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Cardiovascular Research 2000 47(4):688-696; doi:10.1016/S0008-6363(00)00136-X
© 2000 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

Changes in intracellular sodium and pH during ischaemia–reperfusion are attenuated by trimetazidine

Comparison between low- and zero-flow ischaemia

Houda El Banania, Monique Bernardb, Delphine Baetza, Emmanuel Cabanesb, Patrick Cozzoneb, Arnaud Lucienc and Danielle Feuvraya,*

aLaboratoire de Physiologie Cellulaire, Université Paris XI, Orsay, France
bC.R.M.B., Faculté de Médecine Timone, Marseille, France
cI.R.I.S., Courbevoie, France

* Corresponding author. Physiologie Cellulaire, Université Paris Xi, Bât 443, 91405 Orsay Cedex, France. Tel.: +33-1-6915-7898; fax: +33-1-6915-6841 daniellefeuvray{at}ibaic.u-psud.fr

Received 10 December 1999; accepted 2 May 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The aim of this study was to investigate whether trimetazidine (TMZ; 10–6M), which has been shown to inhibit fatty acid oxidation, reduces the ionic imbalance induced by ischaemia and reperfusion, especially through an attenuation in intracellular changes in H+ and Na+. Methods: Isovolumic rat hearts receiving 5.5 mM glucose and 1.2 mM palmitate as metabolic substrates were exposed to zero-flow ischaemia (TI) or low-flow ischaemia (LFI — coronary flow decreased by an average of 90%) (30 min at 37°C) and then reperfused. 23Na nuclear magnetic resonance (NMR) spectroscopy was used to monitor intracellular Na+ (Na+i) and 31P NMR spectroscopy was used to monitor intracellular pH (pHi). Results: During LFI the major effect of TMZ was a significant reduction in intracellular acidosis, whereas during TI the main effect of TMZ was a significant reduction in Na+i gain. In addition, the further gain in Na+i that occurred during the first minutes of reperfusion following TI, and to a far lesser extent following LFI, was suppressed in TMZ-treated hearts and also suppressed when hearts were perfused without fatty acid. In both LFI and TI, TMZ-induced attenuation of ionic imbalance was associated with a significantly improved recovery of ventricular function on reperfusion, as assessed by a lower increase in diastolic pressure and an increased recovery of developed pressure. Conclusion: Our data provide evidence that specific myocardial metabolic modulation plays a significant role in reducing ionic imbalance during ischaemia and reperfusion.

KEYWORDS Acidosis; Intra/extracellular ions; Ischemia; NMR; Reperfusion; Ventricular function

This article is referred to in the Editorial by H.R. Cross (pages 637–639) in this issue.


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Increasing interest in the role of myocardial metabolic modulation during ischaemia and reperfusion over the last 10 years has seen the development of several classes of specific agents. The common link in many of the strategies of metabolic manipulation is the attempt to increase the capacity for glucose oxidation and to limit that of fatty acid oxidation [1]. Among the possible agents that have been proposed as specific modulators of cardiac metabolism are piperazine compounds. This group of compounds includes the agent trimetazidine (1-[2,3,4-trimethoxybenzyl]-piperazine; TMZ). The primary effect of piperazine compounds is an inhibition of the β-oxidation pathway of fatty acid metabolism, which results in an increase in glucose oxidation [2]. In a previous study we have shown that TMZ significantly reduced the ischaemia-induced increase in acyl carnitine levels, determined as indicators of fatty acid utilization [3]. Preservation of mitochondrial oxidative function by inhibition of palmitoylcarnitine oxidation has been implicated in studies with isolated myocytes [4]. Recent work has demonstrated that TMZ stimulates glucose oxidation in the heart, evidently by virtue of a selective inhibition of long-chain fatty acid β-oxidation [5]. TMZ has been found to have anti-anginal effects in the absence of β-adrenergic receptor blockade, or vasodilator properties [6,7]. It has also been shown to have cardioprotective effects during coronary artery bypass graft surgery [8] and catheter angioplasty [9]. However, data regarding the precise mechanism of action of TMZ are incomplete.

Long-chain fatty acids can be detrimental to the ischaemic and reperfused heart because they require more oxygen than does glucose to produce an equivalent amount of ATP [10,11], and also because metabolic products accumulate in ischaemic myocardium [12,13]. Thus, shifting the energy substrate preference away from fatty acid oxidation and toward glucose oxidation may be important during an episode of severe ischaemia, and upon reperfusion. This would reduce accumulation of metabolic products, in particular accumulation of hydrogen ions, since one major source of H+ production during severe ischaemia (and possibly reperfusion) is the uncoupling of glycolysis from glucose oxidation. Indeed, if glucose passing through glycolysis is not subsequently oxidized, H+ is produced from ATP hydrolysis [14]. In contrast, if glucose is oxidized the net production of H+ is zero. In this context, inhibiting fatty acid oxidation would result in greater glucose oxidation by lowering mitochondrial acetyl CoA levels and relieving tonic inhibition of pyruvate dehydrogenase [2]. The accumulation of H+, i.e. decrease in intracellular pH (pHi) during severe ischaemia, is a promoting factor for the imbalance of other cations, especially Na+. Evidence of the involvement of mechanisms that can associate a decrease in pHi with cellular Na+ overload (and consequently Ca2+ overload) through ion transport systems, notably Na+–H+ exchange and possibly other Na+-dependent transporters, is supported by a number of studies [15–17]. In addition, a correlation has been observed between the reperfusion Na+i and the post-ischaemic recovery of ventricular function [15,18,19].

The present experiments were designed to investigate whether trimetazidine reduces the ionic imbalance induced by ischaemia and reperfusion, especially through an attenuation in intracellular changes in H+ and Na+. This was examined in isolated rat hearts perfused with high concentrations of fatty acids as can occur under some pathological conditions [20,21]. Both zero-flow and low-flow models of ischaemia were studied since both the myocardial metabolism and the accumulation of metabolic products during ischaemia are highly dependent upon the severity of the ischaemic insult [2]. 23Na nuclear magnetic resonance (NMR) was used to monitor Na+i and 31P NMR was used to monitor pHi and intracellular high-energy phosphate levels.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
All procedures were in accordance with the regulations laid down by the Ministère de l’Agriculture et de la Forêt, France, for the care and use of laboratory animals.

2.1 Heart perfusions
Male Wistar rats weighing 300–350 g were anaesthetized with thiopentone (5 mg/100 g body weight, intraperitoneally), and the hearts were quickly removed and then Langendorff perfused. Hearts were initially perfused at constant pressure (60 mmHg; 10-min stabilization period) with a modified Krebs–Henseleit bicarbonate perfusate, consisting of (mM): NaCl 118, KCl 5.9, MgSO4 1.2, CaCl2 1.25, NaHCO3 25, glucose 11, gassed with 95% O2–5% CO2 (pH 7.4, 37°C). The range of values for coronary flow was 15.6±0.6 ml/min. Thereafter, and in order to exclude any effect of the different experimental conditions on coronary flow rate, constant-flow perfusion was initiated by setting the flow rate to the level attained at the end of the stabilization period and continued (control perfusion) until inducing ischaemia. All perfusion solutions were filtered (0.8 µm; Millipore) prior to use. Perfusion pressure was monitored with a Statham 23 ID pressure transducer connected by polyethylene tubing to a side arm on the aortic cannula. Isovolumic left ventricular developed pressure was monitored using a fluid-filled latex balloon inserted into the left ventricular cavity via the left atrium. The initial left ventricular end diastolic pressure (LVEDP) was adjusted to 8–10 mmHg. Developed pressure (LVDP) was calculated by subtracting end-diastolic pressure from systolic pressure. Pressure signals were monitored on a Gould 2200 recorder. For all experiments, hearts were allowed to contract spontaneously.

For 23Na NMR measurements, the standard perfusate was modified by inclusion of the shift agent thulium(III)-1,4,7,10-tetraazacyclododecane-N,NI,NII,NIII-tetra-(methylene phosphonate) (TmDOTP5–) (Magnetic Resonance Solutions, Dallas, TX, USA). TmDOTP5– was dissolved in H2O and subsequently added to the perfusate to reach a concentration of 3.5 mmol/l. Since TmDOTP5– is a sodium salt that binds Ca2+ to a significant extent, both [Na+] and [Ca2+] were corrected. NaCl was adjusted to keep the total Na+ content unchanged, and the total Ca2+ content was increased to nominally ≥3.42 mmol/l, resulting in a measured free ionized Ca2+ of 0.85–0.95 mmol/l.

2.2 NMR spectroscopy
The 23Na and 31P spectra were recorded on a Bruker–Nicolet WP-200 spectrometer at 52.9 and 80.9 MHz, respectively, using two selective probes. The spectrometer was equipped with a 4.7 Tesla vertical magnet and the perfused heart was inserted into a 20-mm diameter glass NMR tube in which temperature was maintained at 37°C, as previously described [22]. Each 23Na-NMR spectrum was (1-min time-resolved) obtained by accumulation of 256 consecutive free induction decays (FIDs) using a pulse of 90° and a 0.2-s recycle time using 1-K data points and 2.5-KHz spectral width. The shift agent TmDOTP5– was used to distinguish the Na+o and Na+i resonance of the spectra, and Na+ signals were quantified using the Na+ resonance of a standard solution in a glass capillary. Spectra were processed for the quantitative analysis of the intracellular component (Na+i) and the reference in two steps. First, removal of the overlapping spectral extracellular component was carried out by the HLSVD method [23]. Second, the resulting reference and Na+i peaks were quantified with a time domain fitting routine (AMARES) [24].

31P-NMR spectra were obtained by averaging data obtained from 432 FIDs, using a pulse angle of 45° and a recycle time of 0.7 s (5-min time-resolved spectra) with 2-K data points and 4.5-KHz spectral width [22]. To study changes in pH during the first 5 min of reflow following ischaemia with greater time resolution, 30-s time-resolved spectra were acquired. Prior to Fourier transformation, the free induction decay was multiplied by an exponential function which generated a 20-Hz line broadening. The position and areas of the resonances were determined using the NMR1 software package (NMRi Syracuse, New York, USA). Values for pHi were derived from the chemical shift in the inorganic phosphate resonance, relative to that of phosphocreatine, using a titration curve which was obtained by titrating Pi at 37°C in a solution mimicking the intracellular milieu and which was fitted to the Henderson–Hasselbach equation [25]. Measured areas were corrected for saturation effects by multiplying the peak areas by saturation factors. These factors were determined by comparing the acquired spectra to fully relaxed spectra [26]. Quantitation of the peak areas was done with the aid of an external reference containing an aqueous solution of phenylphosphonic acid.

2.3 Experimental groups and protocol
Spectral and functional parameters were monitored in hearts (n=6) during shift agent (TmDOTP5–) perfusion without any interventions.

The experimental protocol consisted of 20 min of control perfusion, followed by 30 min of total global (TI) or low-flow (LFI; flow being reduced to 10% of the control flow rate) ischaemia, and 30 min of reperfusion at a constant pre-ischaemic coronary flow rate. Two different substrate combinations were used in separate heart groups: 5.5 mM glucose and 1.2 mM palmitate pre-bound to 3% bovine serum albumin (BSA, fraction V) (fat group), or 5.5 mM glucose in the presence of 3% BSA (fat-free group). In another group, 10–6 M trimetazidine (TMZ, MW=339.27) was added to the solution containing fatty acid (fat-TMZ group) and was present throughout. Albumin-containing solutions were dialysed at 4°C against five times the volume of modified Krebs–Henseleit solution containing nominally either 3.42 mmol/l Ca2+ for TmDOTP5–-containing solutions or 1.25 mmol/l Ca2+ for agent-free solutions, for three 8-h periods.

After termination of the protocol, hearts were dried at 80°C for at least 48 h. Intracellular water was assumed to be 2.45 ml/g dry weight [27].

2.4 Statistical analysis
Data are expressed as means±S.E. Results were submitted to an analysis of variance (ANOVA) and comparisons between individual groups’ means were performed using the Student–Newman–Keuls test (SPSS for Windows). Significance was set at P<0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Ventricular function
To assess the stability of the preparation, functional parameters were recorded for hearts perfused with shift agent perfusate for 80 min without any interventions. The preparation was functionally stable, with developed pressures of 74±2 and 69±3 mmHg (fat group values) after 20 and 80 min, respectively. As previously shown [3], the addition of trimetazidine (10–6 M) to the perfusate had no significant effect on pre-ischaemic values of left ventricular developed pressure. Furthermore, TMZ had no effect on spontaneous heart rate either during control perfusion (304±2.7 vs. 301±2.3 beatsper min without TMZ) or during reperfusion.

Figs. 1 and 2Go indicate mean LVDP and LVEDP across time for the experimental groups. For each experimental group, the mean of data includes data obtained from both 31P and 23Na NMR experiments. During LFI (Fig. 1), LVDP decreased to zero for all groups. Upon reperfusion, differences were observed in LVDP recovery, which reached 34% of its pre-ischaemic value after 30 min for the LFIfat group vs. 61% and 78% for the LFIfat-TMZ and LFIfat-free, respectively (no significant difference between the latter two groups). For the three groups, there was no increase in LVEDP during the ischaemic period. A marked increase in LVEDP was observed upon reperfusion in LFIfat hearts (25.5±3.3 mmHg). LVEDP increase was significantly lower in both LFIfat-TMZ and LFIfat-free hearts.


Figure 1
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  		Fig. 1 Diastolic pressure and left ventricular developed pressure during control, 30 min of low-flow ischaemia (LFI) and reperfusion, for LFIfat (-{circ}-, n=14), LFIfat-TMZ (-bullet-, n=12), and LFIfat-free (-{square}-, n=14) hearts. *Indicates P<0.05 for comparison with LFIfat.

 

Figure 2
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  		Fig. 2 Diastolic pressure and left ventricular developed pressure during control, 30 min of zero-flow ischaemia (TI) and reperfusion, for TIfat (-{circ}-, n=16), TIfat-TMZ (-bullet-, n=17), and TIfat-free (-{square}-, n=13) hearts. *Indicates P<0.05 vs. TIfat.

 
As shown in Fig. 2, during no-flow ischaemia (TI) LVDP also decreased to zero in the three groups: TIfat, TIfat-TMZ and TIfat-free. The post-ischaemic LVDP recovery of TIfat-TMZ hearts was significantly greater than that of the TIfat hearts. Development of contracture during the ischaemic period was observed for all groups. However, contracture was not significantly different between TIfat and TIfat-TMZ hearts, while contracture was significantly lower in TIfat-free hearts. The LVEDP of TIfat hearts increased at reperfusion, reaching 161% of the LVEDP at the end of ischaemia. LVEDP also increased upon reperfusion in TIfat-free hearts whereas no increase was observed in the TIfat-TMZ group. However, LVEDP during reperfusion in both TIfat-TMZ and TIfat-free hearts was significantly lower than in TIfat hearts.

3.2 Changes in Na+i
Fig. 3 shows the mean values of Na+i obtained from the 23Na NMR spectra, as recorded during the LFI/reperfusion experiments. Na+i was 10.14±0.16 mM during control perfusion. The increase in Na+i during ischaemia was moderate, reaching approximately 120%, 114% and 108% of control values in the LFIfat, LFIfat-TMZ and LFIfat-free groups respectively (non-significantly different). Mean reperfusion Na+i levels in both LFIfat-TMZ and LFIfat-free groups were significantly below those in the LFIfat group.


Figure 3
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  		Fig. 3 Mean values of Na+i during control, 30-min low-flow ischaemia (LFI) and reperfusion, for LFIfat (-{circ}-, n=7), LFIfat-TMZ (-bullet-, n=7), and LFIfat-free (-{square}-, n=7) hearts. *#{diamondsuit}Indicates P<0.05; *LFIfat-TMZ vs. LFIfat, {diamondsuit}LFIfat-free vs. LFIfat, #LFIfat-free vs. LFIfat-TMZ.

 
Na+i recorded during the zero-flow ischaemia–reperfusion experiments is shown in Fig. 4. In both TIfat and TIfat-free hearts, Na+i similarly increased to substantially above control levels during ischaemia (204 and 230% increase, respectively, at end ischaemia; P=0.56). Upon reperfusion, Na+i decrease occurred rapidly in the TIfat-free hearts whereas, in contrast, Na+i continued to increase in the TIfat hearts. The increase in Na+i in the TIfat group was maintained until 7 min of reperfusion (Na+i max=23.62±1.35 mM) and then it decreased. The increase in Na+i during ischaemia was also observed in the TIfat-TMZ group of hearts. However, the rise was significantly less marked than in the other two groups after 30 min of ischaemia. In those TIfat-TMZ hearts, Na+i decreased immediately upon reperfusion. At late reperfusion, there was no significant difference between the groups.


Figure 4
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  		Fig. 4 Mean values of Na+i during control, 30 min of zero-flow ischaemia (TI) and reperfusion, for TIfat (-{circ}-, n=8), TIfat-TMZ (-bullet-, n=9), and TIfat-free (-{square}-, n=6) hearts. *#{diamondsuit}Indicates P<0.05, *TIfat-TMZ vs. TIfat, {diamondsuit}TIfat-free vs. TIfat, #TIfat-free vs. TIfat-TMZ.

 
3.3 Changes in pHi
No significant differences in control pre-ischaemic pHi values were detected between the different groups of hearts (Fig. 5). In LFIfat and LFIfat-free hearts, the pHi significantly decreased at end ischaemia, to 6.88±0.01 and 6.84±0.02, respectively. In LFIfat-TMZ hearts, pHi decrease was slower, reaching, at end ischaemia, a mean value of 6.94±0.01, which was significantly higher than that observed in the other two groups. During reperfusion, a similar pHi value was reached for the three groups after 5 min, although the initial rates of recovery were different (0.08±0.01, 0.034±0.01 and 0.15±0.01 pH unitsper min over the first 2 min of reperfusion for the LFIfat, *LFIfat-TMZ, and *LFIfat-free hearts, respectively; *P<0.05 vs. LFIfat).


Figure 5
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  		Fig. 5 Mean values of pHi during control, 30 min of low-flow ischaemia (LFI) and reperfusion, for LFIfat (-{circ}-, n=7), LFIfat-TMZ (-bullet-, n=5), and LFIfat-free (-{square}-, n=7) hearts. *Indicates P<0.05 vs. LFIfat; #indicates P<0.05 vs. LFIfat-TMZ.

 
Fig. 6 shows pHi for the zero-flow experiments. The data clearly indicate that during ischaemia, pHi decreased rapidly from control values to levels approaching pH 6. No significant difference in the pHi values was noted between the TIfat, TIfat-TMZ and TIfat-free hearts during the ischaemic period. During reperfusion, a rapid recovery of pHi was observed in the three groups, with no significant difference in the rate of recovery between groups (0.35±0.02, 0.37±0.01 and 0.39±0.01 pH unit per min over the first 3 min of reperfusion for TIfat, TIfat-TMZ and TIfat-free hearts, respectively; see inset in Fig. 6).


Figure 6
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  		Fig. 6 Mean values of pHi during control, 30 min of zero-flow ischaemia (TI) and reperfusion, for TIfat (-{circ}-, n=8), TIfat-TMZ (-bullet-, n=8), and TIfat-free (-{square}-, n=7) hearts. P=0.742 and 0.171 for the comparison of TIfat-TMZ vs. TIfat and TIfat-free vs. TIfat, respectively, after 20 min of ischaemia.

 
3.4 Changes in high-energy phosphates
The decline in PCr and ATP was not significantly affected by including fat in the perfusate for either the low- or zero-flow ischaemic hearts (Fig. 7). However, the presence of TMZ slowed the decrease in ATP in LFI hearts. PCr recovered to significantly higher levels during reperfusion in both LFIfat-free and TIfat-free hearts compared with LFIfat and TIfat hearts, respectively.


Figure 7
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  		Fig. 7 Mean values of PCr and ATP plotted as percent of control values (before ischaemia), during LFI and reperfusion (panel A), and during TI and reperfusion (panel B). -{circ}-: LFIfat and TIfat hearts, n=7 and n=8, respectively; -bullet-: LFIfat-TMZ and TIfat-TMZ hearts, n=5 and n=8, respectively; -{square}-: LFIfat-free and TIfat-free hearts, n=7 for both groups. *P<0.05 vs. LFIfat or vs.TIfat.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The results of the present study demonstrate that trimetazidine (TMZ) exerts markedly different effects on ischaemia/reperfusion-induced ionic imbalance, depending on the degree of severity of ischaemia. These effects were observed at a concentration of TMZ that is within the range of in vivo therapeutic plasma levels [28].

The data clearly indicate that during zero-flow ischaemia, myocardial Na+i increased substantially (approximately two-fold) in both fat- and fat-free perfused rat hearts, whereas during low-flow ischaemia, Na+i increased nominally but not significantly. A similar profile of Na+i accumulation to that in the present work has been described by others in the perfused rat heart submitted to brief periods (20–30 min) of zero-flow ischaemia [18,29]. The most plausible underlying mechanisms for the gain in Na+i during ischaemia are a decrease in Na+ extrusion via the Na+/K+ ATPase and/or an influx of Na+ via Na+–H+ exchange and the voltage-gated Na+ channel (i.e. slowly inactivating or persistent sodium current). However, the relative contribution of these mechanisms is debated and may largely depend on the ischaemic conditions. For example, based on the insensitivity of Na+i to ouabain, one recent study concluded that the Na+/K+ pump may be severely inhibited during ischaemia [30]. The effect of ouabain may however be limited by increases in intracellular K+ which occur rapidly during zero-flow ischaemia [31]. In fact, others have presented data suggesting the pump is stimulated by increases in Na+i which occur during ischaemia and after acidification [see e.g [32]], and that the increase in Na+i during ischaemia is in part the result of increased Na+ entry. This Na+ entry may, at least partly, be mediated by Na+–H+ exchange. The original model of Lazdunski et al. [33] assumed that Na+–H+ would not contribute significantly to the rise in Na+i during ischaemia because of the low pHo. Although low pHo does reduce the activity of Na+–H+ exchange, it has been shown that the exchanger can still operate when pHo<pHi [34]. Several studies have identified this transporter as mediating Na+ influx in ischaemic hearts [18,35], whereas a recent work suggested that it may be inhibited during total ischaemia [36]. In addition to the Na+–H+ exchanger, activation of a Na+–HCO3– cotransporter might also contribute to Na+ influx [37]. Whether or not this cotransporter is involved during ischaemia has, so far, not been investigated. On the other hand, several reports favour the idea that the slowly inactivating component of the Na+ current plays a role in ischaemic (or hypoxic) Na+i rise [38–40].

Our data showed no evidence of available exogenous substrate having any effect on Na+i increase during ischaemia. On the other hand, TMZ significantly reduced the rise in Na+i. This TMZ effect may be related to the recent finding that TMZ decreases the activity of the long-chain isoform of the last enzyme involved in β-oxidation, 3-ketoacyl-CoA thiolase, resulting in an inhibition of long-chain fatty acid oxidation [5]. Also consistent with this finding is our previous work showing that TMZ significantly reduced the ischaemia-induced increase in myocardial long-chain acyl carnitine levels, determined as indicators of fatty acid utilization [3]. In this context, it has been shown that long-chain acyl carnitine such as palmitoyl carnitine, which accumulates in the sarcolemma during ischaemia, markedly increases the slowly inactivating component of Na+ current [41]. Experimental evidence also indicates that palmitoyl carnitine, like ouabain, produces a reversible inhibition of Na+/K+ pump current at micromolar concentrations in isolated guinea-pig ventricular myocytes [42]. Therefore, TMZ limitation of long-chain acyl carnitine accumulation during ischaemia may limit Na+i increase via slowly inactivating Na+ channels and a relative increase in Na+/K+ pump function. This would result in a lesser Na+i increase in TMZ-treated hearts whereas the progression of intracellular acidosis is the same in both TMZ-treated and untreated hearts.

An important finding of the present study is that Na+i continued to increase in untreated hearts receiving palmitate during the first 7 min of reperfusion following total ischaemia, whereas Na+i decreased immediately upon reperfusion in the TMZ-treated hearts as well as in the group that did not receive palmitate. It is worth noting here that most studies measuring Na+i have not involved perfusion with fatty acids [18,29,30]. Our data suggest that some ischaemia-induced alterations in long-chain fatty acid metabolism may be responsible for the additional Na+i increase observed at reperfusion, and which could be attenuated or even suppressed (present results) by TMZ. It is not possible to determine from the present study which of the above mentioned Na+i regulating mechanisms may be altered by ischaemia-induced alterations in fatty acid metabolism. With respect to Na+-dependent pHi regulating mechanisms, there is, so far, no data relating fatty acid metabolism alterations with changes, if any, in their activity. In this regard, no clear information has been provided either by the comparison of the time courses of pHi recovery upon reperfusion between groups, or by the comparison of ATP and PCr levels. Although recent studies [29,40] have concluded that Na+ influx through Na+ channels occurs during ischaemia, we cannot exclude the possibility of these channels participating in Na+i increase upon reperfusion in TIfat hearts. As mentioned above, the decrease in tissue acyl carnitine levels [3] in TIfat-TMZ hearts may well account for either the decrease in persistent Na+ current or maintained Na+/K+ pump activity upon reperfusion, or both. This would explain the TMZ associated Na+i decrease observed upon reperfusion. Furthermore, the present study also showed no prolonged increase in Na+i following ischaemia in TIfat-free hearts in which we have previously demonstrated significantly less acyl carnitine increase than in TIfat hearts at end ischaemia [3].

The moderate Na+i increase during low-flow ischaemia is probably related to a more favorable energy supply-to-demand balance compared with total ischaemia. Both ATP and PCr remained above 50%. During reperfusion, Na+i was significantly lower in both LFIfat-TMZ and LFIfat-free hearts. Although the pHi changes in low-flow ischaemia were not nearly as severe as those observed with zero-flow ischaemia, pHi decrease was markedly attenuated in hearts exposed to TMZ. There may be several explanations for this reduction in pHi decrease by TMZ. One possibility may be a reduction in glycolytic rate. However, measurements of glycolytic flux rates in isolated rat hearts with addition of TMZ to the perfusate have shown no change in anaerobic glucose utilization [43]. However, others have concluded that TMZ inhibits the increase in glycogen utilization after coronary artery ligation in the dog myocardium [44]. Our measurements, in a previous work, of tissue lactate levels in fatty acid-perfused rat hearts at the end of a mild ischaemia (coronary flow decreased by an average of 70%) did not show any significant difference in the presence or absence of TMZ (19.0±2.2 and 25.9±1.6 µmol/g dry weight, respectively). Another possible explanation for the reduced decrease in pHi could be an improved coupling of glycolysis to glucose oxidation, as a consequence of reduced fatty acid oxidation [5]. Indeed, if glucose passing through glycolysis is not subsequently oxidized, H+ is produced from ATP hydrolysis [14]. In contrast, if glucose is oxidized the net production of H+ is zero. It is worth noting that tissue ATP remained at somewhat higher levels during the last 10 min of LFI in TMZ-treated hearts. In addition, or alternatively, decreased LFI-induced intracellular acidosis by TMZ may be related to the positive effect of TMZ on the intracellular H+ buffering capacity of ventricular myocytes [45]. It should be noted here that LFIfat-free hearts exhibited significantly higher rates of pHi recovery during the first 2 min of reperfusion compared with LFIfat hearts. This is in accordance with very recent observations by others [46] showing that high levels of fatty acids decrease the rate of recovery of pHi during reperfusion compared with that of hearts perfused without fatty acid. The authors concluded that the lower rate of pHi recovery resulted from inhibition of glucose oxidation, with an increased production of H+ due to the uncoupling of glycolysis from glucose oxidation, this being associated with a low recovery of cardiac work, in line with the present results. Under conditions of decreased LFI-induced intracellular acidification by TMZ, the activity of Na+–H+ exchange and possibly of Na+–HCO3– cotransporter [22] would then be less stimulated on reperfusion, consistent with our observation of a slower pHi recovery in the LFIfat-TMZ group during the first 2 min of reperfusion. This is associated with lower Na+i levels during reperfusion. Furthermore, the prevention of inhibition of Na+/K+ ATPase activity (mentioned above for TIfat-TMZ/reperfused hearts) may also contribute to protection under low-flow conditions, especially as Na+i increases during reperfusion in the untreated, but not in the TMZ-treated or fat-free reperfused hearts. Given the likely coupling of Na+i and Ca2+i through Na+–Ca2+ exchange, a lesser Na+i build up upon reperfusion would reduce subsequent Ca2+ accumulation [15]. This would then contribute to the lesser increase in diastolic pressure with reperfusion and the improved recovery of ventricular function observed in hearts exposed to TMZ. The significantly higher increase in Na+i during zero-flow ischaemia compared with low-flow, and a further gain in Na+i at the beginning of reperfusion would markedly amplify the cascade of events just described above.

It could be argued that the relatively low level of ionized Ca2+ that has been used in the present work, similar to that used in previous studies by others [18,30] because of Na+ shift agent requirements, may have affected the functional response of the hearts to ischaemia and reperfusion injury. However, the comparison of LVDP recovery after reperfusion following total ischaemia in fat-free perfused hearts (14.8±3.5% of the pre-ischaemic level), with that obtained in an earlier work [47] under similar perfusion conditions except for a higher Ca2+ concentration (1.25 mmol/l), did not show any significant difference (P=0.41) in function recovery after similar durations of ischaemia and reperfusion (19.2±5% of the initial LVDP).

In conclusion, this study indicates that metabolic modulation by TMZ exerts protective effects against ionic disturbances (intracellular H+ and Na+) associated with ischaemia and reperfusion in fatty acid-perfused hearts. TMZ-induced reduction in ionic disturbances depends on the degree of severity of ischaemia. During low-flow ischaemia, the major effect was a significant reduction in intracellular acidosis, whereas during zero-flow ischaemia the main effect was a significant reduction in Na+i gain. Furthermore, the present study indicates that Na+i levels on reperfusion appear to be particularly important, not only under conditions of zero-flow but also after low-flow ischaemia.

Time for primary review 14 days.


   Acknowledgements
 
Servier France generously supplied trimetazidine as well as the funding for other supplies which made this study possible.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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A. Darmellah, C. Rucker-Martin, and D. Feuvray
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