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Cellular Biology

Calpain-Mediated Impairment of Na+/K+–ATPase Activity During Early Reperfusion Contributes to Cell Death After Myocardial Ischemia

Javier Inserte, David Garcia-Dorado, Victor Hernando, Jordi Soler-Soler
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https://doi.org/10.1161/01.RES.0000181170.87738.f3
Circulation Research. 2005;97:465-473
Originally published September 1, 2005
Javier Inserte
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David Garcia-Dorado
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Victor Hernando
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Jordi Soler-Soler
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Abstract

Na+ overload and secondary Ca2+ influx via Na+/Ca2+ exchanger are key mechanisms in cardiomyocyte contracture and necrosis during reperfusion. Impaired Na+/K+–ATPase activity contributes to Na+ overload, but the mechanism has not been established. Because Na+/K+–ATPase is connected to the cytoskeleton protein fodrin through ankyrin, which are substrates of calpains, we tested the hypothesis that calpain mediates Na+/K+–ATPase impairment in reperfused cardiomyocytes. In isolated rat hearts reperfused for 5 minutes after 60 minutes of ischemia, Na+/K+–ATPase activity was reduced by 80%, in parallel with loss of α-fodrin and ankyrin-B and detachment of α1 and α2 subunits of Na+/K+–ATPase from the membrane–cytoskeleton complex. Calpain inhibition with MDL-7943 during reperfusion prevented the loss of these proteins, increased Na+/K+–ATPase activity, attenuated lactate dehydrogenase release, and improved contractile recovery, and these beneficial effects of MDL-7943 were reverted by ouabain. The impairment of Na+/K+–ATPase was not a mere consequence of cell death because it was not altered in hearts in which contracture and cell death had been prevented by contractile blockade with 2,3-butanedione monoxime. In these hearts, concomitant calpain inhibition preserved Na+/K+–ATPase content and function and attenuated cell death occurring on withdrawal of 2,3-butanedione monoxime. In vitro assay showed no detectable degradation of Na+/K+–ATPase subunits after 10 minutes of incubation with activated calpain. Thus, we conclude that calpain activation contributes to the impairment of Na+/K+–ATPase during early reperfusion and that this effect is mainly mediated by degradation of the anchorage of Na+/K+–ATPase to the membrane cytoskeleton.

  • calpain
  • Na+/K+–ATPase
  • Na+/Ca2+ exchanger
  • reperfusion injury

Reperfusion of cardiomyocytes after prolonged ischemia may lead to immediate necrotic cell death that results in a characteristic pathological pattern known as contraction band necrosis.1 Elevated and oscillating cytosolic Ca2+ concentration plays a key role in reperfusion-induced cardiomyocyte necrosis by inducing excessive contractile activation,2 enzyme activation that leads to cytoskeletal and sarcolemmal fragility,3 and by favoring mitochondrial permeability transition.4 One of the main determinants of Ca2+ overload during reperfusion is additional Ca2+ influx through sarcolemmal Na+/Ca2+ exchanger (NCX) operating in reverse mode,5,6 which, in turn, is a consequence of increased cytosolic Na+ concentration.7

Cytosolic accumulation of Na+ during previous ischemia,8 and additional Na+ entry through Na+/H+ exchanger and Na+/HCO3− cotransporter associated with rapid correction of acidosis9,10 and gap junction–mediated propagation of Na+ overload from adjacent cells,11 has been shown to contribute to increase cytosolic Na+ concentration in cardiomyocytes during early reperfusion. However, there is also evidence indicating that impaired Na+ extrusion through the Na+/K+–ATPase may play an important role.12–15 Na+/K+–ATPase activity is highly dependent on myocardial energy, but previous studies have shown that an inadequate energy recovery cannot satisfactorily explain this impairment in cardiomyocytes showing ATP-dependent hypercontracture.16,17 The mechanisms of this Na+/K+–ATPase failure during reperfusion and contribution to Ca2+ overload and cell death have not been completely elucidated.

Na+/K+–ATPase is a transmembrane heterodimer protein composed of α and β subunits. The cytoplasmic domain of α subunit interacts with ankyrin,18 a protein that connects the Na+/K+–ATPase to the fodrin-based membrane skeleton.19 In a recent study, Mohler et al demonstrated reduction in overall protein levels of α1 and α2 Na+/K+–ATPase subunits in cardiomyocytes from mice heterozygous for a null mutation in ankyrin-B.20 Moreover, studies in an epithelial cellular line of renal tubule have suggested that the dissociation of the fodrin–ankyrin complex contributes to the loss of Na+/K+–ATPase polarity after ischemia.21

α-Fodrin and ankyrin are known substrates of calpains, a group of nonlysosomal Ca2+-dependent proteases. Previous studies have demonstrated activation of calpain and degradation of both proteins during reperfusion.22,23 Recently, we have shown, in isolated rat hearts subjected to global ischemia, that inhibition of calpain prevents degradation of structural proteins, including fodrin and ankyrin, by a mechanism that was associated with reduced sarcolemmal fragility and cell death during reperfusion.3

The present study tested the hypothesis that activation of calpain during reperfusion results in proteolysis of ankyrin and fodrin, proteins that form the structural complex that maintains the stability of the Na+/K+–ATPase, resulting in loss of pump activity and, consequently, in Ca2+ entry through reverse mode of NCX, which leads to reperfusion cell injury.

Materials and Methods

Isolated Heart Preparation

The experimental procedures conformed to the Guide for the Care and Use of Laboratory Animals (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; Harlan Iberica, Barcelona, Spain) were anesthetized by IP injection of sodium thiopental (150 mg/kg). The hearts were perfused at constant flow (10 mL/min) in a Langendorff apparatus with a modified Krebs–Henseleit bicarbonate buffer (in mmol/L: 140 NaCl, 24 NaHCO3, 2.7 KCl, 0.4 KH2PO4, 1 MgSO4, 1.8 CaCl2, and 11 glucose) equilibrated with 95% O2–5% CO2 at 37°C. Left ventricular pressure was monitored as previously described.5

Ischemia-Reperfusion Studies

Importance of Na+/K+–ATPase Activity for Myocardial Recovery

After 30 minutes of normoxic perfusion, hearts were subjected to no-flow global ischemia for 30 minutes followed by 30 minutes of reperfusion and allocated to receive 1 of the following during the first 10 minutes of reperfusion (n=6 per group): (1) the inhibitor of Na+/K+–ATPase ouabain at 150 μmol/L; (2) the inhibitor of NCX KB-R7943 at 10 μmol/L (Tocris Ltd); (3) 150 μmol/L ouabain+10 μmol/L KB-R7943; or (4) no drug. In a second set of experiments, hearts were allocated to the same groups of treatment, but ischemia was extended to 60 minutes (n=6 per group).

Effect of Calpain on Na+/K+–ATPase Activity

In 18 hearts submitted to 60 minutes of ischemia, the calpain inhibitor MDL-28170 (Calbiochem) at 10 μmol/L or its vehicle (DMSO) was added to the perfusion media during the 10 minutes before ischemia and the first 10 minutes of reperfusion. To demonstrate that any effect of calpain on Na+/K+–ATPase activity was not a mere consequence of its previously described effect on cell death,3 in 2 additional groups, reperfusion-induced hypercontracture was blocked with the reversible contractile inhibitor 2,3-butanedione monoxime (BDM). Hearts receiving MDL-28170 or its vehicle were perfused with 20 mmol/L BDM during the first 5 minutes of reperfusion (n=18). Four hearts from each group were frozen in liquid nitrogen after 5 minutes of reperfusion for Western blot analysis and determination of calpain and Na+/K+–ATPase activities, and, in 5 additional hearts, reperfusion was prolonged for 30 minutes for measurement of left-ventricular pressure (LVP) and lactate dehydrogenase (LDH) release. To confirm the contribution of calpain to the impaired recovery of Na+/K+–ATPase activity during reperfusion, in an additional group, MDL-28170–treated hearts were perfused with 150 μmol/L ouabain during the first 10 minutes of reperfusion (n=4).

Calpainolysis of Na+/K+–ATPase In Vitro

Proteolysis of Na+/K+–ATPase α subunits, α-fodrin, and ankyrin-B by calpain was examined in the particulate fraction of normoxically perfused hearts obtained as described previously.24 Samples were incubated with 1 μg of rat recombinant m-calpain (Calbiochem) in the presence of 10 mmol/L CaCl2 at 25°C for 5, 10, 30, 60, or 120 minutes. Proteins were analyzed by Western blotting as described below.

Determinations

Quantification of Cell Death

LDH activity was spectrophotometrically measured in the coronary effluent throughout the reperfusion period.5 Because the whole hearts were used to determine enzyme activities and protein contents in cytosolic and membrane fractions, an additional series of experiments was performed for histological analysis. This series included control and MDL-28170–treated hearts (n=5 per group) and 2 hearts treated with 20 mmol/L BDM and reperfused for 5 or 30 minutes. In these hearts, epicardial electrocardiogram was continuously recorded. Hearts were sliced, and 1-μm histological sections were stained with Masson’s trichrome. The remaining slices were incubated with triphenyltetrazolium chloride to quantify necrosis.5

Na+/K+–ATPase Activity Assay

The ouabain-sensitive Na+/K+–ATPase activity of heart homogenates was determined as described previously.24 Generated inorganic phosphate (Pi) was assayed using an ammonium molybdate spectrophotometric assay.25 Ouabain-insensitive ATPase activity assayed in the presence of 2 mmol/L ouabain was subtracted from the value observed in its absence to estimate ouabain-sensitive Na+/K+–ATPase activity.

Calpain Activity Assay

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 AMC release was monitored over 30 minutes at 25°C using excitation and emission wavelengths of 380 nm and 460 nm, respectively. The calpain inhibitor MDL-28170 at 10 μmol/L was used to determine the specificity of the assay.

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 phenylmethylsulfonyl fluoride, and 0.1 dithiothreitol (pH 7.3). For calpain, fodrin, and ankyrin analysis, cytosolic and membrane fractions were separated as described previously.26 The detachment of Na+/K+–ATPase from its cytoskeletal anchorage was analyzed by differential distribution in Triton-soluble and -insoluble cytoskeletal protein fractions as described.27,28 In a parallel experiment, samples from normoxic hearts were stimulated before cellular fractionation by the addition of Ca2+ in the presence or absence of recombinant m-calpain. Control was performed by adding 10 μmol/L MDL-28170.

Proteins were separated by electrophoresis on a 7.5% SDS gel, transferred onto nitrocellulose membrane (Hibond ECL, Amersham), and immunoblotted with antibodies against α1, α2, and α3 (Upstate Biotechnology) Na+/K+–ATPase subunits, α-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 charge-coupled device system (Image Reader LAS-3000, Fujifilm) and image analysis software (Image Gauge, Fujifilm). Equal protein load was confirmed by Ponceau staining.

Statistical Analysis

Differences between groups were assessed by 1-way ANOVA. Changes along time were assessed by repeated-measures ANOVA. Significance was set at a probability value of 0.05. Results are expressed as mean±SEM.

Results

Impairment of Na+/K+–ATPase Activity Exacerbates Lethal Reperfusion Injury

To investigate the role of Na+/K+–ATPase on the mechanisms of cell death during myocardial ischemia/repefusion, isolated rat hearts were subjected to 30 or 60 minutes of global ischemia and reperfused in the presence of different treatments.

At the end of the equilibration period, LV end-diastolic pressure (LVEDP) and LV developed pressure (LVdevP) were 5.8±0.6 and 108.1±6.9 mm Hg, respectively, without differences between groups.

In control hearts submitted to 30 minutes of ischemia, ouabain-sensitive Na+/K+–ATPase activity measured 5 minutes after reperfusion was slightly reduced (76.4±15.2% of values obtained in normoxic hearts, P=not significant [NS]). In these hearts, reperfusion induced an increase in LVEDP with a peak of 98.2±4.1 mm Hg 3 minutes after onset, and LVdevP recovered to 35.4±3.3% of its initial value after 30 minutes of reperfusion. LDH release during this period was 12.5±1.0 U/30 minutes per gdw (Figure 1A). Addition of ouabain to the perfusion buffer during the first 5 minutes of reperfusion was associated with a marked increase in peak LVEDP (134.1±8.1 mm Hg, P=0.003), reduced contractile recovery (5.2±2.2% of preischemic value, P<0.001), and a larger LDH release (43.2±6.3 U/30 minutes per gdw, P<0.001). Addition of the NCX blocker KB-R7943 at 10 μmol/L to the ouabain buffer reverted the detrimental effects of ouabain on peak of LVEDP (P=0.012), functional recovery (28.3±6.4% of preischemic value, P=0.007), and LDH release (19.2±2.2U/30 minutes per gdw, P=0.005). Hearts reperfused with 10 μmol/L KB-R7943 alone showed no significant differences in LVP or LDH release compared with control hearts (Figure 1A).

Figure1
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Figure 1. Peak LVEDP, LVdevP (percentage of baseline values), and cumulative LDH release after 30 minutes of reperfusion in different groups of treatment: control (Control), ouabain (OU), and ouabain+KB-R7943 (OU+KB). A, Hearts submitted to 30 minutes of ischemia. B, Hearts submitted to 60 minutes of ischemia. Data are mean±SEM. *P<0.05 vs control group.

In control hearts subjected to 60 minutes of ischemia, ouabain-sensitive Na+/K+–ATPase activity was markedly reduced after 5 minutes of reperfusion (16.4±7.5% versus basal values, P<0.001). Reperfusion was followed by a more marked increase in LVEDP (peak of 137.2±6.8 mm Hg), poorer recovery of LVdevP (5.8±2.7% of its initial value after 30 minutes of reperfusion), and more severe LDH release (58.5±7.4 U/30 minutes per gdw), as compared with hearts submitted to 30 minutes of ischemia (Figure 1B). The reduction of Na+/K+–ATPase activity was not the consequence of cell death because it was not attenuated when myocyte integrity was preserved during initial reperfusion by preventing reperfusion-induced contracture with the infusion of the contractile inhibitor BDM (Figure 2A). Addition of ouabain to the reperfusion buffer caused no significant additional deterioration of LVEDP, LVdevP, or LDH release. Addition of KB-R7943 to the ouabain-containing buffer reduced peak LVEDP (114.2±3.9 mm Hg, P=0.002), increased LVdevP (25.1±4.7%, P=0.005), and attenuated LDH release (32.4±5.6 U/30 minutes per gdw, P=0.018). Hearts reperfused with 10 μmol/L KB-R7943 alone showed no significant differences in LVP or LDH release compared with hearts coperfused with ouabain (Figure 1B).

Figure2
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Figure 2. Calpain-dependent (A) and ouabain-dependent (B) Na+/K+–ATPase activities (expressed as percentage of values in normoxically perfused hearts [Nx]), cumulative reperfusion-induced LDH release (C), and distribution of m-calpain (80-kDa subunit between membrane and cytosolic fraction) (D) in hearts submitted to 60 minutes of ischemia and 5 minutes of reperfusion and allocated to different treatments: control hearts (Control), MDL-7943–treated hearts (MDL), hearts receiving BDM during the first 5 minutes of reperfusion (BDM), and hearts coperfused with BDM and MDL-7943 (BDM+MDL). Data are presented as mean±SEM. *P<0.05 vs control, $P<0.05 vs BDM.

Loss of Na+/K+–ATPase Anchorage to Membrane Cytoskeleton

Western blot analysis of α1 and α2 subunits of Na+/K+–ATPase in the Triton insoluble fraction of control hearts subjected to 60 minutes of ischemia revealed a significant loss of both isoforms, principally α2, after 5 minutes of reperfusion and a marked increase in the soluble fraction (Figure 3A), indicating dissociation from its cytoskeletal anchorage. As in previous studies,24 we were unable to detect α3 isoform by Western blot in our model. Analysis of the structural proteins α-fodrin and ankyrin-B showed a marked increase in the 145/150-kDa fragments resulting from degradation of α-fodrin and a decrease in the 220-kDa isoform of ankyrin-B (Figure 3B).

Figure3
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Figure 3. Effect of 5 minutes of reperfusion following 60 minutes of ischemia in hearts receiving different treatments during reperfusion. A, Distribution of α1 and α2 Na+/K+–ATPase subunits in the noncytoskeletal soluble fraction (sol.) and in the cytoskeletal insoluble fraction (insol.). B, Levels of calpain-specific α-fodrin breakdown products (145/150 kDa) and ankyrin-B. Data are expressed as percentage of values observed in normoxically perfused hearts (Nx) and presented as mean±SEM of 3 experiments. *P<0.05 vs Nx.

Inhibition of Na+/K+–ATPase Activity in Reperfused Myocardium Is Related to Calpain Activation

In control hearts reperfused after 60 minutes of ischemia, calpain activity was increased after 5 minutes of reperfusion (13.5±0.4 pmol AMC/min per mg protein versus 7.2±0.5 pmol AMC/min per mg protein in normoxically perfused hearts, P<0.001). This activation correlated with translocation of calpain from the cytosol to the membrane fraction without apparent autolysis (Figure 2D). Stimulation of calpain proteolysis in vitro in homogenates from normoxically perfused hearts increased the proportion of the soluble fraction of α subunits of Na+/K+–ATPase to levels comparable to those observed in hearts subjected to reperfusion (see Figure I in the online data supplement available at http://circres.ahajournals.org).

The perfusion with the calpain inhibitor MDL-28170 attenuated calpain activation (8.5±0.9 pmol AMC/min per mg protein, P=NS) and improved recovery of Na+/K+–ATPase activity (P=0.019) (Figure 2B). Western blot analysis showed a marked attenuation in the translocation of calpain to the membrane fraction (Figure 2D) in the degradation of α-fodrin and ankyrin-B (Figure 3B) and in the cytoskeletal detachment of α1 and α2 subunits of Na+/K+–ATPase (Figure 3A) measured after 5 minutes of reperfusion. These effects were associated with a 55% reduction of LDH release (P=0.038), less myocardial necrosis as evaluated by triphenyltetrazolium reaction (30.8±4.1% versus 52.4±5% in control group, P=0.010), markedly reduced extent of contraction band necrosis (online Figure II), and better functional recovery after 30 minutes of reperfusion (25.3±5.7% versus 7.4±3.4% in control hearts, P=0.027). Treatment with MDL-28170 did not prevent the occurrence of reperfusion-induced arrhythmias but reduced their duration during the first 5 minutes of reperfusion (182±19 s versus 249±15 s, P=0.024; online Figure III). The better recovery of Na+/K+–ATPase activity measured in samples from hearts treated with MDL-28170 was confirmed in intact hearts by the addition of ouabain to the medium during the first minutes of reperfusion. Perfusion with ouabain almost completely reverted the reduction of LDH release (49.5±4.3 U/30 minutes per gdw, P=NS versus control hearts) and the improvement in functional recovery (3.2±2.2% of its initial value after 30 minutes of reperfusion, P=NS versus control hearts) achieved with MDL-28170. The improvement of Na+/K+–ATPase activity in hearts treated with MDL-28170 was not a consequence of reduced cell death because treatment with BDM during the first 5 minutes of reperfusion had no effect on calpain and Na+/K+–ATPase activities (13.0±0.7 pmol AMC/min per mg protein and 15.1±9.6% of basal values, P=NS), despite the marked protective effect of MDL-28170 on LDH release while BDM was present (73% reduction, P=0.009) (Figure 2).

Immunoblot analysis demonstrated that the loss of fodrin, ankyrin, and Na+/K+–ATPase from the cytoskeleton during reperfusion was not modified in hearts receiving BDM during the first 5 minutes of reperfusion and that the loss was prevented by MDL-28170 (Figure 3).

Restoration of contractile activity on withdrawal of BDM led to a rapid increase in LVEDP (Figure 4) and massive release of LDH (71.4±11.2 U/30 minutes per gdw versus 62.0±9.5 U/30 minutes per gdw in control hearts, P=NS) (Figure 5). In hearts treated with BDM and MDL-28170, calpain activation was attenuated as in hearts not receiving BDM (8.9±0.6 pmol AMC/min per mg protein, P<0.001); Na+/K+–ATPase activity increased (50.8±9.6%, P=0.030) and resulted in a further reduction of LDH release during BDM infusion (Figure 2B). In these hearts, functional deterioration and LDH release associated with removal of BDM from the reperfusion buffer was clearly attenuated (Figures 4 and 5⇓).

Figure4
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Figure 4. Representative traces of LVP corresponding to control hearts (A) and hearts treated with MDL-7943 hearts (MDL) (B), BDM (C), or both (BDM+MDL) (D). Data are expressed as mean±SEM. *P<0.05 vs control group.

Figure5
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Figure 5. Time course of LDH release during 30 minutes of reperfusion following 60 minutes of ischemia (A) and cumulative LDH release during the 30-minute reperfusion period (B) in control hearts (Control) and in hearts treated with MDL-7943 (MDL), BDM (BDM), or both (BDM+MDL). Data are presented as mean±SEM. *P<0.05 vs control.

Incubation of Na+/K+–ATPase With Calpain In Vitro

Western blot analysis of α1 and α2 subunits of Na+/K+–ATPase and of the known substrates of calpain, fodrin, and ankyrin from samples of normoxically perfused hearts incubated with rat recombinant m-calpain demonstrates that fodrin and ankyrin were importantly degraded after 5 minutes of incubation with calpain. Bands corresponding to α1 and α2 subunits also decreased in the presence of calpain, although much more gradually. The reduction was significant only after 30 minutes of incubation in the case of α1 and after 1 hour in the case of α2 (Figure 6).

Figure6
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Figure 6. Time course of changes in α-fodrin (145/150 kDa) (A), ankyrin (B), α1 Na+/K+–ATPase subunit (C), and α2 Na+/K+–ATPase subunit content (D) in the sarcolemmal fraction of myocardial tissue incubated in the presence of rat recombinant m-calpain. Each value represents the mean±SEM of 3 experiments. *P<0.05 vs preincubation time (0 minutes).

Discussion

Previous studies have shown that Ca2+ influx through reverse-mode NCX contributes importantly to cardiomyocyte hypercontracture and death during the first minutes of reperfusion,5,6 that increased cytosolic Na+ is the key determinant of reverse activity of NCX,7 and that impairment of Na+/K+–ATPase may delay Na+ recovery.12 The present study confirms that Na+/K+–ATPase activity is impaired during early reperfusion after prolonged ischemia and demonstrates for the first time that (1) this impairment is associated with a reduction in the amount of Na+/K+–ATPase protein associated with the membrane–cytoskeletal complex and that both can be prevented by calpain inhibition; (2) the effect of calpain activation on Na+/K+–ATPase is not a mere consequence of a detrimental effect on cell survival mediated by other mechanisms (such as sarcolemmal fragility), because it is still observable when cell death is prevented or delayed by inhibition of reperfusion-induced hypercontracture; and (3) the effect of calpain on Na+/K+–ATPase may be explained by calpain-mediated degradation of the anchorage that fixes Na+/K+–ATPase to the membrane cystoskeleton (ankyrin), and the membrane cytoskeleton itself (fodrin), thus causing a loss of Na+/K+–ATPase anchorage. Altogether, these results demonstrate that calpain activation induced by increased cytosolic Ca2+ concentration during the initial minutes of reperfusion causes an impairment of Na+/K+–ATPase that results in further Ca2+ gain through reverse-mode NCX, thus closing a vicious circle that can eventually lead to cardiomyocyte death.

Impairment of Na+/K+–ATPase During Early Reperfusion After Prolonged Ischemia

Previous studies have shown a decrease in Na+-pump activity in isolated rat heart subjected to ischemia/reperfusion.14,15 The present study confirms the reduction in Na+/K+–ATPase activity during the first minutes of reperfusion and relates its severity with the duration of preceding ischemia. In hearts subjected to 30 minutes of ischemia, a near-complete reactivation of Na+/K+–ATPase occurred within the first 5 minutes of reperfusion. As in previous studies,13,29 inhibition of Na+-pump activity in these hearts with ouabain markedly increased enzyme release and loss of functional recovery. In hearts reperfused after 60 minutes of ischemia, a severe inhibition of Na+/K+–ATPase activity and extensive cell death were observed, and, consistently with previous results,5 inhibition of NCX during the first minutes of reperfusion attenuated cell death and improved functional recovery. Overall, these results indicate that impaired Na+/K+–ATPase activity during the first minutes of reflow contribute to Ca2+ overload in reperfused myocardium, leading to ventricular arrhythmias, to stunning, and, more importantly, to cell death.

Recent studies have described alterations in Na+/K+–ATPase isoform gene expression mediated by oxidative stress30 and an important reduction in the abundance of α2, α3, β1, and β2 isoforms and, to a lesser extent, α1 subunit in isolated rat hearts subjected to ischemia/reperfusion.15 However, the exact mechanism of reduced myocardial Na+/K+–ATPase activity during the initial minutes of reperfusion has not been previously elucidated. Our results agree with previous studies in demonstrating a significant decrease in the protein levels of α1 and, to a greater extent, of α2 isoform attached to the cytoskeleton.

Role of Calpain

Calpain remains inactive before reperfusion because of the acidic pH and increased ionic strength in ischemic myocardium.31,32 However, there is solid evidence documenting calpain activation and degradation of structural proteins during myocardial reperfusion.3,22,23 The present study confirms these previous results, shows that calpain activation is an early event during reperfusion, because it is observed as early as 5 minutes after flow restoration, and supports the hypothesis that activation of the enzyme requires its association to the cell membrane independently of its autolysis.33 The observation that inhibition of calpain with MDL-28170 prevents the cytoskeletal detachment of α1 and α2 subunits of Na+/K+–ATPase in reperfused myocardium and attenuates the reduction in Na+-pump activity indicates that calpain activation is involved in both phenomena. This result is further supported by the fact that the increased extractability of α subunits observed in reperfused hearts is reproduced in vitro in homogenates from normoxically perfused hearts in which calpain activity had been stimulated.

Loss of Na+/K+–ATPase During Reperfusion Is Not a Mere Consequence of Cell Death but Causes It

Because inhibition of calpain activation is associated with reduced cell death, the possibility exists that preservation of Na+/K+–ATPase observed in hearts treated with calpain inhibitors is a mere consequence of increased cell survival rather than its cause. To rule out this possibility, we analyzed Na+/K+–ATPase during initial reperfusion in the presence of BDM. BDM is a reversible inhibitor of actomyosin ATPase and reduces the myofibrillar sensitivity to Ca2+. Through these mechanisms, BDM reduces cross-bridge force production and allows a complete blockade of contractile activity.34 BDM has been consistently shown to prevent hypercontracture and sarcolemmal rupture during the initial minutes of reperfusion.1,2,35,36

Hearts reperfused for 5 minutes in the presence of BDM, in which LDH release and contraction band necrosis were importantly reduced, showed calpain activation, reduction of Na+/K+–ATPase activity, and loss of anchorage of α subunits of Na+/K+–ATPase similar to those observed in hearts reperfused for the same period in the absence of BDM, indicating that these phenomena were not consequences of cell death but preceded it. Interestingly, inhibition of calpain with MDL-28170 in hearts reperfused in the presence of BDM resulted in a significant additional reduction in LDH release, suggesting that calpain activation may contribute to cell death by mechanisms independent of hypercontracture.3

Although prolonged (30 minutes) infusion of BDM limits final infarct size,1,35 infusion of BDM during the first 5 minutes of reperfusion does not prevent the occurrence of hypercontracture and extensive cell death on withdrawal of the drug.36 In the present study, withdrawal of BDM after 5 minutes of reperfusion was immediately followed by a rapid rise in LVEDP and LDH release and development of contraction band necrosis. However, deterioration associated with BDM withdrawal was markedly attenuated in hearts receiving MDL-28170.

Ankyrin, Fodrin, and Na+/K+–ATPase As Calpain Substrates

Ankyrins contain an N-terminal domain that possess a well-characterized binding site for the cytoplasmic domain of the α subunit of Na+/K+–ATPase18 and a central fodrin-binding domain.19 The interaction of ankyrin with fodrin mediates the linkage of Na+/K+–ATPase with the fodrin-based cytoskeleton and stabilizes Na+/K+–ATPase at the plasma membrane. Recently, a decrease in overall protein levels of α1 and α2 Na+/K+–ATPase isoforms has been demonstrated in cardiomyocytes from mice heterozygous for a null mutation in ankyrin-B, which results in a 50% reduction in ankyrin measured by immunoblot.20 These results highlight the importance of the association of Na+/K+–ATPase with the ankyrin-fodrin complex. The results of the present study could be explained by the calpain-mediated proteolysis of the membrane–cytoskeleton complex at the ankyrin-fodrin interface. However, our results are also consistent with proteolytic activity of calpain on Na+/K+–ATPase subunits. In fact, when the amino acid sequences of Na+/K+–ATPase α1 and α2 subunits were subjected to a PEST find analysis (http://www.at.embnet.org/embnet/tools/bio/PESTfind), 3 possible PEST regions (Pro, Glu[Asp], and Ser/Thr) for α1 and 2 for α2 were identified as potential cleavage sites for calpain.37

In our study, in vitro incubation of the particulate fraction obtained from normoxically perfused hearts with calpain showed a partial degradation of α1 and α2 subunits after 30 minutes of incubation. A previous study showed degradation of Na+/K+–ATPase in postmortem brain and no effect of calpain on Na+/K+–ATPase proteolysis in vitro.38 The mechanism responsible for the proteolysis observed in postmortem samples was not identified in this study. A prominent role of direct proteolysis of α subunits by calpain in the loss of the α isoforms during reperfusion appears to be unlikely for several reasons. First, our in vivo results indicate that reperfusion induces a dissociation and redistribution of Na+/K+–ATPase from the membrane–cytoskeletal complex without apparent protein loss by proteolysis. Second, fodrin and ankyrin decreased in parallel with α1 and α2 subunits, and this decrease was pronounced as early as 5 minutes after reperfusion onset. Third, in in vitro experiments, proteolysis of ankyrin at 5 minutes after starting the incubation with active calpain was more pronounced than that observed in reperfused hearts, whereas the loss of the bands corresponding to α1 and α2 subunits was not significant until 30 minutes of incubation.

Degradation of ankyrin could result in the loss of ankyrin-binding proteins other than Na+/K+–ATPase α subunits, with potential effects on intracellular Ca2+. Mohler et al20 demonstrated reduction in the levels of NCX in cardiomyocytes with a null mutation for ankyrin-B, and, more recently, Bano et al39 described the cleavage of NCX3 by calpain in homogenates from brains subjected to ischemia. However, a significant role of altered NCX1 function in myocardial reperfusion injury seems unlikely. In fact, pharmacological inhibition of NCX1 during initial reperfusion has consistently been found to protect myocardium against reperfusion injury after prolonged ischemia,5,6 indicating that, in contrast to what happens with the Na+ pump, NCX1 remains active during the initial minutes of myocardial reperfusion following prolonged, severe ischemia and that this activity, in the reverse “Ca2+ in” mode, is detrimental to cell survival.

Implications

This study supports the hypothesis that the inability of the cell to resume Na+/K+–ATPase activity during early reperfusion because of calpain-mediated detachment of the enzyme from the sarcolemma contributes significantly to Ca2+ influx through reverse mode of NCX and plays a critical role in reperfusion-induced cell death. The study proposes calpain-dependent detachment from the membrane–cytoskeletal complex of α subunits of Na+/K+–ATPase as a new mechanism for explaining the impairment of its activity that occurs during early reperfusion in hearts subjected to prolonged ischemia (Figure 7). Calpain inhibition largely reduces cell death. The fact that calpain inhibition protects less than direct downstream inhibition of reverse mode of NCX, may reflect the fact that Ca2+ overload built up during prior ischemia, altered Ca2+ handling by sarcoplasmic reticulum and/or mitochondria,2 and determinants of Na+ overload others from Na+/K+–ATPase malfunction are important determinants of Ca2+ overload in reperfused myocytes. It cannot be ruled out, however, that calpain-independent mechanisms contribute to Na+/K+–ATPase dysfunction during reperfusion.

Figure7
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Figure 7. Proposed mechanisms by which calpain activation may contribute to immediate cell death during reperfusion. The broken arrow indicates a probably less-important mechanism. [Ca2+]i indicates intracellular Ca2+ concentration; [Na+]i, intracellular Na+ concentration; NHE, Na+/H+ exchanger; NBS, Na+/HCO3− cotrasporter; •, other mechanisms contributing to Na+ overload.

Acknowledgments

This study was partially supported by grants CICYT SAF 2002-0559 and FIS-RECAVA. We appreciate the excellent technical work of Maria Angeles Garcia.

Footnotes

  • Original received May 17, 2005; revision received July 29, 2005; accepted August 1, 2005.

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    Calpain-Mediated Impairment of Na+/K+–ATPase Activity During Early Reperfusion Contributes to Cell Death After Myocardial Ischemia
    Javier Inserte, David Garcia-Dorado, Victor Hernando and Jordi Soler-Soler
    Circulation Research. 2005;97:465-473, originally published September 1, 2005
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    Calpain-Mediated Impairment of Na+/K+–ATPase Activity During Early Reperfusion Contributes to Cell Death After Myocardial Ischemia
    Javier Inserte, David Garcia-Dorado, Victor Hernando and Jordi Soler-Soler
    Circulation Research. 2005;97:465-473, originally published September 1, 2005
    https://doi.org/10.1161/01.RES.0000181170.87738.f3
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