The Na+-Ca2+ Exchanger Is Essential for the Action of Cardiac Glycosides
The widely accepted model to explain the positive inotropic effect of cardiac glycosides invokes altered Na+-Ca2+ exchange activity secondary to Na+ pump inhibition. However, proof of this model is lacking and alternative mechanisms have been proposed. We directly tested the role of the Na+-Ca2+ exchanger in the action of the glycoside ouabain using Na+-Ca2+ exchanger knockout mice. Ablation of the exchanger is embryonic lethal, but contractility can be studied in embryonic heart tubes at day 9.5 postcoitum. Heart tubes isolated from homozygous Na+-Ca2+ exchanger knockout mice (NCX−/−) display surprisingly normal Ca2+ transients. Removal of extracellular Na+ induces Ca2+ overload in wild-type heart tubes but does not alter the Ca2+ transients of NCX−/− heart tubes. Similarly, ouabain, at levels causing Ca2+ overload in wild-type heart tubes, has no effect on NCX−/− heart tubes. We conclude that in embryonic mouse myocytes the Na+-Ca2+ exchanger is absolutely required for the effect of cardiac glycosides on Ca2+i.
The use of digitalis in the treatment of “dropsy” (congestive heart failure) was first described by Withering in 1785.1 Since then, the mechanism of the positive inotropic effect of cardiac glycosides has been an area of intense study. Schatzmann2 was the first to describe inhibition of the Na+,K+-ATPase by a cardiac glycoside, ouabain. It was subsequently suggested that glycoside-induced increases in intracellular Na+ could lead to elevation of internal Ca2+ in cardiac muscle, and an inotropic effect, through the Na+-Ca2+ exchanger (NCX1).3,4⇓ Variations of this model are still used to explain digitalis action, although definitive proof is lacking.
The explanation for the mode of action of cardiac glycosides requires that the Na+ pump is inhibited at therapeutic concentrations of the drug and that [Na+]i subsequently rises. However, some workers report no change in cellular [Na+]i on application of digitalis.5 A variety of alternative mechanisms, independent of Na+ pump inhibition, have been proposed to account for increased intracellular Ca2+. These theories include, for example, a digitalis-induced stimulation of Ca2+ influx through other membrane proteins6–8⇓⇓ and the possibility of an intracellular site of action for cardiac glycosides.9–11⇓⇓
We investigated the role of Na+-Ca2+ exchange in the inotropic effect of cardiac glycosides using an NCX1 knockout mouse model. NCX−/− mice are embryonic lethal at ≈11.0 days postcoitum (dpc).12–14⇓⇓ Nevertheless, NCX−/− heart tubes at 9.5 dpc generate Ca2+ transients,14 presenting a useful model to study excitation-contraction (E-C) coupling in the absence of Na+-Ca2+ exchange.
Materials and Methods
Knockout of the Cardiac Exchanger
We created NCX1 knockout (KO) mice using the cre/loxP system of gene ablation.15,16⇓ A targeting vector was constructed to delete exon 2 of the NCX1 gene, which contains 64% of the coding sequence and includes the translational start site. The targeting vector was electroporated into cultured 129/Sv-LW1 embryonic stem (ES) cells, and neomycin-resistant colonies were screened for site-specific recombination by Southern blot and for loxP integrity by PCR. Selected recombinants were electroporated a second time with a cre recombinase expression vector (GenBank AF334827) to excise both exon 2 and the PGKneo cassette. Appropriate excision was confirmed by a combination of Southern blot and PCR analyses. Selected ES cell NCX+/− knockouts were injected into C57BL/6 blastocysts. Chimeras born from these blastocysts were bred with Black Swiss mice (Taconic), and those that successfully transmitted the targeted NCX1 allele were further propagated in this genetic background. All experiments were performed under approved institutional animal protocols.
SDS-PAGE and Western Blots
Intact heart tubes were dissolved in SDS sample buffer containing 3 mmol/L dithiothreitol and heated in boiling water for 5 minutes. Proteins were separated on 8% SDS-polyacrylamide gels and transferred onto nitrocellulose. To confirm comparable quantities of transferred protein for each heart tube, nitrocellulose was stained with Ponceau S (Sigma) before immunolabeling. The blots were probed with an exchanger-specific monoclonal antibody (R3F1)17 and visualized by chemiluminescence (NEN Life Science).
Isolation of Heart Tubes and Intracellular Ca2+ Measurements
Whole embryos were isolated at 9.5 dpc in preaerated modified Tyrode’s solution containing (in mmol/L) NaCl 136, KCl 5.4, MgCl2 1.0, NaH2PO4 0.33, HEPES 10, glucose 10, and CaCl2 1.0 (pH 7.4) at 37°C. After washing, the embryos were incubated in preaerated Tyrode’s solution containing 3 μmol/L fura-2 acetoxymethyl ester for 10 minutes at 37°C in a rotating water bath. The embryos were washed several times in Tyrode’s solution and incubated at room temperature for 30 minutes to facilitate dye deesterification. Immediately before experiments, heart tubes were excised from the embryos. At 9.5 dpc, NCX+/+, NCX+/− and NCX−/− embryos were of comparable size, and heart tubes showed no differences in development, although differences in vasculature were evident consistent with the findings of Koushik et al.14
For recording intracellular Ca2+, heart tubes were placed in a flow-through chamber (1.0 mL) on the stage of a Nikon Diaphot inverted microscope heated to 26°C. Ca2+ transients were elicited by electrical field stimulation (Grass) of the ventricular segment of heart tubes. Fluorescence was excited at dual wavelengths (335 and 405 nm) and recorded at 510 nm.18 Results are expressed as the ratio of fluorescence excited by the two wavelengths, which is linearly related to Ca2+i. Ratio measurements are resistant to bleaching and motion artifacts.
Results and Discussion
We confirmed the absence of Na+-Ca2+ exchanger protein in hearts of NCX−/− embryos by immunoblot. The blot in Figure 1 presents a pattern of protein bands at 70, 120, and 160 kDa typical of the Na+-Ca2+ exchanger.19 Wild-type (NCX+/+) heart tubes showed clear immunoreaction, whereas there was no reaction against NCX1 in heart tubes from NCX−/− mice. Heterozygote (NCX+/−) heart tubes had intermediate NCX protein levels.
To study E-C coupling, embryonic heart tubes isolated at 9.5 dpc were loaded with the Ca2+ indicator dye fura-2. Both wild-type and knockout embryos showed spontaneous contractions and simultaneous Ca2+ transients. With external field stimulation at 1 Hz, NCX+/+ and NCX−/− heart tubes had similar kinetics for the rise and decay of Ca2+ transients. Ca2+ transient amplitudes in the NCX−/− heart tubes showed a modest decrease (Table). The role of the Na+-Ca2+ exchanger as a Ca2+ efflux mechanism is thought to be critical in normal E-C coupling, and it is surprising that apparently normal Ca2+ transients can be generated in NCX−/− heart tubes (Table and Figure 2), as noted previously.14 Our data suggest that the plasma membrane Ca2+ pump is capable of assuming a primary Ca2+ efflux role in the NCX−/− heart tubes. In the study by Koushik et al,14 the heart tubes from NCX−/− mice did not contract and did not have spontaneous Ca2+ transients, although transients could be elicited by electrical stimulation. Our NCX−/− heart tubes display spontaneous Ca2+ transients and contractile active. The reason for this discrepancy is unclear, because conditions in the two studies are similar, although mouse strains are different.
Replacement of Na+
Removal of the extracellular Na+ surrounding a myocardial cell greatly disturbs fluxes mediated by the Na+-Ca2+ exchanger. Ca2+ efflux through the exchanger no longer occurs, and a marked Ca2+ influx (reverse exchange) is initiated because of the presence of Na+i. Ca2+ overload and contracture ensues. We examined the response of heart tubes to the removal of external Na+ (Figure 2). We chose Li+ as a substitute for Na+ because Li+ is not transported by the Na+-Ca2+ exchanger and is conducted through Na+ channels, thereby maintaining excitability. In NCX+/+ control hearts, the gradual replacement of Na+o by Li+o led to a continuous rise of diastolic Ca2+i and a reduction in the rate of decay of the Ca2+ transient. Ca2+ transients decreased in amplitude and were no longer evident after about 1 minute. In contrast, removal of Na+ did not influence the kinetics of the Ca2+ transient in NCX−/− heart tubes over a time period of 5 minutes, and contractions remained stable. The result is not unexpected but provides a dramatic functional demonstration of the absence of Na+-Ca2+ exchange activity in the NCX−/− heart tubes.
Cardiac glycosides increase contractility through elevation of intracellular Ca2+. The favored explanation has been that the increase in Ca2+ is secondary to a rise in Na+ after Na+ pump inhibition. The presence of the Na+-Ca2+ exchanger is central to this hypothesis. Nevertheless, the role of Na+-Ca2+ exchange as the sole mode of action of glycosides at therapeutic concentrations has been repeatedly questioned.6–11,20,21⇓⇓⇓⇓⇓⇓⇓ We therefore examined the effects of the cardiac glycoside ouabain on Ca2+i in heart tubes from NCX+/+ and NCX−/− mice. Treatment of heart tubes from NCX+/+ mice with 0.03 μmol/L ouabain had a modest effect on Ca2+ transients. At higher concentrations (0.1 μmol/L), the myocytes developed typical signs of Ca2+ overload with an increase in diastolic Ca2+ levels and reduced Ca2+ transient amplitudes (Figure 3). Relaxation was prolonged, consistent with a reduced rate of Ca2+ extrusion. Heart tubes from heterozygote knockouts (NCX+/−) had responses to ouabain similar to those of heart tubes from wild-type NCX+/+ mice (not shown).
In heart tubes from NCX−/− mice, however, ouabain had no effect on diastolic [Ca2+]i or the amplitude or kinetics of the transient (Figure 3). This was true even at elevated ouabain levels (1 μmol/L). These results provide the first direct evidence that the Na+-Ca2+ exchanger is required for the effects of ouabain on myocardial Ca2+. There are two caveats: First, the data were obtained using embryonic tissue and perhaps cannot be generalized to adult myocardium. Two, possibly there are adaptations in NCX−/− heart tubes that modify the response to ouabain. Nevertheless, our results appear to answer a basic question: Na+-Ca2+ exchange activity is essential for the action of cardiac glycosides.
This research was supported by the NIH (HL 48509), the American Heart Association, Western States Affiliate (9950748Y), Köln Fortune, the Deutsche Forschungsgemeinschaft (1496/1-1), and the Laubisch Foundation.
Original received November 29, 2001; revision received December 21, 2001; accepted December 21, 2001.
- ↵Withering W. An Account of the Foxglove, and Some of Its Medical Uses: With Practical Remarks on Dropsy, and Other Diseases Birmingham, UK: M. Swinney; 1785.
- ↵Arnon A, Hamlyn JM, Blaustein MP. Ouabain augments Ca2+ transients in arterial smooth muscle without raising cytosolic Na+. Am J Physiol Heart Circ Physiol. 2000; 279: H679–691.
- ↵Santana LF, Gómez AM, Lederer WJ. Ca2+ flux through promiscuous cardiac Na+ channels: slip-mode conductance. Science. 1998; 279: 1027–1033.
- ↵Dutta S, Goswami S, Datta DK, Lindower JO, Marks BH. The uptake and binding of six radiolabeled cardiac glycosides by guinea-pig hearts and by isolated sarcoplasmic reticulum. J Pharmacol Exp Ther. 1968; 164: 10–21.
- ↵Gervais A, Lane LK, Anner BM, Lindenmayer GE, Schwartz A. A possible molecular mechanism of the action of digitalis: ouabain action on calcium binding to sites associated with a purified sodium-potassium-activated adenosine triphosphatase from kidney. Circ Res. 1977; 40: 8–14.
- ↵Sagawa T, Sagawa K, Kelly JE, Tsushima RG, Wasserstrom JA. Activation of ryanodine receptors by cardiac glycosides. Am J Physiol. 2001; 280: H1201–H1207.
- ↵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.
- ↵Orban PC, Chui D, Marth JD. Tissue- and site-specific DNA recombination in transgenic mice. Proc Natl Acad Sci U S A. 1992; 89: 6861–6865.
- ↵Taki S, Meiering M, Rajewsky K. Targeted insertion of a variable region gene into the immunoglobulin heavy chain locus. Science. 1993; 262: 1268–1271.
- ↵Lee KS, Klaus W. The subcellular basis for the mechanism of inotropic action of cardiac glycosides. Pharmacol Rev. 1971; 23: 193–261.