A Comprehensive TALEN-Based Knockout Library for Generating Human Induced Pluripotent Stem Cell-Based Models for Cardiovascular Diseases.
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.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.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.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.
View details for DOI 10.1161/CIRCRESAHA.116.309948
View details for PubMedID 28246128
View details for PubMedCentralID PMC5429194
Efficient Genome Editing in Induced Pluripotent Stem Cells with Engineered Nucleases In Vitro.
Methods in molecular biology (Clifton, N.J.)
2017; 1521: 55-68
Precision genome engineering is rapidly advancing the application of the induced pluripotent stem cells (iPSCs) technology for in vitro disease modeling of cardiovascular diseases. Targeted genome editing using engineered nucleases is a powerful tool that allows for reverse genetics, genome engineering, and targeted transgene integration experiments to be performed in a precise and predictable manner. However, nuclease-mediated homologous recombination is an inefficient process. Herein, we describe the development of an optimized method combining site-specific nucleases and the piggyBac transposon system for "seamless" genome editing in pluripotent stem cells with high efficiency and fidelity in vitro.
View details for PubMedID 27910041
Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Insights Into Molecular, Cellular, and Functional Phenotypes.
2015; 117 (1): 80-88
Disease models are essential for understanding cardiovascular disease pathogenesis and developing new therapeutics. The human induced pluripotent stem cell (iPSC) technology has generated significant enthusiasm for its potential application in basic and translational cardiac research. Patient-specific iPSC-derived cardiomyocytes offer an attractive experimental platform to model cardiovascular diseases, study the earliest stages of human development, accelerate predictive drug toxicology tests, and advance potential regenerative therapies. Harnessing the power of iPSC-derived cardiomyocytes could eliminate confounding species-specific and interpersonal variations and ultimately pave the way for the development of personalized medicine for cardiovascular diseases. However, the predictive power of iPSC-derived cardiomyocytes as a valuable model is contingent on comprehensive and rigorous molecular and functional characterization.
View details for DOI 10.1161/CIRCRESAHA.117.305365
View details for PubMedID 26089365
- Correction of human phospholamban R14del mutation associated with cardiomyopathy using targeted nucleases and combination therapy NATURE COMMUNICATIONS 2015; 6
- Patient-Specific iPSC-Derived Endothelial Cells Uncover Pathways that Protect against Pulmonary Hypertension in BMPR2 Mutation Carriers CELL STEM CELL 2017; 20 (4): 490-?
Mending a Broken Heart: The Evolution Of Biological Therapeutics.
Heart failure (HF), a common sequela of cardiovascular diseases, remains a staggering clinical problem, associated with high rates of morbidity and mortality worldwide. Advances in pharmacological, interventional, and operative management have improved patient care, but these interventions are insufficient to halt the progression of HF, particularly the end-stage irreversible loss of functional cardiomyocytes. Innovative therapies that could prevent HF progression and improve the function of the failing heart are urgently needed. Following successful preclinical studies, two main strategies have emerged as potential solutions: cardiac gene therapy and cardiac regeneration through stem and precursor cell transplantation. Many potential gene- and cell-based therapies have entered into clinical studies, intending to ameliorate cardiac dysfunction in patients with advanced HF. In this review, we focus on the recent advances in cell- and gene-based therapies in the context of cardiovascular disease, emphasizing the most advanced therapies. The principles and mechanisms of action of gene and cell therapies for HF are discussed along with the limitations of current approaches. Finally, we highlight the emerging technologies that hold promise to revolutionize the biological therapies for cardiovascular diseases. Stem Cells 2017;35:1131-1140.
View details for DOI 10.1002/stem.2602
View details for PubMedID 28233392
Modeling susceptibility to drug-induced long QT with a panel of subject-specific induced pluripotent stem cells
A large number of drugs can induce prolongation of cardiac repolarization and life-threatening cardiac arrhythmias. The prediction of this side effect is however challenging as it usually develops in some genetically predisposed individuals with normal cardiac repolarization at baseline. Here, we describe a platform based on a genetically diverse panel of induced pluripotent stem cells (iPSCs) that reproduces susceptibility to develop a cardiotoxic drug response. We generated iPSC-derived cardiomyocytes from patients presenting in vivo with extremely low or high changes in cardiac repolarization in response to a pharmacological challenge with sotalol. In vitro, the responses to sotalol were highly variable but strongly correlated to the inter-individual differences observed in vivo. Transcriptomic profiling identified dysregulation of genes (DLG2, KCNE4, PTRF, HTR2C, CAMKV) involved in downstream regulation of cardiac repolarization machinery as underlying high sensitivity to sotalol. Our findings offer novel insights for the development of iPSC-based screening assays for testing individual drug reactions.
View details for DOI 10.7554/eLife.19406
View details for Web of Science ID 000393403900001
View details for PubMedID 28134617
View details for PubMedCentralID PMC5279943
Gene Transfer in Cardiomyocytes Derived from ES and iPS Cells.
Methods in molecular biology (Clifton, N.J.)
2017; 1521: 183-193
The advent of human induced pluripotent stem cell (hiPSC) technology has produced patient-specific hiPSC derived cardiomyocytes (hiPSC-CMs) that can be used as a platform to study cardiac diseases and to explore new therapies.The ability to genetically manipulate hiPSC-CMs not only is essential for identifying the structural and/or functional role of a protein but can also provide valuable information regarding therapeutic applications. In this chapter, we describe protocols for culture, maintenance, and cardiac differentiation of hiPSCs. Then, we provide a basic procedure to transduce hiPSC-CMs.
View details for PubMedID 27910049
Patient-Specific iPSC-Derived Endothelial Cells Uncover Pathways that Protect against Pulmonary Hypertension in BMPR2 Mutation Carriers.
Cell stem cell
In familial pulmonary arterial hypertension (FPAH), the autosomal dominant disease-causing BMPR2 mutation is only 20% penetrant, suggesting that genetic variation provides modifiers that alleviate the disease. Here, we used comparison of induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from three families with unaffected mutation carriers (UMCs), FPAH patients, and gender-matched controls to investigate this variation. Our analysis identified features of UMC iPSC-ECs related to modifiers of BMPR2 signaling or to differentially expressed genes. FPAH-iPSC-ECs showed reduced adhesion, survival, migration, and angiogenesis compared to UMC-iPSC-ECs and control cells. The "rescued" phenotype of UMC cells was related to an increase in specific BMPR2 activators and/or a reduction in inhibitors, and the improved cell adhesion could be attributed to preservation of related signaling. The improved survival was related to increased BIRC3 and was independent of BMPR2. Our findings therefore highlight protective modifiers for FPAH that could help inform development of future treatment strategies.
View details for DOI 10.1016/j.stem.2016.08.019
View details for PubMedID 28017794
iPSC-derived cardiomyocytes reveal abnormal TGF-ß signalling in left ventricular non-compaction cardiomyopathy.
Nature cell biology
2016; 18 (10): 1031-1042
Left ventricular non-compaction (LVNC) is the third most prevalent cardiomyopathy in children and its pathogenesis has been associated with the developmental defect of the embryonic myocardium. We show that patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from LVNC patients carrying a mutation in the cardiac transcription factor TBX20 recapitulate a key aspect of the pathological phenotype at the single-cell level and this was associated with perturbed transforming growth factor beta (TGF-β) signalling. LVNC iPSC-CMs have decreased proliferative capacity due to abnormal activation of TGF-β signalling. TBX20 regulates the expression of TGF-β signalling modifiers including one known to be a genetic cause of LVNC, PRDM16, and genome editing of PRDM16 caused proliferation defects in iPSC-CMs. Inhibition of TGF-β signalling and genome correction of the TBX20 mutation were sufficient to reverse the disease phenotype. Our study demonstrates that iPSC-CMs are a useful tool for the exploration of pathological mechanisms underlying poorly understood cardiomyopathies including LVNC.
View details for DOI 10.1038/ncb3411
View details for PubMedID 27642787
Transcriptome Profiling of Patient-Specific Human iPSC-Cardiomyocytes Predicts Individual Drug Safety and Efficacy Responses In Vitro.
Cell stem cell
2016; 19 (3): 311-325
Understanding individual susceptibility to drug-induced cardiotoxicity is key to improving patient safety and preventing drug attrition. Human induced pluripotent stem cells (hiPSCs) enable the study of pharmacological and toxicological responses in patient-specific cardiomyocytes (CMs) and may serve as preclinical platforms for precision medicine. Transcriptome profiling in hiPSC-CMs from seven individuals lacking known cardiovascular disease-associated mutations and in three isogenic human heart tissue and hiPSC-CM pairs showed greater inter-patient variation than intra-patient variation, verifying that reprogramming and differentiation preserve patient-specific gene expression, particularly in metabolic and stress-response genes. Transcriptome-based toxicology analysis predicted and risk-stratified patient-specific susceptibility to cardiotoxicity, and functional assays in hiPSC-CMs using tacrolimus and rosiglitazone, drugs targeting pathways predicted to produce cardiotoxicity, validated inter-patient differential responses. CRISPR/Cas9-mediated pathway correction prevented drug-induced cardiotoxicity. Our data suggest that hiPSC-CMs can be used in vitro to predict and validate patient-specific drug safety and efficacy, potentially enabling future clinical approaches to precision medicine.
View details for DOI 10.1016/j.stem.2016.07.006
View details for PubMedID 27545504
Genomic correction of familial cardiomyopathy in human engineered cardiac tissues.
European heart journal
In this study, we used three-dimensional human engineered cardiac tissue technology to directly show that phospholamban (PLN) R14del mutation impairs cardiac contractility and to demonstrate restoration of contractile properties with targeted genetic correction of this inheritable form of dilated cardiomyopathy.
View details for PubMedID 27450564
Cytokines profile in hypertensive patients with left ventricular remodeling and dysfunction.
Journal of the American Society of Hypertension
2015; 9 (12): 975-984 e3
There is strong evidence that inflammatory mediators play a key role in the progression to heart failure in patients with systemic hypertension (HTN). The present study aimed to identify a set of cytokines that are associated with early left ventricular (LV) remodeling and dysfunction as captured by echocardiography in patients with HTN in a cross-sectional case-control study nested within the FLEMish study on ENvironment, Genes and Health Outcome. We identified three groups of participants from the cohort: normotensive subjects (normotension; n = 30), HTN with normal LV structure and function (HTN [LV-]; n = 30), and HTN with evidence of adverse LV remodeling (HTN [LV+]; n = 50). We measured cytokines using a 63-plex Luminex platform. Using partial least squares-discriminant analysis, we constructed three latent variables from the measured cytokines that explained 35%-45% of the variance between groups. We identified five common cytokines (interleukin 18, monokine induced by gamma interferon, hepatocyte growth factor, epithelial neutrophil-activating peptide 78, and vascular endothelial growth factor D) with a stable signal which had a major impact on the construction of the latent variables. Among these cytokines, after adjustment for confounders, interleukin 18 remained significantly different between HTN participants with and without LV involvement (P = .02). Moreover, granulocyte-macrophage colony-stimulating factor and leptin showed a consistent upward trend in all HTN patients compared with normotensive subjects. In conclusion, in HTN patients with LV remodeling or/and dysfunction, we identified a set of cytokines strongly associated with LV maladaptation. We also found a distinct profile of inflammatory biomarkers that characterize HTN.
View details for DOI 10.1016/j.jash.2015.10.003
View details for PubMedID 26565110
- Cytokines profile in hypertensive patients with left ventricular remodeling and dysfunction JOURNAL OF THE AMERICAN SOCIETY OF HYPERTENSION 2015; 9 (12): 975-984
A Rapid, High-Quality, Cost-Effective, Comprehensive and Expandable Targeted Next-Generation Sequencing Assay for Inherited Heart Diseases.
2015; 117 (7): 603-611
Thousands of mutations across >50 genes have been implicated in inherited cardiomyopathies. However, options for sequencing this rapidly evolving gene set are limited because many sequencing services and off-the-shelf kits suffer from slow turnaround, inefficient capture of genomic DNA, and high cost. Furthermore, customization of these assays to cover emerging targets that suit individual needs is often expensive and time consuming.We sought to develop a custom high throughput, clinical-grade next-generation sequencing assay for detecting cardiac disease gene mutations with improved accuracy, flexibility, turnaround, and cost.We used double-stranded probes (complementary long padlock probes), 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 associated with inherited cardiomyopathies, congenital heart disease, and cardiac development. Multiplexing 11 samples per sequencing run resulted in a mean base pair coverage of 420, of which 97% had >20× coverage and >99% were concordant with known heterozygous single nucleotide polymorphisms. The assay correctly detected germline variants in 24 individuals and revealed several polymorphic regions in miR-499. Total run time was 3 days at an approximate cost of $100 per sample.Accurate, high-throughput detection of mutations across numerous cardiac genes is achievable with complementary long padlock probe technology. Moreover, this format allows facile insertion of additional probes as more cardiomyopathy and congenital heart disease 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 PubMedID 26265630
View details for PubMedCentralID PMC4568077
Phospholamban as a Crucial Determinant of the Inotropic Response of Human Pluripotent Stem Cell-Derived Ventricular Cardiomyocytes and Engineered 3-Dimensional Tissue Constructs
CIRCULATION-ARRHYTHMIA AND ELECTROPHYSIOLOGY
2015; 8 (1): 193-U276
Human (h) embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) serve as a potential unlimited ex vivo source of cardiomyocytes (CMs). However, a well-accepted roadblock has been their immature phenotype. hESC/iPSC-derived ventricular (v) CMs and their engineered cardiac microtissues (hvCMTs) similarly displayed positive chronotropic but null inotropic responses to β-adrenergic stimulation. Given that phospholamban (PLB) is robustly present in adult but poorly expressed in hESC/iPSC-vCMs and its defined biological role in β-adrenergic signaling, we investigated the functional consequences of PLB expression in hESC/iPSC-vCMs and hvCMTs.First, we confirmed that PLB protein was differentially expressed in hESC (HES2, H9)- and iPSC-derived and adult vCMs. We then transduced hES2-vCMs with the recombinant adenoviruses (Ad) Ad-PLB or Ad-S16E-PLB to overexpress wild-type PLB or the pseudophosphorylated point-mutated variant, respectively. As anticipated from the inhibitory effect of unphosphorylated PLB on sarco/endoplasmic reticulum Ca2+-ATPase, Ad-PLB transduction significantly attenuated electrically evoked Ca2+ transient amplitude and prolonged the 50% decay time. Importantly, Ad-PLB-transduced hES2-vCMs uniquely responded to isoproterenol. Ad-S16E-PLB-transduced hES2-vCMs displayed an intermediate phenotype. The same trends were observed with H9- and iPSC-vCMs. Directionally, similar results were also seen with Ad-PLB-transduced and Ad-S16E-transduced hvCMTs. However, Ad-PLB altered neither the global transcriptome nor ICa,L, implicating a PLB-specific effect.Engineered upregulation of PLB expression in hESC/iPSC-vCMs restores a positive inotropic response to β-adrenergic stimulation. These results not only provide a better mechanistic understanding of the immaturity of hESC/iPSC-vCMs but will also lead to improved disease models and transplantable prototypes with adult-like physiological responses.
View details for DOI 10.1161/CIRCEP.114.002049
View details for Web of Science ID 000349873000027
View details for PubMedID 25504561
Effectiveness of gene delivery systems for pluripotent and differentiated cells.
Molecular therapy. Methods & clinical development
2015; 2: 14067-?
Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) assert a great future for the cardiovascular diseases, both to study them and to explore therapies. However, a comprehensive assessment of the viral vectors used to modify these cells is lacking. In this study, we aimed to compare the transduction efficiency of recombinant adeno-associated vectors (AAV), adenoviruses and lentiviral vectors in hESC, hiPSC, and the derived cardiomyocytes. In undifferentiated cells, adenoviral and lentiviral vectors were superior, whereas in differentiated cells AAV surpassed at least lentiviral vectors. We also tested four AAV serotypes, 1, 2, 6, and 9, of which 2 and 6 were superior in their transduction efficiency. Interestingly, we observed that AAVs severely diminished the viability of undifferentiated cells, an effect mediated by induction of cell cycle arrest genes and apoptosis. Furthermore, we show that the transduction efficiency of the different viral vectors correlates with the abundance of their respective receptors. Finally, adenoviral delivery of the calcium-transporting ATPase SERCA2a to hESC and hiPSC-derived cardiomyocytes successfully resulted in faster calcium reuptake. In conclusion, adenoviral vectors prove to be efficient for both differentiated and undifferentiated lines, whereas lentiviral vectors are more applicable to undifferentiated cells and AAVs to differentiated cells.
View details for DOI 10.1038/mtm.2014.67
View details for PubMedID 26052535
Cardiac I-1c Overexpression With Reengineered AAV Improves Cardiac Function in Swine Ischemic Heart Failure
2014; 22 (12): 2038-2045
Cardiac gene therapy has emerged as a promising option to treat advanced heart failure (HF). Advances in molecular biology and gene targeting approaches are offering further novel options for genetic manipulation of the cardiovascular system. The aim of this study was to improve cardiac function in chronic HF by overexpressing constitutively active inhibitor-1 (I-1c) using a novel cardiotropic vector generated by capsid reengineering of adeno-associated virus (BNP116). One month after a large anterior myocardial infarction, 20 Yorkshire pigs randomly received intracoronary injection of either high-dose BNP116.I-1c (1.0 × 10(13) vector genomes (vg), n = 7), low-dose BNP116.I-1c (3.0 × 10(12) vg, n = 7), or saline (n = 6). Compared to baseline, mean left ventricular ejection fraction increased by 5.7% in the high-dose group, and by 5.2% in the low-dose group, whereas it decreased by 7% in the saline group. Additionally, preload-recruitable stroke work obtained from pressure-volume analysis demonstrated significantly higher cardiac performance in the high-dose group. Likewise, other hemodynamic parameters, including stroke volume and contractility index indicated improved cardiac function after the I-1c gene transfer. Furthermore, BNP116 showed a favorable gene expression pattern for targeting the heart. In summary, I-1c overexpression using BNP116 improves cardiac function in a clinically relevant model of ischemic HF.
View details for DOI 10.1038/mt.2014.127
View details for Web of Science ID 000345822600004
View details for PubMedID 25023328
Human-induced pluripotent stem cell models of inherited cardiomyopathies.
Current opinion in cardiology
2014; 29 (3): 214-219
This article provides an overview of the latest advances in in-vitro modeling of inherited cardiomyopathies using human-induced pluripotent stem cells (iPSCs).Inherited cardiomyopathies have been recently modeled by generating iPSCs from patients harboring mutations in genes associated with the pathogenesis of hypertrophic cardiomyopathy, dilated cardiomyopathy, and arrhythmogenic right ventricular cardiomyopathy/dysplasia.Patient-specific iPSCs and their differentiated cardiomyocytes (induced pluripotent stem cell-derived cardiomyocytes) now provide a novel model to study the underlying molecular mechanism of the pathogenesis of familial cardiomyopathies as well as for in-vitro drug screening and drug discovery.
View details for DOI 10.1097/HCO.0000000000000049
View details for PubMedID 24576884
- Rapid and Efficient Conversion of Integration-Free Human Induced Pluripotent Stem Cells to GMP-Grade Culture Conditions. PloS one 2014; 9 (4)
Rapid and efficient conversion of integration-free human induced pluripotent stem cells to GMP-grade culture conditions.
2014; 9 (4)
Data suggest that clinical applications of human induced pluripotent stem cells (hiPSCs) will be realized. Nonetheless, clinical applications will require hiPSCs that are free of exogenous DNA and that can be manufactured through Good Manufacturing Practice (GMP). Optimally, derivation of hiPSCs should be rapid and efficient in order to minimize manipulations, reduce potential for accumulation of mutations and minimize financial costs. Previous studies reported the use of modified synthetic mRNAs to reprogram fibroblasts to a pluripotent state. Here, we provide an optimized, fully chemically defined and feeder-free protocol for the derivation of hiPSCs using synthetic mRNAs. The protocol results in derivation of fully reprogrammed hiPSC lines from adult dermal fibroblasts in less than two weeks. The hiPSC lines were successfully tested for their identity, purity, stability and safety at a GMP facility and cryopreserved. To our knowledge, as a proof of principle, these are the first integration-free iPSCs lines that were reproducibly generated through synthetic mRNA reprogramming that could be putatively used for clinical purposes.
View details for DOI 10.1371/journal.pone.0094231
View details for PubMedID 24718618
Small Molecule-Mediated Directed Differentiation of Human Embryonic Stem Cells Toward Ventricular Cardiomyocytes
STEM CELLS TRANSLATIONAL MEDICINE
2014; 3 (1): 18-31
The generation of human ventricular cardiomyocytes from human embryonic stem cells and/or induced pluripotent stem cells could fulfill the demand for therapeutic applications and in vitro pharmacological research; however, the production of a homogeneous population of ventricular cardiomyocytes remains a major limitation. By combining small molecules and growth factors, we developed a fully chemically defined, directed differentiation system to generate ventricular-like cardiomyocytes (VCMs) from human embryonic stem cells and induced pluripotent stem cells with high efficiency and reproducibility. Molecular characterization revealed that the differentiation recapitulated the developmental steps of cardiovascular fate specification. Electrophysiological analyses further illustrated the generation of a highly enriched population of VCMs. These chemically induced VCMs exhibited the expected cardiac electrophysiological and calcium handling properties as well as the appropriate chronotropic responses to cardioactive compounds. In addition, using an integrated computational and experimental systems biology approach, we demonstrated that the modulation of the canonical Wnt pathway by the small molecule IWR-1 plays a key role in cardiomyocyte subtype specification. In summary, we developed a reproducible and efficient experimental platform that facilitates a chemical genetics-based interrogation of signaling pathways during cardiogenesis that bypasses the limitations of genetic approaches and provides a valuable source of ventricular cardiomyocytes for pharmacological screenings as well as cell replacement therapies.
View details for DOI 10.5966/sctm.2013-0110
View details for Web of Science ID 000330014000004
View details for PubMedID 24324277
Therapeutic cardiac-targeted delivery of miR-1 reverses pressure overload-induced cardiac hypertrophy and attenuates pathological remodeling.
Journal of the American Heart Association
2013; 2 (2)
MicroRNAs (miRNAs) play a key role in the development of heart failure, and recent studies have shown that the muscle-specific miR-1 is a key regulator of cardiac hypertrophy. We tested the hypothesis that chronic restoration of miR-1 gene expression in vivo will regress hypertrophy and protect against adverse cardiac remodeling induced by pressure overload.Cardiac hypertrophy was induced by left ventricular pressure overload in male Sprague-Dawley rats subjected to ascending aortic stenosis. When the hypertrophy was established at 2 weeks after surgery, the animals were randomized to receive either an adeno-associated virus expressing miR-1 (AAV9.miR-1) or green fluorescent protein (GFP) as control (AAV9.GFP) via a single-bolus tail-vein injection. Administration of miR-1 regressed cardiac hypertrophy (left ventricular posterior wall thickness,; 2.32±0.08 versus 2.75±0.07 mm, P<0.001) and (left ventricular septum wall thickness, 2.23±0.06 versus 2.54±0.10 mm, P<0.05) and halted the disease progression compared with control-treated animals, as assessed by echocardiography (fractional shortening, 37.60±5.01% versus 70.68±2.93%, P<0.05) and hemodynamic analyses (end-systolic pressure volume relationship/effective arterial elastance, 1.87±0.46 versus 0.96±0.38, P<0.05) after 7 weeks of treatment. Additionally, miR-1 replacement therapy lead to a marked reduction of myocardial fibrosis, an improvement in calcium handling, inhibition of apoptosis, and inactivation of the mitogen-activated protein kinase signaling pathways, suggesting a favorable effect on preventing the maladaptive ventricular remodeling. We also identified and validated a novel bona fide target of miR-1, Fibullin-2 (Fbln2), a secreted protein implicated in extracellular matrix remodeling.Taken together, our findings suggest that restoration of miR-1 gene expression is a potential novel therapeutic strategy to reverse pressure-induced cardiac hypertrophy and prevent maladaptive cardiac remodeling.
View details for DOI 10.1161/JAHA.113.000078
View details for PubMedID 23612897
AAV9.I-1c delivered via direct coronary infusion in a porcine model of heart failure improves contractility and mitigates adverse remodeling.
Circulation. Heart failure
2013; 6 (2): 310-317
Heart failure is characterized by impaired function and disturbed Ca2+ homeostasis. Transgenic increases in inhibitor-1 activity have been shown to improve Ca2 cycling and preserve cardiac performance in the failing heart. The aim of this study was to evaluate the effect of activating the inhibitor (I-1c) of protein phosphatase 1 (I-1) through gene transfer on cardiac function in a porcine model of heart failure induced by myocardial infarction.Myocardial infarction was created by a percutaneous, permanent left anterior descending artery occlusion in Yorkshire Landrace swine (n=16). One month after myocardial infarction, pigs underwent intracoronary delivery of either recombinant adeno-associated virus type 9 carrying I-1c (n=8) or saline (n=6) as control. One month after myocardial infarction was created, animals exhibited severe heart failure demonstrated by decreased ejection fraction (46.4±7.0% versus sham 69.7±8.5%) and impaired (dP/dt)max and (dP/dt)min. Intracoronary injection of AAV9.I-1c prevented further deterioration of cardiac function and led to a decrease in scar size.In this preclinical model of heart failure, overexpression of I-1c by intracoronary in vivo gene transfer preserved cardiac function and reduced the scar size.
View details for DOI 10.1161/CIRCHEARTFAILURE.112.971325
View details for PubMedID 23271792
Advancing functional engineered cardiac tissues toward a preclinical model of human myocardium.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Cardiac experimental biology and translational research would benefit from an in vitro surrogate for human heart muscle. This study investigated structural and functional properties and interventional responses of human engineered cardiac tissues (hECTs) compared to human myocardium. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs, >90% troponin-positive) were mixed with collagen and cultured on force-sensing elastomer devices. hECTs resembled trabecular muscle and beat spontaneously (1.18±0.48 Hz). Microstructural features and mRNA expression of cardiac-specific genes (α-MHC, SERCA2a, and ACTC1) were comparable to human myocardium. Optical mapping revealed cardiac refractoriness with loss of 1:1 capture above 3 Hz, and cycle length dependence of the action potential duration, recapitulating key features of cardiac electrophysiology. hECTs reconstituted the Frank-Starling mechanism, generating an average maximum twitch stress of 660 μN/mm(2) at Lmax, approaching values in newborn human myocardium. Dose-response curves followed exponential pharmacodynamics models for calcium chloride (EC50 1.8 mM) and verapamil (IC50 0.61 μM); isoproterenol elicited a positive chronotropic but negligible inotropic response, suggesting sarcoplasmic reticulum immaturity. hECTs were amenable to gene transfer, demonstrated by successful transduction with Ad.GFP. Such 3-D hECTs recapitulate an early developmental stage of human myocardium and promise to offer an alternative preclinical model for cardiology research.-Turnbull, I. C., Karakikes, I., Serrao, G. W., Backeris, P., Lee, J.-J., Xie, C., Senyei, G., Gordon, R. E., Li, R. A., Akar, F. G., Hajjar, R. J., Hulot, J.-S., Costa, K. D. Advancing functional engineered cardiac tissues toward a preclinical model of human myocardium.
View details for DOI 10.1096/fj.13-228007
View details for PubMedID 24174427
Interaction of HLA-DR and CD74 at the cell surface of antigen-presenting cells by single particle image analysis
2012; 26 (12): 4886-4896
Major histocompatibility complex (MHC) class II-associated antigen presentation involves an array of interacting molecules. CD74, the cell surface isoform of the MHC class II-associated invariant chain, is one such molecule; its role remains poorly defined. To address this, we have employed a high-resolution single-particle imaging method for quantifying the colocalization of CD74 with human leukocyte antigen (HLA)-DR molecules on human fibroblast cells known for their capacity to function as antigen-presenting cells. We have also examined whether the colocalization induces internalization of HLA-DR using HA(307-319), a "universal" peptide that binds specifically to the peptide-binding groove of all HLA-DR molecules, irrespective of their alleles. We have determined that 25 ± 1.3% of CD74 and 17 ± 0.3% of HLA-DR are colocalized, and the association of CD74 with HLA-DR and the internalization of HLA-DR are both inhibited by HA(307-319). A similar inhibition of HLA-DR internalization was observed in freshly isolated monocyte-derived dendritic cells. A key role of CD74 is to translocate HLA-DR molecules to early endosomes for reloading with peptides prior to recycling to the cell surface. We conclude that CD74 regulates the balance of peptide-occupied and peptide-free forms of MHC class II at the cell surface.
View details for DOI 10.1096/fj.12-211466
View details for Web of Science ID 000311838300012
View details for PubMedID 22889831
Concomitant Intravenous Nitroglycerin With Intracoronary Delivery of AAV1.SERCA2a Enhances Gene Transfer in Porcine Hearts
2012; 20 (3): 565-571
SERCA2a gene therapy improves contractile and energetic function of failing hearts and has been shown to be associated with benefits in clinical outcomes, symptoms, functional status, biomarkers, and cardiac structure in a phase 2 clinical trial. In an effort to enhance the efficiency and homogeneity of gene uptake in cardiac tissue, we examined the effects of nitroglycerin (NTG) in a porcine model following AAV1.SERCA2a gene delivery. Three groups of Göttingen minipigs were assessed: (i) group A: control intracoronary (IC) AAV1.SERCA2a (n = 6); (ii) group B: a single bolus IC injection of NTG (50 µg) immediately before administration of intravenous (IV) AAV1.SERCA2a (n = 6); and (iii) group C: continuous IV NTG (1 µg/kg/minute) during the 10 minutes of AAV1.SERCA2a infusion (n = 6). We found that simultaneous IV infusion of NTG and AAV1.SERCA2a resulted in increased viral transduction efficiency, both in terms of messenger RNA (mRNA) as well as SERCA2a protein levels in the whole left ventricle (LV) compared to control animals. On the other hand, IC NTG pretreatment did not result in enhanced gene transfer efficiency, mRNA or protein levels when compared to control animals. Importantly, the transgene expression was restricted to the heart tissue. In conclusion, we have demonstrated that IV infusion of NTG significantly improves cardiac gene transfer efficiency in porcine hearts.
View details for DOI 10.1038/mt.2011.268
View details for Web of Science ID 000300943700010
View details for PubMedID 22215018
Fetal Cells Traffic to Injured Maternal Myocardium and Undergo Cardiac Differentiation
2012; 110 (1): 82-93
Fetal cells enter the maternal circulation during pregnancy and may persist in maternal tissue for decades as microchimeras.Based on clinical observations of peripartum cardiomyopathy patients and the high rate of recovery they experience from heart failure, our objective was to determine whether fetal cells can migrate to the maternal heart and differentiate to cardiac cells.We report that fetal cells selectively home to injured maternal hearts and undergo differentiation into diverse cardiac lineages. Using enhanced green fluorescent protein (eGFP)-tagged fetuses, we demonstrate engraftment of multipotent fetal cells in injury zones of maternal hearts. In vivo, eGFP+ fetal cells form endothelial cells, smooth muscle cells, and cardiomyocytes. In vitro, fetal cells isolated from maternal hearts recapitulate these differentiation pathways, additionally forming vascular tubes and beating cardiomyocytes in a fusion-independent manner; ≈40% of fetal cells in the maternal heart express Caudal-related homeobox2 (Cdx2), previously associated with trophoblast stem cells, thought to solely form placenta.Fetal maternal stem cell transfer appears to be a critical mechanism in the maternal response to cardiac injury. Furthermore, we have identified Cdx2 cells as a novel cell type for potential use in cardiovascular regenerative therapy.
View details for DOI 10.1161/CIRCRESAHA.111.249037
View details for Web of Science ID 000299023800012
View details for PubMedID 22082491
Shrink-Film Configurable Multiscale Wrinkles for Functional Alignment of Human Embryonic Stem Cells and their Cardiac Derivatives
2011; 23 (48): 5785-?
A biomimetic substrate for cell-culture is fabricated by plasma treatment of a prestressed thermoplastic shrink film to create tunable multiscaled alignment "wrinkles". Using this substrate, the functional alignment of human embryonic stem cell derived cardiomyocytes is demonstrated.
View details for DOI 10.1002/adma.201103463
View details for Web of Science ID 000298084100009
View details for PubMedID 22065428
Delivery of gelfoam-enabled cells and vectors into the pericardial space using a percutaneous approach in a porcine model
2011; 18 (10): 979-985
Intrapericardial drug delivery is a promising procedure, with the ability to localize therapeutics with the heart. Gelfoam particles are nontoxic, inexpensive, nonimmunogenic and biodegradable compounds that can be used to deliver therapeutic agents. We developed a new percutaneous approach method for intrapericardial injection, puncturing the pericardial sac safely under fluoroscopy and intravascular ultrasound (IVUS) guidance. In a porcine model of myocardial infarction (MI), we deployed gelfoam particles carrying either (a) autologous mesenchymal stem cells (MSCs) or (b) an adenovirus encoding enhanced green fluorescent protein (eGFP) 48 h post-MI. The presence of MSCs and viral infection at the infarct zone was confirmed by immunoflourescence and PCR. Puncture was performed successfully in 16 animals. Using IVUS, we successfully determined the size of the pericardial space before the puncture, and safely accessed that space in setting of pericardial effusion and also adhesions induced by the MI. Intrapericardial injection of gelfoam was safe and reliable. Presence of the MSCs and eGFP expression from adenovirus in the myocardium were confirmed after delivery. Our novel percutaneous approach to deliver (stem-) cells or adenovirus was safe and efficient in this pre-clinical model. IVUS-guided delivery is a minimally invasive procedure that seems to be a promising new strategy to deliver therapeutic agents locally to the heart.
View details for DOI 10.1038/gt.2011.52
View details for Web of Science ID 000296145300006
View details for PubMedID 21512506
A Small Molecule Binding to the Coactivator CREB-Binding Protein Blocks Apoptosis in Cardiomyocytes
CHEMISTRY & BIOLOGY
2011; 18 (4): 531-541
As a master transcription factor in cellular responses to external stress, tumor suppressor p53 is tightly regulated. Excessive p53 activity during myocardial ischemia causes irreversible cellular injury and cardiomyocyte death. p53 activation is dependent on lysine acetylation by the lysine acetyltransferase and transcriptional coactivator CREB-binding protein (CBP) and on acetylation-directed CBP recruitment for p53 target gene expression. Here, we report a small molecule ischemin, developed with a structure-guided approach to inhibit the acetyl-lysine binding activity of the bromodomain of CBP. We show that ischemin alters post-translational modifications on p53 and histones, inhibits p53 interaction with CBP and transcriptional activity in cells, and prevents apoptosis in ischemic cardiomyocytes. Our study suggests small molecule modulation of acetylation-mediated interactions in gene transcription as a new approach to therapeutic interventions of human disorders such as myocardial ischemia.
View details for DOI 10.1016/j.chembiol.2010.12.021
View details for Web of Science ID 000290240900018
View details for PubMedID 21513889
Gene Delivery of Sarcoplasmic Reticulum Calcium ATPase Inhibits Ventricular Remodeling in Ischemic Mitral Regurgitation
2010; 3 (5): 627-634
Mitral regurgitation (MR) doubles mortality after myocardial infarction (MI). We have demonstrated that MR worsens remodeling after MI and that early correction reverses remodeling. Sarcoplasmic reticulum Ca(+2)-ATPase (SERCA2a) is downregulated in this process. We hypothesized that upregulating SERCA2a might inhibit remodeling in a surgical model of apical MI (no intrinsic MR) with independent MR-type flow.In 12 sheep, percutaneous gene delivery was performed by using a validated protocol to perfuse both the left anterior descending and circumflex coronary arteries with occlusion of venous drainage. We administered adeno-associated virus 6 (AAV6) carrying SERCA2a under a Cytomegalovirus promoter control in 6 sheep and a reporter gene in 6 controls. After 2 weeks, a standardized apical MI was created, and a shunt was implanted between the left ventricle and left atrium, producing regurgitant fractions of ≈30%. Animals were compared at baseline and 1 and 3 months by 3D echocardiography, Millar hemodynamics, and biopsies. The SERCA2a group had a well-maintained preload-recruitable stroke work at 3 months (decrease by 8±10% vs 42±12% with reporter gene controls; P<0.001). Left ventricular dP/dt followed the same pattern (no change vs 55% decrease; P<0.001). Left ventricular end-systolic volume was lower with SERCA2a (82.6±9.6 vs 99.4±9.7 mL; P=0.03); left ventricular end-diastolic volume, reflecting volume overload, was not significantly different (127.8±6.2 vs 134.3±9.4 mL). SERCA2a sheep showed a 15% rise in antiapoptotic pAkt versus a 30% reduction with the reporter gene (P<0.001). Prohypertrophic activated STAT3 was also 41% higher with SERCA2a than in controls (P<0.001). Proapoptotic activated caspase-3 rose >5-fold during 1 month in both SERCA2a and control animals (P=NS) and decreased by 19% at 3 months, remaining elevated in both groups.In this controlled model, upregulating SERCA2a induced better function and lesser remodeling, with improved contractility, smaller volume, and activation of prohypertrophic/antiapoptotic pathways. Although caspase-3 remained activated in both groups, SERCA2a sheep had increased molecular antiremodeling "tone." We therefore conclude that upregulating SERCA2a inhibits MR-induced post-MI remodeling in this model and thus may constitute a useful approach to reduce the vicious circle of remodeling in ischemic MR.
View details for DOI 10.1161/CIRCHEARTFAILURE.109.891184
View details for Web of Science ID 000281858200010
View details for PubMedID 20634484
KChIP2 attenuates cardiac hypertrophy through regulation of I-to and intracellular calcium signaling
JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY
2010; 48 (6): 1169-1179
Recent evidence shows that the auxiliary subunit KChIP2, which assembles with pore-forming Kv4-subunits, represents a new potential regulator of the cardiac calcium-independent transient outward potassium current (I(to)) density. In hypertrophy and heart failure, KChIP2 expression has been found to be significantly decreased. Our aim was to examine the role of KChIP2 in cardiac hypertrophy and the effect of restoring its expression on electrical remodeling and cardiac mechanical function using a combination of molecular, biochemical and gene targeting approaches. KChIP2 overexpression through gene transfer of Ad.KChIP2 in neonatal cardiomyocytes resulted in a significant increase in I(to)-channel forming Kv4.2 and Kv4.3 protein levels. In vivo gene transfer of KChIP2 in aortic banded adult rats showed that, compared to sham-operated or Ad.beta-gal-transduced hearts, KChIP2 significantly attenuated the developed left ventricular hypertrophy, robustly increased I(to) densities, shortened action potential duration, and significantly altered myocyte mechanics by shortening contraction amplitudes and maximal rates of contraction and relaxation velocities and decreasing Ca(2+) transients. Interestingly, blocking I(to) with 4-aminopyridine in KChIP2-overexpressing adult cardiomyocytes significantly increased the Ca(2+) transients to control levels. One-day-old rat pups intracardially transduced with KChIP2 for two months then subjected to aortic banding for 6-8 weeks (to induce hypertrophy) showed similar echocardiographic, electrical and mechanical remodeling parameters. In addition, in cultured adult cardiomyocytes, KChIP2 overexpression increased the expression of Ca(2+)-ATPase (SERCA2a) and sodium calcium exchanger but had no effect on ryanodine receptor 2 or phospholamban expression. In neonatal myocytes, KChIP2 notably reversed Ang II-induced hypertrophic changes in protein synthesis and MAP-kinase activation. It also significantly decreased calcineurin expression, NFATc1 expression and nuclear translocation and its downstream target, MCiP1.4. Altogether, these data show that KChIP2 can attenuate cardiac hypertrophy possibly through modulation of intracellular calcium concentration and calcineurin/NFAT pathway.
View details for DOI 10.1016/j.yjmcc.2009.12.019
View details for Web of Science ID 000277944700020
View details for PubMedID 20051248
Amniotic Fluid Cells Are More Efficiently Reprogrammed to Pluripotency Than Adult Cells
2010; 12 (2): 117-125
Recently, cultured human adult skin cells were reprogrammed to induced pluripotent stem (iPS) cells, which have characteristics similar to human embryonic stem (hES) cells. Patient-derived iPS cells offer genetic and immunologic advantages for cell and tissue replacement or engineering. The efficiency of generating human iPS cells has been very low; therefore an easily and efficiently reprogrammed cell type is highly desired. Here, we demonstrate that terminally differentiated human amniotic fluid (AF) skin cells provide an accessible source for efficiently generating abundant-induced pluripotent stem (AF-iPS) cells. By induction of pluripotency with the transcription factor quartet (OCT3/4, SOX2, KLF4, and c-MYC) the terminally differentiated, cultured AF skin cells formed iPS colonies approximately twice as fast and yielded nearly a two-hundred percent increase in number, compared to cultured adult skin cells. AF-iPS cells were identical to hES cells for morphological and growth characteristics, antigenic stem cell markers, stem cell gene expression, telomerase activity, in vitro and in vivo differentiation into the three germ layers and for their capacity to form embryoid bodies (EBs) and teratomas. Our findings provide a biological interesting conclusion that these fetal AF cells are more rapidly, easily, and efficiently reprogrammed to pluripotency than neonatal and adult cells. AF-iPS cells may have a "young," more embryonic like epigenetic background, which may facilitate and accelerate pluripotency. The ability to efficiently and rapidly reprogram terminally differentiated AF skin cells and generate induced pluripotent stem cells provides an abundant iPS cell source for various basic studies and a potential for future patient-specific personalized therapies.
View details for DOI 10.1089/cell.2009.0077
View details for Web of Science ID 000276730400001
View details for PubMedID 20677926
Gene Remodeling in Type 2 Diabetic Cardiomyopathy and Its Phenotypic Rescue with SERCA2a
2009; 4 (7)
Diabetes-associated myocardial dysfunction results in altered gene expression in the heart. We aimed to investigate the changes in gene expression profiles accompanying diabetes-induced cardiomyopathy and its phenotypic rescue by restoration of SERCA2a expression.Using the Otsuka Long-Evans Tokushima Fatty rat model of type 2 diabetes and the Agilent rat microarray chip, we analyzed gene expression by comparing differential transcriptional changes in age-matched control versus diabetic hearts and diabetic hearts that received gene transfer of SERCA2a. Microarray expression profiles of selected genes were verified with real-time qPCR and immunoblotting. Our analysis indicates that diabetic cardiomyopathy is associated with a downregulation of transcripts. Diabetic cardiomyopathic hearts have reduced levels of SERCA2a. SERCA2a gene transfer in these hearts reduced diabetes-associated hypertrophy, and differentially modulated the expression of 76 genes and reversed the transcriptional profile induced by diabetes. In isolated cardiomyocytes in vitro, SERCA2a overexpression significantly modified the expression of a number of transcripts known to be involved in insulin signaling, glucose metabolism and cardiac remodeling.This investigation provided insight into the pathophysiology of cardiac remodeling and the potential role of SERCA2a normalization in multiple pathways in diabetic cardiomyopathy.
View details for DOI 10.1371/journal.pone.0006474
View details for Web of Science ID 000268637600021
View details for PubMedID 19649297