Background and Aims Life-threatening arrhythmias are a well-established consequence of reduced cardiac sodium current (INa). Gene therapy approaches to increase INa have demonstrated potential benefits to prevent arrhythmias. However, the development of such therapies is hampered by the large size of sodium channels. In this study, SCN10A-short (S10s), a short transcript encoding the carboxy-terminal domain of the human neuronal sodium channel, was evaluated as a gene therapy target to increase INa and prevent arrhythmias. Methods Adeno-associated viral vector overexpressing S10s was injected into wild type and Scn5a-haploinsufficient mice on which patch-clamp studies, optical mapping, electrocardiogram analyses, and ischaemia reperfusion were performed. In vitro and in silico studies were conducted to further explore the effect of S10s gene therapy in the context of human hearts. Results Cardiac S10s overexpression increased cellular INa, maximal action potential upstroke velocity, and action potential amplitude in Scn5a-haploinsufficient cardiomyocytes. S10s gene therapy rescues conduction slowing in Scn5a-haploinsufficient mice and prevented ventricular tachycardia induced by ischaemia-reperfusion in wild type mice. S10s overexpression increased maximal action potential upstroke velocity in human inducible pluripotent stem cell-derived cardiomyocytes and prevented inducible arrhythmias in simulated human heart models. Conclusions S10s gene therapy may be effective to treat cardiac conduction abnormalities and associated arrhythmias.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) tend to show a mixed population of action potential (AP) types, including atrial-like (A-like) and ventricular-like (V-like) APs. In the present study, we investigated the membrane currents underlying these two AP types in hESC-CMs. These were generated using standard (Std) and retinoic acid (RA)-based differentiation protocols. Patch clamp methodology was used to correlate AP morphology with major cardiac ion currents by applying alternating current and voltage clamp protocols to each cell, and to measure L-type Ca2+ current (ICa,L) and Na+-Ca2+ exchange current (INCX) in detail, whereas Ca2+ transients were measured ratiometrically using Indo-1. A- and V-like APs were found in both Std and RA-treated hESC-CMs and the AP plateau amplitude (APplat), as a measure of fast phase-1 repolarization, appeared the best AP criterion to separate these two AP types. Traditional voltage clamp experiments revealed a significantly smaller ICa,L density in RA-treated hESC-CMs, as well as larger densities of the transient outward and delayed rectifier K+ currents (Ito1 and IK, respectively), without changes in the inward rectifier K+ current (IK1). The APplat showed strong and moderate correlations with the densities of ICa,L and IK, respectively, in the absence of a clear-cut correlation with the density of Ito1. Using pre-recorded, typical A- and V-like APs, AP clamp demonstrated that the ICa,L-mediated Ca2+ influx during the V-like AP in Std hESC-CMs is 3.15 times larger than the influx during the A-like AP in RA-treated hESC-CMs. Ca2+ transients of A-like hESC-CMs have a lower diastolic and systolic level, as well as a lower amplitude, than those of Std hESC-CMs, while their duration is shorter due to enhanced SERCA activity. In conclusion, ICa,L is an important determinant of the differently shaped A- and V-like APs in hESC-CMs. Furthermore, the Ca2+ homeostasis differs between A- and V-like hESC-CMs due to the smaller ICa,L and enhanced SERCA activity during A-like APs, resulting in a strongly reduced Ca2+ influx, which will cause a substantial reduction in INCX, further contributing to the shorter A-like APs.

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Stichting LSH-TKI Introduction In cardiac gene therapy, two prototypic viral vectors are typically considered for long-term transgene expression, i.e. lentivirus (LV) and adeno-associated virus (AAV) vectors. Compared to AAV vectors, LV vectors have the advantages of a larger insert capacity and the ability to confer permanent transgene expression due to integration of the vector genome in the host cell’s chromosomal DNA . However, standard VSV G protein-pseudotyped LV vectors (VSV G-LV vectors) has shown suboptimal transduction of cardiac myocytes in vivo. Purpose In this study, we aimed to improve LV-mediated transduction of cardiomyocytes in vivo by evaluating LV vectors carrying various different envelopes and compared the most potent variants with AAV1 capsid-pseudotyped AAV2 vectors (AAV2/1 vectors). Methods We made a panel of LV vectors pseudotyped with envelope proteins from 76 different viruses carrying an expression cassette for enhanced green fluorescent protein (eGFP). Transduction efficiencies in neonatal rat ventricular myocytes (NRVMs), human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and cardiomyocyte progenitor cells (CMPCs) were determined by fluorescence-activated cell sorting (FACS) as percentage eGFP-positive cells. Male NOD -SCID mice aged 3-6 months were injected directly into the left ventricular myocardium with eGFP-encoding VSV G-LV and AAV2/1 vectors at doses of 1.4 x 10^7 infectious units and 2.0 x 10^10 genome copies, respectively, or PBS as a control. Mice were sacrificed one and four weeks after surgery. Per heart, left ventricular and eGFP-positive area was determined from 40 – 50 slides and the ratio was used as a measure of transduction efficiency. Results Of the panel of pseudotyped LVs none was able to consistently outperform VSV G-LV vectors, which reached transduction efficiencies in vitro of 58% in NRVMs to 96% in hiPSC-CMs and 100% in CMPCs . In AAV2/1 vector-injected hearts, the eGFP expressing area in the left ventricle increased from 1.07% in week 1 to 5.42% in week 4. In contrast, eGFP expression in LV-injected hearts was 0.31% and 0.16% after 1 week and 4 weeks, respectively (figure 1). Conclusion In vitro screening of a panel of pseudotyped LV vectors showed standard VSV G-LV vectors as top performer. Despite encouraging in vitro results and previously reported findings, VSV G-LV vectors was unable to provide for robust in vivo transduction. The observation that none of the 76 different envelopes yielded a meaningful improvement of lentiviral transduction rates together with the discrepancy between in vitro and in vivo transduction efficiencies, suggest that a post-entry block of transduction likely represents an important component of the poor in vivo performance.Figure 1in vivo transduction

We present a physiological basis for the interpretation of multielectrode array-derived, extracellular, electrical signals.

The atrioventricular (AV) conduction axis provides electrical continuity between the atrial and ventricular chambers. The "nodal" cardiomyocytes populating this region (AV canal in the embryo, AV node from fetal stages onward) propagate impulses slowly, ensuring sequential contraction of the chambers. Dysfunction of AV nodal tissue causes severe disturbances in rhythm and contraction, and human models that capture its salient features are limited. Here, we report an approach for the reproducible generation of AV canal cardiomyocytes (AVCMs) with in vivo-like gene expression and electrophysiological profiles. We created the so-called "assembloids" composed of atrial, AVCM, and ventricular spheroids, which effectively recapitulated unidirectional conduction and the "fast-slow-fast" activation pattern typical for the vertebrate heart. We utilized these systems to reveal intracellular calcium mishandling as the basis of LMNA-associated AV conduction block. In sum, our study introduces novel cell differentiation and tissue construction strategies to facilitate the study of complex disorders affecting heart rhythm.

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Dutch Research Councin/NWO Introduction Several unrelated families presenting with a complex, heterogeneous cardiac syndrome including sinoatrial node dysfunction and atrial fibrillation were found to share overlapping deletions in a 1.5 Megabase pair gene desert on chromosome 4q25. These overlapping regions contain evolutionarily conserved CTCF binding sites that modulate the separation of topologically associating domains containing the transcription factor-encoding gene PITX2 and noncoding RNA genes. Aim To understand mechanistically how the deletion in the gene desert causes sinoatrial node dysfunction and atrial arrhythmias. Methods Mice lacking the orthologue of the minimally overlapping genomic region deleted in affected patients were generated. Electrophysiology, morphology, tissue composition, and transcriptomes were analysed in this mouse model. Results These mice recapitulated major morpho-functional features of the syndrome, including sinoatrial node dysfunction and atrial arrhythmogenesis . Out of the 15 genes flanking the gene desert, only Pitx2 expression was deregulated in the sinoatrial node. During development, cardiac Pitx2c expression is normally restricted to the left side where it drives left sinus venosus and left atrial morphogenesis and prevents the formation of a left-sided sinoatrial node. The PITX2 locus has also been associated with atrial fibrillation in GWAS. Immunohistochemistry together with 3D volume analysis showed that Pitx2c was ectopically expressed in half of the pacemaker cardiomyocytes of the developing and postnatal sinoatrial node while its expression in the left atrium was maintained. Key transcription factors that drive pacemaker cell differentiation, Tbx3, Isl1 and Shox2, were lost where Pitx2c was ectopically expressed. Moreover, a conserved long non-coding RNA (Playrr) previously linked to Pitx2 regulation in the developing dorsal mesentery, was downregulated in the sinoatrial node of homozygous mutants. The downregulation of the pacemaker gene program additionally coincided with the activation of the atrial myocardial gene program in half of the pacemaker cardiomyocytes. Conclusion We generated a mouse model of a novel human cardiac syndrome including sinoatrial node dysfunction associated with the deletion of critical CTCF binding sites in a gene desert on chromosome 4q25. This deletion induced ectopic Pitx2c expression in the sinoatrial node alongside a substantial loss of pacemaker cardiomyocyte identity and a gain in a working atrial myocardium-like phenotype in the sinoatrial node of affected mice.Loss of pacemaker cardiomyocyte identitySinoatrial node dysfunction in mutants

Paucity of physiologically relevant cardiac models has limited the widespread application of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes in drug development. Here, we performed comprehensive characterization of hiPSC-derived cardiomyocyte subtypes from 2D and 3D cultures and established a novel 3D model to study impulse initiation and propagation. Directed differentiation approaches were used to generate sinoatrial nodal (SANCM), atrial (ACM) and ventricular cardiomyocytes (VCM). Single cell RNA sequencing established that the protocols yield distinct cell populations in line with expected identities, which was also confirmed by electrophysiological characterization. In 3D EHT cultures of all subtypes, we observed prominent expression of stretch-responsive genes such as NPPA. Response to rate modulating drugs noradrenaline, carbachol and ivabradine were comparable in single cells and EHTs. Differences in the speed of impulse propagation between the subtypes were more pronounced in EHTs compared with 2D monolayers owing to a progressive increase in conduction velocities in atrial and ventricular cardiomyocytes, in line with a more mature phenotype. In a novel binary EHT model of pacemaker-atrial interface, the SANCM end of the tissue consistently paced the EHTs under baseline conditions, which was inhibited by ivabradine. Taken together, our data provide comprehensive insights into molecular and electrophysiological properties of hiPSC-derived cardiomyocyte subtypes, facilitating the creation of next generation composite cardiac models for drug discovery, disease modeling and cell-based regenerative therapies.

Each heartbeat is triggered by the sinoatrial node (SAN), the primary pacemaker of the heart. Studies in animal models have revealed that pacemaker cells share a common progenitor with the (pro)epicardium, and that the pacemaker cardiomyocytes further diversify into ‘transitional’, ‘tail’, and ‘head’ subtypes. However, the underlying molecular mechanisms, especially of human pacemaker cell development, are poorly understood. Here, we performed single cell RNA sequencing (scRNA-seq) and trajectory inference on human induced pluripotent stem cells (hiPSCs) differentiating to SAN-like cardiomyocytes (SANCMs) to construct a roadmap of transcriptional changes and lineage decisions. In differentiated SANCM, we identified distinct clusters that closely resemble different subpopulations of the in vivo SAN. Moreover, the presence of a side population of proepicardial cells suggested their shared ontogeny with SANCM, as also reported in vivo. Our results demonstrate that the divergence of SANCM and proepicardial lineages is determined by WNT signaling. Furthermore, we uncovered roles for TGFβ and WNT signaling in the branching of transitional and head SANCM subtypes, respectively. These findings provide new insights into the molecular processes involved in human pacemaker cell differentiation, opening new avenues for complex disease modeling in vitro and inform approaches for cell therapy-based regeneration of the SAN.

Voltage-gated sodium channels play a critical role in the action potential (AP) upstroke velocity and impulse propagation in the heart. Sodium channel gene therapy is challenged by the relatively large transgene size of Sodium Voltage-Gated Channel Alpha Subunit 5 (SCN5A), the main alpha-subunit of the cardiac sodium channel isoform. Our previous work demonstrated that SCN10Ashort, a small fragment comprising of the C-terminus of SCN10A, increased sodium current when co-expressed with SCN5A. In the present study we therefore explored SCN10Ashort as a novel gene therapy target.

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Research council starting grant 714866 and associated proof-of-concept grant 899422 ZonMW and the Dutch Heart foundation MKMD grant 114021512 and Dutch Heart Foundation Dekker fellowship 2020T023 Rationale: Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes represent an excellent in vitro platform to study cardiac development and model patient-specific diseases. However, their widespread application in drug discovery and regenerative approaches has not yet been realized at least in part due to limited comparative characterization of the available cardiomyocyte subtypes and lack of multicellular models that allow assessment of physiologically relevant parameters. Objective Perform comprehensive characterization of hiPSC-derived sinoatrial nodal cardiomyocytes (SANCM), atrial cardiomyocytes (ACM) and ventricular cardiomyocytes (VCM) from 2D and 3D cultures. Methods SANCM, ACM and VCM were generated using directed differentiation protocols. Electrophysiological analysis was performed by single cell patch-clamp. Subtype-specific differences were further characterized by response to neurohumoral agents noradrenaline, and carbachol and If blocker ivabradine. Next, we generated EHTs to evaluate the effect of 3D culturing on cardiomyocyte subtypes and performed optical mapping. To establish a model to study impulse initiation and propagation in vitro, we generated binary EHTs (BIN-EHTs) composed of heteropolar ends of SANCM and ACM. Results Gene expression analysis and single cell electrophysiology confirmed identities of respective cardiomyocyte subtypes. While response to noradrenaline and carbachol were as expected, ivabradine testing showed the functional presence of pacemaker current If in ACM, besides SANCM. After three weeks in culture as EHTs, cardiac structural genes were markedly upregulated in all groups. Optical mapping demonstrated a three-fold increase in conduction velocities in ACM and VCM while SANCM EHTs retained slower conduction velocities recapitulating in vivo differences. In BIN-EHTs, SANCM end of the EHT consistently paced the tissues under baseline conditions. Upon treatment with ivabradine, cycle length of BIN-EHTs increased and impulse initiation switched to ACM end in the majority of tissues. Conclusions We performed comprehensive characterization of hiPSC-cardiomyocyte subtypes, which recapitulated salient features of their in vivo counterparts. BIN-EHT constructs composed of SANCM and ACM are a valuable rudimentary model for investigating impulse formation and propagation in vitro.

Background: TTN (Titin), the largest protein in humans, forms the molecular spring that spans half of the sarcomere to provide passive elasticity to the cardiomyocyte. Mutations that disrupt the TTN transcript are the most frequent cause of hereditary heart failure. We showed before that TTN produces a class of circular RNAs (circRNAs) that depend on RBM20 to be formed. In this study, we show that the back-splice junction formed by this class of circRNAs creates a unique motif that binds SRSF10 to enable it to regulate splicing. Furthermore, we show that one of these circRNAs (cTTN1) distorts both localization of and splicing by RBM20. Methods: We calculated genetic constraint of the identified motif in 125 748 exomes collected from the gnomAD database. Furthermore, we focused on the highest expressed RBM20-dependent circRNA in the human heart, which we named cTTN1. We used shRNAs directed to the back-splice junction to induce selective loss of cTTN1 in human induced pluripotent stem cell–derived cardiomyocytes. Results: Human genetics suggests reduced genetic tolerance of the generated motif, indicating that mutations in this motif might lead to disease. RNA immunoprecipitation confirmed binding of circRNAs with this motif to SRSF10. Selective loss of cTTN1 in human induced pluripotent stem cell–derived cardiomyocytes induced structural abnormalities, apoptosis, and reduced contractile force in engineered heart tissue. In line with its SRSF10 binding, loss of cTTN1 caused abnormal splicing of important cardiomyocyte SRSF10 targets such as MEF2A and CASQ2 . Strikingly, loss of cTTN1 also caused abnormal splicing of TTN itself. Mechanistically, we show that loss of cTTN1 distorts both localization of and splicing by RBM20. Conclusions: We demonstrate that circRNAs formed from the TTN transcript are essential for normal splicing of key muscle genes by enabling splice regulators RBM20 and SRSF10. This shows that the TTN transcript also has regulatory roles, besides its well-known signaling and structural function. In addition, we demonstrate that the specific sequence created by the back-splice junction of these circRNAs has important functions. This highlights the existence of functionally important sequences that cannot be recognized as such in the human genome but provides an as-yet unrecognized source for functional sequence variation.

Retinoic acid (RA) signaling plays an important role during heart development in establishing anteroposterior polarity, formation of inflow and outflow tract progenitors, and growth of the ventricular compact wall. RA is also utilized as a key ingredient in protocols designed for generating cardiac cell types from pluripotent stem cells (PSCs). This review discusses the role of RA in cardiogenesis, currently available protocols that employ RA for differentiation of various cardiovascular lineages, and plausible transcriptional mechanisms underlying this fate specification. These insights will inform further development of desired cardiac cell types from human PSCs and their application in preclinical and clinical research.

Aims Out‐of‐hospital cardiac arrest (OHCA) mostly results from ventricular tachycardia/ventricular fibrillation (VT/VF), often triggered by acute myocardial infarction (AMI). Sulfonylurea (SU) antidiabetics can block myocardial ATP‐regulated K + channels (K ATP channels), activated during AMI, thereby modulating action potential duration (APD). We studied whether SU drugs impact on OHCA risk, and whether these effects are related to APD changes. Methods We conducted a population‐based case–control study in 219 VT/VF‐documented OHCA cases with diabetes and 697 non‐OHCA controls with diabetes. We studied the association of SU drugs (alone or in combination with metformin) with OHCA risk compared to metformin monotherapy, and of individual SU drugs compared to glimepiride, using multivariable logistic regression analysis. We studied the effects of these drugs on APD during simulated ischaemia using patch‐clamp studies in human induced pluripotent stem cell‐derived cardiomyocytes. Results Compared to metformin, use of SU drugs alone or in combination with metformin was associated with reduced OHCA risk (OR SUdrugs‐alone 0.6 [95% CI 0.4–0.9], OR SUdrugs + metformin 0.6 [95% CI 0.4–0.9]). We found no differences in OHCA risk between SU drug users who suffered OHCA inside or outside the context of AMI. Reduction of OHCA risk compared to glimepiride was found with gliclazide (OR adj 0.5 [95% CI 0.3–0.9]), but not glibenclamide (OR adj 1.3 [95% CI 0.6–2.7]); for tolbutamide, the association with reduced OHCA risk just failed to reach statistical significance (OR adj 0.6 [95% CI 0.3–1.002]). Glibenclamide attenuated simulated ischaemia‐induced APD shortening, while the other SU drugs had no effect. Conclusions SU drugs were associated with reduced OHCA risk compared to metformin monotherapy, with gliclazide having a lower risk than glimepiride. The differential effects of SU drugs are not explained by differential effects on APD.

Electronic pacemakers still face major shortcomings that are largely intrinsic to their hardware-based design. Radical improvements can potentially be generated by gene or cell therapy-based biological pacemakers. Our previous work identified adenoviral gene transfer of Hcn2 and SkM1, encoding a “funny current” and skeletal fast sodium current, respectively, as a potent combination to induce short-term biological pacing in dogs with atrioventricular block. To achieve long-term biological pacemaker activity, alternative delivery platforms need to be explored and optimized. The aim of the present study was therefore to investigate the functional delivery of Hcn2/SkM1 via human cardiomyocyte progenitor cells (CPCs). Nucleofection of Hcn2 and SkM1 in CPCs was optimized and gene transfer was determined for Hcn2 and SkM1 in vitro. The modified CPCs were analyzed using patch-clamp for validation and characterization of functional transgene expression. In addition, biophysical properties of Hcn2 and SkM1 were further investigated in lentivirally transduced CPCs by patch-clamp analysis. To compare both modification methods in vivo, CPCs were nucleofected or lentivirally transduced with GFP and injected in the left ventricle of male NOD-SCID mice. After 1 week, hearts were collected and analyzed for GFP expression and cell engraftment. Subsequent functional studies were carried out by computational modeling. Both nucleofection and lentiviral transduction of CPCs resulted in functional gene transfer of Hcn2 and SkM1 channels. However, lentiviral transduction was more efficient than nucleofection-mediated gene transfer and the virally transduced cells survived better in vivo. These data support future use of lentiviral transduction over nucleofection, concerning CPC-based cardiac gene delivery. Detailed patch-clamp studies revealed Hcn2 and Skm1 current kinetics within the range of previously reported values of other cell systems. Finally, computational modeling indicated that CPC-mediated delivery of Hcn2/SkM1 can generate stable pacemaker function in human ventricular myocytes. These modeling studies further illustrated that SkM1 plays an essential role in the final stage of diastolic depolarization, thereby enhancing biological pacemaker functioning delivered by Hcn2. Altogether these studies support further development of CPC-mediated delivery of Hcn2/SkM1 and functional testing in bradycardia models.

Loss of sinoatrial node or atrioventricular node function may lead to severe bradyarrhythmias, requiring the implantation of an electronic pacemaker. These devices come with several short comings such as lack of adequate autonomic responsiveness, recurrent need for battery replacement, suboptimal cardiac output, and an increasing risk for device-related infections. To overcome these shortcomings, biological pacemakers are under development exploring the use of gene and cell therapy to restore cardiac pacing. In this setting, viral vectors may be used to introduce pacemaker function-related genes to augment spontaneous activity. Moreover, overexpression of transcription factors is being explored to transdifferentiate resident cells towards a pacemaker phenotype. Alternatively, several different stem cells are being investigated as a vehicle for pacemaker function-related genes, or as a source for cells that are being differentiated towards a pacemaker phenotype before transplantation. At present, robust proof-of-concept data has accumulated supporting the initiation of first-in-human testing of short-term biological pacemakers using adenoviral gene transfer. Ongoing research efforts focus on the optimization of long-term biological pacing comparing viral vector-mediated gene transfer to stem cell-based approaches.

Atrial fibrillation (AF) is the most common cardiac arrhythmia. About 5-15% of AF patients have a mutation in a cardiac gene, including mutations inKCNA5, encoding the K(v)1.5 alpha-subunit of the ion channel carrying the atrial-specific ultrarapid delayed rectifier K(+)current (I-Kur). Both loss-of-function and gain-of-function AF-related mutations inKCNA5are known, but their effects on action potentials (APs) of human cardiomyocytes have been poorly studied. Here, we assessed the effects of wild-type and mutant I(Kur)on APs of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). We found that atrial-like hiPSC-CMs, generated by a retinoic acid-based differentiation protocol, have APs with faster repolarization compared to ventricular-like hiPSC-CMs, resulting in shorter APs with a lower AP plateau. Native I-Kur, measured as current sensitive to 50 mu M 4-aminopyridine, was 1.88 +/- 0.49 (mean +/- SEM,n= 17) and 0.26 +/- 0.26 pA/pF (n= 17) in atrial- and ventricular-like hiPSC-CMs, respectively. In both atrial- and ventricular-like hiPSC-CMs, I(Kur)blockade had minimal effects on AP parameters. Next, we used dynamic clamp to inject various amounts of a virtual I-Kur, with characteristics as in freshly isolated human atrial myocytes, into 11 atrial-like and 10 ventricular-like hiPSC-CMs, in which native I(Kur)was blocked. Injection of I(Kur)with 100% density shortened the APs, with its effect being strongest on the AP duration at 20% repolarization (APD(20)) of atrial-like hiPSC-CMs. At I(Kur)densities < 100% (compared to 100%), simulating loss-of-function mutations, significant AP prolongation and raise of plateau were observed. At I(Kur)densities > 100%, simulating gain-of-function mutations, APD(20)was decreased in both atrial- and ventricular-like hiPSC-CMs, but only upon a strong increase in I-Kur. In ventricular-like hiPSC-CMs, lowering of the plateau resulted in AP shortening. We conclude that a decrease in I-Kur, mimicking loss-of-function mutations, has a stronger effect on the AP of hiPSC-CMs than an increase, mimicking gain-of-function mutations, whereas in ventricular-like hiPSC-CMs such increase results in AP shortening, causing their AP morphology to become more atrial-like. Effects of native I(Kur)modulation on atrial-like hiPSC-CMs are less pronounced than effects of virtual I(Kur)injection because I(Kur)density of atrial-like hiPSC-CMs is substantially smaller than that of freshly isolated human atrial myocytes.

The adult mammalian heart has poor regenerative capacity. Loss of functional cardiomyocytes following myocardial infarction leads to the replacement of functional muscle by scar tissue. This has a detrimental effect on cardiac function and may lead to heart failure. Potential regeneration of severe cardiac damage would require replacement of dead and damaged cardiomyocytes by transplantation, recruitment of endogenous progenitor cells, or induction of cardiomyocyte proliferation. For more than a decade, clinical trials to ameliorate the injured heart have been under way. However, after evaluation of the outcome of these trials it is evident that the beneficial effects of these cell-based transplantations are only marginal, and beneficial effects, if any, are not caused by regeneration of cardiomyocytes. In recent years, alternative approaches and various cell sources have been studied and suggested for cardiac repair. Recent advances in these cell-based therapies or strategies to activate endogenous cardiac repair are discussed.