Search

Background Imbalanced transcriptional networks characterize cardiomyocyte stress and result in cardiac remodelling. We hypothesize that re-establishing homeostatic gene networks in cardiomyocytes will prevent further tissue damage. To tackle this challenge, we applied CRISPR-based endogenous gene activation (CRISPRa) in vivo and in vitro. Methods We employ precision transcriptome editing tools based on CRISPR/Cas9 with enzymatically inactive Cas9 (dCas9) fused to transcriptional activators (VPR) to induce target gene expression by directing dCas9VPR to promoter regions by guide RNAs (gRNA). Results Homozygous CRISPRa hiPSC cell lines were generated by targeted integration of a CAG promoter driven dCas9VPR-T2A-tdTomato expression cassette into the AAVS1 locus by CRISPR/Cas9 editing and homology directed repair. Expression of dCas9VPR was evaluated by immunoblotting and co-expressed reporter fluorescence in spontaneously beating hiPSC-CM. We previously identified a crosstalk between WNT signalling and Krueppel-like factor 15 (KLF15) necessary for controlling cardiac homeostasis. We designed and tested 8 non-overlapping gRNAs in the –400 bp region upstream of the KLF15 transcriptional start site (TSS) and tested individual gRNA effectiveness for gene activation in HEK293T cells. Five gRNAs were identified inducing KLF15 transcript levels between 2- and 5-fold compared to non-targeted (NT) gRNA transfected cells (n=3 experiments). The single most effective gRNA was transduced by lentiviral particles into CRISPRa hiPSC-CM increasing KLF15 transcript levels to 1.5-fold compared to NT-gRNA control. Synergistic effects of 3 instead of single gRNA increased KLF15 transcript levels by 3-fold compared to controls (n≥3 experiments). We hypothesized that dCas9VPR expression could be harnessed as an additional option for gene dose titration and we generated hiPSC lines with enhanced dCas9VPR expression (v2.0). We observed up to 5-fold KLF15 gene activation when triple gRNA and v2.0 were combined (n≥4 experiments). Engineered human myocardium (EHM) was generated consisting of CRISPRa cardiomyocytes, fibroblasts and collagen and we observed similar contractility in 4-week cultured EHM suggesting innocuous dCas9VPR and gRNA expression. CRISPRa component expression was maintained over the entire culture period as evaluated by dCas9VPR immunoblotting and KLF15 transcriptional activation (1.4 fold, v1.0 CRISPRa hiPSC-CM, n≥8 tissues) indicating sustained gene activition. Conclusions Targeted gene activation with CRISPR/Cas9 is a precise and effective tool for transcriptional activation in hiPSC-CM. We observed titratability of gene activation by 1.) dCas9VPR expression levels and 2.) single versus multiple gRNA use. We furthermore elucidated general rules for effective gRNA targeting within the 5' TSS of genes of interest which confirmed a dependency of baseline gene activity as a limiting factor for endogenous gene activation. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): German Research Foundation (DFG) - Collaborative Research Center 1002German Center for Cardiovascular Research (DZHK)

Introduction: Ensuring scientific reproducibility and compliance with documentation guidelines of funding bodies and journals is a topic of greatly increasing importance in biomedical research. Failure to comply, or unawareness of documentation standards can have adverse effects on the translation of research into patient treatments, as well as economic implications. In the context of the German Research Foundation-funded collaborative research center (CRC) 1002, an IT-infrastructure sub-project was designed. Its goal has been to establish standardized metadata documentation and information exchange benefitting the participating research groups with minimal additional documentation efforts. Methods: Implementation of the self-developed menoci-based research data platform (RDP) was driven by close communication and collaboration with researchers as early adopters and experts. Requirements analysis and concept development involved in person observation of experimental procedures, interviews and collaboration with researchers and experts, as well as the investigation of available and applicable metadata standards and tools. The Drupal-based RDP features distinct modules for the different documented data and workflow types, and both the development and the types of collected metadata were continuously reviewed and evaluated with the early adopters. Results: The menoci-based RDP allows for standardized documentation, sharing and cross-referencing of different data types, workflows, and scientific publications. Different modules have been implemented for specific data types and workflows, allowing for the enrichment of entries with specific metadata and linking to further relevant entries in different modules. Discussion: Taking the workflows and datasets of the frequently involved experimental service projects as a starting point for (meta-)data types to overcome irreproducibility of research data, results in increased benefits for researchers with minimized efforts. While the menoci-based RDP with its data models and metadata schema was originally developed in a cardiological context, it has been implemented and extended to other consortia at GÃűttingen Campus and beyond in different life science research areas.

Despite known adverse effects of hydroxychloroquine (HCQ) and azithromycin (AZM) on cardiac function, HCQ and AZM have been used as combination therapy in the treatment of COVID-19 patients. Recent clinical data indicate higher complication rates with HCQ/AZM combination treatment in comparison to monotherapy. Here, we used human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to systematically investigate the effects of HCQ and AZM individually and in combination. The clinically observed QT prolongation caused by treatment with HCQ could be recapitulated in iPSC-CMs based on prolonged field potential duration (FPDc). Interestingly, HCQ-induced FPDc prolongation was strongly enhanced by combined treatment with AZM, although AZM alone slightly shortened FPDc in iPSC-CMs. Furthermore, combined treatment with AZM and HCQ leads to higher cardiotoxicity, more severe structural disarrangement, and more pronounced contractile and electrophysiological dysfunctions, compared to respective mono-treatments. First mechanistic insights underlying the synergistic effects of AZM and HCQ on iPSC-CM functionality are provided based on increased Cx43- and Nav1.5-protein levels. Taken together, our results highlight that combined treatment with HCQ and AZM strongly enhances the adverse effects on cardiomyocytes, providing mechanistic evidence for the high mortality in patients receiving HCQ/AZM combination treatment.

Aims Brugada syndrome (BrS) is associated with a pronounced risk to develop sudden cardiac death (SCD). Up to 21% of patients are related to mutations in SCN5A. Studies identified SCN10A as a contributor of BrS. However, the investigation of the human cellular phenotype of BrS in the presence of SCN10A mutations remains lacking. The objective of this study was to establish a cellular model of BrS in presence of SCN10A mutations using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Methods and results Dermal fibroblasts obtained from a BrS patient suffering from SCD harbouring the SCN10A double variants (c.3803G>A and c.3749G>A) and three independent healthy control subjects were reprogrammed to hiPSCs. Human-induced pluripotent stem cells were differentiated into cardiomyocytes (hiPSC-CMs).The hiPSC-CMs from the BrS patient showed a significantly reduced peak sodium channel current (INa) and a significantly reduced ATX II (sea anemone toxin, an enhancer of late INa) sensitive as well as A-887826 (a blocker of SCN10A channel) sensitive late sodium channel current (INa) when compared with the healthy control hiPSC-CMs, indicating loss-of-function of sodium channels. Consistent with reduced INa the action potential amplitude and upstroke velocity (Vmax) were significantly reduced, which may contribute to arrhythmogenesis of BrS. Moreover, Ajmaline effects on action potentials were stronger in BrS-hiPSC-CMs than in healthy control cells. This is in agreement with the higher susceptibility of patients to sodium channel blocking drugs in unmasking BrS. Conclusion Patient-specific hiPSC-CMs are able to recapitulate single-cell phenotype features of BrS with SCN10A mutations and may provide novel opportunities to further elucidate the cellular disease mechanism.

Short QT syndrome (SQTS) is associated with sudden cardiac death and implantable cardioverter-defibrillator (ICD) implantation is recommended in this rare disease. However, only a few SQTS families have been reported in literature with limited follow-up data. In the recent study, we describe the outcome data of 57 SQTS patients receiving ICD implantation. This includes seven SQTS families consecutively admitted to our hospital between 2002 and 2017 as well as patients reported in published literature. Seven SQTS patients admitted to our hospital were followed up. Additionally, 7 studies out of a total of 626 researched articles were identified through systematic database search (PubMed, Web of Science, Cochrane Library, and Cinahl) and their data analyzed according to our model. Complications during a median follow-up time of 67.4 months (IQR 6–162 months) were documented in 31 (54%) patients. Inappropriate shocks were seen in 33% due to T wave oversensing (8.7%), supraventricular tachycardia (19%), lead failure and fracture (21%). Further complications were infection (10%), battery depletion (7%) and psychological distress (3.5%). Appropriate shocks were documented in 19%. Three patients (5%) were treated with s-ICD due to recurrent complications of transvenous ICD. ICD therapy is an effective therapy in SQTS patients. However, it is also associated with significant risk of device-related complications.

Aims Short QT syndrome (SQTS), a disorder associated with characteristic electrocardiogram QT-segment abbreviation, predisposes afflicted patients to sudden cardiac death. Despite some progress in assessing the organ level pathophysiology and genetic changes of the disorder, the understanding of the human cellular phenotype and discovering of an optimal therapy has lagged due to a lack of appropriate human cellular models of the disorder. The aim of this study was to establish a cellular model of SQTS type 5 using human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) and gene-edited cell line using CRISPR/CAS9. Methods and results This study recruited one patient with short QT syndrome type 5 carrying a mutation in CACNb2 gene as well as one healthy control subject. We generated hiPSCs from their skin fibroblasts, and differentiated hiPSCs into cardiomyocytes (hiPSC-CMs) for physiological. Isogenic control hiPSC-CMs generated by the CRISPR/CAS9 technique were also used for the study. The hiPSC-CMs from the patient showed a reduced calcium current (ICa-L) density and shortened action potential duration (APD) compared with healthy control hiPSC-CMs and isogenic hiPSC-CMs. Furthermore, they demonstrated abnormal rhythmic activities. Carbachol increased the arrhythmic events in SQTS significantly but not in healthy and isogenic control cells. Gene and protein expression profiling showed a decreased CACNb2 expression in SQTS cells. Quinidine prolonged the APD and abolished arrhythmic activity. Conclusions Patient-specific hiPSC-CMs are able to recapitulate single-cell phenotype features of SQTS type 5 and provide novel opportunities to further elucidate the cellular disease mechanism and test drug effects. Funding Acknowledgement Type of funding source: None

Summary The generation of homogeneous populations of subtype-specific cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) is crucial in cardiovascular disease modeling as well as in drug discovery and cardiotoxicity screenings. This protocol describes a simple, robust, and efficient monolayer-based differentiation of hiPSCs into defined atrial and ventricular cardiomyocytes. For complete details on the use and execution of this protocol, please refer to Cyganek et al., 2018., Graphical Abstract, Highlights • Subtype-directed differentiation of hiPSCs into atrial and ventricular cardiomyocytes • Simple, robust, and efficient monolayer-based differentiation protocol • Approx. 90%–95% of the intended cardiac subtype within cTNT+ cells can be obtained, The generation of homogeneous populations of subtype-specific cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) is crucial in cardiovascular disease modeling as well as in drug discovery and cardiotoxicity screenings. This protocol describes a simple, robust, and efficient monolayer-based differentiation of hiPSCs into defined atrial and ventricular cardiomyocytes.

Background: Noonan syndrome (NS) is a multisystemic developmental disorder characterized by common, clinically variable symptoms, such as typical facial dysmorphisms, short stature, developmental delay, intellectual disability as well as cardiac hypertrophy. The underlying mechanism is a gain-of-function of the RAS–mitogen-activated protein kinase signaling pathway. However, our understanding of the pathophysiological alterations and mechanisms, especially of the associated cardiomyopathy, remains limited and effective therapeutic options are lacking. Methods: Here, we present a family with two siblings displaying an autosomal recessive form of NS with massive hypertrophic cardiomyopathy as clinically the most prevalent symptom caused by biallelic mutations within the leucine zipper-like transcription regulator 1 ( LZTR1 ). We generated induced pluripotent stem cell–derived cardiomyocytes of the affected siblings and investigated the patient-specific cardiomyocytes on the molecular and functional level. Results: Patients’ induced pluripotent stem cell–derived cardiomyocytes recapitulated the hypertrophic phenotype and uncovered a so-far-not-described causal link between LZTR1 dysfunction, RAS–mitogen-activated protein kinase signaling hyperactivity, hypertrophic gene response and cellular hypertrophy. Calcium channel blockade and MEK inhibition could prevent some of the disease characteristics, providing a molecular underpinning for the clinical use of these drugs in patients with NS, but might not be a sustainable therapeutic option. In a proof-of-concept approach, we explored a clinically translatable intronic CRISPR (clustered regularly interspaced short palindromic repeats) repair and demonstrated a rescue of the hypertrophic phenotype. Conclusions: Our study revealed the human cardiac pathogenesis in patient-specific induced pluripotent stem cell–derived cardiomyocytes from NS patients carrying biallelic variants in LZTR1 and identified a unique disease-specific proteome signature. In addition, we identified the intronic CRISPR repair as a personalized and in our view clinically translatable therapeutic strategy to treat NS-associated hypertrophic cardiomyopathy.

The bioresource (>265 patients with >27,600 biospecimens until December 2019; recruitment ongoing) on severe aortic stenosis is of vital importance to improve the still incomplete understanding of its etiology as well as its transition to heart failure. The bioresource contains various biospecimens, standardised clinical and imaging data sets including transthoracic echocardiography, computed tomography and magnetic resonance imaging of the heart. Biospecimen sampling follows the SOP-driven collection scheme of the German Center for Cardiovascular Research (DZHK) for venous blood and urine [1]. In addition, left-ventricular endomyocardial biopsies, rectal swabs and skin biopsies (for subsequent generation of induced pluripotent stem cells) are collected. Data management includes the use of a professional biospecimen management system as well as a Picture Archiving and Communication System (PACS) for imaging data. A Good Clinical Practice (GCP)-conform software for the management of clinical data and a trusted third party for the management of patient identifying data and pseudonyms are in place. Given these conditions, there is a high reuse-potential for biospecimens and data. Funding statement: Parts of the bioresource were financed by the German Research Foundation (DFG) within the Collaborative Research Center (SFB) 1002.

Brugada syndrome (BrS) is one of the major causes of sudden cardiac death in young people, while the underlying mechanisms are not completely understood. Here, we investigated the pathophysiological phenotypes and mechanisms using induced pluripotent stem cell-derived cardiomyocytes from two BrS patients (BrS-CMs) carrying a heterozygous SCN5A mutation p.S1812X. Compared to cardiomyocytes derived from healthy controls (Ctrl-CMs), BrS-CMs displayed a 50% reduction of INa density, a 69.5% reduction of NaV1.5 expression, and the impaired localization of NaV1.5 and connexin 43 at the cell surface. BrS-CMs exhibited reduced action potential upstroke velocity and conduction slowing. The Ito in BrS-CMs was significantly augmented, and the ICaL window current probability was increased. Our data indicate that the electrophysiological mechanisms underlying arrhythmia in BrS-CMs may involve both depolarization and repolarization disorders. Cilostazol and milrinone showed dramatic inhibitions of Ito in BrS-CMs and alleviated the arrhythmic activity, suggesting their therapeutic potential for BrS patients.

Background and purpose Previous studies revealed that Takotsubo cardiomyopathy (TTC), a transient disorder of ventricular dysfunction affecting predominantly postmenopausal women, is associated with acquired long QT syndrome and arrhythmias, but the exact pathophysiologic mechanism is unknown. Our aim is to investigate the electrophysiological mechanism for QT-prolongation in TTC-patients by using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Methods hiPSC-CMs, which were generated from human skin fibroblasts of three healthy donors, were treated by estradiol (10μM for one week) and a toxic concentration of isoprenaline (Iso, 1mM for 2h). Patch clamp techniques, qPCR and fluorescence-activated cell sorting (FACS) were employed for the study. Key results Iso enhanced late I Na and suppressed I to and thus prolonged the action potential duration (APD), suggesting possible reasons for arrhythmias in TTC. Iso elevated the production of reactive oxygen species (ROS). N -acetylcystein (1mM), a ROS-blocker, abolished the effects of Iso on late I Na and I to . H 2 O 2 (100μM) mimicked Iso effects on late I Na and I to . These data indicate that the effects of Iso were mediated by ROS. Metoprolol (1mM), a beta-blocker, prevented the effects of Iso on late I Na and APD, confirming the adrenoceptor-dependent effects of Iso. Estradiol treatment prevented the APD-prolongation, attenuated the enhancement of I Na , diminished the reduction of I to , suppressed ROS-production induced by Iso and reduced the expression levels of adrenoceptors, suggesting protective effects of estragon against toxic effects of catecholamine. Conclusions Estradiol has protective effects against catecholamine excess and hence reduction in estrogen level may increase the risk of acquired long QT syndrome in TTC.

Background Abnormal activity of muscarinic receptor (M-receptor) activated potassium current (IK,ACh) has been suggested as a potential atrial-specific drug target for antiarrhythmic therapy in patients with atrial fibrillation (AF). Recent work suggests that atrial myocytes derived from human induced pluripotent stem cells (iPSC-CM) hold great potential for in vitro disease modeling and patient-specific drug testing. Here we aim to investigate functional expression of IK,ACh channels in these cells. Methods Atrial differentiation was initiated by temporal WNT and retinoic acid signaling modulation. Action potentials (APs) were recorded using sharp electrode technique in engineered heart muscle preparations (EHM). Currents were measured with voltage-clamp technique in atrial and ventricular iPSC-CM. IK,ACh was activated with a saturating concentration of the non-selective M-receptor agonist carbachol (CCh, 2 μM). Results APs measured in atrial EHMs were shorter compared to their ventricular counterparts (AP duration at 90% repolarization [APD90]: 163.2±3.7 ms, n=6 [EHMs] vs. 210.2±3.1 ms, n=3, p Basal inward-rectifier current, (at −100 mV) in the absence of muscarinic receptor agonists was larger in ventricular iPSC-CM compared to atrial differentiation (−26.1±3.8 pA/pF, n=82/5 [n = cells/line] vs. −15.6±1.6 pA/pF, n=71/5, p CCh-induced current increase, attributed to IK,ACh, was detectable in all myocytes from atrial preparations (−25.2±3.6 pA/pF, n=71/5). The time course of activation with a slow decrease in current amplitude during the continuous presence of CCh resembled measurements in freshly isolated human atrial myocytes. In ventricular iPSC-CM preparations only 13 out of 73 myocytes showed an increase in inward-rectifier current in response to CCh, suggesting that ventricular preparations may contain a minor fraction of atrial myocytes. Of note, in the presence of the selective IK,ACh blocker tertiapin (100 nM), CCh had no effect on basal inward-rectifier current, providing further evidence for the molecular identity of the CCh-induced current increase as IK,ACh. Interestingly, TTP reduced basal current amplitude in CM isolated from atrial EHMs by ∼54% suggesting that agonist-independent, constitutively active IK,ACh contributes to the basal inward-rectifier current amplitude in these myocytes. Conclusion Our data demonstrate that atrial iPSC-CMs express functional IK,ACh-channels, which can be activated by M-receptor agonists. These findings further outline the potential of atrial iPSC-CMs and EHMs as a promising tool for the investigation of channel-associated pathologies such as AF. Acknowledgement/Funding grants from the DFG (VO 1568/3-1, IRTG1816, and SFB1002 project A13), the Else-Kröner-Fresenius Foundation (EKFS 2016_A20) and the DZHK (DZHK GOE MD3)

Short QT syndrome (SQTS) predisposes afflicted patients to sudden cardiac death. Until now, only one drug-quinidine-has been shown to be effective in patients with SQTS type 1(SQTS1). The objective of this study was to use human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from a patient with SQTS1 to search for potentially effective drugs for the treatment of SQTS1 patients. Patch clamp and single-cell contraction measurements were employed to assess drug effects. Ivabradine, mexiletine, and ajmaline but not flecainide, ranolazine, or amiodarone prolonged the action potential duration (APD) in hiPSC-CMs from an SQTS1 patient. Ivabradine, ajmaline, and mexiletine inhibited KCNH2 channel currents significantly, which may underlie their APD-prolonging effects. Under proarrhythmic epinephrine stimulation in spontaneously beating SQTS1 hiPSC-CMs, ivabradine, mexiletine, and ajmaline but not flecainide reduced the epinephrine-induced arrhythmic events. The results demonstrate that ivabradine, ajmaline, and mexiletine may be candidate drugs for preventing tachyarrhythmias in SQTS1 patients.

Background Short QT syndrome ( SQTS ), a disorder associated with characteristic ECG QT ‐segment abbreviation, predisposes affected patients to sudden cardiac death. Despite some progress in assessing the organ‐level pathophysiology and genetic changes of the disorder, the understanding of the human cellular phenotype and discovering of an optimal therapy has lagged because of a lack of appropriate human cellular models of the disorder. The objective of this study was to establish a cellular model of SQTS using human‐induced pluripotent stem cell–derived cardiomyocytes (hi PSC ‐ CM s). Methods and Results This study recruited 1 patient with short QT syndrome type 1 carrying a mutation (N588K) in KCNH 2 as well as 2 healthy control subjects. We generated hi PSC s from their skin fibroblasts, and differentiated hi PSC s into cardiomyocytes (hi PSC ‐ CM s) for physiological and pharmacological studies. The hi PSC ‐ CM s from the patient showed increased rapidly activating delayed rectifier potassium channel current ( I K r ) density and shortened action potential duration compared with healthy control hi PSC ‐ CM s. Furthermore, they demonstrated abnormal calcium transients and rhythmic activities. Carbachol increased the arrhythmic events in SQTS but not in control cells. Gene and protein expression profiling showed increased KCNH 2 expression in SQTS cells. Quinidine but not sotalol or metoprolol prolonged the action potential duration and abolished arrhythmic activity induced by carbachol. Conclusions Patient‐specific hi PSC ‐ CM s are able to recapitulate single‐cell phenotype features of SQTS and provide novel opportunities to further elucidate the cellular disease mechanism and test drug effects.

Background: Limb-Girdle muscular dystrophies (LGMD) are a heritable group of genetically determined disorders with a primary involvement of the pelvic or shoulder girdle musculature with partially cardiac manifestation, such as dilated cardiomyopathy (DCM) and life-threatening tachyarrhythmia. We report here that human induced pluripotent stem cell (hiPSC)–derived cardiomyocytes from a patient with LGMD2I and DCM associated with recurrent ventricular tachycardia displayed ion channel dysfunction and abnormality of calcium homeostasis. Methods: Dermal fibroblasts obtained from a patient with LGMD2I harboring a fukutin-related protein gene mutation (826C>A; Leu276Ile) and 3 healthy donors were reprogrammed to hiPSCs. The hiPSCs were differentiated into cardiomyocytes and used for biological and electrophysiological studies. Results: Compared with hiPSC cardiomyocytes from the healthy donors, the hiPSC cardiomyocytes from the patient exhibited abnormal action potentials characterized by reduced amplitude and upstroke velocity. The peak and late Na channel currents (I Na ) as well as the peak L-type calcium channel currents were significantly reduced. The expression of SCN5A and CACNA1C was reduced in DCM cardiomyocytes, consistent with reduction of I Na and L-type calcium channel currents. In addition, the rapidly activating delayed rectifier potassium current (I Kr ) was reduced, whereas the transient outward current (I to ) and slowly activating delayed rectifier potassium current (I Ks ) were similar in DCM and control cardiomyocytes. Finally, a significant reduction of systolic and diastolic intracellular Ca 2+ concentrations was detected in DCM cardiomyocytes. Conclusions: This study demonstrates that patient-specific hiPSC cardiomyocytes can recapitulate some phenotypic properties of LGMD2I with DCM and provide a platform for studies on the cardiac events in LGMD.

Aims Our aim is to investigate the arrhythmogenic mechanism in arrhythmogenic right ventricular cardiomyopathy (ARVC)-patients by using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Methods and results Human-induced pluripotent stem cell-derived cardiomyocytes were generated from human skin fibroblasts of two healthy donors and an ARVC-patient with a desmoglein-2 (DSG2) mutation. Patch clamp, quantitative polymerase chain reaction, and calcium imaging techniques were employed for the study. The amplitude and maximal upstroke velocity (Vmax) of action potential (AP) in ARVC-cells were smaller than that in healthy donor cells, whereas the resting potential and AP duration (APD) was not changed. The reduced Vmax resulted from decreased peak sodium current. The reason for undetected changes in APD may be the counter-action of reduced transient outward, small conductance Ca2+-activated, adenosine triphosphate-sensitive, Na/Ca exchanger (INCX) currents, and enhanced rapidly delayed rectifier currents. Isoprenaline (Iso) reduced INCX and shortened APD in both donor and ARVC-hiPSC-CMs. However, the effects of Iso in ARVC-cells are significantly larger than that in donor cells. In addition, ARVC-hiPSC-CMs showed more frequently than donor cells arrhythmogenic events induced by adrenergic stimulation. Conclusion Cardiomyocytes derived from the ARVC patient with a DSG2 mutation displayed multiple ion channel dysfunctions and abnormal cellular electrophysiology as well as enhanced sensitivity to adrenergic stimulation. These may underlie the arrhythmogenesis in ARVC patients.

A major research focus in the field of cardiovascular medicine is the prospect of using stem cells for cardiac regeneration. With the advent of induced pluripotent stem cell (iPSC) technology, major efforts are also in process to use iPSCs for modelling heart disease, screening for new drugs, and the development of treatment strategies on a patient-specific level. Here we describe the generation of iPSCs, their efficient differentiation into functional cardiomyocytes, and the power of the iPSC technology for modelling dilative cardiomyopathy on a patient-specific level.

Background Takotsubo syndrome (TTS) is characterized by an acute left ventricular dysfunction and is associated with life-threating complications in the acute phase. The underlying disease mechanism in TTS is still unknown. A genetic basis has been suggested to be involved in the pathogenesis. Objectives The aims of the study were to establish an in vitro induced pluripotent stem cell (iPSC) model of TTS, to test the hypothesis of altered β-adrenergic signaling in TTS iPSC-cardiomyocytes (CMs), and to explore whether genetic susceptibility underlies the pathophysiology of TTS. Methods Somatic cells of patients with TTS and control subjects were reprogrammed to iPSCs and differentiated into CMs. Three-month-old CMs were subjected to catecholamine stimulation to simulate neurohumoral overstimulation. We investigated β-adrenergic signaling and TTS cardiomyocyte function. Results Enhanced β-adrenergic signaling in TTS-iPSC-CMs under catecholamine-induced stress increased expression of the cardiac stress marker NR4A1; cyclic adenosine monophosphate levels; and cyclic adenosine monophosphate–dependent protein kinase A–mediated hyperphosphorylation of RYR2-S2808, PLN-S16, TNI-S23/24, and Cav1.2-S1928, and leads to a reduced calcium time to transient 50% decay. These cellular catecholamine-dependent responses were mainly mediated by β1-adrenoceptor signaling in TTS. Engineered heart muscles from TTS-iPSC-CMs showed an impaired force of contraction and a higher sensitivity to isoprenaline-stimulated inotropy compared with control subjects. In addition, altered electrical activity and increased lipid accumulation were detected in catecholamine-treated TTS-iPSC-CMs, and were confirmed by differentially expressed lipid transporters CD36 and CPT1C. Furthermore, we uncovered genetic variants in different key regulators of cardiac function. Conclusions Enhanced β-adrenergic signaling and higher sensitivity to catecholamine-induced toxicity were identified as mechanisms associated with the TTS phenotype. (International Takotsubo Registry [InterTAK Registry] [InterTAK]; NCT01947621)

Background: Ventricular arrhythmia is a leading cause of cardiac mortality. Most antiarrhythmics present paradoxical proarrhythmic side effects, culminating in a greater risk of sudden death. Methods: We describe a new regulatory mechanism linking mitogen-activated kinase kinase-7 deficiency with increased arrhythmia vulnerability in hypertrophied and failing hearts using mouse models harboring mitogen-activated kinase kinase-7 knockout or overexpression. The human relevance of this arrhythmogenic mechanism is evaluated in human-induced pluripotent stem cell–derived cardiomyocytes. Therapeutic potentials by targeting this mechanism are explored in the mouse models and human-induced pluripotent stem cell–derived cardiomyocytes. Results: Mechanistically, hypertrophic stress dampens expression and phosphorylation of mitogen-activated kinase kinase-7. Such mitogen-activated kinase kinase-7 deficiency leaves histone deacetylase-2 unphosphorylated and filamin-A accumulated in the nucleus to form a complex with Krüppel-like factor-4. This complex leads to Krüppel-like factor-4 disassociation from the promoter regions of multiple key potassium channel genes (Kv4.2, KChIP2, Kv1.5, ERG1, and Kir6.2) and reduction of their transcript levels. Consequent repolarization delays result in ventricular arrhythmias. Therapeutically, targeting the repressive function of the Krüppel-like factor-4/histone deacetylase-2/filamin-A complex with the histone deacetylase-2 inhibitor valproic acid restores K + channel expression and alleviates ventricular arrhythmias in pathologically remodeled hearts. Conclusions: Our findings unveil this new gene regulatory avenue as a new antiarrhythmic target where repurposing of the antiepileptic drug valproic acid as an antiarrhythmic is supported.