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The heterozygous mutation c.155G > T in GNB2 clinically leads to sinus bradycardia and sinus node dysfunction. Here, patient-specific skin fibroblasts of the mutation carrier were used for Sendai virus reprogramming into human induced-pluripotent stem cells (hiPSC). For generating the isogenic control cell line, a CRISPR/Cas9-mediated HDR-repair of the hiPSCs was carried out. Both generated cell lines (GNB2 SV5528, GNB2 K26) maintained a normal karyotype, cell morphology, pluripotency in immunofluoresence and RT-qPCR analysis. Both hiPSC-lines showed differentiation potential into all three germ layers. Differentiated cardiomyocytes of this isogenic set may pave the way for investigating pharmacological rescue strategies for sinus node dysfunction., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [GM is employee and stockholder at DiNAQOR AG. All other authors declare no conflict of interest]., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Aims: Human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCM) could be a helpful tool to study the physiology and diseases of the human atrium. To fulfil this expectation, the electrophysiology of hiPSC-aCM should closely resemble the situation in the human atrium. Data on the contribution of the slowly activating delayed rectifier currents (IKs) to repolarization are lacking for both human atrium and hiPSC-aCM., Methods and Results: Human atrial tissues were obtained from patients with sinus rhythm (SR) or atrial fibrillation (AF). Currents were measured in human atrial cardiomyocytes (aCM) and compared with hiPSC-aCM and used to model IKs contribution to action potential (AP) shape. Action potential was recorded by sharp microelectrodes. HMR-1556 (1 µM) was used to identify IKs and to estimate IKs contribution to repolarization. Less than 50% of hiPSC-aCM and aCM possessed IKs. Frequency of occurrence, current densities, activation/deactivation kinetics, and voltage dependency of IKs did not differ significantly between hiPSC-aCM and aCM, neither in SR nor AF. β-Adrenoceptor stimulation with isoprenaline did not increase IKs neither in aCM nor in hiPSC-aCM. In tissue from SR, block of IKs with HMR-1556 did not lengthen the action potential duration, even when repolarization reserve was reduced by block of the ultra-rapid repolarizing current with 4-aminopyridine or the rapidly activating delayed rectifier potassium outward current with E-4031., Conclusion: I Ks exists in hiPSC-aCM with biophysics not different from aCM. As in adult human atrium (SR and AF), IKs does not appear to relevantly contribute to repolarization in hiPSC-aCM., Competing Interests: Conflict of interest: none declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Retinoic acid (RA) induces an atrial phenotype in human induced pluripotent stem cells (hiPSCs), but expression of atrium-selective currents such as the ultrarapid (I Kur ) and acetylcholine-stimulated K + current is variable and less than in the adult human atrium. We suspected methodological issues and systematically investigated the concentration dependency of RA. RA treatment increased I Kur concentration dependently from 1.1 ± 0.54 pA/pF (0 RA) to 3.8 ± 1.1, 5.8 ± 2.5, and 12.2 ± 4.3 at 0.01, 0.1, and 1 μM, respectively. Only 1 μM RA induced enough I Kur to fully reproduce human atrial action potential (AP) shape and a robust shortening of APs upon carbachol. We found that sterile filtration caused substantial loss of RA. We conclude that 1 μM RA seems to be necessary and sufficient to induce a full atrial AP shape in hiPSC-CM in EHT format. RA concentrations are prone to methodological issues and may profoundly impact the success of atrial differentiation., Competing Interests: Declaration of interests T.E. is consultant and shareholder of Dinaqor AG., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Primary carnitine deficiency (PCD) is an autosomal recessive monogenic disorder caused by mutations in SLC22A5. This gene encodes for OCTN2, which transports the essential metabolite carnitine into the cell. PCD patients suffer from muscular weakness and dilated cardiomyopathy. Two OCTN2-defective human induced pluripotent stem cell lines were generated, carrying a full OCTN2 knockout and a homozygous OCTN2 (N32S) loss-of-function mutation. OCTN2-defective genotypes showed lower force development and resting length in engineered heart tissue format compared with isogenic control. Force was sensitive to fatty acid-based media and associated with lipid accumulation, mitochondrial alteration, higher glucose uptake, and metabolic remodeling, replicating findings in animal models. The concordant results of OCTN2 (N32S) and -knockout emphasizes the relevance of OCTN2 for these findings. Importantly, genome-wide analysis and pharmacological inhibitor experiments identified ferroptosis, an iron- and lipid-dependent cell death pathway associated with fibroblast activation as a novel PCD cardiomyopathy disease mechanism., Competing Interests: Declaration of interests T.E. is a member of the DiNAQOR Scientific Advisory Board and holds shares in DiNAQOR., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Electrical remodeling in human persistent atrial fibrillation is believed to result from rapid electrical activation of the atria, but underlying genetic causes may contribute. Indeed, common gene variants in an enhancer region close to PITX2 (paired-like homeodomain transcription factor 2) are strongly associated with atrial fibrillation, but the mechanism behind this association remains unknown. This study evaluated the consequences of PITX2 deletion (PITX2 -/- ) in human induced pluripotent stem cell-derived atrial cardiomyocytes., Methods: CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) was used to delete PITX2 in a healthy human iPSC line that served as isogenic control. Human induced pluripotent stem cell-derived atrial cardiomyocytes were differentiated with unfiltered retinoic acid and cultured in atrial engineered heart tissue. Force and action potential were measured in atrial engineered heart tissues. Single human induced pluripotent stem cell-derived atrial cardiomyocytes were isolated from atrial engineered heart tissue for ion current measurements., Results: PITX2 -/- atrial engineered heart tissue beats slightly slower than isogenic control without irregularity. Force was lower in PITX2 -/- than in isogenic control (0.053±0.015 versus 0.131±0.017 mN, n=28/3 versus n=28/4, PITX2 -/- versus isogenic control; P <0.0001), accompanied by lower expression of CACNA1C and lower L-type Ca 2+ current density. Early repolarization was weaker (action potential duration at 20% repolarization; 45.5±13.2 versus 8.6±5.3 ms, n=18/3 versus n=12/4, PITX2 -/- versus isogenic control; P <0.0001), and maximum diastolic potential was more negative (-78.3±3.1 versus -69.7±0.6 mV, n=18/3 versus n=12/4, PITX2 -/- versus isogenic control; P =0.001), despite normal inward rectifier currents (both I K1 and I K,ACh ) and carbachol-induced shortening of action potential duration., Conclusions: Complete PITX2 deficiency in human induced pluripotent stem cell-derived atrial cardiomyocytes recapitulates some findings of electrical remodeling of atrial fibrillation in the absence of fast beating, indicating that these abnormalities could be primary consequences of lower PITX2 levels.

The physiological importance of NCX in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is not well characterized but may depend on the relative strength of the current, compared to adult cardiomyocytes, and on the exact spatial arrangement of proteins involved in Ca2+ extrusion. Here, we determined NCX currents and its contribution to action potential and force in hiPSC-CMs cultured in engineered heart tissue (EHT). The results were compared with data from rat and human left ventricular tissue. The NCX currents in hiPSC-CMs were larger than in ventricular cardiomyocytes isolated from human left ventricles (1.3 ± 0.2 pA/pF and 3.2 ± 0.2 pA/pF for human ventricle and EHT, respectively, p < 0.05). SEA0400 (10 µM) markedly shortened the APD90 in EHT (by 26.6 ± 5%, p < 0.05) and, to a lesser extent, in rat ventricular tissue (by 10.7 ± 1.6%, p < 0.05). Shortening in human left ventricular preparations was small and not different from time-matched controls (TMCs; p > 0.05). Force was increased by the NCX block in rat ventricle (by 31 ± 5.4%, p < 0.05) and EHT (by 20.8 ± 3.9%, p < 0.05), but not in human left ventricular preparations. In conclusion, hiPSC-CMs possess NCX currents not smaller than human left ventricular tissue. Robust NCX block-induced APD shortening and inotropy makes EHT an attractive pharmacological model.

Myocardial injury leads to an irreversible loss of cardiomyocytes (CM). The implantation of human engineered heart tissue (EHT) has become a promising regenerative approach. Previous studies exhibited beneficial, dose-dependent effects of human induced pluripotent stem cell (hiPSC)-derived EHT patch transplantation in a guinea pig model in the subacute phase of myocardial injury. Yet, advanced heart failure often results from a chronic remodeling process. Therefore, from a clinical standpoint it is worthwhile to explore the ability to repair the chronically injured heart. In this study human EHT patches were generated from hiPSC-derived CMs (15 × 10 6 cells) and implanted epicardially four weeks after injury in a guinea pig cryo-injury model. Cardiac function was evaluated by echocardiography after a follow-up period of four weeks. Hearts revealed large transmural myocardial injuries amounting to 27% of the left ventricle. EHT recipient hearts demonstrated compact muscle islands of human origin in the scar region, as indicated by a positive staining for human Ku80 and dystrophin, remuscularizing 5% of the scar area. Echocardiographic analysis demonstrated no significant functional difference between animals that received EHT patches and animals in the cell-free control group (fractional area change 36% vs. 34%). Thus, EHT patches engrafted in the chronically injured heart but in contrast to the subacute model, grafts were smaller and EHT patch transplantation did not improve left ventricular function, highlighting the difficulties for a regenerative approach., (Copyright © 2022. Published by Elsevier Ltd.)

Pluripotent stem-cell-derived cardiomyocytes (PSC-CMs) provide an unprecedented opportunity to study human heart development and disease, but they are functionally and structurally immature. Here, we induce efficient human PSC-CM (hPSC-CM) maturation through metabolic-pathway modulations. Specifically, we find that peroxisome-proliferator-associated receptor (PPAR) signaling regulates glycolysis and fatty acid oxidation (FAO) in an isoform-specific manner. While PPARalpha (PPARa) is the most active isoform in hPSC-CMs, PPARdelta (PPARd) activation efficiently upregulates the gene regulatory networks underlying FAO, increases mitochondrial and peroxisome content, enhances mitochondrial cristae formation, and augments FAO flux. PPARd activation further increases binucleation, enhances myofibril organization, and improves contractility. Transient lactate exposure, which is frequently used for hPSC-CM purification, induces an independent cardiac maturation program but, when combined with PPARd activation, still enhances oxidative metabolism. In summary, we investigate multiple metabolic modifications in hPSC-CMs and identify a role for PPARd signaling in inducing the metabolic switch from glycolysis to FAO in hPSC-CMs., Competing Interests: Declaration of interests The authors declare no competing interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Phospholamban (PLN) is the natural inhibitor of the sarco/endoplasmic reticulum Ca 2+ ATP-ase (SERCA2a). Heterozygous PLN p.Arg14del mutation is associated with an arrhythmogenic dilated cardiomyopathy (DCM), whose pathogenesis has been attributed to SERCA2a "superinhibition"., Aim: To test in cardiomyocytes (hiPSC-CMs) derived from a PLN p.Arg14del carrier whether (1) Ca 2+ dynamics and protein localization were compatible with SERCA2a superinhibition and (2) if functional abnormalities could be reverted by pharmacological SERCA2a activation (PST3093)., Methods: Ca 2+ transients (CaT) were recorded at 36 °C in hiPSC-CMs clusters during field stimulation. SERCA2a and PLN where immunolabeled in single hiPSC-CMs. Mutant preparations (MUT) were compared to isogenic wild-type ones (WT), obtained by mutation reversal., Results: WT and MUT differed for the following properties: (1) CaT time to peak (t peak ) and half-time of CaT decay were shorter in MUT; (2) several CaT profiles were identified in WT, "hyperdynamic" ones largely prevailed in MUT; (3) whereas t peak rate-dependently declined in WT, it was shorter and rate-independent in MUT; (4) diastolic Ca 2+ rate-dependently accumulated in WT, but not in MUT. When applied to WT, PST3093 turned all the above properties to resemble those of MUT; when applied to MUT, PST3093 had a smaller or negligible effect. Preferential perinuclear SERCA2a-PLN localization was lost in MUT hiPSC-CMs., Conclusions: Functional data converge to argue for PLN p.Arg14del incompetence in inhibiting SERCA2a in the tested case, thus weakening the rationale for therapeutic SERCA2a activation. Mechanisms alternative to SERCA2a superinhibition should be considered in the pathogenesis of DCM, possibly including dysregulation of Ca 2+ -dependent transcription.

Background: There is ongoing interest in generating cardiomyocytes derived from human induced pluripotent stem cells (hiPSC) to study human cardiac physiology and pathophysiology. Recently we found that norepinephrine-stimulated calcium currents (I Ca ) in hiPSC-cardiomyocytes were smaller in conventional monolayers (ML) than in engineered heart tissue (EHT). In order to elucidate culture specific regulation of β 1 -adrenoceptor (β 1 -AR) responses we investigated whether action of phosphodiesterases (PDEs) may limit norepinephrine effects on I Ca and on cytosolic cAMP in hiPSC-cardiomyocytes. Results were compared to adult human atrial cardiomyocytes., Methods: Adult human atrial cardiomyocytes were isolated from tissue samples obtained during open heart surgery. All patients were in sinus rhythm. HiPSC-cardiomyocytes were dissociated from ML and EHT. Förster-resonance energy transfer (FRET) was used to monitor cytosolic cAMP (Epac1-camps sensor, transfected by adenovirus). I Ca was recorded by whole-cell patch clamp technique. Cilostamide (300 nM) and rolipram (10 μM) were used to inhibit PDE3 and PDE4, respectively. β 1 -AR were stimulated with the physiological agonist norepinephrine (100 μM)., Results: In adult human atrial cardiomyocytes, norepinephrine increased cytosolic cAMP FRET ratio by +13.7 ± 1.5% (n = 10/9, mean ± SEM, number of cells/number patients) and I Ca by +10.4 ± 1.5 pA/pF (n = 15/10). This effect was not further increased in the concomitant presence of rolipram, cilostamide and norepinephrine, indicating saturation by norepinephrine alone. In ML hiPSC-cardiomyocytes, norepinephrine exerted smaller increases in cytosolic cAMP and I Ca (FRET +9.6 ± 0.5% n = 52/21, number of cells/number of ML or EHT, and I Ca  + 1.4 ± 0.2 pA/pF n = 34/7, p < 0.05 each) and both were augmented in the presence of the PDE4 inhibitor rolipram (FRET +16.7 ± 0.8% n = 94/26 and I Ca  + 5.6 ± 1.4 pA/pF n = 11/5, p < 0.05 each). Cilostamide increased the response to norepinephrine on FRET (+12.7 ± 0.5% n = 91/19, p < 0.05), but not on I Ca . In EHT hiPSC-cardiomyocytes, norepinephrine responses on both, FRET and I Ca , were larger than in ML (FRET +12.1 ± 0.3% n = 87/32 and I Ca  + 3.3 ± 0.2 pA/pF n = 13/5, p < 0.05 each). Rolipram augmented the norepinephrine effect on I Ca (+6.2 ± 1.6 pA/pF; p < 0.05 vs. norepinephrine alone, n = 10/4), but not on FRET., Conclusion: Our results show culture-dependent differences in hiPSC-cardiomyocytes. In conventional ML but not in EHT, maximum norepinephrine effects on cytosolic cAMP depend on PDE3 and PDE4, suggesting immaturity when compared to the situation in adult human atrial cardiomyocytes. The smaller I Ca responses to norepinephrine in ML and EHT vs. adult human atrial cardiomyocytes depend at least partially on a non-physiological large impact of PDE4 in hiPSC-cardiomyocytes., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Abstract: Atrial tachypacing is an accepted model for atrial fibrillation (AF) in large animals and in cellular models. Human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CM) provide a novel human source to model cardiovascular diseases. Here, we investigated whether optogenetic tachypacing of atrial-like hiPSC-CMs grown into engineered heart tissue (aEHT) can induce AF-remodeling. After differentiation of atrial-like cardiomyocytes from hiPSCs using retinoic acid, aEHTs were generated from ∼1 million atrial-like hiPSC-CMs per aEHT. AEHTs were transduced with lentivirus expressing channelrhodopsin-2 to enable optogenetic stimulation by blue light pulses. AEHTs underwent optical tachypacing at 5 Hz for 15 seconds twice a minute over 3 weeks and compared with transduced spontaneously beating isogenic aEHTs (1.95 ± 0.07 Hz). Force and action potential duration did not differ between spontaneously beating and tachypaced aEHTs. Action potentials in tachypaced aEHTs showed higher upstroke velocity (138 ± 15 vs. 87 ± 11 V/s, n = 15-13/3; P = 0.018), possibly corresponding to a tendency for more negative diastolic potentials (73.0 ± 1.8 vs. 68.0 ± 1.9 mV; P = 0.07). Tachypaced aEHTs exhibited a more irregular spontaneous beating pattern (beat-to-beat scatter: 0.07 ± 0.01 vs. 0.03 ± 0.004 seconds, n = 15-13/3; P = 0.008). Targeted expression analysis showed higher RNA levels of KCNJ12 [Kir2.2, inward rectifier (IK1); 69 ± 7 vs. 44 ± 4, P = 0.014] and NPPB (NT-proBNP; 39,690 ± 4834 vs. 23,671 ± 3691; P = 0.024). Intermittent tachypacing in aEHTs induces some electrical alterations found in AF and induces an arrhythmic spontaneous beating pattern, but does not affect resting force. Further studies using longer, continuous, or more aggressive stimulation may clarify the contribution of different rate patterns on the changes in aEHT mimicking the remodeling process from paroxysmal to persistent atrial fibrillation., Competing Interests: T. Eschenhagen and A. Hansen are co-founders of EHT Technologies, Hamburg. The other authors report no conflicts of interest., (Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc.)

The reproducibility of stem cell research relies on the constant availability of quality-controlled cells. As the quality of human induced pluripotent stem cells (hiPSCs) can deteriorate in the course of a few passages, cell banking is key to achieve consistent results and low batch-to-batch variation. Here, we provide a cost-efficient route to generate master and working cell banks for basic research projects. In addition, we describe minimal protocols for quality assurance including tests for sterility, viability, pluripotency, and genetic integrity. © 2020 The Authors. Basic Protocol 1: Expansion of hiPSCs Basic Protocol 2: Cell banking of hiPSCs Support Protocol 1: Pluripotency assessment by flow cytometry Support Protocol 2: Thawing control: Viability and sterility Support Protocol 3: Potency, viral clearance, and pluripotency: Spontaneous differentiation and qRT-PCR Support Protocol 4: Identity: Short tandem repeat analysis., (© 2020 The Authors.)

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are commercially available, and cardiac differentiation established routine. Systematic evaluation of several control hiPSC-CM is lacking. We investigated 10 different control hiPSC-CM lines and analyzed function and suitability for drug screening. Five commercial and 5 academic hPSC-CM lines were casted in engineered heart tissue (EHT) format. Spontaneous and stimulated EHT contractions were analyzed, and 7 inotropic indicator compounds investigated on 8 cell lines. Baseline contractile force, kinetics, and rate varied widely among the different lines (e.g., relaxation time range: 118-471 ms). In contrast, the qualitative correctness of responses to BayK-8644, nifedipine, EMD-57033, isoprenaline, and digoxin in terms of force and kinetics varied only between 80% and 93%. Large baseline differences between control cell lines support the request for isogenic controls in disease modeling. Variability appears less relevant for drug screening but needs to be considered, arguing for studies with more than one line., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Animal models are 78% accurate in determining whether drugs will alter contractility of the human heart. To evaluate the suitability of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for predictive safety pharmacology, we quantified changes in contractility, voltage, and/or Ca2+ handling in 2D monolayers or 3D engineered heart tissues (EHTs). Protocols were unified via a drug training set, allowing subsequent blinded multicenter evaluation of drugs with known positive, negative, or neutral inotropic effects. Accuracy ranged from 44% to 85% across the platform-cell configurations, indicating the need to refine test conditions. This was achieved by adopting approaches to reduce signal-to-noise ratio, reduce spontaneous beat rate to ≤ 1 Hz or enable chronic testing, improving accuracy to 85% for monolayers and 93% for EHTs. Contraction amplitude was a good predictor of negative inotropes across all the platform-cell configurations and of positive inotropes in the 3D EHTs. Although contraction- and relaxation-time provided confirmatory readouts forpositive inotropes in 3D EHTs, these parameters typically served as the primary source of predictivity in 2D. The reliance of these "secondary" parameters to inotropy in the 2D systems was not automatically intuitive and may be a quirk of hiPSC-CMs, hence require adaptations in interpreting the data from this model system. Of the platform-cell configurations, responses in EHTs aligned most closely to the free therapeutic plasma concentration. This study adds to the notion that hiPSC-CMs could add value to drug safety evaluation., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society of Toxicology.)

Background and Purpose: Phosphodiesterases (PDEs) are important regulators of β-adrenoceptor signalling in the heart. While PDE4 is the most important isoform that regulates I Ca,L and force in rodent cardiomyocytes, the dominant isoform in adult human cardiomyocytes is PDE3., Experimental Approach: Given the potential of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for biomedical research, this study characterized the contribution of PDE3 and PDE4 isoforms to the regulation of I Ca,L and force in hiPSC-CMs in an engineered heart tissue (EHT) model., Key Results: There was a lower abundance of mRNA for PDE3A and 4A in hiPSC-CM EHT than in non-failing human heart samples. Selective inhibition of PDE3 and 4 with cilostamide and rolipram, respectively, showed that, in hiPSC-CM, PDE4 was the predominant isoform for the regulation of I Ca,L (cilostamide: +1.44-fold; rolipram: +1.77-fold) . Furthermore, in contrast to cilostamide, rolipram decreased the EC 50 of isoprenaline about 15-fold., Conclusion and Implications: The predominance of PDE4 over PDE3 is a peculiarity of hiPSC-CMs and is probably an indicator of immaturity. This finding has implications for the use of hiPSC-CM as pharmacological models to investigate and assess the effects of PDE inhibitors., (© 2020 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Aims: Chronic tachypacing is commonly used in animals to induce cardiac dysfunction and to study mechanisms of heart failure and arrhythmogenesis. Human induced pluripotent stem cells (hiPSC) may replace animal models to overcome species differences and ethical problems. Here, 3D engineered heart tissue (EHT) was used to investigate the effect of chronic tachypacing on hiPSC-cardiomyocytes (hiPSC-CMs)., Methods and Results: To avoid cell toxicity by electrical pacing, we developed an optogenetic approach. EHTs were transduced with lentivirus expressing channelrhodopsin-2 (H134R) and stimulated by 15 s bursts of blue light pulses (0.3 mW/mm2, 30 ms, 3 Hz) separated by 15 s without pacing for 3 weeks. Chronic optical tachypacing did not affect contractile peak force, but induced faster contraction kinetics, shorter action potentials, and shorter effective refractory periods. This electrical remodelling increased vulnerability to tachycardia episodes upon electrical burst pacing. Lower calsequestrin 2 protein levels, faster diastolic depolarization (DD) and efficacy of JTV-519 (46% at 1 µmol/L) to terminate tachycardia indicate alterations of Ca2+ handling being part of the underlying mechanism. However, other antiarrhythmic compounds like flecainide (69% at 1 µmol/L) and E-4031 (100% at 1 µmol/L) were also effective, but not ivabradine (1 µmol/L) or SEA0400 (10 µmol/L)., Conclusion: We demonstrated a high vulnerability to tachycardia of optically tachypaced hiPSC-CMs in EHT and the effective termination by ryanodine receptor stabilization, sodium or hERG potassium channel inhibition. This new model might serve as a preclinical tool to test antiarrhythmic drugs increasing the insight in treating ventricular tachycardia., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent an unlimited source of human CMs that could be a standard tool in drug research. However, there is concern whether hiPSC-CMs express all cardiac ion channels at physiological level and whether they might express non-cardiac ion channels. In a control hiPSC line, we found large, "noisy" outward K + currents, when we measured outward potassium currents in isolated hiPSC-CMs. Currents were sensitive to iberiotoxin, the selective blocker of big conductance Ca 2+ -activated K + current (I BK,Ca ). Seven of 16 individual differentiation batches showed a strong initial repolarization in the action potentials (AP) recorded from engineered heart tissue (EHT) followed by very early afterdepolarizations, sometimes even with consecutive oscillations. Iberiotoxin stopped oscillations and normalized AP shape, but had no effect in other EHTs without oscillations or in human left ventricular tissue (LV). Expression levels of the alpha-subunit (K Ca 1.1) of the BK Ca correlated with the presence of oscillations in hiPSC-CMs and was not detectable in LV. Taken together, individual batches of hiPSC-CMs can express sarcolemmal ion channels that are otherwise not found in the human heart, resulting in oscillating afterdepolarizations in the AP. HiPSC-CMs should be screened for expression of non-cardiac ion channels before being applied to drug research.

Human iPSC-derived engineered heart tissue (hEHT) has been used to remuscularize injured hearts in a guinea pig infarction model. While beneficial effects on cardiac remodeling have been demonstrated, the arrhythmogenic potential of hEHTs is a major concern. We investigated whether hiPSC-derived hEHTs increase the incidence of ventricular arrhythmias. HEHTs were created from human iPSC-derived cardiomyocytes and endothelial cells. Left-ventricular cryo-injury was induced in guinea pigs (n = 37) and telemetry sensors for continuous ECG monitoring were implanted. 7 days following the cryo-injury, hEHTs or cell-free constructs were transplanted into the surviving animals (n = 15 and n = 9). ECGs were recorded over the following 28 days. 10 hEHT animals and 8 control animals survived the observation period and were included in the final analysis. After implantation of hEHTs or cell-free constructs, ventricular arrhythmias (premature ventricular contractions, couplets, triplets and non-sustained ventricular tachycardia) were observed in animals of both groups. The fraction of animals with the respective arrhythmias as well as the rate of arrhythmic events did not differ between groups. Following hEHT implantation, no clinically relevant sustained ventricular tachycardia or ventricular fibrillation was detected. Our telemetric data provides first evidence for the electrical safety of human iPSC-derived EHTs in this experimental model, thereby supporting further development of this approach.

Biological pacemakers could be a promising alternative to electronic pacemakers and human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) may represent a suitable source for implantable cells. To further unravel this potential a thorough understanding of pacemaker function with regard to coupling processes both in the physiological and in the graft-host context is required. Here we developed a 2-component cardiac organoid model with a hiPSC-CM embryoid body (EB) as trigger casted into a rat engineered heart tissue (EHT) as arrhythmic beating substrate. Contractility recordings revealed that the EB controlled the beating activity of the EHT, leading to a regular hiPSC-CM-like beating pattern instead of the irregular beating typically seen in rat EHT. Connectivity was observed with action potential (AP) measurements and calcium transients transmitting from the EB directly into the rat EHT. Immunohistochemistry and genetically labeled hiPSC-CMs demonstrated that EB-derived and rat cells intermingled and formed a transitional zone. Connexin 43 expression followed the same pattern as histological and computer models have indicated for the human sinoatrial node. In conclusion, hiPSC-CM EBs function as a biological pacemaker in a 2-component cardiac organoid model, which provides the possibility to study electrophysiological and structural coupling mechanisms underlying propagation of pacemaker activity., (Copyright © 2019. Published by Elsevier Ltd.)

Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss of relevant amounts of heart muscle mass can lead to the heart failure. Besides heart transplantation, there is no curative treatment option for the end-stage heart failure. In times of organ donor shortage, organ independent treatment modalities are needed. Left-ventricular assist devices are a promising therapy option, however, especially as destination therapy, limited by its side-effects like stroke, infections and bleedings. In recent years, several cardiac repair strategies including stem cell injection, cardiac progenitors or myocardial tissue engineering have been investigated. Recent improvements in cell biology allow for the differentiation of large amounts of cardiomyocytes derived from human induced pluripotent stem cells (iPSC). One of the cardiac repair strategies currently under evaluation is to transplant artificial heart tissue. Engineered heart tissue (EHT) is a three-dimensional in vitro created cardiomyocyte network, with functional properties of native heart tissue. We have created EHT-patches from hiPSC derived cardiomyocytes. Here we present a protocol for the induction of left ventricular myocardial cryoinjury in a guinea pig, followed by implantation of hiPSC derived EHT on the left ventricular wall.

Hypertrophic cardiomyopathy (HCM) is a primary disorder of contractility in heart muscle. To gain mechanistic insight and guide pharmacological rescue, this study models HCM using isogenic pairs of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the E99K-ACTC1 cardiac actin mutation. In both 3D engineered heart tissues and 2D monolayers, arrhythmogenesis was evident in all E99K-ACTC1 hiPSC-CMs. Aberrant phenotypes were most common in hiPSC-CMs produced from the heterozygote father. Unexpectedly, pathological phenotypes were less evident in E99K-expressing hiPSC-CMs from the two sons. Mechanistic insight from Ca 2+ handling expression studies prompted pharmacological rescue experiments, wherein dual dantroline/ranolazine treatment was most effective. Our data are consistent with E99K mutant protein being a central cause of HCM but the three-way interaction between the primary genetic lesion, background (epi)genetics, and donor patient age may influence the pathogenic phenotype. This illustrates the value of isogenic hiPSC-CMs in genotype-phenotype correlations., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Cardiac repolarization abnormalities in drug-induced and genetic long-QT syndrome may lead to afterdepolarizations and life-threatening ventricular arrhythmias. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) should help to overcome the limitations of animal models based on species differences in repolarization reserve. Here, we compared head-to-head the contribution of I Ks (long QT1) and I Kr (long QT2) on action potentials (APs) in human left ventricular (LV) tissue and hiPSC-CM-derived engineered heart tissue (EHT)., Methods: APs were measured with sharp microelectrodes in EHT from 3 different control hiPSC-CM lines and in tissue preparations from failing LV., Results: EHT from hiPSC-CMs showed spontaneous diastolic depolarization and AP generation that were sensitive to low concentrations of ivabradine. I Kr block by E-4031 prolonged AP duration at 90% repolarization with similar half-effective concentration in EHT and LV but larger effect size in EHT (+281 versus +110 ms in LV). Although I Kr block alone evoked early afterdepolarizations and triggered activity in 50% of EHTs, slow pacing, reduced extracellular K + , and blocking of I Kr , I Ks , and I K1 were necessary to induce early afterdepolarizations in LV. In accordance with their clinical safety, moxifloxacin and verapamil did not induce early afterdepolarizations in EHT. In both EHT and LV, I Ks block by HMR-1556 prolonged AP duration at 90% repolarization slightly in the combined presence of E-4031 and isoprenaline., Conclusions: EHT from hiPSC-CMs shows a lower repolarization reserve than human LV working myocardium and could thereby serve as a sensitive and specific human-based model for repolarization studies and arrhythmia, similar to Purkinje fibers. In both human LV and EHT, I Ks only contributed to repolarization under adrenergic stimulation., (© 2018 American Heart Association, Inc.)

Background: Human induced pluripotent stem (iPS) cells have revolutionised research and spark hopes for future tissue replacement therapies. To obtain high cell numbers, iPS cells can be expanded indefinitely. However, as long-term expansion can compromise cell integrity and quality, we set out to assess potential reduction of clonal diversity by inherent growth imbalances., Methods: Using red, green, blue marking as a lentiviral multi-colour clonal cell tracking technology, we marked three different iPS cell lines as well as three other cell lines, assigning a unique fluorescent colour to each cell at one point in culture. Subsequently, we followed the sub-clonal distribution over time by flow cytometry and fluorescence microscopy analysis in regular intervals., Results: In three human iPS cell lines as well as primary human fibroblasts and two widely used human cell lines as controls (K562 and HEK 293 T), we observed a marked reduction in sub-clonal diversity over time of culture (weeks). After 38 passages, all iPS cultures consisted of less than 10 residual clones. Karyotype and function, the latter assessed by cardiomyocyte differentiation and tissue engineering, did not reveal obvious differences., Conclusions: Our results argue for a quick selection of sub-clones with a growth advantage and flag a normally invisible and potentially undesired behaviour of cultured iPS cells, especially when using long-term cultured iPS cells for experiments or even in-vivo applications.

Human induced pluripotent stem cell (hiPSC) cardiomyocytes (CMs) show less negative resting membrane potential (RMP), which is attributed to small inward rectifier currents (I K1 ). Here, I K1 was measured in hiPSC-CMs (proprietary and commercial cell line) cultured as monolayer (ML) or 3D engineered heart tissue (EHT) and, for direct comparison, in CMs from human right atrial (RA) and left ventricular (LV) tissue. RMP was measured in isolated cells and intact tissues. I K1 density in ML- and EHT-CMs from the proprietary line was similar to LV and RA, respectively. I K1 density in EHT-CMs from the commercial line was 2-fold smaller than in the proprietary line. RMP in EHT of both lines was similar to RA and LV. Repolarization fraction and I K,ACh response discriminated best between RA and LV and indicated predominantly ventricular phenotype in hiPSC-CMs/EHT. The data indicate that I K1 is not necessarily low in hiPSC-CMs, and technical issues may underlie low RMP in hiPSC-CMs., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Energy metabolism is a key aspect of cardiomyocyte biology. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising tool for biomedical application, but they are immature and have not undergone metabolic maturation related to early postnatal development. To assess whether cultivation of hiPSC-CMs in 3D engineered heart tissue format leads to maturation of energy metabolism, we analyzed the mitochondrial and metabolic state of 3D hiPSC-CMs and compared it with 2D culture. 3D hiPSC-CMs showed increased mitochondrial mass, DNA content, and protein abundance (proteome). While hiPSC-CMs exhibited the principal ability to use glucose, lactate, and fatty acids as energy substrates irrespective of culture format, hiPSC-CMs in 3D performed more oxidation of glucose, lactate, and fatty acid and less anaerobic glycolysis. The increase in mitochondrial mass and DNA in 3D was diminished by pharmacological reduction of contractile force. In conclusion, contractile work contributes to metabolic maturation of hiPSC-CMs., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a promising tool for drug testing and modelling genetic disorders. Abnormally low upstroke velocity is a current limitation. Here we investigated the use of 3D engineered heart tissue (EHT) as a culture method with greater resemblance to human heart tissue in comparison to standard technique of 2D monolayer (ML) format. I Na was measured in ML or EHT using the standard patch-clamp technique. I Na density was ~1.8 fold larger in EHT (-18.5 ± 1.9 pA/pF; n = 17) than in ML (-10.3 ± 1.2 pA/pF; n = 23; p < 0.001), approaching densities reported for human CM. Inactivation kinetics, voltage dependency of steady-state inactivation and activation of I Na did not differ between EHT and ML and were similar to previously reported values for human CM. Action potential recordings with sharp microelectrodes showed similar upstroke velocities in EHT (219 ± 15 V/s, n = 13) and human left ventricle tissue (LV, 253 ± 7 V/s, n = 25). EHT showed a greater resemblance to LV in CM morphology and subcellular Na V 1.5 distribution. I Na in hiPSC-CM showed similar biophysical properties as in human CM. The EHT format promotes I Na density and action potential upstroke velocity of hiPSC-CM towards adult values, indicating its usefulness as a model for excitability of human cardiac tissue.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) may serve as a new assay for drug testing in a human context, but their validity particularly for the evaluation of inotropic drug effects remains unclear. In this blinded analysis, we compared the effects of 10 indicator compounds with known inotropic effects in electrically stimulated (1.5 Hz) hiPSC-CM-derived 3-dimensional engineered heart tissue (EHT) and human atrial trabeculae (hAT). Human EHTs were prepared from iCell hiPSC-CM, hAT obtained at routine heart surgery. Mean intra-batch variation coefficient in baseline force measurement was 17% for EHT and 49% for hAT. The PDE-inhibitor milrinone did not affect EHT contraction force, but increased force in hAT. Citalopram (selective serotonin reuptake inhibitor), nifedipine (LTCC-blocker) and lidocaine (Na+ channel-blocker) had negative inotropic effects on EHT and hAT. Formoterol (beta-2 agonist) had positive lusitropic but no inotropic effect in EHT, and positive clinotropic, lusitropic, and inotropic effects in hAT. Tacrolimus (calcineurin-inhibitor) had a negative inotropic effect in EHTs, but no effect in hAT. Digoxin (Na+-K+-ATPase-inhibitor) showed a positive inotropic effect only in EHTs, but no effect in hAT probably due to short incubation time. Ryanodine (ryanodine receptor-inhibitor) reduced contraction force in both models. Rolipram and acetylsalicylic acid showed noninterpretable results in hAT. Contraction amplitude and kinetics were more stable over time and less variable in hiPSC-EHTs than hAT. HiPSC-EHT faithfully detected cAMP-dependent and -independent positive and negative inotropic effects, but limited beta-2 adrenergic or PDE3 effects, compatible with an immature CM phenotype., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology.)

Since the advent of the generation of human induced pluripotent stem cells (hiPSCs), numerous protocols have been developed to differentiate hiPSCs into cardiomyocytes and then subsequently assess their ability to recapitulate the properties of adult human cardiomyocytes. However, hiPSC-derived cardiomyocytes (hiPSC-CMs) are often assessed in single-cell assays. A shortcoming of these assays is the limited ability to characterize the physiological parameters of cardiomyocytes, such as contractile force, due to random orientations. This protocol describes the differentiation of cardiomyocytes from hiPSCs, which occurs within 14 d. After casting, cardiomyocytes undergo 3D assembly. This produces fibrin-based engineered heart tissues (EHTs)-in a strip format-that generate force under auxotonic stretch conditions. 10-15 d after casting, the EHTs can be used for contractility measurements. This protocol describes parallel expansion of hiPSCs; standardized generation of defined embryoid bodies, growth factor and small-molecule-based cardiac differentiation; and standardized generation of EHTs. To carry out the protocol, experience in advanced cell culture techniques is required.

Myocardial injury results in a loss of contractile tissue mass that, in the absence of efficient regeneration, is essentially irreversible. Transplantation of human pluripotent stem cell-derived cardiomyocytes has beneficial but variable effects. We created human engineered heart tissue (hEHT) strips from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and hiPSC-derived endothelial cells. The hEHTs were transplanted onto large defects (22% of the left ventricular wall, 35% decline in left ventricular function) of guinea pig hearts 7 days after cryoinjury, and the results were compared with those obtained with human endothelial cell patches (hEETs) or cell-free patches. Twenty-eight days after transplantation, the hearts repaired with hEHT strips exhibited, within the scar, human heart muscle grafts, which had remuscularized 12% of the infarct area. These grafts showed cardiomyocyte proliferation, vascularization, and evidence for electrical coupling to the intact heart tissue in a subset of engrafted hearts. hEHT strips improved left ventricular function by 31% compared to that before implantation, whereas the hEET or cell-free patches had no effect. Together, our study demonstrates that three-dimensional human heart muscle constructs can repair the injured heart., (Copyright © 2016, American Association for the Advancement of Science.)

Analyzing contractile force, the most important and best understood function of cardiomyocytes in vivo is not established in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). This study describes the generation of 3D, strip-format, force-generating engineered heart tissues (EHT) from hiPSC-CM and their physiological and pharmacological properties. CM were differentiated from hiPSC by a growth factor-based three-stage protocol. EHTs were generated and analyzed histologically and functionally. HiPSC-CM in EHTs showed well-developed sarcomeric organization and alignment, and frequent mitochondria. Systematic contractility analysis (26 concentration-response curves) reveals that EHTs replicated canonical response to physiological and pharmacological regulators of inotropy, membrane- and calcium-clock mediators of pacemaking, modulators of ion-channel currents, and proarrhythmic compounds with unprecedented precision. The analysis demonstrates a high degree of similarity between hiPSC-CM in EHT format and native human heart tissue, indicating that human EHTs are useful for preclinical drug testing and disease modeling., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Drug development is time- and cost-intensive and, despite extensive efforts, still hampered by the limited value of current preclinical test systems to predict side effects, including proarrhythmic and cardiotoxic effects in clinical practice. Part of the problem may be related to species-dependent differences in cardiomyocyte biology. Therefore, the event of readily available human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM) has raised hopes that this human test bed could improve preclinical safety pharmacology as well as drug discovery approaches. However, hiPSC-CM are immature and exhibit peculiarities in terms of ion channel function, gene expression, structural organization and functional responses to drugs that limit their present usefulness. Current efforts are thus directed towards improving hiPSC-CM maturity and high-content readouts. Culturing hiPSC-CM as 3-dimensional engineered heart tissue (EHT) improves CM maturity and anisotropy and, in a 24-well format using silicone racks, enables automated, multiplexed high content readout of contractile function. This review summarizes the principal technology and focuses on advantages and disadvantages of this technology and its potential for preclinical drug screening., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Spontaneously beating engineered heart tissue (EHT) represents an advanced in vitro model for drug testing and disease modeling, but cardiomyocytes in EHTs are less mature and generate lower forces than in the adult heart. We devised a novel pacing system integrated in a setup for videooptical recording of EHT contractile function over time and investigated whether sustained electrical field stimulation improved EHT properties. EHTs were generated from neonatal rat heart cells (rEHT, n=96) or human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hEHT, n=19). Pacing with biphasic pulses was initiated on day 4 of culture. REHT continuously paced for 16-18 days at 0.5Hz developed 2.2× higher forces than nonstimulated rEHT. This was reflected by higher cardiomyocyte density in the center of EHTs, increased connexin-43 abundance as investigated by two-photon microscopy and remarkably improved sarcomere ultrastructure including regular M-bands. Further signs of tissue maturation include a rightward shift (to more physiological values) of the Ca(2+)-response curve, increased force response to isoprenaline and decreased spontaneous beating activity. Human EHTs stimulated at 2Hz in the first week and 1.5Hz thereafter developed 1.5× higher forces than nonstimulated hEHT on day 14, an ameliorated muscular network of longitudinally oriented cardiomyocytes and a higher cytoplasm-to-nucleus ratio. Taken together, continuous pacing improved structural and functional properties of rEHTs and hEHTs to an unprecedented level. Electrical stimulation appears to be an important step toward the generation of fully mature EHT., (Copyright © 2014. Published by Elsevier Ltd.)

Omecamtiv mecarbil (OM), a myosin activator, was reported to induce complex concentration- and species-dependent effects on contractile function, and clinical studies indicated a low therapeutic index with diastolic dysfunction at concentrations above 1 mM. To further characterize effects of OM in a human context and under different preload conditions, we constructed a setup that allows isometric contractility analysis of human induced pluripotent stem cell (hiPSC)-derived engineered heart tissues (EHTs). The results were compared with effects of OM on the very same EHTs measured under auxotonic conditions. OM induced a sustained, concentration-dependent increase in time to peak under all conditions (maximally two- to threefold). Peak force, in contrast, was increased by OM only in human, but not rat EHTs and only under isometric conditions, varied between hiPSC lines and showed a biphasic concentration dependency with maximal effects at 1θmM. Relaxation time tended to fall under auxotonic and strongly increased under isometric conditions, again with biphasic concentration dependency. Diastolic tension concentration dependently increased under all conditions. The latter was reduced by an inhibitor of the mitochondrial sodium calcium exchanger (CGP-37157). OM induced increases in mitochondrial oxidation in isolated cardiomyocytes, indicating that OM, an inotrope that does not increase intracellular and mitochondrial Ca2+, can induce mismatch between an increase in ATP and ROS production and unstimulated mitochondrial redox capacity. Taken together, we developed a novel setup well suitable for isometric measurements of EHTs. The effects of OM on contractility and diastolic tension are complex with concentration-, time-, species- and loading-dependent differences. Effects on mitochondrial function require further studies. [ABSTRACT FROM AUTHOR]

Induced pluripotent stem cells (iPSC) constitute a powerful tool to study cardiac physiology and represents a promising treatment strategy to tackle cardiac disease. However, iPSCs remain relatively immature after differentiation. Additionally, engineered heart tissue (EHT) has been investigated as a therapy option in preclinical disease models with promising results, although their vascularization and functionality leave room for improvement. Thymosin β4 (Tβ4) has been shown to promote the differentiation of progenitor cell lines to cardiomyocytes while it also induces angiogenic sprouting and vascular maturation. We examined the potential impact of Tβ4 to enhance maturation of cardiomyocytes from iPSCs. Assessing the expression of transcription factors associated with cardiac differentiation, we were able to demonstrate the increased generation of cells displaying cardiomyocyte characteristics in vitro. Furthermore, we demonstrated, in a zebrafish model of embryonic vascular development, that Tβ4 is crucial for the proper execution of lymphatic and angiogenic vessel sprouting. Finally, utilizing Tβ4-transduced EHTs generated from mice genetically engineered to label endothelial cells in vitro, we show that treatment with Tβ4 promotes vascularization and contractility in EHTs, highlighting Tβ4 as a growth factor improving the formation of cardiomyocytes from iPSC and enhancing the performance of EHTs generated from neonatal cardiomyocytes. [ABSTRACT FROM AUTHOR]

Cardiac tissue engineering aims at repairing the diseased heart and developing cardiac tissues for basic research and predictive toxicology applications. Since the first description of engineered heart tissue 15 years ago, major development steps were directed toward these three goals. Technical innovations led to improved three-dimensional cardiac tissue structure and near physiological contractile force development. Automation and standardization allow medium throughput screening. Larger constructs composed of many small engineered heart tissues or stacked cell sheet tissues were tested for cardiac repair and were associated with functional improvements in rats. Whether these approaches can be simply transferred to larger animals or the human patients remains to be tested. The availability of an unrestricted human cardiac myocyte cell source from human embryonic stem cells or human-induced pluripotent stem cells is a major breakthrough. This review summarizes current tissue engineering techniques with their strengths and limitations and possible future applications. [ABSTRACT FROM AUTHOR]

Intermittent hypoxia and various pharmacological compounds protect the heart from ischemia reperfusion injury in experimental approaches, but the translation into clinical trials has largely failed. One reason may lie in species differences and the lack of suitable human in vitro models to test for ischemia/reperfusion. We aimed to develop a novel hypoxia-reoxygenation model based on three-dimensional, spontaneously beating and work performing engineered heart tissue (EHT) from rat and human cardiomyocytes. Contractile force, the most important cardiac performance parameter, served as an integrated outcome measure. EHTs from neonatal rat cardiomyocytes were subjected to 90 min of hypoxia which led to cardiomyocyte apoptosis as revealed by caspase 3-staining, increased troponin I release (time control vs. 24 h after hypoxia: cTnI 2.7 vs. 6.3 ng/mL, ** p = 0.002) and decreased contractile force (64 ± 6% of baseline) in the long-term follow-up. The detrimental effects were attenuated by preceding the long-term hypoxia with three cycles of 10 min hypoxia (i.e., hypoxic preconditioning). Similarly, [d-Ala2, d-Leu5]-enkephalin (DADLE) reduced the effect of hypoxia on force (recovery to 78 ± 5% of baseline with DADLE preconditioning vs. 57 ± 5% without, p = 0.012), apoptosis and cardiomyocyte stress. Human EHTs presented a comparable hypoxia-induced reduction in force (55 ± 5% of baseline), but DADLE failed to precondition them, likely due to the absence of δ-opioid receptors. In summary, this hypoxia-reoxygenation in vitro model displays cellular damage and the decline of contractile function after hypoxia allows the investigation of preconditioning strategies and will therefore help us to understand the discrepancy between successful conditioning in vitro experiments and its failure in clinical trials. [ABSTRACT FROM AUTHOR]

Background: Cardiac disease modelling using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) requires thorough insight into cardiac cell type differentiation processes. However, current methods to discriminate different cardiac cell types are mostly time-consuming, are costly and often provide imprecise phenotypic evaluation. DNA methylation plays a critical role during early heart development and cardiac cellular specification. We therefore investigated the DNA methylation pattern in different cardiac tissues to identify CpG loci for further cardiac cell type characterization. Results: An array-based genome-wide DNA methylation analysis using Illumina Infinium HumanMethylation450 BeadChips led to the identification of 168 differentially methylated CpG loci in atrial and ventricular human heart tissue samples (n = 49) from different patients with congenital heart defects (CHD). Systematic evaluation of atrial-ventricular DNA methylation pattern in cardiac tissues in an independent sample cohort of non-failing donor hearts and cardiac patients using bisulfite pyrosequencing helped us to define a subset of 16 differentially methylated CpG loci enabling precise characterization of human atrial and ventricular cardiac tissue samples. This defined set of reproducible cardiac tissue-specific DNA methylation sites allowed us to consistently detect the cellular identity of hiPSC-CM subtypes. Conclusion: Testing DNA methylation of only a small set of defined CpG sites thus makes it possible to distinguish atrial and ventricular cardiac tissues and cardiac atrial and ventricular subtypes of hiPSC-CMs. This method represents a rapid and reliable system for phenotypic characterization of in vitro-generated cardiomyocytes and opens new opportunities for cardiovascular research and patient-specific therapy. [ABSTRACT FROM AUTHOR]

The role of DNA methylation in cardiomyocyte physiology and cardiac disease remains a matter of controversy. We have recently provided evidence for an important role of DNMT3A in human cardiomyocyte cell homeostasis and metabolism, using engineered heart tissue (EHT) generated from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes carrying a knockout of the de novo DNA methyltransferase DNMT3A. Unlike isogenic control EHT, knockout EHT displayed morphological abnormalities such as lipid accumulations inside cardiomyocytes associated with impaired mitochondrial metabolism, as well as functional defects and impaired glucose metabolism. Here, we analyzed the role of DNMT3A in the setting of cardiac hypertrophy. We induced hypertrophic signaling by treatment with 50 nM endothelin-1 and 20 μM phenylephrine for one week and assessed EHT contractility, morphology, DNA methylation, and gene expression. While both knockout EHTs and isogenic controls showed the expected activation of the hypertrophic gene program, knockout EHTs were protected from hypertrophy-related functional impairment. Conversely, hypertrophic treatment prevented the metabolic consequences of a loss of DNMT3A, i.e. abolished lipid accumulation in cardiomyocytes likely by partial normalization of mitochondrial metabolism and restored glucose metabolism and metabolism-related gene expression of knockout EHT. Together, these data suggest an important role of DNA methylation not only for cardiomyocyte physiology, but also in the setting of cardiac disease. [Display omitted] • Loss of DNMT3A in cardiomyocytes pathologically affects metabolism and contractility. • Loss of DNMT3A attenuates cardiomyocyte functional impairment in hypertrophy. • Hypertrophic signaling improves mitochondrial metabolism in the absence of DNMT3A. • Hypertrophic signaling rescues glucose metabolism in DNMT3A knockout cardiomyocytes. [ABSTRACT FROM AUTHOR]

Introductions: The feasibility of human engineered heart tissue (hEHT) for cardiac repair was recently shown in a myocardial injury model in guinea pigs. For additional studies towards a possible clinical application hEHT-geometry had to be optimized and cell culture protocols had to be adapted to current good manufacturing practices (cGMP) to conform with recommendations for late preclinical trails. Methods: Cardiomyocytes were differentiated from a human induced pluripotent stem cell line that was reprogrammed under cGMP-conditions (Lonza, TC-1133). Cell culture components for hiPSC expansion, cardiomyocyte differentiation and hEHT generation were adapted to cGMP-grade protocols. To optimize hEHT-geometry for transplantation we created 1.5 x 2.7 cm mesh structured tissue patches in a 6-well format. Mesh structured hEHTs contained 16x106 cardiomyocytes and were cultured under continuous shaking. Cryo-injury was induced in guinea pigs resulting in large transmural scars. Mesh structured hEHT-patches were implanted 7 days after injury and histological analyses were performed 28 days after transplantation. Results: Large mesh structured hEHTs coherently started to beat after 7-10 days in culture. Histological analysis demonstrated homogenous cardiomyocytes distribution throughout the hEHTs. Cardiomyocytes demonstrated regular sarcomere structure and aligned along the force lines. Transplantation of mesh structured hEHTs completely covered the scar and resulted in large human grafts that consisted of densely packed, elongated human cardiomyocytes with regular sarcomere assembly. Four weeks after transplantation, human cardiomyocytes in the graft compared to guinea pig cardiomyocytes in host myocardium were smaller in diameter (6.0 μm vs. 7.4 μm; n=3) and exhibited shorter sarcomere length (1.73 μm vs. 1.86 μm; n=3). Connexin-43 in graft cardiomyocytes was located in small punctae throughout the cardiomyocyte cell membrane. Conclusion: Human engineered heart tissue can be generated under cGMP conditions Mesh structured hEHTs with optimized geometry can lead to complete coverage of the scar area and large grafts in a guinea pig injury model. Transplanted cardiomyocytes are still immature four weeks after transplantation. [ABSTRACT FROM AUTHOR]

Introduction: Genetically induced cardiomyopathy and heart failure are frequent and life-threatening conditions. Recently, we linked genetic deactivation or mutation of the gene PR/SET domain 16 (PRDM16) to cardiomyopathy in patients. Moreover, ablation or mutation of Prdm16 in zebrafish leads to cardiac dysfunction, diminished cardiomyocyte proliferation and increased cardiomyocyte apoptosis. Hypothesis: Here, we explore the molecular mechanisms underlying the PRDM16 associated cardiomyopathy with human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM). Methods: We established a cellular cardiomyopathy model of mono- or biallelic PRDM16 ablation in hiPSC by using the CRISPR/Cas approach. By using a chemical differentiation protocol hiPSC were differentiated into hiPSC derived cardiomyocytes (hiPSC-CM). Functional analysis of hiPSC-CM occurred with the engineered heart tissue (EHT) system. For molecular analysis, we performed a high-throughput mRNA expression screen with RNAseq and subsequent bioinformatics evaluation. Results: Efficient PRDM16 deactivation was confirmed on genomic, mRNA, and protein level. Analysis of hiPSC-CM_PRDM16_hom with the EHT system revealed a ~20% reduction of the maximal beating force but normal beating frequency compared to controls. EHT analysis further showed diminished maximal force development during stimulation with increased calcium concentrations in hiPSC-CM_PRDM16_hom. A high throughput RNAseq screen revealed in hiPSC-CM_PRDM16_hom approx. 500 down-regulated and 350 up-regulated genes compared to controls. Among these, we detected genes related to cell stress and TGF-beta signaling. This observation is in line with previous findings in csp1 mice, which is a hypomorphic Prdm16 mutant model, showing reduced TGF-beta signaling activity in cartilage. Interestingly, other genes that have already been linked to PRDM16 function in adipocytes are not differentially regulated. Conclusions: Our work establishes hiPSC models to explore the function of PRDM16 in cardiomyocytes and other cell types. Initial functional analysis reveals diminished cardiomyocyte beating force and altered TGF-beta signaling after loss of PRDM16. [ABSTRACT FROM AUTHOR]

Introduction: We have previously shown that engineered heart tissue (EHT) from neonatal rat heart cells responds to sustained mechanical or pharmacological stress stimuli similarly as hearts in vivo. Inter alia , EHTs showed a decline in contractile force, prolongation of relaxation, cardiomyocyte hypertrophy, reactivation of fetal genes (e.g. natriuretic peptides) and fibrotic changes. However, human EHTs containing cardiomyocytes obtained by differentiating human induced pluripotent stem cells (hiPSCs) did not respond to stress stimuli. We hypothesized that non-cardiomyocytes might account for this difference and sought a method to produce human EHTs containing all major cardiac cell types. Methods and Results: HiPSCs produced from fibroblasts of a healthy donor were expanded and split into 4 groups. Each group was transduced with lentiviruses encoding different fluorescent proteins. Mesodermal induction was initiated for all groups, but then blue (BFP) cells were differentiated to cardiomyocytes via inhibition of Wnt-signaling, green (Venus) cells were differentiated via VEGF to endothelial cells, orange (mOrange2) cells via TGFβ, bFGF and additional activation of Wnt-signaling to smooth muscle cells, and red (Katushka2) cells via bFGF and additional activation of Wnt-signaling to fibroblasts. All 4 differentiated cell types showed bright fluorescence and were clearly discernible by mRNA-markers and functional or histological parameters: cardiomyocytes (alpha actinin, spontaneous beating), endothelial cells (CD31, tube formation), smooth muscle cells (alpha smooth muscle actin, stress fibers), fibroblasts (periostin, collagen-production). Beating multicellular human EHTs could be successfully produced and the survival of all cell types for several weeks was verified, quantified and visualized by flow cytometry and confocal microscopy. The first multicellular EHTs produced did not develop higher contractile forces than unicellular EHTs (containing only cardiomyocytes). Whether multicellular human EHTs respond differently to stress stimuli than unicellular EHTs, is still to be investigated. Conclusion: Multicellular human EHTs can be produced from color-coded hiPSCs and will enable the investigation of different cell compositions on contractility under basal and stressed conditions. Cell-type specific analyses will be possible following simple FACS-separation by color. [ABSTRACT FROM AUTHOR]