Evidence for a modulatory effect of external potassium ions on ionic current mediated by the cardiac Na+-Ca2+ exchanger

The aim of this study was to investigate whether or not the activity of the cardiac Na(+)-Ca(2+) exchanger might be directly sensitive to external K(+) concentration ([K(+)](e)). Measurements of whole-cell exchanger current (I(NaCa)) were made at 37 degrees C from guinea-pig isolated ventricular myocytes, using whole-cell patch clamp recording with major interfering conductances blocked. Changing [K(+)](e) from 0 to 5mM significantly reduced both outward and inward exchange currents in a time-dependent manner. Various [K(+)](e) between 1 and 15 mM were tested and the inhibitory effect was observed to be concentration-dependent. At steady-state, 5mM [K(+)](e) decreased the density of Ni(2+)-sensitive current by 52.8+/-4.3% (mean+/-S.E.M., n=6) and of 0Na0Ca-sensitive current by 39.0+/-4.4% (n=5). The possibility that the inhibitory effect of external K(+) on I(NaCa) might wholly or in part be secondary to activation of the sarcolemmal Na(+)-K(+) pump was investigated by testing the effect of K(+) addition in the presence of a high concentration of strophanthidin (500 microM). Ni(2+)-sensitive I(NaCa) was still observed to be sensitive to external K(+) (I(NaCa) decreased by 39.4+/-9.4%, n=4), suggesting that the inhibitory effect could occur independently of activation of the Na(+)-K(+) pump. The effect of external K(+) on I(NaCa) was verified using a baby hamster kidney (BHK) cell line stably expressing the cardiac Na(+)-Ca(2+) exchanger isoform, NCX1. Similar to native I(NaCa), NCX1 current was also suppressed by [K(+)](e). However, [K(+)](e) did not alter current amplitude in untransfected BHK cells. The effect of [K(+)](e) on I(NaCa) could not be attributed to simply adding any monovalent cation back to the external solution, since it was not reproduced by application of equimolar Li(+), Cs(+) and TEA(+). Rb(+), however, could mimic the effect of K(+). Collectively, these data suggest that external K(+) at physiologically and pathologically relevant concentrations might be able to modulate directly the activity of the cardiac Na(+)-Ca(2+) exchanger.     

Comparison of Na(+)/Ca(2+) exchanger current and of its response to isoproterenol between acutely isolated and short-term cultured adult ventricular myocytes

The Na(+)/Ca(2+) exchanger protein is present in the cell membrane of many tissue types and plays key roles in Ca(2+) homeostasis, excitation-contraction coupling, and generation of electrical activity in the heart. The use of adult ventricular myocyte cell culture is important to molecular biological approaches to study the roles and modulation of the cardiac Na(+)/Ca(2+) exchanger. Therefore, we characterised the functional expression of the exchanger in adult guinea-pig ventricular myocytes maintained in short-term culture (for 4 days) and compared the response of ionic current (I(NaCa)) carried by the exchanger from acutely isolated and Day 4 cells to beta-adrenoceptor activation with isoproterenol (ISO). Functional activity of the exchanger was assessed by measuring I(NaCa) using whole cell patch clamp, under selective recording conditions. I(NaCa) amplitude measured at both +60 and -100mV declined significantly by Day 1 of cell culture, showing a further small decline by Day 4. However, cell surface area (assessed by measuring membrane capacitance) also declined over this time-frame. I(NaCa) normalised to membrane capacitance (I(NaCa) density) did not differ significantly between acutely isolated and cells cultured for 4 days. However, although ISO (1 microM) increased I(NaCa) in acutely isolated myocytes, it exerted no significant effect on I(NaCa) from Day 4 cells. This was not due to an inherent inability of these cells to respond to ISO, as L-type calcium current amplitude from Day 4 cells was increased by ISO to a similar extent as that from acutely isolated cells. Our data suggest that the functional expression of the Na/Ca exchanger is well maintained during short-term culture of adult ventricular myocytes. The lack of response to ISO of I(NaCa) from Day 4 cells suggests: (a) that, despite a well-maintained I(NaCa) density, cultured adult myocytes may not necessarily be suitable for studies of exchanger modulation by some agonists and (b) that there may exist subtle differences between beta-adrenergic regulation of the exchanger protein and of L-type Ca channels.     

Regulation by endothelin-1 of Na+-Ca2+ exchange current (I(NaCa)) from guinea-pig isolated ventricular myocytes.

The cardiac Na+-Ca2+ exchanger participates in Ca homeostasis, and Na+-Ca2+ exchanger-mediated ionic current (I(NaCa)) also contributes to the regulation of cardiac action potential duration. Moreover, I(NaCa) can contribute to arrhythmogenesis under conditions of cellular Ca overload. Although it has been shown that the peptide hormone endothelin-1 (ET-1) can phosphorylate the cardiac Na+-Ca2+ exchanger via protein kinase C (PKC), little is known about the effect of ET-1 on I(NaCa). In order to examine the effects of ET-1 on I(NaCa), whole-cell patch clamp measurements were made at 378C from guinea-pig isolated ventricular myocytes. With major interfering currents inhibited, I(NaCa) was measured as the current sensitive to nickel (Ni; 10mM) during a descending voltage ramp. ET-1 (10 nM) significantly increased I(NaCa) ( approximately 2-fold at -100 mV). Application of a PKC activator (PMA; 1mM: phorbol 12-myristate 13-acetate), mimicked the effect of ET-1. In contrast, the PKC inhibitor chelerythrine (CLT, 1mM) abolished the stimulatory effect of ET-1. An inactive phorbol ester, 4-alpha-phorbol-12,13-didecanoate (4a-PDD, 1mM) had no effect on I(NaCa). Collectively, these data indicate that ET-1 activated I(NaCa) through a PKC-dependent pathway. In additional experiments, isoprenaline (ISO; which has also been reported to activate I(NaCa) ) was applied. The increase in I(NaCa) density with ISO (1mM) was similar to that induced by ET-1 (10nM). When I(NaCa) was pre-stimulated by ET-1, application of ISO elicited no further increase in current and vice versa. ISO also had no additional effect on I(NaCa) when the cells were pretreated with PMA. Application of CLT did not alter the response of I(NaCa) to ISO. We conclude that ET-1 stimulated ventricular I(NaCa) via a PKC-dependent mechanism under our recording conditions. Concentrations of ET-1 and ISO that stimulated I(NaCa) to similar extents when applied separately were not additive when co-applied. The lack of synergy between the stimulatory effects of ET-1 and ISO may be important in protecting the heart from the potentially deleterious consequences of excessive stimulation of I(NaCa).     

温度和胞内钠、ATP及酸碱度对心室肌细胞钠钙交换电流的调节

心肌缺血损伤过程中,胞内Na^＋、ATP及pH都出现明显变化。钠／钙交换对心肌细胞的钙平衡起重要的调节作用。本实验采用膜片钳全细胞记录豚鼠心室肌细胞钠／钙交换电流,研究温度和胞内Na^＋、ATP及pH对钠／钙交换双向电流的影响。结果表明,温度从22℃升至34℃,钠／钙交换电流增大约4倍,而pH值的改变对钠／钙交换双向电流没有明显的影响。在22～24℃时,同时耗竭胞内ATP和胞内酸化对钠／钙交换双向转运功能影响程度小;而在34—37℃时,同时耗竭胞内ATP和胞内酸化能抑制钠／钙交换双向电流的外向和内向成分,且内向成分抑制程度高于外向成分抑制程度。表明同时耗竭胞内ATP和胞内酸化对钠／钙交换的作用具有温度依赖性。胞内Na^＋超载能使钠／钙交换电流的外向成分增加,但不增加或减少内向电流（即正向转运）成分。因此,胞内酸化及耗竭胞内ATP损伤细胞排钙机制和胞内钠超载通过钠／钙反向交换引起钙内流是引起心肌细胞钙超载的两个独立的重要因素。     

Regulation of cardiac myocyte contractility by phospholemman: Na+/Ca2+ exchange versus Na+ -K+ -ATPase

Phospholemman (PLM) regulates cardiac Na(+)/Ca(2+) exchanger (NCX1) and Na(+)-K(+)-ATPase in cardiac myocytes. PLM, when phosphorylated at Ser(68), disinhibits Na(+)-K(+)-ATPase but inhibits NCX1. PLM regulates cardiac contractility by modulating Na(+)-K(+)-ATPase and/or NCX1. In this study, we first demonstrated that adult mouse cardiac myocytes cultured for 48 h had normal surface membrane areas, t-tubules, and NCX1 and sarco(endo)plasmic reticulum Ca(2+)-ATPase levels, and retained near normal contractility, but alpha(1)-subunit of Na(+)-K(+)-ATPase was slightly decreased. Differences in contractility between myocytes isolated from wild-type (WT) and PLM knockout (KO) hearts were preserved after 48 h of culture. Infection with adenovirus expressing green fluorescent protein (GFP) did not affect contractility at 48 h. When WT PLM was overexpressed in PLM KO myocytes, contractility and cytosolic Ca(2+) concentration ([Ca(2+)](i)) transients reverted back to those observed in cultured WT myocytes. Both Na(+)-K(+)-ATPase current (I(pump)) and Na(+)/Ca(2+) exchange current (I(NaCa)) in PLM KO myocytes rescued with WT PLM were depressed compared with PLM KO myocytes. Overexpressing the PLMS68E mutant (phosphomimetic) in PLM KO myocytes resulted in the suppression of I(NaCa) but had no effect on I(pump). Contractility, [Ca(2+)](i) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the PLMS68E mutant were depressed compared with PLM KO myocytes overexpressing GFP. Overexpressing the PLMS68A mutant (mimicking unphosphorylated PLM) in PLM KO myocytes had no effect on I(NaCa) but decreased I(pump). Contractility, [Ca(2+)](i) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the S68A mutant were similar to PLM KO myocytes overexpressing GFP. We conclude that at the single-myocyte level, PLM affects cardiac contractility and [Ca(2+)](i) homeostasis primarily by its direct inhibitory effects on Na(+)/Ca(2+) exchange.     

A mathematical model of action potential heterogeneity in adult rat left ventricular myocytes.

Mathematical models were developed to reconstruct the action potentials (AP) recorded in epicardial and endocardial myocytes isolated from the adult rat left ventricle. The main goal was to obtain additional insight into the ionic mechanisms responsible for the transmural AP heterogeneity. The simulation results support the hypothesis that the smaller density and the slower reactivation kinetics of the Ca(2+)-independent transient outward K(+) current (I(t)) in the endocardial myocytes can account for the longer action potential duration (APD), and more prominent rate dependence in that cell type. The larger density of the Na(+) current (I(Na)) in the endocardial myocytes results in a faster upstroke (dV/dt(max)). This, in addition to the smaller magnitude of I(t), is responsible for the larger peak overshoot of the simulated endocardial AP. The prolonged APD in the endocardial cell also leads to an enhanced amplitude of the sustained K(+) current (I(ss)), and a larger influx of Ca(2+) ions via the L-type Ca(2+) current (I(CaL)). The latter results in an increased sarcoplasmic reticulum (SR) load, which is mainly responsible for the higher peak systolic value of the Ca(2+) transient [Ca(2+)](i), and the resultant increase in the Na(+)-Ca(2+) exchanger (I(NaCa)) activity, associated with the simulated endocardial AP. In combination, these calculations provide novel, quantitative insights into the repolarization process and its naturally occurring transmural variations in the rat left ventricle.     

Allosteric activation of Na+-Ca2+ exchange by L-type Ca2+ current augments the trigger flux for SR Ca2+ release in ventricular myocytes

The possible contribution of Na(+)-Ca(2+) exchange to the triggering of Ca(2+) release from the sarcoplasmic reticulum in ventricular cells remains unresolved. To gain insight into this issue, we measured the "trigger flux" of Ca(2+) crossing the cell membrane in rabbit ventricular myocytes with Ca(2+) release disabled pharmacologically. Under conditions that promote Ca(2+) entry via Na(+)-Ca(2+) exchange, internal [Na(+)] (10 mM), and positive membrane potential, the Ca(2+) trigger flux (measured using a fluorescent Ca(2+) indicator) was much greater than the Ca(2+) flux through the L-type Ca(2+) channel, indicating a significant contribution from Na(+)-Ca(2+) exchange to the trigger flux. The difference between total trigger flux and flux through L-type Ca(2+) channels was assessed by whole-cell patch-clamp recordings of Ca(2+) current and complementary experiments in which internal [Na(+)] was reduced. However, Ca(2+) entry via Na(+)-Ca(2+) exchange measured in the absence of L-type Ca(2+) current was considerably smaller than the amount inferred from the trigger flux measurements. From these results, we surmise that openings of L-type Ca(2+) channels increase [Ca(2+)] near Na(+)-Ca(2+) exchanger molecules and activate this protein. These results help to resolve seemingly contradictory results obtained previously and have implications for our understanding of the triggering of Ca(2+) release in heart cells under various conditions.     

Voltage-dependent modulation of ion binding and translocation in the cardiac Na(+)-Ca2+ exchange system

The transport of Na+ and Ca2+ ions in the cardiac Na(+)-Ca2+ exchanger can be described as separate events (Khananshvili, D. (1990) Biochemistry 29, 2437-2442). Thus, the Na(+)-Na+ and Ca(2+)-Ca2+ exchange reactions reflect reversible partial reactions of the transport cycle. The effect of diffusion potentials (K(+)-valinomycin) on different modes of the Na(+)-Ca2+ exchanger (Na(+)-Ca2+, Ca(2+)-Ca2+, and Na(+)-Na+ exchanges) were tested in reconstituted proteoliposomes, obtained from the Triton X-100 extracts of the cardiac sarcolemmal membranes. The initial rates of the Nai-dependent 45Ca-uptake (t = 1 s) were measured in EGTA-entrapped proteoliposomes at different voltages. At the fixed values of voltage [45 Ca]o was varied from 4 to 122 microM, and [Na]i was saturating (150 mM). Upon varying delta psi from -94 to +91 mV, the Vmax values were increased from 9.5 +/- 0.5 to 26.5 +/- 1.5 nmol.mg-1.s-1 and the Km from 17.8 +/- 2.5 to 39.1 +/- 5.2 microM, while the Vmax/Km values ranged from only 0.53 +/- 0.08 to 0.73 +/- 0.17 nmol.mg-1.s-1.microM-1. The equilibrium Ca(2+)-Ca2+ exchange was voltage sensitive at very low [Ca]o = [Ca]i = 2 microM, while at saturating [Ca]o = [Ca]i = 200 microM the Ca(2+)-Ca2+ exchange became voltage-insensitive. The rates of the equilibrium Na(+)-Na+ exchange appears to be voltage insensitive at saturating [Na]o = [Na]i = 160 mM. Under the saturating ionic conditions, the rates of the Na(+)-Na+ exchange were at least 2-3-fold slower than the Ca(2+)-Ca2+ exchange. The following conclusions can be drawn. (a) The near constancy of the Vmax/Km for Na(+)-Ca2+ exchange at different voltages is compatible with the ping-pong model proposed previously. (b) The effects of voltage on Vmax of Na(+)-Ca2+ exchange are consistent with the existence of a single charge carrying transport step. (c) It is not yet possible to clearly assign this step to the Na+ or Ca2+ transport half of the cycle although it is more likely that 3Na(+)-transport is a charge carrying step. Thus, the unloaded ion-binding domain contains either -2 or -3 charges (presumably carboxyl groups). (d) The binding of Na+ and Ca2+ appears to be weakly voltage-sensitive. The Ca(2+)-binding site may form a small ion-well (less than 2-3 A).     

Three-dimensional distribution of cardiac Na+-Ca2+ exchanger and ryanodine receptor during development

Mechanisms of cardiac excitation-contraction coupling in neonates are still not clearly defined. Previous work in neonates shows reverse-mode Na(+)-Ca(2+) exchange to be the primary route of Ca(2+) entry during systole and the neonatal sarcoplasmic reticulum to have similar capability as that of adult in storing and releasing Ca(2+). We investigated Na(+)-Ca(2+) exchanger (NCX) and ryanodine receptor (RyR) distribution in developing ventricular myocytes using immunofluorescence, confocal microscopy, and digital image analysis. In neonates, both NCX and RyR clusters on the surface of the cell displayed a short longitudinal periodicity of approximately 0.7 microm. However, by adulthood, both proteins were also found in the interior. In the adult, clusters of NCX on the surface of the cell retained the approximately 0.7-microm periodicity whereas clusters of RyR adopted a longer longitudinal periodicity of approximately 2.0 microm. This suggests that neonatal myocytes also have a peri-M-line RyR distribution that is absent in adult myocytes. NCX and RyR colocalized voxel density was maximal in neonates and declined significantly with ontogeny. We conclude in newborns, Ca(2+) influx via NCX could potentially activate the dense network of peripheral Ca(2+) stores via peripheral couplings, evoking Ca(2+)-induced Ca(2+) release.     

Action potential and contractility changes in [Na(+)](i) overloaded cardiac myocytes: a simulation study

Sodium overload of cardiac cells can accompany various pathologies and induce fatal cardiac arrhythmias. We investigate effects of elevated intracellular sodium on the cardiac action potential (AP) and on intracellular calcium using the Luo-Rudy model of a mammalian ventricular myocyte. The results are: 1) During rapid pacing, AP duration (APD) shortens in two phases, a rapid phase without Na(+) accumulation and a slower phase that depends on [Na(+)](i). 2) The rapid APD shortening is due to incomplete deactivation (accumulation) of I(Ks). 3) The slow phase is due to increased repolarizing currents I(NaK) and reverse-mode I(NaCa), secondary to elevated [Na(+)](i). 4) Na(+)-overload slows the rate of AP depolarization, allowing time for greater I(Ca(L)) activation; it also enhances reverse-mode I(NaCa). The resulting increased Ca(2+) influx triggers a greater [Ca(2+)](i) transient. 5) Reverse-mode I(NaCa) alone can trigger Ca(2+) release in a voltage and [Na(+)](i)-dependent manner. 6) During I(NaK) block, Na(+) and Ca(2+) accumulate and APD shortens due to enhanced reverse-mode I(NaCa); contribution of I(K(Na)) to APD shortening is negligible. By slowing AP depolarization (hence velocity) and shortening APD, Na(+)-overload acts to enhance inducibility of reentrant arrhythmias. Shortened APD with elevated [Ca(2+)](i) (secondary to Na(+)-overload) also predisposes the myocardium to arrhythmogenic delayed afterdepolarizations.     

Cross-signaling between L-type Ca2+ channels and ryanodine receptors in rat ventricular myocytes

Calcium-mediated cross-signaling between the dihydropyridine (DHP) receptor, ryanodine receptor, and Na(+)-Ca2+ exchanger was examined in single rat ventricular myocytes where the diffusion distance of Ca2+ was limited to < 50 nm by dialysis with high concentrations of Ca2+ buffers. Dialysis of the cell with 2 mM Ca(2+)- indicator dye, Fura-2, or 2 mM Fura-2 plus 14 mM EGTA decreased the magnitude of ICa-triggered intracellular Ca2+ transients (Cai-transients) from 500 to 20-100 nM and completely abolished contraction, even though the amount of Ca2+ released from the sarcoplasmic reticulum remained constant (approximately 140 microM). Inactivation kinetics of ICa in highly Ca(2+)-buffered cells was retarded when Ca2+ stores of the sarcoplasmic reticulum (SR) were depleted by caffeine applied 500 ms before activation of ICa, while inactivation was accelerated if caffeine- induced release coincided with the activation of ICa. Quantitative analysis of these data indicate that the rate of inactivation of ICa was linearly related to SR Ca(2+)-release and reduced by > 67% when release was absent. Thapsigargin, abolishing SR release, suppressed the effect of caffeine on the inactivation kinetics of ICa. Caffeine- triggered Ca(2+)-release, in the absence of Ca2+ entry through the Ca2+ channel (using Ba2+ as a charge carrier), caused rapid inactivation of the slowly decaying Ba2+ current. Since Ba2+ does not release Ca2+ but binds to Fura-2, it was possible to calibrate the fluorescence signals in terms of equivalent cation charge. Using this procedure, the amplification factor of ICa-induced Ca2+ release was found to be 17.6 +/- 1.1 (n = 4). The Na(+)-Ca2+ exchange current, activated by caffeine- induced Ca2+ release, was measured consistently in myocytes dialyzed with 0.2 but not with 2 mM Fura-2. Our results quantify Ca2+ signaling in cardiomyocytes and suggest the existence of a Ca2+ microdomain which includes the DHP/ ryanodine receptors complex, but excludes the Na(+)- Ca2+ exchanger. This microdomain appears to be fairly inaccessible to high concentrations of Ca2+ buffers.     

Modulation of late sodium current by Ca2+, calmodulin, and CaMKII in normal and failing dog cardiomyocytes: similarities and differences

Augmented and slowed late Na(+) current (I(NaL)) is implicated in action potential duration variability, early afterdepolarizations, and abnormal Ca(2+) handling in human and canine failing myocardium. Our objective was to study I(NaL) modulation by cytosolic Ca(2+) concentration ([Ca(2+)](i)) in normal and failing ventricular myocytes. Chronic heart failure was produced in 10 dogs by multiple sequential coronary artery microembolizations; 6 normal dogs served as a control. I(NaL) fine structure was measured by whole cell patch clamp in ventricular myocytes and approximated by a sum of fast and slow exponentials produced by burst and late scattered modes of Na(+) channel gating, respectively. I(NaL) greatly enhanced as [Ca(2+)](i) increased from "Ca(2+) free" to 1 microM: its maximum density increased, decay of both exponentials slowed, and the steady-state inactivation (SSI) curve shifted toward more positive potentials. Testing the inhibition of CaMKII and CaM revealed similarities and differences of I(NaL) modulation in failing vs. normal myocytes. Similarities include the following: 1) CaMKII slows I(NaL) decay and decreases the amplitude of fast exponentials, and 2) Ca(2+) shifts SSI rightward. Differences include the following: 1) slowing of I(NaL) by CaMKII is greater, 2) CaM shifts SSI leftward, and 3) Ca(2+) increases the amplitude of slow exponentials. We conclude that Ca(2+)/CaM/CaMKII signaling increases I(NaL) and Na(+) influx in both normal and failing myocytes by slowing inactivation kinetics and shifting SSI. This Na(+) influx provides a novel Ca(2+) positive feedback mechanism (via Na(+)/Ca(2+) exchanger), enhancing contractions at higher beating rates but worsening cardiomyocyte contractile and electrical performance in conditions of poor Ca(2+) handling in heart failure.     

Phospholemman inhibition of the cardiac Na+/Ca2+ exchanger. Role of phosphorylation

We have demonstrated previously that phospholemman (PLM), a 15-kDa integral sarcolemmal phosphoprotein, inhibits the cardiac Na+/Ca2+ exchanger (NCX1). In addition, protein kinase A phosphorylates serine 68, whereas protein kinase C phosphorylates both serine 63 and serine 68 of PLM. Using human embryonic kidney 293 cells that are devoid of both endogenous PLM and NCX1, we first demonstrated that the exogenous NCX1 current (I(NaCa)) was increased by phorbol 12-myristate 13-acetate (PMA) but not by forskolin. When co-expressed with NCX1, PLM resulted in: (i) decreases in I(NaCa), (ii) attenuation of the increase in I(NaCa) by PMA, and (iii) additional reduction in I(NaCa) in cells treated with forskolin. Mutating serine 63 to alanine (S63A) preserved the sensitivity of PLM to forskolin in terms of suppression of I(NaCa), whereas mutating serine 68 to alanine (S68A) abolished the inhibitory effect of PLM on I(NaCa). Mutating serine 68 to glutamic acid (phosphomimetic) resulted in additional suppression of I(NaCa) as compared with wild-type PLM. These results suggest that PLM phosphorylated at serine 68 inhibited I(NaCa). The physiological significance of inhibition of NCX1 by phosphorylated PLM was evaluated in PLM-knock-out (KO) mice. When compared with wild-type myocytes, I(NaCa) was significant larger in PLM-KO myocytes. In addition, the PMA-induced increase in I(NaCa) was significantly higher in PLM-KO myocytes. By contrast, forskolin had no effect on I(NaCa) in wild-type myocytes. We conclude that PLM, when phosphorylated at serine 68, inhibits Na+/Ca2+ exchange in the heart.     

Simulation of the undiseased human cardiac ventricular action potential: model formulation and experimental validation

Cellular electrophysiology experiments, important for understanding cardiac arrhythmia mechanisms, are usually performed with channels expressed in non myocytes, or with non-human myocytes. Differences between cell types and species affect results. Thus, an accurate model for the undiseased human ventricular action potential (AP) which reproduces a broad range of physiological behaviors is needed. Such a model requires extensive experimental data, but essential elements have been unavailable. Here, we develop a human ventricular AP model using new undiseased human ventricular data: Ca(2+) versus voltage dependent inactivation of L-type Ca(2+) current (I(CaL)); kinetics for the transient outward, rapid delayed rectifier (I(Kr)), Na(+)/Ca(2+) exchange (I(NaCa)), and inward rectifier currents; AP recordings at all physiological cycle lengths; and rate dependence and restitution of AP duration (APD) with and without a variety of specific channel blockers. Simulated APs reproduced the experimental AP morphology, APD rate dependence, and restitution. Using undiseased human mRNA and protein data, models for different transmural cell types were developed. Experiments for rate dependence of Ca(2+) (including peak and decay) and intracellular sodium ([Na(+)](i)) in undiseased human myocytes were quantitatively reproduced by the model. Early afterdepolarizations were induced by I(Kr) block during slow pacing, and AP and Ca(2+) alternans appeared at rates >200 bpm, as observed in the nonfailing human ventricle. Ca(2+)/calmodulin-dependent protein kinase II (CaMK) modulated rate dependence of Ca(2+) cycling. I(NaCa) linked Ca(2+) alternation to AP alternans. CaMK suppression or SERCA upregulation eliminated alternans. Steady state APD rate dependence was caused primarily by changes in [Na(+)](i), via its modulation of the electrogenic Na(+)/K(+) ATPase current. At fast pacing rates, late Na(+) current and I(CaL) were also contributors. APD shortening during restitution was primarily dependent on reduced late Na(+) and I(CaL) currents due to inactivation at short diastolic intervals, with additional contribution from elevated I(Kr) due to incomplete deactivation.     

Aldosterone increases voltage-gated sodium current in ventricular myocytes

The role of aldosterone in the pathogenesis of heart failure (HF) is still poorly understood. Recently, aldosterone has been shown to modulate the function of cardiac Ca(2+) and K(+) channels, thus playing a role in the electrical remodeling process. The goal of this work was to investigate the role of aldosterone on the cardiac Na(+) current (I(Na)). We analyzed the effects of aldosterone on I(Na) in isolated adult mouse ventricular myocytes, using the whole cell patch-clamp technique. After 24 h incubation with 1 microM aldosterone, the I(Na) density was significantly increased (+55%), without alteration of the biophysical properties and the cell membrane capacitance. Aldosterone (10 nM) increased the I(Na) by 23%. In 24-h coincubation experiments, with the use of actinomycin D, cycloheximide, or brefeldin A, the effect of aldosterone on I(Na) was abolished. Spironolactone (mineralocorticoid receptor antagonist, 10 microM) prevented the 1 microM aldosterone-dependent I(Na) increase, whereas RU-38486 (glucocorticoid receptor antagonist, 10 microM) did not. The action potential duration (APD) was longer in aldosterone-treated (APD(90): +53%) than in control myocytes. In addition, the L-type Ca(2+) current was also upregulated (+48%). We performed quantitative RT-PCR measurements and Western blots to quantify the mRNA and protein levels of Na(v)1.5 and Ca(v)1.2 (main channels mediating cardiac I(Na) and I(Ca)), but no significant difference was found. In conclusion, this study shows that aldosterone upregulates the cardiac I(Na) and suggest that this phenomenon may contribute to the HF-induced electrical remodeling process that may be reversed by spironolactone.     

Energetics of Na(+)-Ca(2+) exchange in resting cardiac muscle

The energetic effect of extracellular Na(+) removal and readmission (in a nominally Ca(2+)-free perfusate) in Langendorff-perfused ventricles of transgenic mice (TM), which overexpress the sarcolemmal Na(+)-Ca(2+) exchanger; normal mice (NM); young (7-12 days old) rats (YR); and older (13-20 days old) rats (OR) was studied. In all heart muscles, extracellular Na(+) removal induced an increase in heat production (H(1)). Na(+) readmission further increased heat production to a peak value (H(2)) followed by a decrease toward initial values. These effects were more marked in the YR and TM as compared with the OR and NM groups, respectively. Caffeine (1 mM), ryanodine (0.2 microM), and verapamil (1 microM) decreased H(1) and H(2) in both rat groups. EGTA (1 mM) decreased H(1) and H(2) in the YR but not in the OR group. Thapsigargin (1 microM) decreased H(1) and H(2) in all four hearts preparations. A possible interpretation is that Na(+)-Ca(2+) exchange acts as an energy-saving mechanism to prevent Ca(2+) accumulation at the junctional sarcoplasmic reticulum zone (JSR) and thus prevents further release of Ca(2+). Extracellular Na(+) removal lead to Ca(2+) accumulation in the JSR inducing further SR-Ca(2+) release and increased energy release. Na(+) readmission removes the accumulated Ca(2+) at the JSR (cleft) zone by exchanging Ca(2+) with Na(+) producing a transitory increase in energy release due to Na(+)-K pump activation.     

Conformational changes of the Ca(2+) regulatory site of the Na(+)-Ca(2+) exchanger detected by FRET

The Na(+)-Ca(2+) exchanger is a plasma membrane protein expressed at high levels in cardiomyocytes. It extrudes 1 Ca(2+) for 3 Na(+) ions entering the cell, regulating intracellular Ca(2+) levels and thereby contractility. Na(+)-Ca(2+) exchanger activity is regulated by intracellular Ca(2+), which binds to a region (amino acids 371-508) within the large cytoplasmic loop between transmembrane segments 5 and 6. Regulatory Ca(2+) activates the exchanger and removes Na(+)-dependent inactivation. The physiological role of intracellular Ca(2+) regulation of the exchanger is not yet established. Yellow (YFP) and cyan (CFP) fluorescent proteins were linked to the NH(2)- and CO(2)H-termini of the exchanger Ca(2+) binding domain (CBD) to generate a construct (YFP-CBD-CFP) capable of responding to changes in intracellular Ca(2+) concentrations by FRET efficiency measurements. The two fluorophores linked to the CBD are sufficiently close to generate FRET. FRET efficiency was reduced with increasing Ca(2+) concentrations. Titrations of Ca(2+) concentration versus FRET efficiency indicate a K(D) for Ca(2+) of approximately 140 nM, which increased to approximately 400 nM in the presence of 1 mM Mg(2+). Expression of YFP-CBD-CFP in myocytes, generated changes in FRET associated with contraction, suggesting that NCX is regulated by Ca(2+) on a beat-to-beat basis during excitation-contraction coupling.     

Ionic regulatory properties of brain and kidney splice variants of the NCX1 Na(+)-Ca(2+) exchanger.

Ion transport and regulation of Na(+)-Ca(2+) exchange were examined for two alternatively spliced isoforms of the canine cardiac Na(+)-Ca(2+) exchanger, NCX1.1, to assess the role(s) of the mutually exclusive A and B exons. The exchangers examined, NCX1.3 and NCX1.4, are commonly referred to as the kidney and brain splice variants and differ only in the expression of the BD or AD exons, respectively. Outward Na(+)-Ca(2+) exchange activity was assessed in giant, excised membrane patches from Xenopus laevis oocytes expressing the cloned exchangers, and the characteristics of Na(+)(i)- (i.e., I(1)) and Ca(2+)(i)- (i.e., I(2)) dependent regulation of exchange currents were examined using a variety of experimental protocols. No remarkable differences were observed in the current-voltage relationships of NCX1.3 and NCX1.4, whereas these isoforms differed appreciably in terms of their I(1) and I(2) regulatory properties. Sodium-dependent inactivation of NCX1.3 was considerably more pronounced than that of NCX1.4 and resulted in nearly complete inhibition of steady state currents. This novel feature could be abolished by proteolysis with alpha-chymotrypsin. It appears that expression of the B exon in NCX1.3 imparts a substantially more stable I(1) inactive state of the exchanger than does the A exon of NCX1.4. With respect to I(2) regulation, significant differences were also found between NCX1.3 and NCX1.4. While both exchangers were stimulated by low concentrations of regulatory Ca(2+)(i), NCX1.3 showed a prominent decrease at higher concentrations (>1 microM). This does not appear to be due solely to competition between Ca(2+)(i) and Na(+)(i) at the transport site, as the Ca(2+)(i) affinities of inward currents were nearly identical between the two exchangers. Furthermore, regulatory Ca(2+)(i) had only modest effects on Na(+)(i)-dependent inactivation of NCX1.3, whereas I(1) inactivation of NCX1.4 could be completely eliminated by Ca(2+)(i). Our results establish an important role for the mutually exclusive A and B exons of NCX1 in modulating the characteristics of ionic regulation and provide insight into how alternative splicing tailors the regulatory properties of Na(+)-Ca(2+) exchange to fulfill tissue-specific requirements of Ca(2+) homeostasis.     

Effects of sarcoplasmic reticulum Ca2+-ATPase overexpression in postinfarction rat myocytes.

Previous studies in adult myocytes isolated from rat hearts 3 wk after myocardial infarction (MI) demonstrated abnormal contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) homeostasis and decreased sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) expression and activity, but sarcoplasmic reticulum Ca(2+) leak was unchanged. In the present study, we investigated whether SERCA2 overexpression in MI myocytes would restore contraction and [Ca(2+)](i) transients to normal. Compared with sham-operated hearts, 3-wk MI hearts exhibited significantly higher left ventricular end-diastolic and end-systolic volumes but lower fractional shortening and ejection fraction, as measured by M-mode echocardiography. Seventy-two hours after adenovirus-mediated gene transfer, SERCA2 overexpression in 3-wk MI myocytes did not affect Na(+)-Ca(2+) exchanger expression but restored the depressed SERCA2 levels toward those measured in sham myocytes. In addition, the reduced sarcoplasmic reticulum Ca(2+) uptake in MI myocytes was improved to normal levels by SERCA2 overexpression. At extracellular Ca(2+) concentration of 5 mM, the subnormal contraction and [Ca(2+)](i) transient amplitudes in MI myocytes (compared with sham myocytes) were restored to normal by SERCA2 overexpression. However, at 0.6 mM extracellular Ca(2+) concentration, the supernormal contraction and [Ca(2+)](i) transient amplitudes in MI myocytes (compared with sham myocytes) were exacerbated by SERCA2 overexpression. We conclude that SERCA2 overexpression was only partially effective in ameliorating contraction and [Ca(2+)](i) transient abnormalities in our rat model of ischemic cardiomyopathy. We suggest that other Ca(2+) transport pathways, e.g., Na(+)-Ca(2+) exchanger, may also play an important role in contractile and [Ca(2+)](i) homeostatic abnormalities in MI myocytes.