Cardiovascular Research

Cardiovascular Research 1997 34(1):48-54; doi:10.1016/S0008-6363(97)00044-8
© 1997 by European Society of Cardiology

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Copyright © 1997, European Society of Cardiology

The regulation of intracellular pH in the diabetic myocardium

Danielle Feuvray^*

Laboratoire de Physiologie Cellulaire, Université Paris XI, Bât. 443, 91405 Orsay Cedex, France

^* Tel.: +33 (1) 69 15 78 98; fax: +33 (1) 69 15 68 41.

Received 25 October 1996; accepted 13 January 1997

KEYWORDS pH_i regulation; Diabetes; Na⁺/H⁺ exchange; Na⁺- and HCO₃^–-dependent alkalinising transporter; Lactate–H⁺ co-transport

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	1 Introduction

Top 1 Introduction 2 pHi regulation in... 3 pHi regulation in... 4 Concluding remarks References

 
In myocardial cells, as in any given cell, steady-state intracellular pH (pH_i) is strictly maintained within a narrow range at relatively alkaline values. Resting pH_i (7.1–7.2) is determined by the algebraic sum of acid-loading and acid-extruding processes. Whenever acid-loading exceeds acid extrusion, pH_i falls. The degree to which pH_i changes is inversely related to the intracellular buffering power (β_i). The role of intracellular buffering power, which is the first line of defence of a cardiac cell against an intracellular acid–base disturbance, is to moderate pH_i changes produced by acute acid (or alkali) load [1]. However, buffering mechanisms cannot prevent a change in pH_i, but only reduce its amplitude. The regulation of pH_i then largely depends upon the activity of plasma membrane carrier-mediated transport of acid/base equivalents [2, 3]. Intracellular pH is important for the activity of a number of enzymes with optimal pH within the physiological pH range, as well as for the conductivity of ion channels [4], calcium homeostasis, and the efficiency of contractile elements [5, 6].

Despite a vast literature demonstrating myocardial metabolic changes associated with diabetes (for reviews, see Refs. [7, 8]), until recent years relatively few studies have addressed the effects of diabetes on intracellular pH. Yet, disturbances in intracellular pH or in the processes regulating intracellular pH may be expected to occur in diabetic hearts as a result of either altered cellular metabolism and/or cellular and subcellular membrane changes. This review provides a summary of the data obtained with both multicellular and single cell preparations, as well as with isolated hearts from chemically-induced diabetic rats.

	2 pHi regulation in multicellular and single cell preparations

Top 1 Introduction 2 pHi regulation in... 3 pHi regulation in... 4 Concluding remarks References

 
The first study that allowed direct measurement of intracellular pH in cardiac cells of streptozotocin (STZ)-induced diabetic rat hearts was performed by Lagadic-Gossmann et al. in isolated papillary muscles, using pH-sensitive microelectrodes [9]. The results of these authors showed that there were no differences in steady-state pH_i values between diabetic muscle and normal muscle under control bicarbonate-buffered superfusate conditions. However, differences between diabetic and normal muscles were found in the regulation of pH_i in response to induced acidification, though not to alkalinization. The absence of any difference between steady-state pH_i values of normal and diabetic myocardial cells under control bicarbonate buffered conditions has been further documented by more recent works using nuclear magnetic resonance (NMR) in isolated perfused rat hearts [10], as well as using fluorescent probes with pH-dependent fluorescence in isolated myocytes [11]. In papillary muscles, Lagadic-Gossmann et al. [9]used the NH₄Cl method to investigate the problem of pH_i regulation. The effects of external NH₄Cl on pH_i were first described in squid giant axons by Boron and De Weer [12]where, following an initial rapid intracellular alkalinisation due to a rapid influx of NH₃, pH_i recovers as NH₄⁺ passively enters and carries H⁺ into the cell. This results in a secondary slow acidification which has also been described [13]as being at least partly mediated by Cl^–/HCO₃^– exchange, the only acidifying ionic exchanger definitively identified in cardiac cells [14]. These investigators showed that the amplitude of alkalinisation and the time course of pH_i recovery from alkalosis (Fig. 1) were similar in both normal and diabetic papillary muscles. Likewise, recovery from alkalinisation in both preparations was similarly delayed by the disulphonic stilbene derivative, DIDS (Fig. 1), an inhibitor of anion movements [15]. This suggested the participation of Cl^–/HCO₃^– exchange in recovery from alkalosis in rat myocardial cells, whose activity is not changed by diabetes. On the other hand, the amplitude of the acidification induced by the withdrawal of NH₄⁺ (which gives up a hydrogen ion and leaves the cell as NH₃) was markedly increased in diabetic papillary muscles (+67%) as compared to normal muscles. Moreover, there was a marked slowing down of the recovery from acidosis in the diabetics (Fig. 1). The differences in the amplitude of NH₄⁺ withdrawal-induced acidification and in the time course of pH_i recovery from acidification were abolished by amiloride, a known blocker of Na⁺/H⁺ exchanger [16]. Therefore, these findings suggested that diabetes had induced a decrease in the activity of amiloride-sensitive Na⁺/H⁺ exchange, one of the main mechanisms of acid extrusion from cardiac cells, so that pH_i recovery from acidification took place more slowly than in normal papillary muscles.

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Continuous recording of intracellular pH (pHi) of a normal and of a diabetic papillary muscle (D), during addition and subsequent withdrawal of 20 mM NH4Cl in the superfusate. STZ-treated (D; 40 mg/kg b.wt. of STZ) and age-matched control rats were used 3–4 weeks later. Papillary muscles were superfused with Krebs' bicarbonate buffer at 36±1°C. pH measurements were made using resin-filled H+-selective microelectrodes. pHi = change in intracellular pH. *P<0.05 compared with normal papillary muscles. Reproduced from Lagadic-Gossmann et al. [9]with permission.

 
A striking depression of the exchanger was also reported by Pierce and colleagues [17]in a study in which Na⁺/H⁺ exchange was measured as intravesicular H⁺-dependent ²²Na⁺ uptake into sarcolemmal vesicles isolated from hearts of diabetic rats. Although the latter study used diabetic animals 8–10 weeks after the injection of streptozotocin, their conclusions were similar to that of the previous study by Lagadic-Gossmann et al. [9]who used diabetic rats 3–4 weeks after STZ injection. This indicates that these effects of diabetes occur early and are maintained at least for more than 2 months. Because diabetes is essentially a disease of metabolism, L-propionylcarnitine treatment of the diabetic animals was carried out [17]to test the possible involvement of abnormal carnitine metabolism. Abnormalities in carnitine metabolism have been shown in diabetic hearts, and it has been suggested that they may play a role in heart dysfunction [18, 19]. However, no effect of L-propionylcarnitine was observed on the depressed Na⁺/H⁺ exchange in the cardiac sarcolemmal vesicles [17].

The above studies did not measure the degree of diabetes-associated reduction in the activity of the exchanger. This was done more recently in ventricular cells isolated from diabetic rat hearts, using the intracellular fluoroprobe, carboxy-SNARF-1 [20]. In these isolated cells, as in papillary muscle cells [9], pH_i recovery following acidification was significantly slowed down by diabetes. As HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid) was used as extracellular buffer, Na⁺/H⁺ exchange was the main mechanism regulating pH_i following the acid load [21]. In order to determine the effect of diabetes on the acid efflux carried by Na⁺/H⁺ exchange, the authors [20]first estimated the intracellular intrinsic H⁺ buffering power (β_i) over the 6.85–7.45 pH_i range, using the stepwise removal of external NH₄Cl [22]. Their results showed that, whereas diabetes did not change β_i, it significantly decreased acid efflux (J_H^e) through Na⁺/H⁺ exchange (Fig. 2). For example, a 42% reduction in the acid efflux was estimated at pH_i 6.9, when compared to normal.

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 (A) Effects of diabetes on the pHi dependence of intracellular intrinsic buffering power (βi) in rat isolated ventricular myocytes. (B) Effects of diabetes on pHi dependence of the acid efflux carried by Na+/H+ exchange. Reproduced from Le Prigent et al. [20]with permission.

 
However, the mechanism underlying the slowed activity of the exchanger is still unknown. It is possible that a significant decrease in expression of the Na⁺/H⁺ exchanger may be responsible. However, examination of mRNA levels in hearts from diabetic rats 1 or 8 weeks after STZ injection revealed no significant change in comparison to control rats [23]. The activity of the ubiquitous or ‘housekeeping’ form of Na⁺/H⁺ exchanger (the NHE-1 type—i.e., the major form in the myocardium, see Ref. [24]) has been shown to rely upon the inward Na⁺ gradient [25]and upon intracellular and extracellular H⁺ [25, 26]. This exchanger is also known to be controlled by membrane composition and fluidity [27]and by various factors, such as growth factors (for review, see Ref. [28]) and neurotransmitters (for review, see Ref. [14]). In this context, several cellular changes associated with diabetes might account for the depressed activity of Na⁺/H⁺ exchange. For instance, the altered membrane composition [29], through a change in the microenvironment of the exchanger, may induce a shift in its affinity for extracellular and intracellular H⁺ and Na⁺. Although a detailed kinetic analysis was not performed in the study by Pierce et al. [17], it is possible to get an indication that the affinity of the exchanger for at least H⁺ has changed in diabetes. A diabetes-induced change in the pH_e dependence may also appear as a candidate to underlie Na⁺/H⁺ exchange depression. However, most recent investigations are not in favour of such an hypothesis [20]. Indeed, it has been found that an increase or a decrease in the external pH elicited similar relative (to control) changes in steady-state pH_i and in the acid efflux carried by the exchanger, in both normal and diabetic myocytes [20]. It is worth noting that an increase in intracellular Na⁺ activity (a_Naⁱ) has been detected in diabetic papillary muscles [30]. This increase may well contribute to the depressed activity of the exchanger through a decrease in the transmembrane Na⁺ gradient. The increased a_Naⁱ could also play an important and direct inhibitory role here, possibly by competing with H⁺ binding to the internal transport site of the exchanger [31]. However, such a role remains to be demonstrated in cardiac tissue. Another interesting point that came from the study using papillary muscles [30]was that the increase in a_Naⁱ after an acid load was markedly slowed in muscles from diabetic rats. This may have important consequences, particularly on the contractile response to acidosis.

Alterations in intracellular Ca²⁺ (Ca_i²⁺) handling have been demonstrated in diabetic cardiomyopathy (see other articles in this issue). In particular, some recent studies have shown a decrease in basal Ca_i²⁺ level in ventricular myocytes isolated from diabetic rats [11, 32]. Though not yet established in the heart, recent reports have shown that the NHE-1 isoform of the Na⁺/H⁺ exchanger is modulated by intracellular calcium, so that increasing Ca_i²⁺ induces a stimulation of the exchange [33]. It could then be hypothesized that the reduced basal Ca_i²⁺ level after a few weeks of STZ-induced diabetes [11, 32]might be, at least partly, responsible for the depressed Na⁺/H⁺ exchange activity. Most recent experiments carried out with Ca²⁺-buffered cells tend to favour this idea. Indeed, when Ca_i²⁺ was buffered to a low level, the acidic shift of the pH_i dependence for Na⁺/H⁺ exchange was more pronounced in normal than in diabetic myocytes. Moreover, it was noted that the acid efflux carried by the exchange then became identical in both groups [20]. Several recent studies indicate that the control of NHE-1 activity by calcium may go through at least two different pathways: (i) through direct binding of Ca²⁺/calmodulin (CaM) to the exchanger and/or (ii) through phosphorylation by the Ca²⁺/CaM-dependent protein kinase II. With respect to the first pathway, it has been reported that NHE-1 in fibroblasts is a Ca²⁺/CaM-binding protein [34]. Following an increase in Ca_i²⁺ (induced by ionomycin addition), the direct binding of Ca²⁺/CaM to NHE-1 then stimulates the exchange by eliciting an alkaline shift in its pH_i dependence [33]. As to the second pathway, Fliegel and coworkers [35]have shown that the purified protein of the cardiac Na⁺/H⁺ exchange could be directly phosphorylated by CaM kinase II. To our knowledge, the first evidence concerning the regulation, by this kinase, of the exchanger in a cellular system came from recent investigations in cardiac ventricular myocytes [20]. This study demonstrated that inhibition of CaM kinase II under acid load conditions results in a significant reduction in Na⁺/H⁺ exchange activity. This raises the interesting possibility of basal control of cardiac NHE-1 exchange activity by direct CaM-kinase-dependent phosphorylation. Moreover, results suggest that this calcium-dependent regulatory pathway is probably affected by diabetes [20].

Besides the Na⁺/H⁺ antiport, a Na⁺- and HCO₃^–-dependent alkalinising carrier mechanism was described several years ago in mammalian cardiac cells from guinea-pig [21]. It was of interest to determine a possible contribution of the bicarbonate-dependent mechanism alone in the slowing down of pH_i recovery from acidification induced by diabetes. This has very recently been investigated in ventricular myocytes from diabetic rat hearts [36]. The conclusion was that the HCO₃^–-dependent pH_i recovery was not significantly different in the diabetic cell when compared to that recorded in the normal cell. Moreover, the calculation of acid-equivalent efflux carried by the Na⁺- and HCO₃^–-dependent carrier over the 6.75–6.90 pH_i range showed no difference between normal and diabetic cells, indicating that carrier activity is unaffected by diabetes over the pH_i range studied. We have previously seen that acid efflux carried by the Na⁺/H⁺ exchange is markedly reduced in diabetic myocytes following an intracellular acid load [20]. On the contrary, the study just mentioned shows that acid efflux carried by the Na⁺ and HCO₃^– dependent alkalinising carrier remains nearly identical in both normal and diabetic cells [36]. Thus, Le Prigent et al. (unpublished results) have estimated that in normal (adult) myocytes, the contribution of the total efflux of H⁺ equivalents of the Na⁺/H⁺ exchanger and of the Na⁺- and HCO₃^–-dependent carrier to be 69 and 31%, respectively, at pH_i 6.90, and 67% and 33% respectively, at pH_i 6.75. In diabetic adult myocytes, not only is the contribution of the Na⁺-and HCO₃^–-dependent carrier increased up to 38% at pH_i 6.90, but it even becomes predominant over the Na⁺/H⁺ exchanger contribution at pH_i 6.75, reaching 58% of the total acid efflux. In other words, these data suggest that pH_i regulation following an intracellular acid load may become more dependent on Na⁺-and HCO₃^–-dependent alkalinising transporter activity in diabetic cardiac cells.

The participation of a lactate-H⁺ co-transporter, similar to the monocarboxylate/proton carrier found in the membrane of many cell types [37]has also been recognized in the perfused heart [38], isolated sarcolemmal vesicles [39, 37]and cardiac myocytes [40, 41]. This transporter is inhibited by -cyano-4-hydroxycinnamate and stilbene disulphonates such as DBDS (4,4'-dibenzamidostilbene-2,2'-disulphonate) [40, 41]. Studies by Wang et al. [41]have indeed demonstrated the existence of two distinct lactate carriers in heart cells. This demonstration raised the question as to whether both carriers might be present in the same cell, or whether there might be two populations of cells, each with a different lactate carrier. The authors addressed this question by studying lactate transport in a single cell [42]. Their data are consistent with two isoforms of the lactate carrier that are both lactate–H⁺ symports, one sensitive and one insensitive to DBDS. They coexist within a single myocyte and both have a stoichiometry of 1 lactate:1 proton. The proportion of these two isoforms shows little variation between cells of the same species, but there are major differences between guinea-pig and rat. Thus guinea-pig cardiac myocytes contain a greater proportion of the DBDS-sensitive carrier, whereas those from the rat have a greater proportion of the DBDS-insensitive carrier. Whether a diseased state such as diabetes may affect the characteristics and/or the relative proportion of one or other lactate–H⁺ symport isoform in cardiac cells is a question that has so far not been addressed.

	3 pHi regulation in isolated hearts

Top 1 Introduction 2 pHi regulation in... 3 pHi regulation in... 4 Concluding remarks References

 
The initial observation [9]of a change in the regulation of pH_i with a marked decrease in the activity of the Na⁺/H⁺ exchanger in papillary muscles from STZ-diabetic rat hearts prompted further investigations in whole ischaemic hearts. Khandoudi et al. [10]used phosphorus nuclear magnetic resonance (NMR) spectroscopy in isolated working hearts from such diabetic rats to ascertain the influence of pH_i and ion exchange during ischaemia and reperfusion. Myocardial ischaemia involves both inadequate supply of metabolic substrates to the cells and inadequate removal of the products of metabolism, notably protons [43–45]. An accumulation of protons during low-flow or zero-flow ischaemia can have dramatic consequences through activation of the sarcolemmal Na⁺/H⁺ exchange. In view of the direct inhibition of Na⁺/H⁺ exchange by decreased external pH [26], activation of the ionic exchange can only occur, if at all, at the very beginning of an ischaemic episode. Moreover, it will occur particularly when the heart is reperfused at a physiological pH. The pH_i values of diabetic hearts were found, here again, as in papillary muscles [9], to be identical to those of normal hearts under control bicarbonate-buffered perfusate conditions. However, diabetic hearts showed a somewhat slower fall in pH_i during a zero-flow ischaemia, although the mean value reached after 30 min did not differ significantly from that of normal hearts (Fig. 3). The slightly slower fall in pH_i during ischaemia may be explained by a reduced rate of anaerobic glycolysis in those diabetic rats [46]. The main point coming from the study by Khandoudi et al. [10]was that upon reperfusion following ischaemia, pH_i recovery occurred more slowly in the diabetic than in the normal hearts (Fig. 3). This was observed in spite of the absence of any significant difference in coronary flow rate on reperfusion between the two groups of hearts. The possibility of a decrease in proton washout to explain the slow kinetics of recovery can therefore be excluded. The most plausible explanation appeared to be the depressed activity of the Na⁺/H⁺ exchange process previously shown in papillary muscle cells of rats with similar STZ-induced diabetes [9]. Moreover, when the Na⁺/H⁺ exchange process was pharmacologically inhibited by the presence of amiloride in normal hearts, a slow pH_i recovery was observed (Fig. 3). The consequences of these differences in the kinetics of pH_i recovery after reperfusion, on the functional recovery of reperfused hearts, are presented and discussed elsewhere in this issue (see review by D. Feuvray and G.D. Lopaschuk). Briefly, the higher functional recovery on reperfusion, as assessed by the recoveries of aortic flow and stroke volume, was observed for those hearts with slower pH_i recovery. These data, together with those from studies showing that inhibition of the antiporter could reduce Na⁺ accumulation during reperfusion [47, 48], have revealed the determinant role of the Na⁺/H⁺ exchanger in the modulation of the cardiac response to reperfusion. In addition, the diabetic heart thus provided an interesting physiological model for experimentation on the Na⁺/H⁺ exchanger.

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Time courses of changes in intracellular pH (pHi) during ischaemia and reperfusion of normal hearts without amiloride (), with amiloride given before ischaemia and during reperfusion () and of diabetic hearts (). Working heart preparations were submitted to a zero-flow ischaemic period of 30 min at 37°C and then reperfused for 30 min. The time course of pHi decline during ischaemia and of recovery on reperfusion was followed by means of 31P-NMR. *P<0.05 vs. normal hearts without amiloride. Reproduced from Khandoudi et al. [10]with permission.

 
To date, no specific sarcolemmal transport mechanisms other than the Na⁺/H⁺ exchanger have been reported to contribute to pH_i regulation during post-ischaemic reperfusion of diabetic hearts. HCO₃^–-activated acid efflux [21]as well as lactate–H⁺ co-transport [37]were likely candidates. Their possible contribution was recently examined under different experimental perfusion buffer conditions (i.e., HEPES or CO₂/HCO₃^–) [49]. Our study showed that an HCO₃^–-dependent mechanism contributes to pH_i recovery after ischaemia in hearts from diabetic rats, as well as in hearts from normal rats. In addition, when the Na⁺/H⁺ exchanger was pharmacologically blocked in nominally HCO₃^–-free solution, a rapid rise in pH_i still occurred at the very beginning of reperfusion (during the first 2–3 min); this was, however, less abrupt in diabetic hearts. This early rise in pH_i, which could be reduced by supplying external lactate and inhibited by -cyano-4-hydroxycinnamate in the two groups of hearts, suggests that a coupled H⁺–lactate efflux may be a major mechanism for acid extrusion in the initial stage of reperfusion. This mechanism may be less stimulated in diabetic hearts exhibiting a less abrupt initial pH_i recovery, simply because the tissue lactate accumulated at the end of ischaemia was found to be significantly lower in those diabetic hearts than in normal hearts.

The HCO₃^–-dependent pH_i recovery was not shown to require sodium in the study where whole perfused hearts were used [49]. However, recent work using normal and diabetic rat isolated ventricular myocytes has demonstrated the Na⁺-dependency of the HCO₃^–-dependent process [K. Le Prigent, unpublished results]. In whole perfused hearts [49], comparison of the kinetics of pH_i recovery on reperfusion between normal and diabetic hearts receiving HCO₃^–/CO₂ buffer in the presence of a pharmacological block of the Na⁺/H⁺ exchanger seemed to suggest a reduction in the activity of HCO₃^–-activated pH_i regulation in diabetic hearts. However, as reported in the above section, recent experiments performed with single myocytes show that Na⁺- and HCO₃^–-dependent alkalinising transporter activity is unaffected by diabetes over the pH_i range studied (6.8–7.0). In fact, one has to consider that the pH_i recovery at each time point results from the combined activity of several systems. For example, over the 6.2–6.8 pH_i range in the reperfused diabetic hearts receiving HCO₃^–/CO₂ buffer in the absence of an operating Na⁺/H⁺ exchange, the two main systems likely to be involved are Na⁺- and HCO₃^–-dependent co-transport and lactate–H⁺ co-transport. We have seen that the latter may be less important due to less ischaemia-induced tissue lactate accumulation. This may help to explain the slowed-down pH_i recovery in diabetic hearts. Alternatively or together, the possibility cannot be dismissed of a different sensitivity of the diabetic heart, as compared to the normal heart, to neurohormonal modulation of some pH_i-regulating transporters, especially of the bicarbonate-dependent mechanism [50](for review, see Ref. [14]), which may interfere during reperfusion.

	4 Concluding remarks

Top 1 Introduction 2 pHi regulation in... 3 pHi regulation in... 4 Concluding remarks References

 
It has been demonstrated that STZ-induced diabetes mellitus of several weeks duration does not change steady-state pH_i but significantly alters pH_i regulation in cardiac cells by mainly and markedly decreasing the activity of the sarcolemmal Na⁺/H⁺ exchanger. Interestingly, and in contradistinction to the Na⁺/H⁺ exchanger, the activity of the Na⁺- and HCO₃^–-dependent alkalinising transporter remains unchanged in diabetic cardiac cells, at least over the 7.05–6.75 pH_i range studied. Although the mechanism(s) underlying the depressed activity of the Na⁺/H⁺ exchanger is still unknown, several cellular changes associated with diabetes may account for it. Among these, recent evidence has directed attention in favour of altered modulation of the exchanger by intracellular calcium. Whether a calcium-dependent regulatory pathway may be affected because of altered intracellular Ca²⁺ handling related to diabetes, and/or because of alterations in a Ca²⁺/calmodulin-dependent protein kinase II, remains to be clearly demonstrated. This does not rule out any other alterations such as a pre-translational modification of the Na⁺/H⁺ exchanger upon diabetes, or any influence of changes in the membrane composition and environment of the exchanger. Future studies should explore these possibilities. In this respect it is worth noting that the effects of diabetes on Na⁺/H⁺ exchange were reversed towards normal after in vivo administration of insulin to diabetic rats, in papillary muscles [Lagadic-Gossmann et al., unpublished results] as well as in the isolated ischaemic/reperfused heart [Khandoudi et al., unpublished results].

Neurohormonal regulation of cardiac pH_i upon diabetes could be another important aspect to explore in future studies. Indeed, in a diseased state such as diabetes where the concentration of neurohormones, the density of membrane receptors and/or the sensitivity of receptor stimulation may be changed, the activation of pH_i-regulatory mechanisms may be consequently altered. To our knowledge, neurohormonal modulation of pH_i-regulating transporters in diabetic hearts has, so far, not been investigated.

Studies examining pH_i regulation following an ischaemic insult in diabetic hearts have made it possible to highlight the critical role played by the Na⁺/H⁺ exchanger in the modulation of the cardiac response to reperfusion. The diabetic heart thus provides an interesting model for experimentation on the Na⁺/H⁺ exchanger, and has also proved very useful when studying damage associated with ischaemia/reperfusion.

Time for primary review 25 days.

	Acknowledgements

 
Research described in this review was funded by grants from I.N.S.E.R.M. and from the M.E.S.R. Programme DSPT.5 Biologie, Médecine et Santé, to D. Feuvray. The valuable assistance of Mrs. Françoise James in the preparation of this manuscript is gratefully acknowledged.

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