Ioannis Karakikes

All Publications

• Integrative single-cell analysis of cardiogenesis identifies developmental trajectories and non-coding mutations in congenital heart disease. Cell Ameen, M., Sundaram, L., Shen, M., Banerjee, A., Kundu, S., Nair, S., Shcherbina, A., Gu, M., Wilson, K. D., Varadarajan, A., Vadgama, N., Balsubramani, A., Wu, J. C., Engreitz, J. M., Farh, K., Karakikes, I., Wang, K. C., Quertermous, T., Greenleaf, W. J., Kundaje, A. 2022; 185 (26): 4937

  Abstract

  To define the multi-cellular epigenomic and transcriptional landscape of cardiac cellular development, we generated single-cell chromatin accessibility maps of human fetal heart tissues. We identified eight major differentiation trajectories involving primary cardiac cell types, each associated with dynamic transcription factor (TF) activity signatures. We contrasted regulatory landscapes of iPSC-derived cardiac cell types and their invivo counterparts, which enabled optimization of invitro differentiation of epicardial cells. Further, we interpreted sequence based deep learning models of cell-type-resolved chromatin accessibility profiles to decipher underlying TF motif lexicons. De novo mutations predicted to affect chromatin accessibility in arterial endothelium were enriched in congenital heart disease (CHD) cases vs. controls. Invitro studies in iPSCs validated the functional impact of identified variation on the predicted developmental cell types. This work thus defines the cell-type-resolved cis-regulatory sequence determinants of heart development and identifies disruption of cell type-specific regulatory elements in CHD.

  View details for DOI 10.1016/j.cell.2022.11.028

  View details for PubMedID 36563664

• Serine biosynthesis as a novel therapeutic target for dilated cardiomyopathy. European heart journal Perea-Gil, I., Seeger, T., Bruyneel, A. A., Termglinchan, V., Monte, E., Lim, E. W., Vadgama, N., Furihata, T., Gavidia, A. A., Arthur Ataam, J., Bharucha, N., Martinez-Amador, N., Ameen, M., Nair, P., Serrano, R., Kaur, B., Feyen, D. A., Diecke, S., Snyder, M. P., Metallo, C. M., Mercola, M., Karakikes, I. 2022

  Abstract

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

  View details for DOI 10.1093/eurheartj/ehac305

  View details for PubMedID 35728000

• The Unfolded Protein Response as a Compensatory Mechanism and Potential Therapeutic Target in PLN R14del Cardiomyopathy. Circulation Feyen, D. A., Perea-Gil, I., Maas, R. G., Harakalova, M., Gavidia, A. A., Arthur Ataam, J., Wu, T., Vink, A., Pei, J., Vadgama, N., Suurmeijer, A. J., Te Rijdt, W. P., Vu, M., Amatya, P. L., Prado, M., Zhang, Y., Dunkenberger, L., Sluijter, J. P., Sallam, K., Asselbergs, F. W., Mercola, M., Karakikes, I. 2021

  Abstract

  Background: Phospholamban (PLN) is a critical regulator of calcium cycling and contractility in the heart. The loss of arginine at position 14 in PLN (R14del) is associated with dilated cardiomyopathy (DCM) with a high prevalence of ventricular arrhythmias. How the R14 deletion causes DCM is poorly understood and there are no disease-specific therapies. Methods: We used single-cell RNA sequencing to uncover PLN R14del disease-mechanisms in human induced pluripotent stem cells (hiPSC-CMs). We utilized both 2D and 3D functional contractility assays to evaluate the impact of modulating disease relevant pathways in PLN R14del hiPSC-CMs. Results: Modeling of the PLN R14del cardiomyopathy with isogenic pairs of hiPSC-CMs recapitulated the contractile deficit associated with the disease in vitro. Single-cell RNA sequencing revealed the induction of the unfolded protein response pathway (UPR) in PLN R14del compared to isogenic control hiPSC-CMs. The activation of UPR was also evident in the hearts from PLN R14del patients. Silencing of each of the three main UPR signaling branches (IRE1, ATF6, or PERK) by siRNA exacerbated the contractile dysfunction of PLN R14del hiPSC-CMs. We explored the therapeutic potential of activating the UPR with a small molecule activator, BiP protein Inducer X (BiX). PLN R14del hiPSC-CMs treated with BiX showed a dose-dependent amelioration of the contractility deficit of in both 2D cultures and 3D engineered heart tissues without affecting calcium homeostasis. Conclusions: Together, these findings suggest that the UPR exerts a protective effect in the setting of PLN R14del cardiomyopathy and that modulation of the UPR might be exploited therapeutically.

  View details for DOI 10.1161/CIRCULATIONAHA.120.049844

  View details for PubMedID 33928785

• Pharmacological Silencing of MicroRNA-152 Prevents Pressure Overload-Induced Heart Failure. Circulation. Heart failure LaRocca, T. J., Seeger, T. n., Prado, M. n., Perea-Gil, I. n., Neofytou, E. n., Mecham, B. H., Ameen, M. n., Chang, A. C., Pandey, G. n., Wu, J. C., Karakikes, I. n. 2020; 13 (3): e006298

  Abstract

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

  View details for DOI 10.1161/CIRCHEARTFAILURE.119.006298

  View details for PubMedID 32160771

• Translating Genomic Insights into Cardiovascular Medicines: Opportunities and Challenges of CRISPR-Cas9. Trends in cardiovascular medicine Zhang, Y. n., Karakikes, I. n. 2020

  Abstract

  The growing appreciation of human genetics and genomics in cardiovascular disease (CVD) accompanied by the technological breakthroughs in genome editing, particularly the CRISPR-Cas9 technologies, has presented an unprecedented opportunity to explore the application of genome editing tools in cardiovascular medicine. The ever-growing genome-editing toolbox includes an assortment of CRISPR-Cas systems with increasing efficiency, precision, flexibility, and targeting capacity. Over the past decade, the advent of large-scale genotyping technologies and genome-wide association studies (GWAS) has provided powerful tools to identify genotype-phenotype associations for diseases with complex traits. Notably, a growing number of loss-of-function mutations have been associated with favorable CVD risk-factor profiles that may confer protection. Combining the newly gained insights into human genetics with recent breakthrough technologies, such as the CRISPR technology, holds great promise in elucidating novel disease mechanisms and transforming genes into medicines. Nonetheless, translating genetic insights into novel therapeutic avenues remains challenging, and applications of "in body" genome editing for CVD treatment and engineering cardioprotection remain mostly theoretical. Here we highlight the recent advances of the CRISPR-based genome editing toolbox and discuss the potential and challenges of CRISPR-based technologies for translating GWAS findings into genomic medicines.

  View details for DOI 10.1016/j.tcm.2020.06.008

  View details for PubMedID 32603681

• A Novel Recessive Mutation in SPEG Causes Early Onset Dilated Cardiomyopathy. PLoS genetics Levitas, A. n., Muhammad, E. n., Zhang, Y. n., Perea Gil, I. n., Serrano, R. n., Diaz, N. n., Arafat, M. n., Gavidia, A. A., Kapiloff, M. S., Mercola, M. n., Etzion, Y. n., Parvari, R. n., Karakikes, I. n. 2020; 16 (9): e1009000

  Abstract

  Dilated cardiomyopathy (DCM) is a common cause of heart failure and sudden cardiac death. It has been estimated that up to half of DCM cases are hereditary. Mutations in more than 50 genes, primarily autosomal dominant, have been reported. Although rare, recessive mutations are thought to contribute considerably to DCM, especially in young children. Here we identified a novel recessive mutation in the striated muscle enriched protein kinase (SPEG, p. E1680K) gene in a family with nonsyndromic, early onset DCM. To ascertain the pathogenicity of this mutation, we generated SPEG E1680K homozygous mutant human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) using CRISPR/Cas9-mediated genome editing. Functional studies in mutant iPSC-CMs showed aberrant calcium homeostasis, impaired contractility, and sarcomeric disorganization, recapitulating the hallmarks of DCM. By combining genetic analysis with human iPSCs, genome editing, and functional assays, we identified SPEG E1680K as a novel mutation associated with early onset DCM and provide evidence for its pathogenicity in vitro. Our study provides a conceptual paradigm for establishing genotype-phenotype associations in DCM with autosomal recessive inheritance.

  View details for DOI 10.1371/journal.pgen.1009000

  View details for PubMedID 32925938

• Activation of PDGF pathway links LMNA mutation to dilated cardiomyopathy. Nature Lee, J. n., Termglinchan, V. n., Diecke, S. n., Itzhaki, I. n., Lam, C. K., Garg, P. n., Lau, E. n., Greenhaw, M. n., Seeger, T. n., Wu, H. n., Zhang, J. Z., Chen, X. n., Gil, I. P., Ameen, M. n., Sallam, K. n., Rhee, J. W., Churko, J. M., Chaudhary, R. n., Chour, T. n., Wang, P. J., Snyder, M. P., Chang, H. Y., Karakikes, I. n., Wu, J. C. 2019

  Abstract

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

  View details for DOI 10.1038/s41586-019-1406-x

  View details for PubMedID 31316208

• A Premature Termination Codon Mutation in MYBPC3 Causes Hypertrophic Cardiomyopathy via Chronic Activation of Nonsense-Mediated Decay. Circulation Seeger, T., Shrestha, R., Lam, C. K., Chen, C., McKeithan, W. L., Lau, E., Wnorowski, A., McMullen, G., Greenhaw, M., Lee, J., Oikonomopoulos, A., Lee, S., Yang, H., Mercola, M., Wheeler, M., Ashley, E. A., Yang, F., Karakikes, I., Wu, J. C. 2018

  Abstract

  BACKGROUND: Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in myosin binding protein C3 ( MYBPC3) resulting in a premature termination codon (PTC). The underlying mechanisms of how PTC mutations in MYBPC3 lead to the onset and progression of HCM are poorly understood. This study's aim was to investigate the molecular mechanisms underlying the pathogenesis of HCM associated with MYBPC3 PTC mutations by utilizing human isogenic induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).METHODS: Isogenic iPSC lines were generated from patients harboring MYBPC3 PTC mutations (p.R943x; p.R1073P_Fsx4) using genome editing and then differentiated into cardiomyocytes. Comprehensive phenotypical and transcriptome analyses were performed.RESULTS: We observed aberrant calcium handling properties with prolonged decay kinetics and elevated diastolic calcium levels in HCM iPSC-CMs compared to isogenic controls without structural abnormalities or contractile dysfunction. The mRNA expression levels of MYBPC3 were significantly reduced in mutant iPSC-CMs, but the protein levels were comparable among isogenic iPSC-CMs, suggesting that haploinsufficiency of MYBPC3 does not contribute to the pathogenesis of HCM in vitro. Furthermore, truncated MYBPC3 peptides were not detected. At the molecular level, the nonsense-mediated decay (NMD) pathway was activated, and a set of genes involved in major cardiac signaling pathways was dysregulated in HCM iPSC-CMs, indicating an HCM gene signature in vitro. Specific inhibition of the NMD pathway in mutant iPSC-CMs resulted in reversal of the molecular phenotype and normalization of calcium handling abnormalities.CONCLUSIONS: iPSC-CMs carrying MYBPC3 PTC mutations displayed aberrant calcium signaling and molecular dysregulations in the absence of significant haploinsufficiency of MYBPC3 protein. Here we provided the first evidence of the direct connection between the chronically activated NMD pathway and HCM disease development.

  View details for PubMedID 30586709

• A Comprehensive TALEN-Based Knockout Library for Generating Human Induced Pluripotent Stem Cell-Based Models for Cardiovascular Diseases. Circulation research Karakikes, I., Termglinchan, V., Cepeda, D. A., Lee, J., Diecke, S., Hendel, A., Itzhaki, I., Ameen, M., Shrestha, R., Wu, H., Ma, N., Shao, N., Seeger, T., Woo, N. A., Wilson, K. D., Matsa, E., Porteus, M. H., Sebastiano, V., Wu, J. C. 2017

  Abstract

  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

• Efficient Genome Editing in Induced Pluripotent Stem Cells with Engineered Nucleases In Vitro. Methods in molecular biology (Clifton, N.J.) Termglinchan, V., Seeger, T., Chen, C., Wu, J. C., Karakikes, I. 2017; 1521: 55-68

  Abstract

  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. Circulation research Karakikes, I., Ameen, M., Termglinchan, V., Wu, J. C. 2015; 117 (1): 80-88

  Abstract

  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 Karakikes, I., Stillitano, F., Nonnenmacher, M., Tzimas, C., Sanoudou, D., Termglinchan, V., Kong, C., Rushing, S., Hansen, J., Ceholski, D., Kolokathis, F., Kremastinos, D., Katoulis, A., Ren, L., Cohen, N., Gho, J. M., Tsiapras, D., Vink, A., Wu, J. C., Asselbergs, F. W., Li, R. A., Hulot, J., Kranias, E. G., Hajjar, R. J. 2015; 6

  View details for DOI 10.1038/ncomms7955

  View details for Web of Science ID 000353704700007

  View details for PubMedID 25923014

• Empagliflozin Attenuates Arrhythmias in an iPSC-Based Model of Hypertrophy Cardiomyopathy. Circulation. Genomic and precision medicine Arthur Ataam, J., Belbachir, N., Perea-Gil, I., Termglincan, V., Vadgama, N., Garg, P., Ramchandani, R., Gavidia, A. A., Roura, S., Gálvez-Montón, C., Wu, J. C., Bayés-Genis, A., Karakikes, I. 2024: e004526

  View details for DOI 10.1161/CIRCGEN.123.004526

  View details for PubMedID 38752368

• Precision unleashed: tackling DNA mismatch repair for enhanced prime editing. Molecular therapy. Nucleic acids Ramchandani, R., Bharucha, N., Karakikes, I. 2023; 34: 102061

  View details for DOI 10.1016/j.omtn.2023.102061

  View details for PubMedID 38053563

• Identification of constrained sequence elements across 239 primate genomes. Nature Kuderna, L. F., Ulirsch, J. C., Rashid, S., Ameen, M., Sundaram, L., Hickey, G., Cox, A. J., Gao, H., Kumar, A., Aguet, F., Christmas, M. J., Clawson, H., Haeussler, M., Janiak, M. C., Kuhlwilm, M., Orkin, J. D., Bataillon, T., Manu, S., Valenzuela, A., Bergman, J., Rouselle, M., Silva, F. E., Agueda, L., Blanc, J., Gut, M., de Vries, D., Goodhead, I., Harris, R. A., Raveendran, M., Jensen, A., Chuma, I. S., Horvath, J. E., Hvilsom, C., Juan, D., Frandsen, P., Schraiber, J. G., de Melo, F. R., Bertuol, F., Byrne, H., Sampaio, I., Farias, I., Valsecchi, J., Messias, M., da Silva, M. N., Trivedi, M., Rossi, R., Hrbek, T., Andriaholinirina, N., Rabarivola, C. J., Zaramody, A., Jolly, C. J., Phillips-Conroy, J., Wilkerson, G., Abee, C., Simmons, J. H., Fernandez-Duque, E., Kanthaswamy, S., Shiferaw, F., Wu, D., Zhou, L., Shao, Y., Zhang, G., Keyyu, J. D., Knauf, S., Le, M. D., Lizano, E., Merker, S., Navarro, A., Nadler, T., Khor, C. C., Lee, J., Tan, P., Lim, W. K., Kitchener, A. C., Zinner, D., Gut, I., Melin, A. D., Guschanski, K., Schierup, M. H., Beck, R. M., Karakikes, I., Wang, K. C., Umapathy, G., Roos, C., Boubli, J. P., Siepel, A., Kundaje, A., Paten, B., Lindblad-Toh, K., Rogers, J., Marques Bonet, T., Farh, K. K. 2023

  Abstract

  Noncoding DNA is central to our understanding of human gene regulation and complex diseases1,2, and measuring the evolutionary sequence constraint can establish the functional relevance of putative regulatory elements in the human genome3-9. Identifying the genomic elements that have become constrained specifically in primates has been hampered by the faster evolution of noncoding DNA compared to protein-coding DNA10, the relatively short timescales separating primate species11, and the previously limited availability of whole-genome sequences12. Here we construct a whole-genome alignment of 239 species, representing nearly half of all extant species in the primate order. Using this resource, we identified human regulatory elements that are under selective constraint across primates and other mammals at a 5% false discovery rate. We detected 111,318 DNase I hypersensitivity sites and 267,410 transcription factor binding sites that are constrained specifically in primates but not across other placental mammals and validate their cis-regulatory effects on gene expression. These regulatory elements are enriched for human genetic variants that affect gene expression and complex traits and diseases. Our results highlight the important role of recent evolution in regulatory sequence elements differentiating primates, including humans, from other placental mammals.

  View details for DOI 10.1038/s41586-023-06798-8

  View details for PubMedID 38030727

  View details for PubMedCentralID 1891336

• Generation of human induced pluripotent stem cell lines derived from four patients with a pathogenic ALPK3 variant associated with adult-onset hypertrophic cardiomyopathy (HCM). Stem cell research Cheawsamoot, C., Ramchandani, R., Ameen, M., Arthur Ataam, J., Khongphatthanayothin, A., Shotelersuk, V., Karakikes, I. 2023; 73: 103233

  Abstract

  Loss of function variants in ALPK3 have been associated with dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM). However, the underlying pathomechanism remain largely unknown. Here, we generated human iPSC lines from four HCM patients carrying the heterozygous pathogenic variant in ALPK3 (c.2023delC p.Gln675fs). Peripheral blood mononuclear cells (PBMCs) from patients were reprogrammed to induced pluripotent stem cells (iPSCs) with the Sendai virus-based reprogramming method. All four lines display typical iPSC morphology, normal karyotype, expression of pluripotency-associated markers, and trilineage differentiation potential. These iPSC lines represent a valuable resource of ALPK3 patient-derived iPSC lines to the study ALPK3-associated cardiomyopathy.

  View details for DOI 10.1016/j.scr.2023.103233

  View details for PubMedID 37944352

• Transcription factor stoichiometry, motif affinity and syntax regulate single-cell chromatin dynamics during fibroblast reprogramming to pluripotency. bioRxiv : the preprint server for biology Nair, S., Ameen, M., Sundaram, L., Pampari, A., Schreiber, J., Balsubramani, A., Wang, Y. X., Burns, D., Blau, H. M., Karakikes, I., Wang, K. C., Kundaje, A. 2023

  Abstract

  Ectopic expression of OCT4, SOX2, KLF4 and MYC (OSKM) transforms differentiated cells into induced pluripotent stem cells. To refine our mechanistic understanding of reprogramming, especially during the early stages, we profiled chromatin accessibility and gene expression at single-cell resolution across a densely sampled time course of human fibroblast reprogramming. Using neural networks that map DNA sequence to ATAC-seq profiles at base-resolution, we annotated cell-state-specific predictive transcription factor (TF) motif syntax in regulatory elements, inferred affinity- and concentration-dependent dynamics of Tn5-bias corrected TF footprints, linked peaks to putative target genes, and elucidated rewiring of TF-to-gene cis-regulatory networks. Our models reveal that early in reprogramming, OSK, at supraphysiological concentrations, rapidly open transient regulatory elements by occupying non-canonical low-affinity binding sites. As OSK concentration falls, the accessibility of these transient elements decays as a function of motif affinity. We find that these OSK-dependent transient elements sequester the somatic TF AP-1. This redistribution is strongly associated with the silencing of fibroblast-specific genes within individual nuclei. Together, our integrated single-cell resource and models reveal insights into the cis-regulatory code of reprogramming at unprecedented resolution, connect TF stoichiometry and motif syntax to diversification of cell fate trajectories, and provide new perspectives on the dynamics and role of transient regulatory elements in somatic silencing.

  View details for DOI 10.1101/2023.10.04.560808

  View details for PubMedID 37873116

• Non-viral in vivo cytidine base editing in hepatocytes using focused ultrasound targeted microbubbles. Molecular therapy. Nucleic acids Anderson, C. D., Arthur, J. A., Zhang, Y., Bharucha, N., Karakikes, I., Shohet, R. V. 2023; 33: 733-737

  Abstract

  CRISPR-Cas9-based genome editing technologies, such as base editing, have the potential for clinical translation, but delivering nucleic acids into target cells in vivo is a major obstacle. Viral vectors are widely used but come with safety concerns, while current non-viral methods are limited by low transfection efficiency. Here we describe a new method to deliver CRISPR-Cas9 base editing vectors to the mouse liver using focused ultrasound targeted microbubble destruction (FUTMD). We demonstrate, using the example of cytosine base editing of the Pde3b gene, that FUTMD-mediated delivery of cytosine base editing vectors can introduce stop codons (up to ∼2.5% on-target editing) in mouse liver cells in vivo. However, base editing specificity is less than one might hope with these DNA constructs. Our findings suggest that FUTMD-based gene editing tools can be rapidly and transiently deployed to specific organs and sites, providing a powerful platform for the development of non-viral genome editing therapies. Non-viral delivery also reveals greater off-target base exchange in vivo than in vitro.

  View details for DOI 10.1016/j.omtn.2023.07.032

  View details for PubMedID 37662969

  View details for PubMedCentralID PMC10468349

• A deep learning platform to assess drug proarrhythmia risk. Cell stem cell Serrano, R., Feyen, D. A., Bruyneel, A. A., Hnatiuk, A. P., Vu, M. M., Amatya, P. L., Perea-Gil, I., Prado, M., Seeger, T., Wu, J. C., Karakikes, I., Mercola, M. 2022

  Abstract

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

  View details for DOI 10.1016/j.stem.2022.12.002

  View details for PubMedID 36563695

• Isoform changes of action potential regulators in the ventricles of arrhythmogenic phospholamban-R14del humanized mouse hearts. Metabolism: clinical and experimental Rogalska, M. E., Vafiadaki, E., Erpapazoglou, Z., Haghighi, K., Green, L., Mantzoros, C. S., Hajjar, R. J., Tranter, M., Karakikes, I., Kranias, E. G., Stillitano, F., Kafasla, P., Sanoudou, D. 2022: 155344

  Abstract

  Arrhythmogenic cardiomyopathy (ACM) is characterized by life-threatening ventricular arrhythmias and sudden cardiac death and affects hundreds of thousands of patients worldwide. The deletion of Arginine 14 (p.R14del) in the phospholamban (PLN) gene has been implicated in the pathogenesis of ACM. PLN is a key regulator of sarcoplasmic reticulum (SR) Ca2+ cycling and cardiac contractility. Despite global gene and protein expression studies, the molecular mechanisms of PLN-R14del ACM pathogenesis remain unclear. Using a humanized PLN-R14del mouse model and human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs), we investigated the transcriptome-wide mRNA splicing changes associated with the R14del mutation. We identified >200 significant alternative splicing (AS) events and distinct AS profiles were observed in the right (RV) and left (LV) ventricles in PLN-R14del compared to WT mouse hearts. Enrichment analysis of the AS events showed that the most affected biological process was associated with "cardiac cell action potential", specifically in the RV. We found that splicing of 2 key genes, Trpm4 and Camk2d, which encode proteins regulating calcium homeostasis in the heart, were altered in PLN-R14del mouse hearts and human iPSC-CMs. Bioinformatical analysis pointed to the tissue-specific splicing factors Srrm4 and Nova1 as likely upstream regulators of the observed splicing changes in the PLN-R14del cardiomyocytes. Our findings suggest that aberrant splicing may affect Ca2+-homeostasis in the heart, contributing to the increased risk of arrythmogenesis in PLN-R14del ACM.

  View details for DOI 10.1016/j.metabol.2022.155344

  View details for PubMedID 36375644

• De novo and inherited variants in coding and regulatory regions in genetic cardiomyopathies. Human genomics Vadgama, N., Ameen, M., Sundaram, L., Gaddam, S., Genomics England Research Consortium, Gifford, C., Nasir, J., Karakikes, I. 2022; 16 (1): 55

  Abstract

  BACKGROUND: Cardiomyopathies are a leading cause of progressive heart failure and sudden cardiac death; however, their genetic aetiology remains poorly understood. We hypothesised that variants in noncoding regulatory regions and oligogenic inheritance mechanisms may help close the diagnostic gap.METHODS: We first analysed whole-genome sequencing data of 143 parent-offspring trios from Genomics England 100,000 Genomes Project. We used gene panel testing and a phenotype-based, variant prioritisation framework called Exomiser to identify candidate genes in trios. To assess the contribution of noncoding DNVs to cardiomyopathies, we intersected DNVs with open chromatin sequences from single-cell ATAC-seq data of cardiomyocytes. We also performed a case-control analysis in an exome-negative cohort, including 843 probands and 19,467 controls, to assess the association between noncoding variants in known cardiomyopathy genes and disease.RESULTS: In the trio analysis, a definite or probable genetic diagnosis was identified in 21 probands according to the American College of Medical Genetics guidelines. We identified novel DNVs in diagnostic-grade genes (RYR2, TNNT2, PTPN11, MYH7, LZR1, NKX2-5), and five cases harbouring a combination of prioritised variants, suggesting that oligogenic inheritance and genetic modifiers contribute to cardiomyopathies. Phenotype-based ranking of candidate genes identified in noncoding DNV analysis revealed JPH2 as the top candidate. Moreover, a case-control analysis revealed an enrichment of rare noncoding variants in regulatory elements of cardiomyopathy genes (p=.035, OR=1.43, 95% Cl=1.095-1.767) versus controls. Of the 25 variants associated with disease (p< 0.5), 23 are novel and nine are predicted to disrupt transcription factor binding motifs.CONCLUSION: Our results highlight complex genetic mechanisms in cardiomyopathies and reveal novel genes for future investigations.

  View details for DOI 10.1186/s40246-022-00420-0

  View details for PubMedID 36357925

• The potential of CRISPR-Cas9 prime editing for cardiovascular disease research and therapy. Current opinion in cardiology Bharucha, N., Arias, A., Karakikes, I. 2022

  Abstract

  PURPOSE OF REVIEW: The ability to edit any genomic sequence has led to a better understanding of gene function and holds promise for the development of therapies for genetic diseases. This review describes prime editing - the latest CRISPR-Cas9 genome editing technology. Prime editing enables precise and accurate genome editing in terminally differentiated, postmitotic cells like cardiomyocytes, paving the way for therapeutic applications for genetic cardiomyopathies.RECENT FINDINGS: Prime editing has been used to precisely insert up to 40 bases, create deletions up to 80 base pairs, and can perform all 12 possible transition and transversion base mutations with lower indels and off-target effects than other genome editing methods. The development of several software tools has simplified the experimental design and led to increased efficiency of the process. Improvements in methods for in-vivo delivery of the prime editing components should enable this technology to be used to edit the genome in patients.SUMMARY: Prime editing has the potential to revolutionize the future of biomedical research and transform cardiovascular medicine. Improved understanding of the prime editing process and developments in agent design, efficacy and delivery will benefit scientists and patients and could be an effective way to cure cardiovascular diseases.

  View details for DOI 10.1097/HCO.0000000000000985

  View details for PubMedID 35880456

• Mutations in MINAR2 encoding membrane integral NOTCH2-associated receptor 2 cause deafness in humans and mice. Proceedings of the National Academy of Sciences of the United States of America Bademci, G., Lachgar-Ruiz, M., Deokar, M., Zafeer, M. F., Abad, C., Yildirim Baylan, M., Ingham, N. J., Chen, J., Sineni, C. J., Vadgama, N., Karakikes, I., Guo, S., Duman, D., Singh, N., Harlalka, G., Jain, S. P., Chioza, B. A., Walz, K., Steel, K. P., Nasir, J., Tekin, M. 2022; 119 (26): e2204084119

  Abstract

  Discovery of deafness genes and elucidating their functions have substantially contributed to our understanding of hearing physiology and its pathologies. Here we report on DNA variants in MINAR2, encoding membrane integral NOTCH2-associated receptor 2, in four families underlying autosomal recessive nonsyndromic deafness. Neurologic evaluation of affected individuals at ages ranging from 4 to 80 y old does not show additional abnormalities. MINAR2 is a recently annotated gene with limited functional understanding. We detected three MINAR2 variants, c.144G > A (p.Trp48*), c.412_419delCGGTTTTG (p.Arg138Valfs*10), and c.393G > T, in 13 individuals with congenital- or prelingual-onset severe-to-profound sensorineural hearing loss (HL). The c.393G > T variant is shown to disrupt a splice donor site. We show that Minar2 is expressed in the mouse inner ear, with the protein localizing mainly in the hair cells, spiral ganglia, the spiral limbus, and the stria vascularis. Mice with loss of function of the Minar2 protein (Minar2tm1b/tm1b) present with rapidly progressive sensorineural HL associated with a reduction in outer hair cell stereocilia in the shortest row and degeneration of hair cells at a later age. We conclude that MINAR2 is essential for hearing in humans and mice and its disruption leads to sensorineural HL. Progressive HL observed in mice and in some affected individuals and as well as relative preservation of hair cells provides an opportunity to interfere with HL using genetic therapies.

  View details for DOI 10.1073/pnas.2204084119

  View details for PubMedID 35727972

• Generation of a dual edited human induced pluripotent stem cell Myl7-GFP reporter line with inducible CRISPRi/dCas9. Stem cell research Metzl-Raz, E., Bharucha, N., Arthur Ataam, J., Gavidia, A. A., Greenleaf, W. J., Karakikes, I. 2022; 61: 102754

  Abstract

  Temporal regulation of CRISPRi activity is critical for genetic screens. Here, we present an inducible CRISPRi platform enabling selection of iPSC-derived cardiomyocytes and reversible gene knockdown. We targeted a doxycycline-inducible dCas9-KRAB-mCherry cassette into the AAVS1 locus in an MYL7-mGFP reporter iPSC line. A clone with bi-allelic integration displayed minimally leaky CRISPRi activity and strong expression upon addition of doxycycline in iPSCs, iPSC-derived cardiomyocytes, and multilineage differentiated cells. The CRISPRi activity was validated by targeting the MYOCD gene in iPSC cardiomyocytes. In summary, we developed a robust inducible CRISPRi platform to interrogate gene function in human iPSC-derived cardiomyocytes and other cells.

  View details for DOI 10.1016/j.scr.2022.102754

  View details for PubMedID 35325819

• Targeting mAKAPbeta expression as a therapeutic approach for ischemic cardiomyopathy. Gene therapy Martinez, E. C., Li, J., Ataam, J. A., Tokarski, K., Thakur, H., Karakikes, I., Dodge-Kafka, K., Kapiloff, M. S. 1800

  Abstract

  Ischemic cardiomyopathy is a leading cause of death and an unmet clinical need. Adeno-associated virus (AAV) gene-based therapies hold great promise for treating and preventing heart failure. Previously we showed that muscle A-kinase Anchoring Protein beta (mAKAPbeta, AKAP6beta), a scaffold protein that organizes perinuclear signalosomes in the cardiomyocyte, is a critical regulator of pathological cardiac hypertrophy. Here, we show that inhibition of mAKAPbeta expression in stressed adult cardiomyocytes in vitro was cardioprotective, while conditional cardiomyocyte-specific mAKAP gene deletion in mice prevented pathological cardiac remodeling due to myocardial infarction. We developed a new self-complementary serotype 9 AAV gene therapy vector expressing a short hairpin RNA for mAKAPbeta under the control of a cardiomyocyte-specific promoter (AAV9sc.shmAKAP). This vector efficiently downregulated mAKAPbeta expression in the mouse heart in vivo. Expression of the shRNA also inhibited mAKAPbeta expression in human induced cardiomyocytes in vitro. Following myocardial infarction, systemic administration of AAV9sc.shmAKAP prevented the development of pathological cardiac remodeling and heart failure, providing long-term restoration of left ventricular ejection fraction. Our findings provide proof-of-concept for mAKAPbeta as a therapeutic target for ischemic cardiomyopathy and support the development of a translational pipeline for AAV9sc.shmAKAP for the treatment of heart failure.

  View details for DOI 10.1038/s41434-022-00321-w

  View details for PubMedID 35102273

• SARS-CoV-2 Susceptibility and ACE2 Gene Variations Within Diverse Ethnic Backgrounds. Frontiers in genetics Vadgama, N., Kreymerman, A., Campbell, J., Shamardina, O., Brugger, C., Research Consortium, G. E., Deaconescu, A. M., Lee, R. T., Penkett, C. J., Gifford, C. A., Mercola, M., Nasir, J., Karakikes, I. 2022; 13: 888025

  Abstract

  There is considerable variability in the susceptibility and progression for COVID-19 and it appears to be strongly correlated with age, gender, ethnicity and pre-existing health conditions. However, to our knowledge, cohort studies of COVID-19 in clinically vulnerable groups are lacking. Host genetics has also emerged as a major risk factor for COVID-19, and variation in the ACE2 receptor, which facilitates entry of the SARS-CoV-2 virus into the cell, has become a major focus of attention. Thus, we interrogated an ethnically diverse cohort of National Health Service (NHS) patients in the United Kingdom (United Kingdom) to assess the association between variants in the ACE2 locus and COVID-19 risk. We analysed whole-genome sequencing (WGS) data of 1,837 cases who were tested positive for SARS-CoV-2, and 37,207 controls who were not tested, from the UK's 100,000 Genomes Project (100KGP) for the presence of ACE2 coding variants and extract expression quantitative trait loci (eQTLs). We identified a splice site variant (rs2285666) associated with increased ACE2 expression with an overrepresentation in SARS-CoV-2 positive patients relative to 100KGP controls (p = 0.015), and in hospitalised European patients relative to outpatients in intra-ethnic comparisons (p = 0.029). We also compared the prevalence of 288 eQTLs, of which 23 were enriched in SARS-CoV-2 positive patients. The eQTL rs12006793 had the largest effect size (d = 0.91), which decreases ACE2 expression and is more prevalent in controls, thus potentially reducing the risk of COVID-19. We identified three novel nonsynonymous variants predicted to alter ACE2 function, and showed that three variants (p.K26R, p. H378R, p. Y515N) alter receptor affinity for the viral Spike (S) protein. Variant p. N720D, more prevalent in the European population (p < 0.001), potentially increases viral entry by affecting the ACE2-TMPRSS2 complex. The spectrum of genetic variants in ACE2 may inform risk stratification of COVID-19 patients and could partially explain the differences in disease susceptibility and severity among different ethnic groups.

  View details for DOI 10.3389/fgene.2022.888025

  View details for PubMedID 35571054

• Insulin Growth Factor Phenotypes in Heart Failure with Preserved Ejection Fraction, an INSPIRE Registry and CATHGEN Study: IGF axis in HFpEF. Journal of cardiac failure Haddad, F., Ataam, J. A., Amsallem, M., Cauwenberghs, N., Kuznetsova, T., Rosenberg-Hasson, Y., Zamanian, R. T., Karakikes, I., Horne, B. D., Muhlestein, J. B., Kwee, L., Shah, S., Maecker, H., Knight, S., Knowlton, K. 1800

  Abstract

  BACKGROUND: The insulin like growth factor (IGF) axis emerged as an important pathway in heart failure with preserved ejection (HFpEF). We aimed to identify IGF phenotypes associated with HFpEF in the context high-dimensional proteomic profiling.METHODS: From the Intermountain INSPIRE Registry, we identified 96 patients with HFpEF and matched controls. We performed targeted proteomics including IGF-1,2, IGF binding proteins (IGFBP) 1-7 and 111 other proteins (EMD Millipore and ELISA). We used partial least square discriminant analysis (PLS-DA) to identify a set of proteins associated with prevalent HFpEF, pulmonary hypertension (PH) and 5-year-all-cause mortality. K-mean clustering was used to identify IGF phenotypes.RESULTS: Patients with HFpEF had a high prevalence of systemic hypertension (95%) and coronary artery disease (74%). Using PLS-DA, we identified a set of biomarkers including IGF1,2 and IGFBP-1,2,7 that provided a strong discrimination of HFPEF, PH and mortality with an AUC of 0.91, 0.77 and 0.83, respectively. Using K mean clustering, we identified three IGF phenotypes that were independently associated with all-cause 5-year mortality after adjustment for age, NT-proBNP and kidney disease (p=0.004). Multivariable analysis validated the prognostic value of IGFBP-1 and 2 in the CATHGEN biorepository.CONCLUSION: IGF phenotypes were associated with PH and mortality in HFpEF.

  View details for DOI 10.1016/j.cardfail.2021.12.012

  View details for PubMedID 34979242

• Generation of AAVS1 integrated doxycycline-inducible CRISPR-Prime Editor human induced pluripotent stem cell line. Stem cell research Bharucha, N., Ataam, J. A., Gavidia, A. A., Karakikes, I. 2021; 57: 102610

  Abstract

  Prime editing uses the Cas9 nickase fused to a reverse transcriptase to copy a DNA sequence into a specific locus from a 'prime editing' guide RNA (pegRNA), eliminating the need for double-stranded DNA breaks and donor DNA templates. To facilitate prime editing in human induced pluripotent stem cells (iPSCs), we integrated a doxycycline-inducible Prime Editor protein (PE2) into the AAVS1 genomic safe harbor locus. Prime editing of iPSCs resulted in precise insertion of three nucleotides in HEK3 locus with high efficiency, demonstrating the utility of this approach. This engineered cell line can be used to edit a single or multiple genomic loci by introducing a target-specific pegRNA for precise and effective genome editing to facilitate disease modeling and functional genetics studies.

  View details for DOI 10.1016/j.scr.2021.102610

  View details for PubMedID 34875545

• Small-molecule probe reveals a kinase cascade that links stress signaling to TCF/LEF and Wnt responsiveness. Cell chemical biology Cheng, J. n., Tsuda, M. n., Okolotowicz, K. n., Dwyer, M. n., Bushway, P. J., Colas, A. R., Lancman, J. J., Schade, D. n., Perea-Gil, I. n., Bruyneel, A. A., Lee, J. n., Vadgama, N. n., Quach, J. n., McKeithan, W. L., Biechele, T. L., Wu, J. C., Moon, R. T., Si Dong, P. D., Karakikes, I. n., Cashman, J. R., Mercola, M. n. 2021

  Abstract

  Wnt signaling plays a central role in tissue maintenance and cancer. Wnt activates downstream genes through β-catenin, which interacts with TCF/LEF transcription factors. A major question is how this signaling is coordinated relative to tissue organization and renewal. We used a recently described class of small molecules that binds tubulin to reveal a molecular cascade linking stress signaling through ATM, HIPK2, and p53 to the regulation of TCF/LEF transcriptional activity. These data suggest a mechanism by which mitotic and genotoxic stress can indirectly modulate Wnt responsiveness to exert coherent control over cell shape and renewal. These findings have implications for understanding tissue morphogenesis and small-molecule anticancer therapeutics.

  View details for DOI 10.1016/j.chembiol.2021.01.001

  View details for PubMedID 33503403

• Activation of CaMKII Signaling Pathway Contributes to the Pathogenesis of Genetic Hypertrophic Cardiomyopathy Gil, I., Bellbachir, N., Gavidia, A. A., Arthur, J., Zhang, Y., Vadgama, N., Oikonomopoulos, A., Roura, S., Wu, J. C., Bayes-Genis, A., Karakikes, I. LIPPINCOTT WILLIAMS & WILKINS. 2020

  View details for DOI 10.1161/res.127.suppl_1.274

  View details for Web of Science ID 000606541500059

• Disruption of the Genome Architecture at the PRRX1 Locus is Associated With the Pathogenesis of LMNA-related Dilated Cardiomyopathy Zhang, Y., Ameen, M., Gil, I., Arthur, J., Gavidia, A. A., Bharucha, N., Wang, K. C., Karakikes, I. LIPPINCOTT WILLIAMS & WILKINS. 2020

  View details for DOI 10.1161/res.127.suppl_1.MP175Circulation

  View details for Web of Science ID 000606541500326

• Molecular Signatures of Beneficial Class Effects of Statins on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes. Circulation Tian, L., Oikonomopoulos, A., Liu, C., Kitani, T., Shrestha, R., Chen, C. L., Ong, S., Smeets, M., Karakikes, I., Sayed, N., Wu, J. C. 2020; 141 (14): 1208–10

  View details for DOI 10.1161/CIRCULATIONAHA.118.035906

  View details for PubMedID 32250699

• iPSC Modeling of RBM20-Deficient DCM Identifies Upregulation of RBM20 as a Therapeutic Strategy. Cell reports Briganti, F. n., Sun, H. n., Wei, W. n., Wu, J. n., Zhu, C. n., Liss, M. n., Karakikes, I. n., Rego, S. n., Cipriano, A. n., Snyder, M. n., Meder, B. n., Xu, Z. n., Millat, G. n., Gotthardt, M. n., Mercola, M. n., Steinmetz, L. M. 2020; 32 (10): 108117

  Abstract

  Recent advances in induced pluripotent stem cell (iPSC) technology and directed differentiation of iPSCs into cardiomyocytes (iPSC-CMs) make it possible to model genetic heart disease in vitro. We apply CRISPR/Cas9 genome editing technology to introduce three RBM20 mutations in iPSCs and differentiate them into iPSC-CMs to establish an in vitro model of RBM20 mutant dilated cardiomyopathy (DCM). In iPSC-CMs harboring a known causal RBM20 variant, the splicing of RBM20 target genes, calcium handling, and contractility are impaired consistent with the disease manifestation in patients. A variant (Pro633Leu) identified by exome sequencing of patient genomes displays the same disease phenotypes, thus establishing this variant as disease causing. We find that all-trans retinoic acid upregulates RBM20 expression and reverts the splicing, calcium handling, and contractility defects in iPSC-CMs with different causal RBM20 mutations. These results suggest that pharmacological upregulation of RBM20 expression is a promising therapeutic strategy for DCM patients with a heterozygous mutation in RBM20.

  View details for DOI 10.1016/j.celrep.2020.108117

  View details for PubMedID 32905764

• Endogenous Retrovirus-Derived lncRNA BANCR Promotes Cardiomyocyte Migration in Humans and Non-human Primates. Developmental cell Wilson, K. D., Ameen, M. n., Guo, H. n., Abilez, O. J., Tian, L. n., Mumbach, M. R., Diecke, S. n., Qin, X. n., Liu, Y. n., Yang, H. n., Ma, N. n., Gaddam, S. n., Cunningham, N. J., Gu, M. n., Neofytou, E. n., Prado, M. n., Hildebrandt, T. B., Karakikes, I. n., Chang, H. Y., Wu, J. C. 2020

  Abstract

  Transposable elements (TEs) comprise nearly half of the human genome and are often transcribed or exhibit cis-regulatory properties with unknown function in specific processes such as heart development. In the case of endogenous retroviruses (ERVs), a TE subclass, experimental interrogation is constrained as many are primate-specific or human-specific. Here, we use primate pluripotent stem-cell-derived cardiomyocytes that mimic fetal cardiomyocytes in vitro to discover hundreds of ERV transcripts from the primate-specific MER41 family, some of which are regulated by the cardiogenic transcription factor TBX5. The most significant of these are located within BANCR, a long non-coding RNA (lncRNA) exclusively expressed in primate fetal cardiomyocytes. Functional studies reveal that BANCR promotes cardiomyocyte migration in vitro and ventricular enlargement in vivo. We conclude that recently evolved TE loci such as BANCR may represent potent de novo developmental regulatory elements that can be interrogated with species-matching pluripotent stem cell models.

  View details for DOI 10.1016/j.devcel.2020.07.006

  View details for PubMedID 32763147

• Concise Review: Precision Matchmaking: Induced Pluripotent Stem Cells Meet Cardio-Oncology STEM CELLS TRANSLATIONAL MEDICINE Nair, P., Prado, M., Perea-Gil, I., Karakikes, I. 2019; 8 (8): 758–67

  View details for DOI 10.1002/sctm.18-0279

  View details for Web of Science ID 000476835600004

• Treatment of volumetric muscle loss in mice using nanofibrillar scaffolds enhances vascular organization and integration COMMUNICATIONS BIOLOGY Nakayama, K. H., Quarta, M., Paine, P., Alcazar, C., Karakikes, I., Garcia, V., Abilez, O. J., Calvo, N. S., Simmons, C. S., Rando, T. A., Huang, N. F. 2019; 2

  View details for DOI 10.1038/s42003-019-0416-4

  View details for Web of Science ID 000467214900002

• Treatment of volumetric muscle loss in mice using nanofibrillar scaffolds enhances vascular organization and integration. Communications biology Nakayama, K. H., Quarta, M., Paine, P., Alcazar, C., Karakikes, I., Garcia, V., Abilez, O. J., Calvo, N. S., Simmons, C. S., Rando, T. A., Huang, N. F. 2019; 2: 170

  Abstract

  Traumatic skeletal muscle injuries cause irreversible tissue damage and impaired revascularization. Engineered muscle is promising for enhancing tissue revascularization and regeneration in injured muscle. Here we fabricated engineered skeletal muscle composed of myotubes interspersed with vascular endothelial cells using spatially patterned scaffolds that induce aligned cellular organization, and then assessed their therapeutic benefit for treatment of murine volumetric muscle loss. Murine skeletal myoblasts co-cultured with endothelial cells in aligned nanofibrillar scaffolds form endothelialized and aligned muscle with longer myotubes, more synchronized contractility, and more abundant secretion of angiogenic cytokines, compared to endothelialized engineered muscle formed from randomly-oriented scaffolds. Treatment of traumatically injured muscle with endothelialized and aligned skeletal muscle promotes the formation of highly organized myofibers and microvasculature, along with greater vascular perfusion, compared to treatment of muscle derived from randomly-oriented scaffolds. This work demonstrates the potential of endothelialized and aligned engineered skeletal muscle to promote vascular regeneration following transplantation.

  View details for DOI 10.1038/s42003-019-0416-4

  View details for PubMedID 31098403

  View details for PubMedCentralID PMC6505043

• Concise Review: Precision Matchmaking: Induced Pluripotent Stem Cells Meet Cardio-Oncology. Stem cells translational medicine Nair, P., Prado, M., Perea-Gil, I., Karakikes, I. 2019

  Abstract

  As common chemotherapeutic agents are associated with an increased risk of acute and chronic cardiovascular complications, a new clinical discipline, cardio-oncology, has recently emerged. At the same time, the development of preclinical human stem cell-derived cardiovascular models holds promise as a more faithful platform to predict the cardiovascular toxicity of common cancer therapies and advance our understanding of the underlying mechanisms contributing to the cardiotoxicity. In this article, we review the recent advances in preclinical cancer-related cardiotoxicity testing, focusing on new technologies, such as human induced pluripotent stem cell-derived cardiomyocytes and tissue engineering. We further discuss some of the limitations of these technologies and present future directions. Stem Cells Translational Medicine 2019.

  View details for PubMedID 31020786

• Treatment of volumetric muscle loss in mice using nanofibrillar scaffolds enhances vascular organization and integration. Communications biology Nakayama, K. H., Quarta, M. n., Paine, P. n., Alcazar, C. n., Karakikes, I. n., Garcia, V. n., Abilez, O. J., Calvo, N. S., Simmons, C. S., Rando, T. A., Huang, N. F. 2019; 2 (1): 170

  Abstract

  Traumatic skeletal muscle injuries cause irreversible tissue damage and impaired revascularization. Engineered muscle is promising for enhancing tissue revascularization and regeneration in injured muscle. Here we fabricated engineered skeletal muscle composed of myotubes interspersed with vascular endothelial cells using spatially patterned scaffolds that induce aligned cellular organization, and then assessed their therapeutic benefit for treatment of murine volumetric muscle loss. Murine skeletal myoblasts co-cultured with endothelial cells in aligned nanofibrillar scaffolds form endothelialized and aligned muscle with longer myotubes, more synchronized contractility, and more abundant secretion of angiogenic cytokines, compared to endothelialized engineered muscle formed from randomly-oriented scaffolds. Treatment of traumatically injured muscle with endothelialized and aligned skeletal muscle promotes the formation of highly organized myofibers and microvasculature, along with greater vascular perfusion, compared to treatment of muscle derived from randomly-oriented scaffolds. This work demonstrates the potential of endothelialized and aligned engineered skeletal muscle to promote vascular regeneration following transplantation.

  View details for DOI 10.1038/s42003-019-0416-4

  View details for PubMedID 31924993

• AlleleProfileR: A versatile tool to identify and profile sequence variants in edited genomes. PloS one Bruyneel, A. A., Colas, A. R., Karakikes, I., Mercola, M. 2019; 14 (12): e0226694

  Abstract

  Gene editing strategies, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9), are revolutionizing biology. However, quantitative and sensitive detection of targeted mutations are required to evaluate and quantify the genome editing outcomes. Here we present AlleleProfileR, a new analysis tool, written in a combination of R and C++, with the ability to batch process the sequence analysis of large and complex genome editing experiments, including the recently developed base editing technologies.

  View details for DOI 10.1371/journal.pone.0226694

  View details for PubMedID 31877162

• High-Throughput Phenotypic Screening Using Induced Pluripotent Stem Cell Derived Cardiomyocytes Identifies Compounds That Rescue Genetic Dilated Cardiomyopathy Perea-Gil, I., Prado, M., Bruyneel, A. A., McKeithan, W. L., Feyen, D. A., Nair, P., Mercola, M., Karakikes, I. LIPPINCOTT WILLIAMS & WILKINS. 2018: E72

  View details for Web of Science ID 000454198400026

• Mechanosensitive miR-376c Modulates Arrhythmia Susceptibility Via Regulation Of KCNJ2 In hiPSC-derived Cardiomyocytes Wahlquist, C. A., Rojas-Munoz, A., Brunyeel, A. A., Greenhaw, M., Chung, R., Vu, M., Karakikes, I., Mercola, M. LIPPINCOTT WILLIAMS & WILKINS. 2018: E79

  View details for Web of Science ID 000454198400051

• Telomere shortening is a hallmark of genetic cardiomyopathies. Proceedings of the National Academy of Sciences of the United States of America Chang, A. C., Chang, A. C., Kirillova, A., Sasagawa, K., Su, W., Weber, G., Lin, J., Termglinchan, V., Karakikes, I., Seeger, T., Dainis, A. M., Hinson, J. T., Seidman, J., Seidman, C. E., Day, J. W., Ashley, E., Wu, J. C., Blau, H. M. 2018

  Abstract

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

  View details for PubMedID 30150400

• Recent Progress in Genome Editing Approaches for Inherited Cardiovascular Diseases. Current cardiology reports Kaur, B., Perea-Gil, I., Karakikes, I. 2018; 20 (7): 58

  Abstract

  PURPOSE OF REVIEW: This review describes the recent progress in nuclease-based therapeutic applications for inherited heart diseases in vitro, highlights the development of the most recent genome editing technologies and discusses the associated challenges for clinical translation.RECENT FINDINGS: Inherited cardiovascular disorders are passed from generation to generation. Over the past decade, considerable progress has been made in understanding the genetic basis of inherited heart diseases. The timely emergence of genome editing technologies using engineered programmable nucleases has revolutionized the basic research of inherited cardiovascular diseases and holds great promise for the development of targeted therapies. The genome editing toolbox is rapidly expanding, and new tools have been recently added that significantly expand the capabilities of engineered nucleases. Newer classes of versatile engineered nucleases, such as the "base editors," have been recently developed, offering the potential for efficient and precise therapeutic manipulation of the human genome.

  View details for PubMedID 29860642

• SETD7 Drives Cardiac Lineage Commitment through Stage-Specific Transcriptional Activation. Cell stem cell Lee, J. n., Shao, N. Y., Paik, D. T., Wu, H. n., Guo, H. n., Termglinchan, V. n., Churko, J. M., Kim, Y. n., Kitani, T. n., Zhao, M. T., Zhang, Y. n., Wilson, K. D., Karakikes, I. n., Snyder, M. P., Wu, J. C. 2018; 22 (3): 428–44.e5

  Abstract

  Cardiac development requires coordinated and large-scale rearrangements of the epigenome. The roles and precise mechanisms through which specific epigenetic modifying enzymes control cardiac lineage specification, however, remain unclear. Here we show that the H3K4 methyltransferase SETD7 controls cardiac differentiation by reading H3K36 marks independently of its enzymatic activity. Through chromatin immunoprecipitation sequencing (ChIP-seq), we found that SETD7 targets distinct sets of genes to drive their stage-specific expression during cardiomyocyte differentiation. SETD7 associates with different co-factors at these stages, including SWI/SNF chromatin-remodeling factors during mesodermal formation and the transcription factor NKX2.5 in cardiac progenitors to drive their differentiation. Further analyses revealed that SETD7 binds methylated H3K36 in the bodies of its target genes to facilitate RNA polymerase II (Pol II)-dependent transcription. Moreover, abnormal SETD7 expression impairs functional attributes of terminally differentiated cardiomyocytes. Together, these results reveal how SETD7 acts at sequential steps in cardiac lineage commitment, and they provide insights into crosstalk between dynamic epigenetic marks and chromatin-modifying enzymes.

  View details for PubMedID 29499155

• Patient-Specific iPSC-Derived Endothelial Cells Uncover Pathways that Protect against Pulmonary Hypertension in BMPR2 Mutation Carriers CELL STEM CELL Gu, M., Shao, N., Sa, S., Li, D., Termglinchan, V., Ameen, M., Karakikes, I., Sosa, G., Grubert, F., Lee, J., Cao, A., Taylor, S., Ma, Y., Zhao, Z., Chappell, J., Hamid, R., Austin, E. D., Gold, J. D., Wu, J. C., Snyder, M. P., Rabinovitch, M. 2017; 20 (4): 490-?

  View details for DOI 10.1016/j.stem.2016.08.019

  View details for Web of Science ID 000398350800013

• Mending a Broken Heart: The Evolution Of Biological Therapeutics. Stem cells Chen, C., Termglinchan, V., Karakikes, I. 2017

  Abstract

  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 ELIFE Stillitano, F., Hansen, J., Kong, C., Karakikes, I., Faunck-Brentano, C., Geng, L., Scott, S., Reynier, S., Wu, M., Valogne, Y., Desseaux, C., Salem, J., Jeziorowska, D., Zahr, N., Li, R., Iyengar, R., Hajjar, R. J., Hulot, J. 2017; 6

  Abstract

  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.) Stillitano, F., Karakikes, I., Hajjar, R. J. 2017; 1521: 183-193

  Abstract

  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

• Efficient Genome Editing in Induced Pluripotent Stem Cells with Engineered Nucleases In Vitro CARDIAC GENE THERAPY: METHODS AND PROTOCOLS Termglinchan, V., Seeger, T., Chen, C., Wu, J. C., Karakikes, I., Ishikawa, K. 2017; 1521: 55–68

  View details for DOI 10.1007/978-1-4939-6588-5_4

  View details for Web of Science ID 000419725000005

• Molecular and functional resemblance of differentiated cells derived from isogenic human iPSCs and SCNT-derived ESCs. Proceedings of the National Academy of Sciences of the United States of America Zhao, M. T., Chen, H. n., Liu, Q. n., Shao, N. Y., Sayed, N. n., Wo, H. T., Zhang, J. Z., Ong, S. G., Liu, C. n., Kim, Y. n., Yang, H. n., Chour, T. n., Ma, H. n., Gutierrez, N. M., Karakikes, I. n., Mitalipov, S. n., Snyder, M. P., Wu, J. C. 2017

  Abstract

  Patient-specific pluripotent stem cells (PSCs) can be generated via nuclear reprogramming by transcription factors (i.e., induced pluripotent stem cells, iPSCs) or by somatic cell nuclear transfer (SCNT). However, abnormalities and preclinical application of differentiated cells generated by different reprogramming mechanisms have yet to be evaluated. Here we investigated the molecular and functional features, and drug response of cardiomyocytes (PSC-CMs) and endothelial cells (PSC-ECs) derived from genetically relevant sets of human iPSCs, SCNT-derived embryonic stem cells (nt-ESCs), as well as in vitro fertilization embryo-derived ESCs (IVF-ESCs). We found that differentiated cells derived from isogenic iPSCs and nt-ESCs showed comparable lineage gene expression, cellular heterogeneity, physiological properties, and metabolic functions. Genome-wide transcriptome and DNA methylome analysis indicated that iPSC derivatives (iPSC-CMs and iPSC-ECs) were more similar to isogenic nt-ESC counterparts than those derived from IVF-ESCs. Although iPSCs and nt-ESCs shared the same nuclear DNA and yet carried different sources of mitochondrial DNA, CMs derived from iPSC and nt-ESCs could both recapitulate doxorubicin-induced cardiotoxicity and exhibited insignificant differences on reactive oxygen species generation in response to stress condition. We conclude that molecular and functional characteristics of differentiated cells from human PSCs are primarily attributed to the genetic compositions rather than the reprogramming mechanisms (SCNT vs. iPSCs). Therefore, human iPSCs can replace nt-ESCs as alternatives for generating patient-specific differentiated cells for disease modeling and preclinical drug testing.

  View details for PubMedID 29203658

• Passive Stretch Induces Structural and Functional Maturation of Engineered Heart Muscle as Predicted by Computational Modeling. Stem cells (Dayton, Ohio) Abilez, O. J., Tzatzalos, E. n., Yang, H. n., Zhao, M. T., Jung, G. n., Zöllner, A. M., Tiburcy, M. n., Riegler, J. n., Matsa, E. n., Shukla, P. n., Zhuge, Y. n., Chour, T. n., Chen, V. C., Burridge, P. W., Karakikes, I. n., Kuhl, E. n., Bernstein, D. n., Couture, L. A., Gold, J. D., Zimmermann, W. H., Wu, J. C. 2017

  Abstract

  The ability to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes (CMs) makes them an attractive source for repairing injured myocardium, disease modeling, and drug testing. Although current differentiation protocols yield hPSC-CMs to >90% efficiency, hPSC-CMs exhibit immature characteristics. With the goal of overcoming this limitation, we tested the effects of varying passive stretch on engineered heart muscle (EHM) structural and functional maturation, guided by computational modeling.Human embryonic stem cells (hESCs, H7 line) or human induced pluripotent stem cells (hiPSCs, IMR-90 line) were differentiated to human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) in vitro using a small molecule based protocol. hPSC-CMs were characterized by troponin(+) flow cytometry as well as electrophysiological measurements. Afterwards, 1.2 x 10(6) hPSC-CMs were mixed with 0.4 x 10(6) human fibroblasts (IMR-90 line) (3:1 ratio) and Type-I collagen. The blend was cast into custom-made 12-mm long polydimethylsiloxane (PDMS) reservoirs to vary nominal passive stretch of EHMs to 5, 7, or 9 mm. EHM characteristics were monitored for up to 50 days, with EHMs having a passive stretch of 7 mm giving the most consistent formation. Based on our initial macroscopic observations of EHM formation, we created a computational model that predicts the stress distribution throughout EHMs, which is a function of cellular composition, cellular ratio, and geometry. Based on this predictive modeling, we show cell alignment by immunohistochemistry and coordinated calcium waves by calcium imaging. Furthermore, coordinated calcium waves and mechanical contractions were apparent throughout entire EHMs. The stiffness and active forces of hPSC-derived EHMs are comparable to rat neonatal cardiomyocyte-derived EHMs. Three-dimensional EHMs display increased expression of mature cardiomyocyte genes including sarcomeric protein troponin-T, calcium and potassium ion channels, β-adrenergic receptors, and t-tubule protein caveolin-3.Passive stretch affects the structural and functional maturation of EHMs. Based on our predictive computational modeling, we show how to optimize cell alignment and calcium dynamics within EHMs. These findings provide a basis for the rational design of EHMs, which enables future scale-up productions for clinical use in cardiovascular tissue engineering. This article is protected by copyright. All rights reserved.

  View details for PubMedID 29086457

• Patient-Specific iPSC-Derived Endothelial Cells Uncover Pathways that Protect against Pulmonary Hypertension in BMPR2 Mutation Carriers. Cell stem cell Gu, M., Shao, N., Sa, S., Li, D., Termglinchan, V., Ameen, M., Karakikes, I., Sosa, G., Grubert, F., Lee, J., Cao, A., Taylor, S., Ma, Y., Zhao, Z., Chappell, J., Hamid, R., Austin, E. D., Gold, J. D., Wu, J. C., Snyder, M. P., Rabinovitch, M. 2016

  Abstract

  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 Kodo, K., Ong, S., Jahanbani, F., Termglinchan, V., Hirono, K., Inanloorahatloo, K., Ebert, A. D., Shukla, P., Abilez, O. J., Churko, J. M., Karakikes, I., Jung, G., Ichida, F., Wu, S. M., Snyder, M. P., Bernstein, D., Wu, J. C. 2016; 18 (10): 1031-1042

  Abstract

  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 Matsa, E., Burridge, P. W., Yu, K., Ahrens, J. H., Termglinchan, V., Wu, H., Liu, C., Shukla, P., Sayed, N., Churko, J. M., Shao, N., Woo, N. A., Chao, A. S., Gold, J. D., Karakikes, I., Snyder, M. P., Wu, J. C. 2016; 19 (3): 311-325

  Abstract

  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 Stillitano, F., Turnbull, I. C., Karakikes, I., Nonnenmacher, M., Backeris, P., Hulot, J., Kranias, E. G., Hajjar, R. J., Costa, K. D. 2016

  Abstract

  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 Kuznetsova, T., Haddad, F., Knez, J., Rosenberg-Hasson, Y., Sung, J., Cauwenberghs, N., Thijs, L., Karakikes, I., Maecker, H., Mahaffey, K. W., Wu, J. C., Staessen, J. A. 2015; 9 (12): 975-984 e3

  Abstract

  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 Kuznetsova, T., Haddad, F., Knez, J., Rosenberg-Hasson, Y., Sung, J., Cauwenberghs, N., Thijs, L., Karakikes, I., Maecker, H., Mahaffey, K. W., Wu, J. C., Staessen, J. A. 2015; 9 (12): 975-984

  Abstract

  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 Web of Science ID 000367214500014

  View details for PubMedID 26565110

• A Rapid, High-Quality, Cost-Effective, Comprehensive and Expandable Targeted Next-Generation Sequencing Assay for Inherited Heart Diseases. Circulation research Wilson, K. D., Shen, P., Fung, E., Karakikes, I., Zhang, A., Inanloorahatloo, K., Odegaard, J., Sallam, K., Davis, R. W., Lui, G. K., Ashley, E. A., Scharfe, C., Wu, J. C. 2015; 117 (7): 603-611

  Abstract

  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

• 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 Chen, G., Li, S., Karakikes, I., Ren, L., Chow, M. Z., Chopra, A., Keung, W., Yan, B., Chan, C. W., Costa, K. D., Kong, C., Hajjar, R. J., Chen, C. S., Li, R. A. 2015; 8 (1): 193-U276

  Abstract

  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 Rapti, K., Stillitano, F., Karakikes, I., Nonnenmacher, M., Weber, T., Hulot, J., Hajjar, R. J. 2015; 2: 14067-?

  Abstract

  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 MOLECULAR THERAPY Ishikawa, K., Fish, K. M., Tilemann, L., Rapti, K., Aguero, J., Santos-Gallego, C. G., Lee, A., Karakikes, L., Xie, C., Akar, F. G., Shimada, Y. J., Gwathmey, J. K., Asokan, A., McPhee, S., Samulski, J., Samulski, R. J., Sigg, D. C., Weber, T., Kranias, E. G., Hajjar, R. J. 2014; 22 (12): 2038-2045

  Abstract

  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 Karakikes, I., Termglinchan, V., Wu, J. C. 2014; 29 (3): 214-219

  Abstract

  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

• Small Molecule-Mediated Directed Differentiation of Human Embryonic Stem Cells Toward Ventricular Cardiomyocytes STEM CELLS TRANSLATIONAL MEDICINE Karakikes, I., Senyei, G. D., Hansen, J., Kong, C., Azeloglu, E. U., Stillitano, F., Lieu, D. K., Wang, J., Ren, L., Hulot, J., Iyengar, R., Li, R. A., Hajjar, R. J. 2014; 3 (1): 18-31

  Abstract

  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

• Rapid and Efficient Conversion of Integration-Free Human Induced Pluripotent Stem Cells to GMP-Grade Culture Conditions. PloS one Durruthy-Durruthy, J., Briggs, S. F., Awe, J., Ramathal, C. Y., Karumbayaram, S., Lee, P. C., Heidmann, J. D., Clark, A., Karakikes, I., Loh, K. M., Wu, J. C., Hoffman, A. R., Byrne, J., Reijo Pera, R. A., Sebastiano, V. 2014; 9 (4)

  View details for DOI 10.1371/journal.pone.0094231

  View details for PubMedID 24718618

• Rapid and efficient conversion of integration-free human induced pluripotent stem cells to GMP-grade culture conditions. PloS one Durruthy-Durruthy, J., Briggs, S. F., Awe, J., Ramathal, C. Y., Karumbayaram, S., Lee, P. C., Heidmann, J. D., Clark, A., Karakikes, I., Loh, K. M., Wu, J. C., Hoffman, A. R., Byrne, J., Reijo Pera, R. A., Sebastiano, V. 2014; 9 (4)

  Abstract

  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

• 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 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. 2013

  Abstract

  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

• Therapeutic cardiac-targeted delivery of miR-1 reverses pressure overload-induced cardiac hypertrophy and attenuates pathological remodeling. Journal of the American Heart Association Karakikes, I., Chaanine, A. H., Kang, S., Mukete, B. N., Jeong, D., Zhang, S., Hajjar, R. J., Lebeche, D. 2013; 2 (2)

  Abstract

  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 Fish, K. M., Ladage, D., Kawase, Y., Karakikes, I., Jeong, D., Ly, H., Ishikawa, K., Hadri, L., Tilemann, L., Muller-Ehmsen, J., Samulski, R. J., Kranias, E. G., Hajjar, R. J. 2013; 6 (2): 310-317

  Abstract

  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

• Interaction of HLA-DR and CD74 at the cell surface of antigen-presenting cells by single particle image analysis FASEB JOURNAL Karakikes, I., Morrison, I. E., O'Toole, P., Metodieva, G., Navarrete, C. V., Gomez, J., Miranda-Sayago, J. M., Cherry, R. J., Metodiev, M., Fernandez, N. 2012; 26 (12): 4886-4896

  Abstract

  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 MOLECULAR THERAPY Karakikes, I., Hadri, L., Rapti, K., Ladage, D., Ishikawa, K., Tilemann, L., Yi, G., Morel, C., Gwathmey, J. K., Zsebo, K., Weber, T., Kawase, Y., Hajjar, R. J. 2012; 20 (3): 565-571

  Abstract

  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 CIRCULATION RESEARCH Kara, R. J., Bolli, P., Karakikes, I., Matsunaga, I., Tripodi, J., Tanweer, O., Altman, P., Shachter, N. S., Nakano, A., Najfeld, V., Chaudhry, H. W. 2012; 110 (1): 82-93

  Abstract

  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 ADVANCED MATERIALS Chen, A., Lieu, D. K., Freschauf, L., Lew, V., Sharma, H., Wang, J., Diep Nguyen, D., Karakikes, I., Hajjar, R. J., Gopinathan, A., Botvinick, E., Fowlkes, C. C., Li, R. A., Khine, M. 2011; 23 (48): 5785-?

  Abstract

  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 GENE THERAPY Ladage, D., Turnbull, I. C., Ishikawa, K., Takewa, Y., Rapti, K., Morel, C., Karakikes, I., Hadri, L., Mueller-Ehmsen, J., Costa, K. D., Hajjar, R. J., Kawase, Y. 2011; 18 (10): 979-985

  Abstract

  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 Borah, J. C., Mujtaba, S., Karakikes, I., Zeng, L., Muller, M., Patel, J., Moshkina, N., Morohashi, K., Zhang, W., Gerona-Navarro, G., Hajjar, R. J., Zhou, M. 2011; 18 (4): 531-541

  Abstract

  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 CIRCULATION-HEART FAILURE Beeri, R., Chaput, M., Guerrero, J. L., Kawase, Y., Yosefy, C., Abedat, S., Karakikes, I., Morel, C., Tisosky, A., Sullivan, S., Handschumacher, M. D., Gilon, D., Vlahakes, G. J., Hajjar, R. J., Levine, R. A. 2010; 3 (5): 627-634

  Abstract

  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 Jin, H., Hadri, L., Palomeque, J., Morel, C., Karakikes, I., Kaprielian, R., Hajjar, R., Lebeche, D. 2010; 48 (6): 1169-1179

  Abstract

  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 CELLULAR REPROGRAMMING Galende, E., Karakikes, I., Edelmann, L., Desnick, R. J., Kerenyi, T., Khoueiry, G., Lafferty, J., McGinn, J. T., Brodman, M., Fuster, V., Hajjar, R. J., Polgar, K. 2010; 12 (2): 117-125

  Abstract

  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 PLOS ONE Karakikes, I., Kim, M., Hadri, L., Sakata, S., Sun, Y., Zhang, W., Chemaly, E. R., Hajjar, R. J., Lebeche, D. 2009; 4 (7)

  Abstract

  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