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PURPOSE OF REVIEW: Truncating TTN variants (TTNtv) are the most common genetic cause of dilated cardiomyopathy (DCM), but the underlying mechanisms are incompletely understood and effective therapeutic strategies are lacking. Here we review recent data that shed new light on the functional consequences of TTNtv and how these effects may vary with mutation location. RECENT FINDINGS: Whether TTNtv act by haploinsufficiency or dominant negative effects has been hotly debated. New evidence now implicates both mechanisms in TTNtv-related DCM, showing reduced titin content and persistent truncated titin that may be incorporated into protein aggregates. The extent to which aggregate formation and protein quality control defects differ with TTNtv location and contribute to contractile dysfunction is unresolved. TTNtv-associated DCM has a complex etiology that involves varying combinations of wild-type titin deficiency and dominant negative effects of truncated mutant titin. Therapeutic strategies to improve protein handling may be beneficial in some cases.
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Cardiomiopatias , Cardiomiopatia Dilatada , Cardiomiopatias/genética , Cardiomiopatia Dilatada/genética , Conectina/genética , Conectina/metabolismo , Humanos , MutaçãoRESUMO
PURPOSE OF REVIEW: Atrial cardiomyopathy is a frequently encountered but underappreciated clinical entity that is characterized by altered atrial size and function. Although traditionally considered a primary atrial disorder, atrial cardiomyopathy was recently redefined to include secondary atrial remodelling. This conceptual shift has implications for the scope of etiological factors and intervention strategies. Our aim was to evaluate the potential contribution of genetics to atrial cardiomyopathy. RECENT FINDINGS: Although the genetics of atrial cardiomyopathy is relatively unexplored, extensive efforts have been made to identify the genetic underpinnings of atrial fibrillation, which is a common complication of atrial cardiomyopathy. Interestingly, in-silico and functional studies suggest that atrial fibrillation-associated genetic variants mainly act by generating a proarrhythmogenic atrial cardiomyopathic substrate. Investigating the genetic basis of primary defects in atrial structure and function, as well as the genetic contributions to cardiac disorders, comorbidities and lifestyle factors that result in secondary atrial remodelling should expand the spectrum of genetic factors that directly or indirectly cause atrial cardiomyopathy and help to resolve the missing heritability of atrial fibrillation. SUMMARY: Elucidation of the genetic basis of atrial cardiomyopathy may provide new risk markers and facilitate personalized interventions for complications, such as atrial fibrillation, heart failure, and stroke.
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Fibrilação Atrial , Remodelamento Atrial , Cardiomiopatias , Fibrilação Atrial/genética , Remodelamento Atrial/genética , Cardiomiopatias/genética , Predisposição Genética para Doença , Átrios do Coração , Humanos , Acidente Vascular Cerebral/genéticaRESUMO
Echocardiography is an invaluable tool for characterizing cardiac structure and function in vivo. Technological advances in high-frequency ultrasound over the past 3 decades have increased spatial and temporal resolution, and facilitated many important clinical and basic science discoveries. Successful reverse translation of established echocardiographic techniques, including M-mode, B-mode, color Doppler, pulsed-wave Doppler, tissue Doppler and, most recently, myocardial deformation imaging, from clinical cardiology into the basic science laboratory has enabled researchers to achieve a deeper understanding of myocardial phenotypes in health and disease. With high-frequency echocardiography, detailed evaluation of ventricular systolic function in a range of small animal models is now possible. Furthermore, improvements in frame rate and the advent of diastolic strain rate imaging, when coupled with the use of select pulsed-wave Doppler parameters, such as isovolumic relaxation time and E wave deceleration, have enabled nuanced interpretation of ventricular diastolic function. Comparing pulsed-wave Doppler indices of atrioventricular inflow during early and late diastole with parameters that describe the simultaneous myocardial deformation (e.g., tissue Doppler é and á, global longitudinal strain rate and global longitudinal velocity) may yield additional insights related to myocardial compliance. This review will provide a historical perspective of the development of high-frequency echocardiography and consider how ongoing innovation will help future-proof this important imaging modality for 21st century translational research.
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Ecocardiografia/tendências , Animais , Diagnóstico por Imagem/métodos , Ecocardiografia/instrumentação , Ecocardiografia/métodos , Ecocardiografia Doppler de Pulso , Coração/diagnóstico por imagem , Humanos , Camundongos , Pesquisa/instrumentação , Pesquisa/tendências , Peixe-ZebraRESUMO
In endurance athletes, prolonged high intensity exercise participation can have deleterious effects on the myocardium with subsequent structural and electrical remodelling. In a subset of athletes, there is a predilection for atrial involvement and the risk of atrial fibrillation (AF) is increased. The mechanisms underpinning exercise-induced atrial cardiomyopathy have yet to be fully elucidated and the contribution of an individual's genetic makeup is unknown. Some athletes may have rare genetic variants that are sufficient to cause AF irrespective of exercise exposure. In AF-causing variant carriers, the additional haemodynamic stress of exercise on atrial structure and function might accelerate or increase the severity of disease. Variants in genes that lack known links to AF may indirectly promote an arrhythmogenic substrate by affecting threshold levels for exercise-induced myocardial damage and remodelling responses, or by effects on AF-associated co-morbidities, sinus node function, and autonomic nervous system tone. Given the exquisite stress-sensitivity of the atria, mechanosensitive ion channels could plausibly have a key role in mediating exercise effects on atrial structure and function. Knowing an athlete's profile of genetic variants may be useful for AF risk stratification and have implications for clinical management. Pre-participation genetic testing may influence sports choices and facilitate AF prevention.
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Atletas , Fibrilação Atrial/genética , Remodelamento Atrial , Cardiomiopatias/complicações , Exercício Físico , Átrios do Coração/fisiopatologia , Fibrilação Atrial/etiologia , Cardiomiopatias/genética , HumanosRESUMO
PURPOSE OF REVIEW: Truncating variants in the TTN gene (TTNtv) are frequently identified in patients with dilated cardiomyopathy (DCM) but are also present in apparently healthy people in the general population. Consequently, there is considerable uncertainty about what it means for any single individual if a TTNtv is found. The aim of this review is to summarize current evidence implicating TTNtv in DCM pathogenesis and to provide some interpretative guidelines for clinical management. RECENT FINDINGS: Next-generation sequencing studies have recently demonstrated that TTNtv are present in approximately one in five patients with DCM but also in up to 3% of individuals in the general population. These observations question whether TTNtv are sufficient alone to cause DCM and whether some TTNtv may be more deleterious than others. It has been suggested that functional effects of TTNtv can be predicted by their location in the titin protein, with DCM-associated variants typically occurring in the A-band region and/or in exons that are highly utilized across the range of titin isoforms. Recent data from animal and cell models suggest that developmental defects in the structural assembly of titin-deficient sarcomeres provide a template for mechanical stress-induced myocardial dysfunction during later life. SUMMARY: Not all TTNtv are equal, and variants in constitutively expressed exons have the greatest likelihood of pathogenicity. The clinical significance of high-impact TTNtv is likely to differ according to patient context and each individual's unique suite of background genetic factors, comorbidities, and lifestyle factors. TTNtv identified in patients with DCM can be expected to have a major role in disease pathogenesis, but whether unaffected individuals with TTNtv will develop DCM is less certain.
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Genetic variation is an important determinant of atrial fibrillation (AF) susceptibility. Numerous rare variants in protein-coding sequences of genes have been associated with AF in families and in early-onset cases, and chromosomal loci harbouring common risk variants have been mapped in AF cohorts. Many of these loci are in non-coding regions of the human genome and are thought to contain regulatory sequences that modulate gene expression. Disease genes implicated to date have predominantly encoded cardiac ion channels, with predicted mutation effects on the atrial action potential duration. More recent studies have expanded the spectrum of disease-associated genes to include myocardial structural components and have highlighted an unsuspected role for cardiac transcription factors. These paradigm-shifting discoveries suggest that abnormalities of atrial specification arising during cardiac development might provide a template for AF in later adult life. With the escalating pace of variant discovery, there is an increasing need for mechanistic studies not only to evaluate single variants, but also to determine the collective effects of each person's burden of rare and common genetic variants, co-morbidities and lifestyle factors on the atrial substrate for arrhythmogenesis. Elucidation of an individual's genetic predisposition and modifiable environmental risk factors will facilitate personalised approaches to AF treatment.
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Fibrilação Atrial/genética , Predisposição Genética para Doença , Canais Iônicos/genética , Fatores de Transcrição/genética , Fibrilação Atrial/metabolismo , Variação Genética , Humanos , Canais Iônicos/metabolismoRESUMO
The two-pore domain potassium (K(+)) channel TWIK-1 (or K2P1.1) contributes to background K(+) conductance in diverse cell types. TWIK-1, encoded by the KCNK1 gene, is present in the human heart with robust expression in the atria, however its physiological significance is unknown. To evaluate the cardiac effects of TWIK-1 deficiency, we studied zebrafish embryos after knockdown of the two KCNK1 orthologues, kcnk1a and kcnk1b. Knockdown of kcnk1a or kcnk1b individually caused bradycardia and atrial dilation (p<0.001 vs. controls), while ventricular stroke volume was preserved. Combined knockdown of both kcnk1a and kcnk1b resulted in a more severe phenotype, which was partially reversed by co-injection of wild-type human KCNK1 mRNA, but not by a dominant negative variant of human KCNK1 mRNA. To determine whether genetic variants in KCNK1 might cause atrial fibrillation (AF), we sequenced protein-coding regions in two independent cohorts of patients (373 subjects) and identified three non-synonymous variants, p.R171H, p.I198M and p.G236S, that were all located in highly conserved amino acid residues. In transfected mammalian cells, zebrafish and wild-type human TWIK-1 channels had a similar cellular distribution with predominant localization in the endosomal compartment. Two-electrode voltage-clamp experiments using Xenopus oocytes showed that both zebrafish and wild-type human TWIK-1 channels produced K(+) currents that are sensitive to external K(+) concentration as well as acidic pH. There were no effects of the three KCNK1 variants on cellular localization, current amplitude or reversal potential at pH7.4 or pH6. Our data indicate that TWIK-1 has a highly conserved role in cardiac function and is required for normal heart rate and atrial morphology. Despite the functional importance of TWIK-1 in the atrium, genetic variation in KCNK1 is not a common primary cause of human AF.
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Remodelamento Atrial/genética , Estudos de Associação Genética , Átrios do Coração/metabolismo , Frequência Cardíaca/genética , Canais de Potássio de Domínios Poros em Tandem/genética , Adulto , Idoso , Animais , Fibrilação Atrial/genética , Fibrilação Atrial/metabolismo , Fibrilação Atrial/patologia , Fibrilação Atrial/fisiopatologia , Feminino , Expressão Gênica , Técnicas de Inativação de Genes , Variação Genética , Átrios do Coração/anatomia & histologia , Átrios do Coração/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Linhagem , Canais de Potássio de Domínios Poros em Tandem/deficiência , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Transporte Proteico , Fatores de Risco , Peixe-ZebraRESUMO
The two-pore domain potassium channel, K2P3.1 (TASK-1) modulates background conductance in isolated human atrial cardiomyocytes and has been proposed as a potential drug target for atrial fibrillation (AF). TASK-1 knockout mice have a predominantly ventricular phenotype however, and effects of TASK-1 inactivation on atrial structure and function have yet to be demonstrated in vivo. The extent to which genetic variation in KCNK3, that encodes TASK-1, might be a determinant of susceptibility to AF is also unknown. To address these questions, we first evaluated the effects of transient knockdown of the zebrafish kcnk3a and kcnk3b genes and cardiac phenotypes were evaluated using videomicroscopy. Combined kcnk3a and kcnk3b knockdown in 72 hour post fertilization embryos resulted in lower heart rate (p<0.001), marked increase in atrial diameter (p<0.001), and mild increase in end-diastolic ventricular diameter (p=0.01) when compared with control-injected embryos. We next performed genetic screening of KCNK3 in two independent AF cohorts (373 subjects) and identified three novel KCNK3 variants. Two of these variants, present in one proband with familial AF, were located at adjacent nucleotides in the Kozak sequence and reduced expression of an engineered reporter. A third missense variant, V123L, in a patient with lone AF, reduced resting membrane potential and altered pH sensitivity in patch-clamp experiments, with structural modeling predicting instability in the vicinity of the TASK-1 pore. These in vitro data suggest that the double Kozak variants and V123L will have loss-of-function effects on ITASK. Cardiac action potential modeling predicted that reduced ITASK prolongs atrial action potential duration, and that this is potentiated by reciprocal changes in activity of other ion channel currents. Our findings demonstrate the functional importance of ITASK in the atrium and suggest that inactivation of TASK-1 may have diverse effects on atrial size and electrophysiological properties that can contribute to an arrhythmogenic substrate.
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Fibrilação Atrial/genética , Variação Genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Motivos de Aminoácidos , Animais , Fibrilação Atrial/fisiopatologia , Células CHO , Cricetulus , Predisposição Genética para Doença , Átrios do Coração/anatomia & histologia , Átrios do Coração/fisiopatologia , Humanos , Modelos Animais , Modelos Moleculares , Peixe-ZebraRESUMO
The recent exponential increase in human genetic studies due to the advances of next generation sequencing has generated unprecedented numbers of new gene variants. Determining which of these are causative of human disease is a major challenge. In-vitro studies and murine models have been used to study inherited cardiac arrhythmias but have several limitations. Zebrafish models provide an attractive alternative for modeling human heart disease due to similarities in cardiac electrophysiology and contraction, together with ease of genetic manipulation, external development and optical transparency. Although zebrafish cardiac mutants and morphants have been widely used to study loss and knockdown of zebrafish gene function, the phenotypic effects of human dominant-negative gene mutations expressed in transgenic zebrafish have not been evaluated. The aim of this study was to generate and characterize a transgenic zebrafish arrhythmia model harboring the pathogenic human cardiac sodium channel mutation SCN5A-D1275N, that has been robustly associated with a range of cardiac phenotypes, including conduction disease, sinus node dysfunction, atrial and ventricular arrhythmias, and dilated cardiomyopathy in humans and in mice. Stable transgenic fish with cardiac expression of human SCN5A were generated using Tol2-mediated transgenesis and cardiac phenotypes were analyzed using video microscopy and ECG. Here we show that transgenic zebrafish expressing the SCN5A-D1275N mutation, but not wild-type SCN5A, exhibit bradycardia, conduction-system abnormalities and premature death. We furthermore show that SCN5A-WT, and to a lesser degree SCN5A-D1275N, are able to compensate the loss of endogenous zebrafish cardiac sodium channels, indicating that the basic pathways, through which SCN5A acts, are conserved in teleosts. This proof-of-principle study suggests that zebrafish may be highly useful in vivo models to differentiate functional from benign human genetic variants in cardiac ion channel genes in a time- and cost-efficient manner. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes".
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Bradicardia/genética , Sistema de Condução Cardíaco/anormalidades , Canal de Sódio Disparado por Voltagem NAV1.5/biossíntese , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Bradicardia/fisiopatologia , Modelos Animais de Doenças , Frequência Cardíaca , Humanos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Penetrância , FenótipoRESUMO
Dilated cardiomyopathy (DCM) is a common heart muscle disorder that frequently leads to heart failure, arrhythmias, and death. While DCM is often heritable, disease-causing mutations are identified in only ~30% of cases. In a forward genetic mutagenesis screen, we identified a novel zebrafish mutant, heart and head (hahvcc43), characterized by early-onset cardiomyopathy and craniofacial defects. Linkage analysis and next-generation sequencing identified a nonsense variant in the highly conserved scfd1 gene, also known as sly1, that encodes sec1 family domain-containing 1. Sec1/Munc18 proteins, such as Scfd1, are involved in membrane fusion regulating endoplasmic reticulum (ER)/Golgi transport. CRISPR/Cas9-engineered scfd1vcc44 null mutants showed severe cardiac and craniofacial defects and embryonic lethality that recapitulated the phenotype of hahvcc43 mutants. Electron micrographs of scfd1-depleted cardiomyocytes showed reduced myofibril width and sarcomere density, as well as reticular network disorganization and fragmentation of Golgi stacks. Furthermore, quantitative PCR analysis showed upregulation of ER stress response and apoptosis markers. Both heterozygous hahvcc43 mutants and scfd1vcc44 mutants survived to adulthood, showing chamber dilation and reduced ventricular contraction. Collectively, our data implicate scfd1 loss-of-function as the genetic defect at the hahvcc43 locus and provide new insights into the role of scfd1 in cardiac development and function.
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Dilated cardiomyopathy (DCM) is a common heart muscle disorder characterized by ventricular dilation and contractile dysfunction that is associated with significant morbidity and mortality. New insights into disease mechanisms and strategies for treatment and prevention are urgently needed. Truncating variants in the TTN gene, which encodes the giant sarcomeric protein titin (TTNtv), are the most common genetic cause of DCM, but exactly how TTNtv promote cardiomyocyte dysfunction is not known. Although rodent models have been widely used to investigate titin biology, they have had limited utility for TTNtv-related DCM. In recent years, zebrafish (Danio rerio) have emerged as a powerful alternative model system for studying titin function in the healthy and diseased heart. Optically transparent embryonic zebrafish models have demonstrated key roles of titin in sarcomere assembly and cardiac development. The increasing availability of sophisticated imaging tools for assessment of heart function in adult zebrafish has revolutionized the field and opened new opportunities for modelling human genetic disorders. Genetically modified zebrafish that carry a human A-band TTNtv have now been generated and shown to spontaneously develop DCM with age. This zebrafish model will be a valuable resource for elucidating the phenotype modifying effects of genetic and environmental factors, and for exploring new drug therapies.
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BACKGROUND: KCNMA1 encodes the α-subunit of the large-conductance Ca2+-activated K+ channel, KCa1.1, and lies within a linkage interval for atrial fibrillation (AF). Insights into the cardiac functions of KCa1.1 are limited, and KCNMA1 has not been investigated as an AF candidate gene. METHODS: The KCNMA1 gene was sequenced in 118 patients with familial AF. The role of KCa1.1 in normal cardiac structure and function was evaluated in humans, mice, zebrafish, and fly. A novel KCNMA1 variant was functionally characterized. RESULTS: A complex KCNMA1 variant was identified in 1 kindred with AF. To evaluate potential disease mechanisms, we first evaluated the distribution of KCa1.1 in normal hearts using immunostaining and immunogold electron microscopy. KCa1.1 was seen throughout the atria and ventricles in humans and mice, with strong expression in the sinus node. In an ex vivo murine sinoatrial node preparation, addition of the KCa1.1 antagonist, paxilline, blunted the increase in beating rate induced by adrenergic receptor stimulation. Knockdown of the KCa1.1 ortholog, kcnma1b, in zebrafish embryos resulted in sinus bradycardia with dilatation and reduced contraction of the atrium and ventricle. Genetic inactivation of the Drosophila KCa1.1 ortholog, slo, systemically or in adult stages, also slowed the heartbeat and produced fibrillatory cardiac contractions. Electrophysiological characterization of slo-deficient flies revealed bursts of action potentials, reflecting increased events of fibrillatory arrhythmias. Flies with cardiac-specific overexpression of the human KCNMA1 mutant also showed increased heart period and bursts of action potentials, similar to the KCa1.1 loss-of-function models. CONCLUSIONS: Our data point to a highly conserved role of KCa1.1 in sinus node function in humans, mice, zebrafish, and fly and suggest that KCa1.1 loss of function may predispose to AF.
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Fibrilação Atrial/patologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Nó Sinoatrial/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Fibrilação Atrial/genética , Função Atrial/efeitos dos fármacos , Função Atrial/fisiologia , Embrião não Mamífero/metabolismo , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Humanos , Indóis/química , Indóis/metabolismo , Indóis/farmacologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Camundongos , Contração Miocárdica , Linhagem , Polimorfismo Genético , Interferência de RNA , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacologia , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Precision medicine promises to dramatically improve patient outcomes and reduce health care costs through a shift in focus from disease treatment to prevention and individualized therapies. For families with inherited cardiomyopathies, efforts to date have been directed toward discovery and functional characterization of single disease-causing variants. With advances in sequencing, the cataloging of personal genetic variation has been expedited, providing improved insights into the key importance of the genes in which variants occur. These advances have propelled seminal opportunities for successful variant-targeted disease-reversing therapy. New challenges have also emerged-particularly interpretation of the rapidly rising numbers of "variants of unknown significance." For treatments based on patient genotype to be feasible on a wider scale, these obstacles need to be overcome. Here the authors focus on genetics of dilated cardiomyopathy and provide a roadmap for implementing genomic information into future patient management.
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Cardiomiopatia Dilatada/genética , Predisposição Genética para Doença , Testes Genéticos/métodos , Genômica/métodos , Medicina de Precisão/métodos , Cardiomiopatia Dilatada/diagnóstico , Genótipo , Humanos , FenótipoRESUMO
Zebrafish are increasingly used as a vertebrate model to study human cardiovascular disorders. Although heart structure and function are readily visualized in zebrafish embryos because of their optical transparency, the lack of effective tools for evaluating the hearts of older, nontransparent fish has been a major limiting factor. The recent development of high-frequency echocardiography has been an important advance for in vivo cardiac assessment, but it necessitates anesthesia and has limited ability to study acute interventions. We report the development of an alternative experimental ex vivo technique for quantifying heart size and function that resembles the Langendorff heart preparations that have been widely used in mammalian models. Dissected adult zebrafish hearts were perfused with a calcium-containing buffer, and a beat frequency was maintained with electrical stimulation. The impact of pacing frequency, flow rate and perfusate calcium concentration on ventricular performance (including end-diastolic and end-systolic volumes, ejection fraction, radial strain, and maximal velocities of shortening and relaxation) were evaluated and optimal conditions defined. We determined the effects of age on heart function in wild-type male and female zebrafish, and successfully detected hypercontractile and hypocontractile responses after adrenergic stimulation or doxorubicin treatment, respectively. Good correlations were found between indices of cardiac contractility obtained with high-frequency echocardiography and with the ex vivo technique in a subset of fish studied with both methods. The ex vivo beating heart preparation is a valuable addition to the cardiac function tool kit that will expand the use of adult zebrafish for cardiovascular research.
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Envelhecimento/fisiologia , Coração/fisiologia , Perfusão/métodos , Peixe-Zebra/fisiologia , Animais , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/fisiopatologia , Doxorrubicina/efeitos adversos , Eletrocardiografia , Feminino , Ventrículos do Coração/anatomia & histologia , Masculino , Contração Miocárdica , Tamanho do ÓrgãoRESUMO
Background Truncating variants in the TTN gene ( TTNtv) are common in patients with dilated cardiomyopathy (DCM) but also occur in the general population. Whether TTNtv are sufficient to cause DCM or require a second hit for DCM manifestation is an important clinical issue. Methods We generated a zebrafish model of an A-band TTNtv identified in 2 human DCM families in which early-onset disease appeared to be precipitated by ventricular volume overload. Cardiac phenotypes were serially assessed from 0 to 12 months using video microscopy, high-frequency echocardiography, and histopathologic analysis. The effects of sustained hemodynamic stress resulting from an anemia-induced hyperdynamic state were also evaluated. Results Homozygous ttna mutants had severe cardiac dysmorphogenesis and premature death, whereas heterozygous mutants ( ttnatv/+) survived into adulthood and spontaneously developed DCM. Six-month-old ttnatv/+ fish had reduced baseline ventricular systolic function and failed to mount a hypercontractile response when challenged by hemodynamic stress. Pulsed wave and tissue Doppler analysis also revealed unsuspected ventricular diastolic dysfunction in ttnatv/+ fish with prolonged isovolumic relaxation and increased diastolic passive stiffness in the absence of myocardial fibrosis. These defects reduced diastolic reserve under stress conditions and resulted in disproportionately greater atrial dilation than observed in wild-type fish. Conclusions Heterozygosity for A-band titin truncation is sufficient to cause DCM in adult zebrafish. Abnormalities of systolic and diastolic reserve in titin-truncated fish reduce stress tolerance and may contribute to a substrate for atrial arrhythmogenesis. These data suggest that hemodynamic stress may be an important modifiable risk factor in human TTNtv-related DCM.
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Cardiomiopatia Dilatada/genética , Conectina/genética , Hemodinâmica/genética , Estresse Fisiológico/genética , Adaptação Biológica/genética , Adolescente , Adulto , Idoso , Animais , Animais Geneticamente Modificados , Cardiomiopatia Dilatada/patologia , Cardiomiopatia Dilatada/fisiopatologia , Embrião não Mamífero , Feminino , Estudos de Associação Genética , Coração/embriologia , Coração/crescimento & desenvolvimento , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Sarcômeros/patologia , Deleção de Sequência , Volume Sistólico/genética , Adulto Jovem , Peixe-ZebraRESUMO
The zebrafish (Danio rerio) is an increasingly popular model organism in cardiovascular research. Major insights into cardiac developmental processes have been gained by studies of embryonic zebrafish. However, the utility of zebrafish for modeling adult-onset heart disease has been limited by a lack of robust methods for in vivo evaluation of cardiac function. We established a physiological protocol for underwater zebrafish echocardiography using high frequency ultrasound, and evaluated its reliability in detecting altered cardiac function in two disease models. Serial assessment of cardiac function was performed in wild-type zebrafish aged 3 to 12â months and the effects of anesthetic agents, age, sex and background strain were evaluated. There was a varying extent of bradycardia and ventricular contractile impairment with different anesthetic drugs and doses, with tricaine 0.75â mmolâ l-1 having a relatively more favorable profile. When compared with males, female fish were larger and had more measurement variability. Although age-related increments in ventricular chamber size were greater in females than males, there were no sex differences when data were normalized to body size. Systolic ventricular function was similar in both sexes at all time points, but differences in diastolic function were evident from 6â months onwards. Wild-type fish of both sexes showed a reliance on atrial contraction for ventricular diastolic filling. Echocardiographic evaluation of adult zebrafish with diphtheria toxin-induced myocarditis or anemia-induced volume overload accurately identified ventricular dilation and altered contraction, with suites of B-mode, ventricular strain, pulsed-wave Doppler and tissue Doppler indices showing concordant changes indicative of myocardial hypocontractility or hypercontractility, respectively. Repeatability, intra-observer and inter-observer correlations for echocardiographic measurements were high. We demonstrate that high frequency echocardiography allows reliable in vivo cardiac assessment in adult zebrafish and make recommendations for optimizing data acquisition and analysis. This enabling technology reveals new insights into zebrafish cardiac physiology and provides an imaging platform for zebrafish-based translational research.
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Envelhecimento/fisiologia , Ecocardiografia/normas , Cardiopatias/diagnóstico por imagem , Cardiopatias/fisiopatologia , Testes de Função Cardíaca/normas , Peixe-Zebra/fisiologia , Pontos de Referência Anatômicos , Anemia/patologia , Anestesia , Animais , Tamanho Corporal , Toxina Diftérica , Modelos Animais de Doenças , Estudos de Viabilidade , Feminino , Ventrículos do Coração/diagnóstico por imagem , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Masculino , Contração Miocárdica , Miocardite/diagnóstico por imagem , Miocardite/patologia , Miocardite/fisiopatologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Tamanho do Órgão , Padrões de Referência , Reprodutibilidade dos Testes , Função Ventricular EsquerdaRESUMO
Mechanoelectrical feedback may increase arrhythmia susceptibility, but the molecular mechanisms are incompletely understood. This study showed that mechanical stretch altered the localization, protein levels, and function of the cation-selective transient receptor potential channel (TRPC)-6 in atrial endocardial cells in humans, pigs, and mice. In endocardial/myocardial cross-talk studies, addition of media from porcine atrial endocardium (AE) cells altered the calcium (Ca2+) transient characteristics of human-induced pluripotent stem cell-derived cardiomyocytes. These changes did not occur with media from stretched AE cells. Our data suggested that endocardial TRPC-6-dependent paracrine signaling may modulate myocardial Ca2+ homeostasis under basal conditions and protect against stretch-induced atrial arrhythmias.
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Integrin-linked kinase (ILK) is an essential component of the cardiac mechanical stretch sensor and is bound in a protein complex with parvin and PINCH proteins, the so-called ILK-PINCH-parvin (IPP) complex. We have recently shown that inactivation of ILK or ß-parvin activity leads to heart failure in zebrafish via reduced protein kinase B (PKB/Akt) activation. Here, we show that PINCH proteins localize at sarcomeric Z disks and costameres in the zebrafish heart and skeletal muscle. To investigate the in vivo role of PINCH proteins for IPP complex stability and PKB signaling within the vertebrate heart, we inactivated PINCH1 and PINCH2 in zebrafish. Inactivation of either PINCH isoform independently leads to instability of ILK, loss of stretch-responsive anf and vegf expression, and progressive heart failure. The predominant cause of heart failure in PINCH morphants seems to be loss of PKB activity, since PKB phosphorylation at serine 473 is significantly reduced in PINCH-deficient hearts and overexpression of constitutively active PKB reconstitutes cardiac function in PINCH morphants. These findings highlight the essential function of PINCH proteins in controlling cardiac contractility by granting IPP/PKB-mediated signaling.
Assuntos
Proteínas Musculares/fisiologia , Contração Miocárdica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Insuficiência Cardíaca/etiologia , Músculo Esquelético , Miocárdio/química , Miocárdio/enzimologia , Fosforilação , Sarcômeros/química , Peixe-ZebraRESUMO
Yeast frequenin (Frq1), a small N-myristoylated EF-hand protein, activates phosphatidylinositol 4-kinase Pik1. The NMR structure of Ca2+-bound Frq1 complexed to an N-terminal Pik1 fragment (residues 121-174) was determined. The Frq1 main chain is similar to that in free Frq1 and related proteins in the same branch of the calmodulin superfamily. The myristoyl group and first eight residues of Frq1 are solvent-exposed, and Ca2+ binds the second, third, and fourth EF-hands, which associate to create a groove with two pockets. The Pik1 peptide forms two helices (125-135 and 156-169) connected by a 20-residue loop. Side chains in the Pik1 N-terminal helix (Val-127, Ala-128, Val-131, Leu-132, and Leu-135) interact with solvent-exposed residues in the Frq1 C-terminal pocket (Leu-101, Trp-103, Val-125, Leu-138, Ile-152, and Leu-155); side chains in the Pik1 C-terminal helix (Ala-157, Ala-159, Leu-160, Val-161, Met-165, and Met-167) contact solvent-exposed residues in the Frq1 N-terminal pocket (Trp-30, Phe-34, Phe-48, Ile-51, Tyr-52, Phe-55, Phe-85, and Leu-89). This defined complex confirms that residues in Pik1 pinpointed as necessary for Frq1 binding by site-directed mutagenesis are indeed sufficient for binding. Removal of the Pik1 N-terminal region (residues 8-760) from its catalytic domain (residues 792-1066) abolishes lipid kinase activity, inconsistent with Frq1 binding simply relieving an autoinhibitory constraint. Deletion of the lipid kinase unique motif (residues 35-110) also eliminates Pik1 activity. In the complex, binding of Ca2+-bound Frq1 forces the Pik1 chain into a U-turn. Frq1 may activate Pik1 by facilitating membrane targeting via the exposed N-myristoyl group and by imposing a structural transition that promotes association of the lipid kinase unique motif with the kinase domain.