Three New Tools
The contraction of cardiac muscle is initiated by the influx of Ca2+ through voltage-sensitive Ca2+ channels. Some of the Ca2+ entering the cell binds to Ca2+ release channels (ryanodine receptors) on the sarcoplasmic reticulum (SR) and triggers a release of Ca2+ from the SR. After the subsequent contraction, Ca2+ must be removed from the myoplasm to facilitate relaxation. Most Ca2+ is pumped back into the SR, but a significant fraction is extruded from the cell by the Na+-Ca2+ exchanger. In the steady state, the amount of Ca2+ leaving the cell via the exchanger equals the amount of Ca2+ that enters through Ca2+ channels.1,2⇓
This simple framework explains general aspects of cardiac excitation-contraction coupling but, of course, there are complications and controversy. Some of the controversy revolves around the exact role of the Na+-Ca2+ exchanger in this scenario. The exchanger can transport Ca2+ in either direction across the cell membrane and possibly sometimes mediates Ca2+ influx. The direction of net Ca2+ transport is determined by three factors: the Na+ gradient, the Ca2+ gradient, and the membrane potential. During the early phases of the action potential, for example, depolarization favors reversal of the exchanger into the Ca2+ influx mode. On the other hand, Ca2+ will be rising rapidly at this time as a result of channel openings and SR Ca2+ release. An increased intracellular Ca2+ level will push the exchanger back into the Ca2+ efflux mode.
So, does the Na+-Ca2+ exchanger contribute significantly to Ca2+ influx? Attempts to address this question are not straightforward. The primary problem is that experiments to address the issue typically use pharmacological or ionic interventions that by themselves alter the role of the exchanger. Thus, a substantial Ca2+ influx mediated by the exchanger can be readily ascertained experimentally, but the question as to whether this occurs under physiological conditions remains unclear.
Na+ and Ca2+ levels and the membrane potential are all amenable to experimental measurement and manipulation. These values can be used in a straightforward manner to calculate the net direction of Na+-Ca2+ exchange. The problem arises, however, that the exchanger responds to the rapidly changing levels of Na+ and Ca2+ in the local environment and not to the experimentally accessible values of global myoplasmic Na+ and Ca2+. Accurate estimates of subsarcolemmal values of Na+ and Ca2+ have been elusive. Uncertainties are introduced by areas of restricted diffusion and lack of quantitative information on the proximity of the exchanger to Na+ and Ca2+ channels in the sarcolemma and to the SR Ca2+ release channel.
Further impetus to try to understand the role of the Na+-Ca2+ exchanger in excitation-contraction coupling arises in pathophysiological situations. In many models of hypertrophy and heart failure, for example, Na+-Ca2+ exchange activity is upregulated. Additionally, other alterations in heart failure (prolonged action potential, increased Na+i) might increase the importance of the exchanger as a Ca2+ influx pathway. Indeed, the results of several studies have implicated the importance of reverse Na+-Ca2+ exchange in the maintenance of contractility during heart failure. Na+-Ca2+ exchange has also been implicated in the generation of arrhythmias during heart failure, and it remains unclear whether an upregulation of the exchanger is a beneficial adaptation.3,4⇓
Two studies in this issue of Circulation Research provide new tools and new information on these issues. A clever, and technically demanding, approach was used by Weber et al5 to infer the magnitude of the Na+-Ca2+ exchange current, INCX, during the action potential. How is this done? First, the properties of INCX are determined as a function of membrane potential and Ca2+i under steady-state conditions. To do this, the Ca2+i is heavily buffered so no Ca2+ gradients should exist within the myoplasm. Thus, submembrane Ca2+ equals the bulk Ca2+ as measured with a Ca2+ indicator. Then, under nonbuffered conditions, INCX is determined at different times during an action potential by measuring the peak inward INCX tail current upon rapid repolarization to −90 mV. Weber et al5 find that, early in the action potential, INCX is much larger than would be expected based on the level of bulk Ca2+i. That is, the level of submembrane Ca2+ must be greater than that of the bulk Ca2+. From knowledge of the Ca2+ dependence of steady-state exchange currents, the submembrane Ca2+ is quantitated.
The submembrane Ca2+ transient peaked earlier (<32 ms) and to higher levels (>3.2 μmol/L) than the Ca2+ transient of the bulk myoplasm (1.1 μmol/L at 81 ms). Presumably, this is due to regions of restricted diffusion and to rapid fluxes through Ca2+ channels. The results go a long way toward confirming the existence of a submembrane Ca2+ pool. Submembrane Ca2+ levels have been the subject of many hypotheses and speculations in the literature but are rarely quantitated. Weber et al5 are not the first to provide information on submembrane Ca2+ but have certainly pushed quantitation to a new quantum level. It should be remembered that “submembrane Ca2+” in this context refers specifically to that Ca2+ “seen” by the Na+-Ca2+ exchanger and may not equal the Ca2+ in all regions of the diadic cleft.
Armed with knowledge of the time course of submembrane Ca2+, the authors are then able to calculate the magnitude and direction of Na+-Ca2+ exchange during an action potential. The rapid and large rise in submembrane Ca2+ pushes the exchanger into the Ca2+ efflux mode early in the action potential. It is estimated that a modest Ca2+ influx will occur for only 15 ms. If the values of bulk Ca2+i (determined from dye measurements) were used in the calculations, then the reverse exchange would be larger and more prolonged. The results strongly argue against the importance of Na+-Ca2+ exchange as a trigger for SR Ca2+ release.
The study is unlikely to be the last word on the topic. First, conflicting conclusions in the literature need to be resolved. Second, Na+-Ca2+ exchange activity is highly sensitive to experimental conditions. For example, small shifts in the Na+i level can have large effects on the magnitude and direction of exchange activity, and Na+i varies with species and experimental conditions. Also, submembrane concentration of Na+i may differ from bulk Na+i concentration. Weber et al5 do a careful study, but the milieu of their isolated myocytes may have differences from that of intact myocardium. Third, as the authors point out, they are measuring an average submembrane Ca2+. Heterogeneities may exist. For example, there may be individual exchangers in close proximity to an SR Ca2+ release channel. These exchangers may trigger Ca2+ release, especially if the opening of a nearby L-type Ca2+ channel fails or is delayed. Fourth, as with all studies, the Weber et al study requires some approximations and assumptions. For example, are the properties of Na+-Ca2+ exchange under conditions of heavy Ca2+ buffering and in the presence of various drugs really identical to exchange properties under physiological conditions? Even under steady-state conditions, does submembrane Ca2+ really equal bulk Ca2+? Despite these qualifications, a new standard has been set for understanding the action of Na+-Ca2+ exchange during an action potential. Future work should not ignore the implications of this study.
Importantly, the approach of Weber et al5 can now be directly applied to pathophysiological situations. Will the altered conditions present in heart failure bestow the Na+-Ca2+ exchanger with increased importance as a trigger mechanism? We will stay tuned for the answer.
A second study on Na+-Ca2+ exchange by Müller et al,6 also in this issue, provides a new molecular tool. The Na+-Ca2+ exchanger gene (NCX1) has three alternative promoters that are used in a tissue-specific manner.7,8⇓ One of these promoters is responsible for expression of the exchanger in the myocardium as indicated by previous biochemical and in vitro analyses. Müller et al6 create a mouse model to study exchanger expression using a transgene composed of the cardiac-specific promoter of NCX1 linked to a luciferase reporter gene. The transgenic mouse can then be used in a relatively simple manner to quantitate promoter activity. This should be of substantial utility because many reports in the literature document changes in exchanger expression in response to pathophysiological, pharmacological, and hormonal interventions. The study also suggests the possible use of the NCX1 heart promoter for the expression and analysis of other transgenes where early cardiac-restricted expression is desired.
Müller et al6 use this transgenic mouse to study activity of the cardiac-specific promoter during development and during the onset of hypertrophy. In development, NCX1 expression is limited to the heart at early stages (up to day 14 postcoitum) but then occurs in other tissues. Müller et al6 found that luciferase RNA is restricted to the heart throughout development, indicating the absolute tissue specificity of the cardiac promoter. After transaortic constriction of adult transgenic mice to induce hypertrophy (7 days), cardiac luciferase activity was upregulated by 2-fold, consistent with the upregulation of NCX protein as indicated by Western blots.
These initial studies demonstrate the usefulness of these transgenic mice to study exchanger expression. Two caveats are necessary: First, it is possible (although unlikely) that alternative promoters become active in the heart during interventions such as hypertrophy. Second, it would be prudent to carry out quantitative comparisons of expression of luciferase and exchanger protein and activity to learn whether luciferase expression truly parallels exchanger expression. Nevertheless, these promoter/luciferase transgenic mice hold great promise for future studies to determine the importance of various promoter elements and signal transduction pathways and also to monitor the effects of a variety of interventions on Na+-Ca2+ exchanger expression.
The third tool involves the use of embryonic heart tubes from NCX1 knockout mice to study excitation-contraction coupling. This is described in a study9 that was published online in Circulation Research (to appear in print in the next issue) and can be accessed using the journal’s Online First feature. (This work is from the author’s own laboratory. Lest the reader bristle at the obvious conflict of interest, be assured that inclusion of this citation was imposed by the Editor.) The article provides new information on the essential role of Na+-Ca2+ exchange in the action of cardiac glycosides.
Taken together, the various new tools at our disposal promise to help us pinpoint the precise physiological role of the exchanger and how its function affects the diseased heart.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
- ↵Bers DM. Excitation-Contraction Coupling and Cardiac Contractile Force. 2nd ed. Dordrecht, The Netherlands: Kluwer; 2001.
- ↵Goldhaber JI. Sodium-calcium exchange: the phantom menace. Circ Res. 1999; 85: 982–984.
- ↵Barry WH. Na+-Ca2+ exchange in failing myocardium: friend or foe? Circ Res. 2000; 87: 529–531.
- ↵Weber CR, Piacentino V III, Ginsburg KS, Houser SR, Bers DM. Na+-Ca2+ exchange current and submembrane [Ca2+] during the cardiac action potential. Circ Res. 2002; 90: 182–189.
- ↵Müller JG, Isomatsu Y, Koushik SV, O’Quinn M, Xu L, Kappler CS, Hapke E, Zile MR, Conway SJ, Menick DR. Cardiac-specific expression and hypertrophic upregulation of the feline Na+-Ca2+ exchanger gene H1-promoter in a transgenic mouse model. Circ Res. 2002; 90: 158–164.
- ↵Barnes KV, Cheng G, Dawson MM, Menick DR. Cloning of cardiac, kidney, and brain promoters of the feline ncx1 gene. J Biol Chem. 1997; 272: 11510–11517.
- ↵Reuter H, Henderson SA, Han T, Ross RS, Goldhaber JI, Philipson KD. Na+-Ca2+ exchanger is essential for the action of cardiac glycosides. Circ Res. January 3, 2002; 10.1161/hh0302.104562. Available at: http://circres.ahajournals.org/onlinefirst.shtml. Accessed January 9, 2002.