Physiological significance of two common haplotypes of human angiotensinogen using gene targeting in the mouse

2002 ◽  
Vol 11 (3) ◽  
pp. 253-262 ◽  
Author(s):  
Branimir Cvetkovic ◽  
Henry L. Keen ◽  
Xiaoji Zhang ◽  
Deborah Davis ◽  
Baoli Yang ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Association Studies ◽  
Single Copy ◽  
Hypoxanthine Phosphoribosyl Transferase ◽  
Heart Weight ◽  
Human Renin ◽  
Physiological Relevance ◽  
Experimental Mouse ◽  
Mouse Model System

Angiotensinogen (AGT) was the first gene to be genetically linked to hypertension in humans. Analysis of the gene sequence identified a number of polymorphisms, several of which were reported associated with increased blood pressure (BP) or other cardiovascular diseases. One haplotype of the human AGT (hAGT) gene consisting of an allele at the −6 (A vs. G) position in the promoter and the sequence encoding amino acid 235 (Thr vs. Met) attracted the most attention and has been the subject of numerous association studies. In this report, we addressed the physiological relevance of alleles at these two positions using an experimental mouse model system. Transgenic mice were generated by targeting each haplotype [−6G/235Met (GM) and −6A/235Thr (AT)] as a single copy transgene to the mouse hypoxanthine phosphoribosyl transferase locus, allowing direct comparison of the two transgenes in vivo. Our results indicate that both transgenes exhibit the same transcriptional activity and produce similar levels of hAGT protein in the plasma of the transgenic mice. BP analysis was performed in double transgenic mice generated by breeding each hAGT line to mice expressing a human renin gene. A small but significant increase in BP and relative heart weight was demonstrated by mice carrying the GM haplotype. Moreover, compensatory downregulation of endogenous renin expression was more pronounced in mice containing the GM variant. Our findings suggest that the AT and GM haplotypes of the hAGT gene have no effect on gene expression, but may affect the cardiovascular system and the regulation of BP differently.

scholarly journals Cardiac phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase increases glycolysis, hypertrophy, and myocyte resistance to hypoxia

2008 ◽  
Vol 294 (6) ◽  
pp. H2889-H2897 ◽  
Author(s):  
Qianwen Wang ◽  
Rajakumar V. Donthi ◽  
Jianxun Wang ◽  
Alex J. Lange ◽  
Lewis J. Watson ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Cardiac Fibrosis ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Weight Ratio ◽  
Cardiac Metabolism ◽  
Heart Weight ◽  
Acid Oxidation ◽  
Important Regulator

During ischemia and heart failure, there is an increase in cardiac glycolysis. To understand if this is beneficial or detrimental to the heart, we chronically elevated glycolysis by cardiac-specific overexpression of phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) in transgenic mice. PFK-2 controls the level of fructose-2,6-bisphosphate (Fru-2,6-P2), an important regulator of phosphofructokinase and glycolysis. Transgenic mice had over a threefold elevation in levels of Fru-2,6-P2. Cardiac metabolites upstream of phosphofructokinase were significantly reduced, as would be expected by the activation of phosphofructokinase. In perfused hearts, the transgene caused a significant increase in glycolysis that was less sensitive to inhibition by palmitate. Conversely, oxidation of palmitate was reduced by close to 50%. The elevation in glycolysis made isolated cardiomyocytes highly resistant to contractile inhibition by hypoxia, but in vivo the transgene had no effect on ischemia-reperfusion injury. Transgenic hearts exhibited pathology: the heart weight-to-body weight ratio was increased 17%, cardiomyocyte length was greater, and cardiac fibrosis was increased. However, the transgene did not change insulin sensitivity. These results show that the elevation in glycolysis provides acute benefits against hypoxia, but the chronic increase in glycolysis or reduction in fatty acid oxidation interferes with normal cardiac metabolism, which may be detrimental to the heart.

Cardiomyopathy in transgenic mice with cardiac-specific overexpression of serum response factor

2001 ◽  
Vol 280 (4) ◽  
pp. H1782-H1792 ◽  
Author(s):  
Xiaomin Zhang ◽  
Gohar Azhar ◽  
Jianyuan Chai ◽  
Pamela Sheridan ◽  
Koichiro Nagano ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Cardiac Muscle ◽  
Serum Response Factor ◽  
Interstitial Fibrosis ◽  
Weight Ratio ◽  
Heart Weight ◽  
Cardiomyocyte Hypertrophy ◽  
Response Factor ◽  
Serum Response

Serum response factor (SRF), a member of the MCM1, agamous, deficiens, SRF (MADS) family of transcriptional activators, has been implicated in the transcriptional control of a number of cardiac muscle genes, including cardiac α-actin, skeletal α-actin, α-myosin heavy chain (α-MHC), and β-MHC. To better understand the in vivo role of SRF in regulating genes responsible for maintenance of cardiac function, we sought to test the hypothesis that increased cardiac-specific SRF expression might be associated with altered cardiac morphology and function. We generated transgenic mice with cardiac-specific overexpression of the human SRF gene. The transgenic mice developed cardiomyopathy and exhibited increased heart weight-to-body weight ratio, increased heart weight, and four-chamber dilation. Histological examination revealed cardiomyocyte hypertrophy, collagen deposition, and interstitial fibrosis. SRF overexpression altered the expression of SRF-regulated genes and resulted in cardiac muscle dysfunction. Our results demonstrate that sustained overexpression of SRF, in the absence of other stimuli, is sufficient to induce cardiac change and suggest that SRF is likely to be one of the downstream effectors of the signaling pathways involved in mediating cardiac hypertrophy.

Abstract 28: In Vivo Knockdown of Mirna-15b Results in Increased Cardiac Hypertrophy and Fibrosis in Response to Pressure Overload of the Mouse Heart

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Anke J Tijsen ◽  
Ingeborg van der Made ◽  
Elza D van Deel ◽  
Monika Hiller ◽  
Yolan J Reckman ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Cardiac Function ◽  
Cardiac Fibrosis ◽  
Negative Control ◽  
Heart Weight ◽  
Mouse Heart ◽  
Hypertrophic Response ◽  
Wide Range ◽  
Tgfβ Pathway

MiRNAs play an important role in the control of diverse aspects of cardiac function. MiR-15b is highly expressed in the heart and is found consistently upregulated in hypertrophic and failing hearts. To investigate the function of miR-15b in the heart we set out two experiments. In the first experiment we generated two independent transgenic mouse lines that drive miR-15b expression under the αMHC-promotor and show a three and four fold overexpression of miR-15b. Strikingly, both lines show a decrease in heart weight/tibia length of 20% in adult and aged mice when compared to littermate controls. We investigated the response of these transgenic mice to thoracic aorta constriction (TAC) and found no differences in the hypertrophic response or in cardiac function measured by echocardiography between wild-type and transgenic mice. In a second experiment, we inhibited miR-15b using LNA-based antimiRs. In these mice, TAC resulted in an increased hypertrophic response and increased cardiac fibrosis when compared to a negative control antimiR. A wide range of predicted targets of miR-15 belong to the pathways of the TGFβ-superfamily and using a smad-dependent reporter we show that miR-15b inhibits TGFβ-induced Smad activity in HepG2 cells. One of the predicted targets in the TGFβ pathway is TGFβ receptor 1 (TGFβR1), of which the 3’UTR contains six predicted miR-15 binding sites. This suggests that the phenotype in the transgenic mice and after knockdown of miR-15b may be (partly) mediated by repression of TGFβR1. Indeed, in the adult miR-15b transgenic hearts we found a downregulation of TGFβR1 mRNA and protein and we confirmed binding of miR-15 to the TGFβR1 3’UTR by luciferase assays. In conclusion, miR-15b causes a cardiac hypotrophic phenotype at baseline in transgenic mice and inhibition of miR-15b leads to a stronger hypertrophic and fibrotic response after TAC. Furthermore miR-15b inhibits the TGFβ pathway by targeting the TGFβR1 and possibly other targets in this pathway. This research is funded by the Dutch Heart Foundation (NHF grant #2007B077).

Abstract 1828: Dyxin/Lmcd1 Mediates Cardiac Hypertrophy Both In Vitro And In Vivo

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Derk Frank ◽  
Robert Frauen ◽  
Christiane Hanselmann ◽  
Christian Kuhn ◽  
Rainer Will ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Cardiac Hypertrophy ◽  
Neonatal Rat ◽  
Heart Weight ◽  
Cardiomyocyte Hypertrophy ◽  
The Novel ◽  
Rat Cardiomyocytes ◽  
A Genome

In order to identify new molecular mediators of cardiomyocyte hypertrophy, we performed a genome wide mRNA microarray screen of biomechanically stretched neonatal rat cardiomyocytes (NRCM). We found the novel sarcomeric LIM protein Dyxin/Lmcd1 being significantly upregulated (5.6x, p<0.001). Moreover, Dyxin was also significantly induced in several mouse models of myocardial hypertrophy including aortic banding, calcineurin overexpression and angiotensin stimulation, suggesting a potential role as a mediator of cardiac hypertrophy. To further test this hypothesis, we adenovirally overexpressed Dyxin in NRCM which potently induced cellular hypertrophy (150%, p<0.001) and the hypertrophic gene program (ANF, BNP). Consistent with an induction of calcineurin signalling, the calcineurin-responsive gene Rcan1– 4 (MCIP1.4) was found significantly upregulated (3.2x, p<0.001). Conversely, knockdown of Dyxin (−75% on protein level) via miRNA completely blunted the hypertrophic response to hypertrophic stimuli, including stretch and PE (both p<0.001). Furthermore, PE-mediated activation of calcineurin signaling (Upregulation of Rcan1– 4 by 7.3x, p<0.001) was completely blocked by knockdown of Dyxin. To confirm these results in vivo, we next generated transgenic mice with cardiac-restricted overexpression of Dyxin using the α -MHC promoter. Despite normal cardiac function as assessed by echocardiography, adult transgenic mice displayed significant cardiac hypertrophy in morphometrical analyses (3.9 vs. 3.5 mg/g LV/heart weight, n=8–11, p<0.05). This finding was supplemented by a robust induction of the hypertrophic gene program including ANF (3.7-fold, n=6, p=0.01) and α -skeletal actin (2.8-fold, n=6, p<0.05). Likewise, Rcan1– 4 was found upregulated (+112%, n=5, p<0.05), Taken together, we show that the novel sarcomeric z-disc protein Dyxin/Lmcd1 is significantly upregulated in several models of cardiac hypertrophy and potently induces cardiomyocyte hypertrophy both in vitro and in vivo. Mechanistically, Lmcd1/Dyxin appears to signal through the calcineurin pathway.

Persistent Human γ-Globin Expression in Adult Transgenic Mice Following Selective Deletion of Two Repressor Elements Located 3′ to the Aγ-Globin Gene.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1585-1585
Author(s):  
Maria Gazouli ◽  
Elena Katsantoni ◽  
Theodore Kosteas ◽  
Nicholas P. Anagnou
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Globin Gene ◽  
Adult Life ◽  
Single Copy ◽  
Perinatal Period ◽  
Globin Genes ◽  
Cis Acting ◽  
Transgenic Lines

Abstract Adult β-globin gene expression is tightly regulated during development and hematopoiesis. The human globin genes undergoing two developmental switches are regulated by a complex interplay between cis-acting elements and stage-specific trans-acting factors. Understanding the molecular basis of globin gene switching is of particular interest as persistent expression of the fetal γ-globin genes in the adult ameliorates the effects of hemoglobinopathies. Natural occurring deletions within the human β-globin gene cluster lead to specific clinical syndromes characterized by increased production of fetal hemoglobin (HbF) in adult life. These clinical syndromes provide an excellent model to reveal and delineate novel cis-acting elements involved in the developmental control of hemoglobin switching. One major hypothesis, which accounts for these distinct phenotypic features, assumes that silencers located within the Aγ to δ gene region, are deleted in both HPFH and δβ-thalassemias leading to the failure of switching. Previous studies of our laboratory suggested that four elements (Enh, F, O and P) located within the Aγ toδ globin intergenic region, exhibited silencer activity in transient assays (Clin Res 41:308, 1993 and Blood 84:506, 1994) and that the Enh and F elements were capable of down-regulating transcription of the human β-globin locus in an embryonic-specific manner in transgenic mice (Exp Hematol 32:224, 2004). In the present study, we sought to further clarify the in vivo role of the Enh and F elements in the silencing of the fetal Aγ-gene. To this end, we have generated transgenic mice by using cosmid constructs containing the full length human globin LCR linked to the 3.3 kb Aγ gene, lacking both the Enh and F elements. As controls, we used transgenic lines containing the full length LCR linked to the 5.6 kb Aγ-gene construct, which includes both the Enh and F elements, previously shown by us (Blood102:3412, 2003) and others (Nature350:252, 1991) to be autonomously regulated during the perinatal period. Three transgenic lines for the LCR 3.3 kb Aγ-gene construct have been generated. Cosmid integrity and copy numbers (2, 3 and 4 copies respectively) were determined by Southern blot analysis. Expression analysis in adult blood RNA performed by S1 nuclease protection and real-time reverse transcriptase PCR, documented persistence of expression of Aγ-gene in adult life. To further investigate whether the persistence of Aγ-gene expression was not a non-specific effect of the multicopy integrants, we generated a new series of single copy mice by cross-breeding the three transgenic lines with a line expressing the Cre recombinase gene (CAG-Cre). As expected, in the control LCR-5.6 kb Aγ lines, containing the Enh and F elements, the Aγ-globin gene was silenced in all lines tested in the adult stage. In contrast, high levels of Aγ-globin gene expression, similar to those of multicopy integrants were documented in all three generated single copy LCR-3.3 kb Aγ lines, lacking the Enh and F elements. Thus, this study documents directly for the first time the in vivo role of of these two gene-proximal negative regulatory elements on the silencing of the Aγ-gene in the perinatal period and may permit the design of future therapeutic strategies for their exploitation in therapeutic approaches for thalassemias.

scholarly journals A haplotype of human angiotensinogen gene containing −217A increases blood pressure in transgenic mice compared with −217G

2008 ◽  
Vol 295 (6) ◽  
pp. R1849-R1857 ◽  
Author(s):  
Sudhir Jain ◽  
Govindaiah Vinukonda ◽  
Steven N. Fiering ◽  
Ashok Kumar
Keyword(s):  
Blood Pressure ◽  
Transgenic Mice ◽  
Mrna Level ◽  
Human Renin ◽  
Renin Gene ◽  
Plasma Angiotensin ◽  
Double Transgenic Mice ◽  
Agt Gene

The human angiotensinogen (hAGT) gene contains an A/G polymorphism at −217, and frequency of −217A allele is increased in African-American hypertensive patients. The hAGT gene has seven polymorphic sites in the 1.2-kb region of its promoter, and variant −217A almost always occurs with −532T, −793A, and −1074T, whereas variant −217G almost always occurs with −532C, −793G, and −1074G. Since allele −6A is the predominant allele in African-Americans, the AGT gene can be subdivided into two main haplotypes, −6A:−217A (AA) and −6A:−217G (AG). To understand the role of these haplotypes on hAGT gene expression and on blood pressure regulation in an in vivo situation, we have generated double transgenic mice containing human renin gene and either AA or AG haplotype of the hAGT gene using knock-in strategy at the hypoxanthine phosphoribosyltransferase locus. We show here that 1) hAGT mRNA level is increased in the liver by 60% and in the kidney by 40%; and 2) plasma AGT level is increased by ∼40%, and plasma angiotensin II level is increased by ∼50% in male double transgenic mice containing AA haplotype of the hAGT gene compared with the AG haplotype. In addition, systolic blood pressure is increased by 8 mmHg in transgenic mice containing the AA haplotype compared with the AG haplotype. This is the first report to show the effect of polymorphisms in the promoter of a human gene on its transcription in an in vivo situation that ultimately leads to an increase in blood pressure.

Alterations of β-adrenergic signaling and cardiac hypertrophy in transgenic mice overexpressing TGF-β1

2002 ◽  
Vol 283 (3) ◽  
pp. H1253-H1262 ◽  
Author(s):  
Stephan Rosenkranz ◽  
Markus Flesch ◽  
Kerstin Amann ◽  
Claudia Haeuseler ◽  
Heiko Kilter ◽  
...  
Keyword(s):  
Heart Failure ◽  
Growth Factor ◽  
Transgenic Mice ◽  
Cardiac Hypertrophy ◽  
Contractile Function ◽  
Heart Weight ◽  
Adrenergic System ◽  
Adrenergic Signaling ◽  
In Vivo Effects

Transforming growth factor-β1 (TGF-β1) promotes or inhibits cell proliferation and induces fibrotic processes and extracellular matrix production in numerous cell types. Several cardiac diseases are associated with an increased expression of TGF-β1 mRNA, particularly during the transition from stable cardiac hypertrophy to heart failure. In vitro studies suggest a link between TGF-β1 signaling and the β-adrenergic system. However, the in vivo effects of this growth factor on myocardial tissue have been poorly identified. In transgenic mice overexpressing TGF-β1 (TGF-β), we investigated the in vivo effects on cardiac morphology, β-adrenergic signaling, and contractile function. When compared with nontransgenic controls (NTG), TGF-β mice revealed significant cardiac hypertrophy (heart weight, 164 ± 7 vs. 130 ± 3 mg, P < 0.01; heart weight-to-body weight ratio, 6.8 ± 0.3 vs. 5.1 ± 0.1 mg/g, P < 0.01), accompanied by interstitial fibrosis. These morphological changes correlated with an increased expression of hypertrophy-associated proteins such as atrial natriuretic factor (ANF). Furthermore, overexpression of TGF-β1 led to alterations of β-adrenergic signaling as myocardial β-adrenoceptor density increased from 7.3 ± 0.3 to 11.2 ± 1.1 fmol/mg protein ( P < 0.05), whereas the expression of β-adrenoceptor kinase-1 and inhibitory G proteins decreased by 56 ± 9.7% and 58 ± 7.6%, respectively ( P < 0.05). As a consequence of altered β-adrenergic signaling, hearts from TGF-β showed enhanced contractile responsiveness to isoproterenol stimulation. In conclusion, we conclude that TGF-β1 induces cardiac hypertrophy and enhanced β-adrenergic signaling in vivo. The morphological alterations are either induced by direct effects of TGF-β1 or may at least in part result from increased β-adrenergic signaling, which may contribute to excessive catecholamine stimulation during the transition from compensated hypertrophy to heart failure.

scholarly journals Dysregulated human renin expression in transgenic mice carrying truncated genomic constructs: evidence supporting the presence of insulators at the renin locus

2008 ◽  
Vol 295 (3) ◽  
pp. F642-F653 ◽  
Author(s):  
Xiyou Zhou ◽  
Eric T. Weatherford ◽  
Xuebo Liu ◽  
Ella Born ◽  
Henry L. Keen ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Large Scale ◽  
Artificial Chromosome ◽  
Specific Cell ◽  
Distal Enhancer ◽  
Specific Expression ◽  
Tissue Specific ◽  
Human Renin ◽  
Renin Gene

We previously generated transgenic mice carrying a large P1 artificial chromosome (PAC160) encompassing a 160-kb segment containing the human renin gene, two upstream genes, and one downstream gene. We also previously generated mutant PAC160 constructs lacking the distal enhancer and concluded it is required to maintain baseline expression of human renin, but is not required for tissue-specific, cell-specific, and regulated expression of renin in vivo. We now report two additional transgenic lines carrying random truncations of PAC160 upstream of the renin gene. Southern and PCR mapping studies indicate that the truncation break points in the two lines are located ∼10.4 and 2.5 kb upstream of the renin gene causing a deletion of all DNA upstream of the break. We tested the hypothesis that large-scale deletion of DNA upstream of the human renin gene including the enhancer would cause dysregulation of human renin expression. Phenotypically, these truncations cause a severe dysregulation of human renin expression, but remarkably, a preservation of the normal tissue-specific expression of the human ethanolamine kinase 2 (ETNK2) gene which lies immediately downstream of renin. Several functional binding sites for CTCF, a mammalian insulator protein, were identified in and around the renin and ETNK2 loci by gel shift and chromatin immunoprecipitation. We conclude that there are sequences in and around the renin and ETNK2 loci which act as boundaries between neighboring genes which insulate them from each other. The study illustrates the value of taking a much wider genomic perspective when studying mechanisms regulating gene expression.

Using Knockout and Transgenic Mice to Study Neurophysiology and Behavior

1998 ◽  
Vol 78 (4) ◽  
pp. 1131-1163 ◽  
Author(s):  
MARINA R. PICCIOTTO ◽  
KEVIN WICKMAN
Keyword(s):  
Transgenic Mice ◽  
Reverse Genetics ◽  
New Technologies ◽  
Detailed Knowledge ◽  
Central Nervous System Function ◽  
Physiological Relevance ◽  
Nervous System Function ◽  
And Behavior ◽  
Mouse Mutagenesis

Picciotto, Marina R., and Kevin Wickman. Using Knockout and Transgenic Mice to Study Neurophysiology and Behavior. Physiol. Rev. 78: 1131–1163, 1998. — Reverse genetics, in which detailed knowledge of a gene of interest permits in vivo modification of its expression or function, provides a powerful method for examining the physiological relevance of any protein. Transgenic and knockout mouse models are particularly useful for studies of complex neurobiological problems. The primary aims of this review are to familiarize the nonspecialist with the techniques and limitations of mouse mutagenesis, to describe new technologies that may overcome these limitations, and to illustrate, using representative examples from the literature, some of the ways in which genetically altered mice have been used to analyze central nervous system function. The goal is to provide the information necessary to evaluate critically studies in which mutant mice have been used to study neurobiological problems.

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