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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.
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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
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.
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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
Muscle contraction is regulated by the movement of end-to-end-linked troponin-tropomyosin complexes over the thin filament surface, which uncovers or blocks myosin binding sites along F-actin. The N-terminal half of troponin T (TnT), TNT1, independently promotes tropomyosin-based, steric inhibition of acto-myosin associations, in vitro. Recent structural models additionally suggest TNT1 may restrain the uniform, regulatory translocation of tropomyosin. Therefore, TnT potentially contributes to striated muscle relaxation; however, the in vivo functional relevance and molecular basis of this noncanonical role remain unclear. Impaired relaxation is a hallmark of hypertrophic and restrictive cardiomyopathies (HCM and RCM). Investigating the effects of cardiomyopathy-causing mutations could help clarify TNT1's enigmatic inhibitory property. We tested the hypothesis that coupling of TNT1 with tropomyosin's end-to-end overlap region helps anchor tropomyosin to an inhibitory position on F-actin, where it deters myosin binding at rest, and that, correspondingly, cross-bridge cycling is defectively suppressed under diastolic/low Ca2+ conditions in the presence of HCM/RCM lesions. The impact of TNT1 mutations on Drosophila cardiac performance, rat myofibrillar and cardiomyocyte properties, and human TNT1's propensity to inhibit myosin-driven, F-actin-tropomyosin motility were evaluated. Our data collectively demonstrate that removing conserved, charged residues in TNT1's tropomyosin-binding domain impairs TnT's contribution to inhibitory tropomyosin positioning and relaxation. Thus, TNT1 may modulate acto-myosin activity by optimizing F-actin-tropomyosin interfacial contacts and by binding to actin, which restrict tropomyosin's movement to activating configurations. HCM/RCM mutations, therefore, highlight TNT1's essential role in contractile regulation by diminishing its tropomyosin-anchoring effects, potentially serving as the initial trigger of pathology in our animal models and humans.
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Cardiomiopatías/metabolismo , Mutación/genética , Tropomiosina , Troponina T , Actinas/química , Actinas/metabolismo , Animales , Calcio/metabolismo , Diástole/genética , Diástole/fisiología , Proteínas de Drosophila , Humanos , Miocitos Cardíacos/química , Miocitos Cardíacos/metabolismo , Unión Proteica , Ratas , Tropomiosina/química , Tropomiosina/metabolismo , Troponina T/química , Troponina T/genética , Troponina T/metabolismoRESUMEN
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.
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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
microRNAs (miRNAs) are a class of endogenous regulatory RNAs that play a key role in myriad biological processes. Upon transcription, primary miRNA transcripts are sequentially processed by Drosha and Dicer ribonucleases into ~22-24 nt miRNAs. Subsequently, miRNAs are incorporated into the RNA-induced silencing complexes (RISCs) that contain Argonaute (AGO) family proteins and guide RISC to target RNAs via complementary base pairing, leading to post-transcriptional gene silencing by a combination of translation inhibition and mRNA destabilization. Select pre-mRNA splicing factors have been implicated in small RNA-mediated gene silencing pathways in fission yeast, worms, flies and mammals, but the underlying molecular mechanisms are not well understood. Here, we show that SmD1, a core component of the Drosophila small nuclear ribonucleoprotein particle (snRNP) implicated in splicing, is required for miRNA biogenesis and function. SmD1 interacts with both the microprocessor component Pasha and pri-miRNAs, and is indispensable for optimal miRNA biogenesis. Depletion of SmD1 impairs the assembly and function of the miRISC without significantly affecting the expression of major canonical miRNA pathway components. Moreover, SmD1 physically and functionally associates with components of the miRISC, including AGO1 and GW182. Notably, miRNA defects resulting from SmD1 silencing can be uncoupled from defects in pre-mRNA splicing, and the miRNA and splicing machineries are physically and functionally distinct entities. Finally, photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis identifies numerous SmD1-binding events across the transcriptome and reveals direct SmD1-miRNA interactions. Our study suggests that SmD1 plays a direct role in miRNA-mediated gene silencing independently of its pre-mRNA splicing activity and indicates that the dual roles of splicing factors in post-transcriptional gene regulation may be evolutionarily widespread.
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Drosophila melanogaster/genética , Proteínas de Insectos/genética , MicroARNs/genética , Procesamiento Postranscripcional del ARN/genética , Empalme del ARN/genética , Animales , Proteínas Argonautas/genética , Secuencia de Bases , Línea Celular , Proteínas de Drosophila/genética , Regulación de la Expresión Génica , Silenciador del Gen , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Complejo Silenciador Inducido por ARN/genética , Análisis de Secuencia de ARNRESUMEN
In recent years the Drosophila heart has become an established model for many different aspects of human cardiac disease. This model has allowed identification of disease-causing mechanisms underlying congenital heart disease and cardiomyopathies and has permitted the study of underlying genetic, metabolic and age-related contributions to heart function. In this review we discuss methods currently employed in the analysis of the Drosophila heart structure and function, such as optical methods to infer heart function and performance, electrophysiological and mechanical approaches to characterize cardiac tissue properties, and conclude with histological techniques used in the study of heart development and adult structure.
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Envejecimiento/genética , Biología Evolutiva/métodos , Proteínas de Drosophila/biosíntesis , Corazón/crecimiento & desarrollo , Envejecimiento/patología , Animales , Drosophila/genética , Drosophila/crecimiento & desarrollo , HumanosRESUMEN
Neddylation is a posttranslational modification that plays important roles in regulating protein structure and function by covalently conjugating NEDD8, an ubiquitin-like small molecule, to the substrate. Here, we report that Parkinson's disease (PD)-related parkin and PINK1 are NEDD8 conjugated. Neddylation of parkin and PINK1 results in increased E3 ligase activity of parkin and selective stabilization of the 55 kDa PINK1 fragment. Expression of dAPP-BP1, a NEDD8 activation enzyme subunit, in Drosophila suppresses abnormalities induced by dPINK1 RNAi. PD neurotoxin MPP(+) inhibits neddylation of both parkin and PINK1. NEDD8 immunoreactivity is associated with Lewy bodies in midbrain dopaminergic neurons of PD patients. Together, these results suggest that parkin and PINK1 are regulated by neddylation and that impaired NEDD8 modification of these proteins likely contributes to PD pathogenesis.
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Proteínas de Drosophila/genética , Proteínas Serina-Treonina Quinasas/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Células Cultivadas , Neuronas Dopaminérgicas/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Humanos , Inmunohistoquímica , Cuerpos de Lewy/metabolismo , Mesencéfalo/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , Transfección , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Transverse (T)-tubules - vast, tubulated domains of the muscle plasma membrane - are critical to maintain healthy skeletal and heart contractions. How the intricate T-tubule membranes are formed is not well understood, with challenges to systematically interrogate in muscle. We established the use of intact Drosophila larval body wall muscles as an ideal system to discover mechanisms that sculpt and maintain the T-tubule membrane network. A muscle-targeted genetic screen identified specific phosphoinositide lipid regulators necessary for T-tubule organization and muscle function. We show that a PI4KIIIα - Skittles/PIP5K pathway is needed for T-tubule localized PI(4)P to PI(4,5)P 2 synthesis, T-tubule organization, calcium regulation, and muscle and heart rate functions. Muscles deficient for PI4KIIIα or Amphiphysin , the homolog of human BIN1 , similarly exhibited specific loss of transversal T-tubule membranes and dyad junctions, yet retained longitudinal membranes and the associated dyads. Our results highlight the power of live muscle studies, uncovering distinct mechanisms and functions for sub-compartments of the T-tubule network relevant to human myopathy. Summary: T-tubules - vast, tubulated domains of the muscle plasma membrane - are critical to maintain skeletal and heart contractions. Fujita et al . establish genetic screens and assays in intact Drosophila muscles that uncover PI(4,5)P 2 regulation critical for T-tubule maintenance and function. Key Findings: PI4KIIIα is required for muscle T-tubule formation and larval mobility. A PI4KIIIα-Sktl pathway promotes PI(4)P and PI(4,5)P 2 function at T-tubules. PI4KIIIα is necessary for calcium dynamics and transversal but not longitudinal dyads. Disruption of PI(4,5)P 2 function in fly heart leads to fragmented T-tubules and abnormal heart rate.
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CREB-regulated transcription co-activator (CRTC) is activated by Calcineurin (CaN) to regulate gluconeogenic genes. CaN also has roles in cardiac hypertrophy. Here, we explore a cardiac-autonomous role for CRTC in cardiac hypertrophy. In Drosophila, CRTC mutants exhibit severe cardiac restriction, myofibrillar disorganization, fibrosis, and tachycardia. Cardiac-specific CRTC knockdown (KD) phenocopies mutants, and cardiac overexpression causes hypertrophy. CaN-induced hypertrophy in Drosophila is reduced in CRTC mutants, suggesting that CRTC mediates the effects. RNA sequencing (RNA-seq) of CRTC-KD and -overexpressing hearts reveals contraregulation of metabolic genes. Genes with conserved CREB sites include the fly ortholog of Sarcalumenin, a Ca2+-binding protein. Cardiac manipulation of this gene recapitulates the CRTC-KD and -overexpression phenotypes. CRTC KD in zebrafish also causes cardiac restriction, and CRTC KD in human induced cardiomyocytes causes a reduction in Srl expression and increased action potential duration. Our data from three model systems suggest that CaN-CRTC-Sarcalumenin signaling represents an alternate, conserved pathway underlying cardiac function and hypertrophy.
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Transcription is initiated at the core promoter, which confers specific functions depending on the unique combination of core promoter elements. The downstream core promoter element (DPE) is found in many genes related to heart and mesodermal development. However, the function of these core promoter elements has thus far been studied primarily in isolated, in vitro or reporter gene settings. tinman (tin) encodes a key transcription factor that regulates the formation of the dorsal musculature and heart. Pioneering a novel approach utilizing both CRISPR and nascent transcriptomics, we show that a substitution mutation of the functional tin DPE motif within the natural context of the core promoter results in a massive perturbation of Tinman's regulatory network orchestrating dorsal musculature and heart formation. Mutation of endogenous tin DPE reduced the expression of tin and distinct target genes, resulting in significantly reduced viability and an overall decrease in adult heart function. We demonstrate the feasibility and importance of characterizing DNA sequence elements in vivo in their natural context, and accentuate the critical impact a single DPE motif has during Drosophila embryogenesis and functional heart formation.
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Obesity and type 2 diabetes are at epidemic levels and a significant proportion of these patients are diagnosed with left ventricular hypertrophy. CREB R egulated T ranscription C o-activator ( CRTC ) is a key regulator of metabolism in mammalian hepatocytes, where it is activated by calcineurin (CaN) to increase expression of gluconeogenic genes. CaN is known its role in pathological cardiac hypertrophy, however, a role for CRTC in the heart has not been identified. In Drosophila , CRTC null mutants have little body fat and exhibit severe cardiac restriction, myofibrillar disorganization, cardiac fibrosis and tachycardia, all hallmarks of heart disease. Cardiac-specific knockdown of CRTC , or its coactivator CREBb , mimicked the reduced body fat and heart defects of CRTC null mutants. Comparative gene expression in CRTC loss- or gain-of-function fly hearts revealed contra-regulation of genes involved in glucose, fatty acid, and amino acid metabolism, suggesting that CRTC also acts as a metabolic switch in the heart. Among the contra-regulated genes with conserved CREB binding sites, we identified the fly ortholog of Sarcalumenin, which is a Ca 2+ -binding protein in the sarcoplasmic reticulum. Cardiac knockdown recapitulated the loss of CRTC cardiac restriction and fibrotic phenotypes, suggesting it is a downstream effector of CRTC we named thinman ( tmn ). Importantly, cardiac overexpression of either CaN or CRTC in flies caused hypertrophy that was reversed in a CRTC mutant background, suggesting CRTC mediates hypertrophy downstream of CaN, perhaps as an alternative to NFAT. CRTC novel role in the heart is likely conserved in vertebrates as knockdown in zebrafish also caused cardiac restriction, as in fl ies. These data suggest that CRTC is involved in myocardial cell maintenance and that CaN-CRTC- Sarcalumenin/ tmn signaling represents a novel and conserved pathway underlying cardiac hypertrophy.
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Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease (CHD) with a likely oligogenic etiology, but our understanding of the genetic complexities and pathogenic mechanisms leading to HLHS is limited. We performed whole genome sequencing (WGS) on 183 HLHS patient-parent trios to identify candidate genes, which were functionally tested in the Drosophila heart model. Bioinformatic analysis of WGS data from an index family of a HLHS proband born to consanguineous parents prioritized 9 candidate genes with rare, predicted damaging homozygous variants. Of them, cardiac-specific knockdown (KD) of mitochondrial MICOS complex subunit dCHCHD3/6 resulted in drastically compromised heart contractility, diminished levels of sarcomeric actin and myosin, reduced cardiac ATP levels, and mitochondrial fission-fusion defects. These defects were similar to those inflicted by cardiac KD of ATP synthase subunits of the electron transport chain (ETC), consistent with the MICOS complex's role in maintaining cristae morphology and ETC assembly. Five additional HLHS probands harbored rare, predicted damaging variants in CHCHD3 or CHCHD6. Hypothesizing an oligogenic basis for HLHS, we tested 60 additional prioritized candidate genes from these patients for genetic interactions with CHCHD3/6 in sensitized fly hearts. Moderate KD of CHCHD3/6 in combination with Cdk12 (activator of RNA polymerase II), RNF149 (goliath, E3 ubiquitin ligase), or SPTBN1 (ß-Spectrin, scaffolding protein) caused synergistic heart defects, suggesting the likely involvement of diverse pathways in HLHS. Further elucidation of novel candidate genes and genetic interactions of potentially disease-contributing pathways is expected to lead to a better understanding of HLHS and other CHDs.
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Cardiopatías Congénitas , Síndrome del Corazón Izquierdo Hipoplásico , Humanos , Síndrome del Corazón Izquierdo Hipoplásico/genética , Actomiosina , Biología Computacional , Adenosina Trifosfato , Proteínas MitocondrialesRESUMEN
Deciphering the genetic architecture of human cardiac disorders is of fundamental importance but their underlying complexity is a major hurdle. We investigated the natural variation of cardiac performance in the sequenced inbred lines of the Drosophila Genetic Reference Panel (DGRP). Genome-wide associations studies (GWAS) identified genetic networks associated with natural variation of cardiac traits which were used to gain insights as to the molecular and cellular processes affected. Non-coding variants that we identified were used to map potential regulatory non-coding regions, which in turn were employed to predict transcription factors (TFs) binding sites. Cognate TFs, many of which themselves bear polymorphisms associated with variations of cardiac performance, were also validated by heart-specific knockdown. Additionally, we showed that the natural variations associated with variability in cardiac performance affect a set of genes overlapping those associated with average traits but through different variants in the same genes. Furthermore, we showed that phenotypic variability was also associated with natural variation of gene regulatory networks. More importantly, we documented correlations between genes associated with cardiac phenotypes in both flies and humans, which supports a conserved genetic architecture regulating adult cardiac function from arthropods to mammals. Specifically, roles for PAX9 and EGR2 in the regulation of the cardiac rhythm were established in both models, illustrating that the characteristics of natural variations in cardiac function identified in Drosophila can accelerate discovery in humans.
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Drosophila melanogaster , Corazón , Sitios de Carácter Cuantitativo , Animales , Humanos , Drosophila melanogaster/fisiología , Redes Reguladoras de Genes , Variación Genética , Estudio de Asociación del Genoma Completo , Fenotipo , Corazón/fisiologíaRESUMEN
The cross talk between adipose tissue and the heart has an increasing importance for cardiac function under physiological and pathological conditions. This study characterizes the role of fat body lipolysis for cardiac function in Drosophila melanogaster. Perturbation of the function of the key lipolytic enzyme, brummer (bmm), an ortholog of the mammalian ATGL (adipose triglyceride lipase) exclusively in the fly's fat body, protected the heart against starvation-induced dysfunction. We further provide evidence that this protection is caused by the preservation of glycerolipid stores, resulting in a starvation-resistant maintenance of energy supply and adequate cardiac ATP synthesis. Finally, we suggest that alterations of lipolysis are tightly coupled to lipogenic processes, participating in the preservation of lipid energy substrates during starvation. Thus, we identified the inhibition of adipose tissue lipolysis and subsequent energy preservation as a protective mechanism against cardiac dysfunction during catabolic stress.
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Cardiolipin (CL) is a major cardiac mitochondrial phospholipid maintaining regular mitochondrial morphology and function in cardiomyocytes. Cardiac CL production includes its biosynthesis and a CL remodeling process. Here we studied the impact of CL biosynthesis and the enzyme cardiolipin synthase (CLS) on cardiac function. CLS and cardiac CL species were significantly downregulated in cardiomyocytes following catecholamine-induced cardiac damage in mice, accompanied by increased oxygen consumption rates, signs of oxidative stress, and mitochondrial uncoupling. RNAi-mediated cardiomyocyte-specific knockdown of CLS in Drosophila melanogaster resulted in marked cardiac dilatation, severe impairment of systolic performance, and slower diastolic filling velocity assessed by fluorescence-based heart imaging. Finally, we showed that CL72:8 is significantly decreased in cardiac samples from patients with heart failure with reduced ejection fraction (HFrEF). In summary, we identified CLS as a regulator of cardiac function. Considering the cardiac depletion of CL species in HFrEF, pharmacological targeting of CLS may be a promising therapeutic approach.
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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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Fibrilación Atrial/patología , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Nodo Sinoatrial/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Fibrilación Atrial/genética , Función Atrial/efectos de los fármacos , Función Atrial/fisiología , Embrión no Mamífero/metabolismo , Atrios Cardíacos/metabolismo , Atrios Cardíacos/patología , Humanos , Indoles/química , Indoles/metabolismo , Indoles/farmacología , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/antagonistas & inhibidores , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Ratones , Contracción Miocárdica , Linaje , Polimorfismo Genético , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/farmacología , Pez Cebra , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The outermost layer of the vertebrate heart originates from migratory mesothelial cells (epicardium) that give rise to coronary vascular smooth muscles and fibroblasts. The role of the epicardium in myocardial morphogenesis and establishment of normal heart function is still largely unknown. Here, we use Drosophila to investigate non-autonomous influences of epicardial-like tissue surrounding the heart tube on the structural and functional integrity of the myocardium. It has previously been shown that during Drosophila heart formation, mesodermal expression of the homeobox transcription factor even-skipped (eve) is required for specification of a subset of non-myocardial progenitors in the precardiac mesoderm. These progenitors may share some similarities with the vertebrate epicardium. To investigate a non-autonomous epicardial-like influence on myocardial physiology, we studied the consequences of reduced mesodermal Eve expression and epi/pericardial cell numbers on the maturation of the myocardial heart tube, its contractility, and acquisition of a normal heart rhythm in the Drosophila model. Targeting the eve repressor ladybird early (lbe) with the minimal eve mesodermal enhancer efficiently eliminates the mesodermal Eve lineages. These flies exhibit defects in heart structure, including a reduction in systolic and diastolic diameter (akin to 'restrictive cardiomyopathy'). They also exhibit an elevated incidence of arrhythmias and intermittent asystoles, as well as compromised performance under stress. These abnormalities are restored by eve reexpression or by lbe-RNAi co-overexpression. The data suggest that adult heart function in Drosophila is likely to be modulated non-autonomously, possibly by paracrine influences from neighboring cells, such as the epi/pericardium. Thus, Drosophila may serve as a model for finding genetic effectors of epicardial-myocardial interactions relevant to higher organisms.
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Diferenciación Celular/fisiología , Drosophila/fisiología , Animales , Tipificación del Cuerpo/fisiología , Drosophila/anatomía & histología , Drosophila/embriología , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiología , Epitelio/fisiología , Corazón/anatomía & histología , Corazón/embriología , Corazón/fisiología , Frecuencia Cardíaca , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Contracción Miocárdica/fisiología , Pericardio/citología , Pericardio/fisiología , Factores de Transcripción/genética , Factores de Transcripción/fisiologíaRESUMEN
Left-sided congenital heart defects (CHDs) are among the most common forms of congenital heart disease, but a disease-causing gene has only been identified in a minority of cases. Here, we identified a candidate gene for CHDs, KIF1A, that was associated with a chromosomal balanced translocation t(2;8)(q37;p11) in a patient with left-sided heart and aortic valve defects. The breakpoint was in the 5' untranslated region of the KIF1A gene at 2q37, which suggested that the break affected the levels of Kif1A gene expression. Transgenic fly lines overexpressing Kif1A specifically in the heart muscle (or all muscles) caused diminished cardiac contractility, myofibrillar disorganization, and heart valve defects, whereas cardiac knockdown had no effect on heart structure or function. Overexpression of Kif1A also caused increased collagen IV deposition in the fibrous network that normally surrounds the fly heart. Kif1A overexpression in C2C12 myoblasts resulted in specific displacement of the F-actin fibers, probably through a direct interaction with G-actin. These results point to a Kif1A-mediated disruption of F-actin organization as a potential mechanism for the pathogenesis in at least some human CHDs.
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The causal genetic underpinnings of congenital heart diseases, which are often complex and multigenic, are still far from understood. Moreover, there are also predominantly monogenic heart defects, such as cardiomyopathies, with known disease genes for the majority of cases. In this study, we identified mutations in myomesin 2 (MYOM2) in patients with Tetralogy of Fallot (TOF), the most common cyanotic heart malformation, as well as in patients with hypertrophic cardiomyopathy (HCM), who do not exhibit any mutations in the known disease genes. MYOM2 is a major component of the myofibrillar M-band of the sarcomere, and a hub gene within interactions of sarcomere genes. We show that patient-derived cardiomyocytes exhibit myofibrillar disarray and reduced passive force with increasing sarcomere lengths. Moreover, our comprehensive functional analyses in the Drosophila animal model reveal that the so far uncharacterized fly gene CG14964 [herein referred to as Drosophila myomesin and myosin binding protein (dMnM)] may be an ortholog of MYOM2, as well as other myosin binding proteins. Its partial loss of function or moderate cardiac knockdown results in cardiac dilation, whereas more severely reduced function causes a constricted phenotype and an increase in sarcomere myosin protein. Moreover, compound heterozygous combinations of CG14964 and the sarcomere gene Mhc (MYH6/7) exhibited synergistic genetic interactions. In summary, our results suggest that MYOM2 not only plays a critical role in maintaining robust heart function but may also be a candidate gene for heart diseases such as HCM and TOF, as it is clearly involved in the development of the heart.This article has an associated First Person interview with Emilie Auxerre-Plantié and Tanja Nielsen, joint first authors of the paper.
Asunto(s)
Cardiomiopatía Hipertrófica/genética , Conectina/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Estudios de Asociación Genética , Proteínas de la Membrana/genética , Tetralogía de Fallot/genética , Animales , Proteínas de Drosophila/metabolismo , Femenino , Humanos , Locomoción , Masculino , Proteínas de la Membrana/metabolismo , Músculos/metabolismo , Mutación/genética , Miocardio , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Miofibrillas/metabolismo , Miofibrillas/patología , Especificidad de Órganos , Unión Proteica , Mapeo de Interacción de ProteínasRESUMEN
Understanding the effects of microgravity on human organs is crucial to exploration of low-earth orbit, the moon, and beyond. Drosophila can be sent to space in large numbers to examine the effects of microgravity on heart structure and function, which is fundamentally conserved from flies to humans. Flies reared in microgravity exhibit cardiac constriction with myofibrillar remodeling and diminished output. RNA sequencing (RNA-seq) in isolated hearts revealed reduced expression of sarcomeric/extracellular matrix (ECM) genes and dramatically increased proteasomal gene expression, consistent with the observed compromised, smaller hearts and suggesting abnormal proteostasis. This was examined further on a second flight in which we found dramatically elevated proteasome aggregates co-localizing with increased amyloid and polyQ deposits. Remarkably, in long-QT causing sei/hERG mutants, proteasomal gene expression at 1g, although less than the wild-type expression, was nevertheless increased in microgravity. Therefore, cardiac remodeling and proteostatic stress may be a fundamental response of heart muscle to microgravity.