Catalyst Development for High-Temperature Polymer Electrolyte Membrane Fuel Cell (HT-PEMFC) Applications.

A constant increase in global emission standard is causing fuel cell (FC) technology to gain importance. Over the last two decades, a great deal of research has been focused on developing more active catalysts to boost the performance of high-temperature polymer electrolyte membrane fuel cells (HT-PEMFC), as well as their durability. Due to material degradation at high-temperature conditions, catalyst design becomes challenging. Two main approaches are suggested: (i) alloying platinum (Pt) with low-cost transition metals to reduce Pt usage, and (ii) developing novel catalyst support that anchor metal particles more efficiently while inhibiting corrosion phenomena. In this comprehensive review, the most recent platinum group metal (PGM) and platinum group metal free (PGM-free) catalyst development is detailed, as well as the development of alternative carbon (C) supports for HT-PEMFCs.

Performance degradation and mitigation of high temperature polybenzimidazole-based polymer electrolyte membrane fuel cells.

To meet challenges associated with climate changes due to the continuous increase in global energy demand, implementation of hydrogen and fuel cell technologies, especially the polymer electrolyte membrane type, are recognized as potential solutions. The high temperature polymer electrolyte membrane fuel cell based on acid doped polybenzimidazoles has attracted enormous R&D attention due to the simplified construction and operation of the power system. In order to improve the reliability and lifetime of the technology, studies on material degradation and mitigation are essential. The present work is a comprehensive review of the current knowledge on degradation mechanisms of the fuel cell components including the acid loss, polymer oxidation and catalyst instability due to the metal dissolution and carbon support corrosion. The durability results are updated according to the categories of steady state and dynamic operations. Durability protocols, diagnostic techniques and mitigation strategies are also discussed.

Synthesis of Pt-Rare Earth Metal Nanoalloys.

Chemical synthesis of platinum-rare earth metal (Pt-RE) nanoalloys, one of the most active catalysts for the oxygen reduction reaction, has been a formidable challenge, mainly due to the vastly different standard reduction potentials of the two metals and high oxophilicity of the latter. Here we report a universal chemical process to prepare Pt-RE nanoalloys with tunable compositions and particle sizes. Pt and RE metal ions from the most common hydrated metal salts are first atomically embedded into an in situ formed C-N network, yielding a stable compound insensitive to O2 and H2O. The Pt-RE nanoalloys are subsequently obtained by heating the compound under a mild reducing atmosphere (e.g., 3.3% H2/Ar). The key intermediate step of the process is the formation of RE carbodiimides (RE2(CN2)3) along with Pt particles. This synthesis mechanism suggests an efficient strategy to prepare nanoalloys with highly reactive metals.

Electrochemical probing into the active sites of graphitic-layer encapsulated iron oxygen reduction reaction electrocatalysts.

The graphitic-layer encapsulated iron-containing nanoparticles (G@Fe) have been proposed as a potential type of active and stable non-precious metal electrocatalysts (NPMCs) for the oxygen reduction reaction (ORR). However, the contribution of the encapsulated components to the ORR activity is still unclear compared with the well-recognized surface coordinated FeNx/C structure. Using the strong complexing effect of the iron component with anions, cyanide (CN-) in alkaline and thiocyanate (SCN-) in acidic media, the metal containing active sites are electrochemically probed. Three representative catalysts are chosen for a comparison including the as-prepared encapsulated G@Fe, commercial Fe/N/C catalyst with iron-nitrogen coordinated surface functionalities and molecular iron phthalocyanine (FePc) containing well-defined structures and compositions. It was found that all samples showed significant shifts of half-wave potentials indicating that surface Fe coordinated sites in all cases. The G@Fe catalyst showed the weakest poisoning effect (the lowest shifts of half-wave potential) compared to the Fe/N/C and FePc catalysts in both electrolytes. These results could be explained that the encapsulated iron components influence the FeNx/C and/or NxC surface functionality.

Xanthohumol Modulates Calcium Signaling in Rat Ventricular Myocytes: Possible Antiarrhythmic Properties.

Cardiac arrhythmia is a major cause of mortality in cardiovascular pathologies. A host of drugs targeted to sarcolemmal Na+, Ca2+, and K+ channels has had limited success clinically. Recently, Ca2+ signaling has been target of pharmacotherapy based on finding that leaky ryanodine receptors elevate local Ca2+ concentrations causing membrane depolarizations that trigger arrhythmias. In this study, we report that xanthohumol, an antioxidant extracted from hops showing therapeutic effects in other pathologies, suppresses aberrant ryanodine receptor Ca2+ release. The effects of xanthohumol (5-1000 nM) on Ca2+ signaling pathways were probed in isolated rat ventricular myocytes incubated with Fluo-4 AM using the perforated patch-clamp technique. We found that 5-50 nM xanthohumol reduced the frequency of spontaneously occurring Ca2+ sparks (>threefold) and Ca2+ waves in control myocytes and in cells subjected to Ca2+ overload caused by the following: 1) exposure to low K+ solutions, 2) periods of high frequency electrical stimulation, 3) exposures to isoproterenol, or 4) caffeine. At room temperatures, 50-100 nM xanthohumol reduced the rate of relaxation of electrically- or caffeine-triggered Ca2+transients, without suppressing ICa, but this effect was small and reversed by isoproterenol at physiologic temperatures. Xanthohumol also suppressed the Ca2+ content of the SR and its rate of recirculation. The stabilizing effects of xanthohumol on the frequency of spontaneously triggered Ca2+ sparks and waves combined with its antioxidant properties, and lack of significant effects on Na+ and Ca2+ channels, may provide this compound with clinically desirable antiarrhythmic properties.

Regionally diverse mitochondrial calcium signaling regulates spontaneous pacing in developing cardiomyocytes.

The quintessential property of developing cardiomyocytes is their ability to beat spontaneously. The mechanisms underlying spontaneous beating in developing cardiomyocytes are thought to resemble those of adult heart, but have not been directly tested. Contributions of sarcoplasmic and mitochondrial Ca(2+)-signaling vs. If-channel in initiating spontaneous beating were tested in human induced Pluripotent Stem cell-derived cardiomyocytes (hiPS-CM) and rat Neonatal cardiomyocytes (rN-CM). Whole-cell and perforated-patch voltage-clamping and 2-D confocal imaging showed: (1) both cell types beat spontaneously (60-140/min, at 24°C); (2) holding potentials between -70 and 0mV had no significant effects on spontaneous pacing, but suppressed action potential formation; (3) spontaneous pacing at -50mV activated cytosolic Ca(2+)-transients, accompanied by in-phase inward current oscillations that were suppressed by Na(+)-Ca(2+)-exchanger (NCX)- and ryanodine receptor (RyR2)-blockers, but not by Ca(2+)- and If-channels blockers; (4) spreading fluorescence images of cytosolic Ca(2+)-transients emanated repeatedly from preferred central cellular locations during spontaneous beating; (5) mitochondrial un-coupler, FCCP at non-depolarizing concentrations (∼50nM), reversibly suppressed spontaneous pacing; (6) genetically encoded mitochondrial Ca(2+)-biosensor (mitycam-E31Q) detected regionally diverse, and FCCP-sensitive mitochondrial Ca(2+)-uptake and release signals activating during INCX oscillations; (7) If-channel was absent in rN-CM, but activated only negative to -80mV in hiPS-CM; nevertheless blockers of If-channel failed to alter spontaneous pacing.

Flexible sample environment for high resolution neutron imaging at high temperatures in controlled atmosphere.

High material penetration by neutrons allows for experiments using sophisticated sample environments providing complex conditions. Thus, neutron imaging holds potential for performing in situ nondestructive measurements on large samples or even full technological systems, which are not possible with any other technique. This paper presents a new sample environment for in situ high resolution neutron imaging experiments at temperatures from room temperature up to 1100 °C and/or using controllable flow of reactive atmospheres. The design also offers the possibility to directly combine imaging with diffraction measurements. Design, special features, and specification of the furnace are described. In addition, examples of experiments successfully performed at various neutron facilities with the furnace, as well as examples of possible applications are presented. This covers a broad field of research from fundamental to technological investigations of various types of materials and components.

Diversity of mitochondrial Ca²âº signaling in rat neonatal cardiomyocytes: evidence from a genetically directed Ca²âº probe, mitycam-E31Q.

I(Ca)-gated Ca(2+) release (CICR) from the cardiac SR is the main mechanism mediating the rise of cytosolic Ca(2+), but the extent to which mitochondria contribute to the overall Ca(2+) signaling remains controversial. To examine the possible role of mitochondria in Ca(2+) signaling, we developed a low affinity mitochondrial Ca(2+) probe, mitycam-E31Q (300-500 MOI, 48-72h) and used it in conjunction with Fura-2AM to obtain simultaneous TIRF images of mitochondrial and cytosolic Ca(2+) in cultured neonatal rat cardiomyocytes. Mitycam-E31Q staining of adult feline cardiomyocytes showed the typical mitochondrial longitudinal fluorescent bandings similar to that of TMRE staining, while neonatal rat cardiomyocytes had a disorganized tubular or punctuate appearance. Caffeine puffs produced rapid increases in cytosolic Ca(2+) while simultaneously measured global mitycam-E31Q signals decreased more slowly (increased mitochondrial Ca(2+)) before decaying to baseline levels. Similar, but oscillating mitycam-E31Q signals were seen in spontaneously pacing cells. Withdrawal of Na(+) increased global cytosolic and mitochondrial Ca(2+) signals in one population of mitochondria, but unexpectedly decreased it (release of Ca(2+)) in another mitochondrial population. Such mitochondrial Ca(2+) release signals were seen not only during long lasting Na(+) withdrawal, but also when Ca(2+) loaded cells were exposed to caffeine-puffs, and during spontaneous rhythmic beating. Thus, mitochondrial Ca(2+) transients appear to activate with a delay following the cytosolic rise of Ca(2+) and show diversity in subpopulations of mitochondria that could contribute to the plasticity of mitochondrial Ca(2+) signaling.

Direct synthesis of Fe3 C-functionalized graphene by high temperature autoclave pyrolysis for oxygen reduction.

We present a novel approach to direct fabrication of few-layer graphene sheets with encapsulated Fe3 C nanoparticles from pyrolysis of volatile non-graphitic precursors without any substrate. This one-step autoclave approach is facile and potentially scalable for production. Tested as an electrocatalyst, the graphene-based composite exhibited excellent catalytic activity towards the oxygen reduction reaction in alkaline solution with an onset potential of ca. 1.05 V (vs. the reversible hydrogen electrode) and a half-wave potential of 0.83 V, which is comparable to the commercial Pt/C catalyst.

Hollow spheres of iron carbide nanoparticles encased in graphitic layers as oxygen reduction catalysts.

Nonprecious metal catalysts for the oxygen reduction reaction are the ultimate materials and the foremost subject for low-temperature fuel cells. A novel type of catalysts prepared by high-pressure pyrolysis is reported. The catalyst is featured by hollow spherical morphologies consisting of uniform iron carbide (Fe3 C) nanoparticles encased by graphitic layers, with little surface nitrogen or metallic functionalities. In acidic media the outer graphitic layers stabilize the carbide nanoparticles without depriving them of their catalytic activity towards the oxygen reduction reaction (ORR). As a result the catalyst is highly active and stable in both acid and alkaline electrolytes. The synthetic approach, the carbide-based catalyst, the structure of the catalysts, and the proposed mechanism open new avenues for the development of ORR catalysts.

A new method to detect rapid oxygen changes around cells: how quickly do calcium channels sense oxygen in cardiomyocytes?

Acute hypoxia is thought to trigger protective responses that, in tissues like heart and carotid body, include rapid (5-10 s) suppression of Ca(2+) and K(+) channels. To gain insight into the mechanism for the suppression of the cardiac l-type Ca(2+) channel, we measured O2-dependent fluorescence in the immediate vicinity of voltage-clamped cardiac cells subjected to rapid exchange of solutions with different O2 tensions. This was accomplished with an experimental chamber with a glass bottom that was used as a light guide for excitation of a thin ruthenium-based O2-sensitive ORMOSIL coating. Fluorescence imaging showed that steady-state Po2 was well controlled within the entire stream from an electromagnetically controlled solution "puffer" but that changes were slower at the periphery of the stream (τ1/2 ∼ 500 ms) than immediately around the voltage-clamped myocyte (τ1/2 ∼ 225 ms) where, in turn, firmly attached cells produced an additional local delay of 50-100 ms. Performing simultaneous voltage clamp and O2 measurements, we found that acute hypoxia gradually and reversibly suppressed the Ca(2+) channel (CaV1.2). Using Ba(2+) as charge carrier, the suppression was significant after 1.5 s, reached ∼10% after 2.5 s, and was nearly completely reversible in 5 s. The described fluorescence measurements provide the means to check and fine tune solution puffers and suggest that changes in Po2 can be accomplished within ∼200 ms. The rapid and reversible suppression of barium current under hypoxia is consistent with the notion that the cardiac Ca(2+) channel is directly modulated by O2.

Cardiac calcium signalling pathologies associated with defective calmodulin regulation of type 2 ryanodine receptor.

Cardiac ryanodine receptor (RyR2) is a homotetramer of 560 kDa polypeptides regulated by calmodulin (CaM), which decreases its open probability at diastolic and systolic Ca(2+) concentrations. Point mutations in the CaM-binding domain of RyR2 (W3587A/L3591D/F3603A, RyR2(ADA)) in mice result in severe cardiac hypertrophy, poor left ventricle contraction and death by postnatal day 16, suggesting that CaM inhibition of RyR2 is required for normal cardiac function. Here, we report on Ca(2+) signalling properties of enzymatically isolated, Fluo-4 dialysed whole cell clamped cardiac myocytes from 10-15-day-old wild-type (WT) and homozygous Ryr2(ADA/ADA) mice. Spontaneously occurring Ca(2+) spark frequency, measured at -80 mV, was 14-fold lower in mutant compared to WT myocytes. ICa, though significantly smaller in mutant myocytes, triggered Ca(2+) transients that were of comparable size to those of WT myocytes, but with slower activation and decay kinetics. Caffeine-triggered Ca(2+) transients were about three times larger in mutant myocytes, generating three- to four-fold bigger Na(+)-Ca(2+) exchanger NCX currents (INCX). Mutant myocytes often exhibited Ca(2+) transients of variable size and duration that were accompanied by similarly alternating and slowly activating INCX. The data suggest that RyR2(ADA) mutation produces significant reduction in ICa density and ICa-triggered Ca(2+) release gain, longer but infrequently occurring Ca(2+) sparks, larger sarcoplasmic reticulum Ca(2+) loads, and spontaneous Ca(2+) releases accompanied by activation of large and potentially arrhythmogenic inward INCX.

Covalently cross-linked sulfone polybenzimidazole membranes with poly(vinylbenzyl chloride) for fuel cell applications.

Covalently cross-linked polymer membranes were fabricated from poly(aryl sulfone benzimidazole) (SO(2)PBI) and poly(vinylbenzyl chloride) (PVBCl) as electrolytes for high-temperature proton-exchange-membrane fuel cells. The cross-linking imparted organo insolubility and chemical stability against radical attack to the otherwise flexible SO(2)PBI membranes. Steady phosphoric acid doping of the cross-linked membranes was achieved at elevated temperatures with little swelling. The acid-doped membranes exhibited increased mechanical strength compared to both pristine SO(2)PBI and poly[2,2'-(m-phenylene)-5,5'-bibenzimidazole] (mPBI). The superior characteristics of the cross-linked SO(2)PBI membranes allowed higher acid doping levels and, therefore, higher proton conductivity. Fuel-cell tests with the cross-linked membranes demonstrated a high open circuit voltage and improved power performance and durability.

Cardiac progenitor cells engineered with Pim-1 (CPCeP) develop cardiac phenotypic electrophysiological properties as they are co-cultured with neonatal myocytes.

Stem cell transplantation has been successfully used for amelioration of cardiomyopathic injury using adult cardiac progenitor cells (CPC). Engineering of mouse CPC with the human serine/threonine kinase Pim-1 (CPCeP) enhances regeneration and cell survival in vivo, but it is unknown if such apparent lineage commitment is associated with maturation of electrophysiological properties and excitation-contraction coupling. This study aims to determine electrophysiology and Ca(2+)-handling properties of CPCeP using neonatal rat cardiomyocyte (NRCM) co-culture to promote cardiomyocyte lineage commitment. Measurements of membrane capacitance, dye transfer, expression of connexin 43 (Cx43), and transmission of ionic currents (I(Ca), I(Na)) from one cell to the next suggest that a subset of co-cultured CPCeP and NRCM becomes connected via gap junctions. Unlike NRCM, CPCeP had no significant I(Na), but expressed nifedipine-sensitive I(Ca) that could be measured more consistently with Ba(2+) as permeant ion using ramp-clamp protocols than with Ca(2+) and step-depolarization protocols. The magnitude of I(Ca) in CPCeP increased during culture (4-7 days vs. 1-3 days) and was larger in co-cultures with NRCM and with NRCM-conditioned medium, than in mono-cultured CPCeP. I(Ca) was virtually absent in CPC without engineered expression of Pim-1. Caffeine and KCl-activated Ca(2+)-transients were significantly present in co-cultured CPCeP, but smaller than in NRCM. Conversely, ATP-induced (IP(3)-mediated) Ca(2+) transients were larger in CPCeP than in NRCM. I(NCX) and I(ATP) were expressed in equivalent densities in CPCeP and NRCM. These in vitro studies suggest that CPCeP in co-culture with NRCM: a) develop I(Ca) current and Ca(2+) signaling consistent with cardiac lineage, b) form electrical connections via Cx43 gap junctions, and c) respond to paracrine signals from NRCM. These properties may be essential for durable and functional myocardial regeneration under in vivo conditions.

Hypoxic regulation of cardiac Ca2+ channel: possible role of haem oxygenase.

Acute and chronic hypoxias are common cardiac diseases that lead often to arrhythmia and impaired contractility. At the cellular level it is unclear whether the suppression of cardiac Ca(2+) channels (Ca(V)1.2) results directly from oxygen deprivation on the channel protein or is mediated by intermediary proteins affecting the channel. To address this question we measured the early effects of hypoxia (5-60 s, P(O(2)) < 5 mmHg) on Ca(2+) current (I(Ca)) and tested the involvement of protein kinase A (PKA) phosphorylation, Ca(2+)/calmodulin-mediated signalling and the haem oxygenase (HO) pathway in the hypoxic regulation of Ca(V)1.2 in rat and cat ventricular myocytes and HEK-293 cells. Hypoxic suppression of ICa) and Ca(2+) transients was significant within 5 s and intensified in the following 50 s, and was reversible. Phosphorylation by cAMP or the phosphatase inhibitor okadaic acid desensitized I(Ca) to hypoxia, while PKA inhibition by H-89 restored the sensitivity of I(Ca) to hypoxia. This phosphorylation effect was specific to Ca(2+), but not Ba(2+) or Na(+), permeating through the channel. CaMKII inhibitory peptide and Bay K8644 reversed the phosphorylation-induced desensitization to hypoxia. Mutation of CAM/CaMKII-binding motifs of the α(1c) subunit of Ca(V)1.2 fully desensitized the Ca(2+) channel to hypoxia. Rapid application of HO inhibitors (zinc protoporphyrin (ZnPP) and tin protoporphyrin (SnPP)) suppressed the channel in a manner similar to acute hypoxia such that: (1) I(Ca) and I(Ba) were suppressed within 5 s of ZnPP application; (2) PKA activation and CaMKII inhibitors desensitized I(Ca), but not I(Ba), to ZnPP; and (3) hypoxia failed to further suppress I(Ca) and I(Ba) in ZnPP-treated myocytes. We propose that the binding of HO to the CaM/CaMKII-specific motifs on Ca(2+) channel may mediate the rapid response of the channel to hypoxia.

In vitro modeling of ryanodine receptor 2 dysfunction using human induced pluripotent stem cells.

BACKGROUND/AIMS: Induced pluripotent stem (iPS) cells generated from accessible adult cells of patients with genetic diseases open unprecedented opportunities for exploring the pathophysiology of human diseases in vitro. Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is an inherited cardiac disorder that is caused by mutations in the cardiac ryanodine receptor type 2 gene (RYR2) and is characterized by stress-induced ventricular arrhythmia that can lead to sudden cardiac death in young individuals. The aim of this study was to generate iPS cells from a patient with CPVT1 and determine whether iPS cell-derived cardiomyocytes carrying patient specific RYR2 mutation recapitulate the disease phenotype in vitro. METHODS: iPS cells were derived from dermal fibroblasts of healthy donors and a patient with CPVT1 carrying the novel heterozygous autosomal dominant mutation p.F2483I in the RYR2. Functional properties of iPS cell derived-cardiomyocytes were analyzed by using whole-cell current and voltage clamp and calcium imaging techniques. RESULTS: Patch-clamp recordings revealed arrhythmias and delayed afterdepolarizations (DADs) after catecholaminergic stimulation of CPVT1-iPS cell-derived cardiomyocytes. Calcium imaging studies showed that, compared to healthy cardiomyocytes, CPVT1-cardiomyocytes exhibit higher amplitudes and longer durations of spontaneous Ca(2+) release events at basal state. In addition, in CPVT1-cardiomyocytes the Ca(2+)-induced Ca(2+)-release events continued after repolarization and were abolished by increasing the cytosolic cAMP levels with forskolin. CONCLUSION: This study demonstrates the suitability of iPS cells in modeling RYR2-related cardiac disorders in vitro and opens new opportunities for investigating the disease mechanism in vitro, developing new drugs, predicting their toxicity, and optimizing current treatment strategies.

Regulation of cardiac Ca(2+) channel by extracellular Na(+).

Hyponatremia is a predictor of poor cardiovascular outcomes during acute myocardial infarction and in the setting of preexisting heart failure [1]. There are no definitive mechanisms as to how hyponatremia suppresses cardiac function. In this report we provide evidence for direct down-regulation of Ca(2+) channel current in response to low serum Na(+). In voltage-clamped rat ventricular myocytes or HEK 293 cells expressing the L-type Ca(2+) channel, a 15mM drop in extracellular Na(+) suppressed the Ca(2+) current by ∼15%; with maximal suppression of ∼30% when Na(+) levels were reduced to 100mM or less. The suppressive effects of low Na(+) on I(Ca), in part, depended on the substituting monovalent species (Li(+), Cs(+), TEA(+)), but were independent of phosphorylation state of the channel and possible influx of Ca(2+) on Na(+)/Ca(2+) exchanger. Acidification sensitized the Ca(2+) channel current to Na(+) withdrawal. Collectively our data suggest that Na(+) and H(+) may interact with regulatory site(s) at the outer recesses of the Ca(2+) channel pore thereby directly modulating the electro-diffusion of the permeating divalents (Ca(2+), Ba(2+)).

Developmental aspects of cardiac Ca(2+) signaling: interplay between RyR- and IP(3)R-gated Ca(2+) stores.

The dominant mode of intracellular Ca(2+) release in adult mammalian heart is gated by ryanodine receptors (RyRs), but it is less clear whether inositol 1,4,5-trisphosphate (IP(3))-gated Ca(2+) release channels (IP(3)Rs), which are important during embryogenesis, play a significant role during early postnatal development. To address this question, we measured confocal two-dimensional Ca(2+) dependent fluorescence images in acutely isolated neonatal (days 1 to 2) and juvenile (days 8-10) rat cardiomyocytes, either voltage-clamped or permeabilized, where rapid exchange of solution could be used to selectively activate the two types of Ca(2+) release channel. Targeting RyRs with caffeine produced large and rapid Ca(2+) signals throughout the cells. Application of ATP and endothelin-1 to voltage-clamped, or IP(3) to permeabilized, cells produced smaller and slower Ca(2+) signals that were most prominent in subsarcolemmal regions and were suppressed by either the IP(3)R-blocker 2-aminoethoxydiphenylborate or replacement of the biologically active form of IP(3) with its L-stereoisomer. Such IP(3)R-gated Ca(2+) releases were amplified by Ca(2+)-induced Ca(2+) release (CICR) via RyRs since they were also reduced by compounds that block the RyRs (tetracaine) or deplete the Ca(2+) pools they gate (caffeine, ryanodine). Spatial analysis revealed both subsarcolemmal and perinuclear origins for the IP(3)-mediated Ca(2+) release events RyR- and IP(3)R-gated Ca(2+) signals had larger magnitudes in juvenile than in neonatal cardiomyocytes. Ca(2+) signaling was generally quite similar in atrial and ventricular cardiomyocytes but showed divergent development of IP(3)-mediated regulation in juveniles. Our data suggest that an intermediate stage of Ca(2+) signaling may be present in developing cardiomyocytes, where, in addition to RyR-gated Ca(2+) pools, IP(3)-gated Ca(2+) release is sufficiently large in magnitude and duration to trigger or contribute to activation of CICR and cardiac contraction.

beta-adrenergic regulation of a novel isoform of NCX: sequence and expression of shark heart NCX in human kidney cells.

The function, regulation, and molecular structure of the cardiac Na(+)/Ca(2+) exchangers (NCXs) vary significantly among vertebrates. We previously reported that beta-adrenergic suppression of amphibian cardiac NCX1.1 is associated with specific molecular motifs. Here we investigated the bimodal, cAMP-dependent regulation of spiny dogfish shark (Squalus acanthias) cardiac NCX, exploring the effects of molecular structure, host cell environment, and ionic milieu. The shark cardiac NCX sequence (GenBank accession no. DQ 068478) revealed two novel proline/alanine-rich amino acid insertions. Wild-type and mutant shark NCXs were cloned and expressed in mammalian cells (HEK-293 and FlpIn-293), where their activities were measured as Ni(2+)-sensitive Ca(2+) fluxes (fluo 4) and membrane (Na(+)/Ca(2+) exchange) currents evoked by changes in extracellular Na(+) concentration and/or membrane potential. Regardless of Ca(2+) buffering, beta-adrenergic stimulation of cloned wild-type shark NCX consistently produced bimodal regulation (defined as differential regulation of Ca(2+)-efflux and -influx pathways), with suppression of the Ca(2+)-influx mode and either no change or enhancement of the Ca(2+)-efflux mode, closely resembling results from parallel experiments with native shark cardiomyocytes. In contrast, mutant shark NCX, with deletion of the novel region 2 insertion, produced equal suppression of the inward and outward currents and Ca(2+) fluxes, thereby abolishing the bimodal nature of the regulation. Control experiments with nontransfected and dog cardiac NCX-expressing cells showed no cAMP regulation. We conclude that bimodal beta-adrenergic regulation is retained in cloned shark NCX and is dependent on the shark's unique molecular motifs.