Knockout Mice for Pharmacological Screening
Testing the Specificity of Na+-Ca2+ Exchange Inhibitors
The role of the Na+-Ca2+ exchanger as a major determinant of cell Ca2+ is well defined in cardiac tissue, and there has been much effort to develop specific inhibitors of the exchanger. We use a novel system to test the specificity of two putative specific inhibitors, KB-R7943 and SEA0400. The drugs are applied to electrically stimulated heart tubes from control mouse embryos or embryos with the Na+-Ca2+ exchanger knocked out. We monitored effects of the drugs on Ca2+ transients. Both drugs depress the Ca2+ transients at low concentrations even in the absence of any Na+-Ca2+ exchanger. KB-R7943 and SEA0400 are not completely specific and should be used with caution as Na+-Ca2+ exchange inhibitors.
The Na+-Ca2+ exchanger (NCX) is the major Ca2+ efflux mechanism in cardiac muscle.1,2⇓ Mediating cellular Ca2+ efflux is a role of special importance in cardiomyocytes because substantial transsarcolemmal Ca2+ fluxes occur with each contraction. The Na+-Ca2+ exchanger also plays a role in inotropic responses and is upregulated in many studies of hypertrophy and heart failure. The exact roles of the Na+-Ca2+ exchanger are difficult to study because of the multiplicity of Ca2+ flux pathways in cardiomyocytes and the lack of specific inhibitors.
In the present study, we investigate the specificity of two recently described Na+-Ca2+ exchange inhibitors. The first, KB-R7943,3,4⇓ inhibits Na+-Ca2+ exchange activity with an IC50 in the low micromolar range. The inhibitory mechanism of KB-R7943 has been investigated,3,5⇓ and amino acids of the exchanger involved in binding KB-R7943 have been identified.6
In some studies,3,5,7⇓⇓ although not all,8–10⇓⇓ the action of KB-R7943 was more potent on the reverse mode (Ca2+ influx) than the forward mode (Ca2+ efflux) of exchange. Despite this inconsistency, KB-R7943 has been used as a specific inhibitor of reverse-mode Na+-Ca2+ exchange11,12⇓ and has implied a role for reverse-mode exchange in arrhythmogenicity.7,13⇓ Relatively potent effects of KB-R7943 on other transporters and channels have been described. KB-R7943 binds to the norepinephrine transporter,14 blocks or binds to NMDA, K+, Na+, and Ca2+ channels,14,15⇓ and blocks the nicotinic acetylcholine receptor.16
The second drug that we investigate is SEA0400,14,17⇓ described as the most potent known inhibitor of Na+-Ca2+ exchange with an IC50 of ≈20 nmol/L.14 SEA0400 reduced Ca2+-induced damage in cultured astrocytes14 and improved functional recovery after myocardial ischemia (T. Matsuda and A. Baba, unpublished observation, 2002), implicating a role for Na+-Ca2+ exchange in these processes. Moreover, SEA0400 had no effects on several ion channels and transporters and seems to have excellent specificity.
Four laboratories18–21⇓⇓⇓ have found that knockout of the mouse Na+-Ca2+ exchanger, NCX1, is embryonic lethal by 11 days postcoitum (dpc). NCX1 is the only isoform of the exchanger in myocardium, and it might be expected that excitation-contraction coupling would fail in the absence of exchange activity. Koushik et al,19 however, found normal Ca2+ transients in heart tubes from 9.5-dpc NCX−/− embryos. We reproduced this finding and used the NCX1 knockout model to identify the importance of the Na+-Ca2+ exchanger in the response of myocardium to ouabain.21
In this investigation, we use heart tubes from NCX−/− mice to examine the specificity of KB-R7943 and SEA0400. Any effects of drugs on Ca2+ transients in NCX−/− heart tubes must be due to modulation of something other than Na+-Ca2+ exchange.
Materials and Methods
Production of Na+-Ca2+ exchanger knockout mice has been described.21 All experiments were performed under approved institutional animal protocols. As before, the heart tubes from embryos at 9.5 dpc were loaded with fura 2, and Ca2+ transients were measured by fluorescence in a flow-through chamber on an inverted microscope. Heart tubes were paced at 1 Hz at 35°C.
The heart tubes were from embryos that are NCX− /−, NCX+/−, or NCX+/+. However, in several series of experiments, the hemizygous knockouts and wild-type embryos had identical properties. Therefore, we combined data obtained with NCX+ /− and NCX+/+ heart tubes into one group representing NCX1-containing heart tubes and refer to this group as NCX+.
KB-R7943 and SEA0400 were kind gifts from Kanebo and Taisho Pharmaceutical, respectively.
Stock solutions of KB-R7943 and SEA0400 were in DMSO, and we tested whether DMSO by itself altered Ca2+ transients. Figure 1 shows that 0.1% DMSO had no effects on Ca2+ transients in heart tubes from NCX−/− embryos. Figure 1 also demonstrates the excellent stability of the system. No changes were observed in Ca2+ transients over the 10-minute period in DMSO.
Typical effects of KB-R7943 (5 μmol/L, 5 minutes) on Ca2+ transients in embryonic heart tubes are shown in Figure 2A and are summarized in Figure 2B. In NCX+ heart tubes, KB-R7943 had substantial effects on diastolic Ca2+, ΔCa2+, and relaxation time. However, even in the absence of the Na+-Ca2+ exchanger, KB-R7943 significantly reduced ΔCa2+ by 34±8%. Continued exposure to KB-R7943 further reduced the Ca2+ transients. After 10 minutes of exposure to KB-R7943 (Figure 2C), ΔCa2+ was largely eliminated for both the NCX+ and NCX−/− heart tubes.
Effects of SEA0400 (0.1 μmol/L) on the Ca2+ transients of embryonic heart tubes are shown in Figure 3A and are summarized in Figure 3B. The effects are similar to those obtained with KB-R7943, although achieved with much lower concentrations. Both drugs elevated diastolic Ca2+ and reduced ΔCa2+ in NCX+ heart tubes and reduced ΔCa2+ in NCX−/− heart tubes.
The voltage of the field stimulation needed to be increased to maintain Ca2+ transients after application of both drugs. This occurred with both NCX+ and NCX− /− heart tubes. The voltage typically would need to be increased from 30 to 40 or 50 V after 3 minutes of drug exposure.
The usefulness in physiological settings of the Na+-Ca2+ exchange inhibitors KB-R7943 and SEA0400 requires specificity. We compared the effects of the drugs on Ca2+ transients in embryonic heart tubes of control and Na+-Ca2+ exchanger knockout mice. If a drug affects the Ca2+ transient in the absence of exchanger, it is certainly not a specific inhibitor of the exchanger. We have recently used this approach to demonstrate that the presence of the exchanger is required to elicit a response to ouabain or to removal of external Na+.21
KB-R7943 and SEA0400 both depress Ca2+ transients even in the absence of the Na+-Ca2+ exchanger. This depressant effect clearly occurs by a mechanism not involving Na+-Ca2+ exchange. KB-R7943 has already been noted to have multiple other effects,14–16⇓⇓ but SEA0400 had seemed promising as a specific exchange inhibitor.14,17⇓
The differences between the effects of the drugs on myocardium containing or lacking the exchanger is informative. Both drugs elevate diastolic Ca2+ only in the presence of the exchanger. Diastolic Ca2+ in NCX− /− hearts was unaffected. The difference is likely related to a direct inhibitory effect of the drugs on the exchanger. Acute inhibition of the exchanger apparently blocks a component of Ca2+ efflux and the Ca2+ transient never fully relaxes. The fact that both drugs required the field stimulation to be increased for maintenance of Ca2+ transients suggests that ion channels involved in tissue excitation are being affected.
In summary, KB-R7943 and SEA0400 are not specific inhibitors of the Na+-Ca2+ exchanger. Although we observe a lack of specificity, we have not determined the other actions of these drugs on embryonic heart tubes. KB-R7943 and SEA0400 may have uses in Na+-Ca2+ exchange research but should be used with caution.
This research was supported by the NIH (HL 48509), the American Heart Association, Western States Affiliate (9950748Y), Koeln Fortune, Deutsche Forschungsgemeinschaft (1496/1-1), and the Laubisch Foundation.
Original received May 28, 2002; revision received June 13, 2002; accepted June 13, 2002.
- ↵Bers DM. Excitation-Contraction Coupling and Cardiac Contractile Force. 2nd ed. Dordrecht, The Netherlands: Kluwer Academic Publishers; 2001.
- ↵Iwamoto T, Watano T, Shigekawa M. A novel isothiourea derivative selectively inhibits the reverse mode of Na+/Ca2+ exchange in cells expressing NCX1. J Biol Chem. 1996; 271: 22391–22397.
- ↵Elias CL, Lukas A, Shurraw S, Scott J, Omelchenko A, Gross GJ, Hnatowich M, Hryshko LV. Inhibition of Na+/Ca2+ exchange by KB-R7943: transport mode selectivity and antiarrhythmic consequences. Am J Physiol Heart Circ Physiol. 2001; 281: H1334–H1345.
- ↵Iwamoto T, Kita S, Uehara A, Inoue Y, Taniguchi Y, Imanaga I, Shigekawa M. Structural domains influencing sensitivity to isothiourea derivative inhibitor KB-R7943 in cardiac Na+/Ca2+ exchanger. Mol Pharmacol. 2001; 59: 524–531.
- ↵Satoh H, Ginsburg KS, Qing K, Terada H, Hayashi H, Bers DM. KB-R7943 block of Ca2+ influx via Na+/Ca2+exchange does not alter twitches or glycoside inotropy but prevents Ca2+ overload in rat ventricular myocytes. Circulation. 2000; 101: 1441–1446.
- ↵Nishimaru K, Kobayashi M, Matsuda T, Tanaka Y, Tanaka H, Shigenobu K. α-Adrenoceptor stimulation-mediated negative inotropism and enhanced Na+/Ca2+ exchange in mouse ventricle. Am J Physiol Heart Circ Physiol. 2001; 280: H132–H141.
- ↵Woo SH, Morad M. Bimodal regulation of Na+-Ca2+ exchanger by β-adrenergic signaling pathway in shark ventricular myocytes. Proc Natl Acad Sci U S A. 2001; 98: 2023–2028.
- ↵Perez NG, de Hurtado MC, Cingolani HE. Reverse mode of the Na+-Ca2+ exchange after myocardial stretch: underlying mechanism of the slow force response. Circ Res. 2001; 88: 376–382.
- ↵Matsuda T, Arakawa N, Takuma K, Kishida Y, Kawasaki Y, Sakaue M, Takahashi K, Takahashi T, Suzuki T, Ota T, Hamano-Takahashi A, Onishi M, Tanaka Y, Kameo K, Baba A. SEA0400, a novel and selective inhibitor of the Na+-Ca2+ exchanger, attenuates reperfusion injury in the in vitro and in vivo cerebral ischemic models. J Pharmacol Exp Ther. 2001; 298: 249–256.
- ↵Wakimoto K, Kobayashi K, Kuro OM, Yao A, Iwamoto T, Yanaka N, Kita S, Nishida A, Azuma S, Toyoda Y, Omori K, Imahie H, Oka T, Kudoh S, Kohmoto O, Yazaki Y, Shigekawa M, Imai Y, Nabeshima Y, Komuro I. Targeted disruption of Na+/Ca2+ exchanger gene leads to cardiomyocyte apoptosis and defects in heartbeat. J Biol Chem. 2000; 275: 36991–36998.
- ↵Koushik SV, Wang J, Rogers R, Moskophidis D, Lambert NA, Creazzo TL, Conway SJ. Targeted inactivation of the sodium-calcium exchanger (Ncx1) results in the lack of a heartbeat and abnormal myofibrillar organization. FASEB J. 2001; 15: 1209–1211.
- ↵Reuter H, Henderson SA, Han T, Ross RS, Goldhaber JI, Philipson KD. The Na+-Ca2+ exchanger is essential for the action of cardiac glycosides. Circ Res. 2002; 90: 305–308.