Pharmacological Discrimination of Plasmalemmal and Mitochondrial Sodium–Calcium Exchanger in Cardiomyocyte-Derived H9c2 Cells

Abstract

We examined the effects of SEA0400 and CGP-37157 on the plasmalemmal Na⁺–Ca²⁺ exchanger (NCX) and mitochondrial NCX using H9c2 cardiomyocytes loaded with Ca²⁺-sensitive fluorescent probes. The plasmalemmal NCX activity, which was measured as the increase in cytoplasmic Ca²⁺ concentration after application of low Na⁺ extracellular solution, was inhibited by SEA0400 but not by CGP-37157. The mitochondrial NCX activity, which was measured in permeabilized H9c2 cells as the decrease in mitochondrial Ca²⁺ concentration after application of Ca²⁺-free extramitochondrial solution, was inhibited by CGP-37157 but not by SEA0400. These results indicate that SEA0400 and CGP-37157 act as selective inhibitors towards plasmalemmal and mitochondrial NCX, respectively, and provide pharmacological evidence that the plasmalemmal and mitochondrial NCX are distinct molecular entities.

The Na⁺–Ca²⁺ exchanger (NCX) is involved in the physiological and pathophysiological regulation of intracellular Ca²⁺ concentration in various cell types including cardiomyocytes. Although the major role of plasmalemmal NCX in cardiomyocytes is to extrude Ca²⁺ from the cell and maintain low cytoplasmic Ca²⁺ concentration,¹⁾ NCX is also considered to be responsible for the pathogenesis of ischemia–reperfusion and arrhythmia.^2,3) Concerning pharmacological inhibition of NCX, SEA0400 {2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline}, a benzyloxyphenyl compound, was established as a potent inhibitor of NCX with minimum effects on other plasmalemmal ion channels and transporters.^4–6) In voltage-clamped guinea pig ventricular myocytes, 1 µM SEA0400, which inhibited the plasmalemmal NCX current by more than 80%, had no effect on the Na⁺ current, L-type Ca²⁺ current, delayed rectifier K⁺ current, inwardly rectifying K⁺ current.⁵⁾ The involvement of plasmalemmal NCX in ouabain-induced arrhythmogenesis,⁷⁾ ischemia–reperfusion injury^8,9) and automaticity of the pulmonary-vein myocardium¹⁰⁾ was demonstrated with SEA0400.

On the other hand, NCX was revealed to exist not only on the cell membrane but also in the mitochondria of various organs including the heart.¹¹⁾ The mitochondrial NCX is considered to be the main system extruding Ca²⁺ ions from the mitochondria; it extrudes Ca²⁺ ions from the mitochondrial matrix in exchange for cytoplasmic Na⁺, which in turn enters the matrix. Mitochondrial NCX has been postulated to be present on the mitochondrial inner membrane^12,13) and to have functional properties different from those of plasmalemmal NCX, such as transport stoichiometry and ion selectivity,^14–18) but details still remain to be clarified. CGP-37157, a benzothiazepine compound, was reported to inhibit the mitochondrial NCX without affecting the L-type Ca²⁺ channel, the Na⁺–K⁺-ATPase of the cardiac sarcolemma, or the Ca²⁺-ATPase of the cardiac sarcoplasmic reticulum at the concentration of 10 µM.¹⁹⁾ Based on these findings CGP-37157 rapidly became a privileged tool to investigate mitochondrial NCX.^20,21) However, recent studies showed that CGP-37157 may also affect plasmalemmal NCX.^17,22) Concerning SEA0400, whether it affects mitochondrial NCX or not has not yet been examined.

In the present study, we examined the effects of SEA0400 and CGP-37157 on the plasmalemmal and mitochondrial NCX using the rat embryonic heart derived H9c2 cardiomyocytes. We intended to examine the selectivity of these compounds towards the plasmalemmal and mitochondrial NCX, and clarify whether the plasmalemmal and mitochondrial NCX can be pharmacologically discriminated.

MATERIALS AND METHODS

Measurement of Plasmalemmal NCX Activity

For the measurement of plasmalemmal NCX activity, H9c2 cardiomyocytes were preincubated in normal Tyrode solution (143 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 0.33 mM NaH₂PO₄, 10 mM glucose, and 5 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES): pH 7.4 with NaOH) and were loaded with the cytoplasmic Ca²⁺ fluorescence probe indo-1 (5 µM indo-1/AM for 30 min at 37°C). To induce Ca²⁺ influx through the plasmalemmal NCX, the extracellular solution was changed to a low Na⁺ solution (143 mM choline Cl, 3 µM cyclopiazonic acid, 1 µM ouabain, 1 µM atropine, 5.4 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 5.5 mM glucose, 5 mM HEPES, and 0.33 mM NaH₂PO₄: pH 7.4). Ouabain (1 µM) and 3 µM cyclopiazonic acid were present throughout the Ca²⁺ measurements, and 10 µM monensin was present in the normal Tyrode solution before application of low Na⁺ solution.

Measurement of Mitochondrial NCX Activity

For the measurement of mitochondrial NCX activity, H9c2 cardiomyocytes were preincubated with the mitochondrial Ca²⁺ fluorescence probe Rhod 2 (2 µM Rhod 2/AM for 30 min at room temperature). The plasmalemmal membrane was permeabilized by perfusion of digitonin (20 µg/mL) in a Ca²⁺-free extramitochondrial solution containing (in mM) 50 KCl, 80 potassium aspartate, 4 sodium pyruvate, 20 HEPES, 3 MgCl₂, 3 Na₂ATP, 5.8 glucose, and 3 ethylene glycol bis (2-aminoethyl ether)-N,N,N′,N′-tetra acetic acid (EGTA) (pH 7.3 with KOH). On measurement of mitochondrial NCX activity, the Ca²⁺ concentration of the extramitochondrial solution was changed from 300 nM to 0 nM. The basal solution was either the same as that shown above, which contained 10 mM Na⁺, or supplemented with NaCl up to 50 mM Na⁺. The free Ca²⁺ concentration in the extramitochondrial solution was changed according to the experimental protocol. The free Ca²⁺ concentration was adjusted using the software Webmax extended.²³⁾

Measurement of Cytoplasmic and Mitochondrial Ca²⁺

For the measurement of cytoplasmic Ca²⁺ concentration, the cells loaded with Indo-1 were excited at 350 nm and the emission bands 395–415 nm and 470–490 nm were detected with an image separation system (W-VIEW system, Hamamatsu Photonics, Hamamatsu, Japan) and a high-speed cooled CCD camera (HISCA, Hamamatsu Photonics) at a time resolution of 500 ms. The ratio of emission intensity between short wavelength and long wavelength fluorescence was calculated (Aquacosmos software, Hamamatsu Photonics). In situ calibration of indo-1 fluorescence ratio values to intracellular Ca²⁺ concentration was performed as previously described.^24,25) For the measurement of mitochondrial Ca²⁺ concentration, permeabilized cells loaded with Rhod 2 were excited 543 nm and the emission at 580–600 nm were detected at a time resolution of 10 s.

Drugs and Chemicals

SEA0400, kindly provided by Taisho Pharmaceutical Company, Ltd., and CGP-37157 (Tocris, Bristol, U.K.) were dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO in the extracellular solution did not affect the parameters observed.

RESULTS

Measurement of Plasmalemmal NCX Activity

To clarify the effects of SEA0400 and CGP-37157 on plasmalemmal NCX, NCX activities were measured as the increase in cytoplasmic Ca²⁺ concentration in intact H9c2 cells (Fig. 1). Treatment of intact H9c2 cells with a low Na⁺ extracellular solution elicited an increase in cytoplasmic Ca²⁺ concentration which reflects Ca²⁺ influx through the plasmalemmal NCX. The cytoplasmic Ca²⁺ concentration before and after application of low Na⁺ extracellular solution was 127.7±14.2 and 945.3±26.9 nM, respectively (n=35). SEA0400 (1 µM) inhibited the increase in Ca²⁺ concentration induced by low Na⁺ solution (Figs. 1A, C), but CGP-37157 (10 µM) had no effect (Figs. 1B, C). The peak cytoplasmic Ca²⁺ concentration after application of low Na⁺ extracellular solution in the presence of SEA0400 and CGP-37157 was 233.1±27.7 (n=20) and 943.0±32.2 nM (n=15), respectively.

Fig. 1. Analysis of Plasmalemmal NCX in Indo-1-Loaded H9c2 Cells

A, B: Typical recording of the changes in cytoplasmic Ca²⁺ on application of low Na⁺ extracellular solution in intact H9c2 cells in the absence (Aa, Ba) and presence of SEA0400 (1 µM; Ab) or CGP-37157 (10 µM; Bb). C: Summarized results for the effect of inhibitors. The low Na⁺-induced increase in cytoplasmic Ca²⁺ in the presence of SEA0400 or CGP-37157 was expressed as a percentage of that in the absence. Columns and bars represent the mean±S.E.M. from 15 to 20 experiments. Asterisks indicate significant difference from corresponding values in the absence of inhibitors as evaluated by the paired t-test.

Measurement of Mitochondrial NCX Activity

To clarify the effects of SEA0400 and CGP-37157 on mitochondrial NCX, the declining phase of mitochondrial Ca²⁺ concentration was analyzed in permeabilized H9c2 cells. The decline of mitochondrial Ca²⁺ concentration on changing the extramitochondrial Ca²⁺ concentration from 300 nM to 0 nM was dependent on extra-mitochondrial Na⁺ indicating that the decline reflects mitochondrial NCX activity (Fig. 2). CGP-37157 (10 µM) inhibited the declination of mitochondrial Ca²⁺. In contrast, SEA0400 (1 µM) had no effect (Fig. 2).

Fig. 2. Analysis of Mitochondrial NCX in Rhod-2-Loaded Permeabilized H9c2 Cells

A: Typical recordings of the changes in Rhod-2 fluorescence after removal of extramitochondrial Ca²⁺ (300 to 0 nM) under 10 mM Na⁺ in the absence of inhibitors (dashed grey line) and under 50 mM Na⁺ in the absence of inhibitors (solid grey line), in the presence of 10 µM CGP-37157 (solid black line) and in the presence of 1 µM SEA0400 (dashed black line). B: Summarized results for mitochondrial NCX activity. The decrease in fluorescence at 10 min after changing to Ca²⁺-free extramitochondrial solution was expressed as a percentage of the fluorescence under 300 nM Ca²⁺. Columns and bars represent the mean±S.E.M. from 10 to 20 experiments. Asterisks indicate significant difference from corresponding values in control as evaluated by the Dunnett’s test for multiple comparisons.

DISCUSSION

The plasmalemmal and mitochondrial NCX function either in the Ca²⁺ extrusion or Ca²⁺ influx mode, depending on the Na⁺ gradient.^1,26) This enabled us to construct fluorescence-based analyzing systems of plasmalemmal and mitochondrial NCX activities and evaluate the effects of pharmacological agents. Plasmalemmal NCX activity could be elicited by exposure to low Na⁺ extracellular solution of H9c2 cells loaded with the cytoplasmic Ca²⁺ indicator indo-1, and the resulting increase in fluorescence reflected plasmalemmal NCX activity (Fig. 1). To detect mitochondrial NCX activity, we used permeabilized H9c2 cells loaded with the mitochondria selective Ca²⁺ indicator Rhod 2. Exposure of these cells to Ca²⁺-free extra-mitochondrial solution resulted in a decrease in mitochondrial Ca²⁺ concentration; the decrease was dependent on Na⁺ indicating that the decrease indeed reflects mitochondrial NCX activity (Fig. 2). Thus, the present experimental system was shown to be suitable to study mitochondrial Ca²⁺ regulation.

Information concerning the pharmacological properties of plasmalemmal and mitochondrial NCX is limited. CGP-37157 was reported to inhibit the mitochondrial NCX without affecting the major ion channels and transporters. It did not affect the plasmalemmal NCX in isolated cardiac sarcolemmal vesicles.¹⁹⁾ However, CGP-37157 was reported to block the plasmalemmal NCX (NCX 1.1) expressed in Xenopus oocytes.^17,22) In the present study with intact cardiomyocyte-derived H9c2 cells, CGP-37157 (10 µM), which completely inhibited the mitochondrial NCX (Fig. 2), had no effects on plasmalemmal NCX (Fig. 1). These results indicated that CGP-37157 is a selective inhibitor of mitochondrial NCX in intact cardiac myocytes, and thus can be used as a tool for the studies of mitochondrial NCX activity. On the other hand, SEA0400 (1 µM), which markedly inhibited the plasmalemmal NCX (Fig. 1), had no effect on mitochondrial NCX (Fig. 2). This indicates that SEA0400 is a selective inhibitor of plasmalemmal NCX in intact cardiac myocytes, and thus can be used as a tool to obtain information on plasmalemmal NCX function.

The present results provide pharmacological evidence that the plasmalemmal and mitochondrial NCX are distinct molecular entities. The postulated ion transporting region of the plasmalemmal NCX and NCLX is formed by two transmembrane domains called the α1 and α2 repeats.^15,27) NCX and NCLX shares about 62% homology within the α1 and α2 repeats, but not outside these regions.²⁷⁾ The discrimination by SEA0400 and CGP37157 may suggest that their binding sites on these transporters largely involve regions outside of the α1 and α2 repeats. This appears to be consistent with an earlier binding site model for NCX inhibitors of benzyloxyphenyl structure such as SEA0400.¹⁵⁾ In any case, mitochondrial NCX and plasmalemmal NCX could be pharmacologically discriminated, which provides a basis for the development of novel therapeutic agents.

In conclusion, we demonstrated that SEA0400 and CGP-37157 act as selective inhibitors towards plasmalemmal and mitochondrial NCX, respectively, in intact cardiomyocyte-derived H9c2 cells, which provided pharmacological evidence that the plasmalemmal and mitochondrial NCX are distinct molecular entities.

Conflict of Interest

The authors declare no conflict of interest.

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