Abstract 6: Restoration of Impaired Diastolic Function in Hypertrophic Cardiomyopathy Induced Pluripotent Stem Cell-derived Cardiomyocytes by Re-balancing the Calcium Homeostasis

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Haodi Wu ◽  
Huaxiao Yang ◽  
Joe Zhang ◽  
Chi Keung Lam ◽  
June-Wha Rhee ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hypertrophic Cardiomyopathy ◽  
Diastolic Dysfunction ◽  
Diastolic Function ◽  
Calcium Level ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Cellular Mechanism ◽  
Patient Specific ◽  
Induced Pluripotent

Background: Diastolic dysfunction is commonly seen in hypertrophic cardiomyopathy (HCM). However, the cellular mechanism is not fully understood, and no effective treatment so far has been developed. We hypothesize here that HCM patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) can recapitulate the cellular mechanism, and provide us a platform for mechanistic study and for drug screening of diastolic dysfunctions in HCM. Methods and Results: We generated beating iPSC-CMs from healthy individuals and HCM patients carrying familial mutations (MYH7 R663H (n=2 lines) and MYBPC3 R943ter (n=2 lines)). Sarcomere shortening measurement in patterned iPSC-CMs with live cell confocal imaging showed significantly prolonged diastolic phase and slower relaxation velocity in HCM iPSC-CMs compared to WT cells. To elucidate the cellular mechanism, Fura-2 AM ratiometric calcium imaging showed marked elevation of resting calcium level and increased abnormal calcium handlings in HCM iPSC-CMs, which were exaggerated by β-adrenergic activation with isoproterenol. By applying calcium transient and contractile force simultaneous recording, we defined a “risk index of diastolic dysfunction” (measured as transient-contraction gain factor), which was significantly increased in HCM iPSC-CMs. Thus, both elevated basal calcium level and increased calcium sensitivity of myofilament contribute to the abnormal diastolic function in HCM iPSC-CMs. Gene expression profiling of HCM and WT iPSC-CMs indicated that increased calcium channels may underlie the increased basal calcium concentration in HCM cells. Indeed, partially blocking the calcium influx by calcium blockers reset the basal calcium level, attenuated calcium mishandling, and restored the diastolic function in HCM iPSC-CMs. Moreover, re-balancing calcium homeostasis significantly improved long-term survival rate of HCM iPSC-CMs at both basal level and under β-adrenergic stress. Conclusion: The iPSC-CM models carrying patient-specific HCM mutations recapitulated diastolic dysfunction on single cell level. Future studies using these platform may reveal additional novel cellular mechanisms and therapeutic targets of diastolic dysfunction in HCM disease.

scholarly journals Modelling diastolic dysfunction in induced pluripotent stem cell-derived cardiomyocytes from hypertrophic cardiomyopathy patients

2019 ◽  
Vol 40 (45) ◽  
pp. 3685-3695 ◽  
Author(s):  
Haodi Wu ◽  
Huaxiao Yang ◽  
June-Wha Rhee ◽  
Joe Z Zhang ◽  
Chi Keung Lam ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hypertrophic Cardiomyopathy ◽  
Diastolic Dysfunction ◽  
Diastolic Function ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Cellular Mechanisms ◽  
Term Survival ◽  
Induced Pluripotent

Abstract Aims Diastolic dysfunction (DD) is common among hypertrophic cardiomyopathy (HCM) patients, causing major morbidity and mortality. However, its cellular mechanisms are not fully understood, and presently there is no effective treatment. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) hold great potential for investigating the mechanisms underlying DD in HCM and as a platform for drug discovery. Methods and results In the present study, beating iPSC-CMs were generated from healthy controls and HCM patients with DD. Micropatterned iPSC-CMs from HCM patients showed impaired diastolic function, as evidenced by prolonged relaxation time, decreased relaxation rate, and shortened diastolic sarcomere length. Ratiometric Ca2+ imaging indicated elevated diastolic [Ca2+]i and abnormal Ca2+ handling in HCM iPSC-CMs, which were exacerbated by β-adrenergic challenge. Combining Ca2+ imaging and traction force microscopy, we observed enhanced myofilament Ca2+ sensitivity (measured as dF/Δ[Ca2+]i) in HCM iPSC-CMs. These results were confirmed with genome-edited isogenic iPSC lines that carry HCM mutations, indicating that cytosolic diastolic Ca2+ overload, slowed [Ca2+]i recycling, and increased myofilament Ca2+ sensitivity, collectively impairing the relaxation of HCM iPSC-CMs. Treatment with partial blockade of Ca2+ or late Na+ current reset diastolic Ca2+ homeostasis, restored diastolic function, and improved long-term survival, suggesting that disturbed Ca2+ signalling is an important cellular pathological mechanism of DD. Further investigation showed increased expression of L-type Ca2+channel (LTCC) and transient receptor potential cation channels (TRPC) in HCM iPSC-CMs compared with control iPSC-CMs, which likely contributed to diastolic [Ca2+]i overload. Conclusion In summary, this study recapitulated DD in HCM at the single-cell level, and revealed novel cellular mechanisms and potential therapeutic targets of DD using iPSC-CMs.

scholarly journals Intronic CRISPR Repair in a Preclinical Model of Noonan Syndrome–Associated Cardiomyopathy

Circulation ◽  
2020 ◽  
Vol 142 (11) ◽  
pp. 1059-1076
Author(s):  
Ulrich Hanses ◽  
Mandy Kleinsorge ◽  
Lennart Roos ◽  
Gökhan Yigit ◽  
Yun Li ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hypertrophic Cardiomyopathy ◽  
Protein Kinase ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Mitogen Activated Protein Kinase ◽  
Patient Specific ◽  
Mitogen Activated Protein ◽  
Induced Pluripotent ◽  
Protein Kinase Signaling

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.

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

2017 ◽  
Vol 113 (5) ◽  
pp. 531-541 ◽  
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

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

2010 ◽  
Vol 363 (15) ◽  
pp. 1397-1409 ◽  
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

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

2016 ◽  
Vol 2016 ◽  
pp. 1-15 ◽  
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.

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

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.

Sign in / Sign up

Export Citation Format

Share Document