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Background Tachycardia-induced cardiomyopathy (TCM) is a reversible and likely underrecognized form of heart failure. Thus, a better understanding of the TCM-pathophysiology is warranted as the underlying early mechanisms that mediate the progression of TCM remain unclear. Purpose This study aimed to identify the cellular mechanisms of TCM. Methods and results Human induced pluripotent stem cell cardiomyocytes (iPSC-CM) were utilized as a translational human-based model. We performed chronic tachycardic (120 bpm) or normofrequent (control, 60bpm) cell culture pacing to study cellular changes during TCM progression. Already after 24h of tachycardic stimulation of iPSC-CM, we detected a decrease in Ca transient amplitude compared to control (Fura-2, n=49/44 cells/9 differentiations). Diastolic Ca levels and cytosolic Ca elimination were not affected after 24h of tachycardia (n=49/44/9). We detected no difference in sarcoplasmic reticulum (SR) Ca load (assessed via caffeine application) or SERCA activity (Ksys-Kcaff) after 24h of tachycardia (n=13/15/5). However, demonstrating the progress of TCM, 7d of tachycardia resulted in progressive decline of Ca transient amplitude together with an impaired Ca elimination, while diastolic Ca concentration was unchanged (n=73/66/8). These changes may underlie the reduced systolic force and impaired relaxation in TCM. We could explain these results by a significantly reduced SR Ca load and a diminished SERCA activity after 7d tachycardia (n=13/7 vs. 13/4). Using confocal microscopy (Fluo-4) we detected no difference in SR Ca spark frequency after 24h of tachycardia (n=82/66/8), while 7d of tachycardia caused an increase of Ca spark frequency (n=76/79/7), which is a typical hallmark of maladaptive remodeling in HF and likely underlie the reduced SR Ca load. Voltage clamp data of late Na current (INaL) showed no difference in INaL after 24h of stimulation (n=17/6 vs. 19/7), whereas INaL was increased after 7d of tachycardia (n=26/7 vs. 19/6). Accordingly, whole-cell current clamp experiments revealed a prolongation of the action potential after 7d of tachycardia compared to control (n=21/6 vs. 19/5), while no difference of action potential duration could be detected after 24h (n=37/31/8). Resting membrane potential and action potential amplitude were not changed. Finally, we investigated tachycardia-mediated effects on explanted human failing hearts. 8h of tachycardic stimulation (120 bpm) of human failing ventricular trabeculae already compromised systolic force, and diastolic tension and relaxation time were markedly increased compared to control (60bpm, n=8/6 trabeculae /7/6 human hearts). Conclusion This study demonstrates that persistent tachycardia adversely alters cardiomyocyte excitation-contraction coupling via electrophysiological cellular remodeling. Our translational investigation in human myocardium may help to understand the pathophysiology of an underrated but prevalent disease. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Else Kröner-Fresenius-Stiftung (EKFS)Deutsche Gesellschaft für Innere Medizin

Background and aims The Takotsubo syndrome (TTS) is characterised by an acute left ventricular dysfunction without exhibiting signs of stenosis. TTS affects mainly the left ventricle, however, its pathophysiology comprises transient impairments in left atrium functions with a prevalence of atrial fibrillation (AF) of around 18% correlating with higher ventricular heart rhythm disorders. In this study, we aimed to prove the hypothesis that molecular and cellular arrhythmic events contribute to the development of TTS under catecholamine stress and to test if the currently clinically used β-blockers (Metoprolol) or the PDE4 activator MR-L2 are suitable for Takotsubo cardiomyopathy in vitro. Methods and results We generated induced pluripotent stem cell-derived atrial cardiomyocytes (TTS-iPSC-aCMs) from TTS patients, confirmed atrial marker expression (MLC2a, PItX2, NR2F2), and treated them with catecholamines (Iso) to mimic TTS-phenotype. Using a cytosolic Förster resonance energy transfer (FRET) based cAMP sensor, we tested the activity of phosphodiesterases (PDEs) in TTS-iPSC-aCMs and observed that after β-AR stimulation, the strong effects of the PDE4 family in the cytosol of atrial control cells were significantly decreased in aCMs of the TTS patients. This effect was rescued after application of PDE4 activator MR-L2 and is in line with the previously described downregulation of PDE4 in human atrial myocardium of AF patients. In functional studies, Iso-induced increase in systolic Ca2+ transient amplitude was more pronounced in TTS iPSC-aCM compared to controls. These effects were rescued by both, the clinically approved β-blocker Metoprolol and by MR-L2. To analyse arrhythmic events in atrial TTS CMs, we performed confocal microscope Ca2+ measurements and demonstrated that the diastolic sarcoplasmic reticulum Ca2+ leak was increased in iPSC aCMs of TTS patients compared to control under basal conditions and after Iso-treatment. In addition, TTS patients displayed faster Ca2+ kinetics compared to control cells, already under basal conditions. These results were underlined on a molecular level by increased phospholamban phosphorylation in TTS iPSC-aCM. Subsequent treatment with Metoprolol rescued the Ca2+ kinetic parameters and the increased calcium sparks in all cell lines. Conclusion In conclusion, we were able to draw a comprehensive picture on the role of the atrium in the development of arrhythmias in TTS. We found TTS-patient-specific differences with reduced PDE4 activity, elevated arrhythmic events and enhanced reactions to catecholamines, which could be rescued by the clinically approved drug Metoprolol and partly by the PDE4 activator MR-L2. Therefore, Metoprolol has proven to be an effective treatment option for TTS and preliminary data of MR-L2 demonstrate promising effects as a new patient-specific therapeutic target for TTS under catecholamine-stress. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): German centre for cardiovascular research

Introduction Ischemic heart failure persists as a global health problem despite optimized medical and adjunctive device therapies. Loss of cardiomyocytes in the absence of a proliferative response comprise a major contributor to pathological remodeling and death in this patient population. Experimental studies have shown that microRNAs (miRNAs) may be used as a therapeutic option to reinduce adult cardiomyocyte proliferation. Purpose This study thought to evaluate proliferative potential in human cardiomyocytes after overexpression and inhibition of 2019 miRNAs. Methods To identify miRNAs that regulate cardiomyocyte proliferation, we performed functional high-throughput screenings in human iPSC-derived cardiomyocytes (hiPSC-CM) after transient hypoxia. Herein, 2019 miRNA-mimics for overexpression and 2019 anti-miRs for inhibition were individually transfected to examine EdU-incorporation in hiPSC-CM. MiR-mimic-515 and miR-mimic-519e that induced the highest EdU-uptake, were further assessed by immunostaining and molecular methods for markers indicative of early and late mitosis. In addition, RNA-Sequencing in hiPSC-CM after overexpression of miR-515 and miR-519e was performed to examine differential gene expression and miRNA-modulated pathways involved in cardiomyocyte proliferation. Results Using a functional high-throughput screening, we assessed differential proliferative potential of 2019 miRNAs after transient hypoxia by transfecting both miR-inhibitor and miR-mimic libraries in human iPSC-derived cardiomyocytes (hiPSC-CM). Overexpression of 28 miRNAs substantially induced proliferative activity in hiPSC-CM, with an overrepresentation of miRNAs belonging to the C19MC-cluster and adjacent miR-371–373 family. Two of these miRNAs, miR-515 and miR-519e increased markers of early and late mitosis, with an additive cardiomyocyte turnover after transient hypoxia and substantially increased Aurora B-kinase activity in midbodies, indicative of cell division. These findings were supported by molecular studies using qRT-PCR, Western blot, and RNA-Sequencing after overexpression of miR-515 and miR-519e showing substantial alterations of signaling pathways relevant for cardiomyocytes proliferation in human iPSC-CM. Conclusion Collectively, these results support a critical role of miR-515 and miR-519e for induction of proliferation in human cardiomyocytes under hypoxic conditions, such as present in patients with ischemia-driven cardiomyopathy. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): This work was supported by the German Centre for Cardiovascular Research (DZHK), Deutsche Stiftung für Herzforschung (DSHF) and OPO Foundation.

Background Mutations in the splice factor RBM20 account for ∼3% of genetic cardiomyopathies. Mutations at position R634 in the hotspot RS-domain were found to cause dilative cardiomyopathy (DCM) (R634W) or left ventricular non-compaction cardiomyopathy (LVNC) (R634L), but the pathophysiological mechanisms that govern the heterogeneity in phenotype presentation remain unknown. Purpose We aimed here to identify the molecular events caused by the distinct RBM20 mutations from DCM and LVNC patients using a patient-specific induced stem cell model (iPSC) and test if the currently clinically used β-blockers (Metroprolol) are suitable for different RBM20-dependent cardiomyopathies. Methods We generated iPSC-cardiomyocytes of 2 DCM- and 2 LVNC-patients harboring the RBM20-mutations R634W (DCM) or R634L (LVNC). We investigated alternative splicing, sarcomeric regularity, cAMP-level, kinase-specific phosphorylation of Ca2+ players and Ca2+ handling. To investigate the impact of the genetic background, isogenic rescue lines were generated by CRISPR/Cas9. Different clinical drugs as Metoprolol and Verapamil were used to analyze the pharmacological improvement in vitro. Results We investigated the splicing pattern of the 2 RBM20 mutations in DCM and LVNC iPSC-CMs and observed common isoform changes in titin and a 24bp-insertion in the gene RYR2. The Ca2+ handling gene triadin is misspliced in LVNC-CMs, whereas the structural gene LDB3 is misspliced in DCM-CMs. As a possible consequence of splice defects in sarcomeric genes, both DCM and LVNC-CMs exhibited an irregular sarcomeric structure. The Ca2+ handling gene CAMK2δ was predominantly misspliced in LVNC-CMs leading to CAMK2δ-dependent hyperphosphorylation of its target PLN-Thr17 and subsequently to shortened Ca2+ elimination time and weakened response to β-adrenergic stimulation. By contrast, DCM-CMs exhibited increased Ca2+ sparks and decreased systolic and diastolic Ca2+ levels. RBM20 expression itself was decreased in LVNC-CMs, but not in DCM-CMs. This highlights that 2 distinct RBM20 mutations can lead to different pathological Ca2+ phenotypes. Isogenic CRISPR/Cas9 repair of both RBM20 mutations in LVNC and DCM demonstrated a rescue in gene missplicing, sarcomeric regularity and the Ca2+ handling aberrations and underscored the causative nature of the 2 mutations and their diverging effects. Ca2+ channel blockage with Verapamil showed a significant improvement of some of the LVNC disease characteristics compared to commonly clinically used β-blocker Metoprolol and underpins the potential clinical use of this drug in patients with LVNC. Conclusion We show the first iPSC-model of splice-defect associated RBM20-dependent LVNC and DCM. In summary, our results suggest that the molecular aberrations in alternative splicing differ depending on the distinct mutation in RBM20 and lead to shared and differential pathologies. Verapamil could be a good candidate in the treatment of RBM20-dependent LVNC. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Bunderministerium für Bildung und Forschung BMBFGerman Center for Cardiovascular Research DZHK

Atrial fibrillation (AF) is often found in patients with heart failure (HF). Clinical data indicated that the arrhythmic component of AF alone could contribute to left-ventricular (LV) dysfunction. However, the effects of non-tachycardic AF with arrhythmic excitation of the human LV, are unknown. We investigated human LV myocardium from patients with sinus rhythm (SR) or normofrequent AF (mean EF>50%, matched clinical data, derived from septal resections during AVR). In histological analysis we detected no difference between SR (n=17 patients) and AF patients (n=18) regarding the amount and distribution of fibrosis. We isolated human LV cardiomyocytes (CM) and studied cellular Ca-handling (Fura-2). Systolic Ca-transient amplitude of LV CM was reduced in patients suffering from AF (n=8 AF patients vs. 11 SR), while diastolic Ca-levels and Ca-transient kinetics were not significantly changed. These results were confirmed in LV CM from non-failing donors (NF) with AF (n=4 AF patients vs. 8 SR). For the standardized investigation of a normofrequent arrhythmia, we simulated AF in vitro by using arrhythmic (60 bpm, 40% beat-to-beat variability) or rhythmic (60 bpm) field stimulation. Human LV CM from NF SR patients (n=8) showed an impaired Ca-transient amplitude after 24h arrhythmic culture pacing without changes in diastolic Ca and Ca-transient kinetics. For studying a model suitable for more standardized chronic pacing, we utilized human iPSC cardiomyocytes (iPSC-CM) from healthy donors (n=6). After 7 days, arrhythmically paced iPSC-CM exhibited a reduced systolic Ca-transient amplitude, a trend towards a prolonged Ca-elimination time and a reduced sarcoplasmic reticulum Ca-load. Confocal line-scans of arrhythmically paced cells (Fluo-4 AM) showed an increased diastolic Ca-leak from the sarcoplasmic reticulum, possibly underlying the reduced Ca-load. Coupled with the Ca changes, cytosolic Na was elevated after arrhythmia. We found an increased late INa, which could explain the detrimentally altered Ca/Na-interplay. Accordingly, Patch-clamp experiments revealed a prolonged action potential duration after arrhythmia. We further elucidated the underlying mechanisms of this electrophysiological remodeling by showing that oxidative stress (H2O2, LPO) is increased in the LV of patients suffering from AF (n=6 AF patients vs. 6 SR), which was associated with an enhanced NOX2/-4 activity. Consecutively, Ca2+/calmodulin-dependent protein kinase IIδ (CaMKII) was found to be more oxidized (CaMKII-Met281/282) in the LV of AF patients (n=7 AF patients vs. 7 SR) leading to an increased CaMKII activity, which adversely regulated EC-coupling protein phosphorylation including RyR2 hyperphosphorylation. Normofrequent arrhythmia/AF impairs human ventricular EC-coupling via increased oxidative stress and enhanced CaMKII. Thus, this translational study provides the first mechanistic characterization and the potential negative impact of isolated AF on the human LV. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Else Kröner-Fresenius-Stiftung (EKFS) and Deutsche Gesellschaft für Innere Medizin

Background In heart failure (HF) both Ca2+/Calmodulin-dependent protein-kinase II (CaMKII) and late sodium current (INaL) are known to contribute to arrhythmogenesis as they contribute to action-potential (AP) prolongation and the occurrence of early- (EADs) and delayed afterdepolarizations (DADs). Further, augmented CaMKII and INaL maintain a vicious cycle as they both can activate each other. We recently found that the sodium channel isoform NaV1.8 is upregulated in HF and hypertrophy and that it is involved in INaL-generation. In the current study we investigated the effects of NaV1.8-knock-out (KO) on HF-progression and arrhythmogenesis in a CaMKII-overexpressing HF mouse model. Methods/Results CaMKII overexpressing mice (CaMKII+/T) were crossbred with NaV1.8-KO mice (SCN10A−/−). To our surprise knock-out of NaV1.8 in CaMKII+/T mice (SCN10A−/−/CaMKII+/T) significantly improved survival (median survival 103 days vs 74.5 CaMKII+/T, p Conclusion We found a survival benefit by selective knock-out of the neuronal sodium channel isoform NaV1.8 in a proarrhythmic HF mouse model with augmented CaMKII expression. However, in our model NaV1.8-knock-out showed no effects on HF progression, while cellular proarrhythmic triggers were attenuated. Taken together with our findings in IPS-cardiomyocytes treated with the CRSIPR/Cas9 technology NaV1.8 plays a significant role for the generation of INaL and cellular arrhythmogenic triggers in the cardiomyocyte. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Deutsche Stiftung für Herzforschung

Background/Purpose Takotsubo syndrome (TTS) is characterized by acute transient left ventricular dysfunction in the absence of obstructive coronary lesions. We identified a higher sensitivity to catecholamine-induced stress toxicity as mechanism associated with the TTS phenotype in our former study, but the pathogenesis of TTS is still not completely understood. In this study our aim was to prove the hypothesis of an altered phosphodiesterase (PDE)-dependent 3',5'-cyclic adenosine monophosphate (cAMP)-signaling in TTS in patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Methods and results We generated functional TTS-iPSC-CMs and treated them with catecholamines to mimic a TTS-phenotype. To directly address the hypothesis that local cAMP dynamics might be altered in TTS, we used Förster resonance energy transfer (FRET) based cAMP sensors, which are specifically located in the cytosol or at the sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA) micro domain. We demonstrated that β-adrenergic receptor (β-AR) stimulations resulted in stronger cytosolic FRET responses in TTS-CMs compared to controls. In contrast, no differences of cAMP level were observed in the SERCA-PLN micro domain between TTS- and control-iPSC-CMs. To analyze the interplay of β-AR signaling and specific PDE contribution to the cAMP signaling in TTS, specific PDE-inhibitors were used. We were able to show in the cytosol that after β-AR stimulation, the strong effects of the PDE4 family of control cells were significantly decreased in diseased TTS CMs, which is in line with previously described reduced PDE4 activity in failing mouse hearts. In contrast, the contribution of PDE3 to cytoplasmic cAMP degradation was increased in TTS (Figure 1 A). This is in line with increased PDE3A and down-regulated PDE4D protein expression in TTS-iPSC-CMs compared to control cells. Analysis of PDE-dependent cAMP level in the SERCA micro domain show also a significantly reduced PDE4 activity. But the dynamic cytosolic PDE contribution of PDE2 and PDE3 after catecholamine treatment in TTS is lost in SERCA micro domain (Figure1B). Conclusion Our data showed for the first time alterations of local cAMP signaling in healthy and diseased TTS-iPSC-CMs. We demonstrated an isozym shift from PDE4 in control to PDE3 and PDE2 in TTS and identified PDE4 as an important player in the β-adrenergic cAMP signaling in TTS. Therefore, PDE4 activators may be a possible new therapeutic target option in the treatment of TTS. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): DZHK

Background and aim Mutations in the splice factor RBM20 have been identified to account for ∼3% of cardiomyopathies. In particular, the highly conserved RS-domain is a hotspot for disease-associated mutations. Distinct mutations at position 634 in the RS-domain were already described to be associated to dilative cardiomyopathy (DCM) (R634W) or to left ventricular non-compaction cardiomyopathy (LVNC) (R634L), but the molecular mechanisms that govern the heterogenic entity of DCM and LVNC remain largely unknown. We aimed to analyze the molecular driver behind the RBM20 mutation-based DCM and LVNC in a patient-specific stem cell model. Methods Human somatic cells from 2 DCM- and 2 LVNC-patients harboring the RBM20-mutations R634W (DCM) or R634L (LVNC) were reprogrammed into induced pluripotent stem cells (iPSC) and differentiated into functional cardiomyocytes (CM). Gene expression, alternative splicing activity, sarcomeric regularity, cAMP level, kinase-specific phosphorylation of important Ca2+ players, and physiological cardiac functions as Ca2+ homeostasis were analyzed (Fluo3 and Fura4). Isogenic rescue lines were generated by CRISPR/Cas9 technology to analyze the direct impact of the RBM20 mutations to the cardiac phenotype. Results We investigated the role of RBM20 mutations in DCM and LVNC-iPSC-CMs RBM20-splicing and observed common splice defects in titin-isoform-switch or a 24bp insertion in the gene ryanodine receptor 2 (RYR2).. In contrast, the calcium-handling gene Camk2δ was predominantly mis-spliced in LVNC-CMs, whereas the structural gene LDB3 was mis-spliced in DCM-CMs. As a possible consequence of splice defects in sarcomeric genes both DCM and LVNC-CMs exhibited an irregular sarcomeric structure at the Z-disk and M-line. Interestingly, the LVNC-CMs showed faster Ca2+ transient decay time and weakened response to β-adrenergic stimulation. In contrast, the DCM-CMs did exhibit increased Ca2+-sparks and decreased systolic and diastolic Ca2+ highlighting that two distinct missense mutations can lead to different pathological Ca2+ phenotypes. Ca2+ kinetic defects in LVNC-iPSC-CMs were independent of cAMP, but in line with Camk2δ-dependent hyperphosphorylation of the specific target PLN. Isogenic WT-iPSC lines were generated using CRISPR/Cas9 technology and underscored the role of RBM20-mutations in cardiomyopathies as the sarcomeric defects, Ca2+ cycling and leakage were rescued for both LVNC-CMs and DCM-CMs. Conclusion We show the first iPSC-model of splice-defect-associated RBM20-dependent LVNC and DCM. Our data demonstrate that RBM20-R634L induce mis-splicing of Camk2δ leading to hyperphosphorylation of PLN-Thr17 along with increased Ca2+ kinetics in LVNC, whereas RBM20-R634W induced RYR2-dependent Ca2+ leak with disturbed systolic and diastolic Ca2+in DCM. Taken together these results suggest that the molecular aberrations in alternative splicing differ depending on the distinct missense mutation in RBM20. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): BMBF, DZHK German Center for Cardiovascular research

Background/Purpose Dilated cardiomyopathy (DCM) is characterized by left ventricular dilation and contractile dysfunction. Fibrosis is one major phenotypic result in DCM, pointing to the contribution of both, cardiomyocytes (CM) and cardiac fibroblasts (cFB) to DCM. The molecular basis of most DCM cases remains unknown. Nevertheless, it is known that up to 35% of all cases have a family history, linked to mutations in more than 30 gene loci. The aim of this study is to analyse the crosstalk of iPSC-CM and cFB and the underlying genetic and molecular causes in a patient-specific induced pluripotent stem cell (iPSC) model of DCM. Methods and results For this purpose a 4-member family was recruited containing 2 patients (father and daughter) with severe DCM and heart transplantation. iPSCs of all family members were generated and differentiated into iPSC-CMs. All iPSC-CMs express general cardiac markers, e.g. βMHC, α-actinin. Interestingly, αMHC expression was decreased in diseased iPSC-CMs in comparison to control cells. Additionally, the sarcomeric regularity was decreased in diseased iPSC-CMs. As we found significantly increased fibrosis (22%) in explanted myocardium of the diseased father compared to healthy myocardium (8%), both cFB and CM seem to play an important role. From the same myocardium primary cFBs were isolated and shown to express typical cFB markers clearly distinguishing these cells from non-fibroblasts as well as from fibroblasts with different origin. To analyse the contribution of cFBs and CMs to DCM on a functional level, 3D engineered heart muscles (EHMs) were generated in different diseased/healthy cell combinations. EHMs composed of both or either one affected DCM-iPSC-CMs and/or DCM-cFBs in comparison to healthy control EHMs did not produce any measurable force, indicating that the DCM-EHM phenotype is clearly diseased. Evaluation of tissues' viscoelasticity showed that DCM-cFB, DCM-iPSC-CMs and DCM-EHMs were stiffer than healthy control EHMs. Thus these data suggest that apart from the obvious dysfunction of DCM CMs, DCM cFBs clearly contribute to the contractile pathophysiology in DCM EHMs. Furthermore, whole exome sequencing of iPSCs was conducted to identify disease-causing variants. This analyses point towards a new genetic variant in the FLNc gene coding for a protein important in development, stabilization and maintenance of myofibrils. Rescue of this variant by CRISPR Cas9 genome editing will shed more light onto the role of this variant during DCM development in the future. Conclusion Using a ps-iPSC-CM model of a 4-member family with two severe DCM patients, we could demonstrate a clear contribution of both cell types, iPSC-CMs and cFB, to the contractile pathophysiology of DCM. We identified a potentially disease-causing new variant in the FLNc gene, which may contribute to the impaired functionality within the diseased iPSC-CMs and EHM. This makes FLNc a new therapeutic target for DCM. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): IRTG1816

Background The consequence of normofrequent atrial fibrillation (AF) on the ventricle remains largely unknown. Methods and results To elucidate the effects of arrhythmic excitation on human ventricular myocardium we performed contractility experiments using ventricular trabecula from patients with heart failure (HF). Normofrequent AF was simulated using arrhythmic (60 bpm, 40% R-R interval variability) or rhythmic field stimulation (60 bpm). Within 8h of arrhythmic stimulation, human specimen showed an impaired systolic force, while diastolic tension increased pathologically (n=5–7 each/7 HF patients, Fig. 1). The characterization of the ventricular (in-vivo) phenotype in patients with AF was performed by utilizing ventricular myocardium from patients with sinus rhythm (SR) and from patients with AF in the absence of HF (compensated hypertrophy, EF>50%, matched clinical characteristics, LV myocardium obtained from aortic valve replacement surgery). Histological investigation showed increased levels of interstitial fibrosis in myocardium from patients with AF compared to SR (n=10 patients each). Studies of cellular Ca-homeostasis (epifluorescence microscopy, Fura-2) were performed using isolated human ventricular cardiomyocytes. While systolic Ca-transient amplitude (0.5 Hz) was preserved in ventricular cardiomyocytes from patients with AF, we found a significantly prolonged Ca-elimination time (RT80) by 22.0±7.7% and a trend towards increased diastolic Ca-levels (n=17–23 cells/4 patients each). This finding may be explained by a decrease in SERCA2a activity (ksys-kCaff, n=10–12/4 each) and an enhanced phospholamban expression in Western Blot experiments (n=5 patients each). For the standardized investigation of the involved targets/mechanisms mediating the pathological changes upon arrhythmic excitation, we utilized human induced pluripotent stem cell cardiomyocytes (iPSC-CM) from healthy donors for chronic arrhythmic culture stimulation (24h). Arrhythmic paced iPSC-CM showed no changes in systolic Ca-transient amplitude (0.5 Hz), whereas diastolic Ca-levels were increased, which fits nicely to the finding of disturbed trabeculae diastolic function (n=15 cells each). In patch clamp experiments, arrhythmic paced cells showed no alterations of resting membrane potential, upstroke velocity, action-potential amplitude or -duration (n=7–9 cells each). Protein expression levels of key Ca-handling proteins in iPSC-CM as well as regulated genes are already under investigation. Conclusion This study demonstrates that arrhythmic ventricular excitation deteriorates human myocardial contractility early in HF. In biopsies from patients with preserved EF, chronic AF was associated with increased levels of interstitial fibrosis and pathological diastolic Ca-handling, which could be causally confirmed in chronically arrhythmic paced iPSC-CM. Therefore, this study provides first mechanistic characterization of AF mediated effects on the human ventricle.

Background/Purpose Takotsubo syndrome (TTS) is characterized by acute transient left ventricular dysfunction in the absence of obstructive coronary lesions. Although, we identified an enhanced β-adrenergic signaling and higher sensitivity to catecholamine-induced stress toxicity as mechanisms associated with the TTS phenotype in our former study, the pathogenesis of TTS is still not completely understood. Here, we aimed to prove the hypothesis of a phosphodiesterase (PDE)-dependent regulation of 3',5'-cyclic adenosine monophosphate (cAMP) signaling in TTS under catecholamine stress. Methods and results We generated functional TTS induced pluripotent stem cell-derived cardiomyocytes (TTS-iPSC-CMs) from 6 patients and treated the cells with catecholamines to mimic a TTS-phenotype. Using a cytosolic Förster resonance energy transfer (FRET) based cAMP sensor, we could observe that β-adrenergic receptor (β-AR) stimulations led to stronger FRET responses in the cytosol of TTS-CMs as compared to controls. Besides β-ARs, PDEs are main players involved in cAMP signaling in CMs. At basal level TTS-CM show a significantly higher PDE3A and a reduced PDE4D protein expression in the TTS-CMs compared to control. In addition, FRET experiments show that after β-AR stimulation, the strong effects of the PDE4 family in the cytosol of control cells were significantly decreased in TTS-CMs. This is in line with previously described reduced PDE4 activity in failing mouse hearts. By analyzing PDE-dependent cAMP downstream effects as PKA-dependent phosphorylation, we could show an additional increase of PLN phosphorylation (PLN-S16), especially in control, when treating iPSC-CMs with a combination of iso and PDE4 inhibitor. In contrast, in TTS-iPSC-CMs the contribution of the PDE-families PDE2, 3 or 4 to phosphorylation of PLN-S16 was increased over iso alone. This suggests that different PDEs in TTS and control are involved in functional segregation of the SERCA2a microdomain from the cytosol in terms of cAMP downstream effects. To directly address the hypothesis that local cAMP dynamics might be altered in TTS, we used a SERCA micro domain targeted FRET based cAMP sensor. In contrast to the cytosolic cAMP regulation, the PDE4 inhibitor effects in the SERCA2 micro domain were only slightly decreased in TTS. Instead, the contribution of PDE2 to local cAMP degradation was slightly increased. Conclusion Our data show for the first time alterations of local cAMP signaling in healthy and diseased TTS-iPSC-CMs. TTS leads to changes in PDE composition in the cytosol but not significantly in SERCA microdomain. Our results uncover a PDE-dependent altered β-adrenergic signaling as a potential disease cause. This data highlight that TTS-iPSC-CMs can be used to provide a versatile tool for evaluating new treatment options for TTS as therapeutic targets.

Purpose: The ability to generate patient-specific human induced pluripotent stem cells (ps-iPSCs) provides a unique opportunity for modeling heart disease. Dilated cardiomyopathy (DCM) is due to a progressive enlargement of the heart leading finally to heart failure. Mutations in many genes have been implicated in the pathogenesis of DCM including the RNA-binding motif 20 (RBM20). We aimed to generate iPSCs from DCM patients with a RBM20 mutations, and to analyze the functionality and cell biology of cardiomyocytes derived from these ps-iPSCs (ps-iPSC-CM) with regard to the cardiac DCM phenotype. Methods and Results: Dermal fibroblasts from two patients (34y; 75y) with DCM harboring different RBM20 mutations were reprogrammed to ps-iPSCs via lentiviral infection using the STEMCCA system. The ps-iPSCs show pluripotent characteristics by expressing pluripotency markers on mRNA and protein level similar to human embryonic stem cells. Furthermore, bisulfite sequencing analysis showed that the OCT4 and NANOG promoters were significantly demethylated in ps-iPSCs compared to their parental cells. In addition, the generated RBM20-iPSCs are able to differentiate in vitro and in vivo into derivatives of all three germ layers. The cardiac in vitro differentiation efficiency into beating cardiomyocytes of the RBM20-iPSCs is similar to control iPSCs. Electrophysiological studies showed that under basal conditions ps-iPSC-CM exhibited spontaneous action potentials characteristic for pacemaker-, atrial-, ventricular- and Purkinje-like cells in a similar manner as control iPSC-CM. Using Fast Fourier Transformation the sarcomere-associated periodic signal amplitude was analyzed for α-actinin. A significant higher percentage of RBM20-iPSC-CM showed abnormal sarcomeric α-actinin distribution compared to control CM, suggesting disorganized myofilament structure. But no significant difference was observed in cell size between RBM20-iPSC-CMs and control cells. Further, we found a different isoform expression pattern of the cardiac titin in the mutant ps-iPSC-CM compared to the control cells. This demonstrates a RBM20-dependent regulation of organized myofilament structure and titin splicing in an in vitro ps-DCM-iPSC-model. Conclusion: We demonstrate that iPSC-derived cardiomyocytes from young and old DCM patients harboring a RBM20 mutation recapitulate the abnormalities that were found in individuals with DCM caused by the same mutation. The differentiated cardiomyocytes may be used for individual drug testing and development of novel treatments for this inherited disorder.

Background and purpose Takotsubo syndrome (TTS) is characterized by an acute left ventricular dysfunction similar to a myocardial infarction (MI) in the absence of coronary artery stenosis. Patients show symptoms similar to the acute MI with increased biomarkers and blood serum catecholamines. Recently, we developed a patient-specific TTS stem cell model and identified a higher sensitivity to catecholamine-induced stress. Furthermore, familial TTS cases and genetic studies point to a genetic predisposition. The purpose of this study was to analyze a genetic predisposition by characterizing genetic variants in genes associated with cardiac pathologies and their impact on calcium homoeostasis in TTS. Methods and results Whole exome sequencing analysis of a TTS patient discovered 2 missense AHNAK variants in its C-terminal domain and in addition the missense variant F189L in the calcium buffering calsequestrin 2 gene (CASQ2). AHNAK is a 700kDa big nucleoprotein and is involved in the β-adrenergic regulation of the cardiac calcium channel Cav1.2. 3-month old TTS-iPSC-derived cardiomyocytes (CM) were generated and the variants were confirmed by sequencing. We found AHNAK higher expressed in TTS-iPSC-CMs compared to control, whereas no expression alteration was observed for Cav1.2. Since AHNAK is described to act as a repressor towards Cav1.2, which is relieved under β-adrenergic stimulation, we analyzed the effect of AHNAK variants on a potential co-localization and interaction between both proteins. AHNAK and Cav1.2 were shown to co-localize in the cytoplasm as well as the membranes and co-immunoprecipitation experiments confirmed an interaction of AHNAK and Cav1.2 in all tested control- and TTS-iPSC-CMs. On a functional level, we were able to show by patch clamp analysis that Cav1.2 calcium currents are significantly increased in TTS-iPSC-CMs compared to control. The influence of CASQ2-F189L on sarcomeric reticulum (SR) calcium load was analyzed by epifluorescence microscopy using FURA4 and caffeine-applications. We found significantly decreased SR calcium content with an increased fractional release during systole in TTS-iPSC-CMs. To test, whether these variants are the main reason for altered interaction of AHNAK and Cav1.2, calcium currents or SR calcium load in TTS need to be proven in the future by using CRISPR/Cas9-rescued AHNAK/CASQ2 lines. Conclusion Here we show the cardiac functional consequences of AHNAK and CASQ2 missense mutations in TTS-iPSC-CMs with regard to calcium currents and SR calcium load. These results show that AHNAK and CASQ2 variants may predispose to TTS and enable a new therapeutic option for TTS. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Else Kröner-Fresenius Foundation