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.     

Myocardial recovery during post-ischemic reperfusion: Optimal concentrations of Na+ and Ca2+ in the reperfusate and protective effects of amiloride added to cardioplegic solution

The effects of Na⁺ and Ca²⁺ concentrations in the reperfusate on post-ischemic myocardial recovery were examined. Also, the myocardial protective effects of amiloride, an inhibitor of the Na⁺/Ca²⁺ and Na⁺/H⁺ exchange systems, added to cardioplegic solutions were assessed, using an isolated working rat heart perfusion system. Global myocardial ischemia was induced by 30-min normothermic cardioplegic arrest, using St. Thomas’ solution. The concentration of Na⁺ in the reperfusate varied, stepwise, from 75 to 145 mM/l, and that of Ca²⁺, from 0.1 to 2.5 mM/l. In this study post-ischemic functional recovery was best at 110 mM/l Na⁺ and 1.2–1.8 mM/l Ca²⁺ in the reperfusate. A significantly greater postischemic functional recovery and a lower creatine kinase release were observed when amiloride was added to the cardioplegic solution. Ca²⁺ overload via Na⁺/Ca²⁺ and Na⁺/H⁺ exchange systems would, thus, appear to be due, at least in part, to post-ischemic reperfusion injury.     

Slow Ca2+ and Na+ responses induced by isoproterenol and methylxanthines in isolated perfused guinea pig hearts exposed to elevated K+.

The induction of Ca²⁺ channels in cardiac muscles by catecholamines (CA) was studied in a physiological preparation, the isolated perfused guinea pig heart. The heart was rendered inexcitable by elevating external [K⁺] to 27 mm, which depolarizes the cells to about ?40 mV. Isoproterenol (10^?7m) infusion induced excitability within 1 min, i.e., slowlyrising electrical potentials with concomitant contraction in response to stimulation. Their shape and duration was similar to that of the plateau component of the normal action potential, but their maximum rate of rise was only 6 to 12 V/s. The slow responses persisted for over 2 h. Methylxanthines (MX) (caffeine and theophylline, 3 mm) mimicked the catecholamines, except that they were not blocked by propranolol. The slow responses were blocked by: (a) Ca²⁺-free solution, (b) 1 mm Mn²⁺, and (c) verapamil (0.05 μg/ml), suggesting that an inward Ca²⁺ current plays a key role in the response. In hearts perfused with normal Ringer, these conditions greatly depressed the contractions at a time when there was little or no effect on the shape and duration of the action potentials. Sr²⁺ and Ba²⁺ could replace Ca²⁺ in maintaining the response. Na⁺ is also required for the responses, because the magnitude, duration, and rate of rise of the response diminished in lowered [Na⁺]_o; the responses disappeared in 0 to 30 mm Na⁺. Ouabain (10^?5m) suppressed the CA- or MX-induced response. Continuous perfusion with dc-AMP or ATP (10^?4m) slowly induced the slow responses even in the presence of propranolol. The Ca²⁺ ionophore, X-537A, also induced the slow responses, but its effect was blocked by propranolol, suggesting that its action was mediated by endogenous CA release. Metabolic poisons, hypoxia, or ischemia blocked the slow responses. The following conclusions were drawn from the results: (a) the induced slow response is dependent on both Ca²⁺ and Na⁺ ions; (b) the underlying mechanism requires both Na⁺ and Ca²⁺ for its operation; (c) c-AMP is implicated in the control of the activities of this mechanism; (d) the production and/or maintenance of the mechanism is energy dependent; and (e) inactivation of the slow response mechanism results in electromechanical uncoupling.     

An effect of the cardiotoxic protein volvatoxin a on the function and structure of heart muscle cells

Volvatoxin-A, the heat labile cardiotoxin present in the mushroom Volvariella volvacea, causes a competitive, dose and time-dependent inhibition of the Ca²⁺-accumulating activity of a sarcoplasmic-reticulum rich microsomal fraction isolated from guinea pig ventricular muscle. The inhibition is accompanied by an activation of the Ca²⁺-dependent ATPase enzyme. Concentrations of toxin which inhibit the Ca²⁺-transporting activity of the microsomes render them leaky to Ca²⁺, but do not effect the rate of incorporation of P³². Ten μg/ml toxin failed to alter the activity of the Na⁺ + K⁺-activated, ouabain sensitive ATPase enzyme. It damaged the fine morphology of the mitochondria, inhibited the ability of isolated mitochondria to accumulate Ca²⁺, and had little effect on the ability of isolated plasma membranes to bind Ca²⁺. These findings may explain why volvatoxin A increases the diastolic resting tension in heart muscle.     

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.     

Mechanism of Ca2+ resistance in adult heart cells isolated with trypsin plus Ca2+

Ca²⁺-resistant heart cells prepared with trypsin and Ca²⁺ leak Na⁺ and K⁺ more slowly than Ca²⁺-susceptible cells prepared without trypsin and Ca²⁺. The two preparations show similar leak rates for amino acids and nucleotides. Cells prepared with Ca²⁺ alone show low ion leak rates, but the yield of rod-shaped cells is less than half that when trypsin is present. Cells prepared with trypsin alone show high ion leak rates. The Na⁺-K⁺ ATPase activity of Ca²⁺-susceptible cells appears to be approximately three-fold greater than that of Ca²⁺-resistant cells. Imposing a Na⁺-K⁺ leak by the addition of gramicidin D causes no stimulation of Na⁺-K⁺ ATPase in Ca²⁺-susceptible cells, but stimulates the activity of Ca²⁺-resistant cells up to that of the Ca²⁺-susceptible cells. Ca²⁺-resistant cells appear to contain more K⁺ and less Na⁺ than Ca²⁺-susceptible cells. Treatment of Ca²⁺-resistant cells with ouabain (1 mm) for 5 min changes the $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ balance to approximately that of the Ca²⁺-susceptible cells, and induces a similar degree of Ca²⁺ susceptibility. We therefore conclude that treatment with trypsin plus Ca²⁺ confers Ca²⁺ resistance by keeping the permeability of the sarcolemma to Na⁺ and K⁺ sufficiently low to allow the Na⁺-K⁺ ATPase and $\text{Na}{}^{\mathrm{+}}\text{Ca}{}^{\mathrm{+}}$ exchanger to maintain normal gradients of Na⁺, K⁺ and Ca²⁺. The agent responsible for maintaining low ion permeability appears to be Ca²⁺ itself, while trypsin increases the yield and purity of the Ca²⁺-resistant cells.     

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.     

Calcium tolerance of isolated rat heart cells

Freshly isolated adult rat heart cells, which initially show the elongated, rod-shaped morphology typical of heart cells in situ, are almost quantitatively converted to rounded contracture forms by exposure to 1 m Ca²⁺. These Ca²⁺-sensitive cells became Ca²⁺-tolerant following a short period of metabolic activity in a low-Ca²⁺ medium, in that they retain their rod-shaped configuration when challenged with Ca²⁺ after this preincubation step. Tolerance to Ca²⁺ develops in parallel with the establishment of low Na⁺/K⁺ ratios in these cells and both processes are sensitive to ouabain. The initial net uptake of Ca²⁺ is greater in Ca²⁺-sensitive than in Ca²⁺-tolerant cells. These results suggest that contracture in the Ca²⁺-sensitive cells is a consequence of the rapid entry of excessive amounts of Ca²⁺ in exchange for internal Na⁺.     

A mathematical model of vasoreactivity in rat mesenteric arterioles: I. Myoendothelial communication

To study the effect of myoendothelial communication on vascular reactivity, we integrated detailed mathematical models of Ca²⁺ dynamics and membrane electrophysiology in arteriolar smooth muscle (SMC) and endothelial (EC) cells. Cells are coupled through the exchange of Ca²⁺, Cl^?, K⁺, and Na⁺ ions, inositol 1,4,5‐triphosphate (IP₃), and the paracrine diffusion of nitric oxide (NO). EC stimulation reduces intracellular Ca²⁺ ([Ca²⁺ in the SMC by transmitting a hyperpolarizing current carried primarily by K⁺. The NO‐independent endothelium‐derived hyperpolarization was abolished in a synergistic‐like manner by inhibition of EC SK_Ca and IK_Ca channels. During NE stimulation, IP₃diffusing from the SMC induces EC Ca²⁺ release, which, in turn, moderates SMC depolarization and [Ca²⁺]_i elevation. On the contrary, SMC [Ca²⁺]_i was not affected by EC‐derived IP₃. Myoendothelial Ca²⁺ fluxes had no effect in either cell. The EC exerts a stabilizing effect on calcium‐induced calcium release‐dependent SMC Ca²⁺ oscillations by increasing the norepinephrine concentration window for oscillations. We conclude that a model based on independent data for subcellular components can capture major features of the integrated vessel behavior. This study provides a tissue‐specific approach for analyzing complex signaling mechanisms in the vasculature.     

Involvement of an Na+Ca2+ exchange system in genesis of ouabain-induced arrhythmias of cultured myocardial cells

The changes of sarcomere length of cultured myocardial cells during genesis of ouabain-induced fibrillatory beating were measured, and it was found that the sarcomere continued to be in the shortened state throughout the fibrillatory beating. Addition of ouabain caused gradual increases of both the Na content of myocardial cells and the rate of Ca uptake by the cells, and fibrillatory beating appeared to develop when the Na content and the rate of Ca uptake exceeded the normal levels by about 1.5 times and 2.0 times, respectively.Myocardial cells loaded with various concentrations of Na⁺ could be prepared by incubating the cells in Ca²⁺-free medium containing various concentrations of Na⁺, and it was found that a slight increase in the intracellular Na content from the physiological concentration caused an appreciable increase in Ca uptake by the cells. This Ca uptake was achieved by a carrier-mediated Na⁺Ca²⁺ exchange system and the stoichiometry of Na⁺: Ca²⁺ exchange was greater than 2 Na⁺:1 Ca²⁺. From these observations the possible involvement of an Na⁺Ca²⁺ exchange system in genesis of ouabain-induced arrhythmias of cultured myocardial cells is discussed.     

The influence of chronic potassium deficiency on energy production, calcium metabolism and phospholipid composition of isolated heart mitochondria.

Rabbits were maintained for an average of 28 days either on a normal (controls) or a K⁺-deficient diet. In the low potassium group, myocardial contractility was significantly decreased revealing the cardiomyopathy. The K⁺ content of the serum as well as of the myocardial tissue was significantly decreased while the Na⁺ content in serum was higher than in the control animals. Ca²⁺ and Mg²⁺-concentrations remained unchanged. In mitochondria isolated from K⁺-depleted hearts, the state-4-respiration and the rate of Ca²⁺-uptake in the presence of ATP were increased (P < 0.001 or 0.01). Passive Ca²⁺-binding to isolated cardiac mitochondria was significantly higher as compared with controls. These data suggest that mitochondria are able to store and take up more Ca²⁺ in K⁺-deficiency. The mitochondrial phospholipid pattern shows a depression of the phosphatidylcholine content so that the phosphatidylethanolamine portion is relatively increased. A positive correlation was found between the phosphatidylethanolamine content of cardiac mitochondria and the amount of passively bound Ca²⁺. Comparing the fatty acid composition of mitochondrial phospholipids, the linoleic acid content of the cardiolipin fraction was increased in chronic K⁺-deficiency when compared with control animals. The absolute amount of linoleic acid was positively correlated to the mitochondrial state-4-respirations as well as to the ATP-dependent calcium uptake both from K⁺-depleted and normal animals.     

Intracellular [Na<Superscript>+</Superscript>] modulates synergy between Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchanger and L-type Ca<Superscript>2+</Superscript> current in cardiac excitation–contraction coupling during action potentials

Excitation–contraction coupling (ECC) in cardiac myocytes involves triggering of Ca²⁺ release from the sarcoplasmic reticulum (SR) by L-type Ca channels, whose activity is strongly influenced by action potential (AP) profile. The contribution of Ca²⁺ entry via the Na⁺/Ca²⁺ exchanger (NCX) to trigger SR Ca²⁺ release during ECC in response to an AP remains uncertain. To isolate the contribution of NCX to SR Ca²⁺ release, independent of effects on SR Ca²⁺ load, Ca²⁺ release was determined by recording Ca²⁺ spikes using confocal microscopy on patch-clamped rat ventricular myocytes with [Ca²⁺]_i fixed at 150 nmol/L. In response to AP clamps, normalized Ca²⁺ spike amplitudes (ΔF/F ₀) increased sigmoidally and doubled as [Na⁺]_i was elevated from 0 to 20 mmol/L with an EC₅₀ of ~10 mmol/L. This [Na⁺]_i-dependence was independent of I _Na as well as SR Ca²⁺ load, which was unchanged under our experimental conditions. However, NCX inhibition using either KB-R7943 or XIP reduced ΔF/F ₀ amplitude in myocytes with 20 mmol/L [Na⁺]_i, but not with 5 mmol/L [Na⁺]_i. SR Ca²⁺ release was complete before the membrane repolarized to −15 mV, indicating Ca²⁺ entry into the dyad (not reduced extrusion) underlies [Na⁺]_i-dependent enhancement of ECC. Because I _Ca,L inhibition with 50 mmol/L Cd²⁺ abolished Ca²⁺ spikes, our results demonstrate that during cardiac APs, NCX enhances SR Ca²⁺ release by synergistically increasing the efficiency of I _Ca,L-mediated ECC.     

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.     

Role and mechanism of subcellular Ca2+ distribution in the action of two inotropic agents with different toxicity

Pro-arrhythmic risk strongly limits the therapeutic value of current inotropic interventions. Istaroxime (previously PST2744) is a novel inotropic agent, significantly less pro-arrhythmic than digoxin that, in addition to block Na⁺/K⁺ pump, stimulates sarcoplasmic reticulum (SR) Ca²⁺ ATPase (SERCA2). Here we compare istaroxime and digoxin effects to further address the role of SR modulation in reducing the toxicity associated with Na⁺/K⁺ pump blockade. In murine ventricular myocytes both compounds increased cell twitch (inotropy) in a concentration-dependent fashion. At high concentrations digoxin, but not istaroxime, induced unstimulated contractions, a sign of pro-arrhythmic toxicity. To evaluate the mechanism of this difference, we compared the two drugs at concentrations exerting equal inotropy but different toxicity. At these concentrations: (1) the two drugs equally inhibited the Na⁺/K⁺ pump; (2) digoxin induced larger increases in resting Ca²⁺ and in diastolic Ca²⁺ during pacing; (3) neither drug affected the relationship between RyR-mediated SR Ca²⁺ leak and Ca²⁺ content; (4) istaroxime, but not digoxin, enhanced SR Ca²⁺ reuptake rate. In conclusion, digoxin toxicity was associated to larger accumulation of cytosolic Ca²⁺, which did not result from RyR facilitation, but which might ultimately induce it to promote unstimulated Ca²⁺ release. The lower toxicity of Na⁺/K⁺ pump blockade by istaroxime may thus reflect improved Ca²⁺ confinement within the SR, likely to result from concomitant SERCA2 stimulation.     

Excitation-contraction coupling and mitochondrial energetics

Cardiac excitation-contraction (EC) coupling consumes vast amounts of cellular energy, most of which is produced in mitochondria by oxidative phosphorylation. In order to adapt the constantly varying workload of the heart to energy supply, tight coupling mechanisms are essential to maintain cellular pools of ATP, phosphocreatine and NADH. To our current knowledge, the most important regulators of oxidative phosphorylation are ADP, P_i, and Ca²⁺. However, the kinetics of mitochondrial Ca²⁺-uptake during EC coupling are currently a matter of intense debate. Recent experimental findings suggest the existence of a mitochondrial Ca²⁺ microdomain in cardiac myocytes, justified by the close proximity of mitochondria to the sites of cellular Ca²⁺ release, i. e., the ryanodine receptors of the sarcoplasmic reticulum. Such a Ca²⁺ microdomain could explain seemingly controversial results on mitochondrial Ca²⁺ uptake kinetics in isolated mitochondria versus whole cardiac myocytes. Another important consideration is that rapid mitochondrial Ca²⁺ uptake facilitated by microdomains may shape cytosolic Ca²⁺ signals in cardiac myocytes and have an impact on energy supply and demand matching. Defects in EC coupling in chronic heart failure may adversely affect mitochondrial Ca²⁺ uptake and energetics, initiating a vicious cycle of contractile dysfunction and energy depletion. Future therapeutic approaches in the treatment of heart failure could be aimed at interrupting this vicious cycle.     

Na+/K+ pump inhibition induces cell shrinkage in cultured chick cardiac myocytes

Summary Myocardial cell swelling occurs in ischemia and in reperfusion injury before the onset of irreversible injury. Swelling has been attributed to failure of the Na⁺/K⁺ pump and the accumulation of intracellular Na⁺. To evaluate the role of the pump-leak model of cell volume maintenance, short term changes in cell volume in response to Na⁺/K⁺ pump inhibition were studied in aggregates of cultured embryonic chick cardiac myocytes using optical and biochemical methods. Exposure to 100 M ouabain over 20 min induced cell shrinkage of approximately 10%. Cell water was also decreased by Na⁺/K⁺ pump inhibition; incubation for 1 hr either in the presence of 100 M ouaain or in K⁺-free solution reduced cell water by 18.4% and 28.4% respectively. When exposed to ouabain in the absence of extracellular Ca²⁺, the aggregates swelled by approximately 15%, indicating that extracellular Ca²⁺ was required for the ouabain-induced shrinkage to occur. Ouabain still caused shrinkage, however, in the presence of the Ca²⁺ channel blockers verapamil (10 M) and nifedipine (10 M), suggesting that Na⁺/Ca²⁺ exchange, rather than Ca²⁺ channels, is the route for Ca²⁺ influx during Na⁺/K⁺ pump inhibition. Efflux of amino acids (taurine, aspartate, glutamate, glycine and alanine) from confluent monolayers of chick heart cells exposed to ouabain for 20 min was nearly double that observed in control solution. These results suggest that, during Na⁺/K⁺ pump inhibition, chick heart cells can limit accumulation of intracellular sodium by means of Na⁺/Ca²⁺ exchange, and that a rise in intracellular [Ca²⁺], also mediated by Na⁺/Ca²⁺ exchange, promotes the loss of amino acids and ions to cause cell shrinkage. Therefore, swelling during ischemic injury may not result from Na⁺/K⁺ pump failure alone, but may reflect the exhaustion of alternative volume regulatory transport mechanisms.     

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.     

Ionic mechanism of morphological changes of cultured myocardial cells on successive incubation in media without and with Ca2+

(1) Incubation of cultured mouse myocardial cells in medium containing Ca²⁺ after brief pre-incubation in Ca²⁺-free medium caused morphological changes, such as full contraction of myofibrils and balloon formation of the cell membrane (“Ca²⁺ paradox phenomenon”). (2) When myocardial cells were pre-incubated in Ca²⁺-free medium, and then incubated in medium containing various concentrations of Ca²⁺, both the percentage of cells showing morphological changes and the rate of Ca²⁺ uptake increased with increase in the Ca²⁺ concentration. (3) Pre-incubation in medium containing 10^?7m Ca²⁺ or less was necessary for induction of both morphological changes and excess uptake of Ca²⁺ during incubation in medium containing Ca²⁺. (4) When myocardial cells were pre-incubated in Ca²⁺-free medium containing various concentrations of Na⁺, and then incubated in medium containing Ca²⁺, both the percentage of cells showing morphological changes and the rate of Ca²⁺ uptake increased with increase in the Na⁺ concentration in the pre-incubation medium. (5) Various treatments that inhibited excess uptake of Ca²⁺ by the cells inhibited the genesis of morphological changes. Thus the observed morphological changes of myocardial cells were due to excess uptake of Ca²⁺ by the myocardial cells. (6) When myocardial cells were incubated in medium containing various concentrations of Ca²⁺ and a fixed concentration of Na⁺, the intracellular concentration of Na⁺ increased with decrease in the Ca²⁺ concentration of the medium. Cells that had been preloaded with a higher concentration of Na⁺ took up the Ca²⁺ faster. (7) Conditions that inhibited Na⁺ uptake by the cells during pre-incubation without Ca²⁺ inhibited Ca²⁺ uptake by the cells during subsequent incubation with Ca²⁺. These results suggest that Ca²⁺ uptake by myocardial cells during incubation in medium with Ca²⁺ depends upon the intracellular Na⁺ concentration.     

Alloxan effects on mitochondria: study of oxygen consumption,fluxes of Mg2+, Ca2+, K+ and adenine nucleotides,membrane potential and volume change in vitro

Summary Isolated mouse liver mitochondria incubated with alloxan showed stimulated resting (state 4) respiration with succinate, and inhibited resting respiration with pyridine-linked substrates, whereas active (state 3) respiration was decreased with both kinds of substrates. The effects were dependent on the concentration of alloxan, on the energy state, and on transport of inorganic phosphate and uptake of Ca²⁺. Using succinate as substrate, the effects of alloxan on endogenous Mg²⁺, K⁺ and adenine nucleotides, uptake of K⁺, accumulated Ca²⁺, membrane potential and volume were studied in liver mitochondria, and in addition efflux of endogenous K⁺ and accumulated Ca²⁺ were investigated in mouse islet mitochondria. High concentrations of alloxan ( 3 mmol/l) induced efflux of endogenous Mg²⁺, K⁺ and adenine nucleotides, efflux of accumulated Ca²⁺, inhibition of uptake of K⁺, loss of membrane potential, and swelling. Low concentrations of alloxan (< 3 mmol/l) had similar effects only in the presence of added Ca²⁺ and inorganic phosphate. The influence of potentially protective agents was studied mainly with regard to alloxan induced swelling. Complete or partial protection was offered by antimycin A, malonate, La³⁺, Ni²⁺, ruthenium red, mersalyl and N-ethylmaleimide, suggesting requirement for energized transport of Ca²⁺ and uptake of inorganic phosphate. The start of the respiratory changes, decrease of membrane potential and loss of Mg²⁺ preceded the release of accumulated Ca²⁺, which occurred in parallel with efflux of K⁺ and swelling. The loss of Ca²⁺ in association with swelling agrees with data previously obtained using qualitative and quantitative electron microscopy and X-ray microanalysis of islet cells from alloxan-treated mice. Since preceding studies in vivo have shown that alloxan passes across plasma membranes and is taken up in mitochondria of islet cells and hepatocytes, the combined data support the view that alloxan diabetes may be due to mitochondrial damage.     

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.