Instructor, Medicine - Cardiovascular Medicine
Honors & Awards
K99/R00 Pathway to Independence Award, NIH/NHLBI (2012)
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 Web of Science ID 000335060500003
View details for PubMedID 24576884
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
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
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
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
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
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
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 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
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
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
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
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
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
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
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