Steroids Limit Myocardial Edema During Ex Vivo Perfusion of Hearts Donated After Circulatory Death.

BACKGROUND: Normothermic ex vivo heart perfusion (EVHP) has been shown to improve the preservation of hearts donated after circulatory arrest and to facilitate clinical successful transplantation. Steroids are added to the perfusate solution in current clinical EVHP protocols; however, the impact of this approach on donor heart preservation has not been previously investigated. We sought to determine the impact of steroids on the inflammatory response and development of myocardial edema during EVHP. METHODS: Thirteen pigs were anesthetized, mechanical ventilation was discontinued, and a hypoxemic cardiac arrest ensued. A 15-minute warm-ischemic standoff period was observed, and then hearts were resuscitated with a cardioplegic solution. Donor hearts were then perfused ex vivo in a normothermic beating state for 6 hours with 500 mg of methylprednisolone (steroid: n = 5) or without (control: n = 8). RESULTS: The addition of steroids to the perfusate solution reduced the generation of proinflammatory cytokines (interleukin-6, -8, -1ß, and tumor necrosis factor-α) and the development of myocardial edema during EVHP (percentage of weight gain: control = 26% ± 7% versus steroid = 16% ± 10%, p = 0.049). Electron microscopy suggested less endothelial cell edema in the steroid group (injury score: control = 1.8 ± 0.2 versus steroid = 1.2 ± 0.2, p = 0.06), whereas perfusate troponin-I (control = 11.9 ± 1.9 ng/mL versus steroid = 9.5 ± 2.4 ng/mL, p = 0.448) and myocardial function were comparable between the groups. CONCLUSIONS: The addition of methylprednisolone to the perfusion solution minimizes the generation of proinflammatory cytokines and development of myocardial edema during normothermic ex vivo perfusion of hearts donated after circulatory arrest.

A whole blood-based perfusate provides superior preservation of myocardial function during ex vivo heart perfusion.

BACKGROUND: Ex vivo heart perfusion (EVHP) provides the opportunity to resuscitate unused donor organs and facilitates assessments of myocardial function that are required to demonstrate organ viability before transplantation. We sought to evaluate the effect of different oxygen carriers on the preservation of myocardial function during EVHP. METHODS: Twenty-seven pig hearts were perfused ex vivo in a normothermic beating state for 6 hours and transitioned into working mode for assessments after 1 (T1), 3 (T3), and 5 (T5) hours. Hearts were allocated to 4 groups according to the perfusate composition. Red blood cell concentrate (RBC, n = 6), whole blood (RBC+Plasma, n = 6), an acellular hemoglobin-based oxygen carrier (HBOC, n = 8), or HBOC plus plasma (HBOC+Plasma, n = 7) were added to STEEN Solution (XVIVO Perfusion, Goteborg, Sweden) to achieve a perfusate hemoglobin concentration of 40 g/liter. RESULTS: The perfusate composition affected the preservation of systolic (T5 dP/dtmax: RBC+Plasma = 903 ± 99, RBC = 771 ± 77, HBOC+Plasma = 691 ± 82, HBOC = 563 ± 52 mm Hg/sec; p = 0.047) and diastolic (T5 dP/dtmin: RBC+Plasma = -574 ± 48, RBC = -492 ± 63, HBOC+Plasma = -326 ± 32, HBOC = -268 ± 22 mm Hg/sec; p < 0.001) function, and the development of myocardial edema (weight gain: RBC+Plasma = 6.6 ± 0.9, RBC = 6.6 ± 1.2, HBOC+Plasma = 9.8 ± 1.7, HBOC = 16.3 ± 1.9 g/hour; p < 0.001) during EVHP. RBC+Plasma hearts exhibited less histologic evidence of myocyte damage (injury score: RBC+Plasma = 0.0 ± 0.0, RBC = 0.8 ± 0.3, HBOC+Plasma = 2.6 ± 0.2, HBOC = 1.75 ± 0.4; p < 0.001) and less troponin-I release (troponin-I fold-change T1-T5: RBC+Plasma = 7.0 ± 1.7, RBC = 13.1 ± 1.6, HBOC+Plasma = 20.5 ± 1.1, HBOC = 16.7 ± 5.8; p < 0.001). Oxidative stress was minimized by the addition of plasma to RBC and HBOC hearts (oxidized phosphatidylcholine compound fold-change T1-T5: RBC+Plasma = 1.83 ± 0.20 vs RBC = 2.31 ± 0.20, p < 0.001; HBOC+Plasma = 1.23 ± 0.17 vs HBOC = 2.80 ± 0.28, p < 0.001). CONCLUSIONS: A whole blood-based perfusate (RBC+Plasma) minimizes injury and provides superior preservation of myocardial function during EVHP. The beneficial effect of plasma on the preservation of myocardial function requires further investigation.

Extracellular and intracellular proteases in cardiac dysfunction due to ischemia-reperfusion injury.

Various procedures such as angioplasty, thrombolytic therapy, coronary bypass surgery, and cardiac transplantation are invariably associated with ischemia-reperfusion (I/R) injury. Impaired recovery of cardiac function due to I/R injury is considered to be a consequence of the occurrence of both oxidative stress and intracellular Ca(2+)-overload in the myocardium. These changes in the ischemic myocardium appear to activate both extracellular and intracellular proteases which are responsible for the cleavage of extracellular matrix and subcellular structures involved in the maintenance of cardiac function. It is thus intended to discuss the actions of I/R injury on several proteases, with a focus on calpain, matrix metalloproteinases, and cathepsins as well as their role in inducing alterations both inside and outside the cardiomyocytes. In addition, modifications of subcellular organelles such as myofibrils, sarcoplasmic reticulum and sarcolemma as well as extracellular matrix, and the potential regulatory effects of endogenous inhibitors on protease activities are identified. Both extracellular and intracellular proteolytic activities appear to be imperative in determining the true extent of I/R injury and their inhibition seems to be of critical importance for improving the recovery of cardiac function. Thus, both extracellular and intracellular proteases may serve as potential targets for the development of cardioprotective interventions for reducing damage to the heart and retarding the development of contractile dysfunction caused by I/R injury.

µ-Calpain-mediated deregulation of cardiac, brain, and kidney NCX1 splice variants.

µ-Calpain is a Ca(2+)-activated protease abundant in mammalian tissues. Here, we examined the effects of µ-calpain on three alternatively spliced variants of NCX1 using the giant, excised patch technique. Membrane patches from Xenopus oocytes expressing either heart (NCX1.1), kidney (NCX1.3), or brain (NCX1.4) variants of NCX1 were exposed to µ-calpain and their Na(+)-dependent (I(1)) and Ca(2+)-dependent (I(2)) regulatory phenotypes were assessed. For these exchangers, I(1) inactivation is evident as a Na(+)(i)-dependent decay of peak outward currents whereas I(2) regulation manifests as outward current activation by micromolar Ca(2+)(i) concentrations. Notably, with NCX1.1 and NCX1.4 but not in NCX1.3, higher Ca(2+)(i) levels alleviate I(1) inactivation. Our results show that (i) µ-calpain selectively ablates Ca(2+)-dependent (I(2)) regulation leading to a constitutive activation of exchange current, (ii) µ-calpain has much smaller effects on Na(+)-dependent (I(1)) regulation, produced by a slight destabilization of the I(1) state, and (iii) Ca(2+)-dependent regulation (I(2)) and Ca(2+)-mediated alleviation of I(1) appear to be functionally distinct mechanisms, the latter of which is left largely intact after µ-calpain treatment. The ability of µ-calpain to selectively and constitutively activate Na(+)-Ca(2+) exchange currents may have important pathophysiological implications in tissue where these splice variants are expressed.

Role of myosin light chain kinase in cardiotrophin-1-induced cardiac myofibroblast cell migration.

Chemotactic movement of myofibroblasts is recognized as a common means for their sequestration to the site of tissue injury. Following myocardial infarction (MI), recruitment of cardiac myofibroblasts to the infarct scar is a critical step in wound healing. Contractile myofibroblasts express embryonic smooth muscle myosin, α-smooth muscle actin, as well as collagens I and III. We examined the effects of cardiotrophin-1 (CT-1) in the induction of primary rat ventricular myofibroblast motility. Changes in membrane potential (E(m)) and Ca(2+) entry were studied to reveal the mechanisms for induction of myofibroblast migration. CT-1-induced cardiac myofibroblast cell migration, which was attenuated through the inhibition of JAK2 (25 µM AG490), and myosin light chain kinase (20 µM ML-7). Inhibition of K(+) channels (1 mM tetraethylammonium or 100 µM 4-aminopyridine) and nonselective cation channels by 10 µM gadolinium (Gd(3+)) significantly reduced migration in the presence of CT-1. CT-1 treatment caused a significant increase in myosin light chain phosphorylation, which could be inhibited by incubation in Ca(2+)-free conditions or by application of AG490, ML-7, and W7 (100 µM; calmodulin inhibitor). Monitoring myofibroblast membrane potential with potentiometric fluorescent DiBAC(4)(3) dye revealed a biphasic response to CT-1 consisting of an initial depolarization followed by hyperpolarization. Increased intracellular Ca(2+), as assessed by fluo 3, occurred immediately after membrane depolarization and attenuated at the time of maximal hyperpolarization. CT-1 exerts chemotactic effects via multiple parallel signaling modalities in ventricular myofibroblasts, including changes in membrane potential, alterations in intracellular calcium, and activation of a number of intracellular signaling pathways. Further study is warranted to determine the precise role of K(+) currents in this process.

Crystal structures of progressive Ca2+ binding states of the Ca2+ sensor Ca2+ binding domain 1 (CBD1) from the CALX Na+/Ca2+ exchanger reveal incremental conformational transitions.

Na(+)/Ca(2+) exchangers (NCX) constitute a major Ca(2+) export system that facilitates the re-establishment of cytosolic Ca(2+) levels in many tissues. Ca(2+) interactions at its Ca(2+) binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na(+)/Ca(2+) exchange activity. The structure of the Ca(2+)-bound form of CBD1, the primary Ca(2+) sensor from canine NCX1, but not the Ca(2+)-free form, has been reported, although the molecular mechanism of Ca(2+) regulation remains unclear. Here, we report crystal structures for three distinct Ca(2+) binding states of CBD1 from CALX, a Na(+)/Ca(2+) exchanger found in Drosophila sensory neurons. The fully Ca(2+)-bound CALX-CBD1 structure shows that four Ca(2+) atoms bind at identical Ca(2+) binding sites as those found in NCX1 and that the partial Ca(2+) occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca(2+) binding at CBD1. The structures also predict that the primary Ca(2+) pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu(455), which coordinates the primary Ca(2+) pair, produces dramatic reductions of the regulatory Ca(2+) affinity for exchange current, whereas mutagenesis of Glu(520), which coordinates the secondary Ca(2+) pair, has much smaller effects. Furthermore, our structures indicate that Ca(2+) binding only enhances the stability of the Ca(2+) binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca(2+) regulatory function of CBD1, and possibly that for the entire NCX family, is mediated through domain interactions between CBD1 and the adjacent CBD2 at this hinge.

Crystal structure of CBD2 from the Drosophila Na(+)/Ca(2+) exchanger: diversity of Ca(2+) regulation and its alternative splicing modification.

Na(+)/Ca(2+) exchangers (NCXs) promote the extrusion of intracellular Ca(2+) to terminate numerous Ca(2+)-mediated signaling processes. Ca(2+) interaction at two Ca(2+) binding domains (CBDs; CBD1 and CBD2) is important for tight regulation of the exchange activity. Diverse Ca(2+) regulatory properties have been reported with several NCX isoforms; whether the regulatory diversity of NCXs is related to structural differences of the pair of CBDs is presently unknown. Here, we reported the crystal structure of CBD2 from the Drosophila melanogaster exchanger CALX1.1. We show that the CALX1.1-CBD2 is an immunoglobulin-like structure, similar to mammalian NCX1-CBD2, but the predicted Ca(2+) interaction region of CALX1.1-CBD2 is arranged in a manner that precludes Ca(2+) binding. The carboxylate residues that coordinate two Ca(2+) in the NCX1-CBD1 structure are neutralized by two Lys residues in CALX1.1-CBD2. This structural observation was further confirmed by isothermal titration calorimetry. The CALX1.1-CBD2 structure also clearly shows the alternative splicing region forming two adjacent helices perpendicular to CBD2. Our results provide structural evidence that the diversity of Ca(2+) regulatory properties of NCX proteins can be achieved by (1) local structure rearrangement of Ca(2+) binding site to change Ca(2+) binding properties of CBD2 and (2) alternative splicing variation altering the protein domain-domain conformation to modulate the Ca(2+) regulatory behavior.

Characterization of zebrafish (Danio rerio) NCX4: a novel NCX with distinct electrophysiological properties.

Members of the Na+/Ca2+ exchanger (NCX) family are important regulators of cytosolic Ca2+ in myriad tissues and are highly conserved across a wide range of species. Three distinct NCX genes and numerous splice variants exist in mammals, many of which have been characterized in a variety of heterologous expression systems. Recently, however, we discovered a fourth NCX gene (NCX4), which is found exclusively in teleost, amphibian, and reptilian genomes. Zebrafish (Danio rerio) NCX4a encodes for a protein of 939 amino acids and shows a high degree of identity with known NCXs. Although knockdown of NCX4a activity in zebrafish embryos has been shown to alter left-right patterning, it has not been demonstrated that NCX4a functions as a NCX. In this study, we 1) demonstrated, for the first time, that this gene encodes for a novel NCX; 2) characterized the tissue distribution of zebrafish NCX4a; and 3) evaluated its kinetic and transport properties. While ubiquitously expressed, the highest levels of NCX4a expression occurred in the brain and eyes. NCX4a exhibits modest levels of Na+-dependent inactivation and requires much higher levels of regulatory Ca2+ to activate outward exchange currents. NCX4a also exhibited extremely fast recovery from Na+-dependent inactivation of outward currents, faster than any previously characterized wild-type exchanger. While this result suggests that the Na+-dependent inactive state of NCX4a is far less stable than in other NCX family members, this exchanger was still strongly inhibited by 2 microM exchanger inhibitory peptide. We demonstrated that a new putative member of the NCX gene family, NCX4a, encodes for a NCX with unique functional properties. These data will be useful in understanding the role that NCX4a plays in embryological development as well as in the adult, where it is expressed ubiquitously.

Differential sensitivities of the NCX1.1 and NCX1.3 isoforms of the Na+-Ca2+ exchanger to alpha-linolenic acid.

OBJECTIVE: Dietary intake of omega-3 polyunsaturated fatty acids (PUFA) like alpha-linolenic acid (ALA) is antiarrhythmic and cardioprotective. PUFA may also be beneficial in hypertension. Altered Na(+)-Ca(2+) exchanger (NCX) activity has been implicated in arrhythmias, hypertension and heart failure and may be a target for PUFA. Thus, we tested the effects of ALA and other distinct fatty acids on the cardiac (NCX1.1) and vascular (NCX1.3) NCX isoforms. METHODS: HEK293 cells stably expressing NCX isoforms were ramped from +60 to -100 mV (over 1600 ms) in the absence and presence of 25 microM oleic acid (OA, omega-9), linoleic acid (LA, omega-6), ALA (omega-3), or eicosapentaenoic acid (EPA, omega-3). NiCl(2) (5 mM) was used to inhibit and therefore identify the NCX current. The effect of 25 microM ALA on NCX1.1 and NCX1.3 activity was also assessed in adult rat ventricular cardiomyocytes and rabbit aortic vascular smooth muscle cells (VSMC) by measuring [Ca(2+)](i) following substitution of [Na(+)](o) with Li(+). RESULTS: Application of Ni(2+) had no effect in non-transfected cells. ALA and EPA (25 microM) reduced the Ni(2+)-sensitive forward NCX1.1 current (at -100 mV) by 64% and reverse current (at +60 mV) by 57%, and inhibited the Ni(2+)-sensitive NCX1.3 forward and reverse currents by 79% and 76%, respectively. Neither OA nor LA (25 microM) affected the NCX1.1 currents, but both partially inhibited the forward and reverse mode NCX1.3 currents. Inhibition of NCX1.3 by ALA occurred at a much lower IC(50) ( approximately 19 nM) than for NCX1.1 ( approximately 120 nM). In cardiomyocytes and VSMC, ALA significantly reduced the Li(+)-induced rise in intracellular [Ca(2+)]. CONCLUSIONS: NCX1.3 is more sensitive to inhibition by ALA than NCX1.1. In addition, only omega-3 PUFA inhibits NCX1.1, but several classes of fatty acids inhibit NCX1.3. The differential sensitivity of NCX isoforms to fatty acids may have important implications as therapeutic approaches for hypertension, heart failure and arrhythmias.

Cells expressing unique Na+/Ca2+ exchange (NCX1) splice variants exhibit different susceptibilities to Ca2+ overload.

The Na+/Ca2+ exchanger (NCX) NCX1 exhibits tissue-specific alternative splicing. Such NCX splice variants as NCX1.1 and NCX1.3 are also differentially regulated by Na+ and Ca2+, although the physiological implications of these regulatory characteristics are unclear. On the basis of their distinct regulatory profiles, we hypothesized that cells expressing these different splice variants might exhibit unique responses to conditions promoting Ca2+ overload, such as during exposure to cardiac glycosides or simulated ischemia. NCX1.1 or NCX1.3 was expressed in human embryonic kidney (HEK)-293 cells or rat neonatal ventricular cardiomyocytes (NVC), and expression was confirmed by Western blotting and immunocytochemical analyses. HEK-293 cells lacked NCX1 protein before transfection. With use of adenoviral vectors, neonatal cardiomyocytes were induced to overexpress the NCX1.1 splice variant by nearly twofold, whereas the NCX1.3 isoform was expressed on the endogenous NCX1.1 background. Total expression was comparable for NCX1.1 and NCX1.3. Exposure of NVC to ouabain induced a significant increase in cellular Ca2+, an effect that was exaggerated in cells overexpressing NCX1.1, but not NCX1.3. The increase in intracellular Ca2+ was inhibited by 5 microM KB-R7943. Cardiomyocytes overexpressing NCX1.1 also exhibited a greater accumulation of intracellular Ca2+ in response to simulated ischemia than did cells expressing NCX1.3. Similar responses were observed in HEK-293 cells where NCX1.1 was expressed. We conclude that expression of the NCX1.3 splice variant protects against severe Ca2+ overload, whereas NCX1.1 promotes Ca2+ overload in response to cardiac glycosides and ischemic challenges. These results highlight the importance of ionic regulation in controlling NCX1 activity under conditions that promote Ca2+ overload.

Frequency-dependent regulation of cardiac Na(+)/Ca(2+) exchanger.

The activity of the cardiac Na(+)/Ca(2+) exchanger (NCX1.1) undergoes continuous modulation during the contraction-relaxation cycle because of the accompanying changes in the electrochemical gradients for Na(+) and Ca(2+). In addition, NCX1.1 activity is also modulated via secondary, ionic regulatory mechanisms mediated by Na(+) and Ca(2+). In an effort to evaluate how ionic regulation influences exchange activity under pulsatile conditions, we studied the behavior of the cloned NCX1.1 during frequency-controlled changes in intracellular Na(+) and Ca(+) (Na(i)(+) and Ca(i)(2+)). Na(+)/Ca(2+) exchange activity was measured by the giant excised patch-clamp technique with conditions chosen to maximize the extent of Na(+)- and Ca(2+)-dependent ionic regulation so that the effects of variables such as pulse frequency and duration could be optimally discerned. We demonstrate that increasing the frequency or duration of solution pulses leads to a progressive decline in pure outward, but not pure inward, Na(+)/Ca(2+) exchange current. However, when the exchanger is permitted to alternate between inward and outward transport modes, both current modes exhibit substantial levels of inactivation. Changes in regulatory Ca(2+), or exposure of patches to limited proteolysis by alpha-chymotrypsin, reveal that this "coupling" is due to Na(+)-dependent inactivation originating from the outward current mode. Under physiological ionic conditions, however, evidence for modulation of exchange currents by Na(i)(+)-dependent inactivation was not apparent. The current approach provides a novel means for assessment of Na(+)/Ca(2+) exchange ionic regulation that may ultimately prove useful in understanding its role under physiological and pathophysiological conditions.

cDNA cloning and expression of the cardiac Na+/Ca2+ exchanger from Mozambique tilapia (Oreochromis mossambicus) reveal a teleost membrane transporter with mammalian temperature dependence.

The complete cDNA sequence of the tilapia cardiac Na(+)/Ca2+ exchanger (NCX-TL1.0) was determined. The 3.1-kb transcript encodes a protein 957 amino acids in length, with a predicted signal peptide cleaved at residue 31 and two potential N-glycosylation sites in the extracellular N terminus. Hydropathy analysis and sequence comparison predicted a mature protein with nine transmembrane-spanning segments, consistent with the structural topologies of other known mammalian and teleost NCX isoforms. Overall sequence comparison shows high identity to both trout NCX-TR1.0 ( approximately 81%) and mammalian NCX1.1 ( approximately 73%), and phylogenetic analyses confirmed its identity as a member of the NCX1 gene family, expressing exons A, C, D, and F in the alternative splice site. Sequence identity is even higher in the alpha-repeats, the exchanger inhibitory peptide (XIP) site, and Ca(2+)-binding domains, which is reflected in the functional and regulatory properties of tilapia NCX-TL1.0. When NCX-TL1.0 was expressed in Xenopus oocytes and the currents were measured in giant excised patches, they displayed both positive regulation by Ca2+ and Na(+)-dependent inactivation in a manner similar to trout NCX-TR1.0. However, tilapia NCX-TL1.0 exhibited a relatively high sensitivity to temperature compared with trout NCX-TR1.0. Whereas trout NCX-TR1.0 currents displayed activation energies of approximately 7 kJ/mol, tilapia NCX-TL1.0 currents showed mammal-like temperature dependence, with peak and steady-state current activation energies of 53 +/- 9 and 67 +/- 21 kJ/mol, respectively. Using comparative sequence analysis, we highlighted 10 residue positions in the N-terminal domain of the NCX that, in combination, may confer exchanger temperature dependence through subtle changes in protein flexibility. Tilapia NCX-TL1.0 represents the first non-mammalian NCX to exhibit a mammalian temperature dependence phenotype and will prove to be a useful model in defining the interplay between molecular flexibility and stability in NCX function.

Therapeutic potential of novel Na+-Ca2+ exchange inhibitors in attenuating ischemia-reperfusion injury.

The cardiac Na+-Ca2+ exchanger (NCX) plays an essential role in regulating Ca2+ under physiological and pathophysiological conditions. In its forward mode of operation, which predominates under physiological conditions, it extrudes the Ca2+ that enters the cardiac myocyte on a beat-to-beat basis. During ischemia and reperfusion, increased intracellular Na+ leads to a decrease in Ca2+ efflux and enhanced Ca2+ influx via the NCX, potentially leading to Ca2+ overload, which is one of the major pathophysiological mechanisms for ischemia-reperfusion injury. Novel NCX inhibitors discovered in recent years have shown great promise in attenuating ischemia-reperfusion injury.

Adenovirally delivered shRNA strongly inhibits Na+-Ca2+ exchanger expression but does not prevent contraction of neonatal cardiomyocytes.

The cardiac Na(+)-Ca(2+) exchanger (NCX1) is the main mechanism for Ca(2+) efflux in the heart and is thought to serve an essential role in cardiac excitation-contraction (E-C) coupling. The demonstration that an NCX1 gene knock-out is embryonic lethal provides further support for this essential role. However, a recent report employing the Cre/loxP technique for cardiac specific knock-out of NCX1 has revealed that cardiac function is remarkably preserved in these mice, which survived to adulthood. This controversy highlights the necessity for further investigation of NCX1 function in the heart. In this study, we report on a novel approach for depletion of NCX1 in postnatal rat myocytes that utilizes RNA interference (RNAi), administered with high efficiency via adenoviral transfection. Depletion of NCX1 was confirmed by immunocytochemical detection, Western blots and radioisotopic assays of Na(+)-Ca(2+) exchange activity. Exchanger expression was inhibited by up to approximately 94%. Surprisingly, spontaneous beating of these cardiomyocytes was still maintained, although at a lower frequency. Electrical stimulation could elicit a normal beating rhythm, although NCX depleted cells exhibited a depressed Ca(2+) transient amplitude, a depressed rate of Ca(2+) rise and decline, elevated diastolic [Ca(2+)], and shorter action potentials. We also observed a compensatory increase in sarcolemmal Ca(2+) pump expression. Our data support an important, though non-essential, role for the NCX1 in E-C coupling in these neonatal heart cells. Furthermore, this approach provides a valuable means for assessing the role of NCX1 and could be utilized to examine other cardiac proteins in physiological and pathological studies.

Allosteric activation of sodium-calcium exchange by picomolar concentrations of cadmium.

Chinese hamster ovary cells expressing the bovine cardiac Na+-Ca2+ exchanger (NCX1.1) accumulated Cd2+ after a lag period of several tens of seconds. The lag period reflects the progressive allosteric activation of exchange activity by Cd2+ as it accumulates within the cytosol. The lag period was greatly reduced in cells expressing a mutant exchanger, Delta(241-680), that does not require allosteric activation by Ca2+ for activity. Non-transfected cells did not show Cd2+ uptake under the same conditions. In cells expressing NCX1.1, the lag period was nearly abolished following an elevation of the cytosolic Ca2+ concentration. Cytosolic Cd2+ concentrations estimated at 0.5-2 pm markedly stimulated the subsequent uptake of Ca2+ by Na+-Ca2+ exchange. Outward exchange currents in membrane patches from Xenopus oocytes expressing the canine NCX1.1 were rapidly and reversibly stimulated by 3 pm Cd2+ applied at the cytosolic membrane surface. Exchange currents activated by 3 pm Cd2+ were 40% smaller than currents activated by 1 mum cytosolic Ca2+. Current amplitudes declined by 30% and the rate of current development fell sharply upon repetitive applications of Na+ in the presence of 3 pm Cd2+. Cd2+ mimicked the anomalous inhibitory effects of Ca2+ on outward exchange currents generated by the Drosophila exchanger CALX1.1. We conclude that the regulatory sites responsible for allosteric Ca2+ activation bind Cd2+ with high affinity and that Cd2+ mimics the regulatory effects of Ca2+ at concentrations 5 orders of magnitude lower than Ca2+.

SEA0400: a novel sodium-calcium exchange inhibitor with cardioprotective properties.

The cardiac sodium-calcium exchanger (NCX) plays an important role in calcium homeostasis. It is the primary mechanism for removing calcium ions that enter myocytes through L-type calcium channels on a beat-to-beat basis. Its direction of transport is determined by the membrane potential and the ionic concentrations of Na+ and Ca2+, with the forward (or Ca2+-efflux) mode of transport being the dominant mode under physiological conditions. In contrast, the Ca2+-influx mode (or reverse mode) of NCX becomes important in certain pathophysiological conditions, such as myocardial ischemia and reperfusion. Recent discovery of compounds that inhibit the Ca2+-influx mode (or reverse mode) of NCX has generated intense research interest in the pharmacology of NCX. Among the newer NCX inhibitors described to date, 2-[4-[(2,5-difluorophenyl)methoxy]-phenoxy]-5-ethoxyaniline (SEA0400) appears particularly promising in attenuating cardiac, renal, and cerebral ischemia/reperfusion injuries in various experimental models. Moreover, the mixed results that have emerged from clinical trials evaluating the efficacy and safety of inhibitors of the sodium-hydrogen exchanger (an upstream target in relation to the sodium-calcium exchanger) in myocardial protection stimulated interest in evaluating NCX as an alternative therapeutic target. This article reviews the pharmacological profile of SEA0400, as presented in the published literature, and discusses the therapeutic potential of this compound in attenuating myocardial ischemia/reperfusion injury.

Inhibitory profile of SEA0400 [2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline] assessed on the cardiac Na+-Ca2+ exchanger, NCX1.1.

SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline) has recently been described as a potent and selective inhibitor of Na(+)-Ca(2+) exchange in cardiac, neuronal, and renal preparations. The inhibitory effects of SEA0400 were investigated on the cloned cardiac Na(+)-Ca(2+) exchanger, NCX1.1, expressed in Xenopus laevis oocytes to gain insight into its inhibitory mechanism. Na(+)-Ca(2+) exchange currents were measured using the giant excised patch technique using conditions to evaluate both inward and outward currents. SEA0400 inhibited outward Na(+)-Ca(2+) exchange currents with high affinity (IC(50) = 78 +/- 15 and 23 +/- 4 nM for peak and steady-state currents, respectively). Considerably less inhibitory potency (i.e., micromolar) was observed for inward currents. The inhibitory profile was reexamined after proteolytic treatment of excised patches with alpha-chymotrypsin, a procedure that eliminates ionic regulatory mechanisms. After this treatment, an IC(50) value of 1.2 +/- 0.6 microM was estimated for outward currents, whereas inward currents became almost insensitive to SEA0400. The inhibitory effects of SEA0400 on outward exchange currents were evident at both high and low concentrations of regulatory Ca(2+), although distinct features were noted. SEA0400 accelerated the inactivation rate of outward currents. Based on paired pulse experiments, SEA0400 altered the recovery of exchangers from the Na(+)(i)-dependent inactive state, particularly at higher regulatory Ca(2+)(i) concentrations. Finally, the inhibitory potency of SEA0400 was strongly dependent on the intracellular Na(+) concentration. Our data confirm that SEA0400 is the most potent inhibitor of the cardiac Na(+)-Ca(2+) exchanger described to date and provide a reasonable explanation for its apparent transport mode selectivity.

Effects of SEA0400 on mutant NCX1.1 Na+-Ca2+ exchangers with altered ionic regulation.

SEA0400 (SEA) blocks cardiac and neuronal Na+-Ca2+ exchange with the highest affinity of any known inhibitor, yet very little is known about its molecular mechanism of action. Previous data from our lab suggested that SEA stabilizes or modulates the transition of NCX1.1 exchangers into a Na+i-dependent (I1) inactive state. To test this hypothesis, we examined the effects of SEA on mutant exchangers with altered ionic regulatory properties. With mutants where Na+i-dependent inactivation is absent, the effects of SEA were greatly reduced. Conversely, with mutants displaying accelerated Na+i-dependent inactivation, block of NCX1.1 by SEA was either enhanced or unchanged, depending upon the phenotype of the particular mutation. With mutant exchangers where Ca2+i-dependent (I2) inactivation was suppressed, block of exchange currents by SEA was similar to that observed for wild-type NCX1.1. These data strongly support the involvement of I1 inactivation in the inhibitory mechanism of NCX1.1 by SEA, whereas I2 inactivation does not seem to serve an important role. The involvement of processes regulated by intracellular Na+ in the inhibitory mechanism of SEA may prove to be particularly important when considering the potential cardioprotective effects of this agent.

Inhibition of canine (NCX1.1) and Drosophila (CALX1.1) Na(+)-Ca(2+) exchangers by 7-chloro-3,5-dihydro-5-phenyl-1H-4,1-benzothiazepine-2-one (CGP-37157).

The electrophysiological effects of the benzothiazepine 7-chloro-3,5-dihydro-5-phenyl-1H-4,1-benzothiazepine-2-one (CGP-37157) (CGP) were investigated on the canine (NCX1.1) and Drosophila (CALX1.1) plasmalemmal Na+-Ca2+ exchangers. These exchangers were selected for study because they show opposite responses to cytoplasmic regulatory Ca2+, thereby allowing us to examine the role of this regulatory mechanism in the inhibitory effects of CGP. CGP blocked Na+-Ca2+ exchange current mediated by both transporters with moderate potency (IC50 values = approximately 3-17 microM) compared with other recently reported blockers of Na+-Ca2+ exchange [e.g., 2-[4-[2,5-difluorophenyl) methoxy]phenoxy]phenoxy]-5-ethoxyaniline (KB-R7943) and 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea (SEA0400)]. Experiments using alpha-chymotrypsin to remove autoregulation of Na+-Ca2+ exchange showed that block by CGP was reduced, suggesting that part of the effects of this drug may require intact ionic regulatory mechanisms. For NCX1.1, the inhibition produced by CGP was greater for outward Na+-Ca2+ exchange currents compared with inward currents. When CALX1.1 was examined, the extent of inhibition was similar for both inward and outward exchange currents. Although the extent and potency of CGP-mediated inhibition of Na+-Ca2+ exchange are less than those observed with SEA0400 and KB-R7943, our data demonstrate that CGP constitutes a novel class of plasmalemmal Na+-Ca2+ exchange inhibitors. Moreover, the widespread use of CGP as a selective mitochondrial Na+-Ca2+ exchange inhibitor should be reconsidered in light of these additional inhibitory effects.