RESUMEN
BACKGROUND: Mutations of cardiac troponin C (cTnC) can cause dilated cardiomyopathy in humans. METHODS AND RESULTS: Plasmids were constructed such that the reverse tetracycline-controlled transactivator (rtTA) was driven by the cardiac myosin light chain 2 promoter. This heart-specific rtTA bound another bidirectional promoter to express the green fluorescence protein reporter gene and the antisense RNA of cTnC in the presence of doxycycline. A transgenic line of zebrafish (CA17) with cTnC dysfunction was also generated. The heart rates of the embryos in the CA17 line were significantly slower than those of embryos in the control T03 transgenic line at 6 and 12 days post fertilization (dpf). In the CA17 line, cardiac chambers in the F2 embryos were significantly greater and the ventricular ejection fraction was lower than those in the T03 at both 6 and 12 dpf. The mortality rate of F2 adult fish of the CA17 line was also significantly higher (P<0.001). CONCLUSIONS: Using conditional expression of antisense RNA of zebrafish cTnC, a new animal model with phenotypes simulating dilated cardiomyopathy has been created.
Asunto(s)
Cardiomiopatía Dilatada/genética , Técnicas de Silenciamiento del Gen , ARN sin Sentido/biosíntesis , Troponina C/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Función Atrial/genética , Secuencia de Bases , Western Blotting , Miosinas Cardíacas/genética , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/patología , Cardiomiopatía Dilatada/fisiopatología , Modelos Animales de Enfermedad , Genotipo , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Frecuencia Cardíaca/genética , Datos de Secuencia Molecular , Contracción Miocárdica/genética , Miocardio/metabolismo , Miocardio/patología , Cadenas Ligeras de Miosina/genética , Fenotipo , Regiones Promotoras Genéticas , Volumen Sistólico/genética , Transcripción Genética , Troponina C/metabolismo , Función Ventricular/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Lysophosphatidic acid (LPA) is a low-molecular-weight lysophospholipid (LPL), which regulates endothelial cells participating in inflammation processes via interactions with endothelial differentiation gene (Edg) family G protein-coupled receptors. In this study, we attempted to determine which LPA receptors mediate the inflammatory response in human endothelial cells. Introduction of siRNA against LPA1 significantly suppressed LPA-induced ICAM-1 mRNA, total protein, and cell surface expressions, and subsequent U937 monocyte adhesion to LPA-treated human umbilical endothelial cells (HUVECs). By knock down of LPA1 and LPA3 in HUVECs, LPA-enhanced IL-1beta mRNA expression was significantly attenuated. Moreover, LPA1 and LPA3 siRNA also inhibited LPA-enhanced IL-1-dependent long-term IL-8 and MCP-1 mRNA expression, and subsequent THP-1 cell chemotaxis toward LPA-treated HUVEC-conditioned media. These results suggest that the expression of LPA-induced inflammatory response genes is mediated by LPA1 and LPA3. Our findings suggest the possible utilization of LPA1 or LPA3 as drug targets to treat severe inflammation.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Lisofosfolípidos/farmacología , Receptores del Ácido Lisofosfatídico/metabolismo , Adhesión Celular , Células Cultivadas , Quimiocina CCL2/genética , Células Endoteliales/citología , Humanos , Inflamación/genética , Inflamación/metabolismo , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-1/genética , Interleucina-8/genética , Fragmentos de Péptidos/genética , ARN Interferente Pequeño/genéticaRESUMEN
BACKGROUND/PURPOSE: The zebrafish (Danio rerio) is a new animal model for cardiac research. Zebrafish possessing a green fluorescent heart facilitates the dynamic observation of cardiac development, morphology, and function in vivo. However, the effect of an excessive expression of green fluorescent protein (GFP) in cardiac muscle on the heart function of zebrafish has not been reported. METHODS: We cloned a 1.6 kb polymerase chain reaction (PCR) product containing the upstream sequence (870 bp), exon 1 (39 bp), intron 1 (682 bp), and exon 2 (69 bp) of the zebrafish cardiac myosin light chain 2 gene. A germ line-transmitted zebrafish possessing a green fluorescent heart was generated by injecting this PCR product fused with the GFP gene with ends consisting of inverted terminal repeats of an adeno-associated virus. RESULTS: Green fluorescence was intensively and specifically expressed in the myocardial cells located around both the heart chambers. Two lines with different GFP expression were bred (A26 and A277). The luminance of A277 was brighter than that of A26 (1.7-fold). The 4 days postfertilization (dpf) cardiac function and morphology were similar between these two groups. However, the 8 dpf cardiac growth seemed to be retarded in the A277 group. The 8 dpf heart rate, stroke volume, and cardiac output were also significantly lower in the A277 group. CONCLUSION: Excess expression of GFP seems to exert some detrimental effects on zebrafish hearts.
Asunto(s)
Proteínas Fluorescentes Verdes/genética , Corazón/anatomía & histología , Corazón/fisiología , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , InmunohistoquímicaRESUMEN
To develop the first heart-specific tetracycline (Tet)-On system in zebrafish, we constructed plasmids in which the cardiac myosin light chain 2 promoter of zebrafish was used to drive the reverse Tet-controlled transactivator (rtTA) and the green fluorescent protein (GFP) reporter gene was preceded by an rtTA-responsive element. In the zebrafish fibroblast cell-line, rtTA-M2, one of rtTA's derivatives, demonstrated the highest increase in luciferase activity upon doxycycline (Dox) induction. We then generated two germ lines of transgenic zebrafish: line T03 was derived from microinjection of a plasmid containing rtTA-M2 and a plasmid containing a responsive reporter gene, whereas line T21 was derived from microinjection of a single dual plasmid. Results showed that line T21 was superior to line T03 in terms of greater GFP intensity after induction and with of minimal leakiness before induction. The photographic images of induced GFP in the heart of F2 larvae showed that the fluorescent level of GFP was dose-responsive. The level of GFP expressed in the F3 3 days postfertilization larvae that were treated with Dox for 1 hr decreased gradually after the withdrawal of the inducer; and the fluorescent signal disappeared after 5 days. The GFP induction and reduction were also tightly controlled by Dox in the F3 adult fish from line T21. This Tet-On system developed in zebrafish shows much promise for the study of the gene function in a specific tissue at the later developmental stage.
Asunto(s)
Técnicas de Transferencia de Gen , Miocardio/metabolismo , Transgenes , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Miosinas Cardíacas/genética , Línea Celular , Fibroblastos , Genes Reporteros , Plásmidos , Regiones Promotoras Genéticas , Tetraciclina , Transactivadores/genética , Pez Cebra/embriología , Pez Cebra/metabolismoRESUMEN
In response to the lack of a transgenic line of zebrafish labeled with heart-specific fluorescence in vivo to serve as a research model, we cloned a 1.6-kb polymerase chain reaction (PCR) -product containing the upstream sequence (-870 bp), exon 1 (39 bp), intron 1 (682 bp), and exon 2 (69 bp) of the zebrafish cardiac myosin light chain 2 gene, (cmlc2). A germ-line transmitted zebrafish possessing a green fluorescent heart was generated by injecting this PCR product fused with the green fluorescent protein (GFP) gene with ends consisting of inverted terminal repeats of an adeno-associated virus. Green fluorescence was intensively and specifically expressed in the myocardial cells located both around the heart chambers and the atrioventricular canal. Neither the epicardium nor the endocardium showed fluorescent signals. The GFP expression in the transgenic line faithfully recapitulated with the spatial and temporal expression of the endogenous cmlc2. Promoter analysis showed that the fragment consisting of nucleotides from -210 to 34 (-210/34) was sufficient to drive heart-specific expression, with a -210/-73 motif as a basal promoter and a -210/-174 motif as an element involved in suppressing ectopic (nonheart) expression. Interestingly, a germ-line of zebrafish whose GFP appeared ectopically in all muscle types (heart, skeletal, and smooth) was generated by injecting the fragment including a single nucleotide mutation from G to A at -119, evidence that A at -119 combined with neighboring nucleotides to create a consensus sequence for binding myocyte-specific enhancer factor-2.