RESUMEN
All species continuously evolve short open reading frames (sORFs) that can be templated for protein synthesis and may provide raw materials for evolutionary adaptation. We analyzed the evolutionary origins of 7,264 recently cataloged human sORFs and found that most were evolutionarily young and had emerged de novo. We additionally identified 221 previously missed sORFs potentially translated into peptides of up to 15 amino acids-all of which are smaller than the smallest human microprotein annotated to date. To investigate the bioactivity of sORF-encoded small peptides and young microproteins, we subjected 266 candidates to a mass-spectrometry-based interactome screen with motif resolution. Based on these interactomes and additional cellular assays, we can associate several candidates with mRNA splicing, translational regulation, and endocytosis. Our work provides insights into the evolutionary origins and interaction potential of young and small proteins, thereby helping to elucidate this underexplored territory of the human proteome.
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Péptidos , Biosíntesis de Proteínas , Humanos , Sistemas de Lectura Abierta , Péptidos/genética , Proteómica , MicropéptidosRESUMEN
Cellular mRNA-binding proteins (mRBPs) are major posttranscriptional regulators of gene expression. Although many posttranslational modification sites in mRBPs have been identified, little is known about how these modifications regulate mRBP function. Here, we developed quantitative RNA-interactome capture (qRIC) to quantify the fraction of mRBPs pulled down with polyadenylated mRNAs. Combining qRIC with phosphoproteomics allowed us to systematically compare pull-down efficiencies of phosphorylated and nonphosphorylated forms of mRBPs. Almost 200 phosphorylation events affected pull-down efficiency compared with the unmodified mRBPs and thus have regulatory potential. Our data capture known regulatory phosphorylation sites in ELAVL1, SF3B1, and UPF1 and identify potential regulatory sites. Follow-up experiments on the splicing regulator RBM20 revealed multiple phosphorylation sites in the C-terminal disordered region affecting nucleocytoplasmic localization, association with cytoplasmic ribonucleoprotein granules, and alternative splicing. Together, we show that qRIC in conjunction with phosphoproteomics is a scalable method to identify functional posttranslational modification sites in mRBPs.
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Proteoma , Proteínas de Unión al ARN , Fosforilación , Proteoma/genética , Proteoma/metabolismo , ARN/metabolismo , Empalme del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
The heart has the ability to detect and respond to changes in mechanical load through a process called mechanotransduction. In this study, we focused on investigating the role of the cardiac-specific N2B element within the spring region of titin, which has been proposed to function as a mechanosensor. To assess its significance, we conducted experiments using N2B knockout (KO) mice and wildtype (WT) mice, subjecting them to three different conditions: 1) cardiac pressure overload induced by transverse aortic constriction (TAC), 2) volume overload caused by aortocaval fistula (ACF), and 3) exercise-induced hypertrophy through swimming. Under conditions of pressure overload (TAC), both genotypes exhibited similar hypertrophic responses. In contrast, WT mice displayed robust left ventricular hypertrophy after one week of volume overload (ACF), while the KO mice failed to undergo hypertrophy and experienced a high mortality rate. Similarly, swim exercise-induced hypertrophy was significantly reduced in the KO mice. RNA-Seq analysis revealed an abnormal ß-adrenergic response to volume overload in the KO mice, as well as a diminished response to isoproterenol-induced hypertrophy. Because it is known that the N2B element interacts with the four-and-a-half LIM domains 1 and 2 (FHL1 and FHL2) proteins, both of which have been associated with mechanotransduction, we evaluated these proteins. Interestingly, while volume-overload resulted in FHL1 protein expression levels that were comparable between KO and WT mice, FHL2 protein levels were reduced by over 90% in the KO mice compared to WT. This suggests that in response to volume overload, FHL2 might act as a signaling mediator between the N2B element and downstream signaling pathways. Overall, our study highlights the importance of the N2B element in mechanosensing during volume overload, both in physiological and pathological settings.
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Conectina , Mecanotransducción Celular , Ratones Noqueados , Animales , Ratones , Conectina/metabolismo , Conectina/genética , Hipertrofia Ventricular Izquierda/metabolismo , Hipertrofia Ventricular Izquierda/fisiopatología , Hipertrofia Ventricular Izquierda/genética , Miocardio/metabolismo , Miocardio/patología , Masculino , Condicionamiento Físico Animal , Proteínas con Homeodominio LIM/metabolismo , Proteínas con Homeodominio LIM/genética , Modelos Animales de Enfermedad , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas con Dominio LIM/metabolismo , Proteínas con Dominio LIM/genética , Proteínas Quinasas , Péptidos y Proteínas de Señalización IntracelularRESUMEN
Tissue dissociation, a crucial step in single-cell sample preparation, can alter the transcriptional state of a sample through the intrinsic cellular stress response. Here we demonstrate a general approach for measuring transcriptional response during sample preparation. In our method, transcripts made during dissociation are labeled for later identification upon sequencing. We found general as well as cell-type-specific dissociation response programs in zebrafish larvae, and we observed sample-to-sample variation in the dissociation response of mouse cardiomyocytes despite well-controlled experimental conditions. Finally, we showed that dissociation of the mouse hippocampus can lead to the artificial activation of microglia. In summary, our approach facilitates experimental optimization of dissociation procedures as well as computational removal of transcriptional perturbation response.
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ARN , Transcriptoma , Ratones , Animales , Pez Cebra/genética , Análisis de Secuencia de ARN/métodos , Microglía , Análisis de la Célula Individual , Perfilación de la Expresión Génica/métodosRESUMEN
A key question in receptor signaling is how specificity is realized, particularly when different receptors trigger the same biochemical pathway(s). A notable case is the two ß-adrenergic receptor (ß-AR) subtypes, ß1 and ß2, in cardiomyocytes. They are both coupled to stimulatory Gs proteins, mediate an increase in cyclic adenosine monophosphate (cAMP), and stimulate cardiac contractility; however, other effects, such as changes in gene transcription leading to cardiac hypertrophy, are prominent only for ß1-AR but not for ß2-AR. Here, we employ highly sensitive fluorescence spectroscopy approaches, in combination with a fluorescent ß-AR antagonist, to determine the presence and dynamics of the endogenous receptors on the outer plasma membrane as well as on the T-tubular network of intact adult cardiomyocytes. These techniques allow us to visualize that the ß2-AR is confined to and diffuses within the T-tubular network, as opposed to the ß1-AR, which is found to diffuse both on the outer plasma membrane as well as on the T-tubules. Upon overexpression of the ß2-AR, this compartmentalization is lost, and the receptors are also seen on the cell surface. Such receptor segregation depends on the development of the T-tubular network in adult cardiomyocytes since both the cardiomyoblast cell line H9c2 and the cardiomyocyte-differentiated human-induced pluripotent stem cells express the ß2-AR on the outer plasma membrane. These data support the notion that specific cell surface targeting of receptor subtypes can be the basis for distinct signaling and functional effects.
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Membrana Celular/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Imagen Molecular , Miocitos Cardíacos/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Animales , Línea Celular , Membrana Celular/genética , Humanos , Ratones , Ratones Transgénicos , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 2/genéticaRESUMEN
Arginine-serine (RS) domain(s) in splicing factors are critical for protein-protein interaction in pre-mRNA splicing. Phosphorylation of RS domain is important for splicing control and nucleocytoplasmic transport in the cell. RNA-binding motif 20 (RBM20) is a splicing factor primarily expressed in the heart. A previous study using phospho-antibody against RS domain showed that RS domain can be phosphorylated. However, its actual phosphorylation sites and function have not been characterized. Using middle-down mass spectrometry, we identified 16 phosphorylation sites, two of which (S638 and S640 in rats, or S637 and S639 in mice) were located in the RSRSP stretch in the RS domain. Mutations on S638 and S640 regulated splicing, promoted nucleocytoplasmic transport and protein-RNA condensates. Phosphomimetic mutations on S638 and S640 indicated that phosphorylation was not the major cause for RBM20 nucleocytoplasmic transport and condensation in vitro. We generated a S637A knock-in (KI) mouse model (Rbm20S637A ) and observed the reduced RBM20 phosphorylation. The KI mice exhibited aberrant gene splicing, protein condensates, and a dilated cardiomyopathy (DCM)-like phenotype. Transcriptomic profiling demonstrated that KI mice had altered expression and splicing of genes involving cardiac dysfunction, protein localization, and condensation. Our in vitro data showed that phosphorylation was not a direct cause for nucleocytoplasmic transport and protein condensation. Subsequently, the in vivo results reveal that RBM20 mutations led to cardiac pathogenesis. However, the role of phosphorylation in vivo needs further investigation.
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Empalme del ARN , Proteínas de Unión al ARN , Transporte Activo de Núcleo Celular , Animales , Ratones , Miocitos Cardíacos/metabolismo , Fosforilación , Motivos de Unión al ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , RatasRESUMEN
[Figure: see text].
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Caveolina 3/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Miocitos Cardíacos/metabolismo , Simportadores/metabolismo , Potenciales de Acción , Caveolina 3/genética , Células Cultivadas , Humanos , Ácido Láctico/metabolismo , Mutación con Pérdida de Función , Miocitos Cardíacos/fisiología , Unión Proteica , Sodio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
Cardiac protein homeostasis, sarcomere assembly, and integration of titin as the sarcomeric backbone are tightly regulated to facilitate adaptation and repair. Very little is known on how the >3-MDa titin protein is synthesized, moved, inserted into sarcomeres, detached, and degraded. Here, we generated a bifluorescently labeled knockin mouse to simultaneously visualize both ends of the molecule and follow titin's life cycle in vivo. We find titin mRNA, protein synthesis and degradation compartmentalized toward the Z-disk in adult, but not embryonic cardiomyocytes. Originating at the Z-disk, titin contributes to a soluble protein pool (>15% of total titin) before it is integrated into the sarcomere lattice. Titin integration, disintegration, and reintegration are stochastic and do not proceed sequentially from Z-disk to M-band, as suggested previously. Exchange between soluble and integrated titin depends on titin protein composition and differs between individual cardiomyocytes. Thus, titin dynamics facilitate embryonic vs. adult sarcomere remodeling with implications for cardiac development and disease.
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Miocitos Cardíacos/metabolismo , Proteínas Quinasas , Proteostasis/fisiología , Animales , Ratones , Ratones Transgénicos , Microscopía , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Sarcómeros/metabolismo , Análisis de la Célula IndividualRESUMEN
Mutations in the lamin A/C gene (LMNA) cause an autosomal dominant inherited form of dilated cardiomyopathy associated with cardiac conduction disease (hereafter referred to as LMNA cardiomyopathy). Compared with other forms of dilated cardiomyopathy, mutations in LMNA are responsible for a more aggressive clinical course owing to a high rate of malignant ventricular arrhythmias. Gap junctions are intercellular channels that allow direct communication between neighboring cells, which are involved in electrical impulse propagation and coordinated contraction of the heart. For gap junctions to properly control electrical synchronization in the heart, connexin-based hemichannels must be correctly targeted to intercalated discs, Cx43 being the major connexin in the working myocytes. We here showed an altered distribution of Cx43 in a mouse model of LMNA cardiomyopathy. However, little is known on the molecular mechanisms of Cx43 remodeling in pathological context. We now show that microtubule cytoskeleton alteration and decreased acetylation of α-tubulin lead to remodeling of Cx43 in LMNA cardiomyopathy, which alters the correct communication between cardiomyocytes, ultimately leading to electrical conduction disturbances. Preventing or reversing this process could offer a strategy to repair damaged heart. Stabilization of microtubule cytoskeleton using Paclitaxel improved intraventricular conduction defects. These results indicate that microtubule cytoskeleton contributes to the pathogenesis of LMNA cardiomyopathy and that drugs stabilizing the microtubule may be beneficial for patients.
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Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Conexina 43/metabolismo , Lamina Tipo A/genética , Paclitaxel/farmacología , Acetilación/efectos de los fármacos , Animales , Trastorno del Sistema de Conducción Cardíaco/genética , Cardiomiopatías/patología , Conexina 43/genética , Citoesqueleto/metabolismo , Citoesqueleto/patología , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/metabolismo , Uniones Comunicantes/patología , Lamina Tipo A/metabolismo , Masculino , Ratones , Ratones Noqueados , Microtúbulos/metabolismo , Microtúbulos/patología , Mutación , Miocardio/patología , Miocitos Cardíacos/patologíaRESUMEN
Fluorescence recovery after photobleaching (FRAP) has been useful in delineating cardiac myofilament biology, and innovations in fluorophore chemistry have expanded the array of microscopic assays used. However, one assumption in FRAP is the irreversible photobleaching of fluorescent proteins after laser excitation. Here we demonstrate reversible photobleaching regarding the photoconvertible fluorescent protein mEos3.2. We used CRISPR/Cas9 genome editing in human induced pluripotent stem cells (hiPSCs) to knock-in mEos3.2 into the COOH terminus of titin to visualize sarcomeric titin incorporation and turnover. Upon cardiac induction, the titin-mEos3.2 fusion protein is expressed and integrated in the sarcomeres of hiPSC-derived cardiomyocytes (CMs). STORM imaging shows M-band clustered regions of bound titin-mEos3.2 with few soluble titin-mEos3.2 molecules. FRAP revealed a baseline titin-mEos3.2 fluorescence recovery of 68% and half-life of ~1.2 h, suggesting a rapid exchange of sarcomeric titin with soluble titin. However, paraformaldehyde-fixed and permeabilized titin-mEos3.2 hiPSC-CMs surprisingly revealed a 55% fluorescence recovery. Whole cell FRAP analysis in paraformaldehyde-fixed, cycloheximide-treated, and untreated titin-mEos3.2 hiPSC-CMs displayed no significant differences in fluorescence recovery. FRAP in fixed HEK 293T expressing cytosolic mEos3.2 demonstrates a 58% fluorescence recovery. These data suggest that titin-mEos3.2 is subject to reversible photobleaching following FRAP. Using a mouse titin-eGFP model, we demonstrate that no reversible photobleaching occurs. Our results reveal that reversible photobleaching accounts for the majority of titin recovery in the titin-mEos3.2 hiPSC-CM model and should warrant as a caution in the extrapolation of reliable FRAP data from specific fluorescent proteins in long-term cell imaging.
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Diferenciación Celular , Conectina/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Células Madre Pluripotentes Inducidas/metabolismo , Microscopía Fluorescente , Microscopía por Video , Miocitos Cardíacos/metabolismo , Sarcómeros/metabolismo , Adulto , Línea Celular , Conectina/genética , Humanos , Cinética , Proteínas Luminiscentes/metabolismo , Masculino , Proteínas Recombinantes de Fusión/metabolismo , Reproducibilidad de los Resultados , Sarcómeros/genéticaRESUMEN
BACKGROUND: Titin is a giant elastic protein that spans the half-sarcomere from Z-disk to M-band. It acts as a molecular spring and mechanosensor and has been linked to striated muscle disease. The pathways that govern titin-dependent cardiac growth and contribute to disease are diverse and difficult to dissect. METHODS: To study titin deficiency versus dysfunction, the authors generated and compared striated muscle specific knockouts (KOs) with progressive postnatal loss of the complete titin protein by removing exon 2 (E2-KO) or an M-band truncation that eliminates proper sarcomeric integration, but retains all other functional domains (M-band exon 1/2 [M1/2]-KO). The authors evaluated cardiac function, cardiomyocyte mechanics, and the molecular basis of the phenotype. RESULTS: Skeletal muscle atrophy with reduced strength, severe sarcomere disassembly, and lethality from 2 weeks of age were shared between the models. Cardiac phenotypes differed considerably: loss of titin leads to dilated cardiomyopathy with combined systolic and diastolic dysfunction-the absence of M-band titin to cardiac atrophy and preserved function. The elastic properties of M1/2-KO cardiomyocytes are maintained, while passive stiffness is reduced in the E2-KO. In both KOs, we find an increased stress response and increased expression of proteins linked to titin-based mechanotransduction (CryAB, ANKRD1, muscle LIM protein, FHLs, p42, Camk2d, p62, and Nbr1). Among them, FHL2 and the M-band signaling proteins p62 and Nbr1 are exclusively upregulated in the E2-KO, suggesting a role in the differential pathology of titin truncation versus deficiency of the full-length protein. The differential stress response is consistent with truncated titin contributing to the mechanical properties in M1/2-KOs, while low titin levels in E2-KOs lead to reduced titin-based stiffness and increased strain on the remaining titin molecules. CONCLUSIONS: Progressive depletion of titin leads to sarcomere disassembly and atrophy in striated muscle. In the complete knockout, remaining titin molecules experience increased strain, resulting in mechanically induced trophic signaling and eventually dilated cardiomyopathy. The truncated titin in M1/2-KO helps maintain the passive properties and thus reduces mechanically induced signaling. Together, these findings contribute to the molecular understanding of why titin mutations differentially affect cardiac growth and have implications for genotype-phenotype relations that support a personalized medicine approach to the diverse titinopathies.
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Cardiomiopatía Dilatada/metabolismo , Mecanotransducción Celular , Miocitos Cardíacos/metabolismo , Proteínas Quinasas/deficiencia , Sarcómeros/metabolismo , Disfunción Ventricular Izquierda/metabolismo , Disfunción Ventricular Derecha/metabolismo , Animales , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/patología , Cardiomiopatía Dilatada/fisiopatología , Eliminación de Gen , Masculino , Ratones Noqueados , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Atrofia Muscular/genética , Atrofia Muscular/metabolismo , Atrofia Muscular/patología , Miocitos Cardíacos/patología , Fenotipo , Proteínas Quinasas/genética , Sarcómeros/patología , Disfunción Ventricular Izquierda/genética , Disfunción Ventricular Izquierda/patología , Disfunción Ventricular Izquierda/fisiopatología , Disfunción Ventricular Derecha/genética , Disfunción Ventricular Derecha/patología , Disfunción Ventricular Derecha/fisiopatología , Función Ventricular Izquierda , Función Ventricular DerechaRESUMEN
RNA-binding motif protein 20 (RBM20) is a cardiac splice regulator that adapts cardiac filling via its diverse substrates-including the sarcomeric protein titin. The molecular basis and regulation of RBM20-dependent exon exclusion are largely unknown. In tissue culture experiments, we show that the combination of RNA recognition motif (RRM) and C-terminus is necessary and sufficient for RBM20 activity, indicating an important function of the ZnF2 domain in splicing repression. Using splice reporter and in vitro binding assays targeting titin exons 241-243, we identified a minimal genomic segment that is necessary for RBM20-mediated splicing repression of the alternative exon. Here, RBM20 binds the cluster containing most RBM20 binding motifs through its RRM domain and represses the upstream and downstream introns. For subsequent exon exclusion, specific regions upstream, downstream and within the alternative exon 242 are required. Regulation of exon exclusion involves PTB4 as a novel titin splice regulator, which counteracts RBM20 repressor activity in HEK293 cells. Together, these mechanistic insights into the regulation and action of RBM20 and PTB4 provide a basis for the future development of RBM20 modulators that adapt titin elasticity in cardiac disease.
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Conectina/genética , Exones , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Proteínas de Unión al ARN/metabolismo , Empalme Alternativo , Sitios de Unión , Conectina/metabolismo , Células HEK293 , Ribonucleoproteínas Nucleares Heterogéneas/genética , Humanos , Intrones , Proteína de Unión al Tracto de Polipirimidina/genética , Proteínas de Unión al ARN/genética , Globinas beta/genéticaRESUMEN
AIMS: Heart failure with preserved ejection fraction (HFpEF) and pathological cardiac aging share a complex pathophysiology, including extracellular matrix remodelling (EMR). Protease-activated receptor 2 (PAR2) deficiency is associated with EMR. The roles of PAR1 and PAR2 have not been studied in HFpEF, age-dependent cardiac fibrosis, or diastolic dysfunction (DD). METHODS AND RESULTS: Evaluation of endomyocardial biopsies from patients with HFpEF (n = 14) revealed that a reduced cardiac PAR2 expression was associated with aggravated DD and increased myocardial fibrosis (r = -0.7336, P = 0.0028). In line, 1-year-old PAR2-knockout (PAR2ko) mice suffered from DD with preserved systolic function, associated with an increased age-dependent α-smooth muscle actin expression, collagen deposition (1.7-fold increase, P = 0.0003), lysyl oxidase activity, collagen cross-linking (2.2-fold increase, P = 0.0008), endothelial activation, and inflammation. In the absence of PAR2, the receptor-regulating protein caveolin-1 was down-regulated, contributing to an augmented profibrotic PAR1 and transforming growth factor beta (TGF-ß)-dependent signalling. This enhanced TGF-ß/PAR1 signalling caused N-proteinase (ADAMTS3) and C-proteinase (BMP1)-related increased collagen I production from cardiac fibroblasts (CFs). PAR2 overexpression in PAR2ko CFs reversed these effects. The treatment with the PAR1 antagonist, vorapaxar, reduced cardiac fibrosis by 44% (P = 0.03) and reduced inflammation in a metabolic disease model (apolipoprotein E-ko mice). Patients with HFpEF with upstream PAR inhibition via FXa inhibitors (n = 40) also exhibited reduced circulating markers of fibrosis and DD compared with patients treated with vitamin K antagonists (n = 20). CONCLUSIONS: Protease-activated receptor 2 is an important regulator of profibrotic PAR1 and TGF-ß signalling in the heart. Modulation of the FXa/FIIa-PAR1/PAR2/TGF-ß-axis might be a promising therapeutic approach to reduce HFpEF.
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Cardiomiopatías/metabolismo , Fibrosis/metabolismo , Miocardio/metabolismo , Receptor PAR-2 , Anciano , Animales , Cardiomiopatías/patología , Femenino , Fibrosis/patología , Insuficiencia Cardíaca Diastólica/metabolismo , Humanos , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Miocardio/patología , Receptor PAR-2/deficiencia , Receptor PAR-2/genética , Receptor PAR-2/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
PAR-CLIP (photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation) facilitates the identification and mapping of protein/RNA interactions. So far, it has been limited to select cell-lines as it requires efficient 4SU uptake. To increase transcriptome complexity and thus identify additional RNA-protein interaction sites we fused HEK 293 T-Rex cells (HEK293-Y) that express the RNA binding protein YBX1 with PC12 cells expressing eGFP (PC12-eGFP). The resulting hybrids enable PAR-CLIP on a neuronally expanded transcriptome (Fusion-CLIP) and serve as a proof of principle. The fusion cells express both parental marker genes YBX1 and eGFP and the expanded transcriptome contains human and rat transcripts. PAR-CLIP of fused cells versus the parental HEK293-Y identified 768 novel RNA targets of YBX1. We were able to trace the origin of the majority of the short PAR-CLIP reads as they differentially mapped to the human and rat genome. Furthermore, Fusion-CLIP expanded the CAUC RNA binding motif of YBX1 to UCUUUNNCAUC. The fusion of HEK293-Y and PC12-eGFP cells resulted in cells with a diverse genome expressing human and rat transcripts that enabled the identification of novel YBX1 substrates. The technique allows the expansion of the HEK 293 transcriptome and makes PAR-CLIP available to fusion cells of diverse origin.
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Fusión Celular/métodos , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , ARN Mensajero/metabolismo , Proteína 1 de Unión a la Caja Y/química , Proteína 1 de Unión a la Caja Y/metabolismo , Secuencias de Aminoácidos , Animales , Sitios de Unión , Reactivos de Enlaces Cruzados , Perfilación de la Expresión Génica/métodos , Células HEK293 , Humanos , Inmunoprecipitación , Células PC12 , Unión Proteica , RatasRESUMEN
AIMS: In this study, we aimed to clinically and genetically characterize LVNC patients and investigate the prevalence of variants in known and novel LVNC disease genes. INTRODUCTION: Left ventricular non-compaction cardiomyopathy (LVNC) is an increasingly recognized cause of heart failure, arrhythmia, thromboembolism, and sudden cardiac death. We sought here to dissect its genetic causes, phenotypic presentation and outcome. METHODS AND RESULTS: In our registry with follow-up of in the median 61 months, we analysed 95 LVNC patients (68 unrelated index patients and 27 affected relatives; definite familial LVNC = 23.5%) by cardiac phenotyping, molecular biomarkers and exome sequencing. Cardiovascular events were significantly more frequent in LVNC patients compared with an age-matched group of patients with non-ischaemic dilated cardiomyopathy (hazard ratio = 2.481, P = 0.002). Stringent genetic classification according to ACMG guidelines revealed that TTN, LMNA, and MYBPC3 are the most prevalent disease genes (13 patients are carrying a pathogenic truncating TTN variant, odds ratio = 40.7, Confidence interval = 21.6-76.6, P < 0.0001, percent spliced in 76-100%). We also identified novel candidate genes for LVNC. For RBM20, we were able to perform detailed familial, molecular and functional studies. We show that the novel variant p.R634L in the RS domain of RBM20 co-segregates with LVNC, leading to titin mis-splicing as revealed by RNA sequencing of heart tissue in mutation carriers, protein analysis, and functional splice-reporter assays. CONCLUSION: Our data demonstrate that the clinical course of symptomatic LVNC can be severe. The identified pathogenic variants and distribution of disease genes-a titin-related pathomechanism is found in every fourth patient-should be considered in genetic counselling of patients. Pathogenic variants in the nuclear proteins Lamin A/C and RBM20 were associated with worse outcome.
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Hipertrofia Ventricular Izquierda/genética , Mutación/genética , Adulto , Arritmias Cardíacas/genética , Cardiomiopatía Dilatada/genética , Conectina/genética , Muerte Súbita Cardíaca/etiología , Femenino , Predisposición Genética a la Enfermedad/genética , Humanos , Lamina Tipo A/genética , Masculino , Linaje , Proteínas de Unión al ARN/genéticaRESUMEN
Src homology and collagen A (ShcA) is an adaptor protein that binds to tyrosine kinase receptors. Its germ line deletion is embryonic lethal with abnormal cardiovascular system formation, and its role in cardiovascular development is unknown. To investigate its functional role in cardiovascular development in mice, ShcA was deleted in cardiomyocytes and vascular smooth muscle cells by crossing ShcA flox mice with SM22a-Cre transgenic mice. Conditional mutant mice developed signs of severe dilated cardiomyopathy, myocardial infarctions, and premature death. No evidence of a vascular contribution to the phenotype was observed. Histological analysis of the heart revealed aberrant sarcomeric Z-disk and M-band structures, and misalignments of T-tubules with Z-disks. We find that not only the ErbB3/Neuregulin signaling pathway but also the baroreceptor reflex response, which have been functionally associated, are altered in the mutant mice. We further demonstrate that ShcA interacts with Caveolin-1 and the costameric protein plasma membrane Ca(2+)/calmodulin-dependent ATPase (PMCA), and that its deletion leads to abnormal dystrophin signaling. Collectively, these results demonstrate that ShcA interacts with crucial proteins and pathways that link Z-disk and costamere.
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Costameras/metabolismo , Corazón/embriología , Miocitos Cardíacos/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Alelos , Animales , Aorta Torácica/metabolismo , Presión Sanguínea , Supervivencia Celular , Distrofina/metabolismo , Ecocardiografía , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Imagen por Resonancia Magnética , Ratones , Ratones Transgénicos , Microscopía Confocal , Fenotipo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , ARN Interferente Pequeño/metabolismo , Ratas , Receptor ErbB-3/metabolismo , Proteínas Adaptadoras de la Señalización Shc/genética , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de SrcRESUMEN
Core myopathies (CM), the main non-dystrophic myopathies in childhood, remain genetically unexplained in many cases. Heart disease is not considered part of the typical CM spectrum. No congenital heart defect has been reported, and childhood-onset cardiomyopathy has been documented in only two CM families with homozygous mutations of the TTN gene. TTN encodes titin, a giant protein of striated muscles. Recently, heterozygous TTN truncating mutations have also been reported as a major cause of dominant dilated cardiomyopathy. However, relatively few TTN mutations and phenotypes are known, and titin pathophysiological role in cardiac and skeletal muscle conditions is incompletely understood. We analyzed a series of 23 families with congenital CM and primary heart disease using TTN M-line-targeted sequencing followed in selected patients by whole-exome sequencing and functional studies. We identified seven novel homozygous or compound heterozygous TTN mutations (five in the M-line, five truncating) in 17% patients. Heterozygous parents were healthy. Phenotype analysis identified four novel titinopathies, including cardiac septal defects, left ventricular non-compaction, Emery-Dreifuss muscular dystrophy or arthrogryposis. Additionally, in vitro studies documented the first-reported absence of a functional titin kinase domain in humans, leading to a severe antenatal phenotype. We establish that CM are associated with a large range of heart conditions of which TTN mutations are a major cause, thereby expanding the TTN mutational and phenotypic spectrum. Additionally, our results suggest titin kinase implication in cardiac morphogenesis and demonstrate that heterozygous TTN truncating mutations may not manifest unless associated with a second mutation, reassessing the paradigm of their dominant expression.
Asunto(s)
Codón sin Sentido , Conectina/genética , Cardiopatías/genética , Miopatía del Núcleo Central/genética , Adolescente , Conectina/metabolismo , Consanguinidad , Femenino , Genes Recesivos , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Cardiopatías/metabolismo , Cardiopatías/patología , Heterocigoto , Humanos , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Miopatía del Núcleo Central/metabolismo , Miopatía del Núcleo Central/patología , Linaje , Fenotipo , Adulto JovenRESUMEN
Cardiac titin is the main determinant of sarcomere stiffness during diastolic relaxation. To explore whether titin stiffness affects the kinetics of cardiac myofibrillar contraction and relaxation, we used subcellular myofibrils from the left ventricles of homozygous and heterozygous N2B-knockout mice which express truncated cardiac titins lacking the unique elastic N2B region. Compared with myofibrils from wild-type mice, myofibrils from knockout and heterozygous mice exhibit increased passive myofibrillar stiffness. To determine the kinetics of Ca(2+)-induced force development (rate constant kACT), myofibrils from knockout, heterozygous and wild-type mice were stretched to the same sarcomere length (2.3 µm) and rapidly activated with Ca(2+). Additionally, mechanically induced force-redevelopment kinetics (rate constant kTR) were determined by slackening and re-stretching myofibrils during Ca(2+)-mediated activation. Myofibrils from knockout mice exhibited significantly higher kACT, kTR and maximum Ca(2+)-activated tension than myofibrils from wild-type mice. By contrast, the kinetic parameters of biphasic force relaxation induced by rapidly reducing [Ca(2+)] were not significantly different among the three genotypes. These results indicate that increased titin stiffness promotes myocardial contraction by accelerating the formation of force-generating cross-bridges without decelerating relaxation.
Asunto(s)
Conectina/metabolismo , Relajación Muscular/fisiología , Contracción Miocárdica/fisiología , Miofibrillas/metabolismo , Sarcómeros/metabolismo , Eliminación de Secuencia/genética , Animales , Secuencia de Bases/genética , Calcio/metabolismo , Conectina/genética , Cinética , Ratones , Contracción Miocárdica/genética , Miocardio/metabolismo , Miofibrillas/fisiologíaRESUMEN
UNLABELLED: The coxsackievirus and adenovirus receptor (CAR) is a cell contact protein with an important role in virus uptake. Its extracellular immunoglobulin domains mediate the binding to coxsackievirus and adenovirus as well as homophilic and heterophilic interactions between cells. The cytoplasmic tail links CAR to the cytoskeleton and intracellular signaling cascades. In the heart, CAR is crucial for embryonic development, electrophysiology, and coxsackievirus B infection. Noncardiac functions are less well understood, in part due to the lack of suitable animal models. Here, we generated a transgenic mouse that rescued the otherwise embryonic-lethal CAR knockout (KO) phenotype by expressing chicken CAR exclusively in the heart. Using this rescue model, we addressed interspecies differences in coxsackievirus uptake and noncardiac functions of CAR. Survival of the noncardiac CAR KO (ncKO) mouse indicates an essential role for CAR in the developing heart but not in other tissues. In adult animals, cardiac activity was normal, suggesting that chicken CAR can replace the physiological functions of mouse CAR in the cardiomyocyte. However, chicken CAR did not mediate virus entry in vivo, so that hearts expressing chicken instead of mouse CAR were protected from infection and myocarditis. Comparison of sequence homology and modeling of the D1 domain indicate differences between mammalian and chicken CAR that relate to the sites important for virus binding but not those involved in homodimerization. Thus, CAR-directed anticoxsackievirus therapy with only minor adverse effects in noncardiac tissue could be further improved by selectively targeting the virus-host interaction while maintaining cardiac function. IMPORTANCE: Coxsackievirus B3 (CVB3) is one of the most common human pathogens causing myocarditis. Its receptor, the coxsackievirus and adenovirus receptor (CAR), not only mediates virus uptake but also relates to cytoskeletal organization and intracellular signaling. Animals without CAR die prenatally with major cardiac malformations. In the adult heart, CAR is important for virus entry and electrical conduction, but its nonmuscle functions are largely unknown. Here, we show that chicken CAR expression exclusively in the heart can rescue the otherwise embryonic-lethal CAR knockout phenotype but does not support CVB3 infection of adult cardiomyocytes. Our findings have implications for the evolution of virus-host versus physiological interactions involving CAR and could help to improve future coxsackievirus-directed therapies inhibiting virus replication while maintaining CAR's cellular functions.
Asunto(s)
Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/fisiología , Infecciones por Coxsackievirus/prevención & control , Corazón/fisiología , Miocarditis/prevención & control , Replicación Viral , Animales , Western Blotting , Células Cultivadas , Pollos , Infecciones por Coxsackievirus/virología , Enterovirus Humano B/fisiología , Técnica del Anticuerpo Fluorescente , Células HeLa , Corazón/virología , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Miocarditis/virologíaRESUMEN
[This corrects the article DOI: 10.1007/s12551-021-00840-7.].