathways, implicated in cardiac developmental arrhythmogenic abnormalities. In conclusion, circulating EV small RNA cargo of HF patients can alter the electrophysiological properties of cardiomyocytes, many of which are derived, predominantly from the brain. More importantly, the arrhythmic phenotype is triggered when there is a recapitulation of developmental genes induced by the EV-derived miRNAs. While these miRNAs could be identified as putative biomarkers for SCD, targeting these developmental genes may be a fruitful therapeutic approach to rescue electrical remodeling in HF patients.

Photobiomodulation therapy preconditioning modifies nitric oxide pathway and oxidative stress in human-induced pluripotent stem cell-derived ventricular cardiomyocytes treated with doxorubicin.

Doxorubicin (DOX) is an anthracycline antibiotic that exhibits high heart toxicity. Human-induced pluripotent stem cell-derived ventricular cardiomyocytes (hiPSC-vCMs) are important in vitro models for testing drug cardiotoxicity. Photobiomodulation therapy (PBMT) is a non-invasive therapy that stimulates cells growth and self-repair using light irradiation. This study aimed to investigate the in vitro effects of PBMT preconditioning on cardiotoxicity induced by DOX. HiPSC-vCMs were treated with PBMT for 500s, followed by the addition of 2μM DOX. LED irradiation preconditioning parameters were at 660nm with an irradiance of 10mW/cm2, performing 5J/cm2, followed by 24-h DOX exposure (2μM). Human iPSC-vCMs treated with 2μM DOX or irradiated with PBMT composed the second and third groups, respectively. The control group did neither receive PBMT preconditioning nor DOX and was irradiated with a white standard lamp. Cells from all groups were collected to perform mRNA and miRNA expressions quantification. PBMT, when applied before the DOX challenge, restored the viability of hiPSC-vCMs and reduced ROS levels. Although downregulated by DOX, myocardial UCP2 mRNA expression presented marked upregulation after PBMT preconditioning. Expression of eNOS and UCP2 mRNA and NO production were decreased after DOX exposure, and PBMT preconditioning before the DOX challenge reversed these changes. Moreover, our data indicated that PBMT preconditioning lowered the miR-24 expression. Our data suggested that PBMT preconditioning ameliorated in vitro DOX-induced cardiotoxicity on transcription level, restoring NO levels and reducing oxidative stress.

Engineered Maturation Approaches of Human Pluripotent Stem Cell-Derived Ventricular Cardiomyocytes.

Heart diseases such as myocardial infarction and myocardial ischemia are paroxysmal and fatal in clinical practice. Cardiomyocytes (CMs) differentiated from human pluripotent stem cells provide a promising approach to myocardium regeneration therapy. Identifying the maturity level of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is currently the main challenge for pathophysiology and therapeutics. In this review, we describe current maturity indicators for cardiac microtissue and microdevice cultivation technologies that accelerate cardiac maturation. It may provide insights into regenerative medicine, drug cardiotoxicity testing, and preclinical safety testing.

Correlation between frataxin expression and contractility revealed by in vitro Friedreich\u2019s ataxia cardiac tissue models engineered from human pluripotent stem cells

BackgroundFriedreich’s ataxia (FRDA) is an autosomal recessive disease caused by a non-coding mutation in the first intron of the frataxin (FXN) gene that suppresses its expression. Compensatory hypertrophic cardiomyopathy, dilated cardiomyopathy, and conduction system abnormalities in FRDA lead to cardiomyocyte (CM) death and fibrosis, consequently resulting in heart failure and arrhythmias. Murine models have been developed to study disease pathology in the past two decades; however, differences between human and mouse physiology and metabolism have limited the relevance of animal studies in cardiac disease conditions. To bridge this gap, we aimed to generate species-specific, functional in vitro experimental models of FRDA using 2-dimensional (2D) and 3-dimensional (3D) engineered cardiac tissues from FXN-deficient human pluripotent stem cell-derived ventricular cardiomyocytes (hPSC-hvCMs) and to compare their contractile and electrophysiological properties with healthy tissue constructs.MethodsHealthy control and FRDA patient-specific hPSC-hvCMs were derived by directed differentiation using a small molecule-based protocol reported previously. We engineered the hvCMs into our established human ventricular cardiac tissue strip (hvCTS) and human ventricular cardiac anisotropic sheet (hvCAS) models, and functional assays were performed on days 7–17 post-tissue fabrication to assess the electrophysiology and contractility of FRDA patient-derived and FXN-knockdown engineered tissues, in comparison with healthy controls. To further validate the disease model, forced expression of FXN was induced in FXN-deficient tissues to test if disease phenotypes could be rescued.ResultsHere, we report for the first time the generation of human engineered tissue models of FRDA cardiomyopathy from hPSCs: FXN-deficient hvCTS displayed attenuated developed forces (by 70–80%) compared to healthy controls. High-resolution optical mapping of hvCAS with reduced FXN expression also revealed electrophysiological defects consistent with clinical observations, including action potential duration prolongation and maximum capture frequency reduction. Interestingly, a clear positive correlation between FXN expression and contractility was observed (ρ > 0.9), and restoration of FXN protein levels by lentiviral transduction rescued contractility defects in FXN-deficient hvCTS.ConclusionsWe conclude that human-based in vitro cardiac tissue models of FRDA provide a translational, disease-relevant biomimetic platform for the evaluation of novel therapeutics and to provide insight into FRDA disease progression.

Comprehensive In Vitro Proarrhythmia Assay (CiPA) Update from a Cardiac Safety Research Consortium / Health and Environmental Sciences Institute / FDA Meeting.

A Cardiac Safety Research Consortium / Health and Environmental Sciences Institute / FDA-sponsored Think Tank Meeting was convened in Washington, DC, on May 21, 2018, to bring together scientists, clinicians, and regulators from multiple geographic regions to evaluate progress to date in the Comprehensive In Vitro Proarrhythmia Assay (CiPA) Initiative, a new paradigm to evaluate proarrhythmic risk. Study reports from the 4 different components of the CiPA paradigm (ionic current studies, in silico modeling to generate a Torsade Metric Score, human induced pluripotent stem cell-derived ventricular cardiomyocytes, and clinical ECG assessments including J-Tpeakc) were presented and discussed. This paper provides a high-level summary of the CiPA data presented at the meeting.

Structural and Mechanistic Bases of Nuclear Calcium Signaling in Human Pluripotent Stem Cell-Derived Ventricular Cardiomyocytes.

The loss of nonregenerative, terminally differentiated cardiomyocytes (CMs) due to aging or diseases is generally considered irreversible. Human pluripotent stem cells (hPSCs) can self-renew while maintaining their pluripotency to differentiate into all cell types, including ventricular (V) cardiomyocytes (CMs), to provide a potential unlimited ex vivo source of CMs for heart disease modeling, drug/cardiotoxicity screening, and cell-based therapies. In the human heart, cytosolic Ca2+ signals are well characterized but the contribution of nuclear Ca2+ is essentially unexplored. The present study investigated nuclear Ca2+ signaling in hPSC-VCMs. Calcium transient or sparks in hPSC-VCMs were measured by line scanning using a spinning disc confocal microscope. We observed that nuclear Ca2+, which stems from unitary sparks due to the diffusion of cytosolic Ca2+ that are mediated by RyRs on the nuclear reticulum, is functional. Parvalbumin- (PV-) mediated Ca2+ buffering successfully manipulated Ca2+ transient and stimuli-induced apoptosis in hPSC-VCMs. We also investigated the effect of Ca2+ on gene transcription in hPSC-VCMs, and the involvement of nuclear factor of activated T-cell (NFAT) pathway was identified. The overexpression of Ca2+-sensitive, nuclear localized Ca2+/calmodulin-dependent protein kinase II δ B (CaMKIIδ B) induced cardiac hypertrophy through nuclear Ca2+/CaMKIIδB/HDAC4/MEF2 pathway. These findings provide insights into nuclear Ca2+ signal in hPSC-VCMs, which may lead to novel strategies for maturation as well as improved systems for disease modeling, drug discovery, and cell-based therapies.

Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes

Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes

Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes.

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a homogenous population of cells, the iPSC-CMs generated by current differentiation protocols represent a mixture of cells with ventricular-, atrial-, and nodal-like phenotypes, which complicates phenotypic analyses. Here, a method to optically record action potentials specifically from ventricular-like iPSC-CMs is presented. This is achieved by lentiviral transduction with a construct in which a genetically-encoded voltage indicator is under the control of a ventricular-specific promoter element. When iPSC-CMs are transduced with this construct, the voltage sensor is expressed exclusively in ventricular-like cells, enabling subtype-specific optical membrane potential recordings using time-lapse fluorescence microscopy.

Probing flecainide block of INa using human pluripotent stem cell-derived ventricular cardiomyocytes adapted to automated patch-clamping and 2D monolayers

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are emerging tools for applications such as drug discovery and screening for pro-arrhythmogenicity and cardiotoxicity as leading causes for drug attrition. Understanding the electrophysiology (EP) of hPSC-CMs is essential but conventional manual patch-clamping is highly laborious and low-throughput. Here we adapted hPSC-CMs derived from two human embryonic stem cell (hESC) lines, HES2 and H7, for a 16-channel automated planar-recording approach for single-cell EP characterization. Automated current- and voltage-clamping, with an overall success rate of 55.0 ± 11.3%, indicated that 90% of hPSC-CMs displayed ventricular-like action potential (AP) and the ventricular cardiomyocytes (VCMs) derived from the two hESC lines expressed similar levels of INa, ICaL, Ikr and If and similarly lacked Ito and IK1. These well-characterized hPSC-VCMs could also be readily adapted for automated assays of pro-arrhythmic drug screening. As an example, we showed that flecainide (FLE) induced INa blockade, leftward steady-state inactivation shift, slowed recovery from inactivation in our hPSC-VCMs. Since single-cell EP assay is insufficient to predict drug-induced reentrant arrhythmias, hPSC-VCMs were further reassembled into 2D human ventricular cardiac monolayers (hvCMLs) for multi-cellular electrophysiological assessments. Indeed, FLE significantly slowed the conduction velocity while causing AP prolongation. Our RNA-seq data suggested that cell-cell interaction enhanced the maturity of hPSC-VCMs. Taken collectively, a combinatorial approach using single-cell EP and hvCMLs is needed to comprehensively assess drug-induced arrhythmogenicity.

Engineering human ventricular heart muscles based on a highly efficient system for purification of human pluripotent stem cell-derived ventricular cardiomyocytes

BackgroundMost infarctions occur in the left anterior descending coronary artery and cause myocardium damage of the left ventricle. Although current pluripotent stem cells (PSCs) and directed cardiac differentiation techniques are able to generate fetal-like human cardiomyocytes, isolation of pure ventricular cardiomyocytes has been challenging. For repairing ventricular damage, we aimed to establish a highly efficient purification system to obtain homogeneous ventricular cardiomyocytes and prepare engineered human ventricular heart muscles in a dish.MethodsThe purification system used TALEN-mediated genomic editing techniques to insert the neomycin or EGFP selection marker directly after the myosin light chain 2 (MYL2) locus in human pluripotent stem cells. Purified early ventricular cardiomyocytes were estimated by immunofluorescence, fluorescence-activated cell sorting, quantitative PCR, microelectrode array, and patch clamp. In subsequent experiments, the mixture of mature MYL2-positive ventricular cardiomyocytes and mesenchymal cells were cocultured with decellularized natural heart matrix. Histological and electrophysiology analyses of the formed tissues were performed 2 weeks later.ResultsHuman ventricular cardiomyocytes were efficiently isolated based on the purification system using G418 or flow cytometry selection. When combined with the decellularized natural heart matrix as the scaffold, functional human ventricular heart muscles were prepared in a dish.ConclusionsThese engineered human ventricular muscles can be great tools for regenerative therapy of human ventricular damage as well as drug screening and ventricular-specific disease modeling in the future.

Structural and electrophysiological dysfunctions due to increased endoplasmic reticulum stress in a long-term pacing model using human induced pluripotent stem cell-derived ventricular cardiomyocytes

BackgroundLong-term ventricular pacing has deleterious effects and becomes more significant when cumulative percent ventricular pacing (Cum%VP) exceeds 40% of time. However, cellular disturbances and pathways by which pacing leads to myocardial disorders are not well understood. Attempts to resolve these questions have been hampered by difficulties in obtaining human cardiac tissue and the inability to build a longer-lasting (lasting longer than weeks) pacing model in vitro.MethodsHuman induced pluripotent stem cell-derived ventricular cardiomyocytes (VCMs) were cultured in the presence of electrical stimulation for 2 weeks. Quantitative structural and electrophysiological analyses were used to define the functional disturbances of pacing.ResultsCompared to controls, paced VCMs exhibited a remarkable reduction in the contractile protein expression, an increased apoptosis ratio and electrophysiological remodelling in a Cum%VP-dependent manner. Investigation of the protein expression levels revealed that long-term pacing universally activated both ER stress and downstream calpain. Moreover, the inhibition of calpain attenuated the adverse effects on the structural remodelling and increased the ICa, L in paced VCMs.ConclusionsThe results demonstrated that pacing VCMs for 2 weeks in vitro led to a series of structural and electrophysiological dysfunctions. The increased ER stress and downstream calpain could be a central mechanism underlying the disease pathogenesis. This finding could represent a new therapeutic target in the management of long-term pacing patients.

Increasing the physical size and nucleation status of human pluripotent stem cell-derived ventricular cardiomyocytes by cell fusion

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) provide an unlimited source of donor cells for potential cardiac regenerative therapies. However, hPSC-CMs are immature. For instance, hPSC-CMs are only 1/10 of the physical size of their adult counterparts; the majority are mono- rather than bi- or multi-nucleated, which is an evolutionary adaptive feature in metabolically active cells such as adult CMs. Here, we attempted to increase the physical size and nucleation status of hPSC-derived ventricular (V) cardiomyocytes (hPSC-VCMs) using chemically-induced cell fusion, and examined the subsequent functional effects. Polyethylene glycol (PEG) was employed to fuse a 1:1 mixture of lentiviral vectors LV-MLC2v-GFP- or –tdTomato-labeled hPSC-VCMs, such that hPSC-VCMs fused syncytia (FS) were identified as doubly GFP+/tdTomato+ multi-nucleated cells. These microscopically-identified FS were doubled in size as gauged by their capacitance when compared to the control mononucleated hPSC-VCMs using patch-clamp analysis. Reduced automaticity or action potential (AP) firing rate and moderately prolonged AP duration were observed in FS from day 6 post-fusion induction. However, Ca2+ handling, mitochondrial biogenesis and the extent of apoptosis were not significantly altered. We conclude that larger, multi-nucleated hPSC-VCMs FS can be created by chemically-induced cell fusion but global maturation requires additional triggering cues.

Mechanistic basis of excitation-contraction coupling in human pluripotent stem cell-derived ventricular cardiomyocytes revealed by Ca2+ spark characteristics: Direct evidence of functional Ca2+-induced Ca2+ release

Human embryonic stem cells (hESCs) serve as a potential unlimited ex vivo source of cardiomyocytes for disease modeling, cardiotoxicity screening, drug discovery, and cell-based therapies. Despite the fundamental importance of Ca(2+)-induced Ca(2+) release in excitation-contraction coupling, the mechanistic basis of Ca(2+) handling of hESC-derived ventricular cardiomyocytes (VCMs) remains elusive. To study Ca(2+) sparks as unitary events of Ca(2+) handling for mechanistic insights. To avoid ambiguities owing to the heterogeneous nature, we experimented with hESC-VCMs, purified on the basis of zeocin resistance and signature ventricular action potential after LV-MLC2v-tdTomato-T2A-Zeo transduction. Ca(2+) sparks that were sensitive to inhibitors of sarco/endoplasmic reticulum Ca(2+)-ATPase (thapsigargin and cyclopiazonic acid) and ryanodine receptor (RyR; ryanodine, tetracaine) but not inositol trisphosphate receptors (xestospongin C and 2-aminoethyl diphenylborinate) could be recorded. In a permeabilization model, we further showed that RyRs could be sensitized by Ca(2+). Increasing external Ca(2+) dramatically escalated the basal Ca(2+) and spark frequency. Furthermore, RyR-mediated Ca(2+) release sensitized nearby RyRs, leading to compound Ca(2+) sparks. Depolarization or L-type Ca(2+) channel agonist (FPL 64176 and Bay K8644) pretreatment induced an extracellular Ca(2+)-dependent cytosolic Ca(2+) increase and reduced the sarcoplasmic reticulum content. By contrast, removal of external Na(+) or the addition of the Na(+)-Ca(2+) exchanger inhibitor (KB-R7943 and SN-6) had no effect, suggesting that the Na(+)-Ca(2+) exchanger is not involved in triggering sparks. Inhibition of mitochondrial Ca(2+) uptake by carbonyl cyanide m-chlorophenyl hydrazone promoted Ca(2+) waves. Taken collectively, our findings provide the first lines of direct evidence that hESC-VCMs have functional Ca(2+)-induced Ca(2+) release. However, the sarcoplasmic reticulum is leaky and without a mature terminating mechanism in early development.

Effect of engineered anisotropy on the susceptibility of human pluripotent stem cell-derived ventricular cardiomyocytes to arrhythmias

Human (h) pluripotent stem cells (PSC) such as embryonic stem cells (ESC) can be directed into cardiomyocytes (CMs), representing a potential unlimited cell source for disease modeling, cardiotoxicity screening and myocardial repair. Although the electrophysiology of single hESC-CMs is now better defined, their multi-cellular arrhythmogenicity has not been thoroughly assessed due to the lack of a suitable experimental platform. Indeed, the generation of ventricular (V) fibrillation requires single-cell triggers as well as sustained multi-cellular reentrant events. Although native VCMs are aligned in a highly organized fashion such that electrical conduction is anisotropic for coordinated contractions, hESC-derived CM (hESC-CM) clusters are heterogenous and randomly organized, and therefore not representative of native conditions. Here, we reported that engineered alignment of hESC-VCMs on biomimetic grooves uniquely led to physiologically relevant responses. Aligned but not isotropic control preparations showed distinct longitudinal (L) and transverse (T) conduction velocities (CV), resembling the native human V anisotropic ratio (AR = LCV/TCV = 1.8–2.0). Importantly, the total incidence of spontaneous and inducible arrhythmias significantly reduced from 57% in controls to 17–23% of aligned preparations, thereby providing a physiological baseline for assessing arrhythmogenicity. As such, promotion of pro-arrhythmic effect (e.g., spatial dispersion by β adrenergic stimulation) could be better predicted. Mechanistically, such anisotropy-induced electrical stability was not due to maturation of the cellular properties of hESC-VCMs but their physical arrangement. In conclusion, not only do functional anisotropic hESC-VCMs engineered by multi-scale topography represent a more accurate model for efficacious drug discovery and development as well as arrhythmogenicity screening (of pharmacological and genetic factors), but our approach may also lead to future transplantable prototypes with improved efficacy and safety against arrhythmias.