RESUMO
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.
Assuntos
Síndrome de Brugada , Células-Tronco Pluripotentes Induzidas , Canais de Sódio Disparados por Voltagem , Animais , Humanos , Potenciais de Ação , Síndrome de Brugada/genética , Síndrome de Brugada/metabolismo , Estudo de Associação Genômica Ampla , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
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.
Assuntos
Cardiotoxicidade/fisiopatologia , Doxorrubicina/efeitos adversos , Variação Genética/genética , Animais , Modelos Animais de Doenças , Genômica , Humanos , Masculino , CamundongosRESUMO
RATIONALE: The cardiac sodium channel NaV1.5 has a fundamental role in excitability and conduction. Previous studies have shown that sodium channels cluster together in specific cellular subdomains. Their association with intracellular organelles in defined regions of the myocytes, and the functional consequences of that association, remain to be defined. OBJECTIVE: To characterize a subcellular domain formed by sodium channel clusters in the crest region of the myocytes and the subjacent subsarcolemmal mitochondria. METHODS AND RESULTS: Through a combination of imaging approaches including super-resolution microscopy and electron microscopy we identified, in adult cardiac myocytes, a NaV1.5 subpopulation in close proximity to subjacent subsarcolemmal mitochondria; we further found that subjacent subsarcolemmal mitochondria preferentially host the mitochondrial NCLX (Na+/Ca2+ exchanger). This anatomic proximity led us to investigate functional changes in mitochondria resulting from sodium channel activity. Upon TTX (tetrodotoxin) exposure, mitochondria near NaV1.5 channels accumulated more Ca2+ and showed increased reactive oxygen species production when compared with interfibrillar mitochondria. Finally, crosstalk between NaV1.5 channels and mitochondria was analyzed at a transcriptional level. We found that SCN5A (encoding NaV1.5) and SLC8B1 (which encode NaV1.5 and NCLX, respectively) are negatively correlated both in a human transcriptome data set (Genotype-Tissue Expression) and in human-induced pluripotent stem cell-derived cardiac myocytes deficient in SCN5A. CONCLUSIONS: We describe an anatomic hub (a couplon) formed by sodium channel clusters and subjacent subsarcolemmal mitochondria. Preferential localization of NCLX to this domain allows for functional coupling where the extrusion of Ca2+ from the mitochondria is powered, at least in part, by the entry of sodium through NaV1.5 channels. These results provide a novel entry-point into a mechanistic understanding of the intersection between electrical and structural functions of the heart.
Assuntos
Cálcio/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Sinalização do Cálcio , Linhagem Celular , Feminino , Humanos , Cinética , Masculino , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Mitocôndrias Cardíacas/ultraestrutura , Proteínas Mitocondriais/genética , Miócitos Cardíacos/ultraestrutura , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Imagem Individual de Molécula , Trocador de Sódio e Cálcio/genética , Superóxidos/metabolismoRESUMO
[Figure: see text].
Assuntos
Arritmias Cardíacas/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Potenciais de Ação , Animais , Arritmias Cardíacas/metabolismo , Células Cultivadas , Glicogênio Sintase Quinase 3 beta/metabolismo , Células HEK293 , Humanos , Mutação com Perda de Função , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/genética , Miócitos Cardíacos/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Transporte Proteico , Peixe-ZebraRESUMO
Patients with HIV present with a higher prevalence of QT prolongation, of which molecular bases are still not clear. Among HIV proteins, Tat serves as a transactivator that stimulates viral genes expression and is required for efficient HIV replication. Tat is actively secreted into the blood by infected T-cells and affects organs such as the heart. Tat has been shown to alter cardiac repolarization in animal models but how this is mediated and whether this is also the case in human cells is unknown. In the present study, we show that Tat transfection in heterologous expression systems led to a decrease in hERG (underlying cardiac IKr) and human KCNE1-KCNQ1 (underlying cardiac IKs) currents and to an acceleration of their deactivation. This is consistent with a decrease in available phosphatidylinositol-(4,5)-bisphosphate (PIP2). A mutant Tat, unable to bind PIP2, did not reproduce the observed effects. In addition, WT-Tat had no effect on a mutant KCNQ1 which is PIP2-insensitive, further confirming the hypothesis. Twenty-four-hour incubation of human induced pluripotent stem cells-derived cardiomyocytes with Wild-type Tat reduced IKr and accelerated its deactivation. Concordantly, this Tat incubation led to a prolongation of the action potential (AP) duration. Events of AP alternans were also recorded in the presence of Tat, and were exacerbated at a low pacing cycle length. Altogether, these data obtained on human K+ channels both in heterologous expression systems and in human cardiomyocytes suggest that Tat sequesters PIP2, leading to a reduction of IKr and IKs, and provide a molecular mechanism for QT prolongation in HIV-infected patients.
Assuntos
Potenciais de Ação , Fosfatidilinositol 4,5-Difosfato/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Animais , Células COS , Diferenciação Celular , Linhagem Celular , Canal de Potássio ERG1/metabolismo , Fenômenos Eletrofisiológicos , Expressão Gênica , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Canal de Potássio KCNQ1/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transfecção , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
The transplantation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) has shown promise in preclinical models of myocardial infarction, but graft myocardium exhibits incomplete host-graft electromechanical integration and a propensity for pro-arrhythmic behavior. Perhaps contributing to this situation, hPSC-CM grafts show low expression of connexin 43 (Cx43), the major gap junction (GJ) protein, in ventricular myocardia. We hypothesized that Cx43 expression and function could be rescued by engineering Cx43 in hPSC-CMs with a series of phosphatase-resistant mutations at three casein kinase 1 phosphorylation sites (Cx43-S3E) that have been previously reported to stabilize Cx43 GJs and reduce arrhythmias in transgenic mice. However, contrary to our predictions, transgenic Cx43-S3E hPSC-CMs exhibited reduced Cx43 expression relative to wild-type cells, both at baseline and following ischemic challenge. Cx43-S3E hPSC-CMs showed correspondingly slower conduction velocities, increased automaticity, and differential expression of other connexin isoforms and various genes involved in cardiac excitation-contraction coupling. Cx43-S3E hPSC-CMs also had phosphorylation marks associated with Cx43 GJ internalization, a finding that may account for their impaired GJ localization. Taken collectively, our data indicate that the Cx43-S3E mutation behaves differently in hPSC-CMs than in adult mouse ventricular myocytes and that multiple biological factors likely need to be addressed synchronously to ensure proper Cx43 expression, localization, and function.
Assuntos
Caseína Quinase I , Conexina 43 , Miócitos Cardíacos , Adulto , Animais , Humanos , Camundongos , Caseína Quinase I/genética , Conexina 43/genética , Conexinas , Junções Comunicantes , Camundongos Transgênicos , MutaçãoRESUMO
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) recapitulate numerous disease and drug response phenotypes, but cell immaturity may limit their accuracy and fidelity as a model system. Cell culture medium modification is a common method for enhancing maturation, yet prior studies have used complex media with little understanding of individual component contribution, which may compromise long-term hiPSC-CM viability. Here, we developed high-throughput methods to measure hiPSC-CM maturation, determined factors that enhanced viability, and then systematically assessed the contribution of individual maturation medium components. We developed a medium that is compatible with extended culture. We discovered that hiPSC-CM maturation can be sub-specified into electrophysiological/EC coupling, metabolism, and gene expression and that induction of these attributes is largely independent. In this work, we establish a defined baseline for future studies of cardiomyocyte maturation. Furthermore, we provide a selection of medium formulae, optimized for distinct applications and priorities, that promote measurable attributes of maturation.
Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Meios de Cultura , Células Cultivadas , Transcrição Gênica , Técnicas de Cultura de Células/métodosRESUMO
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.
RESUMO
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.
Assuntos
Cardiomiopatias , Células-Tronco Pluripotentes Induzidas , Adulto , Humanos , Antraciclinas/efeitos adversos , Estudos de Casos e Controles , Estudo de Associação Genômica Ampla , Metilação de DNA , Epigênese Genética , DNA , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/genética , Ilhas de CpG , Antibióticos Antineoplásicos , Proteínas de Transporte/genética , Proteínas de Membrana/genéticaRESUMO
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.
Assuntos
Sobreviventes de Câncer , Cardiomiopatias , Neoplasias , Humanos , Criança , Metaloproteinase 8 da Matriz/genética , Metaloproteinase 8 da Matriz/uso terapêutico , Metaloproteinase 9 da Matriz , Antraciclinas/efeitos adversos , Estudos de Casos e Controles , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/complicações , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/genética , Antibióticos Antineoplásicos/efeitos adversos , Miócitos Cardíacos , RNA Mensageiro , Expressão GênicaRESUMO
Brugada syndrome (BrS) is a cardiac arrhythmia disorder associated with sudden death in young adults. With the exception of SCN5A, encoding the cardiac sodium channel NaV1.5, susceptibility genes remain largely unknown. Here we performed a genome-wide association meta-analysis comprising 2,820 unrelated cases with BrS and 10,001 controls, and identified 21 association signals at 12 loci (10 new). Single nucleotide polymorphism (SNP)-heritability estimates indicate a strong polygenic influence. Polygenic risk score analyses based on the 21 susceptibility variants demonstrate varying cumulative contribution of common risk alleles among different patient subgroups, as well as genetic associations with cardiac electrical traits and disorders in the general population. The predominance of cardiac transcription factor loci indicates that transcriptional regulation is a key feature of BrS pathogenesis. Furthermore, functional studies conducted on MAPRE2, encoding the microtubule plus-end binding protein EB2, point to microtubule-related trafficking effects on NaV1.5 expression as a new underlying molecular mechanism. Taken together, these findings broaden our understanding of the genetic architecture of BrS and provide new insights into its molecular underpinnings.
Assuntos
Síndrome de Brugada , Alelos , Síndrome de Brugada/complicações , Síndrome de Brugada/genética , Síndrome de Brugada/metabolismo , Suscetibilidade a Doenças/complicações , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Proteínas Associadas aos Microtúbulos/genética , Mutação , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Adulto JovemRESUMO
Doxorubicin is an anthracycline chemotherapy agent effective in treating a wide range of malignancies, but its use is limited by dose-dependent cardiotoxicity. A recent genome-wide association study identified a SNP (rs2229774) in retinoic acid receptor-γ (RARG) as statistically associated with increased risk of anthracycline-induced cardiotoxicity. Here, we show that human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with rs2229774 and who suffered doxorubicin-induced cardiotoxicity (DIC) are more sensitive to doxorubicin. We determine that the mechanism of this RARG variant effect is mediated via suppression of topoisomerase 2ß (TOP2B) expression and activation of the cardioprotective extracellular regulated kinase (ERK) pathway. We use patient-specific hiPSC-CMs as a drug discovery platform, determining that the RARG agonist CD1530 attenuates DIC, and we confirm this cardioprotective effect in an established in vivo mouse model of DIC. This study provides a rationale for clinical prechemotherapy genetic screening for rs2229774 and a foundation for the clinical use of RARG agonist treatment to protect cancer patients from DIC.
Assuntos
Cardiotoxicidade , Células-Tronco Pluripotentes Induzidas , Animais , Antibióticos Antineoplásicos/efeitos adversos , Doxorrubicina/efeitos adversos , Estudo de Associação Genômica Ampla , Humanos , Camundongos , Miócitos CardíacosRESUMO
AIMS: Several inherited arrhythmic diseases have been linked to single gene mutations in cardiac ion channels and interacting proteins. However, the mechanisms underlying most arrhythmias, are thought to involve altered regulation of the expression of multiple effectors. In this study, we aimed to examine the role of a transcription factor (TF) belonging to the Iroquois homeobox family, IRX5, in cardiac electrical function. METHODS AND RESULTS: Using human cardiac tissues, transcriptomic correlative analyses between IRX5 and genes involved in cardiac electrical activity showed that in human ventricular compartment, IRX5 expression strongly correlated to the expression of major actors of cardiac conduction, including the sodium channel, Nav1.5, and Connexin 40 (Cx40). We then generated human-induced pluripotent stem cells (hiPSCs) derived from two Hamamy syndrome-affected patients carrying distinct homozygous loss-of-function mutations in IRX5 gene. Cardiomyocytes derived from these hiPSCs showed impaired cardiac gene expression programme, including misregulation in the control of Nav1.5 and Cx40 expression. In accordance with the prolonged QRS interval observed in Hamamy syndrome patients, a slower ventricular action potential depolarization due to sodium current reduction was observed on electrophysiological analyses performed on patient-derived cardiomyocytes, confirming the functional role of IRX5 in electrical conduction. Finally, a cardiac TF complex was newly identified, composed by IRX5 and GATA4, in which IRX5 potentiated GATA4-induction of SCN5A expression. CONCLUSION: Altogether, this work unveils a key role for IRX5 in the regulation of human ventricular depolarization and cardiac electrical conduction, providing therefore new insights into our understanding of cardiac diseases.
Assuntos
Potenciais de Ação , Arritmias Cardíacas/genética , Doenças Ósseas/genética , Ventrículos do Coração/metabolismo , Proteínas de Homeodomínio/genética , Hipertelorismo/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Deficiência Intelectual/genética , Mutação com Perda de Função , Miócitos Cardíacos/metabolismo , Miopia/genética , Fatores de Transcrição/genética , Animais , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Doenças Ósseas/metabolismo , Doenças Ósseas/fisiopatologia , Células Cultivadas , Conexinas/genética , Conexinas/metabolismo , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Frequência Cardíaca , Proteínas de Homeodomínio/metabolismo , Humanos , Hipertelorismo/metabolismo , Hipertelorismo/fisiopatologia , Deficiência Intelectual/metabolismo , Deficiência Intelectual/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Miopia/metabolismo , Miopia/fisiopatologia , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma , Proteína alfa-5 de Junções ComunicantesRESUMO
Human induced pluripotent stem cell (hiPSC) culture has become routine, yet the cost of pluripotent cell media, frequent medium changes, and the reproducibility of differentiation have remained restrictive. Here, we describe the formulation of a hiPSC culture medium (B8) as a result of the exhaustive optimization of medium constituents and concentrations, establishing the necessity and relative contributions of each component to the pluripotent state and cell proliferation. The reagents in B8 represent only 3% of the costs of commercial media, made possible primarily by the in-lab generation of three E. coli-expressed, codon-optimized recombinant proteins: fibroblast growth factor 2, transforming growth factor ß3, and neuregulin 1. We demonstrate the derivation and culture of 34 hiPSC lines in B8 as well as the maintenance of pluripotency long term (over 100 passages). This formula also allows a weekend-free feeding schedule without sacrificing capacity for differentiation.
Assuntos
Técnicas de Cultura de Células/economia , Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Bioensaio , Diferenciação Celular , Proliferação de Células , Células Cultivadas , HumanosAssuntos
Arritmias Cardíacas/patologia , Síndrome de Brugada/fisiopatologia , Condutividade Elétrica , Regulação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/patologia , Miócitos Cardíacos/patologia , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Biomarcadores , Síndrome de Brugada/genética , Estudos de Casos e Controles , Perfilação da Expressão Gênica , Ventrículos do Coração , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Fenótipo , Sódio/metabolismoRESUMO
BACKGROUND: Human genetically inherited cardiac diseases have been studied mainly in heterologous systems or animal models, independent of patients' genetic backgrounds. Because sources of human cardiomyocytes (CMs) are extremely limited, the use of urine samples to generate induced pluripotent stem cell-derived CMs would be a noninvasive method to identify cardiac dysfunctions that lead to pathologies within patients' specific genetic backgrounds. The objective was to validate the use of CMs differentiated from urine-derived human induced pluripotent stem (UhiPS) cells as a new cellular model for studying patients' specific arrhythmia mechanisms. METHODS AND RESULTS: Cells obtained from urine samples of a patient with long QT syndrome who harbored the HERG A561P gene mutation and his asymptomatic noncarrier mother were reprogrammed using the episomal-based method. UhiPS cells were then differentiated into CMs using the matrix sandwich method.UhiPS-CMs showed proper expression of atrial and ventricular myofilament proteins and ion channels. They were electrically functional, with nodal-, atrial- and ventricular-like action potentials recorded using high-throughput optical and patch-clamp techniques. Comparison of HERG expression from the patient's UhiPS-CMs to the mother's UhiPS-CMs showed that the mutation led to a trafficking defect that resulted in reduced delayed rectifier K(+) current (IKr). This phenotype gave rise to action potential prolongation and arrhythmias. CONCLUSIONS: UhiPS cells from patients carrying ion channel mutations can be used as novel tools to differentiate functional CMs that recapitulate cardiac arrhythmia phenotypes.
Assuntos
Diferenciação Celular , Síndrome do QT Longo/urina , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Medicina de Precisão/métodos , Potenciais de Ação , Técnicas de Cultura de Células , Células Cultivadas , Técnicas de Reprogramação Celular , Canal de Potássio ERG1 , Eletrocardiografia , Canais de Potássio Éter-A-Go-Go/genética , Feminino , Predisposição Genética para Doença , Ensaios de Triagem em Larga Escala , Humanos , Síndrome do QT Longo/genética , Síndrome do QT Longo/patologia , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Miócitos Cardíacos/patologia , Técnicas de Patch-Clamp , Fenótipo , Células-Tronco Pluripotentes/patologia , Urina/citologia , Adulto JovemRESUMO
Cyclooxygenases (COXs) are important membrane-bound heme containing enzymes important in platelet activation and inflammation. COX-1 is constitutively expressed in most cells whereas COX-2 is an inducible isoform highly expressed in inflammatory conditions. Studies have been carried out to evaluate thiazole derivatives as anti-inflammatory molecules. In this study, we investigated the in vitro and in vivo effects of two novel thiazole derivatives compound 1 (N-[4-(4-hydroxy-3-methoxyphenyl)-1,3-thiazol-2-yl] acetamide) and compound 2 (4-(2-amino-1,3-thiazol-4-yl)-2-methoxyphenol) on prostaglandin E2 (PGE2) production and COX activity in inflammatory settings. Our results reveal a potent inhibition of both compound 1 (IC50 9.01±0.01µM) and 2 (IC50 11.65±6.20µM) (Mean±S.E.M.) on COX-2-dependent PGE2 production. We also determined whether COX-1 activity was inhibited. Using cells stably over-expressing COX-1 and human blood platelets, we showed that compound 1 is a specific inhibitor of COX-1 with IC50 (5.56×10(-8)±2.26×10(-8)µM), whereas compound 2 did not affect COX-1. Both compounds exhibit anti-inflammatory effect in the dorsal air pouch model of inflammation as shows by inhibition of PGE2 secretion. Modeling analysis of docking in the catalytic site of COX-1 or COX-2 further confirmed the difference in the effect of these two compounds. In conclusion, this study contributes to the design of new anti-inflammatory agents and to the understanding of cyclooxygenase inhibition by thiazole.