Myocardial afterload is a key biomechanical regulator of atrioventricular myocyte differentiation in zebrafish

Heart valve development is governed by both genetic and biomechanical inputs. Prior work has demonstrated that oscillating shear stress associated with blood flow is required for normal atrioventricular (AV) valve development. Cardiac afterload is defined as the pressure the ventricle must overcome in order to pump blood throughout the circulatory system. In human patients, conditions of high afterload can cause valve pathology. Whether high afterload adversely affects embryonic valve development remains poorly understood. Here we describe a zebrafish model exhibiting increased myocardial afterload, caused by vasopressin, a vasoconstrictive drug. We show that the application of vasopressin reliably produces an increase in afterload without directly acting on cardiac tissue in zebrafish embryos. We have found that increased afterload alters the rate of growth of the cardiac chambers and causes remodeling of cardiomyocytes. Consistent with pathology seen in patients with clinically high afterload, we see defects in both the form and the function of the valve leaflets. Our results suggest that valve defects are due to changes in atrioventricular myocyte signaling, rather than pressure directly acting on the endothelial valve leaflet cells. Cardiac afterload should therefore be considered a biomechanical factor that particularly impacts embryonic valve development

Il ruolo del miR-19a nello sviluppo del cuore in Danio rerio

La regolazione dello sviluppo cardiaco necessita di un controllo spazio-temporale dell’espressione genica, perciò mutazioni a fattori di trascrizione sono frequentemente associate a malattie cardiovascolari. Uno dei fattori di trascrizione importanti nello sviluppo cardiaco è TBX5 che fa parte della famiglia dei T-box transcription factors. Durante il periodo embrionale esso ha un ruolo nella specificazione cellulare e nella morfogenesi e ha un ruolo nello sviluppo del cuore e nella specificità dell’arto. Mutazioni a suo carico sono state associate alla sindrome di Holt-Horam che si manifesta con un’incidenza di 1 su 100.000 nati. Si tratta di una sindrome autosomica dominante a penetranza completa anche se la maggior parte dei casi clinici studiati si riconduce a mutazioni de novo di Tbx5. Fenotipicamente i soggetti affetti mostrano difetti del setto atrio-ventricolare, anomalie elettro-cardiografiche e ipoplasia dei vasi periferici degli arti superiori. Essendo una malattia rara è necessario un organismo modello e il pesce zebra sembra essere il candidato ideale. Infatti mutazioni a carico di Tbx5 in Zebrafish causano il fenotipo detto heartstring e assenza delle pinne pettorali. Sia aploinsufficienza che duplicazione del gene determinano anomalie cardiache, da qui si deduce che un normale processo cardiogenetico dipende dall’equilibrio dell’ espressione dei geni T-box nei precursori cardiaci. Tbx5 potrebbe regolare una serie di micro-RNA, piccole molecole di 20-22 nt che hanno la funzione di regolare l’espressione genica. I micro-RNA sono una scoperta degli anni ’90 e solo di recente ne è stata chiarita la biogenesi. Vari studi hanno dimostrato che i micro-RNA hanno una funzione importante nei processi cardiogenici e nello sviluppo embrionale in toto. In Zebrafish la perdita di Dicer porta a difetti nella gastrulazione, nella morfogenesi cerebrale, nella somitogenesi e nello sviluppo del cuore. Per verificare la presenza di micro-RNA, sia intra-genici che inter-genici, tra i geni regolati da Tbx5 è stato somministrato il morfolino contro Tbx5 (confrontato con un morfolino di controllo) in embrioni di pesce zebra e sono stati trovati una serie di micro-RNA down-regolati e up-regolati. Dai risultati del deep-sequencing si è distinto il micro-19a che è risultato essere down-regolato di circa 2 volte. In letteratura il micro-19a è poco citato, per quanto riguarda la biologia dello sviluppo si suppone che sia importante nella formazione del sistema nervoso e del sistema endoteliale-vascolare. Il lavoro di tesi si è focalizzato sullo studio del 19a cercando di valutarne il ruolo nelle prime fasi di sviluppo di Zebrafish. Ciò è stato fatto up-regolando e down-regolando i livelli del micro-RNA a dosi scalari. Dai risultati ottenuti, osservando il fenotipo nel pesce e dopo aver fatto una ricerca in letteratura, è stato trovato un possibile target: il gene S1PR1, che codifica per il recettore 1 della sfingosina-1-fosfato. La funzione di questo lipide non è ancora del tutto chiarita nella biologia dello sviluppo, ma bassi livelli provocano nel topo alterazioni del setto atrio-ventricolare mentre alti livelli inibiscono la migrazione cellulare e la trasformazione epitelio-mesenchimale

Post-transcriptional Modulation of Sphingosine-1-Phosphate Receptor 1 by miR-19a Affects Cardiovascular Development in Zebrafish

Sphingosine-1-phosphate is a bioactive lipid and a signaling molecule integrated into many physiological systems such as differentiation, proliferation and migration. In mammals S1P acts through binding to a family of five trans-membrane, G-protein coupled receptors (S1PRs) whose complex role has not been completely elucidated. In this study we use zebrafish, in which seven s1prs have been identified, to investigate the role of s1pr1. In mammals S1PR1 is the most highly expressed S1P receptor in the developing heart and regulates vascular development, but in zebrafish the data concerning its role are contradictory. Here we show that overexpression of zebrafish s1pr1 affects both vascular and cardiac development. Moreover we demonstrate that s1pr1 expression is strongly repressed by miR-19a during the early phases of zebrafish development. In line with this observation and with a recent study showing that miR-19a is downregulated in a zebrafish Holt-Oram model, we now demonstrate that s1pr1 is upregulated in heartstring hearts. Next we investigated whether defects induced by s1pr1 upregulation might contribute to the morphological alterations caused by Tbx5 depletion. We show that downregulation of s1pr1 is able to partially rescue cardiac and fin defects induced by Tbx5 depletion. Taken together, these data support a role for s1pr1 in zebrafish cardiovascular development, suggest the involvement of this receptor in the Tbx5 regulatory circuitry, and further support the crucial role of microRNAs in early phase of zebrafish development

New Insights into Mechanisms of Endocrine-Disrupting Chemicals in Thyroid Diseases: The Epigenetic Way

In recent years, the presence in the environment of chemical compounds with thyroid-disrupting effects is progressively increased. This phenomenon has risen concern for human health as the preservation of thyroid system homeostasis is essential for fetal development and for maintaining psychological and physiological wellbeing. An increasing number of studies explored the role of different classes of toxicants in the occurrence and severity of thyroid diseases, but large epidemiological studies are limited and only a few animal or in vitro studies have attempted to identify the mechanisms of chemical action. Recently, epigenetic changes such as alteration of methylation status or modification of non-coding RNAs have been suggested as correlated to possible deleterious effects leading to different thyroid disorders in susceptible individuals. This review aims to analyze the epigenetic alterations putatively induced by chemical exposures and involved in the onset of frequent thyroid diseases such as thyroid cancer, autoimmune thyroiditis and disruption of fetal thyroid homeostasis

Myocardial Afterload Is a Key Biomechanical Regulator of Atrioventricular Myocyte Differentiation in Zebrafish

Heart valve development is governed by both genetic and biomechanical inputs. Prior work has demonstrated that oscillating shear stress associated with blood flow is required for normal atrioventricular (AV) valve development. Cardiac afterload is defined as the pressure the ventricle must overcome in order to pump blood throughout the circulatory system. In human patients, conditions of high afterload can cause valve pathology. Whether high afterload adversely affects embryonic valve development remains poorly understood. Here we describe a zebrafish model exhibiting increased myocardial afterload, caused by vasopressin, a vasoconstrictive drug. We show that the application of vasopressin reliably produces an increase in afterload without directly acting on cardiac tissue in zebrafish embryos. We have found that increased afterload alters the rate of growth of the cardiac chambers and causes remodeling of cardiomyocytes. Consistent with pathology seen in patients with clinically high afterload, we see defects in both the form and the function of the valve leaflets. Our results suggest that valve defects are due to changes in atrioventricular myocyte signaling, rather than pressure directly acting on the endothelial valve leaflet cells. Cardiac afterload should therefore be considered a biomechanical factor that particularly impacts embryonic valve development

Image_2_Post-transcriptional Modulation of Sphingosine-1-Phosphate Receptor 1 by miR-19a Affects Cardiovascular Development in Zebrafish.PDF

<p>Sphingosine-1-phosphate is a bioactive lipid and a signaling molecule integrated into many physiological systems such as differentiation, proliferation and migration. In mammals S1P acts through binding to a family of five trans-membrane, G-protein coupled receptors (S1PRs) whose complex role has not been completely elucidated. In this study we use zebrafish, in which seven s1prs have been identified, to investigate the role of s1pr1. In mammals S1PR1 is the most highly expressed S1P receptor in the developing heart and regulates vascular development, but in zebrafish the data concerning its role are contradictory. Here we show that overexpression of zebrafish s1pr1 affects both vascular and cardiac development. Moreover we demonstrate that s1pr1 expression is strongly repressed by miR-19a during the early phases of zebrafish development. In line with this observation and with a recent study showing that miR-19a is downregulated in a zebrafish Holt-Oram model, we now demonstrate that s1pr1 is upregulated in heartstring hearts. Next we investigated whether defects induced by s1pr1 upregulation might contribute to the morphological alterations caused by Tbx5 depletion. We show that downregulation of s1pr1 is able to partially rescue cardiac and fin defects induced by Tbx5 depletion. Taken together, these data support a role for s1pr1 in zebrafish cardiovascular development, suggest the involvement of this receptor in the Tbx5 regulatory circuitry, and further support the crucial role of microRNAs in early phase of zebrafish development.</p

Data_Sheet_2_Post-transcriptional Modulation of Sphingosine-1-Phosphate Receptor 1 by miR-19a Affects Cardiovascular Development in Zebrafish.xlsx

<p>Sphingosine-1-phosphate is a bioactive lipid and a signaling molecule integrated into many physiological systems such as differentiation, proliferation and migration. In mammals S1P acts through binding to a family of five trans-membrane, G-protein coupled receptors (S1PRs) whose complex role has not been completely elucidated. In this study we use zebrafish, in which seven s1prs have been identified, to investigate the role of s1pr1. In mammals S1PR1 is the most highly expressed S1P receptor in the developing heart and regulates vascular development, but in zebrafish the data concerning its role are contradictory. Here we show that overexpression of zebrafish s1pr1 affects both vascular and cardiac development. Moreover we demonstrate that s1pr1 expression is strongly repressed by miR-19a during the early phases of zebrafish development. In line with this observation and with a recent study showing that miR-19a is downregulated in a zebrafish Holt-Oram model, we now demonstrate that s1pr1 is upregulated in heartstring hearts. Next we investigated whether defects induced by s1pr1 upregulation might contribute to the morphological alterations caused by Tbx5 depletion. We show that downregulation of s1pr1 is able to partially rescue cardiac and fin defects induced by Tbx5 depletion. Taken together, these data support a role for s1pr1 in zebrafish cardiovascular development, suggest the involvement of this receptor in the Tbx5 regulatory circuitry, and further support the crucial role of microRNAs in early phase of zebrafish development.</p

Data_Sheet_1_Post-transcriptional Modulation of Sphingosine-1-Phosphate Receptor 1 by miR-19a Affects Cardiovascular Development in Zebrafish.zip

<p>Sphingosine-1-phosphate is a bioactive lipid and a signaling molecule integrated into many physiological systems such as differentiation, proliferation and migration. In mammals S1P acts through binding to a family of five trans-membrane, G-protein coupled receptors (S1PRs) whose complex role has not been completely elucidated. In this study we use zebrafish, in which seven s1prs have been identified, to investigate the role of s1pr1. In mammals S1PR1 is the most highly expressed S1P receptor in the developing heart and regulates vascular development, but in zebrafish the data concerning its role are contradictory. Here we show that overexpression of zebrafish s1pr1 affects both vascular and cardiac development. Moreover we demonstrate that s1pr1 expression is strongly repressed by miR-19a during the early phases of zebrafish development. In line with this observation and with a recent study showing that miR-19a is downregulated in a zebrafish Holt-Oram model, we now demonstrate that s1pr1 is upregulated in heartstring hearts. Next we investigated whether defects induced by s1pr1 upregulation might contribute to the morphological alterations caused by Tbx5 depletion. We show that downregulation of s1pr1 is able to partially rescue cardiac and fin defects induced by Tbx5 depletion. Taken together, these data support a role for s1pr1 in zebrafish cardiovascular development, suggest the involvement of this receptor in the Tbx5 regulatory circuitry, and further support the crucial role of microRNAs in early phase of zebrafish development.</p