Abstract 20459: Epigenetic Activation of Phosphodiesterase Subtypes Lead to Compromised Beta-Adrenergic Signaling in Induced Pluripotent Stem Cell-Derived Cardiomyocytes From Dilated Cardiomyopathy Patients

Introduction: Familial dilated cardiomyopathy (DCM) has been modeled by human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). However, the mechanisms of compromised signaling transduction and contractile function in DCM iPSC-CMs are still not well understood. Methods and Results: Beating iPSC-CMs were generated from healthy individuals and DCM patients. RNA-seq and real-time PCR showed strict regulation of the main beta-adrenergic signaling proteins in iPSC-CMs during maturation. Confocal imaging of spontaneous calcium activity and hydrogel-based traction force microscopy (TFM) technology demonstrated beta-adrenergic stimulation induced both inotropic and chronotropic regulation in the contractility of iPSC-CMs. Following extended in vitro maturation of iPSC-CMs, we observed a transition in the beta-adrenergic receptor (beta-AR) subtype dependence from beta-2 AR dominance at early stage (day 30) to beta-1/2 AR co-existence at late stage (day 60). Comparison of the beta-adrenergic responsiveness between iPSC-CMs from DCM patients and their familial controls showed compromised beta-adrenergic signaling in DCM cells. Microarray data and expression profiling indicated up-regulated phosphodiesterases (PDE) 2A, 3A and 5A in DCM iPSC-CMs, which impaired cAMP generation and blunted the beta-adrenergic response. By blocking PDE2A, 3A or 5A, beta-adrenergic signaling reactivity and contractile function in DCM iPSC-CMs were both greatly improved. To further elucidate underlying mechanism of PDE regulation, we conducted chromatin immunoprecipitation (CHIP) assays, which showed significant up-regulation of activation histone marker and down-regulation of repressive histone marker in the PDE promoters during maturation process of DCM iPSC-CMs, which closely recapitulated the epigenetic modulation in the ventricle tissues harvested from DCM patients. Conclusions: Patient-specific DCM iPSC-CMs recapitulated impaired beta-adrenergic responsiveness and contractility in diseased heart. Studies on iPSC-CM model revealed a novel epigenetic mechanism that underlies PDE subtype specific regulation and signaling deficiency in DCM pathogenesis, which may serve as a new therapeutic target in the future.

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3074Analyzing the regulation of a catecholamine-dependent altered cAMP signaling in a patient-specific induced pluripotent stem cell takotsubo-model

European Heart Journal ◽

10.1093/eurheartj/ehz745.0030 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

D Huebscher ◽

T Borchert ◽

G Hasenfuss ◽

V Nikolaev ◽

K Streckfuss-Boemeke

Keyword(s):

Stem Cell ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Left Ventricular ◽

Camp Signaling ◽

Pde4 Inhibitor ◽

Patient Specific ◽

Adrenergic Signaling ◽

Downstream Effects ◽

Induced Pluripotent

Abstract 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.

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Establishment of patient-specific induced pluripotent stem cell line SDUBMSi009-A from a patient with X-linked Lowe syndrome

Stem Cell Research ◽

10.1016/j.scr.2021.102171 ◽

2021 ◽

Vol 51 ◽

pp. 102171

Author(s):

Xiaolin Liu ◽

Yong Liu ◽

Yanyan Ma ◽

Yaoqin Gong ◽

Qiji Liu ◽

...

Keyword(s):

Stem Cell ◽

Cell Line ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Patient Specific ◽

Lowe Syndrome ◽

Stem Cell Line ◽

Induced Pluripotent

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Elucidating arrhythmogenic mechanisms of long-QT syndrome CALM1-F142L mutation in patient-specific induced pluripotent stem cell-derived cardiomyocytes

Cardiovascular Research ◽

10.1093/cvr/cvx006 ◽

2017 ◽

Vol 113 (5) ◽

pp. 531-541 ◽

Cited By ~ 64

Author(s):

Marcella Rocchetti ◽

Luca Sala ◽

Lisa Dreizehnter ◽

Lia Crotti ◽

Daniel Sinnecker ◽

...

Keyword(s):

Stem Cell ◽

Long Qt Syndrome ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Patient Specific ◽

Long Qt ◽

Qt Syndrome ◽

Induced Pluripotent

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Patient-Specific Induced Pluripotent Stem-Cell Models for Long-QT Syndrome

New England Journal of Medicine ◽

10.1056/nejmoa0908679 ◽

2010 ◽

Vol 363 (15) ◽

pp. 1397-1409 ◽

Cited By ~ 813

Author(s):

Alessandra Moretti ◽

Milena Bellin ◽

Andrea Welling ◽

Christian Billy Jung ◽

Jason T. Lam ◽

...

Keyword(s):

Stem Cell ◽

Long Qt Syndrome ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Patient Specific ◽

Cell Models ◽

Long Qt ◽

Qt Syndrome ◽

Induced Pluripotent ◽

Stem Cell Models

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Machine Learning Approach to Automated Quality Identification of Human Induced Pluripotent Stem Cell Colony Images

Computational and Mathematical Methods in Medicine ◽

10.1155/2016/3091039 ◽

2016 ◽

Vol 2016 ◽

pp. 1-15 ◽

Cited By ~ 13

Author(s):

Henry Joutsijoki ◽

Markus Haponen ◽

Jyrki Rasku ◽

Katriina Aalto-Setälä ◽

Martti Juhola

Keyword(s):

Machine Learning ◽

Stem Cell ◽

Pluripotent Stem Cell ◽

Disease Modeling ◽

Induced Pluripotent Stem Cell ◽

Patient Specific ◽

Support Vector ◽

Ips Cell ◽

Cell Technology ◽

Induced Pluripotent

The focus of this research is on automated identification of the quality of human induced pluripotent stem cell (iPSC) colony images. iPS cell technology is a contemporary method by which the patient’s cells are reprogrammed back to stem cells and are differentiated to any cell type wanted. iPS cell technology will be used in future to patient specific drug screening, disease modeling, and tissue repairing, for instance. However, there are technical challenges before iPS cell technology can be used in practice and one of them is quality control of growing iPSC colonies which is currently done manually but is unfeasible solution in large-scale cultures. The monitoring problem returns to image analysis and classification problem. In this paper, we tackle this problem using machine learning methods such as multiclass Support Vector Machines and several baseline methods together with Scaled Invariant Feature Transformation based features. We perform over 80 test arrangements and do a thorough parameter value search. The best accuracy (62.4%) for classification was obtained by using ak-NN classifier showing improved accuracy compared to earlier studies.

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Abstract 142: Modeling Drug-Induced Long QT Syndrome with Patient-Specific Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Circulation Research ◽

10.1161/res.113.suppl_1.a142 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Francesca Stillitano ◽

Ioannis Karakikes ◽

Chi-wai Kong ◽

Brett Martinelli ◽

Ronald Li ◽

...

Keyword(s):

Stem Cell ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Patient Specific ◽

Dependent Manner ◽

Long Qt ◽

Drug Induced ◽

Qt Syndrome ◽

Induced Pluripotent ◽

Dose Dependent

Long QT syndrome (LQTS) is characterized by prolonged cardiac repolarization time and increased risk of ventricular arrhythmia. LQTS can be either inherited or induced notably after drugs intake. Mutations in genes encoding cardiac ion channels have been reported to underlie inherited LQTS. In contrast, drug-induced LQTS (diLQTS) most frequently arises from altered function of the hERG channel; the risk of developing diLQTS varies largely between subjects and most people who have life-threatening diLQTS have no known genetic risk factors. We investigated whether the susceptibility to develop diLQTS observed in vivo can be recapitulated in vitro using patient-specific induced pluripotent stem cell (iPSC) technology. We collected skin fibroblasts from ten subjects who developed significant diLQTS after administration of Sotalol and/or Erythromycin. Ten other individuals who displayed no changes in QT interval after administration of the same drugs, were selected. iPSC were generated by retroviral delivery of Oct4, Sox2, Nanog and Klf4 in 17 of the 20 individuals. We report preliminary results obtained from iPSC-derived cardiomyocytes (iPSC-CMs) of two subjects. All experiments were performed in a blinded fashion without knowledge of the associated clinical phenotype. Cardiac differentiation of iPSC resulted in the generation of spontaneously beating embryoid bodies. iPSC-CMs showed positive staining for TNNT2, ACTN2 and Cx43. Gene expression analysis confirmed the expression of NKX2.5, MLC2v, MYH6 and MYH7, and of the relevant KCNH2 gene. The two lines had similar basal electrophysiological properties as assessed by measurements of action potential (AP) by patch-clamp technique and extracellular field potentials (FP) using micro-electrode array (MEA). E4031, a classical HERG blocker, significantly prolonged the FP duration (FPD) in a dose-dependent manner in both lines (EC50: 30.19 and 51.57 respectively). When both Sotalol and Erythromicin were used, FPD was prolonged in one of the two samples in a dose-dependent manner (EC50Sotalol: 100; EC50Erythr: 9.64) while drug response was blunted in the other cell line. This study suggests that patient-specific iPSC can be used to model the functional abnormalities observed in acquired diLQTS.

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