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1.
Nature ; 572(7769): 335-340, 2019 08.
Article in English | MEDLINE | ID: mdl-31316208

ABSTRACT

Lamin A/C (LMNA) is one of the most frequently mutated genes associated with dilated cardiomyopathy (DCM). DCM related to mutations in LMNA is a common inherited cardiomyopathy that is associated with systolic dysfunction and cardiac arrhythmias. Here we modelled the LMNA-related DCM in vitro using patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Electrophysiological studies showed that the mutant iPSC-CMs displayed aberrant calcium homeostasis that led to arrhythmias at the single-cell level. Mechanistically, we show that the platelet-derived growth factor (PDGF) signalling pathway is activated in mutant iPSC-CMs compared to isogenic control iPSC-CMs. Conversely, pharmacological and molecular inhibition of the PDGF signalling pathway ameliorated the arrhythmic phenotypes of mutant iPSC-CMs in vitro. Taken together, our findings suggest that the activation of the PDGF pathway contributes to the pathogenesis of LMNA-related DCM and point to PDGF receptor-ß (PDGFRB) as a potential therapeutic target.


Subject(s)
Cardiomyopathy, Dilated/genetics , Lamin Type A/genetics , Mutation , Platelet-Derived Growth Factor/metabolism , Receptor, Platelet-Derived Growth Factor beta/metabolism , Signal Transduction , Arrhythmias, Cardiac/metabolism , Arrhythmias, Cardiac/pathology , Calcium/metabolism , Cells, Cultured , Chromatin/chemistry , Chromatin/genetics , Chromatin/metabolism , Chromatin Assembly and Disassembly/genetics , Haploinsufficiency/genetics , Homeostasis , Humans , In Vitro Techniques , Induced Pluripotent Stem Cells/pathology , Models, Biological , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Nonsense Mediated mRNA Decay , RNA, Messenger/analysis , RNA, Messenger/genetics , Single-Cell Analysis
2.
Basic Res Cardiol ; 119(1): 93-112, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38170280

ABSTRACT

In recent years, SGLT2 inhibitors have become an integral part of heart failure therapy, and several mechanisms contributing to cardiorenal protection have been identified. In this study, we place special emphasis on the atria and investigate acute electrophysiological effects of dapagliflozin to assess the antiarrhythmic potential of SGLT2 inhibitors. Direct electrophysiological effects of dapagliflozin were investigated in patch clamp experiments on isolated atrial cardiomyocytes. Acute treatment with elevated-dose dapagliflozin caused a significant reduction of the action potential inducibility, the amplitude and maximum upstroke velocity. The inhibitory effects were reproduced in human induced pluripotent stem cell-derived cardiomyocytes, and were more pronounced in atrial compared to ventricular cells. Hypothesizing that dapagliflozin directly affects the depolarization phase of atrial action potentials, we examined fast inward sodium currents in human atrial cardiomyocytes and found a significant decrease of peak sodium current densities by dapagliflozin, accompanied by a moderate inhibition of the transient outward potassium current. Translating these findings into a porcine large animal model, acute elevated-dose dapagliflozin treatment caused an atrial-dominant reduction of myocardial conduction velocity in vivo. This could be utilized for both, acute cardioversion of paroxysmal atrial fibrillation episodes and rhythm control of persistent atrial fibrillation. In this study, we show that dapagliflozin alters the excitability of atrial cardiomyocytes by direct inhibition of peak sodium currents. In vivo, dapagliflozin exerts antiarrhythmic effects, revealing a potential new additional role of SGLT2 inhibitors in the treatment of atrial arrhythmias.


Subject(s)
Atrial Fibrillation , Benzhydryl Compounds , Glucosides , Induced Pluripotent Stem Cells , Sodium-Glucose Transporter 2 Inhibitors , Humans , Animals , Swine , Myocytes, Cardiac , Sodium-Glucose Transporter 2 Inhibitors/pharmacology , Anti-Arrhythmia Agents/pharmacology , Anti-Arrhythmia Agents/therapeutic use , Action Potentials , Sodium
3.
Eur Heart J ; 43(36): 3477-3489, 2022 09 21.
Article in English | MEDLINE | ID: mdl-35728000

ABSTRACT

AIMS: Genetic dilated cardiomyopathy (DCM) is a leading cause of heart failure. Despite significant progress in understanding the genetic aetiologies of DCM, the molecular mechanisms underlying the pathogenesis of familial DCM remain unknown, translating to a lack of disease-specific therapies. The discovery of novel targets for the treatment of DCM was sought using phenotypic sceening assays in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) that recapitulate the disease phenotypes in vitro. METHODS AND RESULTS: Using patient-specific iPSCs carrying a pathogenic TNNT2 gene mutation (p.R183W) and CRISPR-based genome editing, a faithful DCM model in vitro was developed. An unbiased phenotypic screening in TNNT2 mutant iPSC-derived cardiomyocytes (iPSC-CMs) with small molecule kinase inhibitors (SMKIs) was performed to identify novel therapeutic targets. Two SMKIs, Gö 6976 and SB 203580, were discovered whose combinatorial treatment rescued contractile dysfunction in DCM iPSC-CMs carrying gene mutations of various ontologies (TNNT2, TTN, LMNA, PLN, TPM1, LAMA2). The combinatorial SMKI treatment upregulated the expression of genes that encode serine, glycine, and one-carbon metabolism enzymes and significantly increased the intracellular levels of glucose-derived serine and glycine in DCM iPSC-CMs. Furthermore, the treatment rescued the mitochondrial respiration defects and increased the levels of the tricarboxylic acid cycle metabolites and ATP in DCM iPSC-CMs. Finally, the rescue of the DCM phenotypes was mediated by the activating transcription factor 4 (ATF4) and its downstream effector genes, phosphoglycerate dehydrogenase (PHGDH), which encodes a critical enzyme of the serine biosynthesis pathway, and Tribbles 3 (TRIB3), a pseudokinase with pleiotropic cellular functions. CONCLUSIONS: A phenotypic screening platform using DCM iPSC-CMs was established for therapeutic target discovery. A combination of SMKIs ameliorated contractile and metabolic dysfunction in DCM iPSC-CMs mediated via the ATF4-dependent serine biosynthesis pathway. Together, these findings suggest that modulation of serine biosynthesis signalling may represent a novel genotype-agnostic therapeutic strategy for genetic DCM.


Subject(s)
Cardiomyopathy, Dilated , Molecular Targeted Therapy , Myocytes, Cardiac , Protein Kinase Inhibitors , Serine , Troponin T , Activating Transcription Factor 4/metabolism , Adenosine Triphosphate/metabolism , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Carbazoles/pharmacology , Carbazoles/therapeutic use , Cardiomyopathy, Dilated/drug therapy , Cardiomyopathy, Dilated/genetics , Drug Evaluation, Preclinical/methods , Glucose/metabolism , Glycine/biosynthesis , Glycine/genetics , Humans , Imidazoles/pharmacology , Imidazoles/therapeutic use , Induced Pluripotent Stem Cells/physiology , Mutation , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/enzymology , Phosphoglycerate Dehydrogenase/genetics , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Pyridines/pharmacology , Pyridines/therapeutic use , Serine/antagonists & inhibitors , Serine/biosynthesis , Serine/genetics , Troponin T/genetics , Troponin T/metabolism
4.
Proc Natl Acad Sci U S A ; 115(37): 9276-9281, 2018 09 11.
Article in English | MEDLINE | ID: mdl-30150400

ABSTRACT

This study demonstrates that significantly shortened telomeres are a hallmark of cardiomyocytes (CMs) from individuals with end-stage hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy (DCM) as a result of heritable defects in cardiac proteins critical to contractile function. Positioned at the ends of chromosomes, telomeres are DNA repeats that serve as protective caps that shorten with each cell division, a marker of aging. CMs are a known exception in which telomeres remain relatively stable throughout life in healthy individuals. We found that, relative to healthy controls, telomeres are significantly shorter in CMs of genetic HCM and DCM patient tissues harboring pathogenic mutations: TNNI3, MYBPC3, MYH7, DMD, TNNT2, and TTN Quantitative FISH (Q-FISH) of single cells revealed that telomeres were significantly reduced by 26% in HCM and 40% in DCM patient CMs in fixed tissue sections compared with CMs from age- and sex-matched healthy controls. In the cardiac tissues of the same patients, telomere shortening was not evident in vascular smooth muscle cells that do not express or require the contractile proteins, an important control. Telomere shortening was recapitulated in DCM and HCM CMs differentiated from patient-derived human-induced pluripotent stem cells (hiPSCs) measured by two independent assays. This study reveals telomere shortening as a hallmark of genetic HCM and DCM and demonstrates that this shortening can be modeled in vitro by using the hiPSC platform, enabling drug discovery.


Subject(s)
Cardiomyopathy, Dilated , Cardiomyopathy, Hypertrophic, Familial , Cell Division , Induced Pluripotent Stem Cells , Muscle Proteins , Mutation , Telomere Shortening , Cardiomyopathy, Dilated/genetics , Cardiomyopathy, Dilated/metabolism , Cardiomyopathy, Dilated/pathology , Cardiomyopathy, Hypertrophic, Familial/genetics , Cardiomyopathy, Hypertrophic, Familial/metabolism , Cardiomyopathy, Hypertrophic, Familial/pathology , Female , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/pathology , Male , Muscle Proteins/genetics , Muscle Proteins/metabolism
5.
Curr Heart Fail Rep ; 18(1): 1-11, 2021 02.
Article in English | MEDLINE | ID: mdl-33215357

ABSTRACT

PURPOSE OF REVIEW: Heart failure is among the most prevalent disease complexes overall and is associated with high morbidity and mortality. The underlying aetiology is manifold including coronary artery disease, genetic alterations and mutations, viral infections, adverse immune responses, and cardiac toxicity. To date, no specific therapies have been developed despite notable efforts. This can especially be attributed to hurdles in translational research, mainly due to the lack of proficient models of heart failure limited translation of therapeutic approaches from bench to bedside. RECENT FINDINGS: Human induced pluripotent stem cells (hiPSCs) are rising in popularity, granting the ability to divide infinitely, to hold human, patient-specific genome, and to differentiate into any human cell, including cardiomyocytes (hiPSC-CMs). This brings magnificent promise to cardiological research, providing the possibility to recapitulate cardiac diseases in a dish. Advances in yield, maturity, and in vivo resemblance due to straightforward, low-cost protocols, high-throughput approaches, and complex 3D cultures have made this tool widely applicable. In recent years, hiPSC-CMs have been used to model a wide variety of cardiac diseases, bringing along the possibility to not only elucidate molecular mechanisms but also to test novel therapeutic approaches in the dish. Within the last decade, hiPSC-CMs have been exponentially employed to model heart failure. Constant advancements are aiming at improvements of differentiation protocols, hiPSC-CM maturity, and assays to elucidate molecular mechanisms and cellular functions. However, hiPSC-CMs are remaining relatively immature, and in vitro models can only partially recapitulate the complex interactions in vivo. Nevertheless, hiPSC-CMs have evolved as an essential model system in cardiovascular research.


Subject(s)
Heart Failure , Induced Pluripotent Stem Cells , Cell Differentiation , Heart Failure/therapy , Humans , Myocytes, Cardiac
6.
Circulation ; 139(6): 799-811, 2019 02 05.
Article in English | MEDLINE | ID: mdl-30586709

ABSTRACT

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in myosin-binding protein C3 ( MYBPC3) resulting in a premature termination codon (PTC). The underlying mechanisms of how PTC mutations in MYBPC3 lead to the onset and progression of HCM are poorly understood. This study's aim was to investigate the molecular mechanisms underlying the pathogenesis of HCM associated with MYBPC3 PTC mutations by utilizing human isogenic induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). METHODS: Isogenic iPSC lines were generated from HCM patients harboring MYBPC3 PTC mutations (p.R943x; p.R1073P_Fsx4) using genome editing. Comprehensive phenotypic and transcriptome analyses were performed in the iPSC-CMs. RESULTS: We observed aberrant calcium handling properties with prolonged decay kinetics and elevated diastolic calcium levels in the absence of structural abnormalities or contracile dysfunction in HCM iPSC-CMs as compared to isogenic controls. The mRNA expression levels of MYBPC3 were significantly reduced in mutant iPSC-CMs, but the protein levels were comparable among isogenic iPSC-CMs, suggesting that haploinsufficiency of MYBPC3 does not contribute to the pathogenesis of HCM in vitro. Furthermore, truncated MYBPC3 peptides were not detected. At the molecular level, the nonsense-mediated decay pathway was activated, and a set of genes involved in major cardiac signaling pathways was dysregulated in HCM iPSC-CMs, indicating an HCM gene signature in vitro. Specific inhibition of the nonsense-mediated decay pathway in mutant iPSC-CMs resulted in reversal of the molecular phenotype and normalization of calcium-handling abnormalities. CONCLUSIONS: iPSC-CMs carrying MYBPC3 PTC mutations displayed aberrant calcium signaling and molecular dysregulations in the absence of significant haploinsufficiency of MYBPC3 protein. Here we provided the first evidence of the direct connection between the chronically activated nonsense-mediated decay pathway and HCM disease development.


Subject(s)
Cardiomyopathy, Hypertrophic/genetics , Carrier Proteins/genetics , Codon, Nonsense/genetics , Mutation/genetics , Myocytes, Cardiac/physiology , Pluripotent Stem Cells/physiology , RNA, Messenger/genetics , Calcium Signaling , Cell Differentiation , Cell Line , Disease Progression , Gene Editing , Gene Expression Profiling , Haploinsufficiency , Humans
7.
J Mol Cell Cardiol ; 126: 13-22, 2019 01.
Article in English | MEDLINE | ID: mdl-30445017

ABSTRACT

AIMS: Circulating immune cells have a significant impact on progression and outcome of heart failure. Long non-coding RNAs (lncRNAs) comprise novel epigenetic regulators which control cardiovascular diseases and inflammatory disorders. We aimed to identify lncRNAs regulated in circulating immune cells of the blood of heart failure patients. METHODS AND RESULTS: Next-generation sequencing revealed 110 potentially non-coding RNA transcripts differentially expressed in peripheral blood mononuclear cells of heart failure patients with reduced ejection fraction. The up-regulated lncRNA Heat2 was further functionally characterized. Heat2 expression was detected in whole blood, PBMNCs, eosinophil and basophil granulocytes. Heat2 regulates cell division, invasion, transmigration and immune cell adhesion on endothelial cells. CONCLUSION: Heat2 is an immune cell enriched lncRNA that is elevated in the blood of heart failure patients and controls cellular functions.


Subject(s)
Gene Expression Regulation , Heart Failure/genetics , RNA, Long Noncoding/genetics , Adult , Aged , Case-Control Studies , Cohort Studies , Eosinophils/metabolism , Female , Heart Failure/blood , Humans , Leukocytes, Mononuclear/metabolism , Male , Middle Aged , RNA, Long Noncoding/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism
8.
Circ Res ; 120(10): 1561-1571, 2017 May 12.
Article in English | MEDLINE | ID: mdl-28246128

ABSTRACT

RATIONALE: Targeted genetic engineering using programmable nucleases such as transcription activator-like effector nucleases (TALENs) is a valuable tool for precise, site-specific genetic modification in the human genome. OBJECTIVE: The emergence of novel technologies such as human induced pluripotent stem cells (iPSCs) and nuclease-mediated genome editing represent a unique opportunity for studying cardiovascular diseases in vitro. METHODS AND RESULTS: By incorporating extensive literature and database searches, we designed a collection of TALEN constructs to knockout 88 human genes that are associated with cardiomyopathies and congenital heart diseases. The TALEN pairs were designed to induce double-strand DNA break near the starting codon of each gene that either disrupted the start codon or introduced a frameshift mutation in the early coding region, ensuring faithful gene knockout. We observed that all the constructs were active and disrupted the target locus at high frequencies. To illustrate the utility of the TALEN-mediated knockout technique, 6 individual genes (TNNT2, LMNA/C, TBX5, MYH7, ANKRD1, and NKX2.5) were knocked out with high efficiency and specificity in human iPSCs. By selectively targeting a pathogenic mutation (TNNT2 p.R173W) in patient-specific iPSC-derived cardiac myocytes, we demonstrated that the knockout strategy ameliorates the dilated cardiomyopathy phenotype in vitro. In addition, we modeled the Holt-Oram syndrome in iPSC-cardiac myocytes in vitro and uncovered novel pathways regulated by TBX5 in human cardiac myocyte development. CONCLUSIONS: Collectively, our study illustrates the powerful combination of iPSCs and genome editing technologies for understanding the biological function of genes, and the pathological significance of genetic variants in human cardiovascular diseases. The methods, strategies, constructs, and iPSC lines developed in this study provide a validated, readily available resource for cardiovascular research.


Subject(s)
Cardiovascular Diseases/genetics , Gene Knockout Techniques/methods , Gene Library , Genetic Engineering/methods , Induced Pluripotent Stem Cells/physiology , Base Sequence , Cardiovascular Diseases/therapy , Cells, Cultured , Gene Targeting/methods , Humans , Induced Pluripotent Stem Cells/transplantation
9.
Nature ; 495(7439): 107-10, 2013 Mar 07.
Article in English | MEDLINE | ID: mdl-23426265

ABSTRACT

Ageing is the predominant risk factor for cardiovascular diseases and contributes to a significantly worse outcome in patients with acute myocardial infarction. MicroRNAs (miRNAs) have emerged as crucial regulators of cardiovascular function and some miRNAs have key roles in ageing. We propose that altered expression of miRNAs in the heart during ageing contributes to the age-dependent decline in cardiac function. Here we show that miR-34a is induced in the ageing heart and that in vivo silencing or genetic deletion of miR-34a reduces age-associated cardiomyocyte cell death. Moreover, miR-34a inhibition reduces cell death and fibrosis following acute myocardial infarction and improves recovery of myocardial function. Mechanistically, we identified PNUTS (also known as PPP1R10) as a novel direct miR-34a target, which reduces telomere shortening, DNA damage responses and cardiomyocyte apoptosis, and improves functional recovery after acute myocardial infarction. Together, these results identify age-induced expression of miR-34a and inhibition of its target PNUTS as a key mechanism that regulates cardiac contractile function during ageing and after acute myocardial infarction, by inducing DNA damage responses and telomere attrition.


Subject(s)
Aging/physiology , Gene Expression Regulation , Heart/physiology , MicroRNAs/genetics , Myocardium/metabolism , Aging/genetics , Aging/pathology , Animals , Apoptosis , DNA Damage , Fibrosis/genetics , Fibrosis/pathology , Gene Deletion , Gene Knockout Techniques , Genetic Therapy , Mice , Mice, Inbred C57BL , MicroRNAs/metabolism , Myocardial Infarction/genetics , Myocardial Infarction/pathology , Myocardial Infarction/therapy , Myocardium/cytology , Myocardium/pathology , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Substrate Specificity , Telomere/genetics , Telomere/metabolism
10.
J Mol Cell Cardiol ; 94: 145-152, 2016 05.
Article in English | MEDLINE | ID: mdl-27071338

ABSTRACT

Heart failure due to myocardial infarction is a major cause of mortality. The microRNA (miR) family let-7 is expressed during embryonic development and is up-regulated in differentiated cells. The aim of this study was to study the role of let-7 after acute myocardial infarction (AMI). We designed an antimiR to inhibit the highest expressed members of the let-7 family, let-7 a, b and c. Administration at day 0 and day 2 after AMI resulted in sustained knockdown of let-7 after 28days. Let-7 inhibition prevented deterioration of cardiac functions compared to control treatment which was especially due to improvements in the infarcted, apical cardiac segments. We observed higher contents of fibrosis in the border zone as well as increased numbers of cells positive for TCF21, which is also expressed in epicardial cells. Markers were augmented after let-7 inhibition and let-7 blocked EMT in epicardial cells in vitro. Lineage tracing in TCF21(iCre/+):R26R(tdT) mice showed abundant tomato positive cells in the infarct and border zone. In conclusion, let-7 inhibition resulted in functional benefits due to an increase in recruitment of epicardial cells and EMT.


Subject(s)
Epithelial-Mesenchymal Transition , Gene Expression Regulation , MicroRNAs/genetics , Myocardial Infarction/genetics , Myocardial Infarction/physiopathology , Ventricular Dysfunction/genetics , Animals , Base Sequence , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Lineage , Epithelial-Mesenchymal Transition/genetics , Fibrosis , Male , Mice , Mice, Transgenic , MicroRNAs/chemistry , Myocardial Infarction/pathology
11.
J Physiol ; 594(8): 2085-94, 2016 Apr 15.
Article in English | MEDLINE | ID: mdl-26040259

ABSTRACT

MicroRNAs (miRs) have emerged as potent regulators of pathways in physiological and disease contexts. This review focuses on the role of miRs in ageing of the cardiovascular system. Several miRs have been described to be regulated during ageing and some of these miRs are involved in the regulation of ageing-related processes. We discuss the roles of miR-34, miR-217 and miR-29, which are induced during ageing in the vasculature. The roles of miR-34, miR-29 (age-induced) and miR-18/19, which are decreased during ageing in the heart, are discussed as well. Furthermore, numerous miRs that play a role in diseases associated with ageing, like diabetes, atherosclerosis, hypertension, cardiac hypertrophy and atrial fibrillation, are also briefly discussed. miRs also serve as circulating biomarkers for cardiovascular ageing or ageing-associated diseases. Finally, pharmacological modulation of ageing-related miRs might become a promising strategy to combat cardiovascular ageing in a clinical setting.


Subject(s)
Caenorhabditis elegans/genetics , Cardiovascular Diseases/genetics , Cardiovascular System/growth & development , MicroRNAs/genetics , Animals , Caenorhabditis elegans/growth & development , Caenorhabditis elegans/metabolism , Cardiovascular System/metabolism , Disease Models, Animal , Humans
13.
Circ Res ; 109(10): 1115-9, 2011 Oct 28.
Article in English | MEDLINE | ID: mdl-21903938

ABSTRACT

RATIONALE: Aging represents a major risk factor for coronary artery disease and aortic aneurysm formation. MicroRNAs (miRs) have emerged as key regulators of biological processes, but their role in age-associated vascular pathologies is unknown. OBJECTIVE: We aim to identify miRs in the vasculature that are regulated by age and play a role in age-induced vascular pathologies. METHODS AND RESULTS: Expression profiling of aortic tissue of young versus old mice identified several age-associated miRs. Among the significantly regulated miRs, the increased expression of miR-29 family members was associated with a profound downregulation of numerous extracellular matrix (ECM) components in aortas of aged mice, suggesting that this miR family contributes to ECM loss, thereby sensitizing the aorta for aneurysm formation. Indeed, miR-29 expression was significantly induced in 2 experimental models for aortic dilation: angiotensin II-treated aged mice and genetically induced aneurysms in Fibulin-4(R/R) mice. More importantly, miR-29b levels were profoundly increased in biopsies of human thoracic aneurysms, obtained from patients with either bicuspid (n=79) or tricuspid aortic valves (n=30). Finally, LNA-modified antisense oligonucleotide-mediated silencing of miR-29 induced ECM expression and inhibited angiotensin II-induced dilation of the aorta in mice. CONCLUSION: In conclusion, miR-29-mediated downregulation of ECM proteins may sensitize the aorta to the formation of aneurysms in advanced age. Inhibition of miR-29 in vivo abrogates aortic dilation in mice, suggesting that miR-29 may represent a novel molecular target to augment matrix synthesis and maintain vascular wall structural integrity.


Subject(s)
Aorta/metabolism , Aortic Aneurysm/genetics , MicroRNAs/analysis , MicroRNAs/metabolism , Aging/genetics , Angiotensin II , Animals , Aorta/pathology , Aortic Aneurysm/chemically induced , Aortic Aneurysm/metabolism , Aortic Aneurysm/pathology , Aortic Aneurysm/prevention & control , Biopsy , Dilatation, Pathologic , Disease Models, Animal , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Gene Expression Profiling/methods , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutation , Oligoribonucleotides, Antisense/administration & dosage
14.
Cell Stem Cell ; 30(1): 86-95.e4, 2023 01 05.
Article in English | MEDLINE | ID: mdl-36563695

ABSTRACT

Drug safety initiatives have endorsed human iPSC-derived cardiomyocytes (hiPSC-CMs) as an in vitro model for predicting drug-induced cardiac arrhythmia. However, the extent to which human-defined features of in vitro arrhythmia predict actual clinical risk has been much debated. Here, we trained a convolutional neural network classifier (CNN) to learn features of in vitro action potential recordings of hiPSC-CMs that are associated with lethal Torsade de Pointes arrhythmia. The CNN classifier accurately predicted the risk of drug-induced arrhythmia in people. The risk profile of the test drugs was similar across hiPSC-CMs derived from different healthy donors. In contrast, pathogenic mutations that cause arrhythmogenic cardiomyopathies in patients significantly increased the proarrhythmic propensity to certain intermediate and high-risk drugs in the hiPSC-CMs. Thus, deep learning can identify in vitro arrhythmic features that correlate with clinical arrhythmia and discern the influence of patient genetics on the risk of drug-induced arrhythmia.


Subject(s)
Deep Learning , Induced Pluripotent Stem Cells , Torsades de Pointes , Humans , Arrhythmias, Cardiac/chemically induced , Torsades de Pointes/chemically induced , Induced Pluripotent Stem Cells/physiology , Action Potentials , Myocytes, Cardiac/physiology
15.
J Am Heart Assoc ; 11(7): e023472, 2022 04 05.
Article in English | MEDLINE | ID: mdl-35301863

ABSTRACT

Background Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. However, underlying molecular mechanisms are insufficiently understood. Previous studies suggested that microRNA (miRNA) dependent gene regulation plays an important role in the initiation and maintenance of AF. The 2-pore-domain potassium channel TASK-1 (tandem of P domains in a weak inward rectifying K+ channel-related acid sensitive K+ channel 1) is an atrial-specific ion channel that is upregulated in AF. Inhibition of TASK-1 current prolongs the atrial action potential duration to similar levels as in patients with sinus rhythm. Here, we hypothesize that miRNAs might be responsible for the regulation of KCNK3 that encodes for TASK-1. Methods and Results We selected miRNAs potentially regulating KCNK3 and studied their expression in atrial tissue samples obtained from patients with sinus rhythm, paroxysmal AF, or permanent/chronic AF. MiRNAs differentially expressed in AF were further investigated for their ability to regulate KCNK3 mRNA and TASK-1 protein expression in human induced pluripotent stem cells, transfected with miRNA mimics or inhibitors. Thereby, we observed that miR-34a increases TASK-1 expression and current and further decreases the resting membrane potential of Xenopus laevis oocytes, heterologously expressing hTASK-1. Finally, we investigated associations between miRNA expression in atrial tissues and clinical parameters of our patient cohort. A cluster containing AF stage, left ventricular end-diastolic diameter, left ventricular end-systolic diameter, left atrial diameter, atrial COL1A2 (collagen alpha-2(I) chain), and TASK-1 protein level was associated with increased expression of miR-25, miR-21, miR-34a, miR-23a, miR-124, miR-1, and miR-29b as well as decreased expression of miR-9 and miR-485. Conclusions These results suggest an important pathophysiological involvement of miRNAs in the regulation of atrial expression of the TASK-1 potassium channel in patients with atrial cardiomyopathy.


Subject(s)
Atrial Fibrillation , Induced Pluripotent Stem Cells , MicroRNAs , Nerve Tissue Proteins , Potassium Channels, Tandem Pore Domain , Dilatation , Heart Atria , Humans , Induced Pluripotent Stem Cells/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , Nerve Tissue Proteins/metabolism , Potassium Channels, Tandem Pore Domain/metabolism
16.
Cell Rep Med ; 2(11): 100436, 2021 11 16.
Article in English | MEDLINE | ID: mdl-34841289

ABSTRACT

Cellular morphology has the capacity to serve as a surrogate for cellular state and functionality. However, primary cardiomyocytes, the standard model in cardiovascular research, are highly heterogeneous cells and therefore impose methodological challenges to analysis. Hence, we aimed to devise a robust methodology to deconvolute cardiomyocyte morphology on a single-cell level: C-MORE (cellular morphology recognition) is a workflow from bench to data analysis tailored for heterogeneous primary cells using our R package cmoRe. We demonstrate its utility in proof-of-principle applications such as modulation of canonical hypertrophy pathways and linkage of genotype-phenotype in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). In our pilot study, exposure of cardiomyocytes to blood plasma prior to versus after aortic valve replacement allows identification of a disease fingerprint and reflects partial reversibility following therapeutic intervention. C-MORE is a valuable tool for cardiovascular research with possible fields of application in basic research and personalized medicine.


Subject(s)
Algorithms , Cardiovascular Diseases/pathology , Cardiovascular Diseases/therapy , Liquid Biopsy , Precision Medicine , Single-Cell Analysis , Animals , Aortic Valve Stenosis/pathology , Cell Cycle , Green Fluorescent Proteins/metabolism , Humans , Hypertrophy , Induced Pluripotent Stem Cells/metabolism , Myocytes, Cardiac/pathology , Phenotype , Rats , Reproducibility of Results
17.
Leukemia ; 35(5): 1301-1316, 2021 05.
Article in English | MEDLINE | ID: mdl-32948843

ABSTRACT

Clonal hematopoiesis of indeterminate potential (CHIP) is linked to leukemia gene mutations and associates with an increased risk for coronary artery disease and poor prognosis in ischemic cardiomyopathy. Two recurrently mutated genes in CHIP and adult acute myeloid leukemia (AML) encode for isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2). Global expression of mutant IDH2 in transgenic mice-induced dilated cardiomyopathy and muscular dystrophy. In this retrospective observational study, we investigated whether mutant IDH1/2 predisposes to cardiovascular disease in AML patients. Among 363 AML patients, IDH1 and IDH2 mutations were detected in 26 (7.2%) and 39 patients (10.7%), respectively. Mutant IDH1 patients exhibited a significantly higher prevalence of coronary artery disease (26.1% vs. 6.4%, p = 0.002). Applying inverse probability-weighting analysis, patients with IDH1/2 mutations had a higher risk for a declining cardiac function during AML treatment compared to IDH1/2 wild type patients [left ventricular ejection fraction pretreatment compared to 10 months after diagnosis: 59.2% to 41.9% (p < 0.001) vs 58.5% to 55.4% (p = 0.27), respectively]. Mechanistically, RNA sequencing and immunostaining in hiPS-derived cardiomyocytes indicated that the oncometabolite R-2HG exacerbated doxorubicin mediated cardiotoxicity. Evaluation of IDH1/2 mutation status may therefore help identifying AML patients at risk for cardiovascular complications during cytotoxic treatment.


Subject(s)
Coronary Artery Disease/etiology , Coronary Artery Disease/genetics , Isocitrate Dehydrogenase/genetics , Leukemia, Myeloid, Acute/genetics , Mutation/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/genetics , Coronary Artery Disease/pathology , Female , Genotype , Humans , Leukemia, Myeloid, Acute/pathology , Male , Middle Aged , Prognosis , Propensity Score , Retrospective Studies , Stroke Volume , Ventricular Function, Left/genetics , Young Adult
18.
J Vis Exp ; (158)2020 04 03.
Article in English | MEDLINE | ID: mdl-32310234

ABSTRACT

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single cell resolution. Current state-of-the-art single cell micropatterning techniques involve soft lithography and micro-contact printing, which is a powerful technology, but requires trained engineering skills and certain facility support in microfabrication. These limitations require a more accessible technique. Here, we describe a simple alternative lithography-free method: stencil-based single cell patterning. We provide step-by-step procedures including stencil design, polyacrylamide hydrogel fabrication, stencil-based protein incorporation, and cell plating and culture. This simple method can be used to pattern an array of as many as 2,000 cells. We demonstrate the patterning of cardiomyocytes derived from single human induced pluripotent stem cells (hiPSC) with distinct cell shapes, from a 1:1 square to a 7:1 adult cardiomyocyte-like rectangle. This stencil-based single cell patterning is lithography-free, technically robust, convenient, inexpensive, and most importantly accessible to those with a limited bioengineering background.


Subject(s)
Cell Culture Techniques/methods , Lasers/standards , Humans
19.
Circ Heart Fail ; 13(3): e006298, 2020 03.
Article in English | MEDLINE | ID: mdl-32160771

ABSTRACT

BACKGROUND: MicroRNAs are small, noncoding RNAs that play a key role in gene expression. Accumulating evidence suggests that aberrant microRNA expression contributes to the heart failure (HF) phenotype; however, the underlying molecular mechanisms are not well understood. A better understanding of the mechanisms of action of microRNAs could potentially lead to targeted therapies that could halt the progression or even reverse HF. METHODS AND RESULTS: We found that microRNA-152 (miR-152) expression was upregulated in the failing human heart and experimental animal models of HF. Transgenic mice with cardiomyocyte-specific miR-152 overexpression developed systolic dysfunction (mean difference, -38.74% [95% CI, -45.73% to -31.74%]; P<0.001) and dilated cardiomyopathy. At the cellular level, miR-152 overexpression perturbed mitochondrial ultrastructure and dysregulated key genes involved in cardiomyocyte metabolism and inflammation. Mechanistically, we identified Glrx5 (glutaredoxin 5), a critical regulator of mitochondrial iron homeostasis and iron-sulfur cluster synthesis, as a direct miR-152 target. Finally, a proof-of-concept of the therapeutic efficacy of targeting miR-152 in vivo was obtained by utilizing a locked nucleic acid-based inhibitor of miR-152 (LNA 152) in a murine model of HF subjected to transverse aortic constriction. We demonstrated that animals treated with LNA-152 (n=10) showed preservation of systolic function when compared with locked nucleic acid-control treated animals (n=9; mean difference, 18.25% [95% CI, 25.10% to 11.39%]; P<0.001). CONCLUSIONS: The upregulation of miR-152 expression in the failing myocardium contributes to HF pathophysiology. Preclinical evidence suggests that miR-152 inhibition preserves cardiac function in a model of pressure overload-induced HF. These findings offer new insights into the pathophysiology of HF and point to miR-152-Glrx5 axis as a potential novel therapeutic target.


Subject(s)
Antagomirs/administration & dosage , Gene Silencing , Heart Failure/prevention & control , MicroRNAs/metabolism , Myocytes, Cardiac/metabolism , Animals , Aorta/physiopathology , Aorta/surgery , Case-Control Studies , Disease Models, Animal , Glutaredoxins/genetics , Glutaredoxins/metabolism , Heart Failure/genetics , Heart Failure/metabolism , Heart Failure/physiopathology , Humans , Ligation , Male , Mice, Inbred C57BL , Mice, Transgenic , MicroRNAs/genetics , Mitochondria, Heart/genetics , Mitochondria, Heart/metabolism , Mitochondria, Heart/ultrastructure , Myocytes, Cardiac/ultrastructure , Proof of Concept Study , Stroke Volume , Ventricular Function, Left
20.
Cytotherapy ; 11(8): 992-1001, 2009.
Article in English | MEDLINE | ID: mdl-19929463

ABSTRACT

BACKGROUND AIMS: Previous studies in xenograft models have shown that human peripheral blood progenitor cells (PBPC) mobilized with the CXCR4 antagonist plerixafor (AMD3100) have a higher bone marrow (BM) reconstitution potential than granulocyte-colony-stimulating factor (G-CSF)-mobilized PBPC. METHODS: PBPC obtained during G-CSF-supported mobilization before and after a supplementary administration of AMD3100 from patients with multiple myeloma and non-Hodgkin's lymphoma (n=15; phase II study) were investigated for co-expression of primitive and lineage-associated markers, their proliferative activity in vitro and repopulation potential after clinical transplantation. RESULTS: A significant increase in primitive CD34+ CD38(-) cells was observed in intraindividual comparisons of all patients after administration of G-CSF+AMD3100 (peripheral blood: median 8-fold, range 2,4-fold - 39-fold) compared with G-CSF alone. Using a long-term culture-initiating cell assay, this increase was confirmed. After transplantation of G-CSF+AMD3100-mobilized PBPC, the time to leukocyte reconstitution > 1 x 10(3)/microL and platelet reconstitution > 2 x 10(4)/microL was 14 (10-19 days) and 13 days (10-15 days), respectively. A complete and stable hematologic reconstitution (platelets > 1.5 x 10(5)/microL) was observed in 91% of all patients within 35 days. CONCLUSIONS: An additional application of AMD3100 to a standard G-CSF mobilization regimen leads to a significant increase in primitive PBPC with high repopulation capacity.


Subject(s)
Granulocyte Colony-Stimulating Factor/pharmacology , Hematopoietic Stem Cell Mobilization , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/drug effects , Heterocyclic Compounds/pharmacology , ADP-ribosyl Cyclase 1/metabolism , Adult , Aged , Antigens, CD34/metabolism , Benzylamines , Cell Culture Techniques , Cyclams , Drug Therapy, Combination , Europe , Female , Hematopoietic Stem Cell Transplantation , Humans , Leukocyte Count , Male , Middle Aged , Phenotype , Time Factors
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