Karim Sallam, MD, is trained in Cardiovascular Medicine and Advanced Heart Failure.
- Cardiovascular Medicine
- Advanced Heart Failure
- Cardiovascular Disease
Honors & Awards
Highest Performance in Medicine, James D. Heard Junior Prize (2006)
Compassion and Humanism, Jeffrey Alan Grey Memorial Prize (2006)
Empathetic, Sensitive and Respectful Behavior, Leonard Tow Humanism in Medicine Student Award (2006)
Exemplary Professionalism Award, Stanford Medicine Residency Program (2007/2008)
Fellowship: Stanford University Pain Management Fellowship (2015) CA
Board Certification: American Board of Internal Medicine, Advanced Heart Failure and Transplant Cardiology (2016)
Board Certification: American Board of Internal Medicine, Cardiovascular Disease (2013)
Fellowship: Stanford Hospital and Clinics (2015) CA
Residency: Stanford University Medical Center (2013) CA
Board Certification: American Board of Internal Medicine, Internal Medicine (2009)
Medical Education: University of Pittsburgh Medical Center (2006) PA
Generation of two induced pluripotent stem cell lines carrying the phospholamban R14del mutation for modeling ARVD/C.
Stem cell research
2022; 63: 102834
The phospholamban (PLN) R14del mutation is associated with arrhythmogenic right ventricular dysplasia (ARVD/C). ARVD/C is a cardiac disease characterized by arrhythmias and structural abnormalities in the right ventricle. Because PLN is a regulator of calcium release, this mutation can have deleterious effects on tissue integrity and contraction. This mutation is a trinucleotide (AGA) deletion that leads to an arginine deletion at position 14 of the PLN structure. Here we show two lines carrying this mutation with typical iPSC morphology, pluripotency, karyotype, ability to differentiate into the three germ layers in vitro, and readily availability for studying pathological mechanisms or ARVD/C.
View details for DOI 10.1016/j.scr.2022.102834
View details for PubMedID 35700631
- Modeling Effects of Immunosuppressive Drugs on Human Hearts Using Induced Pluripotent Stem Cell-Derived Cardiac Organoids and Single-Cell RNA Sequencing. Circulation 2022; 145 (17): 1367-1369
Heterozygous LMNA mutation-carrying iPSC lines from three cardiac laminopathy patients.
Stem cell research
1800; 59: 102657
LMNA-related dilated cardiomyopathy (LMNA-DCM) is caused by pathogenic variants in the LMNA gene and is characterized by left ventricular chamber enlargement, reduced systolic function, and arrhythmia. Here, we generated three human induced pluripotent stem cell (iPSC) lines from peripheral blood mononuclear cells (PBMCs) of three DCM patients carrying the same single heterozygous mutation, c.398 G>A, in LMNA. All lines exhibited normal iPSC morphology, expressed high levels of pluripotency markers, showed normal karyotypes, and could differentiate into the three germ layers. These patient-specific iPSC lines can serve as invaluable tools to model in vitro pathological mechanisms of LMNA-DCM.
View details for DOI 10.1016/j.scr.2022.102657
View details for PubMedID 34999423
Generation of two induced pluripotent stem cell lines from Brugada syndrome affected patients carrying SCN5A mutations.
Stem cell research
2021; 57: 102605
SCN5A gene loss-of-function mutations are commonly associated with Brugada syndrome, which represents a risk of lethal arrhythmias and sudden cardiac death. The present report describes the generation of two human induced pluripotent stem cell (iPSC) lines reprogrammed from two Brugada syndrome affected patients carrying SCN5A mutations, c.53506 G>A and c.2102 C>T, respectively. Pluripotency markers, karyotype stability, and differentiation capability into derivatives of the three germ layers were assessed and described in the present report. These lines can be used as a reliable cell model for Brugada syndrome investigations and characterization of leading cellular mechanisms.
View details for DOI 10.1016/j.scr.2021.102605
View details for PubMedID 34856468
Coronary Artery Vasospasm Requiring Cardiac Autotransplantation Yet Controlled With Tobacco.
JACC. Case reports
2021; 3 (9): 1177-1181
Coronary artery vasospasm is typically managed through avoidance of triggers and with symptomatic treatments with calcium channel blockers and long-acting nitrates. Here, we report a rare case of medically refractory coronary artery vasospasm associated with genetic predispositions that initially required cardiac autotransplantation followed paradoxically by nicotine for long-term symptomatic control. (Level of Difficulty: Intermediate.).
View details for DOI 10.1016/j.jaccas.2021.03.018
View details for PubMedID 34401754
Generation of three induced pluripotent stem cell lines from hypertrophic cardiomyopathy patients carrying MYH7 mutations.
Stem cell research
2021; 55: 102455
MYH7 heterozygous mutations are common genetic causes of hypertrophic cardiomyopathy (HCM). HCM is characterized by hypertrophy of the left ventricle and diastolic dysfunction. We generated three human induced pluripotent stem cell (iPSC) lines from three HCM patients each carrying a single heterozygous mutation in MYH7, c.2167C>T, c.4066G>A, and c.5135G>A, respectively. All lines expressed high levels of pluripotent markers, had normal karyotype, and possessed capability of differentiation into derivatives of the three germ layers, which can serve as valuable tools for modeling HCM in vitro and investigating the pathological mechanisms related to MYH7 mutations.
View details for DOI 10.1016/j.scr.2021.102455
View details for PubMedID 34352619
A protocol for transdifferentiation of human cardiac fibroblasts into endothelial cells via activation of innate immunity.
2021; 2 (2): 100556
Endothelial cells (ECs) have emerged as key pathogenic players in cardiac disease due to their proximity with cardiomyocytes. Induced pluripotent stem cells (iPSCs) have been employed to generate ECs. However, it may be more clinically relevant to transdifferentiate fibroblasts into ECs directly without introducing pluripotent or virally driven transcription factors. Here, we present a protocol that describes the direct conversion of human cardiac fibroblasts into ECs by leveraging the innate immune system. Our protocol produces bona fide human ECs with 95%-98% purity by first passage. For complete details on the use and execution of this protocol, please refer to Liu etal. (2020) and Sayed etal. (2015).
View details for DOI 10.1016/j.xpro.2021.100556
View details for PubMedID 34151292
The Unfolded Protein Response as a Compensatory Mechanism and Potential Therapeutic Target in PLN R14del Cardiomyopathy.
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
Generation of three induced pluripotent stem cell lines, SCVIi003-A, SCVIi004-A, SCVIi005-A, from patients with ARVD/C caused by heterozygous mutations in the PKP2 gene.
Stem cell research
2021; 53: 102284
Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an inherited heart disease which can cause life-threatening ventricular arrhythmias and cardiac dysfunction. The autosomal dominant form of ARVD/C is caused by mutations in the cardiac desmosome, such as those in the plakoglobin plakophilin-2 (PKP2) gene. Here, we generated three human induced pluripotent stem cell (iPSC) lines from the peripheral blood mononuclear cells (PBMCs) of three ARVD/C patients carrying pathogenic variants in their PKP2 genes (c.2065_2070delinsG; c.235C>T; c.1725_1728dup). All lines show the typical morphology of pluripotent stem cells, demonstrate high expression of pluripotent markers, display normal karyotype, and differentiate into all three germ layers in vitro. These lines are valuable resources for studying the pathological mechanisms of ARVD/C caused by PKP2 mutation.
View details for DOI 10.1016/j.scr.2021.102284
View details for PubMedID 33743362
Sirolimus Adverse Event Profile in a Non-Clinical Trial Cohort of Heart Transplantation Patients.
Annals of transplantation
2021; 26: e923536
BACKGROUND Sirolimus has been used increasingly in heart transplantation for its ability to reduce acute rejection, prevent the progression of cardiac allograft vasculopathy (CAV), and preserve renal function. We sought to assess the adverse reactions associated with the use of sirolimus compared to mycophenolate mofetil (MMF). MATERIAL AND METHODS We retrospectively reviewed the charts of 221 adult heart transplant patients who received either sirolimus or MMF as part of their immunosuppression from June 1, 2001 to April 1, 2005. Patients were assigned to 2 groups based upon immunosuppression use. The prevalence and types of complications were recorded in each group. RESULTS Sirolimus was received by 109 patients and 112 patients received MMF during the study period. Seventy-seven patients (71%) in the sirolimus group experienced adverse reactions compared to 45 patients (40%) in the MMF group (P<0.01). Compared to MMF, the use of sirolimus was associated with a higher prevalence of elevated triglyceride levels, lower-extremity edema, and oral ulcerations. Sirolimus was discontinued due to adverse reactions in 22% of patients, whereas no patients in the MMF group experienced adverse effects requiring drug discontinuation. CONCLUSIONS Compared to MMF, sirolimus use is associated with a higher prevalence of adverse reactions requiring drug discontinuation, but most patients were able to stay on therapy despite adverse effects.
View details for DOI 10.12659/AOT.923536
View details for PubMedID 33462174
Clinical trial in a dish using iPSCs shows lovastatin improves endothelial dysfunction and cellular cross-talk in LMNA cardiomyopathy.
Science translational medicine
2020; 12 (554)
Mutations in LMNA, the gene that encodes lamin A and C, causes LMNA-related dilated cardiomyopathy (DCM) or cardiolaminopathy. LMNA is expressed in endothelial cells (ECs); however, little is known about the EC-specific phenotype of LMNA-related DCM. Here, we studied a family affected by DCM due to a frameshift variant in LMNA Human induced pluripotent stem cell (iPSC)-derived ECs were generated from patients with LMNA-related DCM and phenotypically characterized. Patients with LMNA-related DCM exhibited clinical endothelial dysfunction, and their iPSC-ECs showed decreased functionality as seen by impaired angiogenesis and nitric oxide (NO) production. Moreover, genome-edited isogenic iPSC lines recapitulated the EC disease phenotype in which LMNA-corrected iPSC-ECs showed restoration of EC function. Simultaneous profiling of chromatin accessibility and gene expression dynamics by combining assay for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA sequencing (RNA-seq) as well as loss-of-function studies identified Kruppel-like factor 2 (KLF2) as a potential transcription factor responsible for the EC dysfunction. Gain-of-function studies showed that treatment of LMNA iPSC-ECs with KLF2 agonists, including lovastatin, rescued the EC dysfunction. Patients with LMNA-related DCM treated with lovastatin showed improvements in clinical endothelial dysfunction as indicated by increased reactive hyperemia index. Furthermore, iPSC-derived cardiomyocytes (iPSC-CMs) from patients exhibiting the DCM phenotype showed improvement in CM function when cocultured with iPSC-ECs and lovastatin. These results suggest that impaired cross-talk between ECs and CMs can contribute to the pathogenesis of LMNA-related DCM, and statin may be an effective therapy for vascular dysfunction in patients with cardiolaminopathy.
View details for DOI 10.1126/scitranslmed.aax9276
View details for PubMedID 32727917
Modeling Secondary Iron Overload Cardiomyopathy with Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.
2020; 32 (2): 107886
Excessive iron accumulation in the heart causes iron overload cardiomyopathy (IOC), which initially presents as diastolic dysfunction and arrhythmia but progresses to systolic dysfunction and end-stage heart failure when left untreated. However, the mechanisms of iron-related cardiac injury and how iron accumulates in human cardiomyocytes are not well understood. Herein, using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we model IOC and screen for drugs to rescue the iron overload phenotypes. Human iPSC-CMs under excess iron exposure recapitulate early-stage IOC, including oxidative stress, arrhythmia, and contractile dysfunction. We find that iron-induced changes in calcium kinetics play a critical role in dysregulation of CM functions. We identify that ebselen, a selective divalent metal transporter 1 (DMT1) inhibitor and antioxidant, could prevent the observed iron overload phenotypes, supporting the role of DMT1 in iron uptake into the human myocardium. These results suggest that ebselen may be a potential preventive and therapeutic agent for treating patients with secondary iron overload.
View details for DOI 10.1016/j.celrep.2020.107886
View details for PubMedID 32668256
HIF1α Regulates Early Metabolic Changes due to Activation of Innate Immunity in Nuclear Reprogramming.
Stem cell reports
2020; 14 (2): 192–200
Innate immune signaling has recently been shown to play an important role in nuclear reprogramming, by altering the epigenetic landscape and thereby facilitating transcription. However, the mechanisms that link innate immune activation and metabolic regulation in pluripotent stem cells remain poorly defined, particularly with regard to key molecular components. In this study, we show that hypoxia-inducible factor 1α (HIF1α), a central regulator of adaptation to limiting oxygen tension, is an unexpected but crucial regulator of innate immune-mediated nuclear reprogramming. HIF1α is dramatically upregulated as a consequence of Toll-like receptor 3 (TLR3) signaling and is necessary for efficient induction of pluripotency and transdifferentiation. Bioenergetics studies reveal that HIF1α regulates the reconfiguration of innate immune-mediated reprogramming through its well-established role in throwing a glycolytic switch. We believe that results from these studies can help us better understand the influence of immune signaling in tissue regeneration and lead to new therapeutic strategies.
View details for DOI 10.1016/j.stemcr.2020.01.006
View details for PubMedID 32048999
Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model.
Journal of visualized experiments : JoVE
The growing number of victims of "stem cell tourism," the unregulated transplantation of stem cells worldwide, has raised concerns about the safety of stem cell transplantation. Although the transplantation of differentiated rather than undifferentiated cells is common practice, teratomas can still arise from the presence of residual undifferentiated stem cells at the time of transplant or from spontaneous mutations in differentiated cells. Because stem cell therapies are often delivered into anatomically sensitive sites, even small tumors can be clinically devastating, resulting in blindness, paralysis, cognitive abnormalities, and cardiovascular dysfunction. Surgical access to these sites may also be limited, leaving patients with few therapeutic options. Controlling stem cell misbehavior is, therefore, critical for the clinical translation of stem cell therapy. External beam radiation offers an effective means of delivering targeted therapy to decrease the teratoma burden while minimizing injury to surrounding organs. Additionally, this method avoids genetic manipulation or viral transduction of stem cells-which are associated with additional clinical safety and efficacy concerns. Here, we describe a protocol to create pluripotent stem cell-derived teratomas in mice and to apply external beam radiation therapy to selectively ablate these tumors in vivo.
View details for PubMedID 30829317
- Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model JOVE-JOURNAL OF VISUALIZED EXPERIMENTS 2019
- Electronic Cigarettes: Where There Is Smoke There Is Disease. Journal of the American College of Cardiology 2019; 74 (25): 3121–23
Modelling diastolic dysfunction in induced pluripotent stem cell-derived cardiomyocytes from hypertrophic cardiomyopathy patients.
European heart journal
Diastolic dysfunction (DD) is common among hypertrophic cardiomyopathy (HCM) patients, causing major morbidity and mortality. However, its cellular mechanisms are not fully understood, and presently there is no effective treatment. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) hold great potential for investigating the mechanisms underlying DD in HCM and as a platform for drug discovery.In the present study, beating iPSC-CMs were generated from healthy controls and HCM patients with DD. Micropatterned iPSC-CMs from HCM patients showed impaired diastolic function, as evidenced by prolonged relaxation time, decreased relaxation rate, and shortened diastolic sarcomere length. Ratiometric Ca2+ imaging indicated elevated diastolic [Ca2+]i and abnormal Ca2+ handling in HCM iPSC-CMs, which were exacerbated by β-adrenergic challenge. Combining Ca2+ imaging and traction force microscopy, we observed enhanced myofilament Ca2+ sensitivity (measured as dF/Δ[Ca2+]i) in HCM iPSC-CMs. These results were confirmed with genome-edited isogenic iPSC lines that carry HCM mutations, indicating that cytosolic diastolic Ca2+ overload, slowed [Ca2+]i recycling, and increased myofilament Ca2+ sensitivity, collectively impairing the relaxation of HCM iPSC-CMs. Treatment with partial blockade of Ca2+ or late Na+ current reset diastolic Ca2+ homeostasis, restored diastolic function, and improved long-term survival, suggesting that disturbed Ca2+ signalling is an important cellular pathological mechanism of DD. Further investigation showed increased expression of L-type Ca2+channel (LTCC) and transient receptor potential cation channels (TRPC) in HCM iPSC-CMs compared with control iPSC-CMs, which likely contributed to diastolic [Ca2+]i overload.In summary, this study recapitulated DD in HCM at the single-cell level, and revealed novel cellular mechanisms and potential therapeutic targets of DD using iPSC-CMs.
View details for DOI 10.1093/eurheartj/ehz326
View details for PubMedID 31219556
Activation of PDGF pathway links LMNA mutation to dilated cardiomyopathy.
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
The Incremental Value of Right Ventricular Size and Strain in the Risk Assessment of Right Heart Failure Post - Left Ventricular Assist Device Implantation.
Journal of cardiac failure
2018; 24 (12): 823–32
BACKGROUND: Right heart failure (RHF) after left ventricular assist device (LVAD) implantation is associated with high morbidity and mortality. Existing risk scores include semiquantitative evaluation of right ventricular (RV) dysfunction. This study aimed to determine whether quantitative evaluation of both RV size and function improve risk stratification for RHF after LVAD implantation beyond validated scores.METHODS AND RESULTS: From 2009 to 2015, 158 patients who underwent implantation of continuous-flow devices who had complete echocardiographic and hemodynamic data were included. Quantitative RV parameters included RV end-diastolic (RVEDAI) and end-systolic area index, RV free-wall longitudinal strain (RVLS), fractional area change, tricuspid annular plane systolic excursion, and right atrial area and pressure. Independent correlates of early RHF (<30 days) were determined with the use of logistic regression analysis. Mean age was 56 ± 13 years, with 79% male; 49% had INTERMACS profiles ≤2. RHF occurred in 60 patients (38%), with 20 (13%) requiring right ventricular assist device. On multivariate analysis, INTERMACS profiles (adjusted odds ratio 2.38 [95% confidence interval [CI] 1.47-3.85]), RVEDAI (1.61 [1.08-2.32]), and RVLS (2.72 [1.65-4.51]) were independent correlates of RHF (all P < .05). Both RVLS and RVEDAI were incremental to validated risk scores (including the EUROMACS score) for early RHF after LVAD (all P < .01).CONCLUSIONS: RV end-diastolic and strain are complementary prognostic markers of RHF after LVAD implantation.
View details for PubMedID 30539717
Genome Editing of Induced PluripotentStem Cells to Decipher CardiacChannelopathy Variant.
Journal of the American College of Cardiology
2018; 72 (1): 62–75
BACKGROUND: The long QT syndrome (LQTS) is an arrhythmogenic disorder of QT interval prolongation that predisposes patients to life-threatening ventricular arrhythmias such as Torsades de pointes and sudden cardiac death. Clinical genetic testing has emerged as the standard of care to identify genetic variants in patients suspected of having LQTS. However, these results are often confounded by the discovery of variants of uncertain significance (VUS), for which there is insufficient evidence of pathogenicity.OBJECTIVES: The purpose of this study was to demonstrate that genome editing of patient-specific induced pluripotent stem cells (iPSCs) can be a valuable approach to delineate the pathogenicity of VUS in cardiac channelopathy.METHODS: Peripheral blood mononuclear cells were isolated from a carrier with a novel missense variant (T983I) in the KCNH2 (LQT2) gene and an unrelated healthy control subject. iPSCs were generated using an integration-free Sendai virus and differentiated to iPSC-derived cardiomyocytes (CMs).RESULTS: Whole-cell patch clamp recordings revealed significant prolongation of the action potential duration (APD) and reduced rapidly activating delayed rectifier K+ current (IKr) density in VUS iPSC-CMs compared with healthy control iPSC-CMs. ICA-105574, a potent IKr activator, enhanced IKr magnitude and restored normal action potential duration in VUS iPSC-CMs. Notably, VUS iPSC-CMs exhibited greater propensity to proarrhythmia than healthy control cells in response to high-risk torsadogenic drugs (dofetilide, ibutilide, and azimilide), suggesting a compromised repolarization reserve. Finally, the selective correction of the causal variant in iPSC-CMs using CRISPR/Cas9 gene editing (isogenic control) normalized the aberrant cellular phenotype, whereas the introduction of the homozygous variant in healthy control cells recapitulated hallmark features of the LQTS disorder.CONCLUSIONS: The results suggest that the KCNH2T983I VUS may be classified as potentially pathogenic.
View details for PubMedID 29957233
- RIGHT HEART MALADAPTIVE PHENOTYPES AND PREDICTION OF RIGHT HEART FAILURE FOLLOWING CONTINUOUS-FLOW LEFT VENTRICULAR ASSIST DEVICE IMPLANTATION ELSEVIER SCIENCE INC. 2018: 652
- Transcriptomic and epigenomic differences in human induced pluripotent stem cells generated from six reprogramming methods NATURE BIOMEDICAL ENGINEERING 2017; 1 (10): 826–37
Transcriptomic and epigenomic differences in human induced pluripotent stem cells generated from six reprogramming methods.
Nature biomedical engineering
2017; 1 (10): 826-837
Many reprogramming methods can generate human induced pluripotent stem cells (hiPSCs) that closely resemble human embryonic stem cells (hESCs). This has led to assessments of how similar hiPSCs are to hESCs, by evaluating differences in gene expression, epigenetic marks and differentiation potential. However, all previous studies were performed using hiPSCs acquired from different laboratories, passage numbers, culturing conditions, genetic backgrounds and reprogramming methods, all of which may contribute to the reported differences. Here, by using high-throughput sequencing under standardized cell culturing conditions and passage number, we compare the epigenetic signatures (H3K4me3, H3K27me3 and HDAC2 ChIP-seq profiles) and transcriptome differences (by RNA-seq) of hiPSCs generated from the same primary fibroblast population by using six different reprogramming methods. We found that the reprogramming method impacts the resulting transcriptome and that all hiPSC lines could terminally differentiate, regardless of the reprogramming method. Moreover, by comparing the differences between the hiPSC and hESC lines, we observed a significant proportion of differentially expressed genes that could be attributed to polycomb repressive complex targets.
View details for DOI 10.1038/s41551-017-0141-6
View details for PubMedID 30263871
View details for PubMedCentralID PMC6155993
Simultaneous ramp right heart catheterization and echocardiography in a ReliantHeart left ventricular assist device.
World journal of cardiology
2017; 9 (1): 55-59
Many clinicians caring for patients with continuous flow left ventricular assist devices (CF-LVAD) use ramp right heart catheterization (RHC) studies to optimize pump speed and also to troubleshoot CF-LVAD malfunction. An investigational device, the ReliantHeart Heart Assist 5 (Houston, TX), provides the added benefit of an ultrasonic flow probe on the outflow graft that directly measures flow through the CF-LVAD. We performed a simultaneous ramp RHC and echocardiogram on a patient who received the above CF-LVAD to optimize pump parameters and investigate elevated flow through the CF-LVAD as measured by the flow probe. We found that the patient's hemodynamics were optimized at their baseline pump speed, and that the measured cardiac output via the Fick principle was lower than that measured by the flow probe. Right heart catheterization may be useful to investigate discrepancies between flow measured by a CF-LVAD and a patient's clinical presentation, particularly in investigational devices where little clinical experience exists. More data is needed to elucidate the correlation between the flow measured by an ultrasonic probe and cardiac output as measured by RHC.
View details for DOI 10.4330/wjc.v9.i1.55
View details for PubMedID 28163837
View details for PubMedCentralID PMC5253195
Patient-Specific Induced Pluripotent Stem Cell-Based Disease Model for Pathogenesis Studies and Clinical Pharmacotherapy.
Circulation. Arrhythmia and electrophysiology
2017; 10 (6)
View details for PubMedID 28630175
Patient-Specific and Genome-Edited Induced Pluripotent Stem Cell-Derived Cardiomyocytes Elucidate Single-Cell Phenotype of Brugada Syndrome.
Journal of the American College of Cardiology
2016; 68 (19): 2086-2096
Brugada syndrome (BrS), a disorder associated with characteristic electrocardiogram precordial ST-segment elevation, predisposes afflicted patients to ventricular fibrillation and sudden cardiac death. Despite marked achievements in outlining the organ level pathophysiology of the disorder, the understanding of human cellular phenotype has lagged due to a lack of adequate human cellular models of the disorder.The objective of this study was to examine single cell mechanism of Brugada syndrome using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).This study recruited 2 patients with type 1 BrS carrying 2 different sodium voltage-gated channel alpha subunit 5 variants as well as 2 healthy control subjects. We generated iPSCs from their skin fibroblasts by using integration-free Sendai virus. We used directed differentiation to create purified populations of iPSC-CMs.BrS iPSC-CMs showed reductions in inward sodium current density and reduced maximal upstroke velocity of action potential compared with healthy control iPSC-CMs. Furthermore, BrS iPSC-CMs demonstrated increased burden of triggered activity, abnormal calcium (Ca(2+)) transients, and beating interval variation. Correction of the causative variant by genome editing was performed, and resultant iPSC-CMs showed resolution of triggered activity and abnormal Ca(2+) transients. Gene expression profiling of iPSC-CMs showed clustering of BrS compared with control subjects. Furthermore, BrS iPSC-CM gene expression correlated with gene expression from BrS human cardiac tissue gene expression.Patient-specific iPSC-CMs were able to recapitulate single-cell phenotype features of BrS, including blunted inward sodium current, increased triggered activity, and abnormal Ca(2+) handling. This novel human cellular model creates future opportunities to further elucidate the cellular disease mechanism and identify novel therapeutic targets.
View details for DOI 10.1016/j.jacc.2016.07.779
View details for PubMedID 27810048
A Rapid, High-Quality, Cost-Effective, Comprehensive and Expandable Targeted Next-Generation Sequencing Assay for Inherited Heart Diseases.
2015; 117 (7): 603-611
Thousands of mutations across >50 genes have been implicated in inherited cardiomyopathies. However, options for sequencing this rapidly evolving gene set are limited because many sequencing services and off-the-shelf kits suffer from slow turnaround, inefficient capture of genomic DNA, and high cost. Furthermore, customization of these assays to cover emerging targets that suit individual needs is often expensive and time consuming.We sought to develop a custom high throughput, clinical-grade next-generation sequencing assay for detecting cardiac disease gene mutations with improved accuracy, flexibility, turnaround, and cost.We used double-stranded probes (complementary long padlock probes), an inexpensive and customizable capture technology, to efficiently capture and amplify the entire coding region and flanking intronic and regulatory sequences of 88 genes and 40 microRNAs associated with inherited cardiomyopathies, congenital heart disease, and cardiac development. Multiplexing 11 samples per sequencing run resulted in a mean base pair coverage of 420, of which 97% had >20× coverage and >99% were concordant with known heterozygous single nucleotide polymorphisms. The assay correctly detected germline variants in 24 individuals and revealed several polymorphic regions in miR-499. Total run time was 3 days at an approximate cost of $100 per sample.Accurate, high-throughput detection of mutations across numerous cardiac genes is achievable with complementary long padlock probe technology. Moreover, this format allows facile insertion of additional probes as more cardiomyopathy and congenital heart disease genes are discovered, giving researchers a powerful new tool for DNA mutation detection and discovery.
View details for DOI 10.1161/CIRCRESAHA.115.306723
View details for PubMedID 26265630
Epigenetic Regulation of Phosphodiesterases 2A and 3A Underlies Compromised ß-Adrenergic Signaling in an iPSC Model of Dilated Cardiomyopathy.
Cell stem cell
2015; 17 (1): 89-100
β-adrenergic signaling pathways mediate key aspects of cardiac function. Its dysregulation is associated with a range of cardiac diseases, including dilated cardiomyopathy (DCM). Previously, we established an iPSC model of familial DCM from patients with a mutation in TNNT2, a sarcomeric protein. Here, we found that the β-adrenergic agonist isoproterenol induced mature β-adrenergic signaling in iPSC-derived cardiomyocytes (iPSC-CMs) but that this pathway was blunted in DCM iPSC-CMs. Although expression levels of several β-adrenergic signaling components were unaltered between control and DCM iPSC-CMs, we found that phosphodiesterases (PDEs) 2A and PDE3A were upregulated in DCM iPSC-CMs and that PDE2A was also upregulated in DCM patient tissue. We further discovered increased nuclear localization of mutant TNNT2 and epigenetic modifications of PDE genes in both DCM iPSC-CMs and patient tissue. Notably, pharmacologic inhibition of PDE2A and PDE3A restored cAMP levels and ameliorated the impaired β-adrenergic signaling of DCM iPSC-CMs, suggesting therapeutic potential.
View details for DOI 10.1016/j.stem.2015.04.020
View details for PubMedID 26095046
Finding the rhythm of sudden cardiac death: new opportunities using induced pluripotent stem cell-derived cardiomyocytes.
2015; 116 (12): 1989-2004
Sudden cardiac death is a common cause of death in patients with structural heart disease, genetic mutations, or acquired disorders affecting cardiac ion channels. A wide range of platforms exist to model and study disorders associated with sudden cardiac death. Human clinical studies are cumbersome and are thwarted by the extent of investigation that can be performed on human subjects. Animal models are limited by their degree of homology to human cardiac electrophysiology, including ion channel expression. Most commonly used cellular models are cellular transfection models, which are able to mimic the expression of a single-ion channel offering incomplete insight into changes of the action potential profile. Induced pluripotent stem cell-derived cardiomyocytes resemble, but are not identical, adult human cardiomyocytes and provide a new platform for studying arrhythmic disorders leading to sudden cardiac death. A variety of platforms exist to phenotype cellular models, including conventional and automated patch clamp, multielectrode array, and computational modeling. Induced pluripotent stem cell-derived cardiomyocytes have been used to study long QT syndrome, catecholaminergic polymorphic ventricular tachycardia, hypertrophic cardiomyopathy, and other hereditary cardiac disorders. Although induced pluripotent stem cell-derived cardiomyocytes are distinct from adult cardiomyocytes, they provide a robust platform to advance the science and clinical care of sudden cardiac death.
View details for DOI 10.1161/CIRCRESAHA.116.304494
View details for PubMedID 26044252
Establishing disease causality for a novel gene variant in familial dilated cardiomyopathy using a functional in-vitro assay of regulated thin filaments and human cardiac myosin.
BMC medical genetics
2015; 16 (1): 97-?
As next generation sequencing for the genetic diagnosis of cardiovascular disorders becomes more widely used, establishing causality for putative disease causing variants becomes increasingly relevant. Diseases of the cardiac sarcomere provide a particular challenge in this regard because of the complexity of assaying the effect of genetic variants in human cardiac contractile proteins.In this study we identified a novel variant R205Q in the cardiac troponin T gene (TNNT2). Carriers of the variant allele exhibited increased chamber volumes associated with decreased left ventricular ejection fraction. To clarify the causal role of this variant, we generated recombinant variant human protein and examined its calcium kinetics as well as the maximally activated ADP release of human β-cardiac myosin with regulated thin filaments containing the mutant troponin T. We found that the R205Q mutation significantly decreased the calcium sensitivity of the thin filament by altering the effective calcium dissociation kinetics.The development of moderate throughput post-genomic assays is an essential step in the realization of the potential of next generation sequencing. Although technically challenging, biochemical and functional assays of human cardiac contractile proteins of the thin filament can be achieved and provide an orthogonal source of information to inform the question of causality for individual variants.
View details for DOI 10.1186/s12881-015-0243-5
View details for PubMedID 26498512
View details for PubMedCentralID PMC4620603
Human induced pluripotent stem cell-derived cardiomyocytes as an in vitro model for coxsackievirus B3-induced myocarditis and antiviral drug screening platform.
2014; 115 (6): 556-566
Rationale: Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. A major causative agent for viral myocarditis is the B3 strain of coxsackievirus, a positive-sense RNA enterovirus. However, human cardiac tissues are difficult to procure in sufficient enough quantities for studying the mechanisms of cardiac-specific viral infection. Objective: This study examined whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy. Methods and Results: Human iPSC-CMs were infected with a luciferase-expressing coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were utilized to characterize virally-infected hiPSC-CMs for alterations in cellular morphology and calcium handling. Viral proliferation in hiPSC-CMs was quantified using bioluminescence imaging. Antiviral compounds including interferon beta 1 (IFNβ1), ribavirin, pyrrolidine dithiocarbamate, and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with reported drug effects in previous studies. Mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways after IFNβ1 treatment. Conclusions: This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to predict antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that can screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion.
View details for DOI 10.1161/CIRCRESAHA.115.303810
View details for PubMedID 25015077
Modeling inherited cardiac disorders.
2014; 78 (4): 784-794
Advances in the understanding and treatment of cardiac disorders have been thwarted by the inability to study beating human cardiac cells in vitro. Induced pluripotent stem cells (iPSCs) bypass this hurdle by enabling the creation of patient-specific iPSC-derived cardiomyocytes (iPSC-CMs). These cells provide a unique platform to study cardiac diseases in vitro, especially hereditary cardiac conditions. To date, iPSC-CMs have been used to successfully model arrhythmic disorders, showing excellent recapitulation of cardiac channel function and electrophysiologic features of long QT syndrome types 1, 2, 3, and 8, and catecholaminergic polymorphic ventricular tachycardia (CPVT). Similarly, iPSC-CM models of dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) have shown robust correlation of predicted morphologic, contractile, and electrical phenotypes. In addition, iPSC-CMs have shown some features of the respective phenotypes for arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C), LEOPARD syndrome, Pompe's disease, and Friedriech's ataxia. In this review, we examine the progress of utilizing iPSC-CMs as a model for cardiac conditions and analyze the potential for the platform in furthering the biology and treatment of cardiac disorders.
View details for PubMedID 24632794
Cardiac stem cell biology: glimpse of the past, present, and future.
2014; 114 (1): 21-27
Cardiac regeneration strategies and de novo generation of cardiomyocytes have long been significant areas of research interest in cardiovascular medicine. In this review, we outline a variety of common cell sources and methods used to regenerate cardiomyocytes and highlight the important role that key Circulation Research articles have played in this flourishing field.
View details for DOI 10.1161/CIRCRESAHA.113.302895
View details for PubMedID 24385505
- Concomitant ECG findings and J wave patterns. Journal of electrocardiology 2013; 46 (5): 399-403
Drug screening using a library of human induced pluripotent stem cell-derived cardiomyocytes reveals disease-specific patterns of cardiotoxicity.
2013; 127 (16): 1677-1691
Cardiotoxicity is a leading cause for drug attrition during pharmaceutical development and has resulted in numerous preventable patient deaths. Incidents of adverse cardiac drug reactions are more common in patients with preexisting heart disease than the general population. Here we generated a library of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with various hereditary cardiac disorders to model differences in cardiac drug toxicity susceptibility for patients of different genetic backgrounds.Action potential duration and drug-induced arrhythmia were measured at the single cell level in hiPSC-CMs derived from healthy subjects and patients with hereditary long QT syndrome, familial hypertrophic cardiomyopathy, and familial dilated cardiomyopathy. Disease phenotypes were verified in long QT syndrome, hypertrophic cardiomyopathy, and dilated cardiomyopathy hiPSC-CMs by immunostaining and single cell patch clamp. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and the human ether-a-go-go-related gene expressing human embryonic kidney cells were used as controls. Single cell PCR confirmed expression of all cardiac ion channels in patient-specific hiPSC-CMs as well as hESC-CMs, but not in human embryonic kidney cells. Disease-specific hiPSC-CMs demonstrated increased susceptibility to known cardiotoxic drugs as measured by action potential duration and quantification of drug-induced arrhythmias such as early afterdepolarizations and delayed afterdepolarizations.We have recapitulated drug-induced cardiotoxicity profiles for healthy subjects, long QT syndrome, hypertrophic cardiomyopathy, and dilated cardiomyopathy patients at the single cell level for the first time. Our data indicate that healthy and diseased individuals exhibit different susceptibilities to cardiotoxic drugs and that use of disease-specific hiPSC-CMs may predict adverse drug responses more accurately than the standard human ether-a-go-go-related gene test or healthy control hiPSC-CM/hESC-CM screening assays.
View details for DOI 10.1161/CIRCULATIONAHA.113.001883
View details for PubMedID 23519760
View details for PubMedCentralID PMC3870148
Natural History of Early Repolarization in the Inferior Leads
ANNALS OF NONINVASIVE ELECTROCARDIOLOGY
2012; 17 (4): 331-339
Though early repolarization (ER) in the inferior leads has been associated with increased cardiovascular risk, its natural history is uncertain. We aimed to study the serial electrocardiographic behavior of inferior ER and understand factors associated with that behavior.We selected electrocardiograms (ECGs) from patients with the greatest amplitude of ER in AVF from ECGs of 29,281 ambulatory patients recorded between 1987 and 1999 at the Palo Alto Veterans Affairs Hospital. Starting from the highest amplitude, we reviewed the ECGs and medical records from the first 85%. From this convenience sample, 36 were excluded for abnormal patterns similar to ER. The remaining 257 patients were searched for another ECG at least 5 months later, of whom, 136 satisfied this criteria. These ECGs were paired for comparison and coded by four interpreters.The average time between the first and second ECGs was 10 years. Of the 136 subjects, 47% retained ER while 53% no longer fulfilled the amplitude criteria. While no significant differences were found in initial heart rate (HR) or time interval between ECGs, those who lost the ER pattern had a greater difference in HR between the ECGs. There was no significant difference in the incidence of cardiovascular events or deaths.In conclusion, the ECG pattern of ER was lost over 10 years in over half of the cohort. The loss of ER was partially explained by changes in HR, but not higher incidence of cardiovascular events or death, suggesting the entity is a benign finding.
View details for DOI 10.1111/j.1542-474X.2012.00537.x
View details for Web of Science ID 000310248100005
View details for PubMedID 23094879
Prognostic Implications of Q Waves and T-Wave Inversion Associated With Early Repolarization
MAYO CLINIC PROCEEDINGS
2012; 87 (7): 614-619
To evaluate the prevalence of early polarization (ER) in a stable population and to evaluate the prognostic significance of the association or absence of Q waves or T-wave inversion (TWI).In this retrospective study performed at the university-affiliated Palo Alto Veterans Affairs Health Care Center from March 1, 1987, through December 31, 1999, we evaluated outpatient electrocardiograms. Vital status and cause of death were determined in all patients, with a mean ± SD follow-up of 7.6±3.8 years.Of the 29,281 patients, 87% were men and 13% were African American. Inferior or lateral ER was present in 664 patients (2.3%): in inferior leads in 185 (0.6%), in lateral leads in 479 (1.6%) , and in both inferior and lateral leads in 163 (0.6%). Only when Q waves or TWI accompanied ER was there an increased risk of cardiovascular death (Cox proportional hazards regression model, 5.0; 95% confidence interval, 3.4-7.2; P<.001).Common patterns of ER without concomitant Q waves or TWI are not associated with increased risk of cardiovascular death; however, when either occurs with ER, there is a hazard ratio of 5.0. These findings confirm that ER is a benign entity; however, the presence of Q waves or TWI with ER is predictive of increased cardiovascular death.
View details for DOI 10.1016/j.mayocp.2012.04.009
View details for PubMedID 22766081
COMPARISON OF EARLY REPOLARIZATION IN INFERIOR AND LATERAL LEADS
61st Annual Scientific Session and Expo of the American-College-of-Cardiology (ACC)/Conference on ACC-i2 with TCT
ELSEVIER SCIENCE INC. 2012: E1940–E1940
View details for Web of Science ID 000302326702151
Embryonic stem cell biology: insights from molecular imaging.
Methods in molecular biology (Clifton, N.J.)
2010; 660: 185-199
Embryonic stem (ES) cells have therapeutic potential in disorders of cellular loss such as myocardial infarction, type I diabetes and neurodegenerative disorders. ES cell biology in living subjects was largely poorly understood until incorporation of molecular imaging into the field. Reporter gene imaging works by integrating a reporter gene into ES cells and using a reporter probe to induce a signal detectable by normal imaging modalities. Reporter gene imaging allows for longitudinal tracking of ES cells within the same host for a prolonged period of time. This has advantages over postmortem immunohistochemistry and traditional imaging modalities. The advantages include expression of reporter gene is limited to viable cells, expression is conserved between generations of dividing cells, and expression can be linked to a specific population of cells. These advantages were especially useful in studying a dynamic cell population such as ES cells and proved useful in elucidating the biology of ES cells. Reporter gene imaging identified poor integration of differentiated ES cells transplanted into host tissue as well as delayed donor cell death as reasons for poor long-term survival in vivo. This imaging technology also confirmed that ES cells indeed have immunogenic properties that factor into cell survival and differentiation. Finally, reporter gene imaging improved our understanding of the neoplastic risk of undifferentiated ES cells in forming teratomas. Despite such advances, much remains to be understood about ES cell biology to translate this technology to the bedside, and reporter gene imaging will certainly play a key role in formulating this understanding.
View details for DOI 10.1007/978-1-60761-705-1_12
View details for PubMedID 20680820