Rapid, High-Quality, Cost-Effective, Comprehensive and Expandable Targeted Next-Generation Sequencing Assay for Inherited Heart Diseases
2015; 117 (7): 603-611
Epigenetic Regulation of Phosphodiesterases 2A and 3A Underlies Compromised beta-Adrenergic Signaling in an iPSC Model of Dilated Cardiomyopathy
CELL STEM CELL
2015; 17 (1): 89-100
Thousands of mutations across more than 50 genes have been implicated in inherited cardiomyopathies. However, options for sequencing this rapidly evolving gene set are limited as many sequencing services and off-the-shelf kits suffer from slow turnaround, inefficient capture of genomic DNA, and/or high cost. Furthermore, customization of these assays to cover emerging targets and to suit individual needs is often expensive and time-consuming.We sought to develop a custom high throughput, clinical-grade next generation sequencing (NGS) assay for detecting cardiac disease gene mutations with improved accuracy, flexibility, turnaround, and cost.We employed double-stranded probes (complementary long "padlock" probes (cLPPs)), an inexpensive and customizable capture technology, to efficiently capture and amplify the entire coding region and flanking intronic and regulatory sequences of 88 genes and 40 microRNAs (miRNA) associated with inherited cardiomyopathies, congenital heart disease (CHD), and cardiac development. Multiplexing 11 samples per sequencing run resulted in a mean base coverage of 420, of which 97% had >20X coverage and >99% were concordant with known heterozygous single nucleotide polymorphisms (SNPs). The assay correctly detected germline variants in 24 individuals and revealed several polymorphic regions in miR-499. Total run time was three days at an approximate cost of $100 per sample.Accurate, high throughput detection of mutations across numerous cardiac genes is achievable with cLPP technology. Moreover, this format allows facile insertion of additional probes as more cardiomyopathy and CHD genes are discovered, giving researchers a powerful new tool for DNA mutation detection and discovery.
View details for DOI 10.1161/CIRCRESAHA.115.306723
View details for Web of Science ID 000360967000006
Finding the Rhythm of Sudden Cardiac Death New Opportunities Using Induced Pluripotent Stem Cell-Derived Cardiomyocytes
2015; 116 (12): 1989-2004
β-adrenergic signaling pathways mediate key aspects of cardiac function. Its dysregulation is associated with a range of cardiac diseases, including dilated cardiomyopathy (DCM). Previously, we established an iPSC model of familial DCM from patients with a mutation in TNNT2, a sarcomeric protein. Here, we found that the β-adrenergic agonist isoproterenol induced mature β-adrenergic signaling in iPSC-derived cardiomyocytes (iPSC-CMs) but that this pathway was blunted in DCM iPSC-CMs. Although expression levels of several β-adrenergic signaling components were unaltered between control and DCM iPSC-CMs, we found that phosphodiesterases (PDEs) 2A and PDE3A were upregulated in DCM iPSC-CMs and that PDE2A was also upregulated in DCM patient tissue. We further discovered increased nuclear localization of mutant TNNT2 and epigenetic modifications of PDE genes in both DCM iPSC-CMs and patient tissue. Notably, pharmacologic inhibition of PDE2A and PDE3A restored cAMP levels and ameliorated the impaired β-adrenergic signaling of DCM iPSC-CMs, suggesting therapeutic potential.
View details for DOI 10.1016/j.stem.2015.04.020
View details for Web of Science ID 000361877600011
Human induced pluripotent stem cell-derived cardiomyocytes as an in vitro model for coxsackievirus B3-induced myocarditis and antiviral drug screening platform.
2014; 115 (6): 556-566
Sudden cardiac death is a common cause of death in patients with structural heart disease, genetic mutations, or acquired disorders affecting cardiac ion channels. A wide range of platforms exist to model and study disorders associated with sudden cardiac death. Human clinical studies are cumbersome and are thwarted by the extent of investigation that can be performed on human subjects. Animal models are limited by their degree of homology to human cardiac electrophysiology, including ion channel expression. Most commonly used cellular models are cellular transfection models, which are able to mimic the expression of a single-ion channel offering incomplete insight into changes of the action potential profile. Induced pluripotent stem cell-derived cardiomyocytes resemble, but are not identical, adult human cardiomyocytes and provide a new platform for studying arrhythmic disorders leading to sudden cardiac death. A variety of platforms exist to phenotype cellular models, including conventional and automated patch clamp, multielectrode array, and computational modeling. Induced pluripotent stem cell-derived cardiomyocytes have been used to study long QT syndrome, catecholaminergic polymorphic ventricular tachycardia, hypertrophic cardiomyopathy, and other hereditary cardiac disorders. Although induced pluripotent stem cell-derived cardiomyocytes are distinct from adult cardiomyocytes, they provide a robust platform to advance the science and clinical care of sudden cardiac death.
View details for DOI 10.1161/CIRCRESAHA.116.304494
View details for Web of Science ID 000357403300008
Cardiac stem cell biology: glimpse of the past, present, and future.
2014; 114 (1): 21-27
Rationale: Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. A major causative agent for viral myocarditis is the B3 strain of coxsackievirus, a positive-sense RNA enterovirus. However, human cardiac tissues are difficult to procure in sufficient enough quantities for studying the mechanisms of cardiac-specific viral infection. Objective: This study examined whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy. Methods and Results: Human iPSC-CMs were infected with a luciferase-expressing coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were utilized to characterize virally-infected hiPSC-CMs for alterations in cellular morphology and calcium handling. Viral proliferation in hiPSC-CMs was quantified using bioluminescence imaging. Antiviral compounds including interferon beta 1 (IFNβ1), ribavirin, pyrrolidine dithiocarbamate, and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with reported drug effects in previous studies. Mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways after IFNβ1 treatment. Conclusions: This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to predict antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that can screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion.
View details for DOI 10.1161/CIRCRESAHA.115.303810
View details for PubMedID 25015077
Modeling inherited cardiac disorders.
2014; 78 (4): 784-794
Cardiac regeneration strategies and de novo generation of cardiomyocytes have long been significant areas of research interest in cardiovascular medicine. In this review, we outline a variety of common cell sources and methods used to regenerate cardiomyocytes and highlight the important role that key Circulation Research articles have played in this flourishing field.
View details for DOI 10.1161/CIRCRESAHA.113.302895
View details for PubMedID 24385505
Concomitant ECG findings and J wave patterns
JOURNAL OF ELECTROCARDIOLOGY
2013; 46 (5): 399-403
Drug screening using a library of human induced pluripotent stem cell-derived cardiomyocytes reveals disease-specific patterns of cardiotoxicity.
2013; 127 (16): 1677-1691
Advances in the understanding and treatment of cardiac disorders have been thwarted by the inability to study beating human cardiac cells in vitro. Induced pluripotent stem cells (iPSCs) bypass this hurdle by enabling the creation of patient-specific iPSC-derived cardiomyocytes (iPSC-CMs). These cells provide a unique platform to study cardiac diseases in vitro, especially hereditary cardiac conditions. To date, iPSC-CMs have been used to successfully model arrhythmic disorders, showing excellent recapitulation of cardiac channel function and electrophysiologic features of long QT syndrome types 1, 2, 3, and 8, and catecholaminergic polymorphic ventricular tachycardia (CPVT). Similarly, iPSC-CM models of dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) have shown robust correlation of predicted morphologic, contractile, and electrical phenotypes. In addition, iPSC-CMs have shown some features of the respective phenotypes for arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C), LEOPARD syndrome, Pompe's disease, and Friedriech's ataxia. In this review, we examine the progress of utilizing iPSC-CMs as a model for cardiac conditions and analyze the potential for the platform in furthering the biology and treatment of cardiac disorders.
View details for PubMedID 24632794
Natural History of Early Repolarization in the Inferior Leads
ANNALS OF NONINVASIVE ELECTROCARDIOLOGY
2012; 17 (4): 331-339
Cardiotoxicity is a leading cause for drug attrition during pharmaceutical development and has resulted in numerous preventable patient deaths. Incidents of adverse cardiac drug reactions are more common in patients with preexisting heart disease than the general population. Here we generated a library of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with various hereditary cardiac disorders to model differences in cardiac drug toxicity susceptibility for patients of different genetic backgrounds.Action potential duration and drug-induced arrhythmia were measured at the single cell level in hiPSC-CMs derived from healthy subjects and patients with hereditary long QT syndrome, familial hypertrophic cardiomyopathy, and familial dilated cardiomyopathy. Disease phenotypes were verified in long QT syndrome, hypertrophic cardiomyopathy, and dilated cardiomyopathy hiPSC-CMs by immunostaining and single cell patch clamp. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and the human ether-a-go-go-related gene expressing human embryonic kidney cells were used as controls. Single cell PCR confirmed expression of all cardiac ion channels in patient-specific hiPSC-CMs as well as hESC-CMs, but not in human embryonic kidney cells. Disease-specific hiPSC-CMs demonstrated increased susceptibility to known cardiotoxic drugs as measured by action potential duration and quantification of drug-induced arrhythmias such as early afterdepolarizations and delayed afterdepolarizations.We have recapitulated drug-induced cardiotoxicity profiles for healthy subjects, long QT syndrome, hypertrophic cardiomyopathy, and dilated cardiomyopathy patients at the single cell level for the first time. Our data indicate that healthy and diseased individuals exhibit different susceptibilities to cardiotoxic drugs and that use of disease-specific hiPSC-CMs may predict adverse drug responses more accurately than the standard human ether-a-go-go-related gene test or healthy control hiPSC-CM/hESC-CM screening assays.
View details for DOI 10.1161/CIRCULATIONAHA.113.001883
View details for PubMedID 23519760
Prognostic Implications of Q Waves and T-Wave Inversion Associated With Early Repolarization
MAYO CLINIC PROCEEDINGS
2012; 87 (7): 614-619
Though early repolarization (ER) in the inferior leads has been associated with increased cardiovascular risk, its natural history is uncertain. We aimed to study the serial electrocardiographic behavior of inferior ER and understand factors associated with that behavior.We selected electrocardiograms (ECGs) from patients with the greatest amplitude of ER in AVF from ECGs of 29,281 ambulatory patients recorded between 1987 and 1999 at the Palo Alto Veterans Affairs Hospital. Starting from the highest amplitude, we reviewed the ECGs and medical records from the first 85%. From this convenience sample, 36 were excluded for abnormal patterns similar to ER. The remaining 257 patients were searched for another ECG at least 5 months later, of whom, 136 satisfied this criteria. These ECGs were paired for comparison and coded by four interpreters.The average time between the first and second ECGs was 10 years. Of the 136 subjects, 47% retained ER while 53% no longer fulfilled the amplitude criteria. While no significant differences were found in initial heart rate (HR) or time interval between ECGs, those who lost the ER pattern had a greater difference in HR between the ECGs. There was no significant difference in the incidence of cardiovascular events or deaths.In conclusion, the ECG pattern of ER was lost over 10 years in over half of the cohort. The loss of ER was partially explained by changes in HR, but not higher incidence of cardiovascular events or death, suggesting the entity is a benign finding.
View details for DOI 10.1111/j.1542-474X.2012.00537.x
View details for Web of Science ID 000310248100005
View details for PubMedID 23094879
Embryonic stem cell biology: insights from molecular imaging.
Methods in molecular biology (Clifton, N.J.)
2010; 660: 185-199
To evaluate the prevalence of early polarization (ER) in a stable population and to evaluate the prognostic significance of the association or absence of Q waves or T-wave inversion (TWI).In this retrospective study performed at the university-affiliated Palo Alto Veterans Affairs Health Care Center from March 1, 1987, through December 31, 1999, we evaluated outpatient electrocardiograms. Vital status and cause of death were determined in all patients, with a mean ± SD follow-up of 7.6±3.8 years.Of the 29,281 patients, 87% were men and 13% were African American. Inferior or lateral ER was present in 664 patients (2.3%): in inferior leads in 185 (0.6%), in lateral leads in 479 (1.6%) , and in both inferior and lateral leads in 163 (0.6%). Only when Q waves or TWI accompanied ER was there an increased risk of cardiovascular death (Cox proportional hazards regression model, 5.0; 95% confidence interval, 3.4-7.2; P<.001).Common patterns of ER without concomitant Q waves or TWI are not associated with increased risk of cardiovascular death; however, when either occurs with ER, there is a hazard ratio of 5.0. These findings confirm that ER is a benign entity; however, the presence of Q waves or TWI with ER is predictive of increased cardiovascular death.
View details for DOI 10.1016/j.mayocp.2012.04.009
View details for Web of Science ID 000306872800003
View details for PubMedID 22766081
Embryonic stem (ES) cells have therapeutic potential in disorders of cellular loss such as myocardial infarction, type I diabetes and neurodegenerative disorders. ES cell biology in living subjects was largely poorly understood until incorporation of molecular imaging into the field. Reporter gene imaging works by integrating a reporter gene into ES cells and using a reporter probe to induce a signal detectable by normal imaging modalities. Reporter gene imaging allows for longitudinal tracking of ES cells within the same host for a prolonged period of time. This has advantages over postmortem immunohistochemistry and traditional imaging modalities. The advantages include expression of reporter gene is limited to viable cells, expression is conserved between generations of dividing cells, and expression can be linked to a specific population of cells. These advantages were especially useful in studying a dynamic cell population such as ES cells and proved useful in elucidating the biology of ES cells. Reporter gene imaging identified poor integration of differentiated ES cells transplanted into host tissue as well as delayed donor cell death as reasons for poor long-term survival in vivo. This imaging technology also confirmed that ES cells indeed have immunogenic properties that factor into cell survival and differentiation. Finally, reporter gene imaging improved our understanding of the neoplastic risk of undifferentiated ES cells in forming teratomas. Despite such advances, much remains to be understood about ES cell biology to translate this technology to the bedside, and reporter gene imaging will certainly play a key role in formulating this understanding.
View details for DOI 10.1007/978-1-60761-705-1_12
View details for PubMedID 20680820