Targeted overexpression of ornithine decarboxylase enhances β-adrenergic agonist-induced cardiac hypertrophy

2001 ◽  
Vol 358 (1) ◽  
pp. 25-32 ◽  
Author(s):  
Lisa M. SHANTZ ◽  
David J. FEITH ◽  
Anthony E. PEGG
Keyword(s):  
Transgenic Mice ◽  
Cardiac Hypertrophy ◽  
Ornithine Decarboxylase ◽  
Adrenergic Stimulation ◽  
Adrenergic Agonist ◽  
Specific Expression ◽  
Simultaneous Treatment ◽  
Constitutive Overexpression ◽  
Heart Induction

These studies were designed to determine the consequences of constitutive overexpression of ornithine decarboxylase (ODC) in the heart. Induction of ODC is known to occur in response to agents that induce cardiac hypertrophy. However, it is not known whether high ODC levels are sufficient for the development of a hypertrophic phenotype. Transgenic mice were generated with cardiac-specific expression of a stable ODC protein using the α-myosin heavy-chain promoter. Founder lines with > 1000-fold overexpression of ODC in the heart were established, resulting in a 50-fold overaccumulation of putrescine, 4-fold elevation in spermidine, a slight increase in spermine and accumulation of large amounts of cadaverine compared with littermate controls. Despite these significant alterations in polyamines, myocardial hypertrophy, as measured by ratio of heart to body weight, did not develop, although atrial natriuretic factor RNA was slightly elevated in transgenic ventricles. However, stimulation of β-adrenergic signalling by isoproterenol resulted in severe hypertrophy and even death in ODC-overexpressing mice without further altering polyamine levels, compared with only a mild hypertrophy in littermates. When β1-adrenergic stimulation was blocked by simultaneous treatment with isoproterenol and the β1 antagonist atenolol, a significant, although reduced, hypertrophy was still present in the hearts of transgenic mice, suggesting that both β1 and β2 adrenergic receptors contribute to the hypertrophic phenotype. Therefore these mice provide a model to study the in vivo co-operativity between high ODC activity and activation of other pathways leading to hypertrophy in the heart.

scholarly journals Loss of β-adrenergic–stimulated phosphorylation of CaV1.2 channels on Ser1700 leads to heart failure

2016 ◽  
Vol 113 (49) ◽  
pp. E7976-E7985 ◽  
Author(s):  
Linghai Yang ◽  
Dao-Fu Dai ◽  
Can Yuan ◽  
Ruth E. Westenbroek ◽  
Haijie Yu ◽  
...  
Keyword(s):  
Heart Failure ◽  
Cardiac Hypertrophy ◽  
Troponin I ◽  
Contractile Function ◽  
Voluntary Exercise ◽  
Adrenergic Stimulation ◽  
Compensatory Mechanisms ◽  
Specific Expression ◽  
Altered Expression

L-type Ca2+ currents conducted by voltage-gated calcium channel 1.2 (CaV1.2) initiate excitation–contraction coupling in the heart, and altered expression of CaV1.2 causes heart failure in mice. Here we show unexpectedly that reducing β-adrenergic regulation of CaV1.2 channels by mutation of a single PKA site, Ser1700, in the proximal C-terminal domain causes reduced contractile function, cardiac hypertrophy, and heart failure without changes in expression, localization, or function of the CaV1.2 protein in the mutant mice (SA mice). These deficits were aggravated with aging. Dual mutation of Ser1700 and a nearby casein-kinase II site (Thr1704) caused accelerated hypertrophy, heart failure, and death in mice with these mutations (STAA mice). Cardiac hypertrophy was increased by voluntary exercise and by persistent β-adrenergic stimulation. PKA expression was increased, and PKA sites Ser2808 in ryanodine receptor type-2, Ser16 in phospholamban, and Ser23/24 in troponin-I were hyperphosphorylated in SA mice, whereas phosphorylation of substrates for calcium/calmodulin-dependent protein kinase II was unchanged. The Ca2+ pool in the sarcoplasmic reticulum was increased, the activity of calcineurin was elevated, and calcineurin inhibitors improved contractility and ameliorated cardiac hypertrophy. Cardio-specific expression of the SA mutation also caused reduced contractility and hypertrophy. These results suggest engagement of compensatory mechanisms, which initially may enhance the contractility of individual myocytes but eventually contribute to an increased sensitivity to cardiovascular stress and to heart failure in vivo. Our results demonstrate that normal regulation of CaV1.2 channels by phosphorylation of Ser1700 in cardiomyocytes is required for cardiovascular homeostasis and normal physiological regulation in vivo.

scholarly journals Overproduction of cardiac S-adenosylmethionine decarboxylase in transgenic mice

2005 ◽  
Vol 393 (1) ◽  
pp. 295-302 ◽  
Author(s):  
Oleg Nisenberg ◽  
Anthony E. Pegg ◽  
Patricia A. Welsh ◽  
Kerry Keefer ◽  
Lisa M. Shantz
Keyword(s):  
Transgenic Mice ◽  
Ornithine Decarboxylase ◽  
Fold Increase ◽  
Adrenergic Stimulation ◽  
Compensatory Mechanisms ◽  
Specific Expression ◽  
Adenosylmethionine Decarboxylase ◽  
Cardiac Phenotype ◽  
Founder Line ◽  
Do So

The present study was designed to provide a better understanding of the role played by AdoMetDC (S-adenosylmethionine decarboxylase), the key rate-controlling enzyme in the synthesis of spermidine and spermine, in controlling polyamine levels and the importance of polyamines in cardiac physiology. The αMHC (α-myosin heavy chain) promoter was used to generate transgenic mice with cardiac-specific expression of AdoMetDC. A founder line (αMHC/AdoMetDC) was established with a >100-fold increase in AdoMetDC activity in the heart. Transgene expression was maximal by 1 week of age and remained constant into adulthood. However, the changes in polyamine levels were most pronounced during the first week of age, with a 2-fold decrease in putrescine and spermidine and a 2-fold increase in spermine. At later times, spermine returned to near control levels, whereas putrescine and spermidine levels remained lower, suggesting that compensatory mechanisms exist to limit spermine accumulation. The αMHC/AdoMetDC mice did not display an overt cardiac phenotype, but there was an increased cardiac hypertrophy after β-adrenergic stimulation with isoprenaline (‘isoproterenol’), as well as a small increase in spermine content. Crosses of the αMHC/AdoMetDC with αMHC/ornithine decarboxylase mice that have a >1000-fold increase in cardiac ornithine decarboxylase were lethal in utero, presumably due to increase in spermine to toxic levels. These findings suggest that cardiac spermine levels are highly regulated to avoid polyamine-induced toxicity and that homoeostatic mechanisms can maintain non-toxic levels even when one enzyme of the biosynthetic pathway is greatly elevated but are unable to do so when two biosynthetic enzymes are increased.

scholarly journals The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 319-329 ◽  
Author(s):  
S Dziennis ◽  
RA Van Etten ◽  
HL Pahl ◽  
DL Morris ◽  
TL Rothstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Heterologous Gene Expression ◽  
Myeloid Cells ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Specific Expression ◽  
High Level

Abstract CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.

In vitro and in vivo analysis of murine lipoprotein lipase gene promoter: tissue-specific expression

1995 ◽  
Vol 268 (2) ◽  
pp. E213-E218 ◽  
Author(s):  
J. M. Gimble ◽  
X. Hua ◽  
F. Wanker ◽  
C. Morgan ◽  
C. Robinson ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Lipoprotein Lipase ◽  
Reporter Gene ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Specific Expression ◽  
Tissue Specific ◽  
Brown Adipose ◽  
Octamer Motif

Lipoprotein lipase, an enzyme of central importance to lipid metabolism, is most abundant in adipose tissues, cardiac and skeletal muscle, and portions of the brain. The current work examined the murine lipoprotein lipase promoter using transient transfection, gel-retention analyses, and transgenic mice. Maximum expression of the luciferase reporter gene in transfected cells was observed with -101 bp of the promoter. Nuclear extracts from tissues expressing lipoprotein lipase contained DNA binding proteins that recognize the CCAAT box (-64 bp) and an octamer motif (-46 bp); this combination of factors was absent in nonexpressing tissues. Transgenic mice from three of five founders prepared with -1,824-bp promoter constructs expressed the luciferase reporter gene at highest levels in brown adipose tissue and brain. These findings suggest that the -1,824-bp promoter region contains sequence elements responsible for the tissue-specific transcription of lipoprotein lipase in vivo.

Identification of DNA elements cooperatively activating proopiomelanocortin gene expression in the pituitary glands of transgenic mice

1992 ◽  
Vol 12 (9) ◽  
pp. 3978-3990
Author(s):  
B Liu ◽  
G D Hammer ◽  
M Rubinstein ◽  
M Mortrud ◽  
M J Low
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Reporter Gene ◽  
Transgene Expression ◽  
Intermediate Lobe ◽  
Mobility Shift ◽  
Specific Expression ◽  
Dna Elements ◽  
Pomc Gene

The proopiomelanocortin (POMC) gene is highly expressed in adult mouse pituitary anterior lobe corticotrophs and intermediate lobe melanotrophs. To identify the DNA elements important for this tissue-specific expression, we analyzed a series of POMC reporter genes in transgenic mice. A DNA fragment containing rat POMC 5'-flanking sequences from -323 to -34 recapitulated both basal pituitary cell-specific and hormonally stimulated expression in adult mice when fused to a heterologous thymidine kinase promoter. Developmental onset of the reporter gene expression lagged by 1 day but otherwise closely paralleled the normal ontogeny of murine POMC gene expression, including corticotroph activation at embryonic day 14.5 (E14.5) followed by melanotroph activation at E15.5 to E16.5. AtT20 corticotroph nuclear protein extracts interacted with three specific regions of the functional POMC promoter in DNase I protection assays. The positions of these protected sites were -107 to -160 (site 1), -182 to -218 (site 2), and -249 to -281 (site 3). Individual deletions of these footprinted sites did not alter transgene expression; however, the simultaneous deletion of sites 2 and 3 prevented transgene expression in both corticotrophs and melanotrophs. Electrophoretic mobility shift and Southwestern (DNA-protein) assays demonstrated that multiple AtT20 nuclear proteins bound to these footprinted sites. We conclude that the sequences between -323 and -34 of the rat POMC gene promoter are both necessary and sufficient for correct spatial, temporal, and hormonally regulated expression in the pituitary gland. Our data suggest that the three footprinted sites within the promoter are functionally interchangeable and act in combination with promoter elements between -114 and -34. The inability of any reporter gene construction to dissociate basal and hormonally stimulated expression suggests that these DNA elements are involved in both of these two characteristics of POMC gene expression in vivo.

Beta-MHC and SMLC1 transgene induction in overloaded skeletal muscle of transgenic mice

1996 ◽  
Vol 270 (4) ◽  
pp. C1111-C1121 ◽  
Author(s):  
J. L. Wiedenman ◽  
I. Rivera-Rivera ◽  
D. Vyas ◽  
G. Tsika ◽  
L. Gao ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Troponin C ◽  
Type I ◽  
Initial Attempt ◽  
Specific Expression ◽  
Slow Type ◽  
Transgenic Lines ◽  
Fast Twitch Muscle ◽  
Mechanical Overload

The hypertrophic responses of white fast-twitch muscle to mechanical overload has been investigated using transgenic mice. After 7 wk of overload, endogenous beta-myosin heavy chain (MHC) and slow myosin light chain 1 and 2 (SMLC1, SMLC2) protein were increased in the overloaded plantaris (OP) muscle compared with sham-operated control plantaris (CP)muscle. Concurrently, the levels of endogenous beta-MHC, SMLC1, SMLC2, and cardiac/slow troponin C (CTnC) mRNA transcripts were significantly increased in OP muscles, whereas skeletal troponin C (sTnC) mRNA transcript levels decreased. As an initial attempt to locate DNA sequence(s) that governs beta-MHC induction in response to mechanical overload, multiple independent transgenic lines harboring four different human beta-MHC transgenes (beta 1286, beta 988, beta 450, beta 141) were generated. Except for transgene beta 141, muscle-specific expression and induction (3- to 22-fold) in OP muscles were observed by measuring chloramphenicol acetyltransferase activity (CAT assay). Induction of a SMLC1 transgene (3920SMLC1) in OP muscles was also observed. Collectively, these in vivo data provide evidence that 1) a mechanical overload inducible element(s) is located between nucleotides -450 and +120 of the human beta-MHC transgene, 2) 3,900 bp of 5' sequence is sufficient to confer mechanical overload induction of a SMLC1 transgene, and 3) the increased expression of slow/type I isomyosin (beta-MHC, SMLC1, SMLC2) in response to mechanical overload is regulated, in part, transcriptionally.

scholarly journals Orai1 Channel Inhibition Preserves Left Ventricular Systolic Function and Normal Ca 2+ Handling After Pressure Overload

Circulation ◽  
2020 ◽  
Vol 141 (3) ◽  
pp. 199-216 ◽  
Author(s):  
Fiona Bartoli ◽  
Marc A. Bailey ◽  
Baptiste Rode ◽  
Philippe Mateo ◽  
Fabrice Antigny ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Systolic Function ◽  
Systolic Dysfunction ◽  
Pressure Overload ◽  
Left Ventricular Systolic Function ◽  
Left Ventricular ◽  
Specific Expression ◽  
Channel Inhibition

Background: Orai1 is a critical ion channel subunit, best recognized as a mediator of store-operated Ca 2+ entry (SOCE) in nonexcitable cells. SOCE has recently emerged as a key contributor of cardiac hypertrophy and heart failure but the relevance of Orai1 is still unclear. Methods: To test the role of these Orai1 channels in the cardiac pathophysiology, a transgenic mouse was generated with cardiomyocyte-specific expression of an ion pore-disruptive Orai1 R91W mutant (C-dnO1). Synthetic chemistry and channel screening strategies were used to develop 4-(2,5-dimethoxyphenyl)-N-[(pyridin-4-yl)methyl]aniline (hereafter referred to as JPIII), a small-molecule Orai1 channel inhibitor suitable for in vivo delivery. Results: Adult mice subjected to transverse aortic constriction (TAC) developed cardiac hypertrophy and reduced ventricular function associated with increased Orai1 expression and Orai1-dependent SOCE (assessed by Mn 2+ influx). C-dnO1 mice displayed normal cardiac electromechanical function and cellular excitation-contraction coupling despite reduced Orai1-dependent SOCE. Five weeks after TAC, C-dnO1 mice were protected from systolic dysfunction (assessed by preserved left ventricular fractional shortening and ejection fraction) even if increased cardiac mass and prohypertrophic markers induction were observed. This is correlated with a protection from TAC-induced cellular Ca 2+ signaling alterations (increased SOCE, decreased [Ca 2+ ] i transients amplitude and decay rate, lower SR Ca 2+ load and depressed cellular contractility) and SERCA2a downregulation in ventricular cardiomyocytes from C-dnO1 mice, associated with blunted Pyk2 signaling. There was also less fibrosis in heart sections from C-dnO1 mice after TAC. Moreover, 3 weeks treatment with JPIII following 5 weeks of TAC confirmed the translational relevance of an Orai1 inhibition strategy during hypertrophic insult. Conclusions: The findings suggest a key role of cardiac Orai1 channels and the potential for Orai1 channel inhibitors as inotropic therapies for maintaining contractility reserve after hypertrophic stress.

scholarly journals Transcription factor CHF1/Hey2 suppresses cardiac hypertrophy through an inhibitory interaction with GATA4

2006 ◽  
Vol 290 (5) ◽  
pp. H1997-H2006 ◽  
Author(s):  
Fan Xiang ◽  
Yasuhiko Sakata ◽  
Lei Cui ◽  
Joey M. Youngblood ◽  
Hironori Nakagami ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transgenic Mice ◽  
Cardiac Hypertrophy ◽  
Marker Genes ◽  
Recognition Sequence ◽  
Inhibitory Interaction ◽  
Hypertrophic Response ◽  
Neonatal Cardiomyocytes

Pathological cardiac hypertrophy is considered a precursor to clinical heart failure. Understanding the transcriptional regulators that suppress the hypertrophic response may have profound implications for the treatment of heart disease. We report the generation of transgenic mice that overexpress the transcription factor CHF1/Hey2 in the myocardium. In response to the α-adrenergic agonist phenylephrine, they show marked attenuation in the hypertrophic response compared with wild-type controls, even though blood pressure is similar in both groups. Isolated myocytes from transgenic mice demonstrate a similar resistance to phenylephrine-induced hypertrophy in vitro, providing further evidence that the protective effect of CHF1/Hey2 is mediated at the myocyte level. Induction of the hypertrophy marker genes ANF, BNP, and β- MHC in the transgenic cells is concurrently suppressed in vivo and in vitro, demonstrating that the induction of hypertrophy-associated genes is repressed by CHF1/Hey2. Transfection of CHF1/Hey2 into neonatal cardiomyocytes suppresses activation of an ANF reporter plasmid by the transcription factor GATA4, which has previously been shown to activate a hypertrophic transcriptional program. Furthermore, CHF1/Hey2 binds GATA4 directly in coimmunoprecipitation assays and inhibits the binding of GATA4 to its recognition sequence within the ANF promoter. Our findings demonstrate that CHF1/Hey2 functions as an antihypertrophic gene, possibly through inhibition of a GATA4-dependent hypertrophic program.

Cardiac hypertrophy and altered β-adrenergic signaling in transgenic mice that express the amino terminus of β-ARK1

2003 ◽  
Vol 285 (5) ◽  
pp. H2201-H2211 ◽  
Author(s):  
Janelle R. Keys ◽  
Emily A. Greene ◽  
Chris J. Cooper ◽  
Sathyamangla V. Naga Prasad ◽  
Howard A. Rockman ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Cardiac Hypertrophy ◽  
Protein Interactions ◽  
Amino Terminus ◽  
Amino Acid Residues ◽  
Protein Protein Interactions ◽  
Gpcr Signaling ◽  
Amino Terminal ◽  
G Protein Coupled

The G protein-coupled receptor (GPCR) kinase β-adrenergic receptor (β-AR) kinase-1 (β-ARK1) is elevated during heart failure; however, its role is not fully understood. β-ARK1 contains several domains that are capable of protein-protein interactions that may play critical roles in the regulation of GPCR signaling. In this study, we developed a novel line of transgenic mice that express an amino-terminal peptide of β-ARK1 that is comprised of amino acid residues 50–145 (β-ARKnt) in the heart to determine whether this domain has any functional significance in vivo. Surprisingly, the β-ARKnt transgenic mice presented with cardiac hypertrophy. Our data suggest that the phenotype was driven via an enhanced β-AR system, as β-ARKnt mice had elevated cardiac β-AR density. Moreover, administration of a β-AR antagonist reversed hypertrophy in these mice. Interestingly, signaling through the β-AR in response to agonist stimulation was not enhanced in these mice. Thus the amino terminus of β-ARK1 appears to be critical for normal β-AR regulation in vivo, which further supports the hypothesis that β-ARK1 plays a key role in normal and compromised cardiac GPCR signaling.

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