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Cardiovascular Research 2006 70(2):364-373; doi:10.1016/j.cardiores.2006.02.017
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Copyright © 2006, European Society of Cardiology

Ischemic preconditioning prevents calpain-mediated impairment of Na+/K+-ATPase activity during early reperfusion

Javier Inserte, David Garcia-Dorado*, Victor Hernando, Ignasi Barba and Jordi Soler-Soler

Servicio de Cardiologia, Hospital Universitari Vall d'Hebron, Passeig Vall d'Hebron, 119-129, 08035 Barcelona, Spain

* Corresponding author. Tel.: +34 93 4894038; fax: +34 93 4894032. Email address: dgdorado{at}vhebron.net

Received 30 September 2005; revised 14 February 2006; accepted 15 February 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 Acknowledgments
 References
 
Objectives We previously demonstrated that ischemic preconditioning (IPC) attenuates calpain activation during reperfusion. Herein, we tested the hypothesis that enhancement of Na+/K+-ATPase activity during early reperfusion as a result of calpain inhibition is involved in the protection afforded by myocardial IPC.

Methods Intracellular Na+ concentration ([Na+]i) measured using 23Na-magnetic resonance spectroscopy, Na+/K+-ATPase activity, detachment of Na+/K+-ATPase {alpha} subunits from the membrane cytoskeleton, degradation of fodrin and ankyrin, and calpain activation were analysed in isolated rat hearts reperfused after 60 min of ischemia with or without previous IPC and different treatments aimed to mimic or blunt the effects of IPC.

Results In non-treated hearts subjected to ischemia (control hearts), reperfusion for 5 min severely reduced Na+/K+-ATPase activity and dissociated {alpha}1 and {alpha}2 subunits of Na+/K+-ATPase from the membrane-cytoskeleton complex in parallel with proteolysis of {alpha}-fodrin and ankyrin-B and calpain activation. IPC accelerated the recovery of [Na+]i, increased Na+/K+-ATPase activity, and prevented dissociation of Na+/K+-ATPase from the membrane-cytoskeleton complex. IPC also prevented {alpha}-fodrin and ankyrin-B loss and calpain activation, effects that were associated with attenuated lactate dehydrogenase (LDH) release and infarct size and improved contractile recovery. These effects of IPC were reproduced by perfusing the hearts with the calpain inhibitor MDL-28170 and by transient stimulation of cAMP-dependent protein kinase (PKA) with CPT-cAMP, and they were reverted by perfusing with the PKA inhibitor H89.

Conclusion The results of the present study are consistent with the hypothesis that enhanced recovery of Na+/K+-ATPase activity during reperfusion as a result of attenuated calpain-mediated detachment of the protein from the membrane-cytoskeleton complex contributes to the protection afforded by IPC.

KEYWORDS Myocardial infarction; Sodium; Calcium; Cell death


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 Acknowledgments
 References
 
The discovery of the phenomenon of ischemic preconditioning (IPC), consisting of a state of increased tolerance to ischemia–reperfusion induced by brief episodes of non-lethal ischemia, has revolutionized research on myocardial ischemia/reperfusion injury during the last two decades. The protection afforded by IPC against cell death secondary to transient ischemia is dramatic and observable in all species investigated as well as in tissues other than myocardium. However, although a large number of studies have identified different triggers and mediators of ischemic preconditioning (IPC), the final mechanisms by which IPC protects the myocardium against cell death secondary to ischemia/reperfusion remain poorly understood [1].

Intracellular Ca2+ overload plays a prominent role in reperfusion-induced cardiomyocyte necrosis by inducing excessive contractile activation [2], enzyme activation leading to cytoskeletal and sarcolemmal fragility [3], and mitochondrial permeability transition [4]. The sarcolemmal Na+/Ca2+-exchanger (NCX) operating in reverse mode is the major factor leading to Ca2+ influx during ischemia and reperfusion. Its contribution to Ca2+ overload during reperfusion is determined by how quickly NCX returns to its forward mode, which depends on the rapidity of membrane repolarization and restoration of the transsarcolemmal Na+ gradient [5]. It has been shown that the kinetics of [Na+]i recovery during reperfusion determines the degree of injury, and that altered function of Na+/K+-ATPase during initial reperfusion importantly contributes to Na+ overload [6]. Several studies have described that IPC alters Na+ kinetics by preserving Na+ efflux via Na+/K+-ATPase and have related this effect to the protection afforded by IPC against reperfusion-induced cell death [7–9], but the mechanism by which IPC enhances recovery of Na+/K+-ATPase activity during reperfusion has not yet been elucidated.

Na+/K+-ATPase is a transmembrane heterodimer protein composed of {alpha} and β subunits. The cytoplasmic domain of {alpha} subunit interacts with ankyrin [10], a protein that connects the Na+/K+-ATPase to the fodrin-based membrane skeleton [11]. Recently, we demonstrated that Na+/K+-ATPase activity is impaired during early reperfusion after prolonged ischemia and that this effect may be explained by calpain-mediated degradation of the anchorage that fixes Na+/K+-ATPase to the membrane cytoskeleton (ankyrin) and the membrane cytoskeleton itself (fodrin) [12]. Previously, Aufricht et al. described that IPC prevents dissociation of Na+/K+-ATPase from its cytoskeletal anchorage in rat renal cortex after ischemia [13], and we have demonstrated in isolated rat hearts that IPC attenuates calpain-mediated degradation of structural proteins including fodrin and ankyrin in a PKA-dependent manner and that the administration of calpain inhibitors mimicked in part the protective effects of IPC against cell death [3].

Therefore, in the present study we hypothesized that calpain inhibition induced by IPC prevents proteolysis of the anchorage of Na+/K+-ATPase, resulting in a rapid restoration of Na+-pump activity and consequently in a fast [Na+]i recovery during early reperfusion and limited reperfusion injury.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Appendix A. Supplementary data
 Acknowledgments
 References
 
2.1 Isolated heart preparation
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.

Male Sprague–Dawley rats (300 to 350 g) were anaesthetized with an intraperitoneal injection of sodium thiopental (150 mg/kg). The hearts were perfused in a Langendorff apparatus at 10 ml/min 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% O2–5% CO2 at 37 °C. Left ventricular pressure (LVP) was monitored as previously described [3]. Left ventricular developed pressure (LVdevP) was calculated as the difference between left ventricular peak systolic pressure and left ventricular end-diastolic pressure (LVEDP).

2.2 Ischemia–reperfusion studies
Fig. 1 shows the different study protocols. Control hearts were perfused normoxically for 40 min and then subjected to 60 min ischemia followed by 30 min of reperfusion. The IPC protocol consisted of two cycles of 5 min of ischemia and 5 min of reperfusion.


Figure 1
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  		Fig. 1 Ischemia–reperfusion protocols. IPC=ischemic preconditioning. TTC=2,3,5-triphenyltetrazolium chloride.

 
The role of calpain inhibition in cardioprotection was studied in hearts in which the membrane-permeable calpain inhibitor MDL-28170 (Calbiochem) at 10 µM or its vehicle (0.01% DMSO) was added to the perfusion media during the 10 min prior to ischemia and the first 10 min of reperfusion. To investigate whether the protective effect of calpain inhibition was additive to IPC, a group of hearts subjected to the IPC protocol were perfused with MDL-28170 for the last 5 min prior to sustained ischemia and the first 10 min of reperfusion.

In a series of experiments aimed at mimicking the effects of IPC, PKA was transiently activated by stimulation with the cell-permeable cAMP analogue 8-(4-chlorophenylthio)-cAMP (CPT-cAMP, Calbiochem) at 25 µM (n=6) added to the KHB for 5 min followed by 5 min of washout before ischemia. In another group, the protection afforded by IPC was reverted by inhibition of PKA during the trigger period with the PKA inhibitor H89 (Calbiochem) at 10 µM dissolved in DMSO (final concentration 0.01%) added to the KHB 10 min before the onset of the first preconditioning-ischemia and during the following preconditioning-reperfusion.

Five hearts from each group were frozen in liquid nitrogen after 5 min of reperfusion for Western blot analysis and determination of calpain and Na+/K+-ATPase activities, and in five additional hearts per group reperfusion was prolonged for 30 min for measurement of LVP, infarct size, and lactate dehydrogenase (LDH) release.

2.3 Determinations
2.3.1 NMR spectroscopy
Intracellular Na+ kinetics were measured in an additional series of control, IPC-, and MDL-treated hearts (n=3 per group) by 23Na NMR. Spectroscopy was performed on a Bruker Advance 400 spectrometer equipped with a 20-mm probe tuned to 23Na and 31P. 23Na NMR spectra were obtained at 105.8 MHz. Each spectrum was the accumulation of 104 free induction decays and lasted for 30 s. The [Na+]i was determined using the shift reagent thulium(III) 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetra(methylenephosphonate) (TmDOTP5 –, Magnetic Resonance Solutions Inc.). TmDOTP5 – at 3.5 mM was added to the perfusate during the equilibration period. Total Ca2+ in the perfusate buffer was increased to partially correct the Ca2+ affinity of TmDOTP5 – [14]. 31P NMR spectra were obtained at 161.9 MHz during equilibration and at the end of the reperfusion period. Each spectrum was the accumulation of 128 scans with a delay of 1.18 s, and lasted for 2.5 min.

In order to rule out that the effects on Na+ recovery were not a mere consequence of differences in cell death, hearts from all groups were reperfused in the presence of the contractile inhibitor 2,3 butanedione monoxime (BDM) at 50 mM [15]. Hearts were reperfused for 10 min instead of 30 min to conserve the non-recirculating shift agent.

2.3.2 Quantification of cell death
LDH activity was spectrophotometrically measured in the coronary effluent throughout the reperfusion period [3]. In an additional series of experiments (n=4), infarct size was measured in cardiac slices after incubation with 1% 2,3,5-triphenyltetrazolium chloride (TTC) at the end of reperfusion [12].

2.3.3 Na+/K+-ATPase activity assay
The ouabain-sensitive Na+/K+ ATPase activity of heart homogenates was determined as described previously [12,16]. Generated Pi was assayed using an ammonium molybdate spectrophotometric assay [17]. ATPase activity assayed in the presence of ouabain was subtracted from the value observed in its absence to estimate ouabain-sensitive Na+/K+-ATPase activity.

2.3.4 Western blot analysis
Frozen hearts were homogenized in buffer containing (in mmol/L) 20 Tris–HCl, 140 NaCl, 1 EDTA, 10 sodium azide, 250 sucrose, 0.5 PMSF, and 0.1 DTT, pH 7.3. For calpain, fodrin, and ankyrin analysis, cytosolic and membrane fractions were separated as described previously [18]. The differential distribution of the {alpha} subunit of Na+/K+-ATPase in Triton X-100-soluble and -insoluble protein fractions was analyzed as described [19,20]. Previous studies have shown that the Triton X-100-insoluble fraction consists primarily of actin cytoskeleton and associated proteins, while the soluble fraction consists of soluble proteins and proteins capable of interacting with the cytoskeleton but which are not associated with the cytoskeleton at the time of extraction [21,22]. Although these studies were performed in tissues other than myocardium (epithelial and renal cortical cells), our results indicate that the separation of proteins not associated with the cytoskeleton by this method can be applied to myocardial tissue (see Fig. I, Supplementary data).

Proteins were separated by electrophoresis on a 7.5% SDS gel and immunoblotted with antibodies against {alpha}1 and {alpha}2 (Upstate Biotechnology) Na+/K+-ATPase subunits, {alpha}-fodrin (Affiniti Research Products), ankyrin-B (Ab-1, Oncogene Research Products) and m-calpain domain I (Calbiochem). Protein bands were detected by chemiluminescence (SuperSignal West Dura Extended Duration Substrate, Pierce) and quantified using a CCD camera system (Image Reader LAS-3000, Fujifilm) and image analysis software (Image Gauge, Fujifilm). Equal protein load was confirmed by Ponceau staining (Figs. 5 and 6Go).


Figure 5
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  		Fig. 5 (Panel A) Distribution of {alpha}1 and {alpha}2 Na+/K+-ATPase subunits in the non-cytoskeletal soluble fraction (sol.) and cytoskeletal insoluble fraction (insol.) measured after 5 min of reperfusion in the different groups of treatment: normoxically perfused hearts (Nx), control (Control), CPT-cAMP (cAMP), H89, ischemic preconditioning (IPC), and MDL-28170 (MDL) treated hearts. Data are expressed as percentage compared with normoxically perfused hearts (Nx) and presented as mean±S.E.M. of three experiments. *P<0.05 vs. Nx. (Panel B) Ponceau staining of the membranes.

 

Figure 6
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  		Fig. 6 (Panel A) Effect of IPC and different pharmacological interventions on the levels of calpain-specific {alpha}-fodrin breakdown products (145/150 kDa) and on degradation of ankyrin-B measured after 5 min of reperfusion. Data are expressed as percentage compared with normoxically perfused hearts (Nx) and presented as mean±S.E.M. of three experiments. *P<0.05 vs. Nx. (Panel B) Ponceau staining of the membranes.

 
2.3.5 Calpain activity assay
Frozen hearts were homogenized with tissue protein extraction reagent (TPER, Pierce) and centrifuged at 15,000 x g for 10 min. Calpain activity was measured by fluorometry (Gemini XS, Molecular Devices) using Suc-Leu-Tyr-7-amino-4-methylcoumarin (Suc-Leu-Tyr-AMC, Calbiochem) as substrate as previously described [3]. Suc-Leu-Tyr-AMC release was monitored over 30 min at 25 °C using excitation and emission wavelengths of 380 nm and 460 nm, respectively. MDL-28170 at 10 µM was used to determine the specificity of the assay.

2.3.6 Statistical analysis
Differences between groups were assessed by means of one-way ANOVA. Changes with time were assessed by repeated-measures ANOVA. Significance was set at a P value of 0.05. Results are expressed as mean±S.E.M.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Appendix A. Supplementary data
 Acknowledgments
 References
 
3.1 Functional recovery and cell death during reperfusion
At the end of the equilibration period, LVEDP and LVdevP were 6.7±0.8 and 104.1±5.5 mm Hg, respectively, without differences between groups.

In control hearts subjected to 60 min of ischemia, reperfusion-induced hypercontracture (defined as peak LVEDP after onset of reperfusion) was 140.1±4.4 mm Hg. LVdevP recovered to 7.3±4.5% of its initial value after 30 min of reperfusion. LDH release during this period was 690.2±50.2 U/30 min/g dry wt (Fig. 2) and infarct size averaged 62.4±6.3% of ventricular mass. IPC attenuated hypercontracture (94.8±3.8 mm Hg, P<0.001), increased functional recovery (46.0±7.3%, P<0.001), and greatly reduced LDH release (160.5±30.4 U/30 min/g dry wt, P<0.001) and infarct size (19.3±4.2% of ventricular mass, P=0.001) in hearts reperfused for 30 min (Fig. 2).


Figure 2
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  		Fig. 2 Hypercontracture (peak LV end diastolic pressure, panel A), LV developed pressure (LvdevP as percentage of baseline values, panel B), cumulative LDH release after 30 min of reperfusion (panel C), and infarct size (as percentage of ventricular mass, panel D) in the different groups of treatment: control (C), CPT-cAMP (cAMP), H89, ischemic preconditioning (IPC), MDL-28170 (MDL). Data are mean±S.E.M. *P<0.05 vs. control group. *P<0.05 vs. IPC group.

 
3.2 Na+ kinetics and Na+/K+-ATPase activity in IPC hearts
During equilibration, 31P NMR spectra showed no differences in the cellular energy status between all groups. Addition of TmDOTP5 – resulted in a 32% decrease in LVdevP without differences between groups. During ischemia, the [Na+]i signal intensity increased at a similar rate in all groups (Fig. 3). As in previous studies [12,15], addition of the contractile inhibitor BDM during reperfusion prevented the development of hypercontracture and LDH release. During reperfusion, the rate of [Na+]i recovery was higher in the IPC group as compared to control hearts (27.6±4.2% of decrease in Na+ signal area per minute during the first 4 min of reperfusion vs. 7.4±2.5%/min), and [Na+]i returned to pre-ischemic values within 6 min but remained elevated after the 10 min of reperfusion in the control group (Fig. 3). After 10 min of reperfusion, hearts recovered cellular energetics without significant differences between control and IPC groups [48.4±5.3% and 45.8±4.1% of ATP recovery with respect to basal values, respectively, and 124.9±2.6% and 116.7±5.2% of phosphocreatine (PCr) recovery, respectively].


Figure 3
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  		Fig. 3 (Panel A) Relative changes in intracellular sodium ([Na+]i) with respect to pre-ischemic values during ischemia (left) and reperfusion (right) in control, preconditioned (IPC) and MDL-28170 (MDL) treated hearts. (Panel B) Average rates of change of [Na+]i during ischemia and during the initial 4 min of reperfusion are derived from values shown in panel A. Data are mean±S.E.M. *P<0.05 vs. control group.

 
Ouabain-sensitive Na+/K+-ATPase activity in nonischemic hearts was 12.4±0.7 µmol Pi/mg protein/h (total ATPase activity was 51.6±2.6 µmol Pi/mg protein/h). In control hearts, Na+/K+-ATPase activity measured 5 min after reperfusion was severely reduced (22.0±5.7% of values obtained in normoxic hearts) while recovery of Na+/K+-ATPase activity in IPC hearts was markedly increased (78.4±8.1% of normoxic hearts, P=0.004 with respect to control hearts; Fig. 4).


Figure 4
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  		Fig. 4 Ouabain-dependent Na+/K+-ATPase activity expressed as percentage of values in normoxically perfused hearts (Nx) in control (Control), CPT-cAMP (cAMP), H89, ischemic preconditioning (IPC), and MDL-28170 (MDL) treated hearts. Data are mean±S.E.M. *P<0.05 vs. Nx. *P<0.05 vs. control group.

 
3.3 IPC prevents loss of Na+/K+-ATPase from its anchorage to the membrane cytoskeleton
Western blot analysis of {alpha}1 and {alpha}2 subunits of Na+/K+-ATPase in Triton-insoluble fractions from control hearts subjected to 60 min of ischemia revealed a significant loss of both isoforms, principally {alpha}2, after 5 min of reperfusion and a marked increase in the soluble fraction (Fig. 5), indicating their dissociation from its cytoskeletal anchorage. Analysis of the structural proteins {alpha}-fodrin and ankyrin-B showed a marked increase in the 145/150-kDa fragments resulting from degradation of {alpha}-fodrin and a decrease in the 220-kDa isoform of ankyrin-B (Fig. 6). IPC significantly attenuated reperfusion-induced loss of cytoskeletal anchorage of Na+/K+-ATPase (decreased Triton-extractable Na+/K+-ATPase fraction, Fig. 5) and degradation of {alpha}-fodrin and ankyrin-B (Fig. 6).

3.4 Preservation of Na+/K+-ATPase activity in IPC myocardium is related to calpain inhibition
In control hearts, calpain activity was increased after 5 min of reperfusion (P<0.001 with respect to normoxically perfused hearts; Fig. 7A). This activation correlated with the translocation of calpain from the cytosol to the membrane fraction (Fig. 7B). IPC significantly attenuated calpain activity (P<0.001 with respect to control hearts) and its translocation to the membrane fraction.


Figure 7
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  		Fig. 7 Calpain activity (panel A) and distribution of m-calpain (80 kDa subunit) between membrane and cytosolic fraction (panel B), in hearts reperfused for 5 min and allocated to the different treatments. Data are mean±S.E.M. *P<0.05 vs. normoxically perfused group (Nx). *P<0.05 vs. control group.

 
Perfusion with the calpain inhibitor MDL-28170 attenuated hypercontracture (P<0.001), reduced LDH release (P<0.001) and infarct size (P=0.026), and improved functional recovery after 30 min of reperfusion (Fig. 2). These effects were associated with an accelerated rate of [Na+]i recovery (20.2±1.7%/min; Fig. 3) and with improved restoration of Na+/K+-ATPase activity (57.9±7.5% of normoxic values, P=0.024 vs. control hearts; Fig. 4). Western blot analysis showed a marked attenuation in the cytoskeletal detachment of {alpha}1 and {alpha}2 subunits of Na+/K+-ATPase (Fig. 5) and in the degradation of {alpha}-fodrin and ankyrin-B (Fig. 6) measured after 5 min of reperfusion. These results correlated with an attenuated calpain activity (P<0.001 vs. control hearts) and prevention of its translocation to the membrane fraction (Fig. 7). The protection observed with the combination of MDL-28170 and IPC did not differ from that afforded by IPC alone (hypercontracture: 89.1±5.4 mm Hg; LDH release: 193±35 U/30 min/g dry wt; functional recovery: 43.1±9.2%, P=ns vs. IPC group).

3.5 Involvement of PKA
Since previous studies have demonstrated that transient PKA activation before the onset of ischemia simulated the beneficial effects of IPC [3,23,24], we analyzed the effect of cAMP on Na+/K+-ATPase activity. Perfusion of control hearts with CPT-cAMP markedly attenuated hypercontracture (P<0.001), LDH release (P<0.001), infarct size (P=0.011), and post-ischemic dysfunction after 30 min of reperfusion (P<0.001) (Fig. 2), and these effects were associated with improved recovery of Na+/K+-ATPase activity (70.3±11.4% of normoxic values, P=0.024 compared with control hearts) (Fig. 4) and preservation of {alpha}1 and {alpha}2 subunits of Na+/K+-ATPase in the Triton-insoluble fraction (Fig. 5). {alpha}-Fodrin and ankyrin-B degradation and calpain activation (P=0.004 vs. control group) (Fig. 6) and its translocation to the membrane fraction (Fig. 7) were attenuated. In contrast, PKA inhibition during the preconditioning protocol abolished the beneficial effects of IPC. Perfusion with H89 induced a significant reduction in the beneficial effects of IPC on hypercontracture (P<0.001), LDH release (P<0.001), infarct size (P=0.004), and functional recovery (P=0.002) (Fig. 2), and these effects were associated with a loss of Na+/K+-ATPase activity (29.5±6.2% of normoxic hearts, P=0.014 vs. IPC group) (Fig. 4), increased cytoskeletal detachment of {alpha}1 and {alpha}2 subunits of Na+/K+-ATPase (Fig. 5), {alpha}-fodrin and ankyrin-B breakdown (Fig. 6), and enhanced calpain activation (P=0.015 vs. IPC group) and translocation to the membrane (Fig. 7).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Appendix A. Supplementary data
 Acknowledgments
 References
 
Previous studies have proposed that preservation of Na+ efflux via Na+/K+-ATPase and limitation of the coupled Ca2+ influx through NCX during the first minutes of reperfusion contribute importantly to the cardioprotective effects of IPC [4,7,8]. In this study, we demonstrate for the first time that IPC attenuated the dissociation of Na+/K+-ATPase protein from the membrane-cytoskeletal complex and that this effect is mediated through a reduction of calpain activation during reperfusion.

4.1 Effect of IPC on intracellular Na+ kinetics
In agreement with previous studies, our results fail to show any effect of IPC on the increase of [Na+]i during sustained ischemia [25–27]. These results are, however, in controversy with the slightly but significant attenuation of [Na+]i in preconditioned rat hearts reported by Imahashi et al. [7] and with the reduction in [Na+]i observed by Liu et al. in newborn rabbit hearts [28]. While reasons for the differences between these and other studies, including the present study, remain unclear, our results are consistent with previous reports suggesting that the mechanisms for the protection afforded by Na+/H+-exchanger inhibitors, which dramatically attenuate [Na+]i overload, and IPC are different [29,30].

In contrast with the absence of a clear effect of IPC on [Na+]i overload during ischemia, there is solid evidence demonstrating that IPC accelerates [Na+]i recovery at the beginning of reperfusion [7,25–27]. Our data are consistent with these studies and show that [Na+]i recovers to baseline values within 6 min in preconditioned hearts but not in control hearts. To rule out the possibility that the observed differences in [Na+]i kinetics could be a mere consequence of the protection against cell death achieved by IPC, in NMR experiments the hearts were perfused in the presence of BDM, an inhibitor of the contractility that has been consistently shown to prevent the development of hypercontracture, rupture of the sarcolemma, and cell death [12,15,31]. While BDM was present there was no LDH release, and ATP and PCr levels recovered to similar levels in both preconditioned and non-preconditioned groups. Overall, these observations rule out the possibility that differences in the extent of cell death or in the energy status may be responsible for the faster recovery of [Na+]i in the IPC group.

4.2 Na+/K+-ATPase activity in IPC hearts
Resumption of Na+/K+-ATPase activity upon reperfusion is the main mechanism that the cell possesses to extrude Na+ accumulated during ischemia and the initial phase of reperfusion [14]. In previous studies, we and others have shown a decrease in Na+-pump activity in isolated rat hearts subjected to ischemia/reperfusion and have related the recovery kinetics of [Na+]i during reperfusion to the influx of Ca2+ through NCX and cell injury [6,12,32]. A previous study proposed that IPC prevents the reduction of Na+/K+-ATPase activity during the early phase of sustained ischemia [33]. However, in the present study, IPC did not modify [Na+]i kinetics during ischemia but induced a faster [Na+]i recovery associated with a rapid restoration of Na+/K+-ATPase activity during the first minutes of reperfusion. Although the marked differences in experimental conditions make it impossible to know the reason for these discrepancies, both studies point out an important role of Na+/K+-ATPase in IPC.

Although enhancement of Na+-pump activity in IPC hearts had been previously described [8,9,13,26,34], the mechanism remained unknown. Lundmark et al. attributed the enhancement to a better preservation of ATP during ischemia in IPC.

However, there is still controversy about the possible preservation of the energy status in IPC hearts, and faster [Na+]i recovery has been reported without differences in high-energy phosphates during ischemia and reperfusion [27,28]. In fact, the differences in Na+ kinetics obtained in our study were observed under conditions of similar levels of ATP and PCr between the IPC and control hearts reperfused in the presence of BDM.

Recently, we showed a significant decrease in the protein levels of {alpha}1 and {alpha}2 isoforms of Na+/K+-ATPase attached to the cytoskeleton in reperfused hearts after prolonged ischemia [12]. The present study confirms these observations and demonstrates that IPC prevents the dissociation of Na+/K+-ATPase isoforms from their cytoskeletal anchorage in intact myocardium, an effect that correlates with the observed preservation of the Na+-pump activity.

4.3 Detachment of Na+K+-ATPase from the sarcolemma-cytoskeletal complex
The localization and stability of Na+/K+-ATPase in the membrane is regulated by direct interaction of ankyrin with the cytoplasmic domain of the alpha subunit of Na+/K+-ATPase and with the underlying fodrin-based cytoskeleton [35]. The importance of the association of Na+/K+-ATPase with the ankyrin–fodrin complex has been demonstrated in cardiomyocytes from mice heterozygous for a null mutation in ankyrin-B, where reduced ankyrin resulted in a decrease in {alpha}1 and {alpha}2 protein levels [36]. In a recent study, we showed that the observed detachment of Na+/K+-ATPase in control hearts is a consequence of calpain-mediated cleavage of its well-known substrates ankyrin and fodrin [12].

The Ca+-dependent cysteine protease calpain remains inactive in the cytosol during ischemia due to the acidic pH [37]. It translocates during myocardial reperfusion to the membrane and is converted to an activated form able to cleave different structural proteins [38]. In the present study, we combined different methods (in vitro assay of calpain activity, formation of specific cleavage fragments of fodrin, and translocation to the membrane fraction) to demonstrate the activation state of calpain. The results confirm our previous observations showing that IPC attenuates calpain activation. The fact that inhibition of calpain with MDL-28170 prevents the cytoskeletal detachment of {alpha}1 and {alpha}2 subunits of Na+/K+-ATPase in reperfused myocardium, reproducing the effects of IPC on kinetics of [Na+]i recovery, Na+/K+ ATPase activity, and loss of Na+/K+-ATPase anchorage, suggest that the inhibition of calpain-mediated degradation of the ankyrin–fodrin complex is involved in the preservation of Na+-pump activity observed in IPC hearts. Furthermore, the observation that reperfusion induced a redistribution of Na+/K+-ATPase subunits from the Triton-insoluble to the Triton-soluble fraction without apparent proteolysis, together with our previously published results obtained incubating Na+/K+-ATPase in vitro with calpain [12], excludes a direct proteolytic effect on Na+/K+-ATPase subunits.

Calpain could also improve Na+ kinetics by limiting Na+ influx during reperfusion. For example, although no direct effect of calpain on the Na+/H+-exchanger has been reported, we cannot rule out an indirect effect on its activity that could contribute to [Na+]i recovery [39].

In agreement with our results and using a similar method to study the dissociation of Na+/K+-ATPase from its cytoskeletal anchorage, Bidmon et al. have described that IPC stabilizes Na+/K+-ATPase in rat renal cortex [20]. However, these authors linked this effect to a marked induction and cytoskeletal distribution of HSP-25, HSP-70 and HSP-90 in preconditioned renal cortex. Although the potential role of HSP was not investigated in the present study, there are marked differences between both experimental models and the time of reperfusion for protein measurements (5 min of reperfusion in our model and 18 to 20 h of reflow in that of Bidmon) that could explain the differences in the mechanisms proposed for Na+/K+-ATPase preservation in IPC.

4.4 Pharmacological simulation and inhibition of IPC
The present study confirms our previous results demonstrating that the inhibitory effect of IPC on calpain activation is dependent on transient PKA activation during the preconditioning phase [3].

The fact that the effect of IPC on calpain activity and its correlation with ankyrin–fodrin degradation, Na+/K+-ATPase detachment, and Na+-pump activity could be reproduced by pharmacological simulation of IPC and were attenuated by treatments that reverted the protective effects of IPC strongly suggests a role of reduced calpain activation and preservation of Na+/K+-ATPase activity in the protective effect of IPC. In a previous study, we proposed reduced sarcolemmal fragility as a result of attenuation of calpain-mediated degradation of structural proteins as one of the end-effector mechanisms of the protective effect of IPC against necrotic cell death [3]. The present study stresses the importance of calpain inhibition during early reperfusion in the genesis of the protection afforded by IPC by showing that calpain proteolysis not only contributes to cell death by increasing sarcolemmal fragility but also by favoring the development of hypercontracture by detaching Na+/K+-ATPase from the membrane-cytoskeletal complex which impairs [Na+]i recovery and favors Ca2+ influx through NCX.

Altogether, the present results provide the first experimental evidence of an important role of attenuated calpain-mediated detachment of Na+/K+-ATPase from the membrane-cytoskeleton as an end-effector mechanism of preconditioning protection.


   Appendix A. Supplementary data
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
Acknowledgments
 References
 
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cardiores.2006.02.017.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 Acknowledgments
References
 
This study was partially supported by grants CICYT SAF 2002-00759 and FIS-RECAVA. We appreciate the excellent technical work of Maria Angeles Garcia.


   Notes
 
* This article was handled by Guest Editor Karin Sipido, University of Leuven, Leuven, Belgium.

Time for primary review 36 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 Acknowledgments
 References
 

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