Effect of acidic reperfusion on prolongation of

Cardiovascular Research (2008) 77, 782–790 doi:10.1093/cvr/cvm082

Effect of acidic reperfusion on prolongation of intracellular acidosis and myocardial salvage ń, Marisol Ruiz-Meana, Javier Inserte, Ignasi Barba, Vıćtor Hernando, Arancha Abella Antonio Rodrı´guez-Sinovas, and David Garcia-Dorado* Servicio de Cardiologıá, Hospital Universitari Vall d’Hebron, Pg. Vall d’ Hebron 119–129, Barcelona 08035, Spain Received 30 July 2007; revised 24 October 2007; accepted 20 November 2007; online publish-ahead-of-print 4 December 2007 This paper was guest edited by Jakob Vinten-Johansen, Emory University Time for primary review: 22 days

Postconditioning; Hypercontracture; Reperfusion therapy; Mitochondrial permeability transition; Gap junctions; Calpain

Aims It has been proposed that intracellular acidosis may be the basis of the cardioprotection of different interventions, including postconditioning. However, contradictory reports exist on the effects of acidic reperfusion on myocardial salvage. Here we characterized the effect of lowering the pH of the reperfusion media (pHo) on intracellular pH (pHi) and cell death. Methods and results The effect of acidic perfusion on reperfusion injury was studied in isolated rat hearts submitted to 40 min of ischaemia and 30 min of reperfusion, and its effect on the Naþ/Ca2þexchanger (NCX) was analysed in isolated myocytes. pHi and phosphocreatine (PCr) were monitored by nuclear magnetic resonance spectroscopy. Lowering pHo to 6.4 during the initial 3 min of reperfusion delayed pHi normalization, improved PCr recovery, and markedly reduced (P , 0.001) lactate dehydrogenase release and infarct size (tetrazolium reaction). This cardioprotection was attenuated as pHo was increased, and was lost at pH0 7.0. Extending acidic reperfusion to the first 15 or 30 min of reflow did not result in further delay of pHi normalization and abolished the protection afforded by the initial 3 min of acidic reperfusion unless the Naþ/Hþ-exchanger (NHE) blocker cariporide was added to the 0 acidic perfusate and HCO2 3 substituted for N-[2-hydroxyethyl]piperazine-N -[2-ethanesulphonic acid]. þ In experiments performed in fura-2-loaded myocytes exposed to low Na buffer adjusted to pH 6.4, the lower Ca2þ uptake indicated an inhibitory effect of acidosis on NCX. Conclusion Acidic reperfusion for 3 min delays normalization of pHi and enhances myocardial salvage, but extending it beyond this period fails to further delay pHi recovery. This is probably due to persisting NHE and Naþ/HCO2 3 -cotransporter activities, and it is detrimental, possibly through prolonged NCX inhibition.

1. Introduction Early reperfusion of ischaemic myocardium is the best strategy to limit infarct size.1 However, reperfusion itself induces additional injury that reduces myocardial salvage.2,3 Several pharmacological strategies applied at the onset of myocardial reperfusion have been shown to limit cell death in experimental studies, but translation to patients with acute myocardial infarction has been so far virtually null.4,5 This has not been the case with ischaemic postconditioning, a different approach in which protection is achieved by means of brief periods of ischaemia during the initial minutes of reperfusion.6 Recently, different studies have reported that postconditioning could be an efficient cardioprotective intervention in patients with acute myocardial infarction submitted to primary percutaneous coronary

* Corresponding author. Tel: þ34 93 489 4038; fax: þ34 93 489 4032. E-mail addresses: [email protected]; [email protected]

interventions.7–9 The mediators of postconditioning protection have not yet been elucidated, but it has been suggested that prolongation of acidosis during reperfusion is determinant for the protective effects of postconditioning.2,10,11 A delay in intracellular pH (pHi) recovery at reperfusion may be protective by preventing contractile activation and development of high mechanical stress caused by hypercontracture in reoxygenated cardiomyocytes while the cells re-establish Naþ and Ca2þ control.12 Moreover, acidosis inhibits other mechanisms that have been consistently been shown to be involved in reperfusion injury, such as mitochondrial permeability transition (MPT) pore opening13,14 and activation of Ca2þ-dependent proteases, e.g. calpain,15 all of them factors that could contribute to explain this paradoxical worsening of injury after pH correction. Therefore, acidic reperfusion appears to be a potential protective strategy with options for being translated to a clinical setting.

Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2007. For permissions please email: [email protected].

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KEYWORDS

Effect of acidic reperfusion

2. Methods The experimental procedures conformed with the Guide for the Care and Use of Laboratory Animals published by the United States National Institute of Health (NIH Publication No. 85–23, revised 1996), and were approved by the Research Commission on Ethics of the Hospital Vall d’Hebron.

2.1 Isolated perfused rat heart preparation Male Sprague-Dawley rats (300–350 g) were anaesthetized with sodium pentobarbital (100 mg/kg). Hearts were removed, mounted into a Langendorff apparatus and perfused with a modified Krebs-Henseleit bicarbonate buffer (KHB, in mM: NaCl 140, NaHCO3 24, KCl 2.7, KH2PO4 0.4, MgSO4 1, CaCl2 1.8, and glucose 11) equilibrated with 95% O2 and 5% CO2 and maintained at 378C as previously described.25 Flow rate was initially set to produce a perfusion pressure of 60 mmHg and was held constant thereafter. Left ventricle (LV) pressure was monitored through the use of a water-filled latex balloon inserted into the LV and inflated to obtain an end-diastolic pressure (LVEDP) between 6 and 8 mmHg. LV developed pressure (LVdevP) was calculated as the difference between LV systolic pressure and LVEDP. Perfusion pressure was continuously recorded using a pressure transducer connected to the perfusion line.

2.2 Experimental protocol In a first set of experiments, the effect of prolongation of acidosis during initial reperfusion against cell death was tested. Hearts were stabilized for 30 min and then subjected to no-flow global ischaemia for 40 min followed by 30 min of reperfusion. Hearts were perfused with KHB at either pH 7.4 (control group, n ¼ 10) or KHB adjusted to pH 6.4 (n ¼ 7), 6.7 (n ¼ 6) and 7.0 (n ¼ 6) for the first 3 min of reperfusion. The pH of the acidic solution was adjusted by lowering bicarbonate concentration and increasing sodium chloride to keep osmolarity constant while maintaining the same gassing mixture containing 5% CO2.20

In a second set of experiments, the effect of the duration of the acidic perfusion on reperfusion injury was analysed in two additional groups of hearts perfused with KHB adjusted to pH 6.4 for the first 15 min (n ¼ 6) or 30 min (n ¼ 5) of reperfusion. In two additional groups of hearts the þ þ activities of Naþ/HCO2 3 cotransporter (NBC) and Na /H exchanger (NHE) were blocked during the first 15 min of reperfusion by using NaHCO2 3 -free HEPES buffer adjusted to pH 7.4 (n ¼ 6) or pH 6.4 (n ¼ 7) and containing 10 mM cariporide.

2.3 Quantification of cell death Lactate dehydrogenase (LDH) activity was spectrophotometrically measured in the coronary effluent throughout the reperfusion period. After reperfusion hearts were cut into four slices and incubated at 378C for 10 min in 1% triphenyltetrazolium chloride (TTC) (pH 7.4) and imaged under white light to outline the area of necrosis as previously described.25

2.4 Nuclear magnetic resonance spectroscopy pHi, phosphocreatine (PCr) and adenosine triphosphate (ATP) kinetics were measured by 31P-NMR(nuclear magnetic resonance) in hearts perfused after 40 min of ischaemia with KHB free of phosphate at pH 7.4 or pH 6.4 during the first 3 min or 15 min of reperfusion. In two additional groups, hearts were reperfused for 15 min with bicarbonate-free HEPES buffer adjusted to pH 7.4 or at pH 6.4 and containing 10 mM cariporide. Spectroscopy was performed on a Bruker Avance 400 spectrometer equipped with a 20-mm probe tuned to 31P. Spectra consisted in the accumulation of 50 scans with a delay of 0.6 s between pulses that lasted for 30 s. The PCr peak was integrated with the software provided by the manufacturer, and pHi was measured by the chemical shift of the inorganic phosphate peak relative to the PCr peak.26 In order to circumvent the difficulties in the measurement of ATP caused by the low signal-to-noise ratio during reperfusion, [ATP] was calculated from the average of four consecutive spectra (corresponding to 2 min of acquisition). The g-ATP peak was measured in the averaged spectra.

2.5 Studies in fura-2-loaded cardiomyocytes Freshly isolated rat cardiac myocytes were obtained from adult rat hearts (300 g Sprague-Dawley male rats) by a collagenase perfusion as previously described14 and plated on laminin-precoated glass bottom culture dishes. For intracellular [Ca2þ] measurements, cells were loaded 30 min at 378C with 2.5 mM of the acetoxymethyl ester of fura-2 (Molecular Probes, USA) in control solution (containing in mM: NaCl 140, KCl 3.6, MgSO4 1.2, CaCl2 1, HEPES 20, and glucose 5, at pH 7.4), washed twice and postincubated for 10 min in the same solution. To stimulate the reverse mode of Naþ/Ca2þ-exchanger (NCX) and induce Ca2þ overload, NaCl was reduced to 70 mM, Ca2þ was increased to 2 mM and mannitol 2.2% (v/v) was added to maintain normo-osmolarity (312 mOsm). Thapsigargin 2 mM and ryanodine 10 mM were added during the induction of cytosolic Ca2þ overload to prevent sarcoplasmic reticulum Ca2þ uptake and release. The effect of acidosis on cytosolic [Ca2þ] was induced by lowering extracellular pH to 6.4.


Early experimental evidence demonstrated that transient reoxygenation with acidic solutions could protect isolated myocytes and papillary muscle against cell death.14,16–18 However, the situation is less clear in intact heart. Most of the studies were performed in isolated hearts subjected to short periods of ischaemia in which the main end point analysed was not lethal injury. In these studies, acidic reperfusion has been shown to reduce the incidence of ventricular fibrillation19,20 and to attenuate myocardial stunning.21,22 Other studies, however, have failed to show a protective effect of acidic reperfusion. Prolongation of acidosis for 15 min has been described to have no beneficial effect on functional recovery after 15 min of ischaemia or on protein release when ischaemia was extended to 60 min,23 and in a working rat heart model subjected to cardioplegic ischaemia, prolonged acidosis (longer than 3 min) exacerbated reperfusion injury.24 The aim of this study was to determine the effect of perfusion with an acidic buffer during early myocardial reperfusion on pHi and cell death and to identify the factors modulating these effects. This information is essential for further research aimed at the therapeutic application of acidic reperfusion.

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Cytosolic [Ca2þ] was monitored throughout 10 min under both pH 7.4 and pH 6.4 using a ratiofluorescence imaging system (QuantiCell 900, Visitech, UK). Cells were alternatively excited at 340 and 380 nm, with a bandwidth of 15 nm, by means of a fast speed monochromator. Emitted light was collected by an air-cooled intensified digital camera, and 340/380 ratios were calculated for each pixel at 1-s intervals from background-subtracted signal intensities in pairs of images consecutively obtained at the two wavelengths. Colour-coded 340/380 ratio images were generated and further calibrated at both pH values to correct for pH-dependent changes in fura-2 affinity.27

2.6 Statistical analysis

3. Results 3.1 Effects of acidic reperfusion on myocardial injury In control hearts subjected to 40 min of ischaemia, LVEDP and LVdevP were 7.2 + 1.6 and 99.8 + 4.0 mmHg, respectively, at the end of the equilibration period. At this time perfusion pressure was 59.3 + 3.6 mmHg and coronary flow 12.4 + 1.3 mL/min. No-flow ischaemia resulted in cessation of contractile activity and in a steep increase in LVEDP with a peak of 73.7 + 10.9 mmHg 13.1 + 0.4 min after the onset of ischaemia. No differences among groups were observed during equilibration and ischaemic periods. During reperfusion, hearts perfused with buffer adjusted to pH 6.4 for the first 3 min showed delayed (5.5 + 0.3 min vs. 3.3 + 0.2 min in control group, P , 0.001; Figure 1A) and attenuated (62.6 + 5.8 mmHg vs.115.1 + 6.0 in control group, P , 0.001; Figure 1B) hypercontracture as well as improved contractile recovery (25.2 + 4.6% of basal values after 30 min of reperfusion vs. 5.6 + 1.5% of basal values in control group, P , 0.001; Figure 1C), reduced increase in perfusion pressure (99.9 + 5.6 mmHg vs. 117.9 + 3.7 mmHg, P , 0.001), less LDH release (42%, P ¼ 0.001;

Figure 1 Influence of perfusion with different pH values during the first 3 min of reperfusion on: (A) the time to development of hypercontracture (in min); (B) magnitude of hypercontracture (mmHg); (C ) left ventricle developed pressure recovery (measured after 30 min of reperfusion and expressed as percentage respect to basal values); (D) time course of lactate dehydrogenase (LDH) release during reperfusion; (E) total LDH released; (F ) infarct size measured by TTC and expressed as percentage of ventricular mass (*P , 0.05 vs. group reperfused at pH 7.4; data are mean + SEM).


Data analyses were performed using SPSS for Windows (v. 10). Results are expressed as mean + SEM. Differences between groups were assessed by means of simple one-way analysis of variance (ANOVA). Changes with time were assessed by multiple ANOVA. If significant differences were observed, a least significant square test was applied as a post hoc test. P , 0.05 was considered to be statistically significant.

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Figure 1D and E), and smaller infarcts (38%, P ¼ 0.012; Figure 1F). Reduction of infarct size by acidosis was further confirmed in two additional groups, control (n ¼ 4) and pH 6.4 perfused hearts (n ¼ 4), in which the reperfusion period was extended to 1 h (49.7 + 5.4% in control group vs. 26.2 + 3.6% in hearts reperfused at pH 6.4, P ¼ 0.011). The protective effects afforded by 3 min of acidic perfusion were progressively smaller as the pH of the perfusate was increased. Protection was maximal at pH 6.4 and was lost at pH 7.0 (Figures 1 and 2).

3.2 Time dependence of the effects of acidic reperfusion Prolongation of perfusion with KHB buffer adjusted to pH 6.4 for 15 or 30 min of reperfusion did not induce further delay in hypercontracture (Figure 2A and D) and was associated with loss of the protective effects on contractile recovery (Figure 2C), hypercontracture (Figure 2B and D), perfusion pressure (Figure 2E), LDH release (Figure 3A and B), or

infarct size (Figure 3C) observed when acidic perfusion was limited to the first 3 min of reperfusion. Reperfusion with bicarbonate-free HEPES buffer adjusted to pH 6.4 and containing cariporide markedly delayed hypercontracture and was still protective. When HEPES buffer was adjusted to pH 7.4 the cardioprotective effects on hypercontracture and cell death were attenuated but still statistically significant (Figures 2 and 3).

3.3 Effect of acidic reperfusion on intracellular pH and energetic recovery pHi was 7.01 + 0.02 under normoxic conditions without differences between groups. Ischaemia induced a progressive pHi reduction that reached its maximum (6.43) after 20 min of zero flow. Reperfusion after 40 min of ischaemia resulted in recovery of pHi within 1.83 + 0.17 min of reperfusion. In hearts perfused with KHB at 6.4 for the first 3 min of reperfusion the onset of pHi recovery was delayed and recovery was complete (pH 7.02 + 0.03 at 4.33 + 0.25 min


Figure 2 Time-dependent effect of acidic reperfusion (pH 6.4) on: (A) development of hypercontracture (in min); (B) magnitude of hypercontracture (mmHg); (C ) left ventricle developed pressure (in % respect to basal values); (D) left ventricle end-diastolic pressure (mmHg) and (E) perfusion pressure (PP, mmHg). A gap between 15 and 30 min of reperfusion has been introduced to show the first minute in more detail. 0þHCP and 15þHCP correspond, respectively, to the groups reperfused for 15 min with bicarbonate-free HEPES buffer containing cariporide in which pH is adjusted to pH 7.4 or 6.4 (*P , 0.05 vs. control group; #P , 0.05 respect to group reperfused at pH 6.4 during the first 3 min of reperfusion; $P , 0.05 pH 6.4, 3-min group vs. pH 7.4 group; †P , 0.05 pH 7.4þHCP group vs. pH 7.4 group; ‡P , 0.05 pH 6.4þHCP group vs. control group; data are mean + SEM).

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of reperfusion, Figure 4). Prolongation of acidic perfusion to the first 15 min of reperfusion did not induce further delay in pHi recovery, except when acidic perfusion was performed with bicarbonate-free HEPES buffer containing cariporide, but even in this case pHi returned to normal values after 8.3 min of reperfusion. Perfusion with HEPES buffer containing cariporide and adjusted to pH 7.4 induced a slight but significant delay in pHi recovery (2.70 + 0.12, P ¼ 0.002). The concentrations of PCr and ATP in control hearts were 75.8 + 4.5 mmol PCr/g dry weight and 30.46 + 0.41 mmol ATP/g dry weight, respectively. No differences in these

Figure 4 Effect of acidic perfusion on intracellular pH (pHi): (A) time course of changes in pHi during ischaemia and reperfusion; (B) detail of pHi curves corresponding to the onset of reperfusion; (C ) time from the onset of reperfusion to maximal pHi recovery in the different treatment groups; pH 7.4þHCP and pH 6.4þHCP correspond, respectively, to groups reperfused for 15 min with bicarbonate-free HEPES buffer containing cariporide in which pH is adjusted to pH 7.4 or 6.4 (*P , 0.05 vs. pH 7.4 group; $P , 0.05 pH 6.4, 3-min group vs. pH 7.4 group; †P , 0.05 pH 7.4 þ HCP group vs. pH 7.4 group; ‡P , 0.05 pH 6.4 þ HCP group vs. control group; }P , 0.05 pH 6.4, 15-min vs. pH 7.4 group; data are mean + SEM).

values were observed among groups during equilibration, or in the kinetics of PCr and ATP depletion during ischaemia. Upon reperfusion, PCr levels quickly recovered to 74.57 + 9.65% of its basal value within a maximum of 2 min after reperfusion onset in control hearts, but then abruptly fell to 42.22 + 1.98%. In hearts reperfused with acidic buffer at pH 6.4, PCr recovered to levels similar to those in the control group after 2 min of reperfusion, but the subsequent decrease was significantly attenuated. When acidic perfusion was restricted to the first 3 min of reperfusion, the initial high levels of PCr were maintained even after normalization of the pH of the perfusate. However, prolongation of acidosis beyond the initial 3 min of reperfusion resulted in a progressive reduction of PCr that eventually reached values similar to those observed in the control group. In hearts


Figure 3 Effects of reperfusion at pH 6.4 on myocardial cell death: (A) time course of lactate dehydrogenase (LDH) release during the first 15 min of reperfusion; (B) total LDH released; (C ) infarct size measured by TTC and expressed as percentage of ventricular mass. pH 7.4 þ HCP and pH 6.4 þ HCP correspond, respectively, to groups reperfused for 15 min with bicarbonate-free HEPES buffer containing cariporide in which pH adjusted to pH 7.4 or 6.4 (*P , 0.05 vs. pH 7.4 group; $P , 0.05 pH 6.4, 3-min group vs. pH 7.4 group; †P , 0.05 pH 7.4 þ HCP group vs. pH 7.4 group; ‡P , 0.05 pH 6.4 þ HCP group vs. control group; data are mean + SEM).

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perfused for 15 min with HEPES buffer adjusted to pH 6.4 containing cariporide, energetic recovery was similar to that observed after only 3 min of acidic reperfusion while reperfusion with HEPES buffer adjusted to pH 7.4 only resulted in a slight (1 min) delay in PCr reduction with respect to the control group (Figure 5B). In the control group, [ATP] recovered to a maximum of 22.64 + 2.61% at minute 4 of reperfusion. Acidic reperfusion and inhibition of NHE and NBC with HEPES buffer adjusted to pH 6.4 containing cariporide showed the best recovery of ATP after 30 min of reperfusion (Figure 5C).

3.4 Effect of acidosis on low sodium-induced Ca21 overload Exposure of myocytes to low extracellular [Naþ] (70 mM) resulted in rapid cytosolic Ca2þ overload in the presence of 2 mM extracellular Ca2þ and pH 7.4. However, lowering extracellular pH to 6.4 significantly reduced the degree

of cytosolic Ca2þ overload under the same conditions (Figure 6).

4. Discussion The results of our study provide for the first time direct evidence, by using NMR measurements of pHi, that in the isolated rat heart there is a critical period of time for acidic reperfusion to be protective. Acidic perfusion during the first 3 min of reperfusion delays normalization of pHi, improves energetic and functional recovery of cardiomyocytes and attenuates hypercontracture and cell death. These protective effects are dependent on the level of acidosis, being maximal at pH 6.4. Prolongation of extracellular acidosis beyond the first 3 min does not result in further prolongation of intracellular acidosis, due to NBC and NHE activity, and abolishes the protection afforded by


Figure 5 Effect of acidic perfusion on phosphocreatine (PCr) and ATP levels: (A) time course of PCr, as percentage of preischaemic values, during ischaemia and reperfusion; (B) detail of PCr recovery corresponding to the first 10 min of reperfusion; (C) time course of ATP recovery, as percentage of preischaemic values, during reperfusion. pH 7.4 þ HCP and pH 6.4 þ HCP correspond, respectively, to groups reperfused for 15 min with bicarbonate-free HEPES buffer containing cariporide in which pH is adjusted to pH 7.4 or 6.4 (*P , 0.05 vs. pH 7.4 group; $P , 0.05 pH 6.4, 3-min group vs. pH 7.4 group; ‡P , 0.05 pH 6.4 þ HCP group vs. control group; }P , 0.05 pH 6.4, 15-min vs. pH 7.4 group; data are mean + SEM).

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4.2 Loss of protection with prolonged acidic perfusion during reperfusion

Figure 6 Effect of acidosis on low Naþ-induced Ca2þ overload: (A) time course of changes in cytosolic Ca2þ concentration ([Ca2þ]i) in isolated cardiomyocytes exposed (the arrow points the onset) to low extracellular Naþ (70 mM) and high Ca2þ (2 mM) buffer to induce Ca2þ overload, and pH adjusted to 7.4 or 6.4, *P , 0.05; (B) [Ca2þ]i before (0 min) and after 10 min of incubation with the Ca2þ-overload induced buffer adjusted to pH 7.4 or pH 6.4 (*P , 0.05 0 min vs. 10 min; $P ¼ 0.01 pH 6.4, 10 min vs. pH 7.4, 10 min; data are mean + SEM).

the initial 3 min of acidic reperfusion, possibly in relation to persistent NCX inhibition.

4.1 Protective effects of short periods of perfusion with acidic buffer during reperfusion Several mechanisms could lead to the pronounced effect of pHi in the development of hypercontracture in reperfused cardiomyocytes. First, prolongation of intracellular acidosis inhibits myofibrillar machinery by reducing the sensitivity of myofibrils to Ca2þ12 and prevents the development of hypercontracture during the initial minute of reperfusion when Ca2þ homeostasis has not been yet restored.28 This effect is similar to the infarct-sparing effect observed with the transient blockade of actin-myosin interaction with 2,3-butanedione monoxime during initial reperfusion.29 Secondly, rapid washout of extracellular Hþ upon reperfusion of ischaemic myocardium creates a transmembrane Hþ gradient resulting in Naþ influx coupled to Hþ extrusion via NHE and NBC. Influx of Naþ increases the intracellular Naþ concentration, and the loss of the Naþ gradient, in turn, favours the reverse mode of NCX and enhances Ca2þ overload.30 In addition, several studies, including the present one, propose that acidosis could prevent Ca2þ entry by inhibition of NCX.31 Other studies described that acidosis also

Protection against cell death induced by 3 min of acidic perfusion was lost when the acidic perfusion was extended to 15 min. After 5.5 min of reperfusion hearts developed hypercontracture, increased coronary resistance, and LDH release, and at the end of reperfusion, infarct size was similar to that obtained in control hearts. Continuation of acidosis for 30 min did not result in further injury. These results are in agreement with the lack of protection previously observed in hearts reperfused with an acidic buffer for 15 min.23,24 Our 31P-NMR data on pHi could help explain the time-dependent reversion of the protective effects of acidosis. Although it is generally assumed that acidic reperfusion prolongs intracellular acidosis as long as extracellular acidosis is present, in our study this was only true for the first 3 min of reperfusion. Beyond this time hearts rapidly normalized pHi despite extracellular acidosis, without significant time differences with respect to hearts in which acidic reperfusion was restricted to the first 3 min. The prolongation of pHi observed in hearts reperfused with a bicarbonate-free HEPES buffer containing the NHE inhibitor cariporide demonstrates that the normalization of pHi while extracellular acidosis is maintained is a consequence of the Naþ influx-dependent mechanisms of pHi correction. The eventual normalization of pHi despite acidic reperfusion with bicarbonate-free HEPES buffer containing cariporide indicates that other mechanisms of correction of acidosis such as washout of metabolite (lactate and CO2) may play an important role during this phase.35 The slight although significant delay in pHi observed in the group perfused with HEPES buffer adjusted to pH 7.4 further supports this notion. The recovery of myocardial pHi despite prolonged acidic reperfusion could explain its failure in producing additional protection. However, the reasons for the loss of protection associated with prolonged extracellular acidosis are not clear. It has been suggested that prolonged acidosis could inhibit essential metabolic pathways and impair energetic recovery.24 However, we observed a fast and complete functional recovery in normoxic hearts perfused with acidic


decreases cytosolic Ca2þ entry via a Ca2þ inward current32 and Ca2þ release from the sarcoplasmic reticulum.33 Supporting this effect on cationic homeostasis, it has been demonstrated that prolongation of extracellular acidosis during the early phase of reoxygenation reduces intracellular Naþ and Ca2þ overload.23 Thirdly, acidosis has been suggested to inhibit calpain activity15 and could thus attenuate Ca2þ-dependent proteolytic injury occurring during reperfusion.25 Recently, it has been proposed that calpain activation contributes to reperfusion-induced cell death not only by increasing membrane fragility but also by favouring Ca2þ entry as consequence of attenuated intracellular Naþ recovery due to calpain degradation of the proteins that stabilize Naþ/Kþ-ATPase to the membranecytoskeletum complex.34 Finally, evidence has accumulated in recent years suggesting that the MPT pore is an important player in reperfusion injury. During reperfusion rapid normalization of pHi may allow MPT triggered by ROS and mitochondrial Ca2þ overload.13 Therefore, prolongation of intracellular acidosis with acidic reperfusion is expected to prevent MPT.


Acknowledgements ´ ngeles Garcıá for her excellent technical We warmly thank Marıá A assistance.

Conflict of interest: none declared.

Funding This study was partially supported by grants of FIS-RECAVA RD06/0014/0025 and CICYT SAF 2005-1758.

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buffer for 30 min upon normalization of extracellular pH (data not shown), which makes unlikely the possibility that acidosis causes irreversible deleterious effects. Alternatively, the detrimental consequences of prolonged acidosis could be explained by an inhibitory effect on NCX. We have previously shown that inhibition of NCX has timedependent consequences on reperfusion-induced cell death.30 Although NCX inhibition is markedly protective during the first minutes of reperfusion by preventing additional Ca2þ entry via reverse mode NCX activity, persistent inhibition of NCX beyond the first 4 min of reperfusion impairs recovery of Ca2þ homeostasis in myocytes that have survived initial reperfusion and is detrimental.30 Although we assume the limitations of our model of isolated myocytes for extrapolation of results to intact heart due to the absence of electrical stimulation and contraction, and the non-physiological low Naþ concentration used to favour reverse mode NCX (as described in previous studies30,36), the attenuated [Ca2þ] gain observed in myocytes in which the sarcoplasmic reticulum has been blocked when exposed to low [Naþ] buffer at low pH is consistent with an inhibitory effect of acidosis on NCX activity. In contrast to our results, reperfusion with blood equilibrated at pH 6.8 for 30 min in a model of coronary artery occlusion in dogs, a more clinically relevant model, resulted in a reduction of infarct size.37 While the reasons for this apparent discrepancy are not known, there are large differences between both experimental models and the methodological conditions used for acidic perfusion. In any case, this observation does not limit the potential clinical relevance of our observation indicating that limiting the time of acidic reperfusion could result in stronger protection. In conclusion, our study supports the hypothesis that prolongation of intracellular acidosis during initial reperfusion protects against ischaemia/reperfusion-induced hypercontracture and cell death. Acidic reperfusion can prolong intracellular acidosis only for the first few minutes of reflow, but beyond that time, it fails to maintain intracellular acidification due to persistent NHE and NBC activity and is detrimental, probably due to impaired NCX-mediated Ca2þ efflux. Despite the limitations inherent to the isolated rat model that preclude direct translation to the clinical setting, these results further support the development of therapeutic strategies aimed against reperfusion injury based on prolongation of intracellular acidosis.

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