Human induced pluripotent stem cell-derived atrial cardiomyocytes recapitulate contribution of the slowly activating delayed rectifier currents IKs to repolarization in the human atrium.

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. ß-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.

How does it feel? Passage of time judgments in speeded RT performance.

The relationship between duration perception and the feeling of time passing (passage of time) is not yet understood. In the present study, we assessed introspective reaction times (RT) and passage of time judgments in a speeded RT task. Task difficulty was manipulated in a numerical comparison task by numerical distance (distance from the number 45) and notation (digit vs. word). The results showed that both effects were reflected in introspective RTs, replicating previous results. Moreover, passage of time judgments showed a very similar pattern, with slower passage of time for more difficult comparisons. These results suggest that in the millisecond range judgments of duration and passage of time largely mirror each other when participants introspect about their own RT performance.

Unlocking Personalized Biomedicine and Drug Discovery with Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Fit for Purpose or Forever Elusive?

In recent decades, drug development costs have increased by approximately a hundredfold, and yet about 1 in 7 licensed drugs are withdrawn from the market, often due to cardiotoxicity. This review considers whether technologies using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could complement existing assays to improve discovery and safety while reducing socioeconomic costs and assisting with regulatory guidelines on cardiac safety assessments. We draw on lessons from our own work to suggest a panel of 12 drugs that will be useful in testing the suitability of hiPSC-CM platforms to evaluate contractility. We review issues, including maturity versus complexity, consistency, quality, and cost, while considering a potential need to incorporate auxiliary approaches to compensate for limitations in hiPSC-CM technology. We give examples on how coupling hiPSC-CM technologies with Cas9/CRISPR genome engineering is starting to be used to personalize diagnosis, stratify risk, provide mechanistic insights, and identify new pathogenic variants for cardiovascular disease.

An arrhythmogenic metabolite in atrial fibrillation.

BACKGROUND: Long-chain acyl-carnitines (ACs) are potential arrhythmogenic metabolites. Their role in atrial fibrillation (AF) remains incompletely understood. Using a systems medicine approach, we assessed the contribution of C18:1AC to AF by analysing its in vitro effects on cardiac electrophysiology and metabolism, and translated our findings into the human setting. METHODS AND RESULTS: Human iPSC-derived engineered heart tissue was exposed to C18:1AC. A biphasic effect on contractile force was observed: short exposure enhanced contractile force, but elicited spontaneous contractions and impaired Ca2+ handling. Continuous exposure provoked an impairment of contractile force. In human atrial mitochondria from AF individuals, C18:1AC inhibited respiration. In a population-based cohort as well as a cohort of patients, high C18:1AC serum concentrations were associated with the incidence and prevalence of AF. CONCLUSION: Our data provide evidence for an arrhythmogenic potential of the metabolite C18:1AC. The metabolite interferes with mitochondrial metabolism, thereby contributing to contractile dysfunction and shows predictive potential as novel circulating biomarker for risk of AF.

Human engineered heart tissue transplantation in a guinea pig chronic injury 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 × 106 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.

Human Engineered Heart Tissue Patches Remuscularize the Injured Heart in a Dose-Dependent Manner.

BACKGROUND: Human engineered heart tissue (EHT) transplantation represents a potential regenerative strategy for patients with heart failure and has been successful in preclinical models. Clinical application requires upscaling, adaptation to good manufacturing practices, and determination of the effective dose. METHODS: Cardiomyocytes were differentiated from 3 different human induced pluripotent stem cell lines including one reprogrammed under good manufacturing practice conditions. Protocols for human induced pluripotent stem cell expansion, cardiomyocyte differentiation, and EHT generation were adapted to substances available in good manufacturing practice quality. EHT geometry was modified to generate patches suitable for transplantation in a small-animal model and perspectively humans. Repair efficacy was evaluated at 3 doses in a cryo-injury guinea pig model. Human-scale patches were epicardially transplanted onto healthy hearts in pigs to assess technical feasibility. RESULTS: We created mesh-structured tissue patches for transplantation in guinea pigs (1.5×2.5 cm, 9-15×106 cardiomyocytes) and pigs (5×7 cm, 450×106 cardiomyocytes). EHT patches coherently beat in culture and developed high force (mean 4.6 mN). Cardiomyocytes matured, aligned along the force lines, and demonstrated advanced sarcomeric structure and action potential characteristics closely resembling human ventricular tissue. EHT patches containing ≈4.5, 8.5, 12×106, or no cells were transplanted 7 days after cryo-injury (n=18-19 per group). EHT transplantation resulted in a dose-dependent remuscularization (graft size: 0%-12% of the scar). Only high-dose patches improved left ventricular function (+8% absolute, +24% relative increase). The grafts showed time-dependent cardiomyocyte proliferation. Although standard EHT patches did not withstand transplantation in pigs, the human-scale patch enabled successful patch transplantation. CONCLUSIONS: EHT patch transplantation resulted in a partial remuscularization of the injured heart and improved left ventricular function in a dose-dependent manner in a guinea pig injury model. Human-scale patches were successfully transplanted in pigs in a proof-of-principle study.

Comprehensive analyses of the inotropic compound omecamtiv mecarbil in rat and human cardiac preparations.

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 µM. 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 1 µM. 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.NEW & NOTEWORTHY We developed a novel setup allowing precise control of preload of EHT and characterized effects of the myosin activator OM. OM not only exerted contraction-slowing and positive inotropic effects but also increased diastolic tension. Effect size and direction varied between species, auxotonic and isometric conditions, concentration, and time. We also observed OM-induced increase of mitochondrial ROS, which has not been observed before and may explain part of the effects on contractility.

RGS3L allows for an M2 muscarinic receptor-mediated RhoA-dependent inotropy in cardiomyocytes.

The role and outcome of the muscarinic M2 acetylcholine receptor (M2R) signaling in healthy and diseased cardiomyocytes is still a matter of debate. Here, we report that the long isoform of the regulator of G protein signaling 3 (RGS3L) functions as a switch in the muscarinic signaling, most likely of the M2R, in primary cardiomyocytes. High levels of RGS3L, as found in heart failure, redirect the Gi-mediated Rac1 activation into a Gi-mediated RhoA/ROCK activation. Functionally, this switch resulted in a reduced production of reactive oxygen species (- 50%) in cardiomyocytes and an inotropic response (+ 18%) in transduced engineered heart tissues. Importantly, we could show that an adeno-associated virus 9-mediated overexpression of RGS3L in rats in vivo, increased the contractility of ventricular strips by maximally about twofold. Mechanistically, we demonstrate that this switch is mediated by a complex formation of RGS3L with the GTPase-activating protein p190RhoGAP, which balances the activity of RhoA and Rac1 by altering its substrate preference in cardiomyocytes. Enhancement of this complex formation could open new possibilities in the regulation of the contractility of the diseased heart.

Hypertrophic signaling compensates for contractile and metabolic consequences of DNA methyltransferase 3A loss in human cardiomyocytes.

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.

An Important Role for DNMT3A-Mediated DNA Methylation in Cardiomyocyte Metabolism and Contractility.

BACKGROUND: DNA methylation acts as a mechanism of gene transcription regulation. It has recently gained attention as a possible therapeutic target in cardiac hypertrophy and heart failure. However, its exact role in cardiomyocytes remains controversial. Thus, we knocked out the main de novo DNA methyltransferase in cardiomyocytes, DNMT3A, in human induced pluripotent stem cells. Functional consequences of DNA methylation-deficiency under control and stress conditions were then assessed in human engineered heart tissue from knockout human induced pluripotent stem cell-derived cardiomyocytes. METHODS: DNMT3A was knocked out in human induced pluripotent stem cells by CRISPR/Cas9gene editing. Fibrin-based engineered heart tissue was generated from knockout and control human induced pluripotent stem cell-derived cardiomyocytes. Development and baseline contractility were analyzed by video-optical recording. Engineered heart tissue was subjected to different stress protocols, including serum starvation, serum variation, and restrictive feeding. Molecular, histological, and ultrastructural analyses were performed afterward. RESULTS: Knockout of DNMT3A in human cardiomyocytes had three main consequences for cardiomyocyte morphology and function: (1) Gene expression changes of contractile proteins such as higher atrial gene expression and lower MYH7/MYH6 ratio correlated with different contraction kinetics in knockout versus wild-type; (2) Aberrant activation of the glucose/lipid metabolism regulator peroxisome proliferator-activated receptor gamma was associated with accumulation of lipid vacuoles within knockout cardiomyocytes; (3) Hypoxia-inducible factor 1α protein instability was associated with impaired glucose metabolism and lower glycolytic enzyme expression, rendering knockout-engineered heart tissue sensitive to metabolic stress such as serum withdrawal and restrictive feeding. CONCLUSION: The results suggest an important role of DNA methylation in the normal homeostasis of cardiomyocytes and during cardiac stress, which could make it an interesting target for cardiac therapy.

Characterization of the PLN p.Arg14del Mutation in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Phospholamban (PLN) is the natural inhibitor of the sarco/endoplasmic reticulum Ca2+ 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) Ca2+ dynamics and protein localization were compatible with SERCA2a superinhibition and (2) if functional abnormalities could be reverted by pharmacological SERCA2a activation (PST3093). METHODS: Ca2+ 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 (tpeak) 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 tpeak rate-dependently declined in WT, it was shorter and rate-independent in MUT; (4) diastolic Ca2+ 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 Ca2+-dependent transcription.

Debt Relief as a Last Resort for the Lender of Last Resort?: Monetary Financing - Doing It Right.

The coronavirus crisis has led to a sharp increase in the debt-to-GDP ratios of the euro area member states. Without external support, access to the capital market could be seriously threatened in the medium term for Italy, but also for other member states. While the Pandemic Emergency Purchase Programme, which is designed as a monetary policy instrument, is regarded by some as a violation of the prohibition of monetary financing, the Next Generation EU recovery fund is likely to direct the fundamental structures of the European Union towards a fiscal union with considerable redistribution elements. This article analyses an alternative strategy, namely debt relief by the European System of Central Banks through an EU debt agency. Such a scheme would be possible without amending the EU treaties and would avoid negative equity at the central banks. The question is under what circumstances would this approach be suitable and proportionate?

[ECB Debt Certificates - New Use for an Old Instrument?] / EZB-Schuldverschreibungen ­ neue Verwendung für ein altes Instrument?

In response to the coronavirus crisis, the central banks of the Eurosystem have further increased their APP bond purchases and supplemented them with purchases under the PEPP programme. Together with other unconventional monetary policy measures, this generated additional liquidity would have to be reduced in the case of sustained higher inflationary pressure. The straightforward solution for the ECB would be to sell the once purchased bonds, as they are the source of the extra liquidity. However, the associated writeoffs would result in financial sector instabilities and interest rate increases on government bonds, which would be particularly problematic for crisis-hit countries. This contribution analyses the issuance of debt certificates by the ECB as an alternative way of absorbing liquidity.

MUSCLEMOTION: A Versatile Open Software Tool to Quantify Cardiomyocyte and Cardiac Muscle Contraction In Vitro and In Vivo.

RATIONALE: There are several methods to measure cardiomyocyte and muscle contraction, but these require customized hardware, expensive apparatus, and advanced informatics or can only be used in single experimental models. Consequently, data and techniques have been difficult to reproduce across models and laboratories, analysis is time consuming, and only specialist researchers can quantify data. OBJECTIVE: Here, we describe and validate an automated, open-source software tool (MUSCLEMOTION) adaptable for use with standard laboratory and clinical imaging equipment that enables quantitative analysis of normal cardiac contraction, disease phenotypes, and pharmacological responses. METHODS AND RESULTS: MUSCLEMOTION allowed rapid and easy measurement of movement from high-speed movies in (1) 1-dimensional in vitro models, such as isolated adult and human pluripotent stem cell-derived cardiomyocytes; (2) 2-dimensional in vitro models, such as beating cardiomyocyte monolayers or small clusters of human pluripotent stem cell-derived cardiomyocytes; (3) 3-dimensional multicellular in vitro or in vivo contractile tissues, such as cardiac "organoids," engineered heart tissues, and zebrafish and human hearts. MUSCLEMOTION was effective under different recording conditions (bright-field microscopy with simultaneous patch-clamp recording, phase contrast microscopy, and traction force microscopy). Outcomes were virtually identical to the current gold standards for contraction measurement, such as optical flow, post deflection, edge-detection systems, or manual analyses. Finally, we used the algorithm to quantify contraction in in vitro and in vivo arrhythmia models and to measure pharmacological responses. CONCLUSIONS: Using a single open-source method for processing video recordings, we obtained reliable pharmacological data and measures of cardiac disease phenotype in experimental cell, animal, and human models.

Intermittent Optogenetic Tachypacing of Atrial Engineered Heart Tissue Induces Only Limited Electrical Remodelling.

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.

Digital Competencies and Attitudes Toward Digital Adherence Solutions Among Elderly Patients Treated With Novel Anticoagulants: Qualitative Study.

BACKGROUND: Nonadherence to medication is a driver of morbidity and mortality, and complex medication regimens in patients with chronic diseases foster the problem. Digital technology might help, but despite numerous solutions being developed, none are currently widely used, and acceptance rates remain low, especially among the elderly. OBJECTIVE: This study aimed to better understand and operationalize how new digital solutions can be evaluated. Particularly, the goal was to identify factors that help digital approaches targeting adherence to become more widely accepted. METHODS: A qualitative study using a conceptual grounded theory approach was conducted. We included patients aged 65 years and older who routinely took new oral anticoagulants. To generate theses about the digital competencies of the target group with daily medication intake, face-to-face interviews were conducted, recorded, and anonymized. After coding the interviews, categories were generated, discussed, and combined with several theses until saturation of the statements was reached. RESULTS: The methodological approach led to the finding that after interviews in 20 of 77 potentially available patients, a saturation of statements was reached. The average patient's age was 75 years, and 50% (10/20) of the subjects were female. The data identified five main coding categories-Diseases and medicine, Technology, Autonomy, Patient narrative, and Attitude toward technologies-each including positive and negative subcategories. Main categories and subcategories were summarized as Adherence Radar, which can be considered as a framework to assess the potential of adherence solutions in the process of prototyping and can be applied to all adherence tools in a holistic manner. CONCLUSIONS: The Adherence Radar can be used to increase the acceptance rate of digital solutions targeting adherence. For a patient-centric design, an app should be adapted to the individual patient's needs. According to our results, this application should be based on gender and educational background as well as the individual physician-patient relationship. If used in a proper, individualized manner, digital adherence solutions could become a new cornerstone for the treatment of chronically ill individuals.

CRISPR/Cas9 editing in human pluripotent stem cell-cardiomyocytes highlights arrhythmias, hypocontractility, and energy depletion as potential therapeutic targets for hypertrophic cardiomyopathy.

Aims: Sarcomeric gene mutations frequently underlie hypertrophic cardiomyopathy (HCM), a prevalent and complex condition leading to left ventricle thickening and heart dysfunction. We evaluated isogenic genome-edited human pluripotent stem cell-cardiomyocytes (hPSC-CM) for their validity to model, and add clarity to, HCM. Methods and results: CRISPR/Cas9 editing produced 11 variants of the HCM-causing mutation c.C9123T-MYH7 [(p.R453C-ß-myosin heavy chain (MHC)] in 3 independent hPSC lines. Isogenic sets were differentiated to hPSC-CMs for high-throughput, non-subjective molecular and functional assessment using 12 approaches in 2D monolayers and/or 3D engineered heart tissues. Although immature, edited hPSC-CMs exhibited the main hallmarks of HCM (hypertrophy, multi-nucleation, hypertrophic marker expression, sarcomeric disarray). Functional evaluation supported the energy depletion model due to higher metabolic respiration activity, accompanied by abnormalities in calcium handling, arrhythmias, and contraction force. Partial phenotypic rescue was achieved with ranolazine but not omecamtiv mecarbil, while RNAseq highlighted potentially novel molecular targets. Conclusion: Our holistic and comprehensive approach showed that energy depletion affected core cardiomyocyte functionality. The engineered R453C-ßMHC-mutation triggered compensatory responses in hPSC-CMs, causing increased ATP production and αMHC to energy-efficient ßMHC switching. We showed that pharmacological rescue of arrhythmias was possible, while MHY7: MYH6 and mutant: wild-type MYH7 ratios may be diagnostic, and previously undescribed lncRNAs and gene modifiers are suggestive of new mechanisms.

Cardiac tissue engineering: state of the art.

The engineering of 3-dimensional (3D) heart muscles has undergone exciting progress for the past decade. Profound advances in human stem cell biology and technology, tissue engineering and material sciences, as well as prevascularization and in vitro assay technologies make the first clinical application of engineered cardiac tissues a realistic option and predict that cardiac tissue engineering techniques will find widespread use in the preclinical research and drug development in the near future. Tasks that need to be solved for this purpose include standardization of human myocyte production protocols, establishment of simple methods for the in vitro vascularization of 3D constructs and better maturation of myocytes, and, finally, thorough definition of the predictive value of these methods for preclinical safety pharmacology. The present article gives an overview of the present state of the art, bottlenecks, and perspectives of cardiac tissue engineering for cardiac repair and in vitro testing.

Deciphering the microRNA signature of pathological cardiac hypertrophy by engineered heart tissue- and sequencing-technology.

Pathological cardiac hypertrophy and fibrosis are modulated by a set of microRNAs, most of which have been detected in biologically complex animal models of hypertrophy by arrays with moderate sensitivity and disregard of passenger strand (previously "star") microRNAs. Here, we aimed at precisely analyzing the microRNA signature of cardiac hypertrophy and fibrosis by RNA sequencing in a standardized in vitro hypertrophy model based on engineered heart tissue (EHT). Spontaneously beating, force-generating fibrin EHTs from neonatal rat heart cells were subjected to afterload enhancement for 7days (AE-EHT), and EHTs without intervention served as controls. AE resulted in reduced contractile force and relaxation velocity, fibrotic changes and reactivation of the fetal gene program. Small RNAs were extracted from control and AE-EHTs and sequencing yielded almost 750 different mature microRNAs, many of which have never been described before in rats. The detection of both arms of the precursor stem-loop (pre-miRNA), namely -3p and -5p miRs, was frequent. 22 abundantly sequenced microRNAs were >1.3× upregulated and 15 abundantly sequenced microRNAs downregulated to <0.77×. Among the upregulated microRNAs were 3 pairs of guide and passenger strand microRNAs (miR-21-5p/-3p, miR-322-5p/-3p, miR-210-3p/-5p) and one single passenger strand microRNA (miR-140-3p). Among downregulated microRNAs were 3 pairs (miR-133a-3p/-5p, miR-30e-5p/3p, miR-30c-5p/-3p). Preincubating EHTs with anti-miR-21-5p markedly attenuated the AE-induced contractile failure, cardiomyocyte hypertrophy and fibrotic response, recapitulating prior results in whole animals. Taken together, AE-induced pathological hypertrophy in EHTs is associated with 37 differentially regulated microRNAs, including many passenger strands. Antagonizing miR-21-5p ameliorates dysfunction in this model.