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Human induced pluripotent stem cells (hiPSCs) are an invaluable tool to study molecular mechanisms on a human background. Culturing stem cells at an oxygen level different from their microenvironmental niche impacts their viability. To understand this mechanistically, dermal skin fibroblasts of 52 probands were reprogrammed into hiPSCs, followed by either hyperoxic (20 % O 2 ) or physioxic (5 % O 2 ) culture and proteomic profiling. Analysis of chromosomal stability by Giemsa-banding revealed that physioxic -cultured hiPSC clones exhibited less pathological karyotypes than hyperoxic (e.g. 6 % vs. 32 % mosaicism), higher pluripotency as evidenced by higher Stage-Specific Embryonic Antigen 3 positivity, higher glucose consumption and lactate production. Global proteomic analysis demonstrated lower abundance of several subunits of NADH:ubiquinone oxidoreductase (complex I) and an underrepresentation of pathways linked to oxidative phosphorylation and cellular senescence. Accordingly, release of the pro-senescent factor IGFBP3 and β-galactosidase staining were lower in physioxic hiPSCs. RNA- and ATAC-seq profiling revealed a distinct hypoxic transcription factor-binding footprint, amongst others higher expression of the HIF1α-regulated target NDUFA4L2 along with increased chromatin accessibility of the NDUFA4L2 gene locus. While mitochondrial DNA content did not differ between groups, physioxic hiPSCs revealed lower polarized mitochondrial membrane potential, altered mitochondrial network appearance and reduced basal respiration and electron transfer capacity. Blue-native polyacrylamide gel electrophoresis coupled to mass spectrometry of the mitochondrial complexes detected higher abundance of NDUFA4L2 and ATP5IF1 and loss of incorporation into complex IV or V, respectively. Taken together, physioxic culture of hiPSCs improved chromosomal stability, which was associated with downregulation of oxidative phosphorylation and senescence and extensive re-wiring of mitochondrial complex composition., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: TE is a shareholder and a member of the scientific advisory board of DiNAQOR, which is not relating to this manuscript. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. 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.)

MYBPC3 is the most frequently affected gene in hypertrophic cardiomyopathy (HCM), which is an autosomal-dominant cardiac disease caused by mutations in sarcomeric proteins. Bi-allelic truncating MYBPC3 mutations are associated with severe forms of neonatal cardiomyopathy. We reprogrammed skin fibroblasts from a HCM patient carrying a heterozygous MYBPC3 truncating mutation into human induced pluripotent stem cells (iPSC) and used CRISPR/Cas9 to generate bi-allelic MYBPC3 truncating mutation and isogenic control hiPSC lines. All lines expressed pluripotency markers, had normal karyotype and differentiated into endoderm, ectoderm and cardiomyocytes in vitro. This set of three lines provides a useful tool to study HCM pathomechanisms., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

The phospholamban (PLN) p.Arg14del mutation causes dilated cardiomyopathy, with the molecular disease mechanisms incompletely understood. Patient dermal fibroblasts were reprogrammed to hiPSC, isogenic controls were established by CRISPR/Cas9, and cardiomyocytes were differentiated. Mutant cardiomyocytes revealed significantly prolonged Ca 2+ transient decay time, Ca 2+ -load dependent irregular beating pattern, and lower force. Proteomic analysis revealed less endoplasmic reticulum (ER) and ribosomal and mitochondrial proteins. Electron microscopy showed dilation of the ER and large lipid droplets in close association with mitochondria. Follow-up experiments confirmed impairment of the ER/mitochondria compartment. PLN p.Arg14del end-stage heart failure samples revealed perinuclear aggregates positive for ER marker proteins and oxidative stress in comparison with ischemic heart failure and non-failing donor heart samples. Transduction of PLN p.Arg14del EHTs with the Ca 2+ -binding proteins GCaMP6f or parvalbumin improved the disease phenotype. This study identified impairment of the ER/mitochondria compartment without SR dysfunction as a novel disease mechanism underlying PLN p.Arg14del cardiomyopathy. The pathology was improved by Ca 2+ -scavenging, suggesting impaired local Ca 2+ cycling as an important disease culprit., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Sulforaphane (SFN) is a phytochemical compound extracted from cruciferous plants, like broccoli or cauliflower. Its isothiocyanate group renders SFN reactive, thus allowing post-translational modification of cellular proteins to regulate their function with the potential for biological and therapeutic actions. SFN and stabilized variants recently received regulatory approval for clinical studies in humans for the treatment of neurological disorders and cancer. Potential unwanted side effects of SFN on heart function have not been investigated yet. The present study characterizes the impact of SFN on cardiomyocyte contractile function in cardiac preparations from neonatal rat, adult mouse and human induced-pluripotent stem cell-derived cardiomyocytes. This revealed a SFN-mediated negative inotropic effect, when administered either acutely or chronically, with an impairment of the Frank-Starling response to stretch activation. A direct effect of SFN on myofilament function was excluded in chemically permeabilized mouse trabeculae. However, SFN pretreatment increased lactate formation and enhanced the mitochondrial production of reactive oxygen species accompanied by a significant reduction in the mitochondrial membrane potential. Transmission electron microscopy revealed disturbed sarcomeric organization and inflated mitochondria with whorled membrane shape in response to SFN exposure. Interestingly, administration of the alternative energy source l-glutamine to the medium that bypasses the uptake route of pyruvate into the mitochondrial tricarboxylic acid cycle improved force development in SFN-treated EHTs, suggesting indeed mitochondrial dysfunction as a contributor of SFN-mediated contractile dysfunction. Taken together, the data from the present study suggest that SFN might impact negatively on cardiac contractility in patients with cardiovascular co-morbidities undergoing SFN supplementation therapy. Therefore, cardiac function should be monitored regularly to avoid the onset of cardiotoxic side effects., (Copyright © 2021 The Author(s). Published by Elsevier B.V. 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.)

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.

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.)

Cardiomyocyte energy metabolism is altered in heart failure, and primary defects of metabolic pathways can cause heart failure. Studying cardiac energetics in rodent models has principal shortcomings, raising the question to which extent human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) can provide an alternative. As metabolic maturation of CM occurs mostly after birth during developmental hypertrophy, the immaturity of hiPSC-CM is an important limitation. Here we shortly review the physiological drivers of metabolic maturation and concentrate on methods to mature hiPSC-CM with the goal to benchmark the metabolic state of hiPSC-CM against in vivo data and to see how far known abnormalities in inherited metabolic disorders can be modeled in hiPSC-CM. The current data indicate that hiPSC-CM, despite their immature, approximately mid-fetal state of energy metabolism, faithfully recapitulate some basic metabolic disease mechanisms. Efforts to improve their metabolic maturity are underway and shall improve the validity of this model., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

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.)

Cardiomyocytes (CMs) generated from human induced pluripotent stem cells (hiPSCs) are under investigation for their suitability as human models in preclinical drug development. Antiarrhythmic drug development focuses on atrial biology for the treatment of atrial fibrillation. Here we used recent retinoic acid-based protocols to generate atrial CMs from hiPSCs and establish right atrial engineered heart tissue (RA-EHT) as a 3D model of human atrium. EHT from standard protocol-derived hiPSC-CMs (Ctrl-EHT) and intact human muscle strips served as comparators. RA-EHT exhibited higher mRNA and protein concentrations of atrial-selective markers, faster contraction kinetics, lower force generation, shorter action potential duration, and higher repolarization fraction than Ctrl-EHTs. In addition, RA-EHTs but not Ctrl-EHTs responded to pharmacological manipulation of atrial-selective potassium currents. RA- and Ctrl-EHTs' behavior reflected differences between human atrial and ventricular muscle preparations. Taken together, RA-EHT is a model of human atrium that may be useful in preclinical drug screening., (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.)