© 2001 by European Society of Cardiology
Copyright © 2001, European Society of Cardiology
Overexpression of the Na/Ca exchanger and reduced SERCa function
Department of Cardiac Medicine, National Heart and Lung Institute, Imperial College, Dovehouse Street, London SW3 6LY, UK
* Corresponding author. Tel.: +44-171-352-8121 ext 3322/2097; fax: +44-207-351-8145
Received 23 January 2001; accepted 30 January 2001
Dear Sir,
We have read with great interest Dr Isenberg's editorial [1] published with our article [2] in Cardiovascular Research. Dr Isenberg's analysis highlights the numerous problems in studying Na/Ca exchanger function and Ca regulation and stresses once again that these areas of physiology are far from being completely understood. We feel, however, that some clarification is required on the following points that arise from his assessment of our article:
Use of AM-forms of fluorescent indicators vs. patch-pipette loading
Dr Isenberg's criticism that the possible compartmentalisation of the AM form of Indo-1 may affect our quantification of Ca fluxes is justified. The issue is not new and there are many unresolved problems with the use of this form of the indicator. Undoubtedly loading of the AM-forms is difficult to control and a possible overloading or loading into sub-cellular compartments cannot be excluded. In this respect, patch-pipette loading should be preferred. However, the reader could be misled to believe that the latter technique should have been used in our experiments. Patch-pipette loading is not a valid alternative to the AM-form of loading when the aim of the experiments is to investigate the physiological role of the Na/Ca exchanger in the intact cell. Na/Ca exchanger function is dependent on extracellular and intracellular [Ca] and [Na]. It is likely that [Ca]pip and [Na]pip modify the normal intracellular concentrations of these two ions. Any Ca buffer (e.g. BAPTA) used in the pipette will also influence the cytoplasmic Ca buffering and so the cellular [Na] and [Ca] will not be physiologically relevant.
Mechanisms by which overexpressed Na/Ca exchanger could augment SR Ca filling
The calculations presented in the editorial [1] on assessing Na/Ca exchanger function during the cardiac cycle are intriguing but depend greatly on the assumption that cytoplasmic [Na] is 9.6 mM. We will comment on this later. There are two further points to make regarding the calculations that may not be immediately apparent to the Reader. Firstly, it is important to note that the model cell is not in a steady-state. The size and time-course of the Ca transient are fixed and, as Dr Isenberg mentions in the legend, the model does not incorporate effects of Ix on other Ca fluxes including the SR. Thus, there is no physiological relationship between one beat to the next. The Na/Ca exchanger normally removes the Ca which enters the cell to trigger Ca release (i.e. L-type Ca current). Assuming that other sarcolemmal Ca extrusion mechanisms have a minimal importance, the net amount of Ca handled by the Na/Ca exchanger must be in the direction of Ca extrusion at the steady-state. This is not considered in Dr Isenberg's analysis. Therefore the second point that needs to be made is that the calculations are only a representation of net Ca fluxes mediated by the Na/Ca exchanger. They do not provide a clue as to the net Ca fluxes across the surface membrane during the cardiac cycle.
In addition to the amount of Ca removed via the Na/Ca exchanger to balance Ca entry via L-type Ca channels, some more Ca is removed by the Na/Ca exchanger to balance Ca entry via the exchanger itself. It is the amount of this Ca that is influenced by the overexpression of the Na/Ca exchanger and can affect the filling state of the SR. If there is more Ca entry, there is more SR Ca uptake to increase SR Ca content. So if 2.5 times more exchanger is present, a significantly larger (2.5 times more?) Ca entry follows. More SR Ca content results in a faster and larger SR Ca release at the next beat to change the Ca transient characteristics in favour of more Ca extrusion. Over several beats a new steady-state SR Ca content is established in myocytes from the heart of transgenic mice (TG) which is greater than in control myocytes. However, even in this condition, a balance between fluxes is required and the integral of Ix at the end of the cardiac cycle should be negative again to balance Ca influx via L-type Ca current. This is an important point not fully presented by Dr Isenberg.
In non-steady-state conditions, when SR Ca levels and Ca fluxes are changing, Dr Isenberg's analysis can be expanded to model the effect of changes to [Na]i (Fig. 1).
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The value for [Na]i used by Dr Isenberg in his model may be incorrect. This value for [Na]i was measured by us in a previous study in which very specific experimental conditions were used [3]. In those experiments, designed to measure Ix in TG myocytes using voltage-ramps, we used a large number of channel blockers and Ca buffers, as have many other investigators [4,5]. We were able to show that in TG myocytes, outward Ix is increased, supporting a direct functional overexpression of the Na/Ca exchanger. In control and TG myocytes we obtained similar values for Erev and [Na]i (calculated from Erev) suggesting that [Na]i is probably equally regulated in the two groups. However we were careful not to imply that the value of [Na]i measured in those very specific conditions is the one present during normal stimulation in intact myocytes. The value of 9.6 mM for [Na]i used by Dr Isenberg in his calculations may therefore not be appropriate. Yao et al., using fluorescent techniques, have measured values for [Na]i around 15 mM in both TG and control myocytes [6]. If we use this value of [Na]i in a similar analysis to that used by Dr Isenberg then the Na/Ca exchanger appears to work in Ca influx mode for a large proportion of time (Fig. 1,c). If 12 mM [Na] is used in the calculations (roughly halfway between [Na] used in a and c) there is still a predominant outward Ix during the cardiac cycle (Fig. 1,b). These calculations are based on using bulk cytoplasmic [Na]. Subsarcolemmal [Na] levels should have been used but these are not known.
In conclusion we believe that there are many fundamental issues of, for example, absolute quantification of subsarcolemmal [Na] and [Ca], the thermodynamics of the Na/Ca exchanger itself [7] and the role of feed-back mechanisms that need to be addressed before we are in the position to be able to model Ca movements accurately.
Application of the experiments in TG mice to the failing heart
In the last paragraph of the editorial, Dr Isenberg seems to agree that an increase in Na/Ca exchanger activity can compensate for a reduction in SERCa activity. He seems to disagree on how such compensation is achieved, particularly if one extrapolates to the situation in human heart failure. Although we made some comparisons with this pathological condition in our article [2] we were careful to state that our conclusions cannot necessarily be applied to the situation in human heart failure because of the complexity of this condition and "the possible different roles of the Ca regulatory mechanisms in the human myocardium". The difficulty lies in assessing the relative roles of such mechanisms in human cardiac muscle and, as Dr Isenberg points out, this needs more experimentation. Our paper produces experimental evidence that Na/Ca exchanger overexpression can compensate for SERCa inhibition and proposes that this could be a mechanism acting during one of the stages of heart failure. Whether this is an important mechanism and/or whether it functions in a different way than is described can only be determined by further experimental evidence.
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- Isenberg G. How can overexpression of Na/Ca exchanger compensate the negative inotropic effects of downregulated SERCa? Cardiovascular Research (2001) 49:1–6.
[Free Full Text] - Terracciano C.M.N, Philipson K.D, MacLeod K.T. Overexpression of the Na+/Ca2+ exchanger and inhibition of the sarcoplasmic reticulum Ca ATPase in ventricular myocytes from transgenic mice. Cardiovascular Research (2001) 49:38–47.
[Abstract/Free Full Text] - Terracciano C.M.N, De Souza A, Philipson K.D, MacLeod K.T. Na+/Ca2+ exchange and sarcoplasmic reticular Ca2+ regulation in ventricular myocytes from transgenic mice overexpressing the Na+/Ca2+ exchanger. Journal of Physiology (1998) 512–3:651–667.
- Kimura J, Miyamae S, Noma A. Identification of sodium-calcium exchange current in single ventricular cells of guinea-pig. Journal of Physiology (1987) 384:199–222.
[Abstract/Free Full Text] - Kimura J, Noma A, Irisawa H. Na-Ca exchange current in mammalian heart cells. Nature (1986) 319:596–597.[CrossRef][Medline]
- Yao A, Su Z, Nonaka A, Zubair I, Lu L, Philipson K.D, Bridge J.H.B, Barry W.H. The effects of overexpression of the Na/Ca exchanger on [Ca2+]i transients in murine ventricular myocytes. Circulation Research (1998) 82:657–665.
[Abstract/Free Full Text] - Fujioka Y, Hiroe K, Matsuoka S. Regulation kinetics of Na+/Ca2+ exchange current in guinea-pig ventricular myocytes. J Physiol (2000) 529:611–623.
[Abstract/Free Full Text]
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