Anthracycline Toxicity: Light at the End of the Tunnel?

Anthracycline-induced cardiotoxicity (AIC) is a serious and common side effect of anthracycline therapy. Identification of genes and genetic variants associated with AIC risk has clinical potential as a cardiotoxicity predictive tool and to allow the development of personalized therapies. In this review, we provide an overview of the function of known AIC genes identified by association studies and categorize them based on their mechanistic implication in AIC. We also discuss the importance of functional validation of AIC-associated variants in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to advance the implementation of genetic predictive biomarkers. Finally, we review how patient-specific hiPSC-CMs can be used to identify novel patient-relevant functional targets and for the discovery of cardioprotectant drugs to prevent AIC. Implementation of functional validation and use of hiPSC-CMs for drug discovery will identify the next generation of highly effective and personalized cardioprotectants and accelerate the inclusion of approved AIC biomarkers into clinical practice.

The Role of MAPRE2 and Microtubules in Maintaining Normal Ventricular Conduction.

BACKGROUND: Brugada syndrome is associated with loss-of-function SCN5A variants, yet these account for only ≈20% of cases. A recent genome-wide association study identified a novel locus within MAPRE2, which encodes EB2 (microtubule end-binding protein 2), implicating microtubule involvement in Brugada syndrome. METHODS: A mapre2 knockout zebrafish model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated protein 9) and validated by Western blot. Larval hearts at 5 days post-fertilization were isolated for voltage mapping and immunocytochemistry. Adult fish hearts were used for ECG, patch clamping, and immunocytochemistry. Morpholinos were injected into embryos at 1-cell stage for knockdown experiments. A transgenic zebrafish line with cdh2 tandem fluorescent timer was used to study adherens junctions. Microtubule plus-end tracking and patch clamping were performed in human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) with MAPRE2 knockdown and knockout, respectively. RESULTS: Voltage mapping of mapre2 knockout hearts showed a decrease in ventricular maximum upstroke velocity of the action potential and conduction velocity, suggesting loss of cardiac voltage-gated sodium channel function. ECG showed QRS prolongation in adult knockout fish, and patch clamping showed decreased sodium current density in knockout ventricular myocytes and arrhythmias in knockout iPSC-CMs. Confocal imaging showed disorganized adherens junctions and mislocalization of mature Ncad (N-cadherin) with mapre2 loss of function, associated with a decrease of detyrosinated tubulin. MAPRE2 knockdown in iPSC-CMs led to an increase in microtubule growth velocity and distance, indicating changes in microtubule dynamics. Finally, knockdown of ttl encoding tubulin tyrosine ligase in mapre2 knockout larvae rescued tubulin detyrosination and ventricular maximum upstroke velocity of the action potential. CONCLUSIONS: Genetic ablation of mapre2 led to a decrease in voltage-gated sodium channel function, a hallmark of Brugada syndrome, associated with disruption of adherens junctions, decrease of detyrosinated tubulin as a marker of microtubule stability, and changes in microtubule dynamics. Restoration of the detyrosinated tubulin fraction with ttl knockdown led to rescue of voltage-gated sodium channel-related functional parameters in mapre2 knockout hearts. Taken together, our study implicates microtubule dynamics in the modulation of ventricular conduction.

Targeting OCT3 attenuates doxorubicin-induced cardiac injury.

Doxorubicin is a commonly used anticancer agent that can cause debilitating and irreversible cardiac injury. The initiating mechanisms contributing to this side effect remain unknown, and current preventative strategies offer only modest protection. Using stem-cell-derived cardiomyocytes from patients receiving doxorubicin, we probed the transcriptomic landscape of solute carriers and identified organic cation transporter 3 (OCT3) (SLC22A3) as a critical transporter regulating the cardiac accumulation of doxorubicin. Functional validation studies in heterologous overexpression models confirmed that doxorubicin is transported into cardiomyocytes by OCT3 and that deficiency of OCT3 protected mice from acute and chronic doxorubicin-related changes in cardiovascular function and genetic pathways associated with cardiac damage. To provide proof-of-principle and demonstrate translational relevance of this transport mechanism, we identified several pharmacological inhibitors of OCT3, including nilotinib, and found that pharmacological targeting of OCT3 can also preserve cardiovascular function following treatment with doxorubicin without affecting its plasma levels or antitumor effects in multiple models of leukemia and breast cancer. Finally, we identified a previously unrecognized, OCT3-dependent pathway of doxorubicin-induced cardiotoxicity that results in a downstream signaling cascade involving the calcium-binding proteins S100A8 and S100A9. These collective findings not only shed light on the etiology of doxorubicin-induced cardiotoxicity, but also are of potential translational relevance and provide a rationale for the implementation of a targeted intervention strategy to prevent this debilitating side effect.

A novel transcription factor combination for direct reprogramming to a spontaneously contracting human cardiomyocyte-like state.

The reprogramming of somatic cells to a spontaneously contracting cardiomyocyte-like state using defined transcription factors has proven successful in mouse fibroblasts. However, this process has been less successful in human cells, thus limiting the potential clinical applicability of this technology in regenerative medicine. We hypothesized that this issue is due to a lack of cross-species concordance between the required transcription factor combinations for mouse and human cells. To address this issue, we identified novel transcription factor candidates to induce cell conversion between human fibroblasts and cardiomyocytes, using the network-based algorithm Mogrify. We developed an automated, high-throughput method for screening transcription factor, small molecule, and growth factor combinations, utilizing acoustic liquid handling and high-content kinetic imaging cytometry. Using this high-throughput platform, we screened the effect of 4960 unique transcription factor combinations on direct conversion of 24 patient-specific primary human cardiac fibroblast samples to cardiomyocytes. Our screen revealed the combination of MYOCD, SMAD6, and TBX20 (MST) as the most successful direct reprogramming combination, which consistently produced up to 40% TNNT2+ cells in just 25 days. Addition of FGF2 and XAV939 to the MST cocktail resulted in reprogrammed cells with spontaneous contraction and cardiomyocyte-like calcium transients. Gene expression profiling of the reprogrammed cells also revealed the expression of cardiomyocyte associated genes. Together, these findings indicate that cardiac direct reprogramming in human cells can be achieved at similar levels to those attained in mouse fibroblasts. This progress represents a step forward towards the clinical application of the cardiac direct reprogramming approach.

Identification of Drug Transporter Genomic Variants and Inhibitors That Protect Against Doxorubicin-Induced Cardiotoxicity.

BACKGROUND: Multiple pharmacogenomic studies have identified the synonymous genomic variant rs7853758 (G > A, L461L) and the intronic variant rs885004 in SLC28A3 (solute carrier family 28 member 3) as statistically associated with a lower incidence of anthracycline-induced cardiotoxicity. However, the true causal variant(s), the cardioprotective mechanism of this locus, the role of SLC28A3 and other solute carrier (SLC) transporters in anthracycline-induced cardiotoxicity, and the suitability of SLC transporters as targets for cardioprotective drugs has not been investigated. METHODS: Six well-phenotyped, doxorubicin-treated pediatric patients from the original association study cohort were recruited again, and human induced pluripotent stem cell-derived cardiomyocytes were generated. Patient-specific doxorubicin-induced cardiotoxicity (DIC) was then characterized using assays of cell viability, activated caspase 3/7, and doxorubicin uptake. The role of SLC28A3 in DIC was then queried using overexpression and knockout of SLC28A3 in isogenic human-induced pluripotent stem cell-derived cardiomyocytes using a CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9). Fine-mapping of the SLC28A3 locus was then completed after SLC28A3 resequencing and an extended in silico haplotype and functional analysis. Genome editing of the potential causal variant was done using cytosine base editor. SLC28A3-AS1 overexpression was done using a lentiviral plasmid-based transduction and was validated using stranded RNA-sequencing after ribosomal RNA depletion. Drug screening was done using the Prestwick Chemical Library (n = 1200), followed by in vivo validation in mice. The effect of desipramine on doxorubicin cytotoxicity was also investigated in 8 cancer cell lines. RESULTS: Here, using the most commonly used anthracycline, doxorubicin, we demonstrate that patient-derived cardiomyocytes recapitulate the cardioprotective effect of the SLC28A3 locus and that SLC28A3 expression influences the severity of DIC. Using Nanopore-based fine-mapping and base editing, we identify a novel cardioprotective single nucleotide polymorphism, rs11140490, in the SLC28A3 locus; its effect is exerted via regulation of an antisense long noncoding RNA (SLC28A3-AS1) that overlaps with SLC28A3. Using high-throughput drug screening in patient-derived cardiomyocytes and whole organism validation in mice, we identify the SLC competitive inhibitor desipramine as protective against DIC. CONCLUSIONS: This work demonstrates the power of the human induced pluripotent stem cell model to take a single nucleotide polymorphism from a statistical association through to drug discovery, providing human cell-tested data for clinical trials to attenuate DIC.

Genetic and Epigenetic Regulation of Human Cardiac Reprogramming and Differentiation in Regenerative Medicine.

Regeneration or replacement of lost cardiomyocytes within the heart has the potential to revolutionize cardiovascular medicine. Numerous methodologies have been used to achieve this aim, including the engraftment of bone marrow- and heart-derived cells as well as the identification of modulators of adult cardiomyocyte proliferation. Recently, the conversion of human somatic cells into induced pluripotent stem cells and induced cardiomyocyte-like cells has transformed potential approaches toward this goal, and the engraftment of cardiac progenitors derived from human embryonic stem cells into patients is now feasible. Here we review recent advances in our understanding of the genetic and epigenetic control of human cardiogenesis, cardiac differentiation, and the induced reprogramming of somatic cells to cardiomyocytes. We also cover genetic programs for inducing the proliferation of endogenous cardiomyocytes and discuss the genetic state of cells used in cardiac regenerative medicine.

Targeting the Microtubule EB1-CLASP2 Complex Modulates NaV1.5 at Intercalated Discs.

[Figure: see text].

Human Induced Pluripotent Stem Cell (hiPSC)-Derived Cells to Assess Drug Cardiotoxicity: Opportunities and Problems.

Billions of US dollars are invested every year by the pharmaceutical industry in drug development, with the aim of introducing new drugs that are effective and have minimal side effects. Thirty percent of in-pipeline drugs are excluded in an early phase of preclinical and clinical screening owing to cardiovascular safety concerns, and several lead molecules that pass the early safety screening make it to market but are later withdrawn owing to severe cardiac side effects. Although the current drug safety screening methodologies can identify some cardiotoxic drug candidates, they cannot accurately represent the human heart in many aspects, including genomics, transcriptomics, and patient- or population-specific cardiotoxicity. Despite some limitations, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a powerful and evolving technology that has been shown to recapitulate many attributes of human cardiomyocytes and their drug responses. In this review, we discuss the potential impact of the inclusion of the hiPSC-CM platform in premarket candidate drug screening.

Cellular model systems to study cardiovascular injury from chemotherapy.

In spite of all the efforts for generating efficient pharmacological treatment options for cancer patients, the unwanted side effect of these substances on the cardiovascular system is becoming a major issue for cancer survivors. The fast pacing oncology field necessitate the quest for more accurate and reliable preclinical screenings. hiPSCs derived cardiomyocytes, endothelial and vascular smooth muscle cells provide unlimited source of physiologically relevant cells that could be used in the screening platforms. Cells derived from hiPSCs can measure drug induced alterations to different aspect of the heart including electrophysiology, contractility and structure. In this review, we will give an overview of the different in vivo and in vitro preclinical drug safety screenings. In following sections, we will focus on hiPSCs derived cardiomyocytes, endothelial and vascular smooth muscle cells and present the current knowledge of the application of these cells in unicellular cardiotoxicity assays. In the final part, we will focus on cardiac organoids as multi cell type platform and their role in cardiotoxicity screening of the chemotherapeutic drugs.

GS-967 and Eleclazine Block Sodium Channels in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

GS-967 and eleclazine (GS-6615) are novel sodium channel inhibitors exhibiting antiarrhythmic effects in various in vitro and in vivo models. The antiarrhythmic mechanism has been attributed to preferential suppression of late sodium current (I NaL). Here, we took advantage of a high throughput automated electrophysiology platform (SyncroPatch 768PE) to investigate the molecular pharmacology of GS-967 and eleclazine on peak sodium current (I NaP) recorded from human induced pluripotent stem cell-derived cardiomyocytes. We compared the effects of GS-967 and eleclazine with the antiarrhythmic drug lidocaine, the prototype I NaL inhibitor ranolazine, and the slow inactivation enhancing drug lacosamide. In human induced pluripotent stem cell-derived cardiomyocytes, GS-967 and eleclazine caused a reduction of I NaP in a frequency-dependent manner consistent with use-dependent block (UDB). GS-967 and eleclazine had similar efficacy but evoked more potent UDB of I NaP (IC50 = 0.07 and 0.6 µM, respectively) than ranolazine (7.8 µM), lidocaine (133.5 µM), and lacosamide (158.5 µM). In addition, GS-967 and eleclazine exerted more potent effects on slow inactivation and recovery from inactivation compared with the other sodium channel blocking drugs we tested. The greater UDB potency of GS-967 and eleclazine was attributed to the higher association rates and moderate unbinding rate of these two compounds with sodium channels. We propose that substantial UDB contributes to the observed antiarrhythmic efficacy of GS-967 and eleclazine. SIGNIFICANCE STATEMENT: We investigated the molecular pharmacology of GS-967 and eleclazine on sodium channels in human induced pluripotent stem cell-derived cardiomyocytes using a high throughput automated electrophysiology platform. Sodium channel inhibition by GS-967 and eleclazine has unique effects, including accelerating the onset of slow inactivation and impairing recovery from inactivation. These effects combined with rapid binding and moderate unbinding kinetics explain potent use-dependent block, which we propose contributes to their observed antiarrhythmic efficacy.

Silencing of MYH7 ameliorates disease phenotypes in human iPSC-cardiomyocytes.

Allele-specific RNA silencing has been shown to be an effective therapeutic treatment in a number of diseases, including neurodegenerative disorders. Studies of allele-specific silencing in hypertrophic cardiomyopathy (HCM) to date have focused on mouse models of disease. We here examine allele-specific silencing in a human-cell model of HCM. We investigate two methods of silencing, short hairpin RNA (shRNA) and antisense oligonucleotide (ASO) silencing, using a human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model. We used cellular micropatterning devices with traction force microscopy and automated video analysis to examine each strategy's effects on contractile defects underlying disease. We find that shRNA silencing ameliorates contractile phenotypes of disease, reducing disease-associated increases in cardiomyocyte velocity, force, and power. We find that ASO silencing, while better able to target and knockdown a specific disease-associated allele, showed more modest improvements in contractile phenotypes. These findings are the first exploration of allele-specific silencing in a human HCM model and provide a foundation for further exploration of silencing as a therapeutic treatment for MYH7-mutation-associated cardiomyopathy.

Pluripotent Stem Cell Modeling of Anticancer Therapy-Induced Cardiotoxicity.

PURPOSE OF REVIEW: In this article, we review the different model systems based on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and how they have been applied to identify the cardiotoxic effects of anticancer therapies. RECENT FINDINGS: Developments on 2D and 3D culture systems enabled the use of hiPSC-CMs as screening platforms for cardiotoxic effects of anticancer therapies such as anthracyclines, monoclonal antibodies, and tyrosine kinase inhibitors. Combined with computational approaches and higher throughput screening technologies, they have also enabled mechanistic studies and the search for cardioprotective strategies. As the population ages and cancer treatments become more effective, the cardiotoxic effects of anticancer drugs become a bigger problem leading to an increased role of cardio-oncology. In the past decade, human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important platform for preclinical drug tests, elucidating mechanisms of action for drugs, and identifying cardioprotective pathways that could be further explored in the development of combined treatments. In this article, we highlight 2D and 3D model systems based on hiPSC-CMs that have been used to study the cardiotoxic effects of anticancer drugs, investigating their mechanisms of action and the potential for patient-specific prediction. We also present some of the important challenges and opportunities in the field, indicating possible future developments and how they could impact the landscape of cardio-oncology.

Passive Stretch Induces Structural and Functional Maturation of Engineered Heart Muscle as Predicted by Computational Modeling.

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 (IMR-90 line) were differentiated to hPSC-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 × 106 hPSC-CMs were mixed with 0.4 × 106 human fibroblasts (IMR-90 line) (3:1 ratio) and type-I collagen. The blend was cast into custom-made 12-mm long polydimethylsiloxane 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 with 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, ß-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. Stem Cells 2018;36:265-277.

Plasminogen Activator Inhibitor Type I Controls Cardiomyocyte Transforming Growth Factor-ß and Cardiac Fibrosis.

BACKGROUND: Fibrosis is the pathological consequence of stress-induced tissue remodeling and matrix accumulation. Increased levels of plasminogen activator inhibitor type I (PAI-1) have been shown to promote fibrosis in multiple organ systems. Paradoxically, homozygous genetic deficiency of PAI-1 is associated with spontaneous age-dependent, cardiac-selective fibrosis in mice. We have identified a novel PAI-1-dependent mechanism that regulates cardiomyocyte-derived fibrogenic signals and cardiac transcriptional pathways during injury. METHODS: Cardiac fibrosis in subjects with homozygous mutation in SERPINE-1 was evaluated with late gadolinium-enhanced cardiac magnetic resonance imaging. A murine cardiac injury model was performed by subcutaneous infusion of either saline or Angiotensin II by osmotic minipumps. We evaluated blood pressure, cardiac function (by echocardiography), fibrosis (with Masson Trichrome staining), and apoptosis (with TUNEL staining), and we performed transcriptome analysis (with RNA sequencing). We further evaluated fibrotic signaling in isolated murine primary ventricular myocytes. RESULTS: Cardiac fibrosis was detected in 2 otherwise healthy humans with complete PAI-1 deficiency because of a homozygous frameshift mutation in SERPINE-1. In addition to its suppressive role during spontaneous cardiac fibrosis in multiple species, we hypothesized that PAI-1 also regulates fibrosis during cardiac injury. Treatment of young PAI-1-/- mice with Angiotensin II induced extensive hypertrophy and fibrotic cardiomyopathy, with increased cardiac apoptosis and both reactive and replacement fibrosis. Although Angiotensin II-induced hypertension was blunted in PAI-1-/- mice, cardiac hypertrophy was accelerated. Furthermore, ventricular myocytes were found to be an important source of cardiac transforming growth factor-ß (TGF-ß) and PAI-1 regulated TGF-ß synthesis by cardiomyocytes in vitro as well as in vivo during cardiac injury. Transcriptome analysis of ventricular RNA after Angiotensin II treatment confirmed that PAI-1 deficiency significantly enhanced multiple TGF-ß signaling elements and transcriptional targets, including genes for extracellular matrix components, mediators of extracellular matrix remodeling, matricellular proteins, and cardiac integrins compared with wild-type mice. CONCLUSIONS: PAI-1 is an essential repressor of cardiac fibrosis in mammals. We define a novel cardiomyocyte-specific regulatory mechanism for TGF-ß production by PAI-1, which explains the paradoxical effect of PAI-1 deficiency in promoting cardiac-selective fibrosis. Thus, PAI-1 is a molecular switch that controls the cardiac TGF-ß axis and its early transcriptional effects that lead to myocardial fibrosis.

Chemically defined generation of human cardiomyocytes.

Existing methods for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require complex, undefined medium constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed an optimized cardiac differentiation strategy, using a chemically defined medium consisting of just three components: the basal medium RPMI 1640, L-ascorbic acid 2-phosphate and rice-derived recombinant human albumin. Along with small molecule-based induction of differentiation, this protocol produced contractile sheets of up to 95% TNNT2(+) cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell and was effective in 11 hiPSC lines tested. This chemically defined platform for cardiac specification of hiPSCs will allow the elucidation of cardiomyocyte macromolecular and metabolic requirements and will provide a minimal system for the study of maturation and subtype specification.

Human induced pluripotent stem cell-derived cardiomyocytes as an in vitro model for coxsackievirus B3-induced myocarditis and antiviral drug screening platform.

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: hiPSC-CMs were infected with a luciferase-expressing coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were used 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 interferonß1, 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 interferonß1 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.

Pravastatin reverses obesity-induced dysfunction of induced pluripotent stem cell-derived endothelial cells via a nitric oxide-dependent mechanism.

AIMS: High-fat diet-induced obesity (DIO) is a major contributor to type II diabetes and micro- and macro-vascular complications leading to peripheral vascular disease (PVD). Metabolic abnormalities of induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from obese individuals could potentially limit their therapeutic efficacy for PVD. The aim of this study was to compare the function of iPSC-ECs from normal and DIO mice using comprehensive in vitro and in vivo assays. METHODS AND RESULTS: Six-week-old C57Bl/6 mice were fed with a normal or high-fat diet. At 24 weeks, iPSCs were generated from tail tip fibroblasts and differentiated into iPSC-ECs using a directed monolayer approach. In vitro functional analysis revealed that iPSC-ECs from DIO mice had significantly decreased capacity to form capillary-like networks, diminished migration, and lower proliferation. Microarray and ELISA confirmed elevated apoptotic, inflammatory, and oxidative stress pathways in DIO iPSC-ECs. Following hindlimb ischaemia, mice receiving intramuscular injections of DIO iPSC-ECs had significantly decreased reperfusion compared with mice injected with control healthy iPSC-ECs. Hindlimb sections revealed increased muscle atrophy and presence of inflammatory cells in mice receiving DIO iPSC-ECs. When pravastatin was co-administered to mice receiving DIO iPSC-ECs, a significant increase in reperfusion was observed; however, this beneficial effect was blunted by co-administration of the nitric oxide synthase inhibitor, N(ω)-nitro-l-arginine methyl ester. CONCLUSION: This is the first study to provide evidence that iPSC-ECs from DIO mice exhibit signs of endothelial dysfunction and have suboptimal efficacy following transplantation in a hindlimb ischaemia model. These findings may have important implications for future treatment of PVD using iPSC-ECs in the obese population.

Screening drug-induced arrhythmia [corrected] using human induced pluripotent stem cell-derived cardiomyocytes and low-impedance microelectrode arrays.

BACKGROUND: Drug-induced arrhythmia is one of the most common causes of drug development failure and withdrawal from market. This study tested whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) combined with a low-impedance microelectrode array (MEA) system could improve on industry-standard preclinical cardiotoxicity screening methods, identify the effects of well-characterized drugs, and elucidate underlying risk factors for drug-induced arrhythmia. hiPSC-CMs may be advantageous over immortalized cell lines because they possess similar functional characteristics as primary human cardiomyocytes and can be generated in unlimited quantities. METHODS AND RESULTS: Pharmacological responses of beating embryoid bodies exposed to a comprehensive panel of drugs at 65 to 95 days postinduction were determined. Responses of hiPSC-CMs to drugs were qualitatively and quantitatively consistent with the reported drug effects in literature. Torsadogenic hERG blockers, such as sotalol and quinidine, produced statistically and physiologically significant effects, consistent with patch-clamp studies, on human embryonic stem cell-derived cardiomyocytes hESC-CMs. False-negative and false-positive hERG blockers were identified accurately. Consistent with published studies using animal models, early afterdepolarizations and ectopic beats were observed in 33% and 40% of embryoid bodies treated with sotalol and quinidine, respectively, compared with negligible early afterdepolarizations and ectopic beats in untreated controls. CONCLUSIONS: We found that drug-induced arrhythmias can be recapitulated in hiPSC-CMs and documented with low impedance MEA. Our data indicate that the MEA/hiPSC-CM assay is a sensitive, robust, and efficient platform for testing drug effectiveness and for arrhythmia screening. This system may hold great potential for reducing drug development costs and may provide significant advantages over current industry standard assays that use immortalized cell lines or animal models.

Nilotinib-induced alterations in endothelial cell function recapitulate clinical vascular phenotypes independent of ABL1.

Nilotinib is a highly effective treatment for chronic myeloid leukemia but has been consistently associated with the development of nilotinib-induced arterial disease (NAD) in a subset of patients. To date, which cell types mediate this effect and whether NAD results from on-target mechanisms is unknown. We utilized human induced pluripotent stem cells (hiPSCs) to generate endothelial cells and vascular smooth muscle cells for in vitro study of NAD. We found that nilotinib adversely affects endothelial proliferation and migration, in addition to increasing intracellular nitric oxide. Nilotinib did not alter endothelial barrier function or lipid uptake. No effect of nilotinib was observed in vascular smooth muscle cells, suggesting that NAD is primarily mediated through endothelial cells. To evaluate whether NAD results from enhanced inhibition of ABL1, we generated multiple ABL1 knockout lines. The effects of nilotinib remained unchanged in the absence of ABL1, suggesting that NAD results from off- rather than on-target signaling. The model established in the present study can be applied to future mechanistic and patient-specific pharmacogenomic studies.