doi: 10.1161/CIRCRESAHA.108.178095
© 2008 American Heart Association, Inc.
Editorials |
The Ins and Outs of Calcium in Heart Failure
From The Johns Hopkins University, School of Medicine, Division of Cardiology, Baltimore, Md.
Correspondence to Brian O'Rourke, PhD, The Johns Hopkins University, Institute of Molecular Cardiobiology, 720 Rutland Ave, 1059 Ross Bldg, Baltimore, MD 21205-2195. E-mail bor{at}jhmi.edu
See related article, pages 1398–1405
Key Words: excitation–contraction coupling • sodium–calcium exchange • SERCA2A • SEA-0400 • KB-R7943 • NCX inhibitors
Cardiac excitation–contraction coupling is akin to a biophysical juggling act that involves fast and slow ion movements overlapping in time and space across several compartments. In response to sarcolemmal depolarization, Ca2+ release from the large intracellular store in the sarcoplasmic reticulum (SR) is triggered by a rush of Ca2+ from the extracellular space through L-type Ca2+ channels into the very tiny junctional cleft (12 nm) to activate SR Ca2+ release channels in a tightly controlled signal amplification step. Local inactivation of the L-type Ca2+ channel mediated by the large junctional Ca2+ spike (peaking at >50 µmol/L) provides a mechanism of negative feedback on beat-to-beat Ca2+ influx; thus, an abrupt change in the size of cytosolic Ca2+ transient will impact the trigger for subsequent beats and reciprocally influence the SR Ca2+ content in a process known as autoregulation.1
Ca2+ removal processes, although kinetically slower, also play a major role in shaping the Ca2+ dynamics, and, ultimately, the contraction, of the myocyte. The 2 main pathways, Ca2+ reuptake into the SR by the Ca2+ ATPase (SERCA2A) and Ca2+ efflux across the sarcolemma by the Na+/Ca2+ exchanger (NCX), compete for the job of removing Ca2+ ions in a species-dependent manner. In ventricular myocytes from rats and mice, 
90% of the Ca2+ decline during a transient is mediated by SERCA2A, whereas NCX contributes only 7% to Ca2+ removal (the remainder is taken up by mitochondria or extruded from the cell by the sarcolemmal Ca2+ ATPase).2 In most large animals (eg, rabbits, dogs, cats, ferrets), some small animals (eg, guinea pigs and hamsters), and humans,3 Ca2+ removal by SERCA2A accounts for only 60% to 70% of the total, whereas NCX contributes 25% to 30%.4
When the amplitude of the Ca2+ transient reaches a steady state, Ca2+ influx on each heartbeat must be matched by an equal amount of Ca2+ efflux from the myocyte, or else intracellular Ca2+ overload or depletion will occur. This is primarily accomplished by the so-called forward-mode action of NCX, a 3:1 exchange of extracellular Na+ for intracellular Ca2+. However, because the exchanger is electrogenic, and is also driven by the gradients of Na+ and Ca2+ across the sarcolemma, the driving forces can switch to Ca2+ entry and Na+ efflux (reverse-mode NCX) when the membrane potential is more positive than the NCX reversal potential, which can occur during the early repolarization phase5 and the plateau of the cardiac action potential.6 One of the more difficult challenges for cellular physiologists has been to determine the direction and magnitude of NCX current and Ca2+ transport during excitation–contraction coupling, when the membrane potential, Na+, and Ca2+ gradients are all changing simultaneously. In this context, the ideal tool for probing the role of NCX in the beat-to-beat competition among Ca2+ influx, Ca2+ release, SR Ca2+ uptake, and sarcolemmal Ca2+ efflux would be a tool that could rapidly inhibit NCX in a selective and reversible manner. To date, the perfect NCX inhibitor has not been available: problems include issues of i) selectivity; for example, the inorganic divalent cation Ni2+ effectively blocks NCX but also inhibits voltage-gated Ca2+ and Na+ channels and ii) permeability; the charged exchange inhibitor peptide (XIP), patterned after the autoinhibitory region of the cytoplasmic regulatory loop of the exchanger, is impermeable to membranes and so can only be applied acutely to excised membrane patches or slowly via a patch-clamp pipette.
Pharmacological agents designed to inhibit NCX, such as KB-R7943,7 also lack selectivity, inhibiting various ionic currents including INa, ICa,L, IK, IK1,8 as well as ITRPC,9 with IC50 values (<10 µmol/L) close to that for NCX inhibition. Although it has been extensively used to investigate the role of NCX in ischemia/reperfusion injury, the lack of selectivity of KB-R7943 makes interpretation of the results quite difficult, because many of the alternate targets of the compound could theoretically contribute to the observed actions. The more recently developed SEA-04008 also inhibits ICa with micromolar affinity10; however, it is more potent than KB-R7943 against NCX (IC500.11 µmol/L),10 providing incentive to use it judiciously to readdress the central question about the role of NCX in excitation–contraction coupling in normal and diseased hearts.
An important application for NCX inhibitors is to ascertain the contribution of the exchanger to altered Ca2+ handling in heart failure,11 the subject of the report by Ozdemir et al12 in this issue of Circulation Research. Numerous studies in human3 and animal models13,14 of heart failure have reported that the relative contribution of SERCA2A decreases and that of NCX increases during Ca2+ removal in myocytes isolated from failing hearts. For example, in the canine pacing-induced heart failure model that we have studied, there is a decrease in the SERCA2A activity and an increase in the NCX activity15,16 such that the normal 69:25% contributions of SERCA2A and NCX to total Ca2+ removal change to 35:58%. Qualitatively similar remodeling takes place in many models of heart failure, but the extent of the decrease in SERCA2A and the increase in NCX varies among different animal models. This shift away from reloading the SR and toward Ca2+ extrusion from the cell is thought to contribute to diminished SR Ca2+ content in heart failure.13–15 On the other hand, there is evidence that the slightly longer action potentials and increased intracellular Na+ load of failing heart cells promotes more reverse-mode Ca2+ entry during the action potential, which can contribute to contraction.17 Therefore, it is of paramount importance to determine whether the inhibition of NCX increases or decreases SR Ca2+ load in failing cardiomyocytes. Along these lines, we have previously investigated whether selective inhibition of NCX with XIP can improve the defects in SR Ca2+ loading in a canine pacing-induced heart failure model.18 We found that whereas partial inhibition of NCX (by 30%) had only modest effects on SR Ca2+ load and the frequency dependence of the Ca2+ transient in normal cells, the effect in failing cells was dramatic, with full restoration of the SR Ca2+ content to normal levels and conversion of the flat frequency response of the Ca2+ transient (characteristic of heart failure) to a positive staircase. Moreover, partial inhibition of NCX in the failing group also restored the amplitude and rate of rise of the transient to normal levels. Another interesting effect of XIP treatment in the canine model was that the rate of Ca2+ removal via SERCA2A was enhanced on inhibition of NCX. This suggested that regulation of the pump by Ca2+ may be a factor in the positive inotropic effect of XIP, but it remains to be determined whether similar effects are present in other models of heart failure.
The study by Ozdemir et al12 addresses several key issues regarding the potential clinical utility of SEA-0400 as an inotropic agent in cardiac myocytes. First, it examines the selectivity, mode dependence, and efficacy of NCX inhibition, both with and without internal Ca2+ buffering. Whereas previous studies have suggested that NCX inhibitor compounds were somewhat selective for the reverse-mode of NCX, the present study demonstrates that this depends on the particular experimental conditions used; with normal Na+ and Ca2+ gradients and minimal intracellular buffering, SEA-0400 was about equally effective at blocking both modes of the exchanger at 0.3 or 1 µmol/L concentrations. ICa,L was partially inhibited, but the extent also depended on whether or not Ca2+ was buffered. ICa,L block was greater with less buffering, indicating that part of the Ca2+ channel inhibition was indirectly related to the NCX-mediated changes in intracellular Ca2+. SEA-0400 significantly inhibited forward-mode NCX and increased SR Ca2+ content and cell shortening in normal cardiomyocytes from both pigs and mice. In contrast, no positive inotropic effect was observed in a cardiomyopathic mouse knockout model of myosin LIM protein (MLP–/–), a scaffold protein of the actin-based cytoskeleton, even though relaxation of the twitch was slowed. In contrast, SEA-0400 significantly improved contractility in a mouse model of heart failure induced by transverse aortic constriction.
An important conclusion from the study by Ozdemir et al12 is that all models of heart failure are not equivalent, and a potential therapeutic strategy effective in one may not be useful in another. Thus, it behooves us to further examine the particular Ca2+ handling alterations in each case and to examine the effects of NCX inhibition in both large and small animal models. The Ca2+ handling properties of myocytes in the MLP–/– model have been investigated previously. Two groups19,20 reported a decrease in the amplitude of the Ca2+ transient and cell shortening in myocytes from MLP–/– mice with no change in ICa,L or Ca2+ spark properties. In contrast, Su et al21 found an increase in Ca2+ transient amplitude (almost doubled) and SR Ca2+ content (increased 21%), and a faster Ca2+ decay rate in MLP–/– myocytes, but decreased fractional shortening, suggesting that defective Ca2+ handling was not responsible for the depressed contractility in this model. Based on the latter findings, it is perhaps not surprising then that NCX inhibition did not mediate a positive inotropic effect in the MLP–/– model, because the primary defect may be myofilament disorganization.21 In the transverse aortic constriction model, SEA-0400 was an effective positive inotropic agent,12 consistent with reports that SERCA2A protein is decreased22,23 and NCX mRNA24 is increased by modest amounts, although a detailed characterization of the rate constants for these processes in the transverse aortic constriction model has not been reported.
A skeptic could also question the relevance of using rat or mouse models to study the role of NCX in normal or pathophysiological conditions. As mentioned above, the contribution of NCX to total Ca2+ removal in these species is roughly 4 times less than in larger mammals and humans; hence, the role of NCX has already been minimized by evolution. Of course, the overwhelming advantage of the mouse is the ability to manipulate its genome, yet transgenic knockout of NCX in this case has led to surprising results. Genetic ablation of NCX leads to embryonic lethality,25 indicating that it plays an essential role in development, but conditional NCX knockout using a Cre/Lox expression system showed that only a modest global functional deficit was present under normal conditions26 despite elimination of NCX1 in 80% to 90% of the myocytes (the Cre recombinase efficacy was not 100%). The explanation for why cardiac function is not severely compromised in NCX knockout mice is that marked remodeling of both the action potential and ICa,L occur to substantially decrease the magnitude of Ca2+ influx during each beat, without having a major effect on the ability to trigger Ca2+ release.26 Apparently, in adult myocytes, the smaller net uptake of Ca2+ over many beats is adequately extruded by the sarcolemmal Ca2+ pump, although in embryonic myotubes from NCX knockout mice, this capacity may be exceeded when Ca2+ influx is enhanced.27 Although this makes it difficult to use the knockout to define the role of NCX in normal myocytes, it speaks to the critical requirement for balancing Ca2+ influx and efflux, accomplished by a remarkably plasticity extending beyond acute autoregulation of Ca2+ fluxes. This is underscored by opposite adaptations leading to increased Ca2+ influx in NCX-overexpressing transgenic mice.28
In summary, the study by Ozdemir et al12 provides valuable insight into the potential for NCX inhibitors as inotropic agents in heart failure and confirms that SEA-0400 is approximately equipotent at blocking both the forward and reverse modes of NCX when intracellular Ca2+ is minimally buffered, with the net effect being to increase SR Ca2+ content in pig and mouse myocytes. However, the efficacy of agents like SEA-0400 in heart failure may be completely dependent on the specific alterations in Ca2+ handling present in the model. Moreover, the possible benefits must be carefully assessed in light of the importance of NCX in the beat-to-beat removal of Ca2+ from the myocyte, which can only be determined by further studies in large animals.
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Acknowledgments |
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Sources of Funding
The author is supported by NIH grants R37 HL54598, P01 HL081427, R33 HL087345.
Disclosures
None.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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 Top References |
|---|
1. Eisner DA, Trafford AW, Diaz ME, Overend CL, O'Neill SC. The control of Ca release from the cardiac sarcoplasmic reticulum: regulation versus autoregulation. Cardiovasc Res. 1998; 38: 589–604.
2. Bassani JW, Bassani RA, Bers DM. Relaxation in rabbit and rat cardiac cells: species-dependent differences in cellular mechanisms. J Physiol. 1994; 476: 279–293.
3. Pieske B, Maier LS, Bers DM, Hasenfuss G. Ca2+ handling and sarcoplasmic reticulum Ca2+ content in isolated failing and nonfailing human myocardium. Circ Res. 1999; 85: 38–46.
4. Bers DM. Excitation-Contraction Coupling and Cardiac Contractile Force. 2nd ed. Dordrecht, The Netherlands: Kluwer Academic Publishers; 2001.
5. Weber CR, Piacentino V III, Ginsburg KS, Houser SR, Bers DM. Na(+)-Ca(2+) exchange current and submembrane [Ca(2+)] during the cardiac action potential. Circ Res. 2002; 90: 182–189.
6. Armoundas AA, Hobai IA, Tomaselli GF, Winslow RL, O'Rourke B. Role of sodium-calcium exchanger in modulating the action potential of ventricular myocytes from normal and failing hearts. Circ Res. 2003; 93: 46–53.
7. 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.
8. Tanaka H, Nishimaru K, Aikawa T, Hirayama W, Tanaka Y, Shigenobu K. Effect of SEA0400, a novel inhibitor of sodium-calcium exchanger, on myocardial ionic currents. Br J Pharmacol. 2002; 135: 1096–1100.[CrossRef][Medline] [Order article via Infotrieve]
9. Kraft R. The Na+/Ca2+ exchange inhibitor KB-R7943 potently blocks TRPC channels. Biochem Biophys Res Commun. 2007; 361: 230–236.[CrossRef][Medline] [Order article via Infotrieve]
10. Birinyi P, Acsai K, Banyasz T, Toth A, Horvath B, Virag L, Szentandrassy N, Magyar J, Varro A, Fulop F, Nanasi PP. Effects of SEA0400 and KB-R7943 on Na+/Ca2+ exchange current and L-type Ca2+ current in canine ventricular cardiomyocytes. Naunyn Schmiedebergs Arch Pharmacol. 2005; 372: 63–70.[CrossRef][Medline] [Order article via Infotrieve]
11. Hobai IA, O'Rourke B. The potential of Na+/Ca2+ exchange blockers in the treatment of cardiac disease. Expert Opin Investig Drugs. 2004; 13: 653–664.[CrossRef][Medline] [Order article via Infotrieve]
12. Ozdemir S, Bito V, Holemans P, Vinet L, Mercadier J-J, Varro A, Sipido KR. Pharmacological inhibition of Na/Ca exchange results in increased cellular Ca2+ load attributable to the predominance of forward mode block. Circ Res. 2008; 102: 1398–1405.
13. Pogwizd SM, Qi M, Yuan W, Samarel AM, Bers DM. Upregulation of Na(+)/Ca(2+) exchanger expression and function in an arrhythmogenic rabbit model of heart failure. Circ Res. 1999; 85: 1009–1019.
14. Weisser-Thomas J, Kubo H, Hefner CA, Gaughan JP, McGowan BS, Ross R, Meyer M, Dillmann W, Houser SR. The Na+/Ca2+ exchanger/SR Ca2+ ATPase transport capacity regulates the contractility of normal and hypertrophied feline ventricular myocytes. J Card Fail. 2005; 11: 380–387.[CrossRef][Medline] [Order article via Infotrieve]
15. O'Rourke B, Kass DA, Tomaselli GF, Kaab S, Tunin R, Marban E. Mechanisms of altered excitation-contraction coupling in canine tachycardia-induced heart failure. I. Experimental studies. Circ Res. 1999; 84: 562–570.
16. Hobai IA, O'Rourke B. Enhanced Ca(2+)-activated Na(+)-Ca(2+) exchange activity in canine pacing-induced heart failure. Circ Res. 2000; 87: 690–698.
17. Piacentino V III, Weber CR, Chen X, Weisser-Thomas J, Margulies KB, Bers DM, Houser SR. Cellular basis of abnormal calcium transients of failing human ventricular myocytes. Circ Res. 2003; 92: 651–658.
18. Hobai IA, Maack C, O'Rourke B. Partial inhibition of sodium/calcium exchange restores cellular calcium handling in canine heart failure. Circ Res. 2004; 95: 292–299.
19. Minamisawa S, Hoshijima M, Chu G, Ward CA, Frank K, Gu Y, Martone ME, Wang Y, Ross J Jr, Kranias EG, Giles WR, Chien KR. Chronic phospholamban-sarcoplasmic reticulum calcium ATPase interaction is the critical calcium cycling defect in dilated cardiomyopathy. Cell. 1999; 99: 313–322.[CrossRef][Medline] [Order article via Infotrieve]
20. Esposito G, Santana LF, Dilly K, Cruz JD, Mao L, Lederer WJ, Rockman HA. Cellular and functional defects in a mouse model of heart failure. Am J Physiol Heart Circ Physiol. 2000; 279: H3101–H3112.
21. Su Z, Yao A, Zubair I, Sugishita K, Ritter M, Li F, Hunter JJ, Chien KR, Barry WH. Effects of deletion of muscle LIM protein on myocyte function. Am J Physiol Heart Circ Physiol. 2001; 280: H2665–H2673.
22. Shimura M, Minamisawa S, Takeshima H, Jiao Q, Bai Y, Umemura S, Ishikawa Y. Sarcalumenin alleviates stress-induced cardiac dysfunction by improving Ca2+ handling of the sarcoplasmic reticulum. Cardiovasc Res. 2008; 77: 362–370.
23. Kiriazis H, Sato Y, Kadambi VJ, Schmidt AG, Gerst MJ, Hoit BD, Kranias EG. Hypertrophy and functional alterations in hyperdynamic phospholamban-knockout mouse hearts under chronic aortic stenosis. Cardiovasc Res. 2002; 53: 372–381.
24. Spruill LS, Baicu CF, Zile MR, McDermott PJ. Selective translation of mRNAs in the left ventricular myocardium of the mouse in response to acute pressure overload. J Mol Cell Cardiol. 2008; 44: 69–75.[CrossRef][Medline] [Order article via Infotrieve]
25. 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.
26. Pott C, Philipson KD, Goldhaber JI. Excitation-contraction coupling in Na+-Ca2+ exchanger knockout mice: reduced transsarcolemmal Ca2+ flux. Circ Res. 2005; 97: 1288–1295.
27. Reuter H, Henderson SA, Han T, Mottino GA, Frank JS, Ross RS, Goldhaber JI, Philipson KD. Cardiac excitation-contraction coupling in the absence of Na(+)-Ca2+ exchange. Cell Calcium. 2003; 34: 19–26.[CrossRef][Medline] [Order article via Infotrieve]
28. Pott C, Goldhaber JI, Philipson KD. Homozygous overexpression of the Na+-Ca2+ exchanger in mice: evidence for increased transsarcolemmal Ca2+ fluxes. Ann N Y Acad Sci. 2007; 1099: 310–314.[CrossRef][Medline] [Order article via Infotrieve]
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