NCX Current Research Articles

Regulation of Ca2+ and Na+ in Normal and Failing Cardiac Myocytes

Ca(2+) in cardiac myocytes regulates contractility and relaxation, and Ca(2+) and Na (+)regulation are linked via Na(+)/Ca(2+) exchange (NCX). Heart failure (HF) is accompanied by contractile dysfunction and arrhythmias, both of which may be due to altered cellular Ca(2+) handling. Smaller Ca(2+) transient and sarcoplasmic reticulum (SR) Ca(2+) content cause systolic dysfunction in HF. The reduced SR Ca(2+) content is due to: (a) reduced SR Ca(2+)-ATPase function (which also contributes to diastolic dysfunction), (b) increased expression and function of NCX (which competes with SR Ca(2+)-ATPase during relaxation, but preserves diastolic function), and (c) enhanced diastolic SR Ca(2+) leak. Relative contributions of these may vary with HF etiology and stage. Triggered arrhythmias (e.g., delayed afterdepolarizations [DADs]) are prominent in HF. DADs are due to spontaneous SR Ca(2+) release and consequent activation of transient inward NCX current, which in HF allows DADs to more readily trigger arrhythmogenic action potentials. Thus NCX and Na(+) are critical in systolic and diastolic function and arrhythmias. [Na(+)](i) is elevated in HF, which may limit SR unloading and provide some Ca(2+) influx during the HF action potential, thus limiting the depression of systolic function. High [Na(+)](i) in HF is due to enhanced Na(+) influx. Cellular Na(+)/K(+)-ATPase (NKA) function appears unaltered, despite reduced NKA expression. This dichotomy led us to test NKA regulation by phospholemman (PLM). We find that PLM regulates NKA in a manner analogous to phospholamban regulation of SR Ca(2+)-ATPase (i.e., inhibition that is relieved by PLM phosphorylation). We measured intermolecular FRET between PLM and NKA, which is reduced upon PLM phosphorylation. The lower expression level of more phosphorylated PLM in HF may explain the above dichotomy. Thus, altered Ca(2+) and Na(+) handling contributes to altered contractile function and arrhythmogenesis in HF.

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

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. 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(+). 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+)]. 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.

Response to “β-adrenergic stimulation does not activate Na+/Ca2+ exchange current in guinea pig, mouse, and rat ventricular myocytes”

To the Editor : We read the report of Lin et al. ([1][1]) with great interest. The authors suggest that observed increases in the Ni2+-sensitive bidirectional current after exposure of the mouse, rat, and guinea pig myocytes to isoproterenol were in fact not attributable to changes in the Na+/Ca2+

Fundamental importance of Na+-Ca2+ exchange for the pacemaking mechanism in guinea-pig sino-atrial node.

Na+-Ca2+ exchange (NCX) current has been suggested to play a role in cardiac pacemaking, particularly in association with Ca2+ release from the sarcoplasmic reticulum (SR) that occurs just before the action potential upstroke. The present experiments explore in more detail the contribution of NCX to pacemaking. Na+-Ca2+ exchange current was inhibited by rapid switch to low-Na+ solution (with Li+ replacing Na+) within the time course of a single cardiac cycle to avoid slow secondary effects. Rapid switch to low-Na+ solution caused immediate cessation of spontaneous action potentials. ZD7288 (3 microM), to block I(f) (funny current) channels, slowed but did not stop the spontaneous activity, and tetrodotoxin (10 microM), to block Na+ channels, had little effect, but in the presence of either of these agents, rapid switch to low-Na+ solution again caused immediate cessation of spontaneous action potentials. Spontaneous electrical activity was also stopped following loading of the cells with the Ca2+ chelators BAPTA and EGTA, and by exposure to the NCX inhibitor KB-R7943 (5 microM). When rapid switch to low-Na+ solution caused cessation of spontaneous activity, this was found (using confocal microscopy, with fluo-4 as the Ca2+ probe) to be accompanied by an initial fall in cytosolic [Ca2+], with subsequent appearance of Ca2+ waves. Inhibition of SR Ca2+ uptake with cyclopiazonic acid (CPA, 30 microM) slowed but did not stop spontaneous activity. Rapid switch to low-Na+ solution in the presence of CPA caused abolition of spontaneous Ca2+ transients and a progressive rise in cytosolic [Ca2+]. With ratiometric fluorescence methods (indo-5F as the Ca2+ probe), the minimum level of [Ca2+] between beats was found to be approximately 225 nM, and abolition of beating with nifedipine, acetylcholine or adenosine caused a fall in cytosolic [Ca2+] below this level. These observations support the hypothesis that NCX current is essential for normal pacemaker activity under the conditions of our experiments. A continuous depolarizing influence of current through the NCX protein might result from maintained electrogenic NCX (with 3:1 stoichiometry, supported by a cytosolic [Ca2+] that normally does not fall below 225 nM between beats) and/or from a novel, recently suggested role of the NCX protein to allow a Na+ leak pathway.

Angiotensin II and angiotensin II receptor blocker modulate the arrhythmogenic activity of pulmonary veins.

Angiotensin II receptor blockers (AIIRBs) have been shown to prevent atrial fibrillation. The pulmonary veins (PVs) are the most important focus for the generation of atrial fibrillation. The aim of this study was to evaluate whether angiotensin II or AIIRB may change the arrhythmogenic activity of the PVs. Conventional microelectrodes and whole-cell patch clamps were used to investigate the action potentials (APs) and ionic currents in isolated rabbit PV tissue and single cardiomyocytes before and after administering angiotensin II or losartan (AIIRB). In the tissue preparations, angiotensin II induced delayed after-depolarizations (1, 10, and 100 nM) and accelerated the automatic rhythm (10 and 100 nM). Angiotensin II (100 nM) prolonged the AP duration and increased the contractile force (10 and 100 nM). Losartan (1 and 10 microM) inhibited the automatic rhythm. Losartan (10 microM) prolonged the AP duration and reduced the contractile force (1 and 10 microM). Angiotensin II reduced the transient outward potassium current (I(to)) but increased the L-type calcium, delayed rectifier potassium (I(K)), transient inward (I(ti)), pacemaker, and Na(+)-Ca(2+) exchanger (NCX) currents in the PV cardiomyocytes. Losartan decreased the I(to), I(K), I(ti), and NCX currents. In conclusion, angiotensin II and AIIRB modulate the PV electrical activity, which may play a role in the pathophysiology of atrial fibrillation.

Intermittent hypoxia protects cardiomyocytes against ischemia-reperfusion injury-induced alterations in Ca2+ homeostasis and contraction via the sarcoplasmic reticulum and Na+/Ca2+ exchange mechanisms

We have previously demonstrated that intermittent high-altitude (IHA) hypoxia significantly attenuates ischemia-reperfusion (I/R) injury-induced excessive increase in resting intracellular Ca(2+) concentrations ([Ca(2+)](i)). Because the sarcoplasmic reticulum (SR) and Na(+)/Ca(2+) exchanger (NCX) play crucial roles in regulating [Ca(2+)](i) and both are dysfunctional during I/R, we tested the hypothesis that IHA hypoxia may prevent I/R-induced Ca(2+) overload by maintaining Ca(2+) homeostasis via SR and NCX mechanisms. We thus determined the dynamics of Ca(2+) transients and cell shortening during preischemia and I/R injury in ventricular cardiomyocytes from normoxic and IHA hypoxic rats. IHA hypoxia did not affect the preischemic dynamics of Ca(2+) transients and cell shortening, but it significantly suppressed the I/R-induced increase in resting [Ca(2+)](i) levels and attenuated the depression of the Ca(2+) transients and cell shortening during reperfusion. Moreover, IHA hypoxia significantly attenuated I/R-induced depression of the protein contents of SR Ca(2+) release channels and/or ryanodine receptors (RyRs) and SR Ca(2+) pump ATPase (SERCA2) and SR Ca(2+) release and uptake. In addition, a delayed decay rate time constant of Ca(2+) transients and cell shortening of Ca(2+) transients observed during ischemia was accompanied by markedly inhibited NCX currents, which were prevented by IHA hypoxia. These findings indicate that IHA hypoxia may preserve Ca(2+) homeostasis and contraction by preserving RyRs and SERCA2 proteins as well as NCX activity during I/R.

Relative abundance of α2 Na + pump isoform influences Na +–Ca 2+ exchanger currents and Ca 2+ transients in mouse ventricular myocytes

In the mouse, genetic reduction in the Na +, K +-ATPase α1 or α2 isoforms results in different functional phenotypes: heterozygous α2 isolated hearts are hypercontractile, whereas heterozygous α1 hearts are hypocontractile. We examined Na +/Ca 2+ exchange (NCX) currents in voltage clamped myocytes (pipette [Na +] = 15 mM) induced by abrupt removal of extracellular Na +. In wild-type (WT) myocytes, peak exchanger currents were 0.59 ± 0.04 pA/pF (mean ± S.E.M., n =1 0). In α1 +/– myocytes (α2 isoform increased by 54%), NCX current was reduced to 0.33 ± 0.05 ( n = 9, P < 0.001) indicating a lower subsarcolemmal [Na +]. In α2 +/– myocytes (α2 isoform reduced by 54%), the NCX current was increased to 0.89 ± 0.11 ( n = 8, P = 0.03). The peak sarcolemmal Na + pump currents activated by abrupt increase in [K +] o to 4 mM in voltage clamped myocytes in which the Na + pump had been completely inhibited for 5 min by exposure to 0 [K +] o were similar in α1 +/– (0.86 ± 0.12, n = 10) and α2 +/– myocytes (0.94 ± 0.08 pA/pF, n = 16), and were slightly but insignificantly reduced relative to WT (1.03 ± 0.05, n = 24). The fluo-3 [Ca 2+] i transient ( F/ F o) in WT myocytes paced at 0.5 Hz was 2.18 ± 0.09, n = 34, was increased in α2 +/– myocytes ( F/ F o = 2.56 ± 0.14, n = 24, P = 0.02), and was decreased in α1 +/– myocytes ( F/ F o = 1.93 ± 0.08, n = 28, P < 0.05). Thus the α2 isoform rather than the α1 appears to influence Na +/Ca 2+ exchanger currents [Ca 2+] i transients, and contractility. This finding is consistent with the proposal that α2 isoform of the Na pump preferentially alters [Na +] in a subsarcolemmal micro-domain adjacent to Na +/Ca 2+ exchanger molecules and SR Ca 2+ release sites.

Na+/Ca2+ exchange activity in neonatal rabbit ventricular myocytes.

Much less is known about the contributions of the Na(+)/Ca(2+) exchanger (NCX) and sarcoplasmic reticulum (SR) Ca(2+) pump to cell relaxation in neonatal compared with adult mammalian ventricular myocytes. Based on both biochemical and molecular studies, there is evidence of a much higher density of NCX at birth that subsequently decreases during the next 2 wk of development. It has been hypothesized, therefore, that NCX plays a relatively more important role for cytosolic Ca(2+) decline in neonates as well as, perhaps, a role in excitation-contraction coupling in reverse mode. We isolated neonatal ventricular myocytes from rabbits in four different age groups: 3, 6, 10, and 20 days of age. Using an amphotericin-perforated patch-clamp technique in fluo-3-loaded myocytes, we measured the caffeine-induced inward NCX current (I(NCX)) and the Ca(2+) transient. We found that the integral of I(NCX), an indicator of SR Ca(2+) content, was greatest in myocytes from younger age groups when normalized by cell surface area and that it decreased with age. The velocity of Ca(2+) extrusion by NCX (V(NCX)) was linear with [Ca(2+)] and did not indicate saturation kinetics until [Ca(2+)] reached 1-3 microM for each age group. There was a significantly greater time delay between the peaks of I(NCX) and the Ca(2+) transient in myocytes from the youngest age groups. This observation could be related to structural differences in the subsarcolemmal microdomains as a function of age.

Myocardial infarction causes increased expression but decreased activity of the myocardial Na+-Ca2+ exchanger in the rabbit.

Na+-Ca2+ exchanger (NCX) protein levels and activity were measured in myocardium from the basal region of the left ventricle of rabbit hearts with significant left ventricular dysfunction (LVD), 8-9 weeks after an apical infarction. NCX protein abundance was higher in the tissue homogenates (121 +/- 11%) and purified membrane fractions (143 +/- 12%) in the LVD compared to the sham-operated (sham) group. NCX mRNA was also higher in the LVD group (126%). Lower NCX protein expression was observed in the membrane fractions from the epicardium compared to the endocardium in both the sham and LVD groups. Transmembrane currents were recorded in isolated cardiomyocytes by single-electrode voltage clamp; [Ca2+]i was measured using Fura-2. Rapid application of 10 mmol l-1 caffeine was used to induce Ca2+ release from the sarcoplasmic reticulum. The subsequent NCX-mediated Ca2+ efflux rate constant was lower (70% of sham) in the LVD group. NCX currents were measured in cardiomyocytes dialysed with 250 nM Ca2+ (50 mmol l-1 EGTA). A lower NCX current (75% of sham) was observed in the LVD group. Lower NCX activity was also observed in cardiomyocytes isolated from the epicardium compared to the endocardium; a transmural difference that was also seen in the LVD group. Reduced activity despite increased protein expression may result from reduced Ca2+ sensitivity of the allosteric regulation of NCX. However, measurements indicated increased Ca2+ sensitivity in the LVD group. Cardiomyocytes from LVD hearts displayed a marked reduction in the transverse tubule area (59% of sham) and the surface area/volume ratio (80% of sham). Disrupted transverse tubule structure may contribute to the decrease in NCX activity despite increased protein expression in LVD.

Effect of cardiac glycosides on action potential characteristics and contractility in cat ventricular myocytes: role of calcium overload.

There is increasing evidence that cardiac glycosides act through mechanisms distinct from inhibition of the sodium pump but which may contribute to their cardiac actions. To more fully define differences between agents indicative of multiple sites of action, we studied changes in contractility and action potential (AP) configuration in cat ventricular myocytes produced by six cardiac glycosides (ouabain, ouabagenin, dihydroouabain, actodigin, digoxin, and resibufogenin). AP shortening was observed only with ouabain and actodigin. There was extensive inotropic variability between agents, with some giving full inotropic effects before automaticity occurred whereas others produced minimal inotropy before toxicity. AP shortening was not a result of alterations in calcium current or the inward rectifier potassium current, but correlated with an increase in steady-state outward current (Iss), which was sensitive to KB-R7943, a Na+-Ca2+ exchange (NCX) inhibitor. Interestingly, Iss was observed following exposure to ouabain and dihydroouabain, suggesting that an additional mechanism is operative with dihydroouabain that prevents AP shortening. Further investigation into differences in inotropy between ouabagenin, dihydroouabain and ouabain revealed almost identical responses under AP voltage clamp. Thus all agents appear to act on the sodium pump and thereby secondarily increase the outward reverse mode NCX current, but the extent of AP duration shortening and positive inotropy elicited by each agent is limited by development of their toxic actions. The quantitative differences between cardiac glycosides suggest that mechanisms independent of sodium pump inhibition may result from an altered threshold for calcium overload possibly involving direct or indirect effects on calcium release from the sarcoplasmic reticulum.

Cardiac submembrane [Na+] transients sensed by Na+-Ca2+ exchange current.

Na+ influx via INa during cardiac action potentials can raise bulk [Na+]i by 10 to 15 micromol/L. However, larger rises in submembrane [Na+] ([Na+]sm) local to Na+-Ca2+ exchangers (NCX) could enhance Ca2+ influx via NCX (and Ca2+-induced Ca2+ release). We tested whether INa could increase [Na+]sm, using NCX current (INCX) as a biosensor in rabbit ventricular myocytes (with [Ca2+]i buffered, [Na+]i=10 mmol/L, and other currents blocked). We measured INCX as early as 5 ms after INa. Prior INa activation did not affect INCX at physiological membrane potentials (Em=-100 to +50 mV), but for Em >+50 mV (where INCX is especially sensitive to [Na+]i), INCX shifted outward. At 5 ms and +100 mV, INa shifted INCX outward by 0.23 A/F (corresponding to Delta[Na+]sm=0.24 mmol/L). The effect of INa dissipated with a time constant of approximately 15 ms. Thus, the impact of INa on NCX is almost undetectable at physiological Em and short lived. This suggests that INa effects on excitation-contraction coupling (via outward INCX) are minimal and limited to early during the action potential. However, local Delta[Na+]sm during INa may be 60 times higher than bulk Delta[Na+]i.

Protein kinase A hyperphosphorylation increases basal current but decreases beta-adrenergic responsiveness of the sarcolemmal Na+-Ca2+ exchanger in failing pig myocytes.

The sodium-calcium exchanger (NCX) protein is the major cardiac calcium extrusion mechanism and is upregulated in heart failure (HF). NCX expression level and functional activity as regulated by beta-adrenergic receptor (beta-AR) stimulation in swine with and without tachycardia-induced heart failure were studied. The Ni2+-sensitive NCX current was measured in myocytes from HF and control animals in the basal state or in the presence of isoproterenol, forskolin, 8-Br-cAMP, okadaic acid, or protein phosphatase type 1. Western blot analysis revealed a significant increase in both the 120-kDa (29%) and 80-kDa (69%) fragments in HF (P<0.05 versus control). Despite this modest increase in protein, the basal peak outward NCX current was increased almost 5-fold in HF (P<0.05 versus control). Stimulation with isoproterenol, however, increased the control currents to a significantly greater extent than HF (500% increase in control versus 100% increase in HF, P<0.01); peak stimulated current was not different in HF and control. This reduction in responsiveness to beta-AR stimulation was refractory to forskolin, 8-Br-cAMP, or okadaic acid stimulation. In vitro protein kinase A back-phosphorylation revealed higher phosphorylation capacity of NCX protein in control versus HF, consistent with increased phosphorylation in vivo (hyperphosphorylation) in HF. Protein phosphatase type 1 exposure resulted in a significant reduction (73%) in peak basal current in HF (compared with no significant difference in controls), confirming that the increased basal NCX current in HF is predominantly attributable to hyperphosphorylation. NCX expression and activity are thus increased in HF, although beta-AR responsiveness is decreased because of NCX hyperphosphorylation.

Stoichiometry of Na+-Ca2+ exchange is 3:1 in guinea-pig ventricular myocytes.

In single guinea-pig ventricular myocytes, we examined the stoichiometry of Na(+)-Ca(2+) exchange (NCX) by measuring the reversal potential (E(NCX)) of NCX current (I(NCX)) and intracellular Ca(2+) concentration ([Ca(2+)](i)) with the whole-cell voltage-clamp technique and confocal microscopy, respectively. With given ionic concentrations in the external and pipette solutions, the predicted E(NCX) were -73 and -11 mV at 3:1 and 4:1 stoichiometries, respectively. E(NCX) measured were -69 +/- 2 mV (n = 11), -47 +/- 1 mV (n = 14) and -15 +/- 1 mV (n = 15) at holding potentials (HP) of -73, -42 and -11 mV, respectively. Thus, E(NCX) almost coincided with HP, indicating that [Ca(2+)](i) and/or [Na(+)](i) changed due to I(NCX) flow. Shifts of E(NCX) (deltaE(NCX)) were measured by changing [Ca(2+)](o) or [Na(+)](o). The measured values of deltaE(NCX) were almost always smaller than those expected theoretically at a stoichiometry of either 3:1 or 4:1. Using indo-1 fluorescence, [Ca(2+)](i) measured under the whole-cell voltage-clamp supported a 3:1 but not 4:1 stoichiometry. To prevent Ca(2+) accumulation, we inhibited I(NCX) with Ni(2+) and re-examined E(NCX) during washing out Ni(2+). With HP at predicted E(NCX) at a 3:1 stoichiometry, E(NCX) developed was close to predicted E(NCX) and did not change with time. However, with HP at predicted E(NCX) for a 4:1 stoichiometry, E(NCX) developed initially near a predicted E(NCX) for a 3:1 stoichiometry and shifted toward E(NCX) for a 4:1 stoichiometry with time. We conclude that the stoichiometry of cardiac NCX is 3:1.

Temperature dependence of cloned mammalian and salmonid cardiac Na+/Ca2+ exchanger isoforms

The cardiac Na(+)/Ca(2+) exchanger (NCX), an important regulator of cytosolic Ca(2+) concentration in contraction and relaxation, has been shown in trout heart sarcolemmal vesicles to have high activity at 7 degrees C relative to its mammalian isoform. This unique property is likely due to differences in protein structure. In this study, outward NCX currents (I(NCX)) of the wild-type trout (NCX-TR1.0) and canine (NCX 1.1) exchangers expressed in oocytes were measured to explore the potential contributions of regulatory vs. transport mechanisms to this observation. cRNA was transcribed in vitro from both wild-type cDNA and was injected into Xenopus oocytes. I(NCX) of NCX-TR1.0 and NCX1.1 were measured after 3-4 days over a temperature range of 7-30 degrees C using the giant excised patch technique. The I(NCX) for both isoforms exhibited Na(+)-dependent inactivation and Ca(2+)-dependent positive regulation. The I(NCX) of NCX1.1 exhibited typical mammalian temperature sensitivities with Q(10) values of 2.4 and 2.6 for peak and steady-state currents, respectively. However, the I(NCX) of NCX-TR1.0 was relatively temperature insensitive with Q(10) values of 1.2 and 1.1 for peak and steady-state currents, respectively. I(NCX) current decay was fit with a single exponential, and the resultant rate constant of inactivation (lambda) was determined as a function of temperature. As expected, lambda decreased monotonically with temperature for both isoforms. Although lambda was significantly greater in NCX1.1 compared with NCX-TR1.0 at all temperatures, the effect of temperature on lambda was not different between the two isoforms. These data suggest that the disparities in I(NCX) temperature dependence between these two exchanger isoforms are unlikely due to differences in their inactivation kinetics. In addition, similar differences in temperature dependence were observed in both isoforms after alpha-chymotrypsin treatment that renders the exchanger in a deregulated state. These data suggest that the differences in I(NCX) temperature dependence between the two isoforms are not due to potential disparities in either the I(NCX) regulatory mechanisms or structural differences in the cytoplasmic loop but are likely predicated on differences within the transmembrane segments.

Enhanced Ca(2+)-activated Na(+)-Ca(2+) exchange activity in canine pacing-induced heart failure.

Defective excitation-contraction coupling in heart failure is generally associated with both a reduction in sarcoplasmic reticulum (SR) Ca(2+) uptake and a greater dependence on transsarcolemmal Na(+)-Ca(2+) exchange (NCX) for Ca(2+) removal. Although a relative increase in NCX is expected when SR function is impaired, few and contradictory studies have addressed whether there is an absolute increase in NCX activity. The present study examines in detail NCX density and function in left ventricular midmyocardial myocytes isolated from normal or tachycardic pacing-induced failing canine hearts. No change of NCX current density was evident in myocytes from failing hearts when intracellular Ca(2+) ([Ca(2+)](i)) was buffered to 200 nmol/L. However, when [Ca(2+)](i) was minimally buffered with 50 micromol/L indo-1, Ca(2+) extrusion via NCX during caffeine application was doubled in failing versus normal cells. In other voltage-clamp experiments in which SR uptake was blocked with thapsigargin, both reverse-mode and forward-mode NCX currents and Ca(2+) transport were increased >2-fold in failing cells. These results suggest that, in addition to a relative increase in NCX function as a consequence of defective SR Ca(2+) uptake, there is an absolute increase in NCX function that depends on [Ca(2+)](i) in the failing heart.

Diminished function and expression of the cardiac Na+-Ca2+ exchanger in diabetic rats: implication in Ca2+ overload.

1. The present work was carried out in order to determine whether a decrease in cardiac Na+-Ca2+ exchanger (NCX) activity observed in diabetes is caused by a reduction in NCX protein and mRNA levels and to elucidate the significance of this decrease in alterations in [Ca2+]i homeostasis in diabetic cardiomyocytes. 2. The NCX current was significantly reduced in ventricular myocytes freshly isolated from streptozotocin-induced diabetic rat hearts, and its current density was about 55 % of age-matched controls. 3. Diabetes resulted in a 30 % decrease in cardiac protein and mRNA levels of NCX1, a NCX isoform which is expressed at high levels in the heart. 4. The reduced NCX current and the decreased protein and mRNA levels of NCX1 in diabetes were prevented by insulin therapy. 5. Although both diastolic and peak systolic [Ca2+]i were not different between the two groups of myocytes, increasing external Ca2+ concentration to high levels greatly elevated diastolic [Ca2+]i in diabetic myocytes. Inhibition of NCX by reduction in extracellular Na+ by 50 % could produce a marked rise in diastolic [Ca2+]i in control myocytes in response to high Ca2+, as seen in diabetic myocytes. However, cyclopiazonic acid, an inhibitor of sarcoplasmic reticulum Ca2+ pump ATPase, did not modify the high Ca2+-induced changes in diastolic [Ca2+]i in either control or diabetic myocytes. 6. Only in papillary muscles from diabetic rats did the addition of high Ca2+ cause a marked rise in resting tension signifying a partial contracture that was possibly due to an increase in diastolic [Ca2+]i. 7. In conclusion, the diminished NCX function in diabetic myocytes shown in this study results in part from the decreased levels of cardiac NCX protein and mRNA. We suggest that this impaired NCX function may play an important role in alterations in Ca2+ handling when [Ca2+]i rises to pathological levels.