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1.
Am J Physiol Heart Circ Physiol ; 320(1): H133-H143, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33216635

RESUMO

The usage of flavored electronic nicotine delivery systems (ENDS) is popular, specifically in the teen and young adult age-groups. The possible cardiac toxicity of the flavoring aspect of ENDS is largely unknown. Vaping, a form of electronic nicotine delivery, uses "e-liquid" to generate "e-vapor," an aerosolized mixture of nicotine and/or flavors. We report our investigation into the cardiotoxic effects of flavored e-liquids. E-vapors containing flavoring aldehydes such as vanillin and cinnamaldehyde, as indicated by mass spectrometry, were more toxic in HL-1 cardiomyocytes than fruit-flavored e-vapor. Exposure of human induced pluripotent stem cell-derived cardiomyocytes to cinnamaldehyde or vanillin-flavored e-vapor affected the beating frequency and prolonged the field potential duration of these cells more than fruit-flavored e-vapor. In addition, vanillin aldehyde-flavored e-vapor reduced the human ether-à-go-go-related gene (hERG)-encoded potassium current in transfected human embryonic kidney cells. In mice, inhalation exposure to vanillin aldehyde-flavored e-vapor for 10 wk caused increased sympathetic predominance in heart rate variability measurements. In vivo inducible ventricular tachycardia was significantly longer, and in optical mapping, the magnitude of ventricular action potential duration alternans was significantly larger in the vanillin aldehyde-flavored e-vapor-exposed mice than in controls. We conclude that the widely popular flavored ENDS are not harm free, and they have a potential for cardiac harm. More studies are needed to further assess their cardiac safety profile and long-term health effects.NEW & NOTEWORTHY The use of electronic nicotine delivery systems (ENDS) is not harm free. It is not known whether ENDS negatively affect cardiac electrophysiological function. Our study in cell lines and in mice shows that ENDS can compromise cardiac electrophysiology, leading to action potential instability and inducible ventricular arrhythmias. Further investigations are necessary to assess the long-term cardiac safety profile of ENDS products in humans and to better understand how individual components of ENDS affect cardiac toxicity.


Assuntos
Sistemas Eletrônicos de Liberação de Nicotina , Aromatizantes/toxicidade , Frequência Cardíaca/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Nicotina/toxicidade , Agonistas Nicotínicos/toxicidade , Taquicardia Ventricular/induzido quimicamente , Vaping/efeitos adversos , Potenciais de Ação/efeitos dos fármacos , Administração por Inalação , Animais , Cardiotoxicidade , Canal de Potássio ERG1/metabolismo , Feminino , Aromatizantes/administração & dosagem , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Nicotina/administração & dosagem , Agonistas Nicotínicos/administração & dosagem , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/fisiopatologia , Fatores de Tempo
2.
Mol Genet Genomics ; 296(4): 823-836, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33876311

RESUMO

Next-generation sequencing platforms are being increasingly applied in clinical genetic settings for evaluation of families with suspected heritable disease. These platforms potentially improve the diagnostic yield beyond that of disease-specific targeted gene panels, but also increase the number of rare or novel genetic variants that may confound precise diagnostics. Here, we describe a functional testing approach used to interpret the results of whole exome sequencing (WES) in a family presenting with syncope and sudden death. One individual had a prolonged QT interval on electrocardiogram (ECG) and carried a diagnosis of long QT syndrome (LQTS), but a second individual did not meet criteria for LQTS. Filtering WES results for uncommon variants with arrhythmia association identified four for further analyses. In silico analyses indicated that two of these variants, KCNH2 p.(Cys555Arg) and KCNQ1 p.(Arg293Cys), were likely to be causal in this family's LQTS. We subsequently performed functional characterization of these variants in a heterologous expression system. The expression of KCNQ1-Arg293Cys did not show a deleterious phenotype but KCNH2-Cys555Arg demonstrated a loss-of-function phenotype that was partially dominant. Our stepwise approach identified a precise genetic etiology in this family, which resulted in the establishment of a LQTS diagnosis in the second individual as well as an additional asymptomatic family member, enabling personalized clinical management. Given its ability to aid in the diagnosis, the application of functional characterization should be considered as a value adjunct to in silico analyses of WES.


Assuntos
Canal de Potássio ERG1/genética , Síndrome do QT Longo/genética , Síndrome do QT Longo/fisiopatologia , Proteínas Quinases Ativadas por AMP/genética , Substituição de Aminoácidos/genética , Análise Mutacional de DNA/métodos , Eletrocardiografia , Família , Feminino , Testes Genéticos/métodos , Células HEK293 , Testes de Função Cardíaca/métodos , Humanos , Canal de Potássio KCNQ1/genética , Pessoa de Meia-Idade , Mutação , Linhagem , Fenótipo , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Proteínas Serina-Treonina Quinases/genética , Sequenciamento do Exoma
3.
Am J Physiol Heart Circ Physiol ; 318(2): H212-H222, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31834838

RESUMO

The cardiac potassium IKs current is carried by a channel complex formed from α-subunits encoded by KCNQ1 and ß-subunits encoded by KCNE1. Deleterious mutations in either gene are associated with hereditary long QT syndrome. Interactions between the transmembrane domains of the α- and ß-subunits determine the activation kinetics of IKs. A physical and functional interaction between COOH termini of the proteins has also been identified that impacts deactivation rate and voltage dependence of activation. We sought to explore the specific physical interactions between the COOH termini of the subunits that confer such control. Hydrogen/deuterium exchange coupled to mass spectrometry narrowed down the region of interaction to KCNQ1 residues 352-374 and KCNE1 residues 70-81, and provided evidence of secondary structure within these segments. Key mutations of residues in these regions tended to shift voltage dependence of activation toward more depolarizing voltages. Double-mutant cycle analysis then revealed energetic coupling between KCNQ1-I368 and KCNE1-D76 during channel activation. Our results suggest that the proximal COOH-terminal regions of KCNQ1 and KCNE1 participate in a physical and functional interaction during channel opening that is sensitive to perturbation and may explain the clustering of long QT mutations in the region.NEW & NOTEWORTHY Interacting ion channel subunits KCNQ1 and KCNE1 have received intense investigation due to their critical importance to human cardiovascular health. This work uses physical (hydrogen/deuterium exchange with mass spectrometry) and functional (double-mutant cycle analyses) studies to elucidate precise and important areas of interaction between the two proteins in an area that has eluded structural definition of the complex. It highlights the importance of pathogenic mutations in these regions.


Assuntos
Citoplasma/metabolismo , Canal de Potássio KCNQ1/química , Canal de Potássio KCNQ1/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Clonagem Molecular , Cricetinae , Deutério/metabolismo , Fenômenos Eletrofisiológicos , Células HEK293 , Humanos , Hidrogênio/metabolismo , Canal de Potássio KCNQ1/genética , Síndrome do QT Longo/genética , Síndrome do QT Longo/metabolismo , Mutação , Plasmídeos/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética
4.
J Biol Chem ; 293(31): 12120-12136, 2018 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-29907571

RESUMO

Synonymous nucleotide variation is increasingly recognized as a factor than can affect protein expression, but the underlying mechanisms are incompletely understood. Here, we investigated whether synonymous changes could affect expression of the potassium voltage-gated channel subfamily H member 2 (KCNH2) gene, encoding the human ether-a-go-go-related gene (hERG) ion channel, which is linked to hereditary cardiac arrhythmia. We examined a previously described synthetic version (hERG-codon modified (CM)) with synonymous substitutions designed to reduce GC content, rare codons, and mRNA secondary structure relative to the native construct (hERG-NT). hERG-CM exhibited lower protein expression than hERG-NT in HEK293T cells. We found that the steady-state abundance of hERG-NT mRNA was greater than hERG-CM because of an enhanced transcription rate and increased mRNA stability for hERG-NT. Translation of hERG-CM was independently reduced, contributing to the overall greater synthesis of hERG-NT channel protein. This was partially offset, however, by a higher aggregation of a newly synthesized hERG-NT channel, resulting in nonfunctional protein. Regional mRNA analyses of chimeras of hERG-NT and hERG-CM revealed that synonymous changes in the 5' segments of the coding region had the greatest influence on hERG synthesis at both the mRNA and protein levels. Taken together, these results indicate that synonymous nucleotide variations within the coding region, particularly in the 5' region of the hERG mRNA, can affect both transcription and translation. These findings support the notion that greater attention should be given to the effects of synonymous genetic variation when analyzing hERG DNA sequences in the study of hereditary cardiac disease.


Assuntos
Canal de Potássio ERG1/química , Nucleotídeos/química , Biossíntese de Proteínas , RNA Mensageiro/química , Mutação Silenciosa , Transcrição Gênica , Composição de Bases , Códon/genética , Códon/metabolismo , Canal de Potássio ERG1/genética , Canal de Potássio ERG1/metabolismo , Células HEK293 , Humanos , Potenciais da Membrana/genética , Conformação de Ácido Nucleico , Nucleotídeos/genética , Nucleotídeos/metabolismo , Técnicas de Patch-Clamp , Plasmídeos/química , Plasmídeos/metabolismo , Agregados Proteicos , Domínios Proteicos , Engenharia de Proteínas , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transfecção
5.
J Cardiovasc Electrophysiol ; 28(9): 1070-1082, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28544109

RESUMO

INTRODUCTION: Genetic mutations in KCNH2, which encodes hERG, the alpha subunit of the potassium channel responsible for the IKr current, cause long QT syndrome (LQTS), an inherited cardiac arrhythmia disorder. Electrophysiology techniques are used to correlate genotype with molecular phenotype to determine which mutations identified in patients diagnosed with LQTS are disease causing, and which are benign. These investigations are usually done using heterologous expression in cell lines, and often, epitope fusion tags are used to enable isolation and identification of the protein of interest. METHODS AND RESULTS: Here, we demonstrate through electrophysiology techniques and immunohistochemistry, that both N-terminal and C-terminal myc fusion tags may perturb hERG protein channel expression and kinetics of the IKr current. We also characterize the impact of 2 previously reported inadvertent cDNA variants on hERG channel expression and half-life. CONCLUSION: Our results underscore the importance of careful characterization of the impact of epitope fusion tags and of confirming complete sequence accuracy prior to genotype-phenotype studies for ion channel proteins such as hERG.


Assuntos
DNA/genética , Canal de Potássio ERG1/genética , Regulação da Expressão Gênica , Síndrome do QT Longo/genética , Mutação , Análise Mutacional de DNA , Canal de Potássio ERG1/metabolismo , Eletroforese em Gel de Poliacrilamida , Técnicas Eletrofisiológicas Cardíacas , Genótipo , Humanos , Immunoblotting , Síndrome do QT Longo/metabolismo , Síndrome do QT Longo/patologia , Potenciais da Membrana , Fenótipo
6.
Pacing Clin Electrophysiol ; 40(6): 703-712, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28370132

RESUMO

BACKGROUND: Two genetic variants in SCN5A, encoding the Nav1.5 Na+ channel α-subunit, were found in a 5-month-old girl who died suddenly in her sleep. The first variant is a missense mutation, resulting in an amino acid change (Q1832E), which has been described (but not characterized) in a patient with Brugada syndrome. The second is a nonsense mutation that produces a premature stop codon and a C-terminal truncation (R1944Δ). METHODS AND RESULTS: To investigate their functional relevance with patch clamp experiments in transfected HEK-293 cells. The Q1832E mutation drastically reduced Nav1.5 current density. The R1944Δ C-terminal truncation had negligible effects on Nav1.5 current density. Neither of the mutations affected the voltage dependence of steady activation and inactivation or influenced the late Na+ current or the recovery from inactivation. Biochemical and immunofluorescent approaches demonstrated that the Q1832E mutation caused severe trafficking defects. Polymerase chain reaction cloning and sequencing the victim's genomic DNA allowed us to determine that the two variants were in trans. We investigated the functional consequences by coexpressing Nav1.5(Q1832E) and Nav1.5(R1944Δ), which led to a significantly reduced current amplitude relative to wild-type. CONCLUSIONS: These sudden infant death syndrome (SIDS)-related variants caused a severely dysfunctional Nav1.5 channel, which was mainly due to trafficking defects caused by the Q1832E mutation. The decreased current density is likely to be a major contributing factor to arrhythmogenesis in Brugada syndrome and the sudden death of this SIDS victim.


Assuntos
Ativação do Canal Iônico/genética , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Sódio/metabolismo , Morte Súbita do Lactente/genética , Morte Súbita Cardíaca , Feminino , Marcadores Genéticos/genética , Predisposição Genética para Doença/genética , Células HEK293 , Humanos , Incidência , Lactente , Mutação/genética , Fatores de Risco , Taxa de Sobrevida
7.
J Neurosci ; 35(1): 396-408, 2015 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-25568131

RESUMO

Fragile X syndrome (FXS) is the leading cause of both intellectual disability and autism resulting from a single gene mutation. Previously, we characterized cognitive impairments and brain structural defects in a Drosophila model of FXS and demonstrated that these impairments were rescued by treatment with metabotropic glutamate receptor (mGluR) antagonists or lithium. A well-documented biochemical defect observed in fly and mouse FXS models and FXS patients is low cAMP levels. cAMP levels can be regulated by mGluR signaling. Herein, we demonstrate PDE-4 inhibition as a therapeutic strategy to ameliorate memory impairments and brain structural defects in the Drosophila model of fragile X. Furthermore, we examine the effects of PDE-4 inhibition by pharmacologic treatment in the fragile X mouse model. We demonstrate that acute inhibition of PDE-4 by pharmacologic treatment in hippocampal slices rescues the enhanced mGluR-dependent LTD phenotype observed in FXS mice. Additionally, we find that chronic treatment of FXS model mice, in adulthood, also restores the level of mGluR-dependent LTD to that observed in wild-type animals. Translating the findings of successful pharmacologic intervention from the Drosophila model into the mouse model of FXS is an important advance, in that this identifies and validates PDE-4 inhibition as potential therapeutic intervention for the treatment of individuals afflicted with FXS.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Modelos Animais de Doenças , Síndrome do Cromossomo X Frágil/enzimologia , Plasticidade Neuronal/fisiologia , Inibidores da Fosfodiesterase 4/farmacologia , Animais , Animais Geneticamente Modificados , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Drosophila , Feminino , Síndrome do Cromossomo X Frágil/tratamento farmacológico , Síndrome do Cromossomo X Frágil/genética , Masculino , Camundongos , Camundongos Knockout , Plasticidade Neuronal/efeitos dos fármacos , Inibidores da Fosfodiesterase 4/uso terapêutico
8.
Hum Mutat ; 36(8): 764-73, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25914329

RESUMO

Hereditary long QT syndrome is caused by deleterious mutation in one of several genetic loci, including locus LQT2 that contains the KCNH2 gene (or hERG, human ether-a-go-go related gene), causing faulty cardiac repolarization. Here, we describe and characterize a novel mutation, p.Asp219Val in the hERG channel, identified in an 11-year-old male with syncope and prolonged QT interval. Genetic sequencing showed a nonsynonymous variation in KCNH2 (c.656A>T: amino acid p.Asp219Val). p.Asp219Val resides in a region of the channel predicted to be unstructured and flexible, located between the PAS (Per-Arnt-Sim) domain and its interaction sites in the transmembrane domain. The p.Asp219Val hERG channel produced K(+) current that activated with modest changes in voltage dependence. Mutant channels were also slower to inactivate, recovered from inactivation more readily and demonstrated a significantly accelerated deactivation rate compared with the slow deactivation of wild-type channels. The intermediate nature of the biophysical perturbation is consistent with the degree of severity in the clinical phenotype. The findings of this study demonstrate a previously unknown role of the proximal N-terminus in deactivation and support the hypothesis that the proximal N-terminal domain is essential in maintaining slow hERG deactivation.


Assuntos
Canais de Potássio Éter-A-Go-Go/genética , Síndrome do QT Longo/genética , Mutação , Criança , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/química , Canais de Potássio Éter-A-Go-Go/metabolismo , Células HEK293 , Humanos , Síndrome do QT Longo/fisiopatologia , Masculino , Modelos Moleculares , Estrutura Terciária de Proteína
9.
Hum Mutat ; 40(3): 357, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30740826
10.
FASEB J ; 27(8): 3039-53, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23608144

RESUMO

Human ether-á-gogo-related gene (HERG) encodes a potassium channel that is highly susceptible to deleterious mutations resulting in susceptibility to fatal cardiac arrhythmias. Most mutations adversely affect HERG channel assembly and trafficking. Why the channel is so vulnerable to missense mutations is not well understood. Since nothing is known of how mRNA structural elements factor in channel processing, we synthesized a codon-modified HERG cDNA (HERG-CM) where the codons were synonymously changed to reduce GC content, secondary structure, and rare codon usage. HERG-CM produced typical IKr-like currents; however, channel synthesis and processing were markedly different. Translation efficiency was reduced for HERG-CM, as determined by heterologous expression, in vitro translation, and polysomal profiling. Trafficking efficiency to the cell surface was greatly enhanced, as assayed by immunofluorescence, subcellular fractionation, and surface labeling. Chimeras of HERG-NT/CM indicated that trafficking efficiency was largely dependent on 5' sequences, while translation efficiency involved multiple areas. These results suggest that HERG translation and trafficking rates are independently governed by noncoding information in various regions of the mRNA molecule. Noncoding information embedded within the mRNA may play a role in the pathogenesis of hereditary arrhythmia syndromes and could provide an avenue for targeted therapeutics.


Assuntos
Canais de Potássio Éter-A-Go-Go/fisiologia , Ativação do Canal Iônico/fisiologia , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Composição de Bases/genética , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Códon/genética , DNA Complementar/genética , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/química , Canais de Potássio Éter-A-Go-Go/genética , Células HEK293 , Humanos , Immunoblotting , Ativação do Canal Iônico/genética , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Microscopia de Fluorescência , Mutação , Técnicas de Patch-Clamp , Estrutura Secundária de Proteína , Transporte Proteico/genética , Transporte Proteico/fisiologia , RNA Mensageiro/genética
11.
J Genet Couns ; 23(5): 849-59, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24664857

RESUMO

Genetic testing is becoming increasingly available for cardiac channelopathies, such as long QT syndrome and Brugada syndrome, which can lead to sudden cardiac death. Test results can be used to shape an individual's medical management and to identify at-risk family members. In our qualitative study, all participants had a personal or family history of a diagnosed cardiac arrhythmia syndrome or sudden cardiac death. Open-ended interviews were conducted individually and in focus groups. Interviews were audio recorded, transcribed verbatim, and analyzed using a qualitative grounded-theory approach. Of 50 participants, 37 described their motivations for pursuing genetic testing for long QT syndrome or another cardiac channelopathy. Participants' motivations included: to find an explanation for a family member's sudden death, to relieve uncertainty regarding a diagnosis, to guide future medical management, to allay concern about children or other family members, and to comply with recommendations of physicians or family members. Perceived reasons not to pursue genetic testing included denial, fear, and lack of information. The genetic counseling and informed consent process can be enhanced by understanding and addressing an individual's internal and external motivations either for or against pursuing genetic testing.


Assuntos
Doenças Cardiovasculares/genética , Morte Súbita Cardíaca , Testes Genéticos/estatística & dados numéricos , Motivação , Humanos , Anamnese
12.
Biochim Biophys Acta ; 1823(8): 1273-84, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22613764

RESUMO

The HERG (human ether-a-go-go related gene) potassium channel aids in the repolarization of the cardiomyocyte membrane at the end of each action potential. We have previously shown that sustained protein kinase A or C (PKA and PKC) activity specifically enhances channel synthesis over the course of hours to days in heterologous expression and cardiac myocytes. The kinase-mediated augmentation of the channel is post-transcriptional and occurs near or at the endoplasmic reticulum. Here we report our further investigations into the mechanisms of kinase-mediated augmentation of HERG channel protein. We show that HERG channel phosphorylation alone is not sufficient for the PKA-dependent increase to occur. In vitro translation studies indicate that an additional factor is required for the process. Pharmacologic inhibitors suggest that the channel augmentation is not due to kinase-mediated alteration in proteasome or lysosome activity. PKA activation had no effect on stability of HERG mRNA and polyribosomal profiling showed that kinase activity did not elevate translation from low to high rates. Transcriptional inhibition results suggest that the additional cellular factor is a PKA-regulated protein. Together, these findings suggest that PKA-mediated augmentation of HERG abundance is more complex than previously appreciated involving enhancement of already active translation rates, phosphorylation of the channel protein and at least one other cyclic-AMP/PKA-responsive protein. Further exploration of molecular components of this regulatory pathway will be necessary to determine exact mechanism and the biomedical impact of this process in vivo.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Canais de Potássio Éter-A-Go-Go/genética , Regulação da Expressão Gênica , Proteínas 14-3-3/metabolismo , AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Dactinomicina/farmacologia , Canal de Potássio ERG1 , Ativação Enzimática , Canais de Potássio Éter-A-Go-Go/biossíntese , Células HEK293 , Humanos , Leupeptinas/farmacologia , Fosforilação , Inibidores de Proteassoma/farmacologia , Processamento de Proteína Pós-Traducional , Inibidores da Síntese de Proteínas/farmacologia , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica
13.
Mol Genet Genomic Med ; 11(1): e2093, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36369844

RESUMO

BACKGROUND: Friedreich's ataxia (FRDA) is an autosomal recessive disease, whereby homozygous inheritance of an expanded GAA trinucleotide repeat expansion in the first intron of the FXN gene leads to transcriptional repression of the encoded protein frataxin. FRDA is a progressive neurodegenerative disorder, but the primary cause of death is heart disease which occurs in 60% of the patients. Several functions of frataxin have been proposed, but none of them fully explain why its deficiency causes the FRDA phenotypes nor why the most affected cell types are neurons and cardiomyocytes. METHODS: To investigate, we generated iPSC-derived neurons (iNs) and cardiomyocytes (iCMs) from an FRDA patient and upregulated FXN expression via lentivirus without altering genomic GAA repeats at the FXN locus. RESULTS: RNA-seq and differential gene expression enrichment analyses demonstrated that frataxin deficiency affected the expression of glycolytic pathway genes in neurons and extracellular matrix pathway genes in cardiomyocytes. Genes in these pathways were differentially expressed when compared to a control and restored to control levels when FRDA cells were supplemented with frataxin. CONCLUSIONS: These results offer novel insight into specific roles of frataxin deficiency pathogenesis in neurons and cardiomyocytes.


Assuntos
Ataxia de Friedreich , Células-Tronco Pluripotentes Induzidas , Humanos , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Ataxia de Friedreich/patologia , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Expressão Gênica , Neurônios/metabolismo , Neurônios/patologia , Frataxina
14.
Pilot Feasibility Stud ; 9(1): 47, 2023 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-36949526

RESUMO

BACKGROUND: The COVID-19 pandemic forced healthcare institutions and many clinical research programs to adopt telehealth modalities in order to mitigate viral spread. With the expanded use of telehealth, there is the potential to increase access to genomic medicine to medically underserved populations, yet little is known about how best to communicate genomic results via telehealth while also ensuring equitable access. NYCKidSeq, a multi-institutional clinical genomics research program in New York City, launched the TeleKidSeq pilot study to assess alternative forms of genomic communication and telehealth service delivery models with families from medically underserved populations. METHODS: We aim to enroll 496 participants between 0 and 21 years old to receive clinical genome sequencing. These individuals have a neurologic, cardiovascular, and/or immunologic disease. Participants will be English- or Spanish-speaking and predominantly from underrepresented groups who receive care in the New York metropolitan area. Prior to enrollment, participants will be randomized to either genetic counseling via videoconferencing with screen-sharing or genetic counseling via videoconferencing without screen-sharing. Using surveys administered at baseline, results disclosure, and 6-months post-results disclosure, we will evaluate the impact of the use of screen-sharing on participant understanding, satisfaction, and uptake of medical recommendations, as well as the psychological and socioeconomic implications of obtaining genome sequencing. Clinical utility, cost, and diagnostic yield of genome sequencing will also be assessed. DISCUSSION: The TeleKidSeq pilot study will contribute to innovations in communicating genomic test results to diverse populations through telehealth technology. In conjunction with NYCKidSeq, this work will inform best practices for the implementation of genomic medicine in diverse, English- and Spanish-speaking populations.

15.
J Biol Chem ; 286(24): 21927-36, 2011 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-21536683

RESUMO

Human ether-a-go-go-related gene product (HERG) is a cardiac potassium channel commonly implicated in the pathogenesis of the long QT syndrome, type 2 (LQT2). LQT2 mutations typically have incomplete penetrance and affect individuals at various stages of their lives; this may mirror variations in intracellular signaling and HERG regulation. Previous work showed that sustained protein kinase A (PKA) activity augments HERG protein abundance by a mechanism that includes enhanced protein translation. To investigate the subcellular site of this regulation, we generated site-specific probes to the cytoplasmic surface of the endoplasmic reticulum (ER), the presumed locale of channel synthesis. Real-time FRET-based indicators demonstrated both cAMP and PKA activity at the ER. A PKA inhibitor targeted to the ER surface (termed p4PKIg) completely abolished PKA-mediated augmentation of HERG in HEK293 cells as well as rat neonatal cardiomyocytes. Immunofluorescence co-localization, targeted FRET-based PKA biosensors, phospho-specific antibodies, and in vivo phosphorylation experiments confirmed that p4PKIg is preferentially active at the ER surface rather than the plasma membrane. Rerouting this inhibitor to the outer mitochondrial membrane diminishes its ability to block cAMP-dependent HERG induction. Our results support a model where PKA-dependent regulation of HERG synthesis occurs at the ER surface. Furthermore, reagents generated for this study provide novel experimental tools to probe compartmentalized cAMP/PKA signaling within cells.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Retículo Endoplasmático/metabolismo , Canais de Potássio Éter-A-Go-Go/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Colforsina/farmacologia , AMP Cíclico/metabolismo , Canal de Potássio ERG1 , Inibidores Enzimáticos/farmacologia , Transferência Ressonante de Energia de Fluorescência/métodos , Células HEK293 , Humanos , Microscopia de Fluorescência/métodos , Canais de Potássio/química , Ligação Proteica
16.
Anal Bioanal Chem ; 403(5): 1303-9, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22392372

RESUMO

Three synthetic peptides, derived from the human potassium channel proteins Ether-a-go-go-related gene (HERG), KCNQ1, and KCNE1, were investigated by hydrogen deuterium exchange coupled with electron-transfer dissociation mass spectrometry at single residue resolution. Each amino acid residue in the first half of the HERG peptide incorporated deuterons with a higher rate than those in the second half of the peptide, consistent with the nuclear magnetic resonance structure of this peptide, with amino acids 1-10 being a flexible coil, whereas amino acids 11-24 are a stable amphipathic helix. The binding interface of KCNQ1 and KCNE1 was determined by comparing the difference of sequential fragment ions before and after binding. The residues determined to be involved in binding were consistent with a cysteine cross-linking study and confirmed by double mutant cycle analysis.


Assuntos
Canal de Potássio KCNQ1/química , Canal de Potássio KCNQ1/metabolismo , Peptídeos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Sequência de Aminoácidos , Deutério/química , Humanos , Canal de Potássio KCNQ1/genética , Espectrometria de Massas , Dados de Sequência Molecular , Peptídeos/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Ligação Proteica
17.
Pacing Clin Electrophysiol ; 35(1): 3-16, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21951015

RESUMO

BACKGROUND: The hereditary Long QT Syndrome is a common cardiac disorder where ventricular repolarization is delayed, abnormally prolonging the QTc interval on electrocardiograms. LQTS is linked to various genetic loci, including the KCNH2 (HERG) gene that encodes the α-subunit of the cardiac potassium channel that carries I(Kr). Here, we report and characterize a novel pathologic missense mutation, G816V HERG, in a patient with sudden cardiac death. METHODS: Autopsy-derived tissue sample was used for DNA extraction and sequencing from an unexpected sudden death victim. The G816V HERG mutation was studied using heterologous expression in mammalian cell culture, whole cell patch clamp, confocal immunofluorescence, and immunochemical analyses. RESULTS: The mutant G816V HERG channel has reduced protein expression and shows a trafficking defective phenotype that is incapable of carrying current when expressed at physiological temperatures. The mutant channel showed reduced cell surface localization compared to wild-type HERG (WT HERG) but the mutant and wild-type subunits are capable of interacting. Expression studies at reduced temperatures enabled partial rescue of the trafficking defect with appearance of potassium currents, albeit with reduced current density and altered voltage-dependent activation. Lastly, we examined a potential role for hypokalemia as a contributory factor to the patient's lethal arrhythmia by possible low-potassium-induced degradation of WT HERG and haplo-insufficiency of G816V HERG. CONCLUSION: The G816V mutation in HERG causes a trafficking defect that acts in a partially dominant negative manner. This intermediate severity defect agrees with the mild clinical presentation in other family members harboring the same mutation. Possible hypokalemia in the proband induced WT HERG degradation combined with haplo-insufficiency may have further compromised repolarization reserve and contributed to the lethal arrhythmia.


Assuntos
Canais de Potássio Éter-A-Go-Go/genética , Predisposição Genética para Doença/genética , Síndrome do QT Longo/genética , Adulto , Canal de Potássio ERG1 , Feminino , Humanos , Mutação/genética , Fenótipo
19.
J Community Genet ; 13(6): 629-639, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36203036

RESUMO

Currently, no standardized system exists for evaluating and testing at-risk family members of decedents with abnormal post-mortem genetic testing in cases of sudden unexpected death (SUD). The goal of this study was to evaluate the outcomes of referrals made by an urban medical examiner's office to a multi-disciplinary cardiogenetics clinic. Relatives of decedents with pathogenic/likely pathogenic (P/LP) variants or variants of unknown significance (VUS) in genes known to be associated with cardiomyopathies and/or arrhythmias were identified by the New York City Office of Chief Medical Examiner and referred to the Cardiogenetics Clinic at Montefiore Medical Center. Familial referrals of 15 decedents (median 15 years, range 2 days to 57 years) were evaluated. Variants in 13 genes were identified among decedents (9 arrhythmia, 5 cardiomyopathy). P/LP variants were identified in both arrhythmia (RYR2, SCN5A) and cardiomyopathy syndrome (MYBPC3 (2), MYH7) genes. Thirty-two family members were referred, and 14 variants were detected. One pathogenic (MYBPC3) and two likely pathogenic (RYR2, MYH7) mutations were identified. Referral of at-risk family members of decedents who experienced SUD based on informative post-mortem genetic testing for cardiac and genetic evaluation is warranted, as family studies help to reclassify variants and prevent additional sudden death.

20.
Int J Biochem Cell Biol ; 143: 106137, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34923139

RESUMO

Friedreich ataxia is an autosomal recessive congenital neurodegenerative disease caused by a deficiency in the frataxin protein and is often diagnosed in young adulthood. An expansion of guanine-adenine-adenine repeats in the first intron of the FXN gene leads to decreased frataxin expression. Frataxin plays an essential role in mitochondrial metabolism. Most Friedreich ataxia patients are diagnosed with left ventricular hypertrophic cardiomyopathy, and 60% of patients die with hypertrophic cardiomyopathy. However, the mitochondrial anatomy in Friedreich ataxia hypertrophic cardiomyopathy is still poorly understood. We investigated mitochondrial fission, fusion, and function using biochemical, microscopy, and computational stochastic analysis in human induced pluripotent stem cell derived cardiomyocytes from a patient with Friedreich ataxia hypertrophic cardiomyopathy and a healthy individual. We found a significantly higher mitochondrial footprint, decreased mitochondrial fission protein dynamin-related protein, and mitochondrial fission rate over fusion with more giant mitochondrial clusters in human induced pluripotent stem cell derived cardiomyocytes from a patient with Friedreich ataxia hypertrophic cardiomyopathy, compared to an unaffected individual. We also found significantly depolarized mitochondrial membrane potential and higher reactive oxygen species levels in Friedreich ataxia human induced pluripotent stem cell cardiomyocytes. Our results show that frataxin's depletion may dampen the mitochondrial fission machinery by reducing dynamin-related protein1. The loss of mitochondrial fission might lead to elevated reactive oxygen species and depolarized mitochondrial membrane potential, which may cause oxidative damage in Friedreich ataxia hypertrophic cardiomyopathy. Further investigations are needed to identify the mechanism of downregulating dynamin-related protein1 due to the frataxin deficiency in Friedreich ataxia hypertrophic cardiomyopathy.


Assuntos
Cardiomiopatia Hipertrófica/genética , Dinaminas/metabolismo , Ataxia de Friedreich/genética , Mitocôndrias/metabolismo , Doenças Neurodegenerativas/genética , Adolescente , Cardiomiopatia Hipertrófica/patologia , Criança , Feminino , Ataxia de Friedreich/patologia , Humanos , Masculino
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