RESUMEN
Transcription initiates at the core promoter, which contains distinct core promoter elements. Here, we highlight the complexity of transcriptional regulation by outlining the effect of core promoter-dependent regulation on embryonic development and the proper function of an organism. We demonstrate in vivo the importance of the downstream core promoter element (DPE) in complex heart formation in Drosophila. Pioneering a novel approach using both CRISPR and nascent transcriptomics, we show the effects of mutating a single core promoter element within the natural context. Specifically, we targeted the downstream core promoter element (DPE) of the endogenous tin gene, encoding the Tinman transcription factor, a homologue of human NKX2-5 associated with congenital heart diseases. The 7â bp substitution mutation results in massive perturbation of the Tinman regulatory network that orchestrates dorsal musculature, which is manifested as physiological and anatomical changes in the cardiac system, impaired specific activity features, and significantly compromised viability of adult flies. Thus, a single motif can have a critical impact on embryogenesis and, in the case of DPE, functional heart formation.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Regulación del Desarrollo de la Expresión Génica , Corazón , Regiones Promotoras Genéticas , Factores de Transcripción , Animales , Regiones Promotoras Genéticas/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Corazón/embriología , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Drosophila melanogaster/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Proteína Homeótica Nkx-2.5/genética , Proteína Homeótica Nkx-2.5/metabolismo , Mutación/genética , Desarrollo Embrionario/genética , Humanos , Transcripción Genética , Proteínas Represoras , TransactivadoresRESUMEN
Heart diseases are the most common causes of morbidity and death in humans. Using cardiac-specific RNAi-silencing in Drosophila, we knocked down 7061 evolutionarily conserved genes under conditions of stress. We present a first global roadmap of pathways potentially playing conserved roles in the cardiovascular system. One critical pathway identified was the CCR4-Not complex implicated in transcriptional and posttranscriptional regulatory mechanisms. Silencing of CCR4-Not components in adult Drosophila resulted in myofibrillar disarray and dilated cardiomyopathy. Heterozygous not3 knockout mice showed spontaneous impairment of cardiac contractility and increased susceptibility to heart failure. These heart defects were reversed via inhibition of HDACs, suggesting a mechanistic link to epigenetic chromatin remodeling. In humans, we show that a common NOT3 SNP correlates with altered cardiac QT intervals, a known cause of potentially lethal ventricular tachyarrhythmias. Thus, our functional genome-wide screen in Drosophila can identify candidates that directly translate into conserved mammalian genes involved in heart function.
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Drosophila melanogaster/fisiología , Modelos Animales , Animales , Cardiomiopatías/genética , Cardiomiopatías/fisiopatología , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Femenino , Estudio de Asociación del Genoma Completo , Corazón/embriología , Corazón/fisiología , Humanos , Masculino , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas , Interferencia de ARNRESUMEN
Establishing a catalog of Congenital Heart Disease (CHD) genes and identifying functional networks would improve our understanding of its oligogenic underpinnings. Our studies identified protein biogenesis cofactors Nascent polypeptide-Associated Complex (NAC) and Signal-Recognition-Particle (SRP) as disease candidates and novel regulators of cardiac differentiation and morphogenesis. Knockdown (KD) of the alpha- (Nacα) or beta-subunit (bicaudal, bic) of NAC in the developing Drosophila heart disrupted cardiac developmental remodeling resulting in a fly with no heart. Heart loss was rescued by combined KD of Nacα with the posterior patterning Hox gene Abd-B. Consistent with a central role for this interaction in cardiogenesis, KD of Nacα in cardiac progenitors derived from human iPSCs impaired cardiac differentiation while co-KD with human HOXC12 and HOXD12 rescued this phenotype. Our data suggest that Nacα KD preprograms cardioblasts in the embryo for abortive remodeling later during metamorphosis, as Nacα KD during translation-intensive larval growth or pupal remodeling only causes moderate heart defects. KD of SRP subunits in the developing fly heart produced phenotypes that targeted specific segments and cell types, again suggesting cardiac-specific and spatially regulated activities. Together, we demonstrated directed function for NAC and SRP in heart development, and that regulation of NAC function depends on Hox genes.
Asunto(s)
Ribosomas , Partícula de Reconocimiento de Señal , Animales , Humanos , Partícula de Reconocimiento de Señal/metabolismo , Ribosomas/metabolismo , Corazón , Genes Homeobox , Drosophila/genética , Drosophila/metabolismo , Péptidos/metabolismoRESUMEN
Cardiomyopathy is a common disease of cardiac muscle that negatively affects cardiac function. HDAC3 commonly functions as corepressor by removing acetyl moieties from histone tails. However, a deacetylase-independent role of HDAC3 has also been described. Cardiac deletion of HDAC3 causes reduced cardiac contractility accompanied by lipid accumulation, but the molecular function of HDAC3 in cardiomyopathy remains unknown. We have used powerful genetic tools in Drosophila to investigate the enzymatic and nonenzymatic roles of HDAC3 in cardiomyopathy. Using the Drosophila heart model, we showed that cardiac-specific HDAC3 knockdown (KD) leads to prolonged systoles and reduced cardiac contractility. Immunohistochemistry revealed structural abnormalities characterized by myofiber disruption in HDAC3 KD hearts. Cardiac-specific HDAC3 KD showed increased levels of whole-body triglycerides and increased fibrosis. The introduction of deacetylase-dead HDAC3 mutant in HDAC3 KD background showed comparable results with wild-type HDAC3 in aspects of contractility and Pericardin deposition. However, deacetylase-dead HDAC3 mutants failed to improve triglyceride accumulation. Our data indicate that HDAC3 plays a deacetylase-independent role in maintaining cardiac contractility and preventing Pericardin deposition as well as a deacetylase-dependent role to maintain triglyceride homeostasis.
Asunto(s)
Cardiomiopatías , Modelos Animales de Enfermedad , Proteínas de Drosophila , Drosophila melanogaster , Histona Desacetilasas , Animales , Cardiomiopatías/enzimología , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Cardiomiopatías/fisiopatología , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Técnicas de Silenciamiento del Gen , Corazón/fisiología , Histona Desacetilasas/deficiencia , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Histonas/química , Histonas/metabolismo , Miocardio/metabolismo , Triglicéridos/metabolismo , HomeostasisRESUMEN
Genetics is a significant factor contributing to congenital heart disease (CHD), but our understanding of the genetic players and networks involved in CHD pathogenesis is limited. Here, we searched for de novo copy number variations (CNVs) in a cohort of 167 CHD patients to identify DNA segments containing potential pathogenic genes. Our search focused on new candidate disease genes within 19 deleted de novo CNVs, which did not cover known CHD genes. For this study, we developed an integrated high-throughput phenotypical platform to probe for defects in cardiogenesis and cardiac output in human induced pluripotent stem cell (iPSC)-derived multipotent cardiac progenitor (MCPs) cells and, in parallel, in the Drosophila in vivo heart model. Notably, knockdown (KD) in MCPs of RPL13, a ribosomal gene and SON, an RNA splicing cofactor, reduced proliferation and differentiation of cardiomyocytes, while increasing fibroblasts. In the fly, heart-specific RpL13 KD, predominantly at embryonic stages, resulted in a striking 'no heart' phenotype. KD of Son and Pdss2, among others, caused structural and functional defects, including reduced or abolished contractility, respectively. In summary, using a combination of human genetics and cardiac model systems, we identified new genes as candidates for causing human CHD, with particular emphasis on ribosomal genes, such as RPL13. This powerful, novel approach of combining cardiac phenotyping in human MCPs and in the in vivo Drosophila heart at high throughput will allow for testing large numbers of CHD candidates, based on patient genomic data, and for building upon existing genetic networks involved in heart development and disease.
Asunto(s)
Variaciones en el Número de Copia de ADN , Cardiopatías Congénitas/genética , Miocardio/citología , Proteínas de Neoplasias/genética , Proteínas Ribosómicas/genética , Animales , Células Cultivadas , Estudios de Cohortes , Modelos Animales de Enfermedad , Drosophila , Femenino , Redes Reguladoras de Genes , Humanos , Células Madre Pluripotentes Inducidas/química , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/patología , Masculino , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/química , Miocitos Cardíacos/citología , Miocitos Cardíacos/patología , Estudios RetrospectivosRESUMEN
[This corrects the article DOI: 10.1371/journal.pgen.1007386.].
RESUMEN
Recent evidence suggests that the presence of more than one pathogenic mutation in a single patient is more common than previously anticipated. One of the challenges hereby is to dissect the contribution of each gene mutation, for which animal models such as Drosophila can provide a valuable aid. Here, we identified three families with mutations in ADD3, encoding for adducin-γ, with intellectual disability, microcephaly, cataracts and skeletal defects. In one of the families with additional cardiomyopathy and steroid-resistant nephrotic syndrome (SRNS), we found a homozygous variant in KAT2B, encoding the lysine acetyltransferase 2B, with impact on KAT2B protein levels in patient fibroblasts, suggesting that this second mutation might contribute to the increased disease spectrum. In order to define the contribution of ADD3 and KAT2B mutations for the patient phenotype, we performed functional experiments in the Drosophila model. We found that both mutations were unable to fully rescue the viability of the respective null mutants of the Drosophila homologs, hts and Gcn5, suggesting that they are indeed pathogenic in flies. While the KAT2B/Gcn5 mutation additionally showed a significantly reduced ability to rescue morphological and functional defects of cardiomyocytes and nephrocytes (podocyte-like cells), this was not the case for the ADD3 mutant rescue. Yet, the simultaneous knockdown of KAT2B and ADD3 synergistically impaired kidney and heart function in flies as well as the adhesion and migration capacity of cultured human podocytes, indicating that mutations in both genes may be required for the full clinical manifestation. Altogether, our studies describe the expansion of the phenotypic spectrum in ADD3 deficiency associated with a homozygous likely pathogenic KAT2B variant and thereby identify KAT2B as a susceptibility gene for kidney and heart disease in ADD3-associated disorders.
Asunto(s)
Proteínas de Unión a Calmodulina/genética , Drosophila/genética , Mutación , Factores de Transcripción p300-CBP/genética , Anomalías Múltiples/genética , Adolescente , Adulto , Animales , Proteínas de Unión a Calmodulina/deficiencia , Línea Celular , Células Cultivadas , Análisis Mutacional de ADN , Proteínas de Drosophila/genética , Femenino , Cardiopatías/genética , Homocigoto , Humanos , Fallo Renal Crónico/genética , Masculino , Linaje , FenotipoRESUMEN
PURPOSE OF REVIEW: Our understanding of the fundamental cellular and molecular factors leading to atrial fibrillation (AF) remains stagnant despite significant advancement in ablation and device technologies. Diagnosis and prevention strategies fall behind that of treatment, but expanding knowledge in AF genetics holds the potential to drive progress. We aim to review how an understanding of the genetic contributions to AF can guide an approach to individualized risk stratification and novel avenues in drug discovery. RECENT FINDINGS: Rare familial forms of AF identified monogenic contributions to the development of AF. Genome-wide association studies (GWAS) further identified single-nucleotide polymorphisms (SNPs) suggesting polygenic and multiplex nature of this common disease. Polygenic risk scores accounting for the multitude of associated SNPs that each confer mildly elevated risk have been developed to translate genetic information into clinical practice, though shortcomings remain. Additionally, novel laboratory methods have been empowered by recent genetic findings to enhance drug discovery efforts. AF is increasingly recognized as a disease with a significant genetic component. With expanding sequencing technologies and accessibility, polygenic risk scores can help identify high risk individuals. Advancement in digital health tools, artificial intelligence and machine learning based on standard electrocardiograms, and genomic driven drug discovery may be integrated to deliver a sophisticated level of precision medicine in this modern era of emphasis on prevention. Randomized, prospective studies to demonstrate clinical benefits of these available tools are needed to validate this approach.
Asunto(s)
Fibrilación Atrial , Inteligencia Artificial , Fibrilación Atrial/genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Genómica , Humanos , Estudios ProspectivosRESUMEN
Mutations in the Parkin gene (PARK2) have been linked to a recessive form of Parkinson's disease (PD) characterized by the loss of dopaminergic neurons in the substantia nigra. Deficiencies of mitochondrial respiratory chain complex I activity have been observed in the substantia nigra of PD patients, and loss of Parkin results in the reduction of complex I activity shown in various cell and animal models. Using co-immunoprecipitation and proximity ligation assays on endogenous proteins, we demonstrate that Parkin interacts with mitochondrial Stomatin-like protein 2 (SLP-2), which also binds the mitochondrial lipid cardiolipin and functions in the assembly of respiratory chain proteins. SH-SY5Y cells with a stable knockdown of Parkin or SLP-2, as well as induced pluripotent stem cell-derived neurons from Parkin mutation carriers, showed decreased complex I activity and altered mitochondrial network morphology. Importantly, induced expression of SLP-2 corrected for these mitochondrial alterations caused by reduced Parkin function in these cells. In-vivo Drosophila studies showed a genetic interaction of Parkin and SLP-2, and further, tissue-specific or global overexpression of SLP-2 transgenes rescued parkin mutant phenotypes, in particular loss of dopaminergic neurons, mitochondrial network structure, reduced ATP production, and flight and motor dysfunction. The physical and genetic interaction between Parkin and SLP-2 and the compensatory potential of SLP-2 suggest a functional epistatic relationship to Parkin and a protective role of SLP-2 in neurons. This finding places further emphasis on the significance of Parkin for the maintenance of mitochondrial function in neurons and provides a novel target for therapeutic strategies.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Proteínas de la Membrana/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Anciano , Animales , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Neuronas Dopaminérgicas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Complejo I de Transporte de Electrón/metabolismo , Femenino , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Persona de Mediana Edad , Mitocondrias/metabolismo , Mutación , Neuronas/metabolismo , Enfermedad de Parkinson/genética , Sustancia Negra/metabolismoRESUMEN
The epidemic of obesity and diabetes is causing an increased incidence of dyslipidemia-related heart failure. While the primary etiology of lipotoxic cardiomyopathy is an elevation of lipid levels resulting from an imbalance in energy availability and expenditure, increasing evidence suggests a relationship between dysregulation of membrane phospholipid homeostasis and lipid-induced cardiomyopathy. In the present study, we report that the Drosophila easily shocked (eas) mutants that harbor a disturbance in phosphatidylethanolamine (PE) synthesis display tachycardia and defects in cardiac relaxation and are prone to developing cardiac arrest and fibrillation under stress. The eas mutant hearts exhibit elevated concentrations of triglycerides, suggestive of a metabolic, diabetic-like heart phenotype. Moreover, the low PE levels in eas flies mimic the effects of cholesterol deficiency in vertebrates by stimulating the Drosophila sterol regulatory element-binding protein (dSREBP) pathway. Significantly, cardiac-specific elevation of dSREBP signaling adversely affects heart function, reflecting the cardiac eas phenotype, whereas suppressing dSREBP or lipogenic target gene function in eas hearts rescues the cardiac hyperlipidemia and heart function disorders. These findings suggest that dysregulated phospholipid signaling that alters SREBP activity contributes to the progression of impaired heart function in flies and identifies a potential link to lipotoxic cardiac diseases in humans.
Asunto(s)
Drosophila melanogaster/fisiología , Homeostasis/fisiología , Metabolismo de los Lípidos/fisiología , Fosfolípidos/metabolismo , Transducción de Señal , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Corazón/fisiología , Homeostasis/genética , Metabolismo de los Lípidos/genética , Masculino , Mutación , Proteínas de Unión a los Elementos Reguladores de Esteroles/genética , Estrés FisiológicoRESUMEN
Cardiac hypertrophy is initiated as an adaptive response to sustained overload but progresses pathologically as heart failure ensues. Here we report that genetic loss of APJ, a G-protein-coupled receptor, confers resistance to chronic pressure overload by markedly reducing myocardial hypertrophy and heart failure. In contrast, mice lacking apelin (the endogenous APJ ligand) remain sensitive, suggesting an apelin-independent function of APJ. Freshly isolated APJ-null cardiomyocytes exhibit an attenuated response to stretch, indicating that APJ is a mechanosensor. Activation of APJ by stretch increases cardiomyocyte cell size and induces molecular markers of hypertrophy. Whereas apelin stimulates APJ to activate Gαi and elicits a protective response, stretch signals in an APJ-dependent, G-protein-independent fashion to induce hypertrophy. Stretch-mediated hypertrophy is prevented by knockdown of ß-arrestins or by pharmacological doses of apelin acting through Gαi. Taken together, our data indicate that APJ is a bifunctional receptor for both mechanical stretch and the endogenous peptide apelin. By sensing the balance between these stimuli, APJ occupies a pivotal point linking sustained overload to cardiomyocyte hypertrophy.
Asunto(s)
Cardiomegalia/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Adipoquinas , Animales , Aorta/patología , Apelina , Receptores de Apelina , Arrestinas/deficiencia , Arrestinas/genética , Arrestinas/metabolismo , Presión Sanguínea , Cardiomegalia/patología , Cardiomegalia/fisiopatología , Cardiomegalia/prevención & control , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Péptidos y Proteínas de Señalización Intercelular/deficiencia , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Masculino , Mecanorreceptores/metabolismo , Mecanotransducción Celular/efectos de los fármacos , Mecanotransducción Celular/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/genética , Transducción de Señal/efectos de los fármacos , beta-ArrestinasRESUMEN
The inflow tracts of the embryonic Drosophila cardiac tube, termed ostia, arise in its posterior three segments from cardiac cells that co-express the homeotic transcription factor Abdominal-A (abdA), the orphan nuclear receptor Seven-up (Svp), and the signaling molecule Wingless (Wg). To define the roles of these factors in inflow tract development, we assessed their function in inflow tract formation. We demonstrate, using several criteria, that abdA, svp, and wg are each critical for normal inflow tract formation. We further show that Wg acts in an autocrine manner to impact ostia fate, and that it mediates this effect at least partially through the canonical Wg signaling pathway. By contrast, neither wg expression nor Wg signaling are sufficient for inflow tract formation when expressed in anterior Svp cells that do not normally form inflow tracts in the embryo. Instead, ectopic abd-A expression throughout the cardiac tube is required for the formation of ectopic inflow tracts, indicating that autocrine Wg signaling must be supplemented by additional Hox-dependent factors to effect inflow tract formation. Taken together, these studies define important cellular and molecular events that contribute to cardiac inflow tract development in Drosophila. Given the broad conservation of the cardiac regulatory network through evolution, our studies provide insight into mechanisms of cardiac development in higher animals.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila melanogaster/embriología , Regulación del Desarrollo de la Expresión Génica , Corazón/embriología , Transducción de Señal , Animales , Aorta/embriología , Cruzamientos Genéticos , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Perfilación de la Expresión Génica , Genes Homeobox/genética , Genes de Insecto , Marcadores Genéticos , Genotipo , Homocigoto , Hibridación in Situ , Proteínas Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Factores de Transcripción/metabolismo , Proteína Wnt1/metabolismoRESUMEN
KEY POINTS: Changes in gene expression that occur within hours of exposure to hypoxia in in vivo skeletal muscles remain unexplored. Two hours of hypoxia caused significant down-regulation of extracellular matrix genes followed by a shift at 6 h to altered expression of genes associated with the nuclear lumen while respiratory and blood gases were stabilized. Enrichment analysis of mRNAs classified by stability rates suggests an attenuation of post-transcriptional regulation within hours of hypoxic exposure, where PI3K-Akt signalling was suggested to have a nodal role by pathway analysis. Experimental measurements and bioinformatic analyses suggested that the dephosphorylation of Akt after 2 h of hypoxic exposure might deactivate RNA-binding protein BRF1, hence resulting in the selective degradation of mRNAs. ABSTRACT: The effects of acute hypoxia have been widely studied, but there are few studies of transcriptional responses to hours of hypoxia in vivo, especially in hypoxia-tolerant tissues like skeletal muscles. We used RNA-seq to analyse gene expression in plantaris muscles while monitoring respiration, arterial blood gases, and blood glucose in mice exposed to 8% O2 for 2 or 6 h. Rapid decreases in blood gases and a slower reduction in blood glucose suggest stress, which was accompanied by widespread changes in gene expression. Early down-regulation of genes associated with the extracellular matrix was followed by a shift to genes associated with the nuclear lumen. Most of the early down-regulated genes had mRNA half-lives longer than 2 h, suggesting a role for post-transcriptional regulation. These transcriptional changes were enriched in signalling pathways in which the PI3K-Akt signalling pathway was identified as a hub. Our analyses indicated that gene targets of PI3K-Akt but not HIF were enriched in early transcriptional responses to hypoxia. Among the PI3K-Akt targets, 75% could be explained by a deactivation of adenylate-uridylate-rich element (ARE)-binding protein BRF1, a target of PI3K-Akt. Consistent decreases in the phosphorylation of Akt and BRF1 were experimentally confirmed following 2 h of hypoxia. These results suggest that the PI3K-Akt signalling pathway might play a role in responses induced by acute hypoxia in skeletal muscles, partially through the dephosphorylation of ARE-binding protein BRF1.
Asunto(s)
Hipoxia/genética , Músculo Esquelético/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Animales , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Hipoxia/metabolismo , Masculino , Ratones Endogámicos C57BL , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
Cardiac drug discovery is hampered by the reliance on non-human animal and cellular models with inadequate throughput and physiological fidelity to accurately identify new targets and test novel therapeutic strategies. Similarly, adverse drug effects on the heart are challenging to model, contributing to costly failure of drugs during development and even after market launch. Human induced pluripotent stem cell derived cardiac tissue represents a potentially powerful means to model aspects of heart physiology relevant to disease and adverse drug effects, providing both the human context and throughput needed to improve the efficiency of drug development. Here we review emerging technologies for high throughput measurements of cardiomyocyte physiology, and comment on the promises and challenges of using iPSC-derived cardiomyocytes to model disease and introduce the human context into early stages of drug discovery. This article is part of a Special Issue entitled: Cardiomyocyte biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.
Asunto(s)
Fármacos Cardiovasculares/farmacología , Descubrimiento de Drogas/métodos , Cardiopatías/tratamiento farmacológico , Ensayos Analíticos de Alto Rendimiento , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Animales , Fármacos Cardiovasculares/toxicidad , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Predisposición Genética a la Enfermedad , Cardiopatías/inducido químicamente , Cardiopatías/genética , Cardiopatías/metabolismo , Cardiopatías/patología , Cardiopatías/fisiopatología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Fenotipo , Medición de RiesgoRESUMEN
The heart is a muscle with high energy demands. Hence, most patients with mitochondrial disease produced by defects in the oxidative phosphorylation (OXPHOS) system are susceptible to cardiac involvement. The presentation of mitochondrial cardiomyopathy includes hypertrophic, dilated and left ventricular noncompaction, but the molecular mechanisms involved in cardiac impairment are unknown. One of the most frequent OXPHOS defects in humans frequently associated with cardiomyopathy is cytochrome c oxidase (COX) deficiency caused by mutations in COX assembly factors such as Sco1 and Sco2. To investigate the molecular mechanisms that underlie the cardiomyopathy associated with Sco deficiency, we have heart specifically interfered scox expression, the single Drosophila Sco orthologue. Cardiac-specific knockdown of scox reduces fly lifespan, and it severely compromises heart function and structure, producing dilated cardiomyopathy. Cardiomyocytes with low levels of scox have a significant reduction in COX activity and they undergo a metabolic switch from OXPHOS to glycolysis, mimicking the clinical features found in patients harbouring Sco mutations. The major cardiac defects observed are produced by a significant increase in apoptosis, which is dp53-dependent. Genetic and molecular evidence strongly suggest that dp53 is directly involved in the development of the cardiomyopathy induced by scox deficiency. Remarkably, apoptosis is enhanced in the muscle and liver of Sco2 knock-out mice, clearly suggesting that cell death is a key feature of the COX deficiencies produced by mutations in Sco genes in humans.
Asunto(s)
Apoptosis , Cardiomiopatías/enzimología , Modelos Animales de Enfermedad , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Miocardio/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Cardiomiopatías/fisiopatología , Drosophila/enzimología , Drosophila/genética , Proteínas de Drosophila/genética , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Ageing in humans is associated with a significant increase in the prevalence of cardiovascular disease. We still do not fully understand the molecular mechanisms underpinning this correlation. However, a number of insights into which genes control cardiac ageing have come from studying hearts of the fruit fly, Drosophila melanogaster. The fly's simple heart tube has similar molecular structure and basic physiology to the human heart. Also, both fly and human hearts experience significant age-related morphological and functional decline. Studies on the fly heart have highlighted the involvement of key nutrient sensing, ion channel and sarcomeric genes in cardiac ageing. Many of these genes have also been implicated in ageing of the mammalian heart. Genes that increase oxidative stress, or are linked to cardiac hypertrophy or neurodegenerative diseases in mammals also affect cardiac ageing in the fruit fly. Moreover, fly studies have demonstrated the potential of exercise and statins to treat age-related cardiac disease. These results show the value of Drosophila as a model to discover the genetic causes of human cardiac ageing.
Asunto(s)
Envejecimiento/genética , Drosophila melanogaster/genética , Corazón/crecimiento & desarrollo , Miocardio/metabolismo , Envejecimiento/metabolismo , Envejecimiento/fisiología , Animales , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , HumanosRESUMEN
The Drosophila heart tube represents a structure that similarly to vertebrates' primary heart tube exhibits a large lumen; the mechanisms promoting heart tube morphology in both Drosophila and vertebrates are poorly understood. We identified Multiplexin (Mp), the Drosophila orthologue of mammalian Collagen-XV/XVIII, and the only structural heart-specific protein described so far in Drosophila, as necessary and sufficient for shaping the heart tube lumen, but not that of the aorta. Mp is expressed specifically at the stage of heart tube closure, in a polarized fashion, uniquely along the cardioblasts luminal membrane, and its absence results in an extremely small heart tube lumen. Importantly, Mp forms a protein complex with Slit, and interacts genetically with both slit and robo in the formation of the heart tube. Overexpression of Mp in cardioblasts promotes a large heart lumen in a Slit-dependent manner. Moreover, Mp alters Slit distribution, and promotes the formation of multiple Slit endocytic vesicles, similarly to the effect of overexpression of Robo in these cells. Our data are consistent with Mp-dependent enhancement of Slit/Robo activity and signaling, presumably by affecting Slit protein stabilization, specifically at the lumen side of the heart tube. This activity results with a Slit-dependent, local reduction of F-actin levels at the heart luminal membrane, necessary for forming the large heart tube lumen. Consequently, lack of Mp results in decreased diastolic capacity, leading to reduced heart contractility, as measured in live fly hearts. In summary, these findings show that the polarized localization of Mp controls the direction, timing, and presumably the extent of Slit/Robo activity and signaling at the luminal membrane of the heart cardioblasts. This regulation is essential for the morphogenetic changes that sculpt the heart tube in Drosophila, and possibly in forming the vertebrates primary heart tube.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/genética , Colágeno/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Morfogénesis/genética , Proteínas del Tejido Nervioso/metabolismo , Receptores Inmunológicos/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Aorta/crecimiento & desarrollo , Aorta/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Colágeno/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Corazón/anatomía & histología , Corazón/crecimiento & desarrollo , Proteínas del Tejido Nervioso/genética , Receptores Inmunológicos/genética , Transducción de Señal , Proteínas RoundaboutRESUMEN
Amyloid-like inclusions have been associated with Huntington's disease (HD), which is caused by expanded polyglutamine repeats in the Huntingtin protein. HD patients exhibit a high incidence of cardiovascular events, presumably as a result of accumulation of toxic amyloid-like inclusions. We have generated a Drosophila model of cardiac amyloidosis that exhibits accumulation of PolyQ aggregates and oxidative stress in myocardial cells, upon heart-specific expression of Huntingtin protein fragments (Htt-PolyQ) with disease-causing poly-glutamine repeats (PolyQ-46, PolyQ-72, and PolyQ-102). Cardiac expression of GFP-tagged Htt-PolyQs resulted in PolyQ length-dependent functional defects that included increased incidence of arrhythmias and extreme cardiac dilation, accompanied by a significant decrease in contractility. Structural and ultrastructural analysis of the myocardial cells revealed reduced myofibrillar content, myofibrillar disorganization, mitochondrial defects and the presence of PolyQ-GFP positive aggregates. Cardiac-specific expression of disease causing Poly-Q also shortens lifespan of flies dramatically. To further confirm the involvement of oxidative stress or protein unfolding and to understand the mechanism of PolyQ induced cardiomyopathy, we co-expressed expanded PolyQ-72 with the antioxidant superoxide dismutase (SOD) or the myosin chaperone UNC-45. Co-expression of SOD suppressed PolyQ-72 induced mitochondrial defects and partially suppressed aggregation as well as myofibrillar disorganization. However, co-expression of UNC-45 dramatically suppressed PolyQ-72 induced aggregation and partially suppressed myofibrillar disorganization. Moreover, co-expression of both UNC-45 and SOD more efficiently suppressed GFP-positive aggregates, myofibrillar disorganization and physiological cardiac defects induced by PolyQ-72 than did either treatment alone. Our results demonstrate that mutant-PolyQ induces aggregates, disrupts the sarcomeric organization of contractile proteins, leads to mitochondrial dysfunction and increases oxidative stress in cardiomyocytes leading to abnormal cardiac function. We conclude that modulation of both protein unfolding and oxidative stress pathways in the Drosophila heart model can ameliorate the detrimental PolyQ effects, thus providing unique insights into the genetic mechanisms underlying amyloid-induced cardiac failure in HD patients.
Asunto(s)
Amiloidosis/genética , Enfermedad de Huntington/genética , Proteínas Asociadas a Microtúbulos/genética , Péptidos/genética , Pliegue de Proteína , Amiloide/metabolismo , Amiloide/toxicidad , Amiloidosis/complicaciones , Amiloidosis/metabolismo , Amiloidosis/patología , Animales , Modelos Animales de Enfermedad , Proteínas de Drosophila , Drosophila melanogaster/genética , Drosophila melanogaster/fisiología , Regulación de la Expresión Génica , Corazón/fisiopatología , Humanos , Proteína Huntingtina , Enfermedad de Huntington/complicaciones , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Mutación , Estrés Oxidativo/genética , Transducción de Señal , Superóxido Dismutasa/biosíntesisRESUMEN
Diets high in carbohydrates have long been linked to progressive heart dysfunction, yet the mechanisms by which chronic high sugar leads to heart failure remain poorly understood. Here we combine diet, genetics, and physiology to establish an adult Drosophila melanogaster model of chronic high sugar-induced heart disease. We demonstrate deterioration of heart function accompanied by fibrosis-like collagen accumulation, insulin signaling defects, and fat accumulation. The result was a shorter life span that was more severe in the presence of reduced insulin and P38 signaling. We provide evidence of a role for hexosamine flux, a metabolic pathway accessed by glucose. Increased hexosamine flux led to heart function defects and structural damage; conversely, cardiac-specific reduction of pathway activity prevented sugar-induced heart dysfunction. Our data establish Drosophila as a useful system for exploring specific aspects of diet-induced heart dysfunction and emphasize enzymes within the hexosamine biosynthetic pathway as candidate therapeutic targets.