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Rhiannon R Baggott,
Tamer M A Mohamed,
Delvac Oceandy,
Marylouisa Holton,
Marie Cécile Blanc,
Sandrine C Roux-Soro,
Sarah Brown,
James E Brown, Elizabeth J Cartwright,
Weiguang Wang,
Ludwig Neyses,
Angel L Armesilla
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ABSTRACT: Cancer is caused by defects in the signalling mechanisms that govern cell proliferation and apoptosis. It is well known that calcium-dependent signalling pathways play a critical role in cell regulation. A tight control of calcium homeostasis by transporters and channel proteins is required to assure a proper functioning of the calcium-sensitive signal transduction pathways that regulate cell growth and apoptosis. The plasma membrane calcium ATPase 2 (PMCA2) has been recently identified as a negative regulator of apoptosis that can play a significant role in cancer progression by conferring cells resistance to apoptosis. We have previously reported an inhibitory interaction between PMCA2 and the calcium-activated signalling molecule calcineurin in breast cancer cells. Here, we demonstrate that disruption of the PMCA2/calcineurin interaction in a variety of human breast cancer cells results in activation of the calcineurin/NFAT pathway, upregulation in the expression of the pro-apoptotic protein Fas Ligand and in a concomitant loss of cell viability. Reduction in cell viability is the consequence of an increase in cell apoptosis. Impairment of the PMCA2/calcineurin interaction enhances paclitaxel-mediated cytotoxicity of breast tumoral cells. Our results suggest that therapeutic modulation of the PMCA2/calcineurin interaction might have important clinical applications to improve current treatments for breast cancer patients.
Carcinogenesis 09/2012; · 5.70 Impact Factor
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Wei Liu,
Min Zi,
Ronald Naumann,
Susanne Ulm,
Jiawei Jin,
Domenico M Taglieri,
Sukhpal Prehar,
Junhong Gui,
Hoyee Tsui,
Rui-Ping Xiao,
Ludwig Neyses,
R John Solaro,
Yunbo Ke, Elizabeth J Cartwright,
Ming Lei,
Xin Wang
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ABSTRACT: Stress-induced hypertrophic remodeling is a critical pathogenetic process leading to heart failure. Although many signal transduction cascades are demonstrated as important regulators to facilitate the induction of cardiac hypertrophy, the signaling pathways for suppressing hypertrophic remodeling remain largely unexplored. In this study, we identified p21-activated kinase 1 (Pak1) as a novel signaling regulator that antagonizes cardiac hypertrophy.
Hypertrophic stress applied to primary neonatal rat cardiomyocytes (NRCMs) or murine hearts caused the activation of Pak1. Analysis of NRCMs expressing constitutively active Pak1 or in which Pak1 was silenced disclosed that Pak1 played an antihypertrophic role. To investigate the in vivo role of Pak1 in the heart, we generated mice with a cardiomyocyte-specific deletion of Pak1 (Pak1(cko)). When subjected to 2 weeks of pressure overload, Pak1(cko) mice developed greater cardiac hypertrophy with attendant blunting of JNK activation compared with controls, and these knockout mice underwent the transition into heart failure when prolonged stress was applied. Chronic angiotensin II infusion also caused increased cardiac hypertrophy in Pak1(cko) mice. Moreover, we discovered that the Pak1 activator FTY720, a sphingosine-like analog, was able to prevent pressure overload-induced hypertrophy in wild-type mice without compromising their cardiac functions. Meanwhile, FTY720 failed to exert such an effect on Pak1(cko) mice, suggesting that the antihypertrophic effect of FTY720 likely acts through Pak1 activation.
These results, for the first time, establish Pak1 as a novel antihypertrophic regulator and suggest that it may be a potential therapeutic target for the treatment of cardiac hypertrophy and heart failure.
Circulation 11/2011; 124(24):2702-15. · 14.74 Impact Factor
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Tamer M A Mohamed,
Delvac Oceandy,
Min Zi,
Sukhpal Prehar,
Nasser Alatwi,
Yanwen Wang,
Mohamed A Shaheen,
Riham Abou-Leisa,
Celine Schelcher,
Zeinab Hegab,
Florence Baudoin,
Michael Emerson,
Mamas Mamas,
Giulietta Di Benedetto,
Manuela Zaccolo,
Ming Lei, Elizabeth J Cartwright,
Ludwig Neyses
[show abstract]
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ABSTRACT: Identification of the signaling pathways that regulate cyclic nucleotide microdomains is essential to our understanding of cardiac physiology and pathophysiology. Although there is growing evidence that the plasma membrane Ca(2+)/calmodulin-dependent ATPase 4 (PMCA4) is a regulator of neuronal nitric-oxide synthase, the physiological consequence of this regulation is unclear. We therefore tested the hypothesis that PMCA4 has a key structural role in tethering neuronal nitric-oxide synthase to a highly compartmentalized domain in the cardiac cell membrane. This structural role has functional consequences on cAMP and cGMP signaling in a PMCA4-governed microdomain, which ultimately regulates cardiac contractility. In vivo contractility and calcium amplitude were increased in PMCA4 knock-out animals (PMCA4(-/-)) with no change in diastolic relaxation or the rate of calcium decay, showing that PMCA4 has a function distinct from beat-to-beat calcium transport. Surprisingly, in PMCA4(-/-), over 36% of membrane-associated neuronal nitric-oxide synthase (nNOS) protein and activity was delocalized to the cytosol with no change in total nNOS protein, resulting in a significant decrease in microdomain cGMP, which in turn led to a significant elevation in local cAMP levels through a decrease in PDE2 activity (measured by FRET-based sensors). This resulted in increased L-type calcium channel activity and ryanodine receptor phosphorylation and hence increased contractility. In the heart, in addition to subsarcolemmal calcium transport, PMCA4 acts as a structural molecule that maintains the spatial and functional integrity of the nNOS signaling complex in a defined microdomain. This has profound consequences for the regulation of local cyclic nucleotide and hence cardiac β-adrenergic signaling.
Journal of Biological Chemistry 09/2011; 286(48):41520-9. · 4.77 Impact Factor
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ABSTRACT: The plasma membrane calcium ATPases (PMCA) are a family of genes which extrude Ca(2+) from the cell and are involved in the maintenance of intracellular free calcium levels and/or with Ca(2+) signalling, depending on the cell type. In the cardiovascular system, Ca(2+) is not only essential for contraction and relaxation but also has a vital role as a second messenger in signal transduction pathways. A complex array of mechanisms regulate intracellular free calcium levels in the heart and vasculature and a failure in these systems to maintain normal Ca(2+) homeostasis has been linked to both heart failure and hypertension. This article focuses on the functions of PMCA, in particular isoform 4 (PMCA4), in the heart and vasculature and the reported links between PMCAs and contractile function, cardiac hypertrophy, cardiac rhythm and sudden cardiac death, and blood pressure control and hypertension. It is becoming clear that this family of calcium extrusion pumps have essential roles in both cardiovascular health and disease.
Science China. Life sciences 08/2011; 54(8):691-8. · 2.02 Impact Factor
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Emmanuel Eroume A Egom,
Tamer M A Mohamed,
Mamas A Mamas,
Ying Shi,
Wei Liu,
Debora Chirico,
Sally E Stringer,
Yunbo Ke,
Mohamed Shaheen,
Tao Wang,
Sanoj Chacko,
Xin Wang,
R John Solaro,
Farzin Fath-Ordoubadi, Elizabeth J Cartwright,
Ming Lei
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ABSTRACT: We investigated whether plasma long-chain sphingoid base (LCSB) concentrations are altered by transient cardiac ischemia during percutaneous coronary intervention (PCI) in humans and examined the signaling through the sphingosine-1-phosphate (S1P) cascade as a mechanism underlying the S1P cardioprotective effect in cardiac myocytes. Venous samples were collected from either the coronary sinus (n = 7) or femoral vein (n = 24) of 31 patients at 1 and 5 min and 12 h, following induction of transient myocardial ischemia during elective PCI. Coronary sinus levels of LCSB were increased by 1,072% at 1 min and 941% at 5 min (n = 7), while peripheral blood levels of LCSB were increased by 579% at 1 min, 617% at 5 min, and 436% at 12 h (n = 24). In cultured cardiac myocytes, S1P, sphingosine (SPH), and FTY720, a sphingolipid drug candidate, showed protective effects against CoCl induced hypoxia/ischemic cell injury by reducing lactate dehydrogenase activity. Twenty-five nanomolars of FTY720 significantly increased phospho-Pak1 and phospho-Akt levels by 56 and 65.6% in cells treated with this drug for 15 min. Further experiments demonstrated that FTY720 triggered nitric oxide release from cardiac myocytes is through pertussis toxin-sensitive phosphatidylinositol 3-kinase/Akt/endothelial nitric oxide synthase signaling. In ex vivo hearts, ischemic preconditioning was cardioprotective in wild-type control mice (Pak1(f/f)), but this protection appeared to be ineffective in cardiomyocyte-specific Pak1 knockout (Pak1(cko)) hearts. The present study provides the first direct evidence of the behavior of plasma sphingolipids following transient cardiac ischemia with dramatic and early increases in LCSB in humans. We also demonstrated that S1P, SPH, and FTY720 have protective effects against hypoxic/ischemic cell injury, likely a Pak1/Akt1 signaling cascade and nitric oxide release. Further study on a mouse model of cardiac specific deletion of Pak1 demonstrates a crucial role of Pak1 in cardiac protection against ischemia/reperfusion injury.
AJP Heart and Circulatory Physiology 06/2011; 301(4):H1487-95. · 3.71 Impact Factor
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ABSTRACT: In the heart, Ca(2+) is crucial for the regulation of contraction and intracellular signaling, processes, which are vital to the functioning of the healthy heart. Ca(2+) -activated signaling pathways must function against a background of large, rapid, and tightly regulated changes in intracellular free Ca(2+) concentrations during each contraction and relaxation cycle. This review highlights a number of proteins that regulate signaling Ca(2+) in both normal and pathological conditions including cardiac hypertrophy and heart failure, and discusses how these pathways are not regulated by the marked elevation in free intracellular calcium ([Ca(2+) ](i)) during contraction but require smaller sustained increases in Ca(2+) concentration. In addition, we present published evidence that the pool of Ca(2+) that regulates signaling is compartmentalized into distinct cellular microdomains and is thus distinct from that regulating contraction.
BioFactors 05/2011; 37(3):175-81. · 4.93 Impact Factor
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Min Zi,
Tomomi E. Kimura,
Wei Liu,
Jiawei Jin,
Jonathan Higham,
Sanjay Kharche,
Guoliang Hao,
Ying Shi,
Weijian Shen,
Sukhpal Prehar,
Aleksandr Mironov,
Ludwig Neyses,
Marti F. A. Bierhuizen,
Mark R. Boyett,
Henggui Zhang,
Ming Lei, Elizabeth J. Cartwright,
Xin Wang
[show abstract]
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ABSTRACT: Connexin 43 (Cx43) is the predominant isoform of gap junction proteins in the working myocardium. In the heart, mitogen-activated
protein (MAP) kinases are implicated in regulating Cx43 remodeling; however, their precise roles remain obscure. Mitogen-activated
protein kinase kinase 4 (MKK4) is a critical component of the stress-activated MAP kinase signaling pathway. We have previously
demonstrated that MKK4 antagonizes cardiomyocyte hypertrophy. Herein, we investigate the role of MKK4 in regulating Cx43 expression
in cardiomyocytes. We found that knockdown of MKK4 expression or inhibition of its kinase activity in neonatal rat cardiomyocytes
(NRCMs) significantly reduced phenylephrine-induced Cx43 expression. Furthermore, two activator protein-1 (AP-1) binding elements
in the Cx43 promoter region were identified as being responsible for the MKK4-regulated Cx43 expression. Consistently, we
also detected heterogeneously reduced Cx43 expression and attenuated zonula occludens-1 (ZO-1) content in the hearts of MKK4
cardiomyocyte-specific knockout mice (MKK4cko) following pressure overload. To test whether heterogeneously reduced Cx43 expression
contributes to ventricular arrhythmic vulnerability, MKK4cko and control mice were subjected to pressure overload followed
by programmed electrical stimulation (PES). 6 of 13 MKK4cko mice, but none of the controls, developed ventricular tachycardia.
Epicardial activation mapping recorded from the MKK4cko hypertrophied heart showed ventricular activation delay. Mathematical
models have simulated that the spatially heterogeneous decrease in Cx43 causes slowed ventricular conduction and fragmented
wave propagations leading to re-entrant excitations. Collectively, these data reveal a novel role for MKK4 in regulating Cx43
expression and preventing hypertrophy-associated arrhythmogenesis.
Journal of Biological Chemistry 03/2011; · 4.77 Impact Factor
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Min Zi,
Tomomi E Kimura,
Wei Liu,
Jiawei Jin,
Jonathan Higham,
Sanjay Kharche,
Guoliang Hao,
Ying Shi,
Weijian Shen,
Sukhpal Prehar,
Aleksandr Mironov,
Ludwig Neyses,
Marti F A Bierhuizen,
Mark R Boyett,
Henggui Zhang,
Ming Lei, Elizabeth J Cartwright,
Xin Wang
[show abstract]
[hide abstract]
ABSTRACT: Connexin 43 (Cx43) is the predominant isoform of gap junction proteins in the working myocardium. In the heart, mitogen-activated protein (MAP) kinases are implicated in regulating Cx43 remodeling; however, their precise roles remain obscure. Mitogen-activated protein kinase kinase 4 (MKK4) is a critical component of the stress-activated MAP kinase signaling pathway. We have previously demonstrated that MKK4 antagonizes cardiomyocyte hypertrophy. Herein, we investigate the role of MKK4 in regulating Cx43 expression in cardiomyocytes. We found that knockdown of MKK4 expression or inhibition of its kinase activity in neonatal rat cardiomyocytes (NRCMs) significantly reduced phenylephrine-induced Cx43 expression. Furthermore, two activator protein-1 (AP-1) binding elements in the Cx43 promoter region were identified as being responsible for the MKK4-regulated Cx43 expression. Consistently, we also detected heterogeneously reduced Cx43 expression and attenuated zonula occludens-1 (ZO-1) content in the hearts of MKK4 cardiomyocyte-specific knockout mice (MKK4cko) following pressure overload. To test whether heterogeneously reduced Cx43 expression contributes to ventricular arrhythmic vulnerability, MKK4cko and control mice were subjected to pressure overload followed by programmed electrical stimulation (PES). 6 of 13 MKK4cko mice, but none of the controls, developed ventricular tachycardia. Epicardial activation mapping recorded from the MKK4cko hypertrophied heart showed ventricular activation delay. Mathematical models have simulated that the spatially heterogeneous decrease in Cx43 causes slowed ventricular conduction and fragmented wave propagations leading to re-entrant excitations. Collectively, these data reveal a novel role for MKK4 in regulating Cx43 expression and preventing hypertrophy-associated arrhythmogenesis.
Journal of Biological Chemistry 03/2011; 286(20):17821-30. · 4.77 Impact Factor
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ABSTRACT: There is little doubt that members of mitogen-activated protein kinase (MAPK) families play key roles in the transition from adaptive hypertrophic remodeling to heart failure. Mitogen-activated protein kinase kinase 7 (MKK7) is a critical component of stress-activated MAP kinase signaling pathway. The role of MKK7 plays in mediating cardiac remodeling in response to load stress has yet to be defined. Herein, we investigate the role of MKK7 in regulating cardiac remodeling in response to pressure overload. We generated and examined the phenotype of mice with cardiomyocyte-specific deletion of the mkk7 gene (MKK7(cko)). Following one week of pressure overload, MKK7(cko) mice exhibited characteristic phenotypes of heart failure evidenced by deterioration in ventricular function and pulmonary congestion. Cell death assays revealed an increased prevalence of cardiomyocyte apoptosis in the MKK7(cko) heart, in which elevated p53 levels and attenuated expression of manganese superoxide dismutase (MnSOD) were found. Moreover, extensive interstitial fibrosis was discovered in the knockout heart likely attributable to upregulation of transforming growth factor β (TGF-β) signaling. These results reveal an essential role of MKK7 in cardiomyocytes for protecting the heart from hypertrophic insults thereby preventing the transition to heart failure.
Journal of Molecular and Cellular Cardiology 01/2011; 50(4):702-11. · 5.17 Impact Factor
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ABSTRACT: Calcium has been unequivocally regarded as a key signal messenger in almost every cell type. Calcium regulates a number of important cellular functions including cell growth, myofilament contraction, cell survival and apoptosis as well as gene transcription. A complex regulatory mechanism of cellular calcium is needed to fine tune the precise calcium concentration in each subcellular location and also to transmit the signals carried by the calcium pool to the correct end target. In this article we will review the recently emerging role of the plasma membrane calcium/calmodulin dependent ATPase isoform 4 (PMCA4) in regulating calcium signalling. We will then focus on the function of this molecule in cardiomyocytes, in which PMCA4 forms protein-protein interactions with several key signalling molecules. Recent evidence has shown in vivo physiological functionalities and possible clinical implications of the PMCA4 signalling complex. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.
Biochimica et Biophysica Acta 12/2010; 1813(5):974-8. · 4.66 Impact Factor
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Tomomi E Kimura,
Jiawei Jin,
Min Zi,
Sukhpal Prehar,
Wei Liu,
Delvac Oceandy,
Jun-ichi Abe,
Ludwig Neyses,
Arthur H Weston, Elizabeth J Cartwright,
Xin Wang
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ABSTRACT: Mitogen-activated protein kinase (MAPK) pathways provide a critical connection between extrinsic and intrinsic signals to cardiac hypertrophy. Extracellular signal-regulated protein kinase (ERK)5, an atypical MAPK is activated in the heart by pressure overload. However, the role of ERK5 plays in regulating hypertrophic growth and hypertrophy-induced apoptosis is not completely understood.
Herein, we investigate the in vivo role and signaling mechanism whereby ERK5 regulates cardiac hypertrophy and hypertrophy-induced apoptosis.
We generated and examined the phenotypes of mice with cardiomyocyte-specific deletion of the erk5 gene (ERK5(cko)). In response to hypertrophic stress, ERK5(cko) mice developed less hypertrophic growth and fibrosis than controls. However, increased apoptosis together with upregulated expression levels of p53 and Bad were observed in the mutant hearts. Consistently, we found that silencing ERK5 expression or specific inhibition of its kinase activity using BIX02189 in neonatal rat cardiomyocytes (NRCMs) reduced myocyte enhancer factor (MEF)2 transcriptional activity and blunted hypertrophic responses. Furthermore, the inhibition of MEF2 activity in NRCMs using a non-DNA binding mutant form of MEF2 was found to attenuate the ERK5-regulated hypertrophic response.
These results reveal an important function of ERK5 in cardiac hypertrophic remodeling and cardiomyocyte survival. The role of ERK5 in hypertrophic remodeling is likely to be mediated via the regulation of MEF2 activity.
Circulation Research 03/2010; 106(5):961-70. · 9.49 Impact Factor
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ABSTRACT: The array of contraceptives currently available is clearly inadequate and does not meet consumer demands since it is estimated that up to a quarter of all pregnancies worldwide are unintended. There is, therefore, an overwhelming global need to develop new effective, safe, ideally non-hormonal contraceptives for both male and female use. The contraceptive field, unlike other areas such as cancer, has a dearth of new targets. We have addressed this issue and propose that isoform 4 of the plasma membrane calcium ATPase is a potentially exciting novel target for fertility control. The plasma membrane calcium ATPase is a ubiquitously expressed calcium pump whose primary function in the majority of cells is to extrude calcium to the extracellular milieu. Two isoforms of this gene family, PMCA1 and PMCA4, are expressed in spermatozoa, with PMCA4 being the predominant isoform. Although this gene is ubiquitously expressed, its function is highly tissue-specific. Genetic deletion of PMCA4, in PMCA4 knockout mice, led to 100% infertility specifically in the male mutant mice due to a selective defect in sperm motility. It is important to note that the gene deletion did not affect normal mating characteristics in these mice. This phenotype was mimicked in wild-type sperm treated with the non-specific PMCA inhibitor 5-(and 6-) carboxyeosin diacetate succinimidyl ester; a proof-of-principle that inhibition of PMCA4 has potential importance in the control of fertility. This review outlines the potential for PMCA4 to be a novel target for fertility control by acting to inhibit sperm motility. It will outline the characteristics that make this target drugable and will describe methodologies to identify and validate novel inhibitors of this target.
Handbook of experimental pharmacology 01/2010;
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ABSTRACT: The close relationship between signaling pathways regulating tumor growth and cardiac hypertrophy has attracted considerable interest. Although the involvement of proto-oncogenes in positively modulating myocardial hypertrophy has long been recognized, little is known about factors that counterregulate them. In this article, we review the novel tumor suppressor Ras-association domain family protein isoform 1A (RASSF1A), which strongly inhibits the prohypertrophic Ras-Raf1-ERK1/2 pathway in the heart. RASSF1A interacts with a number of important signaling molecules regulating cell growth, survival, and apoptosis; therefore, it serves as a key adaptor molecule that integrates the upstream stimuli and transduces them to the selective downstream effectors.
Trends in cardiovascular medicine 11/2009; 19(8):262-7. · 4.37 Impact Factor
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Delvac Oceandy,
Adam Pickard,
Sukhpal Prehar,
Min Zi,
Tamer M A Mohamed,
Peter J Stanley,
Florence Baudoin-Stanley,
Raja Nadif,
Stella Tommasi,
Gerd P Pfeifer,
Angel L Armesilla, Elizabeth J Cartwright,
Ludwig Neyses
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ABSTRACT: Ras signaling regulates a number of important processes in the heart, including cell growth and hypertrophy. Although it is known that defective Ras signaling is associated with Noonan, Costello, and other syndromes that are characterized by tumor formation and cardiac hypertrophy, little is known about factors that may control it. Here we investigate the role of Ras effector Ras-association domain family 1 isoform A (RASSF1A) in regulating myocardial hypertrophy.
A significant downregulation of RASSF1A expression was observed in hypertrophic mouse hearts, as well as in failing human hearts. To further investigate the role of RASSF1A in cardiac (patho)physiology, we used RASSF1A knock-out (RASSF1A(-)(/)(-)) mice and neonatal rat cardiomyocytes with adenoviral overexpression of RASSF1A. Ablation of RASSF1A in mice significantly enhanced the hypertrophic response to transverse aortic constriction (64.2% increase in heart weight/body weight ratio in RASSF1A(-)(/)(-) mice compared with 32.4% in wild type). Consistent with the in vivo data, overexpression of RASSF1A in cardiomyocytes markedly reduced the cellular hypertrophic response to phenylephrine stimulation. Analysis of molecular signaling events in isolated cardiomyocytes indicated that RASSF1A inhibited extracellular regulated kinase 1/2 activation, likely by blocking the binding of Raf1 to active Ras.
Our data establish RASSF1A as a novel inhibitor of cardiac hypertrophy by modulating the extracellular regulated kinase 1/2 pathway.
Circulation 09/2009; 120(7):607-16. · 14.74 Impact Factor
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Wei Liu,
Min Zi,
Jiawei Jin,
Sukhpal Prehar,
Delvac Oceandy,
Tomomi E Kimura,
Ming Lei,
Ludwig Neyses,
Arthur H Weston, Elizabeth J Cartwright,
Xin Wang
[show abstract]
[hide abstract]
ABSTRACT: Mitogen-activated protein kinase kinase (MKK)4 is a critical member of the mitogen-activated protein kinase family. It is able to activate the c-Jun NH(2)-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase in response to environmental stresses. JNK and p38 are strongly implicated in pathological cardiac hypertrophy and heart failure; however, the regulatory mechanism whereby the upstream kinase MKK4 activates these signaling cascades in the heart is unknown. To elucidate the biological function of MKK4, we generated mice with a cardiac myocyte-specific deletion of mkk4 (MKK4(cko) mice). In response to pressure overload or chronic beta-adrenergic stimulation, upregulated NFAT (nuclear factor of activated T-cell) transcriptional activity associated with exacerbated cardiac hypertrophy and the appearance of apoptotic cardiomyocytes were observed in MKK4(cko) mice. However, when subjected to swimming exercise, MKK4(cko) mice displayed a similar level of physiological cardiac hypertrophy compared to controls (MKK4(f/f)). In addition, we also discovered that MKK4 expression was significantly reduced in heart failure patients. In conclusion, this study demonstrates for the first time that MKK4 is a key mediator which prevents the transition from an adaptive response to maladaptive cardiac hypertrophy likely involving the regulation of the NFAT signaling pathway.
Circulation Research 04/2009; 104(7):905-14. · 9.49 Impact Factor
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Tamer M A Mohamed,
Delvac Oceandy,
Sukhpal Prehar,
Nasser Alatwi,
Zeinab Hegab,
Florence M Baudoin,
Adam Pickard,
Aly O Zaki,
Raja Nadif, Elizabeth J Cartwright,
Ludwig Neyses
[show abstract]
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ABSTRACT: The cardiac neuronal nitric-oxide synthase (nNOS) has been described as a modulator of cardiac contractility. We have demonstrated previously that isoform 4b of the sarcolemmal calcium pump (PMCA4b) binds to nNOS in the heart and that this complex regulates beta-adrenergic signal transmission in vivo. Here, we investigated whether the nNOS-PMCA4b complex serves as a specific signaling modulator in the heart. PMCA4b transgenic mice (PMCA4b-TG) showed a significant reduction in nNOS and total NOS activities as well as in cGMP levels in the heart compared with their wild type (WT) littermates. In contrast, PMCA4b-TG hearts showed an elevation in cAMP levels compared with the WT. Adult cardiomyocytes isolated from PMCA4b-TG mice demonstrated a 3-fold increase in Ser(16) phospholamban (PLB) phosphorylation as well as Ser(22) and Ser(23) cardiac troponin I (cTnI) phosphorylation at base line compared with the WT. In addition, the relative induction of PLB phosphorylation and cTnI phosphorylation following isoproterenol treatment was severely reduced in PMCA4b-TG myocytes, explaining the blunted physiological response to the beta-adrenergic stimulation. In keeping with the data from the transgenic animals, neonatal rat cardiomyocytes overexpressing PMCA4b showed a significant reduction in nitric oxide and cGMP levels. This was accompanied by an increase in cAMP levels, which led to an increase in both PLB and cTnI phosphorylation at base line. Elevated cAMP levels were likely due to the modulation of cardiac phosphodiesterase, which determined the balance between cGMP and cAMP following PMCA4b overexpression. In conclusion, these results showed that the nNOS-PMCA4b complex regulates contractility via cAMP and phosphorylation of both PLB and cTnI.
Journal of Biological Chemistry 03/2009; 284(18):12091-8. · 4.77 Impact Factor
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Elizabeth J Cartwright
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ABSTRACT: Determining gene function by using transgenesis in the mouse has been immensely popular amongst researchers since the introduction of the techniques in the 1980s. However, we are still a long way from knowing the function of the majority of the genes in the genome. It is becoming increasingly accepted that there needs to be a coherent programme of systematic mutation of every protein-coding gene in the mouse genome. The target is therefore to generate approximately 20,000-25,000 gene knockout mouse models. This is an ambitious aim but one which is vital to enhance our understanding of physiology, development and disease. Recently a number of programmes have been established to meet this aim. A particular feature of these large co-ordinated methods for generating genetically modified mice is that they may obviate the need for individual researchers to perform the onerous process of generating the mutant mouse of their gene of interest - in the future we may be able to order our mouse model of interest with the same ease with which we order laboratory reagents and supplies today.
Methods in molecular biology (Clifton, N.J.) 02/2009; 561:275-83.
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ABSTRACT: The plasma membrane calcium/calmodulin-dependent ATPase (PMCA) is a ubiquitously expressed calcium-extruding enzymatic pump. In the majority of cells the main function of PMCA is as the only system to extrude calcium from the cytosol, however, in the excitable cells of the heart it has only a minor role in the bulk removal of calcium compared to the sodium-calcium exchanger. There is increasing evidence to suggest that PMCA has an additional role as a potential modulator of a number of signal transduction pathways. Of key interest in the heart is the functional interaction between the calcium/calmodulin-dependent enzyme neuronal nitric oxide synthase (nNOS) and isoform 4 of PMCA. Nitric oxide production from nNOS is known to be important in the regulation of excitation-contraction (EC) coupling and subsequently contractility. This article will focus on recent evidence suggesting that PMCA4 has a regulatory role in the nitric oxide signaling pathway in the heart.
Annals of the New York Academy of Sciences 04/2007; 1099:247-53. · 3.15 Impact Factor
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Delvac Oceandy, Elizabeth J Cartwright,
Michael Emerson,
Sukhpal Prehar,
Florence M Baudoin,
Min Zi,
Nasser Alatwi,
Luigi Venetucci,
Kai Schuh,
Judith C Williams,
Angel L Armesilla,
Ludwig Neyses
[show abstract]
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ABSTRACT: Neuronal nitric oxide synthase (nNOS) has recently been shown to be a major regulator of cardiac contractility. In a cellular system, we have previously shown that nNOS is regulated by the isoform 4b of plasma membrane calcium/calmodulin-dependent ATPase (PMCA4b) through direct interaction mediated by a PDZ domain (PSD 95, Drosophilia Discs large protein and Zona occludens-1) on nNOS and a cognate ligand on PMCA4b. It remains unknown, however, whether this interaction has physiological relevance in the heart in vivo.
We generated 2 strains of transgenic mice overexpressing either human PMCA4b or PMCA ct120 in the heart. PMCA ct120 is a highly active mutant form of the pump that does not interact with or modulate nNOS function. Calcium was extruded normally from PMCA4b-overexpressing cardiomyocytes, but in vivo, overexpression of PMCA4b reduced the beta-adrenergic contractile response. This attenuated response was not observed in ct120 transgenic mice. Treatment with a specific nNOS inhibitor (N omega-propyl-L-arginine) reduced the beta-adrenergic response in wild-type and ct120 transgenic mice to levels comparable to those of PMCA4b transgenic animals. No differences in lusitropic response were observed in either transgenic strain compared with wild-type littermates.
These data demonstrate the physiological relevance of the interaction between PMCA4b and nNOS and suggests its signaling role in the heart.
Circulation 02/2007; 115(4):483-92. · 14.74 Impact Factor
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Natalie Burkard,
Adam G Rokita,
Susann G Kaufmann,
Matthias Hallhuber,
Rongxue Wu,
Kai Hu,
Ulrich Hofmann,
Andreas Bonz,
Stefan Frantz, Elizabeth J Cartwright,
Ludwig Neyses,
Lars S Maier,
Sebastian K G Maier,
Thomas Renné,
Kai Schuh,
Oliver Ritter
[show abstract]
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ABSTRACT: The role of the neuronal NO synthase (nNOS or NOS1) enzyme in the control of cardiac function still remains unclear. Results from nNOS(-/-) mice or from pharmacological inhibition of nNOS are contradictory and do not pay tribute to the fact that probably spatial confinement of the nNOS enzyme is of major importance. We hypothesize that the close proximity of nNOS and certain effector molecules like L-type Ca(2+)-channels has an impact on myocardial contractility. To test this, we generated a new transgenic mouse model allowing conditional, myocardial specific nNOS overexpression. Western blot analysis of transgenic nNOS overexpression showed a 6-fold increase in nNOS protein expression compared with noninduced littermates (n=12; P<0.01). Measuring of total NOS activity by conversion of [(3)H]-l-arginine to [(3)H]-l-citrulline showed a 30% increase in nNOS overexpressing mice (n=18; P<0.05). After a 2 week induction, nNOS overexpression mice showed reduced myocardial contractility. In vivo examinations of the nNOS overexpressing mice revealed a 17+/-3% decrease of +dp/dt(max) compared with noninduced mice (P<0.05). Likewise, ejection fraction was reduced significantly (42% versus 65%; n=15; P<0.05). Interestingly, coimmunoprecipitation experiments indicated interaction of nNOS with SR Ca(2+)ATPase and additionally with L-type Ca(2+)- channels in nNOS overexpressing animals. Accordingly, in adult isolated cardiac myocytes, I(Ca,L) density was significantly decreased in the nNOS overexpressing cells. Intracellular Ca(2+)-transients and fractional shortening in cardiomyocytes were also clearly impaired in nNOS overexpressing mice versus noninduced littermates. In conclusion, conditional myocardial specific overexpression of nNOS in a transgenic animal model reduced myocardial contractility. We suggest that nNOS might suppress the function of L-type Ca(2+)-channels and in turn reduces Ca(2+)-transients which accounts for the negative inotropic effect.
Circulation Research 02/2007; 100(3):e32-44. · 9.49 Impact Factor
