Role of Na+:Ca2+ Exchange Current in Cs+-Induced Early Afterdepolarizations in Purkinje Fibers

Na⁺:Ca²⁺ Exchanger and EADs. Introduction: The ionic mechanisms for early afterdepolarizations (EADs) have not been fully clarified. It has been suggested that L-type Ca²⁺ current (I_caL) is the primary current generating EADs that occur near the plateau level (E-EADs) of the membrane potential (Vm) when I_caL is enhanced. The purpose of these studies was to determine accurately the range of Vm at which EADs occur in Purkinje fibers with K⁺ currents blocked by Cs⁺ and to investigate the importance of Na⁺:Ca²⁺ exchange current (I_Na:ca) as opposed to l_CaL and other currents in the generation of EADs occurring later during repolarization (L-EADs). Methods and Results: Shortened Purkinje strands from dogs and guinea pigs were superfused with physiologic solution containing Cs⁺ (3.6 mM) and a low [K⁺]_o (3.0 or 2.0 mM) to induce EADs. The Vm of origin of EADs and their evolution were measured with the aid of phase plane plots of the rate of repolarization against Vm. L-EADs occurred over a wide range of Vm (?35 to ?90 mV), generally more negative in guinea pig than in dog. Elevation of [Ca²⁺]_o, from 1.8 to 3.6 mM suppressed L-EADs within a few cycles, and they returned with continued exposure. After repeated exposures to high [Ca^:2+]₀, L-EADs migrated toward less negative Vm when |Ca²⁺]₀, was reestablished to 1.8 mM in the presence of Cs⁺. Reduction of [Na⁺]₀ from 147.5 to 112.5 mM by substitution with Li⁺ or sucrose also rapidly depressed L-EADs. Conclusions: The observation of Cs⁺-induced L-EADs over a wide range of Vm indicates that there is not a single inward gated current as a common ionic mechanism for L-EADs but does not exclude an important role for I_Na:Ca, which can operate over a wide range of Vm. The rapid suppression of L-EADs with elevated [Ca²⁺]_o, and reduced [Na⁺]_o, and the migration of EADs to more positive Vm after exposures to high |Ca²⁺]_o, are compatible with I_Nc:Ca as the major charge carrier for L-EADs.     

Role of Calcium Loading in Early Afterdepolarizations Generated by Cs+ in Canine and Guinea Pig Purkinje Fibers

Ca²⁺-Loading and EAD. introduction: Our previous observations indicate that the Na²⁺:Ca²⁺ exchange current (I_Na:Ca) plays an important role in early afterdepolarizations occurring at more negative Vm (L-EAD). The purpose of these studies was to examine the role of Ca²⁺-loading, which stimulates I_Na:Ca, in generation of L-EAD. Methods and Results: Purkinje strands and preparations of ventricular myocardium from dogs and guinea pigs were superfused with oxygenated physiologic buffer solutions at 37°C, To induce EADs, [K⁺]₀ was reduced to 2.0 to 3.0 mM and [Cs⁺]₀, (3.6 to 4.0 mM) was added at slow rates of ≤ 0.3 Hz. Isometric contraction in canine Purkinje strands and guinea pig papillary muscles doubled in 1-hour exposure to Cs⁺ and low [K⁺]₀ at slow rates, and the uptake of ⁴⁵Ca²⁺ was approximately doubled after 30 minutes. Forty-three percent of Purkinje fibers developed L-EAD after a latent period of 17 to 123 minutes of exposure. Ouabain (0.2 μM) suppressed LEAD within 10 minutes reversibly. Ca²⁺-loading (low [Na⁺]₀ or high [Ca²⁺]₀ for 5 to 10 minutes before exposure to Cs⁺, low [K⁺]₀, and slow rates resulted in rapid development of L-EAD in all preparations during subsequent exposure. In Ca²⁺-loaded preparations, delayed afterdepolarizations (DADs) as well as L-EADs developed. Conclusion: Reduction of K⁺ currents with Cs⁺, low [K⁺]₀, and slow rates induced L-EAD in a fraction of Purkinje fibers after a latent period during which Ca²⁺-loading of the sarcoplasmic reticulum occurred, while fibers preloaded with Ca²⁺ developed L-EAD rapidly and uniformly. These findings indicate that Ca²⁺-loading is a critical condition for the development of L-EAD. Early suppression of L-EAD by ouabain suggests a dependence of L-EAD on low [Na⁺]₁. These findings implicate I_Na:Ca in the generation of L-EAD.     

Schlüsselrolle des Ca2+ in der Herzinsuffizienz und mögliche neue therapeutische Ansatzpunkte

Chronic heart failure is characterized by distinct alterations in intracellular Ca²⁺ homeostasis leading to perturbations of excitation-contraction coupling. Systolic Ca²⁺ transients are typically lowered with diastolic Ca²⁺ levels being increased. Recent studies showed that these alterations of Ca²⁺ cycling are tightly linked to a reduced expression and activity of SERCA2a in heart failure as well as to an increased diastolic leakage of ryanodine receptors. In addition to that, the late inward current for Na⁺ ions (late I_Na) is increased and leads to an intracellular accumulation of Na⁺. The driving force for the Na⁺/Ca²⁺ exchanger (NCX) is thus reduced, compromising diastolic Ca²⁺ elimination out of the cytosol. This review article outlines the decisive role of Ca²⁺ cycling alterations in the pathogenesis of heart failure and focuses on new insights into underlying pathomechanisms. Furthermore, new therapeutic options are summarized and the stage of development with regard to their clinical application is analyzed.     

Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism

Regulation of ion-transport in the Na⁺/Ca²⁺ exchanger (NCX) occurs via its cytoplasmic Ca²⁺-binding domains, CBD1 and CBD2. Here, we present a mechanism for NCX activation and inactivation based on data obtained using NMR, isothermal titration calorimetry (ITC) and small-angle X-ray scattering (SAXS). We initially determined the structure of the Ca²⁺-free form of CBD2-AD and the structure of CBD2-BD that represent the two major splice variant classes in NCX1. Although the apo-form of CBD2-AD displays partially disordered Ca²⁺-binding sites, those of CBD2-BD are entirely unstructured even in an excess of Ca²⁺. Striking differences in the electrostatic potential between the Ca²⁺-bound and -free forms strongly suggest that Ca²⁺-binding sites in CBD1 and CBD2 form electrostatic switches analogous to C₂-domains. SAXS analysis of a construct containing CBD1 and CBD2 reveals a conformational change mediated by Ca²⁺-binding to CBD1. We propose that the electrostatic switch in CBD1 and the associated conformational change are necessary for exchanger activation. The response of the CBD1 switch to intracellular Ca²⁺ is influenced by the closely located cassette exons. We further propose that Ca²⁺-binding to CBD2 induces a second electrostatic switch, required to alleviate Na⁺-dependent inactivation of Na⁺/Ca²⁺ exchange. In contrast to CBD1, the electrostatic switch in CBD2 is isoform- and splice variant-specific and allows for tailored exchange activities.     

Extracellulary administered lysophosphatidylcholine causes Ca2+ efflux from freshly isolated adult rat cardiomyocytes

It has previously been reported that ischemia and reperfusion of the heart cause accumulation of lyophosphatidylcholine (LPC) within the myocardium. While it is known that LPC causes the transient increase of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) during contraction of cardiac cells, little is known about the mechanism for decreasing [Ca²⁺]_i in cardiomyocytes during LPC accumulation. Since cumulative elevation in [Ca²⁺]_i leads to irreversible injury to cardiomyocytes, elevated [Ca²⁺]_i must be restored to an unstimulated level to maintain cell functions. In the present study, we therefore examined the effect of LPC on Ca²⁺ efflux from freshly isolated adult rat cardiomyocytes. LPC stimulated the efflux of ⁴⁵Ca²⁺ from the cells in a concentration‐dependent manner (10^–7 M – 10^–5 M). Other lysophospholipids, which are generated from phopholipids of the cell membrane, failed to induce ⁴⁵Ca²⁺ efflux from the cells. Dilazep and K‐7259, which are known to inhibit the increase in [Ca²⁺]_i caused by LPC, likewise reduced ⁴⁵Ca²⁺ efflux caused by LPC addition. Furthermore, the LPC‐stimulated ⁴⁵Ca²⁺ efflux was not affected by removal of extracellular Ca²⁺, but was dependent on the presence of extracellular Na⁺. On the other hand, inhibitors of Na⁺/Ca²⁺ exchange, amiloride and 5‐(N,N‐dimethyl)‐amiloride, inhibited LPC induced ⁴⁵Ca²⁺ efflux. These results suggest that LPC stimulates extracellular Na⁺‐dependent ⁴⁵Ca²⁺ efflux from freshly isolated adult rat cardiomyocytes, probably through Na⁺/Ca²⁺ exchange on the plasma membrane of cells. Received: 3 March 1997, Returned for revision: 7 April 1997, Revision received: 1 August 1997, Accepted: 5 September 1997     

GABA uptake-dependent Ca2+ signaling in developing olfactory bulb astrocytes

We studied GABAergic signaling in astrocytes of olfactory bulb slices using confocal Ca²⁺ imaging and two-photon Na⁺ imaging. GABA evoked Ca²⁺ transients in astrocytes that persisted in the presence of GABA_A and GABA_B receptor antagonists, but were suppressed by inhibition of GABA uptake by SNAP 5114. Withdrawal of external Ca²⁺ blocked GABA-induced Ca²⁺ transients, and depletion of Ca²⁺ stores with cyclopiazonic acid reduced Ca²⁺ transients by approximately 90%. This indicates that the Ca²⁺ transients depend on external Ca²⁺, but are mainly mediated by intracellular Ca²⁺ release, conforming with Ca²⁺-induced Ca²⁺ release. Inhibition of ryanodine receptors did not affect GABA-induced Ca²⁺ transients, whereas the InsP₃ receptor blocker 2-APB inhibited the Ca²⁺ transients. GABA also induced Na⁺ increases in astrocytes, potentially reducing Na⁺/Ca²⁺ exchange. To test whether reduction of Na⁺/Ca²⁺ exchange induces Ca²⁺ signaling, we inhibited Na⁺/Ca²⁺ exchange with KB-R7943, which mimicked GABA-induced Ca²⁺ transients. Endogenous GABA release from neurons, activated by stimulation of afferent axons or NMDA application, also triggered Ca²⁺ transients in astrocytes. The significance of GABAergic Ca²⁺ signaling in astrocytes for control of blood flow is demonstrated by SNAP 5114-sensitive constriction of blood vessels accompanying GABA uptake. The results suggest that GABAergic signaling is composed of GABA uptake-mediated Na⁺ rises that reduce Na⁺/Ca²⁺ exchange, thereby leading to a Ca²⁺ increase sufficient to trigger Ca²⁺-induced Ca²⁺ release via InsP₃ receptors. Hence, GABA transporters not only remove GABA from the extracellular space, but may also contribute to intracellular signaling and astrocyte function, such as control of blood flow.     

Sarcoplasmic Reticulum Function in Murine Ventricular Myocytes Overexpressing SR CaATPase

To examine the effects of the overexpression of sarcoplasmic reticulum (SR) CaATPase on function of the SR and Ca²⁺homeostasis, we measured [Ca²⁺]_itransients (fluo-3), and L-type Ca²⁺currents (I_Ca,L), Na/Ca exchanger currents (I_Na/Ca), and SR Ca²⁺content with voltage clamp in ventricular myocytes isolated from wild type (WT) mice and transgenic (SRTG) mice. The amplitude of [Ca²⁺]_itransients was insignificantly increased in SRTG myocytes, while the diastolic [Ca²⁺]_itended to be lower. The initial and terminal declines of [Ca²⁺]_itransients were significantly accelerated in SRTG myocytes, implying a functional upregulation of the SR CaATPase. We examined the functional contribution of only the SR CaATPase to the initial and the terminal phase of the decline of [Ca²⁺]_i, by abruptly inhibiting Na/Ca exchange with a rapid switcher device. The rate of [Ca²⁺] decline mediated by the SR CaATPase was increased by 40% in SRTG compared with WT myocytes. The function of the L-type Ca²⁺channel was unchanged in SRTG myocytes, while INa/Ca density was slightly (10%) decreased. Measured SR Ca²⁺content was significantly increased by 29% in SRTG myocytes. Thus, overexpression of SR CaATPase markedly accelerates the decline of [Ca²⁺]_itransients, and induces an increase in SR Ca²⁺content, with some downregulation of the Na/Ca exchanger.     

Cenário Disfuncional dos Principais Componentes Responsáveis pelo Equilíbrio do Trânsito de Cálcio Miocárdico na Insuficiência Cardíaca Induzida por Estenose Aórtica

Background Maladaptive cardiac remodelling is characterized by diastolic and systolic dysfunction, culminating in heart failure. In this context, the dysfunctional scenario of cardiac calcium (Ca²⁺) handling has been poorly studied. An experimental model of aortic stenosis has been extensively used to improve knowledge about the key mechanisms of cardiac pathologic remodelling.Objective To understand the dysfunctional process of the major components responsible for Ca²⁺ balance and its influence on cardiac function in heart failure induced by aortic stenosis.Methods Male 21-day-old Wistar rats were distributed into two groups: control (sham; n= 28) and aortic stenosis (AoS; n= 18). Cardiac function was analysed by echocardiogram, isolated papillary muscle, and isolated cardiomyocytes. In the papillary muscle assay, SERCA2a and L-type Ca²⁺ channel activity was evaluated. The isolated cardiomyocyte assay evaluated Ca²⁺ handling. Ca²⁺ handling protein expression was analysed by western blot. Statistical significance was set at p <0.05.Results Papillary muscles and cardiomyocytes from AoS hearts displayed mechanical malfunction. AoS rats presented a slower time to the Ca²⁺ peak, reduced Ca²⁺ myofilament sensitivity, impaired sarcoplasmic reticulum Ca²⁺ influx and reuptake ability, and SERCA2a and L-type calcium channel (LTCC) dysfunction. Moreover, AoS animals presented increased expression of SERCA2a, LTCCs, and the Na⁺/Ca²⁺ exchanger.Conclusion Systolic and diastolic heart failure due to supravalvular aortic stenosis was paralleled by impairment of cellular Ca²⁺ influx and inhibition of sarcoplasmic reticulum Ca²⁺ reuptake due to LTCC and SERCA2a dysfunction, as well as changes in Ca²⁺ handling and expression of the major proteins responsible for cellular Ca²⁺ homeostasis.     

NCLX is an essential component of mitochondrial Na+/Ca2+ exchange

Mitochondrial Ca²⁺ efflux is linked to numerous cellular activities and pathophysiological processes. Although it is established that an Na⁺-dependent mechanism mediates mitochondrial Ca²⁺ efflux, the molecular identity of this transporter has remained elusive. Here we show that the Na⁺/Ca²⁺ exchanger NCLX is enriched in mitochondria, where it is localized to the cristae. Employing Ca²⁺ and Na⁺ fluorescent imaging, we demonstrate that mitochondrial Na⁺-dependent Ca²⁺ efflux is enhanced upon overexpression of NCLX, is reduced by silencing of NCLX expression by siRNA, and is fully rescued by the concomitant expression of heterologous NCLX. NCLX-mediated mitochondrial Ca²⁺ transport was inhibited, moreover, by CGP-37157 and exhibited Li⁺ dependence, both hallmarks of mitochondrial Na⁺-dependent Ca²⁺ efflux. Finally, NCLX-mediated mitochondrial Ca²⁺ exchange is blocked in cells expressing a catalytically inactive NCLX mutant. Taken together, our results converge to the conclusion that NCLX is the long-sought mitochondrial Na⁺/Ca²⁺ exchanger.     

Nongenomic Actions of Thyroid Hormone and Intracellular Calcium Metabolism

Nongenomic actions of thyroid hormone include several that involve or require calcium. Actions of thyroid hormone at the plasma or intracellular membranes include stimulation of membrane glucose transport and of the Na⁺/H⁺ antiporter (exchanger) by mechanisms that require liberation of intracellular calcium and stimulation of the cell membrane and sarcoplasmic reticulum calcium pumps (Ca²⁺-ATPases). These pumps not only transport Ca²⁺, but also are regulated by the intracellular calmodulin-Ca²⁺ complex (plasma membrane/sarcolemma) or calmodulin-dependent protein kinase II phosphorylation of phospholamban (sarcoplasmic reticulum). Intracellular calcium ion concentration may also be subject to regulation by other nongenomic effects of iodothyronines, such as those on the Na⁺/H⁺ antiporter or sodium current, that secondarily affect the Na⁺/Ca²⁺ exchanger. Certain of these nongenomic actions of thyroid hormone, e.g., Na⁺/H⁺ exchanger, Ca²⁺-ATPase, are now recognized to begin at a recently described hormone receptor on a heterodimeric structural membrane protein, integrin αvβ3. The thyroid hormone signal at this receptor is further transduced by the mitogen-activated protein kinase (MAPK; extracellular regulated kinase1/2, ERK1/2) pathway.     

Role of mitochondrial dysfunction in cardiac glycoside toxicity

Cardiac glycosides, which inhibit the plasma membrane Na⁺ pump, are one of the four categories of drug recommended for routine use to treat heart failure, yet their therapeutic window is limited by toxic effects. Elevated cytoplasmic Na⁺ ([Na⁺]_i) compromises mitochondrial energetics and redox balance by blunting mitochondrial Ca²⁺ ([Ca²⁺]_m) accumulation, and this impairment can be prevented by enhancing [Ca²⁺]_m. Here, we investigate whether this effect underlies the toxicity and arrhythmogenic effects of cardiac glycosides and if these effects can be prevented by suppressing mitochondrial Ca²⁺ efflux, via inhibition of the mitochondrial Na⁺/Ca²⁺ exchanger (mNCE). In isolated cardiomyocytes, ouabain elevated [Na⁺]_i in a dose-dependent way, blunted [Ca²⁺]_m accumulation, decreased the NADH/NAD + redox potential, and increased reactive oxygen species (ROS). Concomitant treatment with the mNCE inhibitor CGP-37157 ameliorated these effects. CGP-37157 also attenuated ouabain-induced cellular Ca²⁺ overload and prevented delayed afterdepolarizations (DADs). In isolated perfused hearts, ouabain's positive effects on contractility and respiration were markedly potentiated by CGP-37157, as were those mediated by β-adrenergic stimulation. Furthermore, CGP-37157 inhibited the arrhythmogenic effects of ouabain in both isolated perfused hearts and in vivo. The findings reveal the mechanism behind cardiac glycoside toxicity and show that improving mitochondrial Ca²⁺ retention by mNCE inhibition can mitigate these effects, particularly with respect to the suppression of Ca²⁺-triggered arrhythmias, while enhancing positive inotropic actions. These results suggest a novel strategy for the treatment of heart failure.     

Investigation of dose-dependent effects of berberine against renal ischemia/reperfusion injury in experimental diabetic rats

BackgroundIschemia–reperfusion injury causes various severe morphological and functional changes in diabetic patients. To date, numerous antidiabetic and antioxidant agents have been used for treatment of the disease-related changes.ObjectivesWe aimed to examine effective therapeutic doses or doses of berberine against renal ischemia/reperfusion injury (IRI) in a streptozotocin (STZ)-induced diabetic rat model by histopathological and biochemical analysis.MethodsThirty male Sprague Dawley rats were treated with STZ injection for the development of diabetes, and divided into the following groups: STZ-induced diabetic group (STZ); IRI-induced diabetic group (STZ + IRI); 50 mg/kg berberine (BRB) treated diabetic group after inducing IRI (STZ + IRI + BRB₁); 100 mg/kg BRB treated diabetic group after IRI (STZ + IRI + BRB₂); 150 mg/kg BRB treated diabetic group after IRI (STZ + IRI + BRB₃). Bilateral renal ischemia model was applied for 45 min, then reperfusion was allowed for 14 days in STZ-induced diabetic rats. Renal injury was detected histopathologically. Blood urea nitrogen (BUN), creatinine and lactate dehydrogenase (LDH) levels were measured in serum using the ELISA method. Total antioxidant status (TAS) and total oxidant status (TOS) of renal tissue was studied by spectrophotometric assay. Oxidative stress index (OSI) was calculated as TOS-to-TAS ratio. Tumor necrosis factor alpha (TNF-α), C-reactive protein (CRP), Na⁺/K⁺-ATPase (sodium pump), and Ca²⁺-ATPase (calcium ATPase) enzyme levels were measured in tissues using the ELISA method. Anti-apoptotic Bax and pro-apoptotic Bcl-2 protein levels were detected by Western blot analysis. All data were evaluated statistically.ResultsThe highest histopathological score was detected in the STZ + IRI group compared to the other group. BRB administration at the doses of 100 mg/kg and 150 mg/kg markedly improved renal injury. BUN and creatinine levels significantly increased in the STZ + IRI group compared to the STZ group (p < 0.001). 100 mg/kg and 150 mg/kg BRB administration significantly decreased those levels (p < 0.01). The highest TOS and the lowest TAS levels were detected in the STZ + IRI group (p < 0.001). IRI markedly aggravated inflammation via increasing levels of TNF-α and CRP (<0.001), and caused apoptosis via inducing Bcl-2 protein, and suppressing Bax protein (p < 0.001). BRB administration at the doses of 100 mg/kg and 150 mg/kg showed anti-oxidant, anti-inflammatory and anti-apoptotic effects (p < 0.01). The LDH enzyme, was used as a necrosis marker, was higher in the STZ + IRI group than other groups. BRB administration at all of the doses, resulted in the decline of LDH enzyme level (p < 0.001). Ca²⁺-ATPase and Na⁺/K⁺-ATPase enzyme activities decreased in the STZ + IRI group compared to the STZ group (p < 0.001), while BRB administration at the doses of 100 mg/kg and 150 mg/kg significantly increased those of enzyme activities, respectively (p < 0.05).ConclusionIschemia with diabetes caused severe histopathological and biochemical damage in renal tissue. The high doses of berberine markedly improved histopathological findings, regulated kidney function via decreasing BUN and creatinine levels, and rearranged intercellular ion concentration via increasing Na⁺/K⁺-ATPase and Ca^2+? ATPase levels. Berberine showed anti-oxidant, anti-apoptotic, and anti-inflammatory effects. According to these data, we suggest that berberine at the doses of 100 and 150 mg may be used as a potential therapeutic agent to prevent renal ischemic injury.     

Castration reduces mRNA levels for calcium regulatory proteins in rat heart

Sex-related differences in the cardiac phenotype have been well established. This study was designed to determine whether androgens regulate myocardial gene expression and play a role in the sex-related differences in the myocardial phenotype. Gonadectomized male rats were treated with testosterone, and myocardial gene expression was examined in whole heart using quantitative real-time PCR. Gonadectomy produced a substantial decrease in mRNA levels for the androgen receptor, Na⁺/Ca²⁺ exchanger, L-type calcium channel, and β₁-adrenergic receptor (β₁AR). Supplementation of testosterone in castrates produced a fivefold increase in androgen receptor mRNA levels. Testosterone treatment of castrates produced almost a sixfold increase in Na⁺/Ca²⁺ exchanger mRNA, a tenfold increase in L-type calcium channel mRNA accumulation, and a fourfold increase in β₁AR mRNA levels. Increased calcium channel expression, β₁AR expression, and Na⁺/Ca²⁺ exchanger expression together may alter cytosolic calcium. These results provide the first evidence that testoster-one regulates expression of myocardial calcium regulating genes and thus may play a role in modulating the cardiac phenotype in males.     

Regulation of Sodium–Calcium Exchange and Mitochondrial Energetics by Bcl-2 in the Heart of Transgenic Mice

Our previous work in cultured cells has shown that the maintenance of mitochondrial Ca²⁺homeostasis is essential for cell survival, and that the anti-apoptotic protein Bcl-2 is able to maintain a threshold level of mitochondrial Ca²⁺by the inhibition of permeability transition. To test whether Bcl-2 also affects the mitochondrial Na⁺–Ca²⁺exchange (NCE), a major efflux pathway for mitochondrial Ca²⁺, studies using transgenic mice that overexpress Bcl-2 in the heart have been performed. NCE activity was determined as the Na⁺-dependent Ca²⁺efflux in the isolated mitochondria. Overexpression of Bcl-2 led to a significant reduction of NCE activity as well as increased resistance to permeability transition in the mitochondria of transgenic heart. This was accompanied by increased matrix Ca²⁺level, enhanced formation of NADH and enhanced oxidation of pyruvate, an NAD⁺-linked substrate. Furthermore, there was induction of cellular Ca²⁺transport proteins including the Na⁺–Ca²⁺exchanger of the sarcolemma (NCX). Bcl-2 not only stimulates NCX expression in the sarcolemma but also attenuates the Na⁺–Ca²⁺exchange in the mitochondria. These results are consistent with the protection by Bcl-2 against apoptosis in heart following ischemia/reperfusion.     

The Links Between Cellular Ca2+ and Na+/H+ Exchange in the Pathophysiology of Essential Hypertension

This review focuses on the mechanisms whereby the cytosolic Ca²⁺ regulates the ubiquitous Na⁺/H⁺ exchanger (NHE-1) and how these regulatory processes might modify the behavior of NHE-1 in essential hypertension. The pH setpoint for activation of the Na⁺/H⁺ exchanger is controlled by two interrelated and Ca²⁺-dependent pathways, namely, the protein kinase/phosphatase cascade and Ca²⁺/calmodulin. The cytoplasmic domain of NHE-1 contains elements responsive to serine/theorine kinases and a high affinity binding site to Ca²⁺/calmodulin. Phosphorylation of NHE-1 or the binding of the Ca²⁺/calmodulin complex to its binding site promotes an alkaline shift in the pH setpoint for the exchanger. It is suggested that, in essential hypertension, an increased cellular Ca²⁺ load or an enhanced external Ca²⁺ entry stimulate the NHE-1 through protein kinase/phosphatase and Ca²⁺/calmodulin systems, thereby increasing its activity. Am J Hypertens 1996;9:703–707     

Crystal structure of Ca2+/H+ antiporter protein YfkE reveals the mechanisms of Ca2+ efflux and its pH regulation

Ca²⁺ efflux by Ca²⁺ cation antiporter (CaCA) proteins is important for maintenance of Ca²⁺ homeostasis across the cell membrane. Recently, the monomeric structure of the prokaryotic Na⁺/Ca²⁺ exchanger (NCX) antiporter NCX_Mj protein from Methanococcus jannaschii shows an outward-facing conformation suggesting a hypothesis of alternating substrate access for Ca²⁺ efflux. To demonstrate conformational changes essential for the CaCA mechanism, we present the crystal structure of the Ca²⁺/H⁺ antiporter protein YfkE from Bacillus subtilis at 3.1-Å resolution. YfkE forms a homotrimer, confirmed by disulfide crosslinking. The protonated state of YfkE exhibits an inward-facing conformation with a large hydrophilic cavity opening to the cytoplasm in each protomer and ending in the middle of the membrane at the Ca²⁺-binding site. A hydrophobic “seal” closes its periplasmic exit. Four conserved α-repeat helices assemble in an X-like conformation to form a Ca²⁺/H⁺ exchange pathway. In the Ca²⁺-binding site, two essential glutamate residues exhibit different conformations compared with their counterparts in NCX_Mj, whereas several amino acid substitutions occlude the Na⁺-binding sites. The structural differences between the inward-facing YfkE and the outward-facing NCX_Mj suggest that the conformational transition is triggered by the rotation of the kink angles of transmembrane helices 2 and 7 and is mediated by large conformational changes in their adjacent transmembrane helices 1 and 6. Our structural and mutational analyses not only establish structural bases for mechanisms of Ca²⁺/H⁺ exchange and its pH regulation but also shed light on the evolutionary adaptation to different energy modes in the CaCA protein family.     

Moderate heart dysfunction in mice with inducible cardiomyocyte-specific excision of the Serca2 gene

The sarco(endo)plasmic reticulum calcium ATPase 2 (SERCA2) transports Ca²⁺ from cytosol into the sarcoplasmic reticulum (SR) of cardiomyocytes, thereby maintaining the store of releasable Ca²⁺ necessary for contraction. Reduced SERCA function has been linked to heart failure, and loss of SERCA2 in the adult mammalian heart would be expected to cause immediate severe myocardial contractile dysfunction and death. We investigated heart function in adult mice with an inducible cardiomyocyte-specific excision of the Atp2a2 (Serca2) gene (SERCA2 KO). Seven weeks after induction of Serca2 gene excision, the mice displayed a substantial reduction in diastolic function with a 5-fold increase in the time constant of isovolumetric pressure decay (tau). However, already at 4 weeks following gene excision less than 5% SERCA2 protein was found in myocardial tissue. Surprisingly, heart function was only moderately impaired at this time point. Tissue Doppler imaging showed slightly reduced peak systolic tissue velocity and a less than 2-fold increase in tau was observed. The SR Ca²⁺ content was dramatically reduced in cardiomyocytes from 4-week SERCA2 KO mice, and Ca²⁺ transients were predominantly generated by enhanced Ca²⁺ flux through L-type Ca²⁺ channels and the Na⁺-Ca²⁺ exchanger. Moreover, equivalent increases in cytosolic [Ca²⁺] in control and SERCA2 KO myocytes induced greater cell shortening in SERCA2 KO, suggesting enhanced myofilament responsiveness. Our data demonstrate that SR-independent Ca²⁺ transport mechanisms temporarily can prevent major cardiac dysfunction despite a major reduction of SERCA2 in cardiomyocytes.     

Ranolazine improves diastolic dysfunction in isolated myocardium from failing human hearts--role of late sodium current and intracellular ion accumulation

The goal of this study was to test the hypothesis that the novel anti-ischemic drug ranolazine, which is known to inhibit late I_Na, could reduce intracellular [Na⁺]_i and diastolic [Ca²⁺]_i overload and improve diastolic function. Contractile dysfunction in human heart failure (HF) is associated with increased [Na⁺]_i and elevated diastolic [Ca²⁺]_i. Increased Na⁺ influx through voltage-gated Na⁺ channels (late I_Na) has been suggested to contribute to elevated [Na⁺]_i in HF. In isometrically contracting ventricular muscle strips from end-stage failing human hearts, ranolazine (10 µmol/L) did not exert negative inotropic effects on twitch force amplitude. However, ranolazine significantly reduced frequency-dependent increase in diastolic tension (i.e., diastolic dysfunction) by ~ 30% without significantly affecting sarcoplasmic reticulum (SR) Ca²⁺ loading. To investigate the mechanism of action of this beneficial effect of ranolazine on diastolic tension, Anemonia sulcata toxin II (ATX-II, 40 nmol/L) was used to increase intracellular Na⁺ loading in ventricular rabbit myocytes. ATX-II caused a significant rise in [Na⁺]_i typically seen in heart failure via increased late I_Na. In parallel, ATX-II significantly increased diastolic [Ca²⁺]_i. In the presence of ranolazine the increases in late I_Na, as well as [Na⁺]_i and diastolic [Ca²⁺]_i were significantly blunted at all stimulation rates without significantly decreasing Ca²⁺ transient amplitudes or SR Ca²⁺ content. In summary, ranolazine reduced the frequency-dependent increase in diastolic tension without having negative inotropic effects on contractility of muscles from end-stage failing human hearts. Moreover, in rabbit myocytes the increases in late I_Na, [Na⁺]_i and [Ca²⁺]_i caused by ATX-II, were significantly blunted by ranolazine. These results suggest that ranolazine may be of therapeutic benefit in conditions of diastolic dysfunction due to elevated [Na⁺]_i and diastolic [Ca²⁺]_i.     

Differences in time course of myocardial mRNA expression in non-infarcted myocardium after myocardial infarction

In non-infarcted myocardium after myocardial infarction, the change of cardiac phenotypic modulation of contractile protein, extracellular matrix and intracellular Ca²⁺ transport protein, such as sarcoplasmic reticulum Ca²⁺(SR-Ca²⁺)-ATPase, Na⁺-Ca²⁺ exchanger, have a important role during cardiac remodeling. However, the time course in this gene expression in the adjacent and remote left ventricular, or right ventricular myocardium after myocardial infarction has not been well examined. The purpose of this study was to examine the left ventricular function and regional cardiac gene expression after myocardial infarction. Myocardial infarction was produced in Wistar rats by the ligation of the left anterior descending coronary artery. After 3 weeks, 2 months and 4 months from myocardial infarction, we performed Doppler echocardiography and measured the systolic and diastolic function. Then, we analyzed the contractile protein, extracellular matrix and intracellular Ca ²⁺ transport protein mRNAs of cardiac tissues in the adjacent and the remote noninfarcted myocardium, and right ventricular myocardium by Northern blot hybridization. Fractional shortening of infarcted heart progressively decreased. Peak early diastolic filling wave (E wave) velocity increased, and the deceleration rate of the E wave velocity was more rapid in myocardial infarction areas. Atrial filling wave (A wave) velocity decreased, resulting in a marked increase in the ration of E wave to A wave velocity. Expression of myocardial α-skeletal actin, β-MHC and ANP mRNA, or collagen I and III mRNA were higher at 3 weeks after myocardial infarction. SR Ca²⁺-ATPase mRNA in the adjacent non-infarcted myocardium was decreased at 2 months, and that in remote myocardium was decreased at 4 months after infarction. Na⁺-Ca²⁺ exchanger mRNA levels were increased at 3 weeks, but was decreased at 2 months in the adjacent non-infarcted myocardium and at 4 months in the remote myocardium. These findings suggest that the compensation for myocardial infarction by myocardial gene expression in non-infarcted myocardium may occur at an early phase after myocardial infarction, and myocardial dysfunction may begin from adjacent to remote non-infarcted myocardium during progressive cardiac remodeling. Received: 9 August 1999, Returned for revision: 16 September 1999, Revision received: 5 January 2000, Accepted: 26 January 2000