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Stem cells represent an attractive resource for cardiac regeneration. However, the survival and function of transplanted stem cells is poor and remains a major challenge for the development of effective therapies. As two main cell types currently under investigation in heart repair, mesenchymal stromal cells (MSCs) indirectly support endogenous regenerative capacities after transplantation, while induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) functionally integrate into the damaged myocardium and directly contribute to the restoration of its pump function. These two cell types are exposed to a common microenvironment with many stressors in ischemic heart tissue. This review summarizes the research progress on the mechanisms and challenges of MSCs and iPSC-CMs in post-MI heart repair, introduces several randomized clinical trials with 3D-mapping-guided cell therapy, and outlines recent findings related to the factors that affect the survival and function of stem cells. We also discuss the future directions for optimization such as biomaterial utilization, cell combinations, and intravenous injection of engineered nucleus-free MSCs. • IPSC-CMs and MSCs have emerged as the most promising options for ischemic myocardial repair. • The survival and function of transplanted stem cells is poor and remains a major challenge for the development of effective therapies. • Three stress conditions affect the survival and function of stem cells in the ischemic heart: low oxygen/nutrients, metabolic disorders, and inflammation. • Future studies may focus on the selection of patients, the optimization of stem cells, the combination of cells with biomaterials, and repeated intravenous delivery. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H2S) belongs to the family of gasotransmitters and can modulate a myriad of biological signaling pathways. Among others, its cardioprotective effects, through antioxidant, anti-inflammatory, anti-fibrotic, and proangiogenic activities, are well-documented in experimental studies. Cardiorespiratory failure, predominantly cardiomyopathy, is a life-threatening complication that is the number one cause of death in patients with Duchenne muscular dystrophy (DMD). Although recent data suggest the role of H2S in ameliorating muscle wasting in murine and Caenorhabditis elegans models of DMD, possible cardioprotective effects have not yet been addressed. In this review, we summarize the current understanding of the role of H2S in animal models of cardiac dysfunctions and cardiac cells. We highlight that DMD may be amenable to H2S supplementation, and we suggest H2S as a possible factor regulating DMD-associated cardiomyopathy. [ABSTRACT FROM AUTHOR]

Stem cell–secreted extracellular vesicles (EVs) play essential roles in intercellular communication and restore cardiac function in animal models of ischemic heart disease. However, few studies have used EVs derived from clinical-grade stem cells and their derivatives with stable quality. Moreover, there is little information on the mechanism and time course of the multifactorial effect of EV therapy from the acute to the chronic phase, the affected cells, and whether the effects are direct or indirect. Induced pluripotent stem cell–derived cardiomyocytes (iPSCM) were produced using a clinical-grade differentiation induction system. EVs were isolated from the conditioned medium by ultracentrifugation and characterized in silico, in vitro, and in vivo. A rat model of myocardial infarction was established by left anterior descending artery ligation and treated with iPSCM-derived EVs. iPSCM-derived EVs contained microRNAs and proteins associated with angiogenesis, antifibrosis, promotion of M2 macrophage polarization, cell proliferation, and antiapoptosis. iPSCM-derived EV treatment improved left ventricular function and reduced mortality in the rat model by improving vascularization and suppressing fibrosis and chronic inflammation in the heart. EVs were uptaken by cardiomyocytes, endothelial cells, fibroblasts, and macrophages in the cardiac tissues. The pleiotropic effects occurred due to the direct effects of microRNAs and proteins encapsulated in EVs and indirect paracrine effects on M2 macrophages. Clinical-grade iPSCM-derived EVs improve cardiac function by regulating various genes and pathways in various cell types and may have clinical potential for treating ischemic heart disease. [ABSTRACT FROM AUTHOR]

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. V 1/2 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 window current. 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 (APD 90) 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 APD 90 , and treatment with 5μM PHT significantly decreased APD 90 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. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H2S) belongs to the family of gasotransmitters and can modulate a myriad of biological signaling pathways. Among others, its cardioprotective effects, through antioxidant, anti-inflammatory, anti-fibrotic, and proangiogenic activities, are well-documented in experimental studies. Cardiorespiratory failure, predominantly cardiomyopathy, is a life-threatening complication that is the number one cause of death in patients with Duchenne muscular dystrophy (DMD). Although recent data suggest the role of H2S in ameliorating muscle wasting in murine and Caenorhabditis elegans models of DMD, possible cardioprotective effects have not yet been addressed. In this review, we summarize the current understanding of the role of H2S in animal models of cardiac dysfunctions and cardiac cells. We highlight that DMD may be amenable to H2S supplementation, and we suggest H2S as a possible factor regulating DMD-associated cardiomyopathy.

In this article, we consider data complexity in the context of calcium transient signal data collected from induced pluripotent stem cell‐derived cardiomyocytes. We present a novel way to measure data complexity based on the nearest neighbour searching method. Data complexity here is seen as overlapping and mixed data classes in addition to a relatively great number of data cases. Complexity affects classification results, which were run with nearest neighbour searching, feedforward artificial neural networks and random forests for seven genetic cardiological disease classes and healthy controls. The data are obtained from individuals carrying mutations for genetic cardiac diseases with induced pluripotent stem cell (iPSC) technology and the diseases include hypertrophic cardiomyopathy with two different founder gene mutations, dilated cardiomyopathy, long QT syndrome type 1 and 2, Brugada syndrome, a severe genetic ventricular arrhythmia (CPVT) and healthy controls. The data are from calcium transients from spontaneously beating iPSC‐derived cardiomyocytes cultured in a biotechnology laboratory. When the genotype of the iPSC‐derived cardiomyocytes is the same as the donor of the tissue sample and based on the characteristics of the calcium transients, it was possible to classify the seven diseases and healthy controls with machine learning. Peak data first detected before actual pre‐processing from calcium transient signals corresponded to beats (repeating excitation–contraction coupling) of induced stem cell‐derived cardiomyocytes and formed the basis of classification. During pre‐processing of the calcium transient signals, we found that such techniques among others as even strong outlier cleaning or class size balancing by generating artificial cases improved only slightly or not at all classification accuracies. Therefore, the current data set was sufficiently complicated for our data complexity study. Random forests produced the best classification accuracies, 68% for all eight classes. [ABSTRACT FROM AUTHOR]

Background: Most of the long QT syndrome (LQTS) stems from pathogenic variants in KCNQ1, KCNH2, or SCN5A. However, ∼10%-20% of LQTS index cases remain genotype-negative.Objective: The purpose of this study was to identify and characterize functionally a novel LQTS genetic substrate in a multigenerational, "genotype-negative" LQTS pedigree.Methods: The patient was a 40-year-old woman with a history of syncope, seizures, ventricular fibrillation, and a family history of LQTS and sudden death. Commercial genetic testing of all LQTS-causative genes was negative. Genome sequencing was performed on 6 affected family members. Patient-specific and CRISPR/Cas9 "gene-corrected" isogenic control induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated.Results: No ultrarare, nonsynonymous heterozygous variants cosegregated among the 6 LQTS phenotype-positive individuals. Instead, a deep intronic KCNH2 variant (c.3331-316G>T) was present in all affected individuals. Reverse transcription polymerase chain reaction analysis of patient-specific iPSC-CM-derived RNA revealed that c.3331-316G>T creates a novel 89 base-pair exon that results in a frameshift variant (p.S1112Pfs∗171). Action potential duration (APD90) was significantly longer in p.S1112Pfs∗171-iPSC-CMs (602.4 ± 12.2 ms; n =70) compared to isogenic control iPSC-CMs (425.7 ± 9.3 ms; n = 61; P <.0001). Further, field potential duration was significantly longer in p.S1112Pfs∗171-iPSC-CMs (358.9 ± 7.7 ms; n = 65) compared to isogenic control iPSC-CMs (282.2 ± 10.8 ms; n = 51; P <.0001).Conclusion: A novel deep intronic KCNH2 variant was identified in a multigenerational, genetically elusive LQTS pedigree. The iPSC-CMs establish that the variant is the monogenetic cause for this family's LQTS. Deep intronic variants within the 2 most common LQTS-susceptibility genes should be considered in patients with seemingly genetically elusive LQTS. [ABSTRACT FROM AUTHOR]

Abstract Background Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) are regarded as promising cell type for cardiac cell replacement therapy, but it is not known whether the developmental stage influences their persistence and functional integration in the host tissue, which are crucial for a long-term therapeutic benefit. To investigate this, we first tested the cell adhesion capability of murine iPSC-CM in vitro at three different time points during the differentiation process and then examined cell persistence and quality of electrical integration in the infarcted myocardium in vivo. Methods To test cell adhesion capabilities in vitro, iPSC-CM were seeded on fibronectin-coated cell culture dishes and decellularized ventricular extracellular matrix (ECM) scaffolds. After fixed periods of time, stably attached cells were quantified. For in vivo experiments, murine iPSC-CM expressing enhanced green fluorescent protein was injected into infarcted hearts of adult mice. After 6–7 days, viable ventricular tissue slices were prepared to enable action potential (AP) recordings in transplanted iPSC-CM and surrounding host cardiomyocytes. Afterwards, slices were lysed, and genomic DNA was prepared, which was then used for quantitative real-time PCR to evaluate grafted iPSC-CM count. Results The in vitro results indicated differences in cell adhesion capabilities between day 14, day 16, and day 18 iPSC-CM with day 14 iPSC-CM showing the largest number of attached cells on ECM scaffolds. After intramyocardial injection, day 14 iPSC-CM showed a significant higher cell count compared to day 16 iPSC-CM. AP measurements revealed no significant difference in the quality of electrical integration and only minor differences in AP properties between d14 and d16 iPSC-CM. Conclusion The results of the present study demonstrate that the developmental stage at the time of transplantation is crucial for the persistence of transplanted iPSC-CM. iPSC-CM at day 14 of differentiation showed the highest persistence after transplantation in vivo, which may be explained by a higher capability to adhere to the extracellular matrix.

Novel treatment strategies for cardiac tissue regeneration are heading for the use of engineered cardiac tissue made from induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Despite the proven cardiogenic phenotype of these cells, a significant lack of structural and functional properties of mature myocytes prevents safe integration into the diseased heart. To date, maturation processes of cardiomyocytes remain largely unknown but may comprise biophysical cues from the immediate cell environment. Mechanosensing is one critical ability of cells to react to environmental changes. Accordingly, the surrounding substrate stiffness, comprised of extracellular matrix (ECM), cells, and growth surface, critically influences the myocyte’s physiology, as known from deleterious remodeling processes in fibrotic hearts. Conversely, the mechanical properties during culture of iPSC-CMs may impact on their structural and functional maturation. Here, we tested the hypothesis that the environmental stiffness influences structural and functional properties of iPSC-CMs and investigated the effect of different substrate stiffnesses on cell contractility, excitation-contraction (EC) coupling, and intercellular coupling. Culture surfaces with defined stiffnesses ranging from rigid glass with 25GPa to PDMS of physiological softness were coated with ECM proteins and seeded with murine iPSC-CMs. Using confocal imaging, cardiac protein expression was assessed. Ca2+ handling and contractile properties were analyzed on different substrate stiffnesses. Intercellular coupling via gap junctions was investigated by fluorescence recovery after photobleaching (FRAP). Our data revealed greater organization of L-type Ca2+ channels and ryanodine receptors and increased EC-coupling gain, demonstrating structural and functional maturation in cells grown on soft surfaces. In addition, increased shortening and altered contraction dynamics revealed increased myofilament Ca2+ sensitivity in phase-plane loops. Moreover, connexin 43 expression was significantly increased in iPSC-CMs grown on soft surfaces leading to improved intercellular coupling. Taken together, our results demonstrate that soft surfaces with stiffnesses in the physiological range improve the expression pattern and interaction of cardiac proteins relevant for EC-coupling. In parallel, soft substrates influence contractile properties and improve intercellular coupling in iPSC-CMs. We conclude that the mechanical stiffness of the cell environment plays an important role in driving iPSC-CMs toward further maturation by inducing adaptive responses.

Novel treatment strategies for cardiac tissue regeneration are heading for the use of engineered cardiac tissue made from induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Despite the proven cardiogenic phenotype of these cells, a significant lack of structural and functional properties of mature myocytes prevents safe integration into the diseased heart. To date, maturation processes of cardiomyocytes remain largely unknown but may comprise biophysical cues from the immediate cell environment. Mechanosensing is one critical ability of cells to react to environmental changes. Accordingly, the surrounding substrate stiffness, comprised of extracellular matrix (ECM), cells, and growth surface, critically influences the myocyte's physiology, as known from deleterious remodeling processes in fibrotic hearts. Conversely, the mechanical properties during culture of iPSC-CMs may impact on their structural and functional maturation. Here, we tested the hypothesis that the environmental stiffness influences structural and functional properties of iPSC-CMs and investigated the effect of different substrate stiffnesses on cell contractility, excitation-contraction (EC) coupling, and intercellular coupling. Culture surfaces with defined stiffnesses ranging from rigid glass with 25GPa to PDMS of physiological softness were coated with ECM proteins and seeded with murine iPSC-CMs. Using confocal imaging, cardiac protein expression was assessed. Ca2+ handling and contractile properties were analyzed on different substrate stiffnesses. Intercellular coupling via gap junctions was investigated by fluorescence recovery after photobleaching (FRAP). Our data revealed greater organization of L-type Ca2+ channels and ryanodine receptors and increased EC-coupling gain, demonstrating structural and functional maturation in cells grown on soft surfaces. In addition, increased shortening and altered contraction dynamics revealed increased myofilament Ca2+ sensitivity in phase-plane loops. Moreover, connexin 43 expression was significantly increased in iPSC-CMs grown on soft surfaces leading to improved intercellular coupling. Taken together, our results demonstrate that soft surfaces with stiffnesses in the physiological range improve the expression pattern and interaction of cardiac proteins relevant for EC-coupling. In parallel, soft substrates influence contractile properties and improve intercellular coupling in iPSC-CMs. We conclude that the mechanical stiffness of the cell environment plays an important role in driving iPSC-CMs toward further maturation by inducing adaptive responses. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is an X‐linked disease affecting male and rarely adult heterozygous females, resulting in death by the late 20s to early 30s. Previous studies reported depressed left ventricular function in DMD patients which may result from deranged intracellular Ca2+‐handling. To decipher the mechanism(s) underlying the depressed LV function, we tested the hypothesis that iPSC‐CMs generated from DMD patients feature blunted positive inotropic response to β‐adrenergic stimulation. To test the hypothesis, [Ca2+]i transients and contractions were recorded from healthy and DMD‐CMs. While in healthy CMs (HC) isoproterenol caused a prominent positive inotropic effect, DMD‐CMs displayed a blunted inotropic response. Next, we tested the functionality of the sarcoplasmic reticulum (SR) by measuring caffeine‐induced Ca2+ release. In contrast to HC, DMD‐CMs exhibited reduced caffeine‐induced Ca2+ signal amplitude and recovery time. In support of the depleted SR Ca2+ stores hypothesis, in DMD‐CMs the negative inotropic effects of ryanodine and cyclopiazonic acid were smaller than in HC. RNA‐seq analyses demonstrated that in DMD CMs the RNA‐expression levels of specific subunits of the L‐type calcium channel, the β1‐adrenergic receptor (ADRβ1) and adenylate cyclase were down‐regulated by 3.5‐, 2.8‐ and 3‐fold, respectively, which collectively contribute to the depressed β‐adrenergic responsiveness. [ABSTRACT FROM AUTHOR]

Background: Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) are regarded as promising cell type for cardiac cell replacement therapy, but it is not known whether the developmental stage influences their persistence and functional integration in the host tissue, which are crucial for a long-term therapeutic benefit. To investigate this, we first tested the cell adhesion capability of murine iPSC-CM in vitro at three different time points during the differentiation process and then examined cell persistence and quality of electrical integration in the infarcted myocardium in vivo. Methods: To test cell adhesion capabilities in vitro, iPSC-CM were seeded on fibronectin-coated cell culture dishes and decellularized ventricular extracellular matrix (ECM) scaffolds. After fixed periods of time, stably attached cells were quantified. For in vivo experiments, murine iPSC-CM expressing enhanced green fluorescent protein was injected into infarcted hearts of adult mice. After 6–7 days, viable ventricular tissue slices were prepared to enable action potential (AP) recordings in transplanted iPSC-CM and surrounding host cardiomyocytes. Afterwards, slices were lysed, and genomic DNA was prepared, which was then used for quantitative real-time PCR to evaluate grafted iPSC-CM count. Results: The in vitro results indicated differences in cell adhesion capabilities between day 14, day 16, and day 18 iPSC-CM with day 14 iPSC-CM showing the largest number of attached cells on ECM scaffolds. After intramyocardial injection, day 14 iPSC-CM showed a significant higher cell count compared to day 16 iPSC-CM. AP measurements revealed no significant difference in the quality of electrical integration and only minor differences in AP properties between d14 and d16 iPSC-CM. Conclusion: The results of the present study demonstrate that the developmental stage at the time of transplantation is crucial for the persistence of transplanted iPSC-CM. iPSC-CM at day 14 of differentiation showed the highest persistence after transplantation in vivo, which may be explained by a higher capability to adhere to the extracellular matrix. [ABSTRACT FROM AUTHOR]

In adult cardiomyocytes (CMs), the type 2 ryanodine receptor (RYR2) is an indispensable Ca2+ release channel that ensures the integrity of excitation-contraction coupling, which is fundamental for every heartbeat. However, the role and importance of RYR2 during human embryonic cardiac development are still poorly understood. Here, we generated two human induced pluripotent stem cell (iPSC)-based RYR2 knockout (RYR2–/–) lines using the CRISPR/Cas9 gene editing technology. We found that RYR2–/–-iPSCs could differentiate into CMs with the efficiency similar to control-iPSCs (Ctrl-iPSCs); however, the survival of iPSC-CMs was markedly affected by the lack of functional RYR2. While Ctrl-iPSC-CMs exhibited regular Ca2+ handling, we observed significantly reduced frequency and intense abnormalities of Ca2+ transients in RYR2–/–-iPSC-CMs. Ctrl-iPSC-CMs displayed sensitivity to extracellular Ca2+ ([Ca2+ ]o) and caffeine in a concentration-dependent manner, while RYR2–/–-iPSC-CMs showed inconsistent reactions to [Ca2+ ]o and were insensitive to caffeine, indicating there is no RYR2-mediated Ca2+ release from the sarcoplasmic reticulum (SR). Instead, compensatory mechanism for calcium handling in RYR2–/–-iPSC-CMs is partially mediated by the inositol 1,4,5-trisphosphate receptor (IP3R). Similar to Ctrl-iPSC-CMs, SR Ca2+ refilling in RYR2–/–-iPSC-CMs is mediated by SERCA. Additionally, RYR2–/–-iPSC-CMs showed a decreased beating rate and a reduced peak amplitude of L-type Ca2+ current. These findings demonstrate that RYR2 is not required for CM lineage commitment but is important for CM survival and contractile function. IP3R-mediated Ca2+ release is one of the major compensatory mechanisms for Ca2+ cycling in human CMs with the RYR2 deficiency.

Recent advances in human induced pluripotent stem cell-derived cardiomyocytes (iPSCM) field offer a novel platform for modeling cardiac metabolism, heart diseases drug candidates screening and cardiac toxicity assessments. These workflows require a fully functional characterization of iPSCMs. Here we report a step by step protocol for iPSCM metabolic characterization. The described assays cover analysis of small metabolites involved in a vital metabolic pathways.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are promising candidates for disease modeling and therapeutic purposes, however, non-viral intracellular delivery in these cells remains challenging. Gold nanoparticle (AuNP)-sensitized photoporation creates transient pores in the cell membrane by vapor nanobubble formation, allowing diffusion of extracellular biomolecules. This non-viral technique was employed to test and optimize its distinct physical mode of action in iPSC-CMs. Photoporation optimization was aimed at achieving high delivery rates while minimizing cell death. Various AuNP concentrations, in conjunction with different laser fluences, were explored to facilitate the intracellular delivery of 10 kDa and 150 kDa FITC-labelled dextran as model macromolecules. Cardiomyocyte viability was assessed using the CellTiter-Glo® viability assay, while the delivery efficiency was quantified through flow cytometry. On 30 day-old cardiomyocytes, AuNP photoporation was able to yield ∼60 % delivery efficiency while maintaining a high cell viability (∼70 %). Overall, higher AuNP concentrations resulted in greater delivery efficiencies, albeit at the expense of lower cell viability. Finally, photoporation was capable of patterning a geometric shape, demonstrating its exceptional selective resolution in delivering molecules to spatially restricted regions of the cell culture. In conclusion, AuNP-photoporation exhibits considerable potential as an effective and gentle non-viral method for intracellular delivery in iPSC-CMs.•AuNP-photoporation is a non-viral intracellular delivery method suitable for iPSC-CMs with high efficiency and cell viability•This method is capable of spatially resolved intracellular delivery with excellent resolution., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: KB declares financial interest in the company Trince which markets the LumiPore photoporation technology, (© 2024 The Authors. Published by Elsevier B.V.)

Duchenne muscular dystrophy (DMD) is an X‐linked progressive muscle degenerative disease, caused by mutations in the dystrophin gene and resulting in death because of respiratory or cardiac failure. To investigate the cardiac cellular manifestation of DMD, we generated induced pluripotent stem cells (iPSCs) and iPSC‐derived cardiomyocytes (iPSC‐CMs) from two DMD patients: a male and female manifesting heterozygous carrier. Dystrophin mRNA and protein expression were analysed by qRT‐PCR, RNAseq, Western blot and immunofluorescence staining. For comprehensive electrophysiological analysis, current and voltage clamp were used to record transmembrane action potentials and ion currents, respectively. Microelectrode array was used to record extracellular electrograms. X‐inactive specific transcript (XIST) and dystrophin expression analyses revealed that female iPSCs underwent X chromosome reactivation (XCR) or erosion of X chromosome inactivation, which was maintained in female iPSC‐CMs displaying mixed X chromosome expression of wild type (WT) and mutated alleles. Both DMD female and male iPSC‐CMs presented low spontaneous firing rate, arrhythmias and prolonged action potential duration. DMD female iPSC‐CMs displayed increased beat rate variability (BRV). DMD male iPSC‐CMs manifested decreased If density, and DMD female and male iPSC‐CMs showed increased ICa,L density. Our findings demonstrate cellular mechanisms underlying electrophysiological abnormalities and cardiac arrhythmias in DMD. [ABSTRACT FROM AUTHOR]

Aims: The aim of the present study is to develop in vitro experimental analytical method for the electrophysiological properties of allogeneic induced pluripotent stem cell-derived cardiomyocytes (CMs) in cardiac conduction defect model.Methods and results: Cardiomyocytes were derived from rat induced pluripotent stem cells CMs (riPSC-CMs) using an embryoid body-based differentiation method with the serial application of growth factors including activin-A, bone morphogenetic protein 4 (BMP-4), and inhibitor of wnt production 2 (IWP-2). Flow cytometry analysis showed that 74.0 ± 2.7% of riPSC-CMs expressed cardiac troponin-T (n = 3). Immunostaining analysis revealed organized sarcomeric structure in riPSC-CMs and the expression of connexin 43 between riPSC-CMs and neonatal rat ventricular CMs (NRVMs). Ca2+ transient recordings revealed the simultaneous excitement of riPSC-CMs and NRVMs, and prolonged Ca2+ transient duration of riPSC-CMs as compared with NRVMs (731 ± 15.9 vs. 610 ± 7.72 ms, P < 0.01, n = 3). Isolated NRVMs were cultured in two discrete regions to mimic cardiac conduction defects on multi-electrode array dish, and riPSC-CMs were seeded in the channel between the two discrete regions. Membrane potential imaging with di-8-ANEPPS discerned the propagation of the electrical impulse from one NRVM region to the other through a riPSC-CM pathway. This pathway had significantly longer action potential duration as compared with NRVMs. Electrophysiological studies using a multi-electrode array platform demonstrated the longer conduction time and functional refractory period of the riPSC-CM pathway compared with the NRVM pathway.Conclusion: Using an in vitro experimental system to mimic cardiac conduction defect, transplanted allogeneic riPSC-CMs showed electrical coupling between two discrete regions of NRVMs. Electrophysiological testing using our platform will enable electrophysiological screening prior to transplantation of stem cell-derived CMs. [ABSTRACT FROM AUTHOR]

Two-pore K+ (K2p) channels have been described in modulating background conductance as leak channels in different physiological systems. In the heart, the expression of K2p channels is heterogeneous with equivocation regarding their functional role. Our objective was to determine the K2p expression profile and their physiological and pathophysiological contribution to cardiac electrophysiology. Induced pluripotent stem cells (iPSCs) generated from humans were differentiated into cardiomyocytes (iPSC-CMs). mRNA was isolated from these cells, commercial iPSC-CM (iCells), control human heart ventricular tissue (cHVT), and ischemic (iHF) and nonischemic heart failure tissues (niHF). We detected 10 K2p channels in the heart. Comparing quantitative PCR expression of K2p channels between human heart tissue and iPSC-CMs revealed K2p1.1, K2p2.1, K2p5.1, and K2p17.1 to be higher expressed in cHVT, whereas K2p3.1 and K2p13.1 were higher in iPSC-CMs. Notably, K2p17.1 was significantly lower in niHF tissues compared with cHVT. Action potential recordings in iCells after K2p small interfering RNA knockdown revealed prolongations in action potential depolarization at 90% repolarization for K2p2.1, K2p3.1, K2p6.1, and K2p17.1. Here, we report the expression level of 10 human K2p channels in iPSC-CMs and how they compared with cHVT. Importantly, our functional electrophysiological data in human iPSC-CMs revealed a prominent role in cardiac ventricular repolarization for four of these channels. Finally, we also identified K2p17.1 as significantly reduced in niHF tissues and K2p4.1 as reduced in niHF compared with iHF. Thus, we advance the notion that K2p channels are emerging as novel players in cardiac ventricular electrophysiology that could also be remodeled in cardiac pathology and therefore contribute to arrhythmias. [ABSTRACT FROM AUTHOR]

Cardiomyopathy is a pathological condition characterized by cardiac pump failure due to myocardial dysfunction and the major cause of advanced heart failure requiring heart transplantation. Although optimized medical therapies have been developed for heart failure during the last few decades, some patients with cardiomyopathy exhibit advanced heart failure and are refractory to medical therapies. Desmosome, which is a dynamic cell-to-cell junctional component, maintains the structural integrity of heart tissues. Genetic mutations in desmosomal genes cause arrhythmogenic cardiomyopathy (AC), a rare inheritable disease, and predispose patients to sudden cardiac death and heart failure. Recent advances in sequencing technologies have elucidated the genetic basis of cardiomyopathies and revealed that desmosome-related cardiomyopathy is concealed in broad cardiomyopathies. Among desmosomal genes, mutations in PKP2 (which encodes PKP2) are most frequently identified in patients with AC. PKP2 deficiency causes various pathological cardiac phenotypes. Human cardiomyocytes differentiated from patient-derived induced pluripotent stem cells (iPSCs) in combination with genome editing, which allows the precise arrangement of the targeted genome, are powerful experimental tools for studying disease. This review summarizes the current issues associated with practical medicine for advanced heart failure and the recent advances in disease modeling using iPSC-derived cardiomyocytes targeting desmosome-related cardiomyopathy caused by PKP2 deficiency., Competing Interests: Conflict-of-interest statement: Department of Medical Therapeutics for Heart Failure is a Joint Research Department with TOA EIYO Pharmaceutical Company. Dr. Higo reports grants from TOA EIYO Pharmaceutical Company, during the conduct of the study., (©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved.)

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) hold enormous potential in many fields of cardiovascular research. Overcoming many of the limitations of their embryonic counterparts, the application of iPSC-CMs ranges from facilitating investigation of familial cardiac disease and pharmacological toxicity screening to personalized medicine and autologous cardiac cell therapies. The main factor preventing the full realization of this potential is the limited maturity of iPSC-CMs, which display a number of substantial differences in comparison to adult cardiomyocytes. Excitation-contraction coupling, a fundamental property of cardiomyocytes, is often described in iPSC-CMs as being more analogous to neonatal than adult cardiomyocytes. With calcium handling linked, directly or indirectly, to almost all other properties of cardiomyocytes, a solid understanding of this process will be crucial to fully realizing the potential of this technology.Here we discuss the implications of differences in excitation-contraction coupling when considering the potential applications of iPSC-CMs in a number of areas as well as detailing the current understanding of this fundamental process in these cells.

Patients with HIV present with a higher prevalence of QT prolongation, of which molecular bases are still not clear. Among HIV proteins, Tat serves as a transactivator that stimulates viral genes expression and is required for efficient HIV replication. Tat is actively secreted into the blood by infected T-cells and affects organs such as the heart. Tat has been shown to alter cardiac repolarization in animal models but how this is mediated and whether this is also the case in human cells is unknown. In the present study, we show that Tat transfection in heterologous expression systems led to a decrease in hERG (underlying cardiac I Kr ) and human KCNE1-KCNQ1 (underlying cardiac I Ks ) currents and to an acceleration of their deactivation. This is consistent with a decrease in available phosphatidylinositol-(4,5)-bisphosphate (PIP 2 ). A mutant Tat, unable to bind PIP 2 , did not reproduce the observed effects. In addition, WT-Tat had no effect on a mutant KCNQ1 which is PIP 2 -insensitive, further confirming the hypothesis. Twenty-four-hour incubation of human induced pluripotent stem cells-derived cardiomyocytes with Wild-type Tat reduced I Kr and accelerated its deactivation. Concordantly, this Tat incubation led to a prolongation of the action potential (AP) duration. Events of AP alternans were also recorded in the presence of Tat, and were exacerbated at a low pacing cycle length. Altogether, these data obtained on human K + channels both in heterologous expression systems and in human cardiomyocytes suggest that Tat sequesters PIP 2 , leading to a reduction of I Kr and I Ks , and provide a molecular mechanism for QT prolongation in HIV-infected patients. [ABSTRACT FROM AUTHOR]

Induced pluripotent stem cells (i PSCs) were first generated 10 years ago. Their ability to differentiate into any somatic cell type of the body including cardiomyocytes has already made them a valuable resource for modelling cardiac disease and drug screening. Initially human i PSCs were used mostly to model known disease phenotypes; more recently, and despite a number of recognised shortcomings, they have proven valuable in providing fundamental insights into the mechanisms of inherited heart disease with unknown genetic cause using surprisingly small cohorts. In this review, we summarise the progress made with human i PSCs as cardiac disease models with special focus on the latest mechanistic insights and related challenges. Furthermore, we suggest emerging solutions that will likely move the field forward. [ABSTRACT FROM AUTHOR]

Background: Heart failure with ejection fraction (HFpEF) is a syndrome resulting from several co-morbidities in which specific mediators are unknown. The platelet proteome responds to disease processes. We hypothesize that the platelet proteome will change composition in patients with HFpEF and may uncover mediators of the syndrome.Methods and Results: Proteomic changes were assessed in platelets from hospitalized subjects with symptoms of HFpEF (n = 9), the same subjects several weeks later without symptoms (n = 7) and control subjects (n = 8). Mass spectrometry identified 6102 proteins with five scans with peptide probabilities of ≥0.85. Of the 6102 proteins, 165 were present only in symptomatic subjects, 78 were only found in outpatient subjects and 157 proteins were unique to the control group. The S100A8 protein was identified consistently in HFpEF samples when compared with controls. We validated the fining that plasma S100A8 levels are increased in subjects with HFpEF (654 ± 391) compared to controls (352 ± 204) in an external cohort (p = 0.002). Recombinant S100A8 had direct effects on the electrophysiological and calcium handling profile in human induced pluripotent stem cell-derived cardiomyocytes.Conclusions: Platelets may harbor proteins associated with HFpEF. S100A8 is present in the platelets of subjects with HFpEF and increased in the plasma of the same subjects. We further established a bedside-to-bench translational system that can be utilized as a secondary screen to ascertain whether the biomarkers may be an associated finding or causal to the disease process. S100A8 has been linked with other cardiovascular disease such as atherosclerosis and risk for myocardial infarction, stroke, or death. This is the first report on association of S100A8 with HFpEF. [ABSTRACT FROM AUTHOR]

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a cardiac channelopathy causing ventricular tachycardia following adrenergic stimulation. Pathogenic variants in RYR2-encoded ryanodine receptor 2 (RYR2) cause CPVT1 and cluster into domains I-IV, with the most N-terminal domain involving residues 77-466. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated for RYR2-F13L, -L14P, -R15P, and -R176Q variants. Isogenic control iPSCs were generated using CRISPR-Cas9/PiggyBac. Fluo-4 Ca 2+ imaging assessed Ca 2+ handling with/without isoproterenol (ISO), nadolol (Nad), and flecainide (Flec) treatment. CPVT1 iPSC-CMs displayed increased Ca 2+ sparking and Ca 2+ transient amplitude following ISO compared with control. Combined Nad treatment/ISO stimulation reduced Ca 2+ amplitude and sparking in variant iPSC-CMs. Molecular dynamic simulations visualized the structural role of these variants. We provide the first functional evidence that these most proximal N-terminal localizing variants alter calcium handling similar to CPVT1. These variants are located at the N-terminal domain and the central domain interface and could destabilize the RYR2 channel promoting Ca 2+ leak-triggered arrhythmias., Competing Interests: Conflict of interests M.J.A. is a consultant for Abbott, ARMGO Pharma, Boston Scientific, Daiichi Sankyo, Invitae, LQT Therapeutics, and Medtronic. M.J.A. and/or Mayo Clinic are involved in an equity/royalty relationship with AliveCor, Anumana, Pfizer, and UpToDate. However, none of these entities were involved in this study in any manner. A.R.M. and Columbia University own shares in ARMGO Pharma, a biotech company developing RyR-targeted therapeutics., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Rationale: Aging in the heart is a gradual process, involving continuous changes in cardiovascular cells, including cardiomyocytes (CMs), namely cellular senescence. These changes finally lead to adverse organ remodeling and resulting in heart failure. This study exploits CMs from human induced pluripotent stem cells (iCMs) as a tool to model and characterize mechanisms involved in aging. Methods and Results: Human somatic cells were reprogrammed into human induced pluripotent stem cells and subsequently differentiated in iCMs. A senescent-like phenotype (SenCMs) was induced by short exposure (3 hours) to doxorubicin (Dox) at the sub-lethal concentration of 0.2 µM. Dox treatment induced expression of cyclin-dependent kinase inhibitors p21 and p16, and increased positivity to senescence-associated beta-galactosidase when compared to untreated iCMs. SenCMs showed increased oxidative stress, alteration in mitochondrial morphology and depolarized mitochondrial membrane potential, which resulted in decreased ATP production. Functionally, when compared to iCMs, SenCMs showed, prolonged multicellular QTc and single cell APD, with increased APD variability and delayed afterdepolarizations (DADs) incidence, two well-known arrhythmogenic indexes. These effects were largely ascribable to augmented late sodium current (I NaL ) and reduced delayed rectifier potassium current (Ikr). Moreover sarcoplasmic reticulum (SR) Ca 2+ content was reduced because of downregulated SERCA2 and increased RyR2-mediated Ca 2+ leak. Electrical and intracellular Ca 2+ alterations were mostly justified by increased CaMKII activity in SenCMs. Finally, SenCMs phenotype was furtherly confirmed by analyzing physiological aging in CMs isolated from old mice in comparison to young ones. Conclusions: Overall, we showed that SenCMs recapitulate the phenotype of aged primary CMs in terms of senescence markers, electrical and Ca 2+ handling properties and metabolic features. Thus, Dox-induced SenCMs can be considered a novel in vitro platform to study aging mechanisms and to envision cardiac specific anti-aging approach in humans., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Various types of stem cells and nonstem cells have been shown to differentiate or transdifferentiate into cardiomyocytes by way of coculture with appropriate inducer cells. Here we describe a method to induce cardiac differentiation in induced pluripotent stem (iPS) cells through the use of coculture with previously differentiated iPS cell-derived cardiomyocytes (iCMs). This differentiation process can be achieved without the use of exogenous pathway inhibitors and morphogens., (© 2020. Springer Science+Business Media New York.)

Recent advances in cardiac tissue engineering have shown that human induced-pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) cultured in a three-dimensional (3D) micro-environment exhibit superior physiological characteristics compared with their two-dimensional (2D) counterparts. These 3D cultured hiPSC-CMs have been used for drug testing as well as cardiac repair applications. However, the fabrication of a cardiac scaffold with optimal biomechanical properties and high biocompatibility remains a challenge. In our study, we fabricated an aligned polycaprolactone (PCL)-Gelatin coaxial nanofiber patch using electrospinning. The structural, chemical, and mechanical properties of the patch were assessed by scanning electron microscopy (SEM), immunocytochemistry (ICC), Fourier-transform infrared spectroscopy (FTIR)-spectroscopy, and tensile testing. hiPSC-CMs were cultured on the aligned coaxial patch for 2 weeks and their viability [lactate dehydrogenase (LDH assay)], morphology (SEM, ICC), and functionality [calcium cycling, multielectrode array (MEA)] were assessed. Furthermore, particle image velocimetry (PIV) and MEA were used to evaluate the cardiotoxicity and physiological functionality of the cells in response to cardiac drugs. Nanofibers patches were comprised of highly aligned core-shell fibers with an average diameter of 578 ± 184 nm. Acellular coaxial patches were significantly stiffer than gelatin alone with an ultimate tensile strength of 0.780 ± 0.098 MPa, but exhibited gelatin-like biocompatibility. Furthermore, hiPSC-CMs cultured on the surface of these aligned coaxial patches (3D cultures) were elongated and rod-shaped with well-organized sarcomeres, as observed by the expression of cardiac troponin-T and α-sarcomeric actinin. Additionally, hiPSC-CMs cultured on these coaxial patches formed a functional syncytium evidenced by the expression of connexin-43 (Cx-43) and synchronous calcium transients. Moreover, MEA analysis showed that the hiPSC-CMs cultured on aligned patches showed an improved response to cardiac drugs like Isoproterenol (ISO), Verapamil (VER), and E4031, compared to the corresponding 2D cultures. Overall, our results demonstrated that an aligned, coaxial 3D cardiac patch can be used for culturing of hiPSC-CMs. These biomimetic cardiac patches could further be used as a potential 3D in vitro model for "clinical trials in a dish" and for in vivo cardiac repair applications for treating myocardial infarction., (Copyright © 2020 Kumar, Sridharan, Palaniappan, Dougherty, Czirok, Isai, Mergaye, Angelos, Powell and Khan.)

In adult cardiomyocytes (CMs), the type 2 ryanodine receptor (RYR2) is an indispensable Ca 2+ release channel that ensures the integrity of excitation-contraction coupling, which is fundamental for every heartbeat. However, the role and importance of RYR2 during human embryonic cardiac development are still poorly understood. Here, we generated two human induced pluripotent stem cell (iPSC)-based RYR2 knockout (RYR2 -/- ) lines using the CRISPR/Cas9 gene editing technology. We found that RYR2 -/- -iPSCs could differentiate into CMs with the efficiency similar to control-iPSCs (Ctrl-iPSCs); however, the survival of iPSC-CMs was markedly affected by the lack of functional RYR2. While Ctrl-iPSC-CMs exhibited regular Ca 2+ handling, we observed significantly reduced frequency and intense abnormalities of Ca 2+ transients in RYR2 -/- -iPSC-CMs. Ctrl-iPSC-CMs displayed sensitivity to extracellular Ca 2+ ([Ca 2+ ] o ) and caffeine in a concentration-dependent manner, while RYR2 -/- -iPSC-CMs showed inconsistent reactions to [Ca 2+ ] o and were insensitive to caffeine, indicating there is no RYR2-mediated Ca 2+ release from the sarcoplasmic reticulum (SR). Instead, compensatory mechanism for calcium handling in RYR2 -/- -iPSC-CMs is partially mediated by the inositol 1,4,5-trisphosphate receptor (IP3R). Similar to Ctrl-iPSC-CMs, SR Ca 2+ refilling in RYR2 -/- -iPSC-CMs is mediated by SERCA. Additionally, RYR2 -/- -iPSC-CMs showed a decreased beating rate and a reduced peak amplitude of L-type Ca 2+ current. These findings demonstrate that RYR2 is not required for CM lineage commitment but is important for CM survival and contractile function. IP3R-mediated Ca 2+ release is one of the major compensatory mechanisms for Ca 2+ cycling in human CMs with the RYR2 deficiency., (Copyright © 2020 Luo, Li, Künzel, Henze, Cyganek, Strano, Poetsch, Schubert and Guan.)

Recent developments of novel targeted therapies are contributing to the increased long-term survival of cancer patients; however, drug-induced cardiotoxicity induced by cancer drugs remains a serious problem in clinical settings. Nevertheless, there are few in vitro cell-based assays available to predict this toxicity, especially from the aspect of morphology. Here, we developed a simple two-dimensional (2D) morphological assessment system, 2DMA, to predict drug-induced cardiotoxicity in cancer patients using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with image-based high-content analysis in a high-throughput manner. To assess the effects of drugs on cardiomyocytes, we treated iPSC-CMs with 28 marketed pharmaceuticals and measured two key parameters: number of cell nuclei and sarcomere morphology. Drugs that significantly perturbed these two parameters at concentrations ≤30 times the human C max value were regarded as positive in the test. Based on these criteria, the sensitivity and specificity of the 2DMA system were 81% and 100%, respectively. Moreover, the translational predictability of 2DMA was comparable with that of a three-dimensional cardiotoxicity assay. RNA sequencing further revealed that the expression levels of several genes related to sarcomere components decreased following treatment with sunitinib, suggesting that inhibition of the synthesis of proteins that comprise the sarcomere contributes to drug-induced sarcomere disruption. Based on these features, the 2DMA system provides mechanistic insight with high predictability of cancer drug-induced cardiotoxicity in humans, and could thus contribute to the reduction of drug attrition rates at early stages of drug development. • Drug-induced change in number of cell nuclei and sarcomere morphology was evaluated. • This assay showed 81% sensitivity and 100% specificity using 28 approved drugs. • Predictability of the assay was comparable with 3D structural cardiotoxicity assay. • Most of the sarcomere genes were downregulated upon treatment with sunitinib. • This assay can help accurately predict cancer drug-induced cardiotoxicity. [ABSTRACT FROM AUTHOR]

Recent advances in human induced pluripotent stem cell-derived cardiomyocytes (iPSCM) field offer a novel platform for modeling cardiac metabolism, heart diseases drug candidates screening and cardiac toxicity assessments. These workflows require a fully functional characterization of iPSCMs. Here we report a step by step protocol for iPSCM metabolic characterization. The described assays cover analysis of small metabolites involved in a vital metabolic pathways., (© 2019 The Authors.)

Induced pluripotent stem cells (iPSCs) were first generated by Yamanaka and colleagues over a decade ago. Since then, iPSCs have been successfully differentiated into many distinct cell types, enabling tissue-, disease-, and patient-specific in vitro modelling. Cardiovascular disease is the greatest cause of mortality worldwide but encompasses rarer disorders of conduction and myocardial function for which a cellular model of study is ideal. Although methods to differentiate iPSCs into beating cardiomyocytes (iPSC-CMs) have recently been adequately optimized and commercialized, the resulting cells remain largely immature with regards to their structure and function, demonstrating fetal gene expression, disorganized morphology, reliance on predominantly glycolytic metabolism and contractile characteristics that differ from those of adult cardiomyocytes. As such, disease modelling using iPSC-CMs may be inaccurate and of limited utility. However, this limitation is widely recognized, and numerous groups have made substantial progress in addressing this problem. This review highlights successful methods that have been developed for the maturation of human iPSC-CMs using small molecules, environmental manipulation and 3-dimensional (3D) growth approaches., Competing Interests: Conflict-of-interest statement: Authors have no conflicts to disclose.

Two-pore K + (K 2p ) channels have been described in modulating background conductance as leak channels in different physiological systems. In the heart, the expression of K 2p channels is heterogeneous with equivocation regarding their functional role. Our objective was to determine the K 2p expression profile and their physiological and pathophysiological contribution to cardiac electrophysiology. Induced pluripotent stem cells (iPSCs) generated from humans were differentiated into cardiomyocytes (iPSC-CMs). mRNA was isolated from these cells, commercial iPSC-CM (iCells), control human heart ventricular tissue (cHVT), and ischemic (iHF) and nonischemic heart failure tissues (niHF). We detected 10 K 2p channels in the heart. Comparing quantitative PCR expression of K 2p channels between human heart tissue and iPSC-CMs revealed K 2p 1.1, K 2p 2.1, K 2p 5.1, and K 2p 17.1 to be higher expressed in cHVT, whereas K 2p 3.1 and K 2p 13.1 were higher in iPSC-CMs. Notably, K 2p 17.1 was significantly lower in niHF tissues compared with cHVT. Action potential recordings in iCells after K 2p small interfering RNA knockdown revealed prolongations in action potential depolarization at 90% repolarization for K 2p 2.1, K 2p 3.1, K 2p 6.1, and K 2p 17.1. Here, we report the expression level of 10 human K 2p channels in iPSC-CMs and how they compared with cHVT. Importantly, our functional electrophysiological data in human iPSC-CMs revealed a prominent role in cardiac ventricular repolarization for four of these channels. Finally, we also identified K 2p 17.1 as significantly reduced in niHF tissues and K 2p 4.1 as reduced in niHF compared with iHF. Thus, we advance the notion that K 2p channels are emerging as novel players in cardiac ventricular electrophysiology that could also be remodeled in cardiac pathology and therefore contribute to arrhythmias. NEW & NOTEWORTHY Two-pore K + (K 2p ) channels are traditionally regarded as merely background leak channels in myriad physiological systems. Here, we describe the expression profile of K 2p channels in human-induced pluripotent stem cell-derived cardiomyocytes and outline a salient role in cardiac repolarization and pathology for multiple K 2p channels., (Copyright © 2017 the American Physiological Society.)

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) hold enormous potential in many fields of cardiovascular research. Overcoming many of the limitations of their embryonic counterparts, the application of iPSC-CMs ranges from facilitating investigation of familial cardiac disease and pharmacological toxicity screening to personalized medicine and autologous cardiac cell therapies. The main factor preventing the full realization of this potential is the limited maturity of iPSC-CMs, which display a number of substantial differences in comparison to adult cardiomyocytes. Excitation-contraction (EC) coupling, a fundamental property of cardiomyocytes, is often described in iPSC-CMs as being more analogous to neonatal than adult cardiomyocytes. With Ca(2+) handling linked, directly or indirectly, to almost all other properties of cardiomyocytes, a solid understanding of this process will be crucial to fully realizing the potential of this technology. Here, we discuss the implications of differences in EC coupling when considering the potential applications of human iPSC-CMs in a number of areas as well as detailing the current understanding of this fundamental process in these cells.