Indirect data suggest that delayed recovery of intracellular pH (pHi) during reperfusion is involved in postconditioning protection, and calpain activity has been shown to be pH-dependent. We sought to characterize the effect of ischaemic postconditioning on pHi recovery during reperfusion and on calpain-dependent proteolysis, an important mechanism of myocardial reperfusion injury.
Isolated Sprague–Dawley rat hearts were submitted to 40 min of ischaemia and different reperfusion protocols of postconditioning and acidosis. pHi was monitored by 31P-NMR spectroscopy. Myocardial cell death was determined by lactate dehydrogenase (LDH) and triphenyltetrazolium staining, and calpain activity by western blot measurement of α-fodrin degradation. In control hearts, pHi recovered within 1.5 ± 0.24 min of reperfusion. Postconditioning with 6 cycles of 10 s ischaemia–reperfusion delayed pHi recovery slightly to 2.5 ± 0.2 min and failed to prevent calpain-mediated α-fodrin degradation or to elicit protection. Lowering perfusion flow to 50% during reperfusion cycles or shortening the cycles (12 cycles of 5 s ischemia–reperfusion) resulted in a further delay in pHi recovery (4.1 ± 0.2 and 3.5 ± 0.3 min, respectively), attenuated α-fodrin proteolysis, improved functional recovery, and reduced LDH release (47 and 38%, respectively, P < 0.001) and infarct size (36 and 32%, respectively, P < 0.001). This cardioprotection was identical to that produced by lowering the pH of the perfusion buffer to 6.4 during the first 2 min of reperfusion or by calpain inhibition with MDL-28170.
These results provide direct evidence that postconditioning protection depends on prolongation of intracellular acidosis during reperfusion and indicate that inhibited calpain activity could contribute to this protection.
Postconditioning, a novel strategy of cardioprotection consisting of the application of brief cycles of ischaemia–reflow at the onset of reperfusion, represents a promising approach to limit infarct size in the clinical setting.1,2 Pilot studies have found postconditioning to be effective in patients with acute myocardial infarction submitted to primary percutaneous coronary interventions.3–5 However, although it has been proposed that postconditioning shares with ischaemic preconditioning some classical signal transduction pathways and effectors,6 the mechanisms leading to its protective effects against lethal reperfusion injury are not well understood. Recently, prolongation of acidosis during reperfusion has been proposed to play a determinant role.7–10 The contribution of intracellular acidosis to the cardioprotection elicited by postconditioning has been related to the activation of Akt and extracellular signal-regulated kinases (ERK) pathways,9 and the prevention of mitochondrial permeability transition pore (mPTP) formation during the early phase of reperfusion.10 A delay in intracellular pH (pHi) recovery during initial reperfusion may also prevent hypercontracture,11 gap junction-mediated propagation of hypercontracture,12 or activation of the Ca2+-dependent proteases calpains.13 We have previously described that calpain-mediated proteolysis contributes to cardiomyocyte cell death by inducing sarcolemmal fragility and detachment and dysfunction of Na+/K+-ATPase,14,15 and that inhibition of calpain is an end effector of preconditioning.14
Previous studies in different experimental models had demonstrated that transient reperfusion with either respiratory or metabolic acidosis could be protective,11,16–18 and different groups, including ours, have shown that prolongation of extracellular acidosis for just 2–3 min during initial reperfusion was sufficient to protect the myocardium.10,12,19 However, the evidence of delayed pHi recovery in postconditioning was so far indirect, mainly due to the lack of direct measurement of pHi in postconditioned myocardium.9,10
The aim of this study was to demonstrate, by direct measurement of pHi, the role of delayed pHi recovery in postconditioning protection and the potential contribution of limited calpain activation.
The experimental procedures conformed to 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.
Isolated perfused rat heart preparation
Male Sprague–Dawley rats (300–350 g) were anaesthetized with sodium pentobarbital (100 mg/kg). Hearts were removed, mounted onto 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 as described previously.14 Flow rate was initially adjusted 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. Temperature was constantly measured via a thermo-probe inserted in the pulmonary artery and maintained at 37 ± 0.2°C.
In control group, hearts were stabilized for 30 min and then subjected to no-flow global ischaemia for 40 min followed by 60 min of reperfusion (n = 8). Postconditioning was achieved by three different protocols commenced immediately after ischaemia and consisting of 6 cycles of 10 s reperfusion/10 s occlusion (post10 group, n = 7), the same number and duration of cycles but with flow rate adjusted to 50% of basal value during the reperfusion cycles (post10 low-flow group, n = 6)) or 12 cycles of 5 s reperfusion/5 s occlusion (post5 group, n = 8). In a control group, hearts were reperfused for 2 min at reduced flow rate (control low-flow group, n = 6), and in an acidic reperfusion group, hearts were perfused with KHB adjusted at pH 6.4 for the first 2 min of reperfusion (n = 8). 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 To examine the effect of calpain inhibition on reperfusion injury, in an additional group (n = 6), the membrane-permeable calpain inhibitor MDL-28170 (Calbiochem) at 10 µM was added to the perfusion media during the 10 min prior to ischaemia and the first 10 min of reperfusion (Figure 1).
Intracellular pH was measured by 31P-NMR in hearts perfused with KHB free of phosphate. 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. Phosphocreatine (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.21 Time to recovery of pHi was defined as the interval from the onset of reperfusion to the time to reach pHi 7.0.
Lactate washout during reperfusion was measured in the coronary effluent from hearts allocated to control, post10, and post5 groups and corresponding to 0–2 and 2–5 min of reperfusion. Samples were freeze-dried, dissolved into 600 µL of D2O containing 1 mM trimethyl-sylil-tetradeuteropropionic acid (TSP) as chemical shift and concentration standard. 1H-NMR spectra were acquired in a 5 mm probe and consisted in the accumulation of 16 fully relaxed scans; lactate concentration was measured by comparing the areas of the peak of lactate at 1.35 ppm to that of TSP.
The effect of the different study protocols on calpain activation was analysed in an additional series of hearts (three hearts per group) reperfused for 5 min with KHB buffer containing 20 mM of the reversible contractile inhibitor 2,3-butanedione monoxime (BDM). BDM prevents cell death by reducing the mechanical tension caused by the excessive contractile activation occurring at the onset of reperfusion. It was added to discard the possibility that any variation on calpain activity was not a mere consequence of differences in cell death.15 Calpain activity from frozen and homogenized hearts was evaluated by western blotting using a monoclonal antibody to α-fodrin (Affinity Research Products), which recognizes the 145/150 kDa fragments resulting from calpain-mediated α-fodrin proteolysis, as described previously.14
Quantification of cell death
Lactate dehydrogenase (LDH) activity was spectrophotometrically measured in the coronary effluent throughout the reperfusion period. After 60 min of reperfusion, hearts were cut into four slices and incubated at 37°C for 10 min in 1% triphenyltetrazolium chloride (pH 7.4) and imaged under white light to outline the area of necrosis as described previously.15,22
Data analysis was performed using SPSS for Windows. Results are expressed as mean ± SEM. Significant differences were assessed by means of one-way ANOVA. If significant differences were observed, least significant square test was applied as post hoc test. A value of P < 0.05 was considered to be statistically significant.
In control hearts subjected to 40 min of ischaemia, LVEDP and LvdevP (calculated as the difference between LV systolic pressure and LVEDP) were, respectively, 6.4 ± 1.5 and 92.6 ± 5.8 mmHg at the end of the equilibration period. At this time, perfusion pressure was 59.3 ± 3.6 mmHg and coronary flow 12.2 ± 1.2 mL/min. No-flow ischaemia resulted in cessation of contractile activity and in a steep increase in LVEDP with a peak of 79.8 ± 8.4 mmHg 12.8 ± 0.5 min after the onset of ischaemia. No differences among groups were observed during the equilibration and ischaemic periods. In all hearts reperfusion induced a rapid increase in LVEDP (hypercontracture) that was accompanied by LDH release, functional recovery (LVdevP) was incomplete, and at the end of the reperfusion period, triphenyltetrazolium chloride reaction disclosed areas of myocardial necrosis. However, the magnitude of these parameters was different in the different groups.
Intracellular pH, phosphocreatine, lactate washout, and effect of postconditioning
Myocardial pHi was 7.02 ± 0.04 before ischaemia. Ischaemia induced a progressive pHi reduction that reached its maximum (6.42 ± 0.01) after 20 min without differences between groups. After 40 min of ischaemia, reperfusion resulted in a rapid rise in pHi, which recovered to normal values within 1.50 ± 0.24 min (Figure 2). pHi recovery during reperfusion was delayed in all postconditioned groups, but the extent of prolongation of intracellular acidosis was dependent on the postconditioning protocol. In the group postconditioned with cycles of 10 s (post10 group), pHi recovered within 2.50 ± 0.20 min, while in hearts with reduced flow rate during the reperfusion cycles (post10 low-flow group) and in the group subjected to 5 s cycles postconditioning protocol (post 5 group), recovery of pHi was further delayed and completed at 4.08 ± 0.25 and 3.89 ± 0.12 min, respectively (P < 0.001). Control hearts reperfused at reduced flow rate showed a non-significant trend towards a delay in pHi recovery (2.09 ± 0.24 min). In hearts perfused with KHB adjusted at pH 6.4 for the first 2 min of reperfusion, the time course of pHi normalization was very similar to that observed in the post10 low-flow group or in the post5 group. In these hearts, pHi recovery was completed at 3.47 ± 0.31 min (P < 0.001).
Ischaemia induced a rapid exhaustion of PCr without differences among groups. After 60 min of reperfusion, PCr recovered only partially in control hearts (23.77 ± 5.70%) without significant differences with respect to the post10 group (31.36 ± 5.19%, P = ns). However, the content of PCr significantly increased in post10 low-flow group (54.06 ± 4.52%, P = 0.006), post5 group (48.66 ± 5.68%, P = 0.021), and in hearts reperfused at pH 6.4 (52.40 ± 5.70%, P = 0.012).
Measurement of lactate in the coronary effluent demonstrated a close correlation between total lactate released during the first 2 min of reperfusion and the time to pHi recovery (23.48 ± 0.83 µM in control group, 11.4 ± 1.70 µM in post10 group, and 5.29 ± 1.39 µM in post5 group, r2 = 0.92, P < 0.001). Although lactate washout is not the only determinant of pHi, this result supports the notion that metabolite washout contributes to the rate of pHi recovery during reperfusion.
Effect of postconditioning on calpain activation
Control hearts showed a marked increase in the 145/150 kDa fragments resulting from specific calpain-mediated proteolysis of α-fodrin. A similar degree of proteolysis was observed in hearts subjected to treatments that did not delay pHi recovery beyond 2 min: 10 s postconditioning protocol or reperfusion at reduced flow without postconditioning. However, hearts submitted to reperfusion protocols resulting in significantly delayed pHi recovery, i.e. those perfused with reduced flow rate during the 10 s reperfusion periods, or subjected to cycles of 5 s, and those in which the pH of the perfusion buffer was adjusted to 6.4 during the 2 initial minutes of reperfusion, showed a markedly attenuated α-fodrin degradation (Figure 3). These differences in the degradation of α-fodrin were not the consequence of the differences in the magnitude of cell death since, as in previous studies,15 reperfusion with BDM prevented the development of hypercontracture and LDH release.
Recovery of intracellular pH, calpain activation, and myocardial injury
During reperfusion, in hearts subjected to the 10 s postconditioning protocol, there was a delay in hypercontracture (P = 0.023) and a non-significant trend towards reduction in LDH release (18%, P = 0.086) and infarct size (12%, P = 0.116). However, hearts perfused with reduced flow rate during the 10 s reperfusion periods (post10 low-flow group) or subjected to cycles of 5 s (post5 group) showed delayed (P < 0.001; Figure 4A) and attenuated (P < 0.001; Figures 4B) hypercontracture, as well as improved contractile recovery (23.5 ± 3.6 and 20.4 ± 3.2% of basal values after 60 min of reperfusion in post10 low-flow group and post5 group, respectively, vs. 7.0 ± 1.1% of basal values in control group, P < 0.001; Figure 4C), reduced LDH release (47% in post10 low-flow group and 38% in post5 group, P < 0.001; Figure 5A), and infarct size (36% in post10 low-flow group and 32% in post5 group, P < 0.001; Figure 5B). In control hearts, reperfusion at reduced flow rate during the first 2 min of reperfusion (control low-flow group) did not result in significant differences with respect to control group in any measured parameter, while 2 min of acidic perfusion fully mimicked the cardioprotective effects afforded by postconditioning in either post10 low-flow group or post5 group. Regression plot analysis including all groups of treatment disclosed a close correlation between infarct size and the time to pHi recovery (r = 0.96, P < 0.001; Figure 6). The effects of treatments causing delayed pHi recovery on calpain activity, extent of necrosis, and functional recovery were mimicked by pharmacological inhibition of calpain with MDL-28170 (Figures 4 and 5). MDL-28170 treatment did not modify the kinetics of pHi recovery during reperfusion (Figure 2).
This study demonstrates, by using NMR spectroscopy in the isolated rat heart, that the effectiveness of postconditioning in limiting infarct size depends on its ability to delay pHi recovery during reperfusion, and that prolongation of intracellular acidosis during reperfusion by either postconditioning or by acidic perfusion limits myocardial necrosis and improves the contractile recovery at least in part through attenuation of calpain activation.
It has been suggested that prolongation of acidosis during reperfusion is determinant for the protective effects of postconditioning.7–10 However, the available evidence on the role of delayed pHi recovery in postconditioning protection was only indirect, and the mechanism of protection by acidosis remained unknown. Cardioprotective effects of postconditioning were blunted by the perfusion of an alkalotic buffer, and pH of the coronary effluent in isolated hearts with regional ischaemia was lower during postconditioned perfusion.9,10 The present study analyses for the first time the time course of pHi recovery during postconditioning and the relation between the delay in pHi recovery during reperfusion and the effectiveness in inducing cardioprotection. For this purpose, three postconditioning protocols were used in which different flow rates during the reperfusion cycles or different duration of cycles were expected to alter the rate of pHi recovery. In our study, a widely used postconditioning protocol consisting in 6 cycles of 10 s, delayed normalization of pHi by only 1 min with respect to control hearts, and failed to induce cardioprotection in the isolated rat heart in agreement with the lack of protection observed in a previous study using the same protocol of postconditioning,23 although there was a non-significant trend towards reduction in LDH release and infarct size. In contrast, postconditioning protocols that delayed recovery of pHi by more than 2 min were clearly protective. Moreover, perfusion with acidic buffer during the first 2 min of reperfusion mimicked these protective effects. The fact that only postconditioning protocols that significantly delay pHi recovery during initial reperfusion are protective, together with the narrow inverse correlation between the magnitude of the delay and the extent of cell death, demonstrate the critical role of prolongation of intracellular acidosis in postconditioning protection. Previous studies have proposed that the duration of cycles of ischaemia–reperfusion of a postconditioning protocol must be shorter in models perfused with crystalloid buffer due to the many-fold higher coronary flow than in in vivo models, which allows faster normalization of intracellular milieu during the reperfusion periods and, in turn, accelerates pH recovery.10
Normalization of pHi during reperfusion is the consequence of the combined action of different transport systems, including Na+/H+-exchanger, Na+/HCO3− symport, and H+-coupled lactate efflux.24 These systems appear to be redundant to a large extent, since inhibition of one of them does not result in significant delay in pHi recovery. For example, inhibition of Na+/H+-exchanger by using selective drugs applied at the onset of reperfusion has a very small effect on pHi recovery.25,26 In contrast, simultaneous inhibition of Na+/H+-exchanger and Na+/HCO3− symport delay normalization of pHi.11,19,26 The strong correlation observed in our study between the delay in pHi recovery and the levels of lactate measured in the coronary effluent during the first 2 min of reperfusion supports the notion that washout of lactate, H+, and CO2 contributes to pHi correction19,24 and, therefore, that the maximal effective length of postconditioning cycles critically depends on coronary flow. Reduced catabolite washout results in attenuated transmembrane H+ gradient and decreased activity of Na+/H+-exchanger and Na+/HCO3− symport.
The protective effect of prolongation of intracellular acidosis during the first minutes of reperfusion has been solidly demonstrated in different models, including isolated cardiomyocytes11 and intact hearts.16,18,19,27 Different cardioprotective actions of delayed recovery of acidosis have been proposed, although their relative importance has not been established. Intracellular acidosis inhibits contractility28 and prevents Ca2+-dependent hypercontracture during initial reperfusion.11,17,18 Low pHi also inhibits opening of mPTP29 and this explains why mPTP remains closed during ischaemia, despite the presence of oxidative stress, Ca2+ overload, and ATP depletion.30 Acidosis has also been proposed to inhibit calpain activity13 and could thus attenuate Ca2+-dependent proteolytic injury occurring during reperfusion.14,15 Calpains hydrolyse proteins from the sarcolemma and the cytoskeleton including α-fodrin and ankyrin. This causes sarcolemmal fragility and detachment of the Na+-pump from its anchorage to the fodrin-based membrane skeleton, inducing Na+-pump failure and aggravating Na+ overload and secondary Ca2+ influx via reverse Na+/Ca2+-exchanger that may play a critical role in cell death.14,15 In this context, the effect of postconditioning on calpain activity can be at the same time a cause and a consequence of reduced Ca2+ overload. In addition, it has been shown that calpain-mediated Bid cleavage can induce cytochrome c release and mediate apoptotic and energetic cell death.31,32 Although cell death during early reperfusion occurs mainly in the form of necrosis, the potential role of calpain-mediated Bid cleavage as mechanism for the protective effect of prolonged acidosis and postconditioning needs to be investigated.
Finally, the existence of a link between delayed acidosis and other pathways reported to be involved in the cardioprotective effects of postconditioning, specially pathways associated with RISK kinases, has been suggested.9 Perfusion with alkalotic buffer blunted phosphorylation of Akt and ERK in postconditioning myocardium. Although pHi was not measured, the potential direct effect of alkalosis could not be excluded in that study and the molecular nature of any putative link between a prolonged acidosis and RISK activation is completely unknown. Further investigation would be necessary to demonstrate a potential relation between delayed acidosis and the activation of RISK pathway.
The present study does not exclude a significant contribution of mechanisms other than inhibition of calpain activation in the protective effects of delayed pHi recovery (Figure 7).9–12,19 However, our results demonstrate that postconditioning limits calpain activation during early reperfusion as consequence of the prolongation of acidosis, and indicate that this effect could play an important role in the protection afforded by postconditioning. In addition, our results support the potential value of cardioprotective strategies based on interfering with the rapid normalization of pHi during reperfusion therapy or inhibiting calpain activation.
Fondo Investigación Sanitaria (FIS-RECAVA RD06/0014/0025); Comisión Interministerial en Ciencia y Tecnología (CICYT SAF 2005-1758), Spanish Society of Cardiology (Basic Research Grant 2007).
Conflict of interest: none declared.
We thank María Ángeles García for her excellent technical assistance.