Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
Notes
Fluorescence Analysis of the Mitochondrial Effect of a Plasmalemmal Na+/Ca2+ Exchanger Inhibitor, SEA0400, in Permeabilized H9c2 Cardiomyocytes
Iyuki Namekata Shogo HamaguchiNaoko Iida-TanakaTaichi KusakabeKeisuke KatoToru KawanishiHikaru Tanaka
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2017 年 40 巻 9 号 p. 1551-1555

詳細
Abstract

We investigated the effect on mitochondrial Ca2+ of SEA0400, an inhibitor of the Na+/Ca2+ exchanger (NCX) which reduces mitochondrial Ca2+ overload during myocardial ischemia, in digitonin-permeabilized H9c2 cells expressing the mitochondrial-targeted Ca2+ indicator, yellow cameleon 3.1. The elevation of mitochondrial Ca2+ concentration caused by an increase in extramitochondrial Ca2+ concentration was inhibited by carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) or ruthenium red, but enhanced by CGP-37157, a mitochondrial NCX inhibitor. SEA0400 had no effect on mitochondrial Ca2+ under normal and ischemic conditions. Thus, the mitochondria-protective effects of SEA0400 could be explained by inhibition of plasmalemmal NCX but not mitochondrial NCX.

The Na+–Ca2+ exchanger (NCX) is involved in the regulation of Ca2+ concentration in the myocardium. The plasmalemmal NCX basically functions in the forward (transplasmalemmal Ca2+ efflux) mode, but under pathological conditions such as ischemia, it is postulated to function in the reverse (Ca2+ influx) mode and contribute to cellular damage.1) SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline) is a selective inhibitor of NCX with minimum effects on other plasmalemmal transporters and ion channels,25) and thus have been used as a potent pharmacological tool in NCX research. We have previously examined the effects of SEA0400 on a coronary-perfused myocardial ischemia–reperfusion model and found that SEA0400 enhances the recovery of contractile force and action potential duration after reperfusion.6) SEA0400 attenuated cytoplasmic and mitochondrial Ca2+ overload and preserved tissue ATP content and mitochondrial membrane potential during ischemia.7) These results suggested that inhibition of plasmalemmal NCX protects the mitochondria through attenuation of Ca2+ overload during myocardial ischemia. However, the following issues relevant to this hypothesis have not yet been clarified. Firstly, can the attenuation of the rise in mitochondrial Ca2+ be attributed to the attenuation of the rise in cytoplasmic Ca2+ by SEA0400? Secondly, can the preservation of the mitochondrial membrane potential be attributed to the attenuation of the rise in mitochondrial Ca2+ by SEA0400? Thirdly, does SEA0400 have any direct effect on mitochondrial Ca2+ transport pathways including the mitochondrial NCX, which has been considered to have pivotal roles in intracellular Ca2+ handling?8)

In the present study, to obtain information to answer these questions, we constructed a fluorescence-based system for the analysis of mitochondrial Ca2+ concentration. We used the rat embryonic heart derived H9c2 cells expressing the mitochondria-targeted yellow cameleon 3.1, a protein Ca2+ indicator based on fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP).9) After permeabilizing the cells with digitonin, the extramitochondrial environment could be manipulated by changing the cell perfusate and the resulting changes in mitochondrial Ca2+ concentration monitored with yellow cameleon fluorescence ratio. We studied the regulation of mitochondrial Ca2+ under normal and ischemic condition, and examined the effect of pharmacological agents including SEA0400.

MATERIALS AND METHODS

H9c2 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 100 unit/mL penicillin, 0.1 mg/mL streptomycin and 10% fetal bovine serum. H9c2 cells have been used for various studies on myocardial ischemia including mitochondrial Ca2+ regulation.10,11) Concerning the mitochondrial NCX (NCXL12,13)), we confirmed the expression of the mRNA in H9c2 cells by real-time PCR according to the referential sequence (NM 001017488). Full-length cDNA for mitochondria-targeted yellow cameleon 3.19) was introduced into H9c2 cells by lipofection (Lipofectamine 2000; Thermo Fisher Scientific, Waltham, MA, U.S.A.). For the measurement of mitochondrial function, the plasmalemma was permeabilized by perfusion with digitonin (20 µg/mL) in a Ca2+-free solution containing (in mM) 50 KCl, 80 potassium aspartate, 4 sodium pyruvate, 3 Na2ATP, 20 N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), 3 MgCl2/6H2O, 5.8 glucose, and 3 ethylene glycol bis (2-aminoethyl ether)-N,N,N′,N′-tetra acetic acid (EGTA) (pH 7.3 with KOH). Experimental ischemia was produced by changing the perfusion solution to an ischemia-mimetic solution of the following composition (mM): 50 KCl, 80 potassium aspartate, 20 HEPES, 3 MgCl2/6H2O, 3 EGTA, 5 NaCN, 5 NaCl (pH 6.5 with KOH). The free Ca2+ concentration in the extramitochondrial solution was changed according to the experimental protocol. The adjustment of free Ca2+ concentration was performed using the software Webmax extended.14)

Tetramethylrhodamine ethyl ester (TMRE) was imaged under excitation at 514 nm and emission at 580 to 600 nm. The mitochondrial membrane potential was evaluated with JC-1 fluorescence (0.5 µg/mL) under excitation at 488 nm and detection of the emission at wavelength longer than 515 nm. The yellow cameleon expressed in the cells was excited at 440 nm, and emissions at 480±30 nm (CFP) and 535±25 nm (YFP) were detected by a high-speed cooled CCD camera (HISCA, Hamamatsu Photonics, Japan) at a time resolution of 5 s, and ratioed after correction of background fluorescence (Aquacosmos software, Hamamatsu Photonics). The changes in cameleon fluorescence ratio was considered to reflect changes in intramitochondrial Ca2+ and not those in extramitochondrial sites such as the endoplasmic reticulum and the goldi apparatus because the changes in fluorescence ratio following increases in Ca2+ were almost completely inhibited by carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) (Fig. 2B) or ruthenium red (Fig. 2C).

Ruthenium red (Sigma, St. Louis, MO, U.S.A.) was dissolved in water. SEA0400 (synthesized according to the reported method15)), FCCP (Sigma, St. Louis, MO, U.S.A.) and CGP37157 (Tocris, Bristol, U.K.) were dissolved in dimethyl sulfoxide (DMSO). All data were expressed as the mean±standard error of the mean (S.E.M). The data were analyzed by the one-way ANOVA followed by Dunnett’s multiple tests. A p value less than 0.05 was considered statistically significant.

RESULTS

Confocal microscopic images of H9c2 cells expressing the mitochondria-targeted Ca2+ indicator, yellow cameleon, showed the presence of elongated cylindrical mitochondria throughout the cytoplasm. The intracellular localization of yellow cameleon fluorescence highly correlated with that of the mitochondria marker TMRE (Fig. 1A). In permeabilized H9c2 cells, elevation of extramitochondrial Ca2+ concentration resulted in a concentration-dependent increase in mitochondrial Ca2+ concentration (Fig. 1B); the yellow cameleon fluorescence ratio under 0, 100, 300 and 600 nM Ca2+ was 1.24±0.02, 1.33±0.04, 2.00±0.04 and 2.30±0.04, respectively (n=4). FCCP (1 µM), a mitochondria uncoupler, induced a decrease in mitochondrial Ca2+ concentration under 300 nM extramitochondrial Ca2+ concentration (Fig. 1C); the fluorescence ratio before and after the addition of FCCP was 1.98±0.02 and 1.65±0.01 (57.3±0.84%), respectively. Elevation of extramitochondrial Ca2+ concentration resulted in concentration-dependent decrease in mitochondrial membrane potential (Fig. 1D); the fluorescence intensity of the membrane potential-sensitive mitochondria marker JC-1 under 100, 300, and 1000 nM Ca2+ was 83.5±1.4, 45.2±0.9 and 35.0±3.9%, respectively, of that under Ca2+-free condition.

Fig. 1. Effects of Extramitochondrial Ca2+ on Mitochondrial Ca2+ and Membrane Potential in H9c2 Cells

A: Confocal images of mitochondria-targeted yellow cameleon (CFP; a), TMRE (b), and their merge (c). B: Typical recording of the changes in yellow cameleon fluorescence ratio (YFP/CFP) under various extramitochondrial Ca2+ concentrations. C: Typical recording of yellow cameleon fluorescence ratio in permeabilized H9c2 cells before and after application of FCCP (1 µM) under 300 nM extramitochondrial Ca2+ concentration. D: Typical recordings of JC-1 signal in permeabilized H9c2 cells on application of extramitochondrial solution with 300 nM Ca2+.

The increase in mitochondrial Ca2+ concentration on elevation of extramitochondrial Ca2+ concentration was reproducible (Fig. 2A). The increase in mitochondrial Ca2+ was markedly inhibited by 1 µM FCCP (Figs. 2B, F), which eliminates the driving force of the Ca2+ uniporter, or by 1 µM ruthenium red (Figs. 2C, F), an inhibitor of the mitochondrial Ca2+ uniporter. CGP-37157 (10 µM), a mitochondrial NCX inhibitor, rather enhanced the increase in Ca2+ concentration (Figs. 2D, F). SEA0400 (1 µM) had no effect (Figs. 2E, F). Also under an ischemia-mimetic condition, elevation of extramitochondrial Ca2+ concentration induced an increase in mitochondrial Ca2+ concentration (Fig. 3A). Ruthenium red reduced, CGP-37157 enhanced, and SEA0400 had no effect on the increase in mitochondrial Ca2+ concentration under ischemic condition (Fig. 3E).

Fig. 2. Effects of Pharmacological Agents on Mitochondrial Ca2+ Concentration in Permeabilized H9c2 Cells

The permeabilized H9c2 cell was exposed to solutions containing 300 nM Ca2+ (indicated by black bars) for 5 min, and after 10 min perfusion with Ca2+ free solution, [Ca2+] was elevated again. Before the second addition of Ca2+, H9c2 cells were treated with pharmacological agents (indicated by white bars). Effects of 1 µM FCCP (B), 1 µM ruthenium red (C), 10 µM CGP-37157 (D), and 1 µM SEA0400 (E) on mitochondrial Ca2+ influx. Summarized data of the changes in yellow cameleon fluorescence ratio on the second application of 300 nM Ca2+ solution (n=6–7; F). Asterisks indicate significant differences (p<0.05) from the corresponding control values.

Fig. 3. Effects of Pharmacological Agents on Mitochondrial Ca2+ Concentration in Permeabilized H9c2 Cells under Ischemic Condition

The permiabilized H9c2 cell was exposed to solutions containing 300 nM Ca2+ (indicated by black bars) for 5 min, and after 10 min perfusion with Ca2+ free solution, [Ca2+] was elevated again. Before the second addition of Ca2+, H9c2 cells were treated with an ischemia-mimetic solution and pharmacological agents (indicated by white bars). Effects of 1 µM ruthenium red (B), 10 µM CGP-37157 (C), 1 µM SEA0400 (D) on mitochondrial Ca2+ dynamics under ischemia condition. Summarized data of the changes in yellow cameleon fluorescence ratio after 5 min of the second application of 300 nM Ca2+ solution under ischemia condition (n=6–7; E). Asterisks indicate significant differences (p<0.05) from the corresponding control values.

DISCUSSION

The present study was undertaken to obtain information on the direct and indirect effects of SEA0400 on mitochondrial Ca2+ concentration and membrane potential under normal and ischemic conditions in H9c2 cardiomyocytes. The co-localization of yellow cameleon fluorescence with that of TMRE (Fig. 1) proved that the Ca2+ indicator was indeed expressed in the mitochondria. Increases in the extramitochondrial Ca2+ concentration resulted in increases in mitochondrial Ca2+ concentration both under normal and ischemic conditions. These results indicate that mitochondrial Ca2+ concentration is dependent on cytoplasmic Ca2+ concentration under both normal and ischemic conditions. Thus, the attenuation of the rise in mitochondrial Ca2+ in ischemic cardiomyocytes7) could be attributed to the attenuation of the rise in cytoplasmic Ca2+ by SEA0400.

The increases in extramitochondrial Ca2+ concentration not only caused increases in mitochondrial Ca2+ concentration but also reduced the mitochondrial membrane potential. On the other hand, reduction of the membrane potential by FCCP resulted in decreases in mitochondrial Ca2+ concentration (Fig. 1). These results indicate that the decrease in mitochondrial membrane potential could be the result but not the cause of rise in mitochondrial Ca2+ concentration. Thus, the preservation of the mitochondrial membrane potential in ischemic cardiomyocytes by SEA04007) could be attributed to the attenuation of the rise in mitochondrial Ca2+ concentration.

Concerning the mechanisms for mitochondrial Ca2+ regulation (Fig. 2), the inhibition of the rise in mitochondrial Ca2+ by ruthenium red and FCCP indicated that Ca2+ influx occurs through the Ca2+ uniporter. The enhancement of the rise in mitochondrial Ca2+ by CGP37157 indicated that mitochondrial NCX is involved in Ca2+ efflux rather than influx through the mitochondria inner membrane. These agrees with earlier results obtained with isolated mitochondria16) and intact cardiomyocytes.17) The effects of pharmacological agents, inhibition by ruthenium red and enhancement by CGP377157, were the same under normal and ischemic conditions (Fig. 3). SEA0400, at a concentration of 1 µM which shows marked inhibition of the plasmalemmal NCX in various cells including H9c2 cells,18) had no effect on mitochondrial Ca2+ both under normal and ischemic conditions. This indicates that SEA0400 inhibits neither the Ca2+ uniporter nor the mitochondrial NCX, and has no direct effect on mitochondrial Ca2+ transport pathways. Thus, the mitochondria protective effect of SEA04007) could not be attributed to its direct effect on mitochondrial Ca2+ transport.

Concerning the mechanism for the mitochondria protective effect of SEA0400 in ischemic cardiomyocytes,6,7) the conclusion drawn from the present results is that SEA0400 reduced mitochondrial Ca2+ overload through inhibition of plasmalemmal NCX and the resulting attenuation of the increase in cytoplasmic Ca2+ concentration. This conclusion is supported by studies showing that ruthenium red, which directly inhibited Ca2+ influx to mitochondria during ischemia (Fig. 3), improves contractile recovery following ischemia–reperfusion.19) Also, mitochondrial NCX inhibition, which induced an increase in mitochondrial Ca2+ during ischemia (Fig. 3), was reported to produce deleterious effects during ischemia–reperfusion including reduced resynthesis of energy phosphates.20) Thus, selective inhibition of plasmalemmal NCX, but not mitochondrial NCX, appears to be an effective therapeutic strategy against myocardial ischemia–reperfusion injury.

Acknowledgments

The authors express their thanks to Dr. Atsushi Miyawaki (RIKEN Brain Science Institute, Japan) for kindly providing yellow cameleon 3.1. This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers JP20890233, JP15K08247.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES
 
© 2017 The Pharmaceutical Society of Japan
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