Erratum: Design of a Proteolytically Stable Sodium-Calcium Exchanger 1 Activator Peptide for In Vivo Studies

The cardiac sodium–calcium exchanger (NCX1) is important for normal Na+- and Ca2+-homeostasis and cardiomyocyte relaxation and contraction. It has been suggested that NCX1 activity is reduced by phosphorylated phospholemman (pSer68-PLM); however its direct interaction with PLM is debated. Disruption of the potentially inhibitory pSer68-PLM-NCX1 interaction might be a therapeutic strategy to increase NCX1 activity in cardiac disease. In the present study, we aimed to analyze the binding affinities and kinetics of the PLM-NCX1 and pSer68-PLM-NCX1 interactions by surface plasmon resonance (SPR) and to develop a proteolytically stable NCX1 activator peptide for future in vivo studies. The cytoplasmic parts of PLM (PLMcyt) and pSer68-PLM (pSer68-PLMcyt) were found to bind strongly to the intracellular loop of NCX1 (NCX1cyt) with similar KD values of 4.1 ± 1.0 nM and 4.3 ± 1.9 nM, but the PLMcyt-NCX1cyt interaction showed higher on/off rates. To develop a proteolytically stable NCX1 activator, we took advantage of a previously designed, high-affinity PLM binding peptide (OPT) that was derived from the PLM binding region in NCX1 and that reverses the inhibitory PLM (S68D)-NCX1 interaction in HEK293. We performed N- and C-terminal truncations of OPT and identified PYKEIEQLIELANYQV as the minimum sequence required for pSer68-PLM binding. To increase peptide stability in human serum, we replaced the proline with an N-methyl-proline (NOPT) after identification of N-terminus as substitution tolerant by two-dimensional peptide array analysis. Mass spectrometry analysis revealed that the half-life of NOPT was increased 17-fold from that of OPT. NOPT pulled down endogenous PLM from rat left ventricle lysate and exhibited direct pSer68-PLM binding in an ELISA-based assay and bound to pSer68-PLMcyt with a KD of 129 nM. Excess NOPT also reduced the PLMcyt-NCX1cyt interaction in an ELISA-based competition assay, but in line with that NCX1 and PLM form oligomers, NOPT was not able to outcompete the physical interaction between endogenous full length proteins. Importantly, cell-permeable NOPT-TAT increased NCX1 activity in cardiomyocytes isolated from both SHAM-operated and aorta banded heart failure (HF) mice, indicating that NOPT disrupted the inhibitory pSer68-PLM-NCX1 interaction. In conclusion, we have developed a proteolytically stable NCX1-derived PLM binding peptide that upregulates NCX1 activity in SHAM and HF cardiomyocytes.

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The Effect of a Novel Highly Selective Inhibitor of the Sodium/Calcium Exchanger (NCX) on Cardiac Arrhythmias in In Vitro and In Vivo Experiments

PLoS ONE ◽

10.1371/journal.pone.0166041 ◽

2016 ◽

Vol 11 (11) ◽

pp. e0166041 ◽

Cited By ~ 23

Author(s):

Zsófia Kohajda ◽

Nikolett Farkas-Morvay ◽

Norbert Jost ◽

Norbert Nagy ◽

Amir Geramipour ◽

...

Keyword(s):

Cardiac Arrhythmias ◽

Selective Inhibitor ◽

Sodium Calcium Exchanger ◽

In Vivo Experiments ◽

Sodium Calcium

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Egr-1 negatively regulates expression of the sodium?calcium exchanger-1 in cardiomyocytes in vitro and in vivo

Cardiovascular Research ◽

10.1016/j.cardiores.2004.09.026 ◽

2005 ◽

Vol 65 (1) ◽

pp. 187-194 ◽

Cited By ~ 24

Author(s):

C WANG ◽

S DOSTANIC ◽

N SERVANT ◽

L CHALIFOUR

Keyword(s):

Sodium Calcium Exchanger ◽

Sodium Calcium

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Phorbol esters downregulate expression of the sodium/calcium exchanger in renal epithelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.1995.269.2.c457 ◽

1995 ◽

Vol 269 (2) ◽

pp. C457-C463 ◽

Cited By ~ 13

Author(s):

L. Smith ◽

H. Porzig ◽

H. W. Lee ◽

J. B. Smith

Keyword(s):

Epithelial Cells ◽

Phorbol Esters ◽

Sodium Calcium Exchanger ◽

Myristate Acetate ◽

Renal Epithelial Cells ◽

Kinase Substrate ◽

Kinase C ◽

Sodium Calcium ◽

Pkc Alpha

The Na+/Ca2+ exchanger (NCE) contributes to Ca2+ reabsorption by connecting tubules of the nephron. A line of renal epithelial cells from monkey kidney (LLC-MK2) was used to investigate the regulation of NCE expression. After the activation of protein kinase C (PKC) by phorbol myristate acetate (PMA), NCE activity decreased exponentially by 75% in 48 h (half time approximately 19 h). PMA decreased NCE mRNA by 85% in 24 h. The decrease in NCE transcript preceded the downregulation of NCE activity. NCE protein was quantified with a monoclonal antibody to cardiac NCE. PMA decreased the binding of 3H-labeled antibody to cell sonicates by 40% in 24 h. Immunoblots show that PMA produced a marked and extended increase in membrane-associated PKC-alpha, although PMA depleted total PKC-alpha by 65% in 24 h. In vivo 32P labeling of myristolated alanine-rich C kinase substrate, a specific PKC substrate, confirmed that PMA produced a rapid and extended activation of PKC. 4 alpha-PMA, a stereoisomer of PMA that neither binds nor activates PKC, had no effect on NCE activity or transcript. These findings indicate that activation of PKC with phorbol esters downregulates NCE mRNA, protein, and activity in renal epithelial cells.

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Abstract 93: Genetic Deletion of Slc8b1, the Mitochondrial Sodium/Calcium Exchanger, Causes Sudden Cardiac Death and Overexpression Protects Against Myocardial Infarction and Pressure-Overload Heart Failure

Circulation Research ◽

10.1161/res.117.suppl_1.93 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Timothy S Luongo ◽

Mary Nwokedi ◽

Jonathan P Lambert ◽

Erhe Gao ◽

April C Carpenter ◽

...

Keyword(s):

Myocardial Infarction ◽

Heart Failure ◽

Pressure Overload ◽

Tamoxifen Treatment ◽

Lv Function ◽

Sodium Calcium Exchanger ◽

Cell Death Pathways ◽

Sodium Calcium ◽

First Time

Mitochondrial calcium ( m Ca 2+ ) signaling is critical for both energy production and the activation of cell death pathways in the heart. Further, m Ca 2+ overload is hypothesized to be a significant contributor to the development and progression of heart failure (HF). The mitochondrial sodium/calcium exchanger (mNCX) is hypothesized to be the primary mechanism of m Ca 2+ efflux, but to date no study has genetically confirmed its identity or function in an in vivo system. To investigate the role of mNCX in HF, we generated mutant mice with loxP sites flanking exons 5-7 of the candidate gene, Slc8b1 (also known as NCLX) , and crossed them with a tamoxifen (tamox)-inducible cardiac-specific Cre mouse to delete mNCX in the adult heart (mNCX-cKO). Cardiomyocytes isolated from mNCX-cKO mice displayed a significant reduction in m Ca 2+ efflux rate and Ca 2+ uptake capacity. Tamoxifen-induced ablation of mNCX resulted in sudden death with only 54% of mice surviving 8d post-tamoxifen treatment (Fig 1). Assessment of mNCX-cKO hearts 2d post-tamox revealed significant remodeling characterized by dilation and a decrease in %EF (Fig 2). Next, we generated a conditional, cardiac-specific mNCX overexpression mouse model (mNCX-Tg) to evaluate if increased m Ca 2+ efflux would alter the progression of HF. mNCX-Tg and controls were subjected to both myocardial infarction (LCA ligation) and pressure-overload induced HF (transverse aortic constriction). mNCX-Tg mice displayed preserved LV function, structure and a reduction in HF indices in both models. For the first time we show that mNCX is essential for m Ca 2+ efflux in cardiomyocytes and that mNCX represents a novel therapeutic target in HF.

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Transient reversal of the sodium/calcium exchanger boosts presynaptic calcium and synaptic transmission at a cerebellar synapse

Journal of Neurophysiology ◽

10.1152/jn.00854.2012 ◽

2013 ◽

Vol 109 (6) ◽

pp. 1669-1680 ◽

Cited By ~ 10

Author(s):

Chris J. Roome ◽

Emmet M. Power ◽

Ruth M. Empson

Keyword(s):

Synaptic Transmission ◽

Information Transfer ◽

Calcium Influx ◽

Calcium Entry ◽

Parallel Fiber ◽

Sodium Calcium Exchanger ◽

Reverse Mode ◽

Sodium Calcium ◽

Presynaptic Calcium

The sodium/calcium exchanger (NCX) is a widespread transporter that exchanges sodium and calcium ions across excitable membranes. Normally, NCX mainly operates in its “forward” mode, harnessing the electrochemical gradient of sodium ions to expel calcium. During membrane depolarization or elevated internal sodium levels, NCX can instead switch the direction of net flux to expel sodium and allow calcium entry. Such “reverse”-mode NCX operation is frequently implicated during pathological or artificially extended periods of depolarization, not during normal activity. We have used fast calcium imaging, mathematical simulation, and whole cell electrophysiology to study the role of NCX at the parallel fiber-to-Purkinje neuron synapse in the mouse cerebellum. We show that nontraditional, reverse-mode NCX activity boosts the amplitude and duration of parallel fiber calcium transients during short bursts of high-frequency action potentials typical of their behavior in vivo. Simulations, supported by experimental manipulations, showed that accumulation of intracellular sodium drove NCX into reverse mode. This mechanism fueled additional calcium influx into the parallel fibers that promoted synaptic transmission to Purkinje neurons for up to 400 ms after the burst. Thus we provide the first functional demonstration of transient and fast NCX-mediated calcium entry at a major central synapse. This unexpected contribution from reverse-mode NCX appears critical for shaping presynaptic calcium dynamics and transiently boosting synaptic transmission, and is likely to optimize the accuracy of cerebellar information transfer.

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Cerebral microvascular nNOS responds to lowered oxygen tension through a bumetanide-sensitive cotransporter and sodium-calcium exchanger

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01074.2007 ◽

2008 ◽

Vol 294 (5) ◽

pp. H2166-H2173 ◽

Cited By ~ 11

Author(s):

Holly D. Bauser-Heaton ◽

Jin Song ◽

H. Glenn Bohlen

Keyword(s):

Oxygen Tension ◽

Neuronal Damage ◽

No Synthase ◽

Sodium Calcium Exchanger ◽

No Formation ◽

Sodium Calcium ◽

Dilatory Response ◽

Hypoxic State ◽

Arteriolar Diameter

Na+ cotransporters have a substantial role in neuronal damage during brain hypoxia. We proposed these cotransporters have beneficial roles in oxygen-sensing mechanisms that increase periarteriolar nitric oxide (NO) concentration ([NO]) during mild to moderate oxygen deprivation. Our prior studies have shown that cerebral neuronal NO synthase (nNOS) is essential for [NO] responses to decreased oxygen tension and that endothelial NO synthase (eNOS) is of little consequence. In this study, we explored the mechanisms of three specific cotransporters known to play a role in the hypoxic state: KB-R7943 for blockade of the Na+/Ca2+ exchanger, bumetanide for the Na+-K+-2Cl− cotransporter, and amiloride for Na+/H+ cotransporters. In vivo measurements of arteriolar diameter and [NO] at normal and locally reduced oxygen tension in the rat parietal cortex provided the functional analysis. As previously found for intestinal arterioles, bumetanide-sensitive cotransporters are primarily responsible for sensing reduced oxygen because the increased [NO] and dilation were suppressed. The Na+/Ca2+ exchanger facilitated increased NO formation because blockade also suppressed [NO] and dilatory responses to decreased oxygen. Amiloride-sensitive Na+/H+ cotransporters did not significantly contribute to the microvascular regulation. To confirm that nNOS rather than eNOS was primarily responsible for NO generation, eNOS was suppressed with the fusion protein cavtratin for the caveolae domain of eNOS. Although the resting [NO] decreased and arterioles constricted as eNOS was suppressed, most of the increased NO and dilatory response to oxygen were preserved because nNOS was functional. Therefore, nNOS activation secondary to Na+-K+-2Cl− cotransporter and Na+/Ca2+ exchanger functions are key to cerebral vascular oxygen responses.

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Functional alterations after cardiac sodium-calcium exchanger overexpression in heart failure

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01324.2005 ◽

2006 ◽

Vol 291 (2) ◽

pp. H488-H495 ◽

Cited By ~ 7

Author(s):

Götz Münch ◽

Kai Rosport ◽

Christine Baumgartner ◽

Zhongmin Li ◽

Silvia Wagner ◽

...

Keyword(s):

Heart Failure ◽

Gene Transfer ◽

Left Ventricular ◽

Contractile Dysfunction ◽

Sodium Calcium Exchanger ◽

Cell Shortening ◽

Sodium Calcium ◽

Adenoviral Delivery ◽

Fractional Shortening

The sodium-calcium exchanger (NCX) is discussed as one of the key proteins involved in heart failure. However, the causal role and the extent to which NCX contributes to contractile dysfunction during heart failure are poorly understood. NCX overexpression was induced by infection with an adenovirus coding for NCX, which coexpressed green fluorescence protein (GFP) (AdNCX) by ex vivo gene transfer to nonfailing and failing rabbit cardiomyocytes. Myocardial gene transfer in rabbits in vivo was achieved by adenoviral delivery via aortic cross-clamping. Peak cell shortening of cardiomyocytes was determined photo-optically. Hemodynamic parameters in vivo were determined by echocardiography (fractional shortening) and tip catheter [maximal first derivative of left ventricular (LV) pressure (dP/d tmax); maximal negative derivative of LV pressure (−dP/d tmax)]. Peak cell shortening was depressed after NCX gene delivery in isolated nonfailing and in failing cardiomyocytes. In nonfailing rabbits in vivo, basal systolic contractility (fractional shortening and dP/d tmax) and maximum rate of LV relaxation (−dP/d tmax) in vivo were largely unaffected after NCX overexpression. However, during heart failure, long-term NCX overexpression over 2 wk significantly improved fractional shortening and dP/d tmax compared with AdGFP-infected rabbits, both without inotropic stimulation and after β-adrenergic stimulation with isoproterenol. −dP/d tmax was also improved after NCX overexpression in the failing rabbits group. These results indicate that short-term effects of NCX overexpression impair contractility of isolated failing and nonfailing rabbit cardiomyocytes. NCX overexpression over 2 wk in vivo does not seem to affect myocardial contractility in nonfailing rabbits. Interestingly, in vivo overexpression of NCX decreased the progression of systolic and diastolic contractile dysfunction and improved β-adrenoceptor-mediated contractile reserve in heart failure in rabbits in vivo.

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