The sarcolemmal Na+/H+ exchanger is a major H+ extrusion system and plays an important role in the restoration of pHi after an acidic load in myocardial cells (1,2). In general, ischemia causes cellular acidosis in cardiomyocytes by disturbing energy metabolism. This cellular acidosis activates the Na+/H+ exchange, which leads to intracellular Na+ overload, and then consecutive Ca2+ overload through the reverse-mode activation of Na+/Ca2+ exchange (1,3). Ca2+ overload provokes severe arrhythmia, cardiac functional disease, and lethal myocardial cell injury (4-7). Thus inhibition of Na+/H+ exchange, which would lead to prevention of Ca2+ overload, is a promising therapeutic approach against myocardial ischemic and reperfusion injury (8,9). Many studies report that Na+/H+ exchange inhibitors are effective in preventing myocardial ischemia/reperfusion injury, such as mechanical dysfunction, arrhythmia, myocardial stunning, or myocardial infarction, in the in vivo and in vitro models (10-20).
Conversely, the cardioprotective effects of Na+/H+ exchange inhibitors such as amiloride or its derivatives are relatively weak, and moreover, these compounds have unacceptable adverse effects on cardiac functions, which are possibly attributed to their lack of specificity for Na+/H+ exchange inhibition (21). For example, some amiloride derivatives were shown to block the Ca2+ current, Na+ current, K+ current, Na+/Ca2+ exchange, or Na+/K+ ATPase (21-24). Hence Pierce et al. (21) demonstrated that the use of amiloride or its derivatives in cells or perfused tissue experiments is normally not recommended because of their nonselectivity.
In this study, a newly synthesized compound, SM-20550 [N-(aminoiminomethyl)-1,4-dimethyl-1H-indole-2-carboxamide methanesulfonic acid, the chemical structure shown in Fig. 1], was first investigated for its effect on Na+/H+ and Na+/Ca2+ exchange activities in rat cardiomyocytes, and its effect on the binding affinity with several channels and receptors in membrane preparations. Second, we evaluated the cardioprotective effect of SM-20550 on ischemia/reperfusion injury in isolated perfused rat hearts.
MATERIALS AND METHODS
Male Sprague-Dawley (SD) and Wistar/ST rats were purchased from Crea Japan Inc. (Tokyo, Japan) and Nippon SLC (Shizuoka, Japan), respectively. All experiments were approved by the Institutional Animal Care and Use Committee at Sumitomo Pharmaceuticals Research Center (Osaka, Japan).
Preparation of cardiac myocytes
Cardiac myocytes were isolated by the enzymatic digestion method reported by Isenberg and Klockner (25) and Yamamoto et al. (26). In brief, rats (6-7 weeks old) were anesthetized with diethyl ether, and hearts were removed and mounted on a modified Langendorff perfusion system for the retrograde perfusion of the coronary circulation. Hearts were perfused first with normal Krebs solution (composition in mM: NaCl, 112; KCl, 4.7; CaCl2, 2.2; MgCl2, 1.2; NaHCO3, 25; NaH2PO4, 1.2; glucose, 14; bubbled with mixed gas of 95% O2 and 5% CO2) for 5 min; second with calcium-free Krebs solution (same as Krebs solution except calcium not added) for 10 min; third with calcium-free Krebs solution containing 0.015% collagenase (mixture of YK-102, Yakult, Tokyo, Japan, and Type IA, Sigma, St. Louis, MO, U.S.A.) for 5-20 min; and finally with Kraftbruhe solution (composition in mM: KCl, 70; KH2PO4, 2; glutamic acid monopotassium, 70; taurine, 20; glucose, 11; EGTA, 0.5; HEPES, 10; pH adjusted to 7.4 with Tris) for 5 min. The right ventricular wall was suspended in Kraftbruhe solution, and myocytes were dispersed by gently stirring. After the cells sedimented, suspension buffer was displaced by calcium-free HEPES solution (composition in mM: NaCl, 127; KCl, 5.9; MgCl2, 11; taurine, 20; glucose, 14; HEPES, 10; pH adjusted to 7.4 with Tris), and then normal HEPES solution (composition in mM: NaCl, 137; KCl, 5.9; CaCl2, 2.2; MgCl2, 1.2; glucose, 14; HEPES, 10; pH adjusted to 7.4 with Tris) was added step-wise to make the final Ca2+ concentration of 2.2 mM.
Measurement of pHi and Na+/H+ exchange inhibitory activity
For the measurement of pHi, myocytes were loaded with the membrane-permeable acetoxymethyl (AM) ester form of the pH-sensitive fluorescent indicator 3′-O-acetyl-2′,7′-bis(carboxyethyl)-4(5)-carboxyfluorescein, diacetoxymethyl ester (BCECF-AM; Wako Chemicals, Osaka, Japan, final concentration of 3 μM) for 30 min at room temperature. Myocytes loaded with BCECF-AM were then allowed to settle on a laminin-coated glass coverslip at the bottom of a small chamber, which was mounted on the stage of an inverted microscope (Nikon, Tokyo, Japan). After adhering to the coverslip, myocytes were superfused (3 ml/min) with normal HEPES solution. Intracellular BCECF was illuminated at 450 and 490 nm, and the BCECF ratio (490 nm/450 nm) of emitted light signal at 530 nm was measured with a fluorescence image analyzer (Argus-50; Hamamatsu Photonics, Hamamatsu, Japan). The emission intensity ratio (BCECF ratio) was used as an index of pHi(11,13).
According to the methods of Scholz et al. (11), Moffat and Karmazyn (13), Nakanishi et al. (27), and Loh et al. (28), the Na+/H+ exchange inhibition activity was determined by the inhibition of pHi recovery from acidosis. Intracellular acidification was produced by an NH4Cl prepulse technique; the cells were first perfused with normal HEPES buffer containing 20 mM NH4Cl and then with normal HEPES buffer. This measurement was performed under HCO3−-free conditions, in which the pHi recovery from acidosis is restricted to Na+/H+ exchange. After the BCECF ratio had recovered to the normal value, the acidification procedure was repeated again with drug or vehicle. The percentage recovery of the BCECF ratio from the negative peak at the second NH4Cl prepulse against the first NH4Cl prepulse was determined at each time course and plotted (BCECF ratio recovery). As BCECF ratio recovery rate was linear until 2 min from the peak of acidosis (negative peak of BCECF ratio), the percentage BCECF ratio recovery at 2 min from the peak of acidosis with drugs against that with vehicle was determined as the Na+/H+ exchange inhibitory action of drugs.
Measurement of [Ca]i and Na+/Ca2+ exchange inhibitory activity
The cardiac myocytes were loaded with the membrane-permeable AM form of the Ca2+-sensitive fluorescent indicator fura 2 (fura 2-AM; Wako Chemicals, Osaka, Japan; final concentration of 5 μM) for 20 min at room temperature. Myocytes loaded with fura 2-AM were then allowed to settle on a laminin-coated glass coverslip at the bottom of a small chamber, which was mounted on the stage of an inverted microscope (Nikon). After adhering to the coverslip, myocytes were superfused (3 ml/min) with normal HEPES solution. Intracellular fura 2 was illuminated at 340 and 380 nm, and the fura-2 ratio (340 nm/380 nm) of emitted light signal at 500 nm was measured with an image fluorescent analyzer (Argus-50). Because fura-2, when loaded as the ester form, is known to localize variably to sarcoplasmic reticulum or mitochondria (29), the precise quantification of [Ca]i is impossible; therefore the fura-2 ratio was used to index [Ca]i.
The Na+/Ca2+ exchange activity was determined by Na+-dependent Ca2+ influx and was measured according to the methods of Ziegelstein et al. (30) and Atsma et al. (31) as the increase of fura-2 ratio after perfusion of Na+-free normal HEPES buffer (NaCl was eliminated and displaced with equivalent choline chloride). During the measurement of the Na+/Ca2+ exchange activity, myocytes were stimulated by square wave pulse at a frequency of 1 Hz, 30-50% above of threshold voltage with 1 ms duration, supplied by an electrical stimulator through a pair of silver-plate electrodes placed between the myocytes. The Na+-dependent Ca2+ influx was induced by perfusion of Na+-free normal HEPES buffer for 3 min (Na+-free treatment), and then normal HEPES buffer was perfused for 5 min. This episode was repeated 3 times in the same cell, and the drug to be tested was added to the perfusion buffer during the third Na+-free treatment. The percentage of peak fura-2 ratio at the third Na+-free treatment against the second Na+-free treatment was determined as the Na+/Ca2+ exchange inhibitory action of the drug. The cells were determined to be not damaged by preserved peak fura-2 ratio at the fourth Na+-free treatment after three episodes performed for confirmation.
Displacement of specific radioligand bindings
The affinity of SM-20550 for several receptors and channels was determined by radioligand binding assays at MDS Panlabs Taiwan, Ltd. (Taipei, Taiwan). Membrane preparations from rat cortex, rat brain, hamster pancreas, rabbit adrenal gland, guinea pig lung, A10 cells, U266 cells, and human recombinant cells were prepared by the methods described previously (32-52). The radioligand binding activities of Ca2+, K+, or Na+ channels and α- or β-adrenoceptors, angiotensin, endothelin, or muscarinic receptors using the membrane preparations were measured as previously described (32-52). Membrane preparations and specific radioligands used in the experiments are listed in Table 1.
Isolated perfused rat hearts
Male Wistar/ST rats (8-10 weeks) were killed by cervical dislocation, the thorax was opened, and the hearts were quickly removed and placed in Krebs-Henseleit buffer (composition in mM: NaCl, 119; NaHCO3, 24.9; KCl, 4.7; KH2PO4, 1.2; CaCl2, 1.3; MgSO4, 1.2; glucose, 11). Hearts were then cannulated via the aorta and perfused with Krebs-Henseleit buffer, continuously bubbled with a 95% O2 and 5% CO2 gas mixture, at a constant flow rate of 10 ml/min using a Langendorff perfusion technique. The entire system was maintained at 37°C.
The coronary perfusion pressure (CPP) was measured through a pressure transducer (TP-300T; Nihon Koden, Tokyo, Japan) connected to the aortic cannula. A water-filled latex balloon attached to a polyethylene cannula was inserted into the left ventricle and connected to another pressure transducer (TP-300T) for the measurement of the left ventricular pressure (LVP). The balloon volume was adjusted to provide the left ventricular end-diastolic pressure (LVEDP) of −10-10 mm Hg. The isovolumic left ventricular developed pressure (LVDP) was calculated as the peak systolic LVP minus LVEDP. Heart rate and LVdP/dt (first derivative of LVP) were obtained from LVP. All parameters were recorded on a linearly recording thermostylus oscillograph (WR3101; Graphtec, Yokohama, Japan).
After 20-30 min of equilibration, global ischemia was produced by shutting off the perfusion. Global ischemia was maintained for 40 min, and the hearts were reperfused for 20 min. Drugs were administered for 5 min before ischemia and 20 min throughout the reperfusion period. In one group, hearts were perfused under normal conditions for ∼90 min.
At the end of the experiments, 15 ml of ice-cold sucrose-histidine buffer (0.35 M sucrose, 5 mM histidine) was infused through the aortic cannula to eliminate the perfusate from the vascular space, according to the method of Alto and Dhalla (53). A small part of the left ventricle was dried 8 h at 95°C for tissue analysis of ion contents.
The creatine phosphokinase (CPK) activity in the perfusate collected from the coronary effluent was measured spectrophotometrically with an assay kit (CPK II test WAKO; Wako chemicals) and a spectrophotometer (U-2000; Hitachi, Tokyo, Japan).
Tissue cations (Na+ and Ca2+) were extracted with acetate and trichloroacetate, and the ion contents were measured by the method of Sparrow and Johnstone (54) with an atomic spectrophotometer (Z-9000; Hitachi).
SM-20550 was dissolved in distilled water. EIPA (Sigma) was dissolved in 10% (vol/vol) dimethyl sulfoxide.
The concentrations of SM-20550 and EIPA necessary to inhibit pHi recovery from acidosis by 50% were determined (IC50 values) and used as Na+/H+ exchange inhibitory activities. LVDP, LVEDP, CPP, heart rate (HR), CPK release, and tissue Na+ or Ca2+ contents in isolated perfused hearts are expressed as mean ± SEM, and statistical analysis was performed with Williams' test (55). Differences of p < 0.05 were considered statistically significant.
Na+/H+ exchange inhibitory activity in rat cardiomyocytes
The Na+/H+ exchange activities in rat isolated cardiomyocytes were measured by recovery of pHi from acidosis induced by an NH4Cl prepulse technique. This measurement was performed under HCO3−-free conditions, in which the pHi recovery from acidosis is restricted to Na+/H+ exchange. An NH4Cl prepulse induced immediate pHi decrease after moderate pHi recovery to the resting level. Then the same cell preparations were subjected to a second NH4Cl prepulse, with test compounds included in the perfusion buffers. EIPA, a known Na+/H+ exchange inhibitor, and SM-20550 (Fig. 2) concentration-dependently suppressed the pHi recovery from acidosis. The IC50 values were 1 × 10−8M for SM-20550 and 1 × 10−7M for EIPA. The IC50 value of EIPA was in agreement with the results reported by Scholz et al. (10,12), and the Na+/H+ exchange inhibitory activity of SM-20550 was 10 times more potent than that of EIPA in cardiomyocytes.
Na+/Ca2+ exchange inhibitory activity in rat cardiomyocytes
The Na+/Ca2+ exchange activity was measured by Na+-dependent Ca2+ influx in isolated rat cardiomyocytes, induced by exposing myocytes to Na+-free HEPES-based buffer. The fura-2 ratio immediately increased after Na+-free treatment, and was restored to normal resting level after the following perfusion with Na+. A well-known Na+/Ca2+ exchange inhibitor, LaCl3 at 10−5M, inhibited ∼50% of the Na+-free-induced [Ca]i increase (data not shown) in this model, which was similar to the result reported by Jacob et al. (56). Thus this model was proved to be appropriate for measuring the effect of the test compound on Na+/Ca2+ exchange activity. The results are shown in Table 2. SM-20550 at concentrations of 10−8-10−6M (∼100 times higher than the IC50 of Na+/H+ exchange activity) did not affect the Na+-free-induced [Ca]i increase. Therefore SM-20550 has no effect on the cardiac Na+/Ca2+ exchanger.
Binding affinity with several receptors or channels
The affinity of SM-20550 for several channels or receptors was tested by radioligand binding assay. Among those tested, SM-20550 had slight affinity for only three receptors and two channels [α1- and α2-adrenergic receptors, L-type Ca2+ channels (benzothiazepine- and phenylalkylamine-sensitive Ca2+ channels), and Na+ channel]. Their IC50 values were 6 × 10−6, 2 × 10−6, 4 × 10−6, 7 × 10−6, and 4 × 10−6M, respectively. To other channels and receptors, L-type Ca2+ channel (dihydropyridine-sensitive Ca2+ channel), N-type Ca2+ channel, A-type K+ channel [KA], adenosine triphosphate (ATP)-sensitive K+ channel [KATP], voltage-dependent K+ channel [KV], Ca2+-activated K+ channel (low conductance) [SKCa], adenosine (A1 and A2A) receptors, β-adrenergic receptor, angiotensin (AT1 and AT2) receptors, endothelin (ETA and ETB) receptors, and muscarinic receptor, SM-20550 did not show any affinity at high concentration (10−5M).
Ischemia/reperfusion injury in isolated rat perfused hearts
SM-20550 (3 × 10−9 to 10−7M) had no effect on normoxic, preischemic values of the CPP, HR, or LVP (Figs. 3 and 4). The abnormal increase of CPP after ischemic reperfusion was suppressed by the treatment with SM-20550 (3 × 10−9 to 10−7M) in a concentration-dependent manner (Fig. 4). In the vehicle-treated group, the LVEDP increased during ischemia and reperfusion, but SM-20550 (10−8-10−7M) administration reduced the LVEDP increase during reperfusion (Fig. 3). Furthermore, SM-20550 at 10−7M significantly inhibited the LVEDP increase during the ischemic period (Fig. 3). SM-20550 also improved the postischemic LV contractile dysfunction [decrease in LVDP (Fig. 3) and LVdP/dt (data not shown) after reperfusion]. SM-20550 improved the decrease in HR at an early phase of reperfusion, but there was no significant difference between vehicle- and SM-20550-treated groups, at the end of reperfusion (Fig. 4).
Administration of SM-20550 (3 × 10−9 to 10−7M) significantly reduced the release of CPK into the coronary effluent during reperfusion (Fig. 5). The tissue contents of Na+ and Ca2+ were increased after ischemia/reperfusion as compared with those after normal perfusion, but were markedly reduced by SM-20550 administration (Fig. 6).
In this study, we have characterized a newly synthesized compound, SM-20550, as a potent and highly selective Na+/H+ exchange inhibitor, and investigated the effect of this compound on ischemia/reperfusion injury in perfused rat hearts.
To examine the effect of SM-20550 and EIPA on Na+/H+ exchange in cardiomyocytes, the experiments were performed in HCO3−-free solution. Under this condition, pHi recovery from acidosis is exclusively mediated by acid extrusion of the Na+/H+ exchange. SM-20550 concentration-dependently inhibited the recovery from acidosis by an NH4Cl prepulse in rat cardiomyocytes, and its IC50 was 10−8M, ∼10 times lower than that of EIPA, a well-known Na+/H+ exchange inhibitor (10,12). Hence, SM-20550 is a potent Na+/H+ exchange inhibitor.
Recent evidence has suggested that the Na+/Ca2+ exchange represents an important pathway to provoke intracellular Ca2+ overload during myocardial ischemia and reperfusion (56). Therefore we evaluated the influence of SM-20550 on this exchange system in rat cardiomyocytes. SM-20550 expressed no influence on the Na+/Ca2+ exchange at the concentration ranges of Na+/H+ exchange inhibition and pharmacologic effectiveness, which was similar to that of Hoe642 (11), another well-established Na+/H+ exchange inhibitor.
Several cardioprotective drugs are known to interact at various points in the key processes leading to ischemic heart injury. These include β-adrenoceptor antagonists, α-adrenoceptor antagonists, ACE inhibitors, endothelin antagonists, adenosine-related agents, Ca2+ channel blockers, Na+ channel blockers, and K+ channel openers (7,57-59). We investigated the effect of SM-20550 on these receptors or channels using radioligand binding assays. Although SM-20550 had little affinity for α1-, α2-receptors, or Ca2+ and Na+ channels, the IC50 values were within 10−6-10−5M, > 100 times higher than that of Na+/H+ exchange inhibitory activity. SM-20550 revealed no affinity to other common channels or receptors at 10−5M. Accordingly, we excluded the possibility that the cardioprotection by SM-20550 may be attributed to interaction with other channels or receptors. Altogether from these results, SM-20550 is characterized as a highly specific and extremely potent Na+/H+ exchange inhibitor.
In isolated perfused rat heart, SM-20550 revealed protection against ischemia/reperfusion injury, that is, improvement of postischemic recovery of the LV function, prevention of the coronary circulation disorder, and reduction of the intracellular enzyme release. In addition, the cardioprotective effect of this compound was associated concomitantly with attenuation of abnormal tissue Na+ and Ca2+ accumulation during postischemic reperfusion. The pharmacologically effective concentration ranges of SM-20550 were approximately within the concentration ranges of the Na+/H+ exchange inhibition. We also investigated the effect of EIPA for further confirmation, and a similar cardioprotective effect was observed in this model (data not shown), which was in agreement with previous studies (12,19). Thus a pharmacologic intervention at the step of Na+/H+ exchange would be beneficial in the protection against myocardial dysfunction during ischemia and reperfusion.
Moreover, the concentration of SM-20550 necessary to reduce tissue Ca2+ content or CPK release was 3 nM, whereas the concentration needed to improve cardiac function was 10 nM. These results suggest that the effect of SM-20550 on preventing Ca2+ overload precedes improvement of cardiac function. Therefore, our results strongly supported the hypothesis that Na+/H+ exchange inhibitors prevent cardiac ischemia/reperfusion injury through suppression of Na+ and Ca2+ overload (1). Conversely, SM-20550 markedly reduced cardiac Ca2+ contents after ischemia/reperfusion, but the concentration relation of this effect was not clear. As described by Alto and Dhalla (53), perfusion of heart with sucrose-histidine buffer eliminated the cations from vascular space, and this method is usually accepted as a measurement of cardiac ion contents (53); therefore we used this method in measuring the ion contents. It is not easy to explain the reason for a poor concentration dependency of SM-20550 on Ca2+ accumulation, but it may be partially due to the indirect measurement of the cardiac intercellular ion content in myocytes, also including contamination of extracellular space or other cell types.
Furthermore, in this study, SM-20550 at concentrations sufficient to have a cardioprotective effect did not affect normal, nonischemic, HR, LVP, or CPP in isolated rat hearts. It would certainly be a greater advantage of SM-20550 not to affect the normal hemodynamics, because occasionally it would involve risk to give cardio-depressing drugs to patients with ischemic heart diseases.
In conclusion, our results suggest that SM-20550 is a potent and selective Na+/H+ exchange inhibitor, showing a highly efficacious myocardial protective effect in ischemia/reperfusion injury without directly affecting the cardiac function or coronary vascular tone. In addition, our findings support the hypothesis that the Na+/H+ exchange system is likely to play a major role in the pathophysiology of development of ischemia/reperfusion injury (2-9), and moreover, the cardioprotection observed with SM-20550 is likely to be attributed to the Na+/H+ exchange inhibition.
Acknowledgment: We thank Ms. Yuka Yamana for her excellent technical assistance with the Na+/H+ and Na+/Ca2+ exchange activity studies.
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