Functional Validation of Doxorubicin-Induced Cardiotoxicity-Related Genes.

Background: Genome-wide association studies and candidate gene association studies have identified more than 180 genetic variants statistically associated with anthracycline-induced cardiotoxicity (AIC). However, the lack of functional validation has hindered the clinical translation of these findings. Objectives: The aim of this study was to functionally validate all genes associated with AIC using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Methods: Through a systemic literature search, 80 genes containing variants significantly associated with AIC were identified. Additionally, 3 more genes with potential roles in AIC (GSTM1, CBR1, and ERBB2) were included. Of these, 38 genes exhibited expression in human fetal heart, adult heart, and hiPSC-CMs. Using clustered regularly interspaced short palindromic repeats/Cas9-based genome editing, each of these 38 genes was systematically knocked out in control hiPSC-CMs, and the resulting doxorubicin-induced cardiotoxicity (DIC) phenotype was assessed using hiPSC-CMs. Subsequently, functional assays were conducted for each gene knockout on the basis of hypothesized mechanistic implications in DIC. Results: Knockout of 26 genes increased the susceptibility of hiPSC-CMs to DIC. Notable genes included efflux transporters (ABCC10, ABCC2, ABCB4, ABCC5, and ABCC9), well-established DIC-associated genes (CBR1, CBR3, and RAC2), and genome-wide association study-discovered genes (RARG and CELF4). Conversely, knockout of ATP2B1, HNMT, POR, CYBA, WDR4, and COL1A2 had no significant effect on the in vitro DIC phenotype of hiPSC-CMs. Furthermore, knockout of the uptake transporters (SLC28A3, SLC22A17, and SLC28A1) demonstrated a protective effect against DIC. Conclusions: The present findings establish a comprehensive platform for the functional validation of DIC-associated genes, providing insights for future studies in DIC variant associations and potential mechanistic targets for the development of cardioprotective drugs.

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.

Altered Peripheral Blood Gene Expression in Childhood Cancer Survivors With Anthracycline-Induced Cardiomyopathy - A COG-ALTE03N1 Report.

Background Anthracycline-induced cardiomyopathy is a leading cause of premature death in childhood cancer survivors, presenting a need to understand the underlying pathogenesis. We sought to examine differential blood-based mRNA expression profiles in anthracycline-exposed childhood cancer survivors with and without cardiomyopathy. Methods and Results We designed a matched case-control study (Children's Oncology Group-ALTE03N1) with mRNA sequencing on total RNA from peripheral blood in 40 anthracycline-exposed survivors with cardiomyopathy (cases) and 64 matched survivors without (controls). DESeq2 identified differentially expressed genes. Ingenuity Pathway Analyses (IPA) and Gene Set Enrichment Analyses determined the potential roles of altered genes in biological pathways. Functional validation was performed by gene knockout in human-induced pluripotent stem cell-derived cardiomyocytes using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) technology. Median age at primary cancer diagnosis for cases and controls was 8.2 and 9.7 years, respectively. Thirty-six differentially expressed genes with fold change ≥±2 were identified; 35 were upregulated. IPA identified "hepatic fibrosis" and "iron homeostasis" pathways to be significantly modulated by differentially expressed genes, including toxicology functions of myocardial infarction, cardiac damage, and cardiac dilation. Leading edge analysis from Gene Set Enrichment Analyses identified lactate dehydrogenase A (LDHA) and cluster of differentiation 36 (CD36) genes to be significantly upregulated in cases. Interleukin 1 receptor type 1, 2 (IL1R1, IL1R2), and matrix metalloproteinase 8, 9 (MMP8, MMP9) appeared in multiple canonical pathways. LDHA-knockout human-induced pluripotent stem cell-derived cardiomyocytes showed increased sensitivity to doxorubicin. Conclusions We identified differential mRNA expression profiles in peripheral blood of anthracycline-exposed childhood cancer survivors with and without cardiomyopathy. Upregulation of LDHA and CD36 genes suggests metabolic perturbations in a failing heart. Dysregulation of proinflammatory cytokine receptors IL1R1 and IL1R2 and matrix metalloproteinases, MMP8 and MMP9 indicates structural remodeling that accompanies the clinical manifestation of symptomatic cardiotoxicity.

Identification of novel hypermethylated or hypomethylated CpG sites and genes associated with anthracycline-induced cardiomyopathy.

Anthracycline-induced cardiomyopathy is a leading cause of late morbidity in childhood cancer survivors. Aberrant DNA methylation plays a role in de novo cardiovascular disease. Epigenetic processes could play a role in anthracycline-induced cardiomyopathy but remain unstudied. We sought to examine if genome-wide differential methylation at 'CpG' sites in peripheral blood DNA is associated with anthracycline-induced cardiomyopathy. This report used participants from a matched case-control study; 52 non-Hispanic White, anthracycline-exposed childhood cancer survivors with cardiomyopathy were matched 1:1 with 52 survivors with no cardiomyopathy. Paired ChAMP (Chip Analysis Methylation Pipeline) with integrated reference-based deconvolution of adult peripheral blood DNA methylation was used to analyze data from Illumina HumanMethylation EPIC BeadChip arrays. An epigenome-wide association study (EWAS) was performed, and the model was adjusted for GrimAge, sex, interaction terms of age at enrollment, chest radiation, age at diagnosis squared, and cardiovascular risk factors (CVRFs: diabetes, hypertension, dyslipidemia). Prioritized genes were functionally validated by gene knockout in human induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) using CRISPR/Cas9 technology. DNA-methylation EPIC array analyses identified 32 differentially methylated probes (DMP: 15 hyper-methylated and 17 hypo-methylated probes) that overlap with 23 genes and 9 intergenic regions. Three hundred and fifty-four differential methylated regions (DMRs) were also identified. Several of these genes are associated with cardiac dysfunction. Knockout of genes EXO6CB, FCHSD2, NIPAL2, and SYNPO2 in hiPSC-CMs increased sensitivity to doxorubicin. In addition, EWAS analysis identified hypo-methylation of probe 'cg15939386' in gene RORA to be significantly associated with anthracycline-induced cardiomyopathy. In this genome-wide DNA methylation profile study, we observed significant differences in DNA methylation at the CpG level between anthracycline-exposed childhood cancer survivors with and without cardiomyopathy, implicating differential DNA methylation of certain genes could play a role in pathogenesis of anthracycline-induced cardiomyopathy.

Nutritional requirements of human induced pluripotent stem cells.

The nutritional requirements for human induced pluripotent stem cell (hiPSC) growth have not been extensively studied. Here, building on our prior work that established the suitable non-basal medium components for hiPSC growth, we develop a simplified basal medium consisting of just 39 components, demonstrating that many ingredients of DMEM/F12 are either not essential or are at suboptimal concentrations. This new basal medium along with the supplement, which we call BMEM, enhances the growth rate of hiPSCs over DMEM/F12-based media, supports derivation of multiple hiPSC lines, and allows differentiation to multiple lineages. hiPSCs cultured in BMEM consistently have enhanced expression of undifferentiated cell markers such as POU5F1 and NANOG, along with increased expression of markers of the primed state and reduced expression of markers of the naive state. This work describes titration of the nutritional requirements of human pluripotent cell culture and identifies that suitable nutrition enhances the pluripotent state.

HiPSC-derived cardiomyocyte to model Brugada syndrome: both asymptomatic and symptomatic mutation carriers reveal increased arrhythmogenicity.

Brugada syndrome is an inherited cardiac arrhythmia disorder that is mainly associated with mutations of the cardiac voltage-gated sodium channel alpha subunit 5 (SCN5A) gene. The clinical symptoms include ventricular fibrillation and an increased risk of sudden cardiac death. Human-induced pluripotent stem cell (hiPSC) lines were derived from symptomatic and asymptomatic individuals carrying the R1913C mutation in the SCN5A gene. The present work aimed to observe the phenotype-specific differences in hiPSC-derived cardiomyocytes (CMs) obtained from symptomatic and asymptomatic mutation carriers. In this study, CM electrophysiological properties, beating abilities and calcium parameters were measured. Mutant CMs exhibited higher average sodium current densities than healthy CMs, but the differences were not statistically significant. Action potential durations were significantly shorter in CMs from the symptomatic individual, and a spike-and-dome morphology of action potential was exclusively observed in CMs from the symptomatic individual. More arrhythmias occurred in mutant CMs at single cell and cell aggregate levels compared with those observed in wild-type CMs. Moreover, there were no major differences in ionic currents or intracellular calcium dynamics between the CMs of asymptomatic and symptomatic individuals after the administration of adrenaline and flecainide.In conclusion, mutant CMs were more prone to arrhythmia than healthy CMs but did not explain why only one of the mutation carriers was symptomatic.

Data analytics for cardiac diseases.

In the present research we tackled the classification of seven genetic cardiac diseases and control subjects by using an extensive set of machine learning algorithms with their variations from simple K-nearest neighbor searching method to support vector machines. The research was based on calcium transient signals measured from induced pluripotent stem cell-derived cardiomyocytes. All in all, 55 different machine learning alternatives were used to model eight classes by applying the principle of 10-fold crossvalidation with the peak data of 1626 signals. The best classification accuracy of approximately 69% was given by random forests, which can be seen high enough here to show machine learning to be potential for the differentiation of the eight disease classes.

On computational classification of genetic cardiac diseases applying iPSC cardiomyocytes.

BACKGROUND: Cardiomyocytes differentiated from human induced pluripotent stem cells (iPSC-CMs) can be used to study genetic cardiac diseases. In patients these diseases are manifested e.g. with impaired contractility and fatal cardiac arrhythmias, and both of these can be due to abnormal calcium transients in cardiomyocytes. Here we classify different genetic cardiac diseases using Ca2+ transient data and different machine learning algorithms. METHODS: By studying calcium cycling of disease-specific iPSC-CMs and by using calcium transients measured from these cells it is possible to classify diseases from each other and also from healthy controls by applying machine learning computation on the basis of peak attributes detected from calcium transient signals. RESULTS: In the current research we extend our previous study having Ca-transient data from four different genetic diseases by adding data from two additional diseases (dilated cardiomyopathy and long QT Syndrome 2). We also study, in the light of the current data, possible differences and relations when machine learning modelling and classification accuracies were computed by using either leave-one-out test or 10-fold cross-validation. CONCLUSIONS: Despite more complex classification tasks compared to our earlier research and having more different genetic cardiac diseases in the analysis, it is still possible to attain good disease classification results. As excepted, leave-one-out test and 10-fold cross-validation achieved virtually equal results.

hiPSC-Derived Cardiomyocyte Model of LQT2 Syndrome Derived from Asymptomatic and Symptomatic Mutation Carriers Reproduces Clinical Differences in Aggregates but Not in Single Cells.

Mutations in the HERG gene encoding the potassium ion channel HERG, represent one of the most frequent causes of long QT syndrome type-2 (LQT2). The same genetic mutation frequently presents different clinical phenotypes in the family. Our study aimed to model LQT2 and study functional differences between the mutation carriers of variable clinical phenotypes. We derived human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) from asymptomatic and symptomatic HERG mutation carriers from the same family. When comparing asymptomatic and symptomatic single LQT2 hiPSC-CMs, results from allelic imbalance, potassium current density, and arrhythmicity on adrenaline exposure were similar, but a difference in Ca2+ transients was observed. The major differences were, however, observed at aggregate level with increased susceptibility to arrhythmias on exposure to adrenaline or potassium channel blockers on CM aggregates derived from the symptomatic individual. The effect of this mutation was modeled in-silico which indicated the reactivation of an inward calcium current as one of the main causes of arrhythmia. Our in-vitro hiPSC-CM model recapitulated major phenotype characteristics observed in LQT2 mutation carriers and strong phenotype differences between LQT2 asymptomatic vs. symptomatic were revealed at CM-aggregate level.

Modeling of LMNA-Related Dilated Cardiomyopathy Using Human Induced Pluripotent Stem Cells.

Dilated cardiomyopathy (DCM) is one of the leading causes of heart failure and heart transplantation. A portion of familial DCM is due to mutations in the LMNA gene encoding the nuclear lamina proteins lamin A and C and without adequate treatment these patients have a poor prognosis. To get better insights into pathobiology behind this disease, we focused on modeling LMNA-related DCM using human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM). Primary skin fibroblasts from DCM patients carrying the most prevalent Finnish founder mutation (p.S143P) in LMNA were reprogrammed into hiPSCs and further differentiated into cardiomyocytes (CMs). The cellular structure, functionality as well as gene and protein expression were assessed in detail. While mutant hiPSC-CMs presented virtually normal sarcomere structure under normoxia, dramatic sarcomere damage and an increased sensitivity to cellular stress was observed after hypoxia. A detailed electrophysiological evaluation revealed bradyarrhythmia and increased occurrence of arrhythmias in mutant hiPSC-CMs on ß-adrenergic stimulation. Mutant hiPSC-CMs also showed increased sensitivity to hypoxia on microelectrode array and altered Ca2+ dynamics. Taken together, p.S143P hiPSC-CM model mimics hallmarks of LMNA-related DCM and provides a useful tool to study the underlying cellular mechanisms of accelerated cardiac degeneration in this disease.

Scaling and correlation properties of RR and QT intervals at the cellular level.

We study complex scaling properties of RR and QT intervals of electrocardiograms (ECGs) with their equivalences at the cellular level, that is, inter-beat intervals (IBI) and field potential durations (FPD) of spontaneously beating human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) aggregates. Our detrended fluctuation analysis and Poincaré plots reveal remarkable similarities between the ECG and hiPSC-CM data. In particular, no statistically significant difference was found in the short- and long-term scaling exponents α1 and α2 of RR and QT intervals and their cellular equivalences. Previously unknown scaling properties of FPDs of hiPSC-CM aggregates reveal that the increasing scaling exponent of QT intervals as a function of the time scale, is an intrinsic feature at the cellular level.

Low extracellular potassium prolongs repolarization and evokes early afterdepolarization in human induced pluripotent stem cell-derived cardiomyocytes.

Long QT syndrome (LQTS) is characterized by a prolonged QT-interval on electrocardiogram and by increased risk of sudden death. One of the most common and potentially life-threatening electrolyte disturbances is hypokalemia, characterized by low concentrations of K+ Using a multielectrode array platform and current clamp technique, we investigated the effect of low extracellular K+ concentration ([K+]Ex) on the electrophysiological properties of hiPSC-derived cardiomyocytes (CMs) generated from a healthy control subject (WT) and from two symptomatic patients with type 1 of LQTS carrying G589D (LQT1A) or IVS7-2A>G mutation (LQT1B) in KCNQ1 The baseline prolongations of field potential durations (FPDs) and action potential durations (APDs) were longer in LQT1-CMs than in WT-CMs. Exposure to low [K+]Ex prolonged FPDs and APDs in a concentration-dependent fashion. LQT1-CMs were found to be more sensitive to low [K+]Ex compared to WT-CMs. At baseline, LQT1A-CMs had more prolonged APDs than LQT1B-CMs, but low [K+]Ex caused more pronounced APD prolongation in LQT1B-CMs. Early afterdepolarizations in the action potentials were observed in a subset of LQT1A-CMs with further prolonged baseline APDs and triangular phase 2 profiles. This work demonstrates that the hiPSC-derived CMs are sensitive to low [K+]Ex and provide a platform to study acquired LQTS.

An automated fabrication strategy to create patterned tubular architectures at cell and tissue scales.

The use of materials to impose tissue-like architecture at cell resolution will be important if engineered functional replacements for damaged cardiovascular, pulmonary, renal or digestive tissues are to be authentically engineered. Here, we demonstrate a coordinated system for the fabrication and subsequent culture of tubular tissues composed of multiple layers, cell-types and materials with physiological dimensions and defined architectures at cell resolution. We developed an automated tube fabricator that rolls 2D-matrices into 3D-tubular constructs directly from cells, hydrogels and scaffold biomaterials. Coordinated use of surface modification strategies allows 2D cell sheets and cell/biomaterial composites (i.e. hydrogels or electrospun scaffolds) to be fabricated which may be transferred into a perfusion bioreactor in a rapid and standardized procedure. To exemplify our strategy we fabricated structures resembling human mammary artery and gut; these can be imaged in situ and real-time electrical resistance measurements performed of the vessel walls, allowing non-invasive assessment of viability and functionality. Our system allows patterning at cellular resolution with variable tissue thickness, length, luminal diameter, and constituent biomaterial. This inherent flexibility will allow the recapitulation of the complex hierarchical biological architectures and generate functionality found natively in vivo.

Combined hydrogels that switch human pluripotent stem cells from self-renewal to differentiation.

The ability of materials to define the architecture and microenvironment experienced by cells provides new opportunities to direct the fate of human pluripotent stem cells (HPSCs) [Robinton DA, Daley GQ (2012) Nature 481(7381):295-305]. However, the conditions required for self-renewal vs. differentiation of HPSCs are different, and a single system that efficiently achieves both outcomes is not available [Giobbe GG, et al. (2012) Biotechnol Bioeng 109(12):3119-3132]. We have addressed this dual need by developing a hydrogel-based material that uses ionic de-cross-linking to remove a self-renewal permissive hydrogel (alginate) and switch to a differentiation-permissive microenvironment (collagen). Adjusting the timing of this switch can preferentially steer the HPSC differentiation to mimic lineage commitment during gastrulation to ectoderm (early switch) or mesoderm/endoderm (late switch). As an exemplar differentiated cell type, we showed that directing early lineage specification using this single system can promote cardiogenesis with increased gene expression in high-density cell populations. This work will facilitate regenerative medicine by allowing in situ HPSC expansion to be coupled with early lineage specification within defined tissue geometries.