Regulation of Ncx1 Gene Expression in the Normal and Hypertrophic Heart (original) (raw)
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Mechanisms of Development, 2001
The Na 1-Ca 21 exchanger (NCX) regulates intracellular calcium homeostasis. We report on an upstream region of the rat NCX1 multipartite promoter that is active in cardiac myocytes. Although inactive in most non-cardiac cell lines, its activity can be rescued by cotransfection with GATA-4 and-6, but not GATA-5 transcription factors. In transgenic mice and similar to endogenous NCX1 mRNA expression, the upstream promoter region directs uniform b-galactosidase expression in cardiac myocytes from ,7.75 dpc. In adult mouse hearts, promoter activity is, however, significantly reduced and heterogeneous, except in the conduction system (sinoatrial and atrioventricular node, atrioventricular bundles). The upstream NCX1 promoter region thus directs appropriate spatial and temporal control of cardiac expression throughout development. Published by Elsevier Science Ireland Ltd.
Journal of Biological Chemistry, 2006
The Na ؉-Ca 2؉ exchanger (NCX1) is up-regulated in hypertrophy and is often found up-regulated in end-stage heart failure. Studies have shown that the change in its expression contributes to contractile dysfunction. We have previously shown that the 1831-bp Ncx1 H1 (1831Ncx1) promoter directs cardiac-specific expression of the exchanger in both development and in the adult, and is sufficient for the up-regulation of Ncx1 in response to pressure overload. Here, we utilized adenoviral mediated gene transfer and transgenics to identify minimal regions and response elements that mediate Ncx1 expression in the heart. We demonstrate that the proximal 184 bp of the Ncx1 H1 (184Ncx1) promoter is sufficient for expression of reporter genes in adult cardiomyocytes and for the correct spatiotemporal pattern of Ncx1 expression in development but not for up-regulation in response to pressure overload. Mutational analysis revealed that both the ؊80 CArG and the ؊50 GATA elements were required for expression in isolated adult cardiomyocytes. Chromatin immunoprecipitation assays in adult cardiocytes demonstrate that SRF and GATA4 are associated with the proximal region of the endogenous Ncx1 promoter. Transgenic lines were established for the 1831Ncx1 promoter-luciferase containing mutations in the ؊80 CArG or ؊50 GATA element. No luciferase activity was detected during development, in the adult, or after pressure overload in any of the ؊80 CArG transgenic lines. The Ncx1 ؊50 GATA mutant promoter was sufficient for driving the normal spatiotemporal pattern of Ncx1 expression in development and for up-regulation in response to pressure overload but importantly, expression was no longer cardiac restricted. This work is the first in vivo study that demonstrates which cis elements are important for Ncx1 regulation.
Developmental Dynamics, 2001
Ouabain-induced inhibition of early heart development indicated that Na/K-AT-Pase plays an important role in maintaining normal ionic balances during differentiation of cardiomyocytes (Linask and Gui [1995] Dev Dyn 203: 93-105). Inhibition of the sodium pump is generally accepted to affect the activity of the Na ؉ -Ca ؉؉ exchanger (NCX) to increase intracellular [Ca ؉؉ ]. These previous findings suggested that Ca ؉؉ signaling may be an important modulator during differentiation of cardiomyocytes. In order to identify a connection between heart development and NCX-mediated Ca ؉؉ regulation, we determined the embryonic spatiotemporal protein expression pattern of NCX-1 during early developmental stages. In both chick and mouse embryos, NCX-1 (the cardiac NCX isoform) is asymmetrically expressed during gastrulation; in the right side of the Hensen's node in the chick, in the right lateral mesoderm in the mouse. At slightly later stages, NCX-1 is expressed in the heart fields at comparable stages of heart development, in the chick at stage 7 and in the mouse at embryonic day (ED) 7.5. By ED 8 in the mouse, the exchanger protein displays a rostrocaudal difference in cardiac expression and an outer curvature-inner curvature ventricular difference. By ED 9.5, cardiac expression has increased from that seen at ED8 and NCX-1 is distributed throughout the myocardium consistent with the possibility that it is important in regulating initial cardiac contractile function. Only a low level of expression is detected in inflow and outflow regions. To substantiate a role for the involvement of calcium-mediated signaling, using pharmacologic approaches, ionomycin (a Ca ؉؉ ionophore) was shown to perturb cardiac cell differentiation in a manner similar to ouabain as assayed by cNkx2.5 and sarcomeric myosin heavy chain expression. In addition, we show that an inhibitor of NCX, KB-R7943, can similarly and adversely affect early cardiac development at stage 4/5 and arrests cardiac cell contractility in 12-somite embryos. Thus, based upon NCX-1 protein expression patterns in the embryo, experimental Ca ؉؉ modulation, and inhibition of NCX activity by KB-R7943, these results suggest an early and central role for calcium-mediated signaling in cardiac cell differentiation and NCX's regulation of the initial heartbeats in the embryo.
European Journal of Heart Failure, 2007
α 1-adrenergic stimulation and mechanical load are considered crucial for the expression of sarcolemmal Na + /Ca 2+ exchanger (NCX1). However, the interaction between these processes is unknown. We investigated electrically stimulated (1 Hz, 1.75 mmol/L Ca 2+) rabbit ventricular trabeculae at physiological preload under stimulation by the selective α 1-agonist phenylephrine (PE, 10 μmol/L). Using quantitative real-time PCR, downregulation of mRNA to 76.5% (p b 0.05) was found, while B-type natriuretic peptide (BNP) was increased to 569.5% (p b 0.05) compared to control. These changes were abolished in the presence of both the α 1-blocker prazosin (13 μmol/L) and the PKC inhibitor GF109203X (1 μmol/L). Furthermore, no changes in NCX mRNA levels under the influence of PE were found in unstretched trabeculae or in unstretched isolated rabbit myocytes (24 h), while BNP was increased in both preparations. In addition, since the α 1-adrenergic effect could be Ca 2+-dependent we tested increased extracellular Ca 2+ (3.0 mmol/L) in stretched trabeculae and found downregulation of NCX1 to 75.2% (p b 0.05). α 1-stimulation decreases NCX1 mRNA in rabbit myocardium via PKC. This is critically load-dependent and may be mediated by changes in [Ca 2+ ]. In hypertrophy and heart failure, distinct phenotypes with respect to NCX1 expression may result from the interaction between mechanical load and α 1-adrenergic stimulation.
Myocardial Function With Reduced Expression of the Sodium-Calcium Exchanger
Journal of Cardiac Failure, 2010
Background-The complete removal of the cardiac sodium-calcium exchanger (NCX1) is associated with embryonic lethality while its overexpression is linked to heart failure. To determine whether or not a reduced expression of NCX1 is compatible with normal heart structure and function, we studied two knockout mouse models with reduced levels of NCX1: a heterozygous global knockout (HG-KO) with a 50% level of NCX1 expression in all myocytes, and a ventricular specific KO (V-KO) with NCX1 expression in only 10-20% of the myocytes.
Functional Adult Myocardium in the Absence of Na+-Ca2+ Exchange: Cardiac-Specific Knockout of NCX1
Circulation Research, 2004
The excitation-contraction coupling cycle in cardiac muscle is initiated by an influx of Ca 2ϩ through voltage-dependent Ca 2ϩ channels. Ca 2ϩ influx induces a release of Ca 2ϩ from the sarcoplasmic reticulum and myocyte contraction. To maintain Ca 2ϩ homeostasis, Ca 2ϩ entry is balanced by efflux mediated by the sarcolemmal Na ϩ -Ca 2ϩ exchanger. In the absence of Na ϩ -Ca 2ϩ exchange, it would be expected that cardiac myocytes would overload with Ca 2ϩ . Using Cre/loxP technology, we generated mice with a cardiac-specific knockout of the Na ϩ -Ca 2ϩ exchanger, NCX1. The exchanger is completely ablated in 80% to 90% of the cardiomyocytes as determined by immunoblot, immunofluorescence, and exchange function. Surprisingly, the NCX1 knockout mice live to adulthood with only modestly reduced cardiac function as assessed by echocardiography. At 7.5 weeks of age, measures of contractility are decreased by 20% to 30%. We detect no adaptation of the myocardium to the absence of the Na ϩ -Ca 2ϩ exchanger as measured by both immunoblots and microarray analysis. Ca 2ϩ transients of isolated myocytes from knockout mice display normal magnitudes and relaxation kinetics and normal responses to isoproterenol. Under voltage clamp conditions, the current through L-type Ca 2ϩ channels is reduced by 50%, although the number of channels is unchanged. An abbreviated action potential may further reduce Ca 2ϩ influx. Rather than upregulate other Ca 2ϩ efflux mechanisms, the myocardium appears to functionally adapt to the absence of the Na ϩ -Ca 2ϩ exchanger by limiting Ca 2ϩ influx. The magnitude of Ca 2ϩ transients appears to be maintained by an increased gain of sarcoplasmic reticular Ca 2ϩ release. The myocardium of the NCX1 knockout mice undergoes a remarkable adaptation to maintain near normal cardiac function. (Circ Res. 2004;95: 604-611.)
Mapping the in vitro interactome of cardiac sodium (Na(+) )-calcium (Ca(2+) ) exchanger 1 (NCX1)
Proteomics, 2017
NCX1 is an antiporter membrane protein encoded by the SLC8A1 gene. In the heart, it maintains cytosolic Ca(2+) homeostasis, serving as the primary mechanism for Ca(2+) extrusion during relaxation. Dysregulation of NCX1 is observed in end-stage human heart failure. In this study we used affinity purification coupled with mass spectrometry in rat left ventricle lysates to identify novel NCX1 interacting proteins in the heart. Two screens were conducted using: 1) anti-NCX1 against endogenous NCX1 and 2) anti-His with His-TF-NCX1cyt recombinant protein as bait. The respective methods identified 112 and 350 protein partners, of which several were known NCX1 partners from the literature, and 29 occurred in both screens. Ten novel protein partners (DYRK1A, PPP2R2A, SNTB1, DMD, RABGGTA, DNAJB4, BAG3, PDE3A, POPDC2, STK39) were validated for binding to NCX1, and two partners (DYRK1A, SNTB1) increased NCX1 activity when expressed in HEK293 cells. A cardiac NCX1 protein-protein interaction map...
Modulation of expression of Na + /Ca 2+ exchanger in heart of rat and mouse under stress
Acta Physiologica, 2007
The Na + /Ca 2+ exchanger (NCX) is a major Ca 2+ extrusion system in the plasma membrane of cardiomyocytes and an important component participating on the excitation-contraction coupling process in muscle cells. NCX1 isoform is the most abundant in the heart and is known to be changed after development of ischaemia or myocardial infarction. Objective of this study was to investigate the effect of stress factors (immobilization, cold and short-term hypoxia) on the expression of NCX1, in vivo, in the heart of rat and mouse. Methods: We compared gene expression and protein levels of control and stressed animals. The activity of NCX was measured by the whole cell configuration using the patch clamp. We also measured physiological parameters of the heart in physiological conditions and under ischaemiareperfusion to compare response of control and stressed hearts. Results: We have found that only strong stress stimulus (hypoxia, immobilization) applied repeatedly for several days elevated the NCX1 mRNA level. Cold, which is a weaker stressor that activates mainly sympathoneural, and only marginally adrenomedullary system did not affect the gene expression of NCX1. Thus, from these results it appears that hormones produced by the adrenal medulla (mainly adrenaline) might be involved in this process. To study possible mechanism of the NCX1 regulation by stress, we focused on the possible role of the hypothalamo-pituitary-adrenocortical pathway in the activation of catecholamine synthesis in the adrenal medulla. We have already published that cortisol affects activity, but not the gene expression of NCX1. In this work, we used corticotropin-releasing hormone (CRH) knockout mice, where secretion of corticosterone and subsequently adrenaline is significantly suppressed. As no increase in NCX1 mRNA was observed in CRH knockout mice due to immobilization stress, we proposed that adrenaline (probably regulated via corticosterone) is involved in the regulation of NCX1 gene expression during stress. Conclusions: The gene expression and protein levels of the NCX1 are increased by the strong stress stimuli, e.g. hypoxia, or immobilization stress. The activity of NCX1 is decreased. Based on these results, we assume that the gene expression of NCX is increased as a consequence of suppressed activity of this transport system.
Cardiovascular Research, 2013
The complex molecular mechanisms underlying spontaneous cardiac pacemaking are not fully understood. Recent findings point to a coordinated interplay between intracellular Ca 2+ cycling and plasma membrane-localized cation transport determining the origin and periodicity of pacemaker potentials. The sodium-calcium exchanger (NCX1) is a key sarcolemmal protein for the maintenance of calcium homeostasis in the heart. Here, we investigated the contribution of NCX1 to cardiac pacemaking. Methods and results We used an inducible and sinoatrial node-specific Cre transgene to create mice lacking NCX1 selectively in cells of the cardiac pacemaking and conduction system (cpNCX1 KO). RT-PCR and immunolabeling experiments confirmed the precise tissue-specific and temporally controlled deletion. Ablation of NCX1 resulted in a progressive slowing of heart rate accompanied by severe arrhythmias. Isolated sinoatrial tissue strips displayed a significantly decreased and irregular contraction rate underpinning a disturbed intrinsic pacemaker activity. Mutant animals displayed a gradual increase in the heart-to-body weight ratio and developed ventricular dilatation; however, their ventricular contractile performance was not significantly affected. Pacemaker cells from cpNCX1 KO showed no NCX1 activity in response to caffeine-induced Ca 2+ release, determined by Ca 2+ imaging. Regular spontaneous Ca 2+ discharges were frequently seen in control, but only sporadically in knockout (KO) cells. The majority of NCX1 KO cells displayed an irregular and a significantly reduced frequency of spontaneous Ca 2+ signals. Furthermore, Ca 2+ transients measured during electrical field stimulation were of smaller magnitude and decelerated kinetics in KO cells. Conclusions Our results establish NCX1 as a critical target for the proper function of cardiac pacemaking.
Physiological Genomics, 2010
In myocardial disease, elevated expression and activity of Na+/H+ exchanger isoform 1 (NHE1) are detrimental. To better understand the involvement of NHE1, transgenic mice with elevated heart-specific NHE1 expression were studied. N-line mice expressed wild-type NHE1, and K-line mice expressed activated NHE1. Cardiac morphology, interstitial fibrosis, and cardiac function were examined by histological staining and echocardiography. Differences in gene expression between the N-line or K-line and nontransgenic littermates were probed with genechip analysis. We found that NHE1 K-line (but not N-line) hearts developed hypertrophy, including elevated heart weight-to-body weight ratio and increased cross-sectional area of the cardiomyocytes, interstitial fibrosis, as well as depressed cardiac function. N-line hearts had modest changes in gene expression (50 upregulations and 99 downregulations, P < 0.05), whereas K-line hearts had a very strong transcriptional response (640 upregulatio...