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Cardiovascular Research 2004 64(1):105-114; doi:10.1016/j.cardiores.2004.06.001
© 2004 by European Society of Cardiology
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Copyright © 2004, European Society of Cardiology

Ischemic preconditioning attenuates calpain-mediated degradation of structural proteins through a protein kinase A-dependent mechanism*

Javier Inserte, David Garcia-Dorado*, Marisol Ruiz-Meana, Luis Agulló, Pilar Pina and Jordi Soler-Soler

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

* Corresponding author. Tel.: +34-93-489-4038; fax: +34-93-489-4032. E-mail address: dgdorado{at}vhebron.net

Received 9 January 2004; revised 17 May 2004; accepted 1 June 2004


   Abstract
Top
 Abstract
1. Methods
 2. Results
 3. Discussion
 4. Conclusion
 References
 
Objectives: It has been shown that sarcolemmal rupture can occur during reenergization in cardiomyocytes in which previous ischemia has induced sarcolemmal fragility by calpain-dependent hydrolysis of structural proteins. We tested the hypothesis that attenuated calpain activation contributes to the protection against reperfusion-induced cell death afforded by ischemic preconditioning (IPC), and investigated the involvement of protein kinase A (PKA) in this effect. Methods: Calpain activity and degradation of different structural proteins were studied along with the extent of necrosis in isolated rat hearts submitted to 60 min of ischemia and 30 min of reperfusion with or without previous IPC (two cycles of 5 min ischemia–5 min reperfusion), and the ability of different treatments to mimic or blunt the effects of IPC were analyzed. Results: IPC accelerated ATP depletion and rigor onset during ischemia but reduced LDH release during reperfusion by 69% (P<0.001). At the end off reperfusion, calpain activity was reduced by 66% (P<0.001) in IPC, and calpain-dependent degradation of sarcolemmal proteins was attenuated. Addition of the calpain inhibitor MDL-28170 mimicked the effects of IPC on protein degradation and reduced LDH release by 48% (P<0.001). The effects of IPC on calpain, {alpha}-fodrin, and LDH release were blunted by the application of the PKA inhibitor H89 or alprenolol during IPC, while transient stimulation of PKA with CPT-cAMP or isoproterenol before ischemia attenuated calpain activation, {alpha}-fodrin degradation, and markedly reduced LDH release (P<0.001). In hearts exposed to Na+-free perfusion, IPC attenuated calpain activation by 67% (P<0.001) and reduced by 56% (P<0.001) LDH release associated to massive edema occurring during Na+ readmission without modifying its magnitude. Conclusion: These results are consistent with PKA-dependent attenuation of calpain-mediated degradation of structural proteins being an end-effector mechanism of the protection afforded by IPC.

KEYWORDS Preconditioning; Ischemia; Reperfusion injury; Protein kinase A; Calpain

Although the triggers and mediators of the protective effect of ischemic preconditioning (IPC) against cell death secondary to ischemia–reperfusion have been partially elucidated, its end-effectors remain unknown [1]. Sarcolemmal rupture and cell death may occur during the initial minutes of reperfusion in cardiomyocytes that have successfully resumed ATP synthesis [2,3]. Excessive contractile activation, cell edema, and sarcolemmal and cytoskeletal fragility have been shown to play important roles in sarcolemmal rupture [3,4]. Reperfusion-induced edema is attenuated by IPC, but a cause–effect relationship between this effect and protection against cell death could not be demonstrated [5]. In a recent study in isolated cardiomyocytes, Armstrong et al. [6] showed that IPC failed to modify sarcolemmal bleb formation but increased the resistance of cells to osmotic challenge.

In this study, we investigated the possibility that at least part of the protective effect of IPC against cell death occurring during reperfusion is exerted by limiting sarcolemmal fragility caused by reperfusion. Alpha-fodrin, a 227-kDa tetrameric, membrane-bound protein that forms the backbone of the membrane protein skeleton [7], is degraded during reperfusion [8,9], and {alpha}-fodrin degradation has been associated with membrane fragility [10–12]. Alpha-fodrin is a substrate for calpain [8,13], a group of Ca2+-dependent proteases, and previous studies have shown that IPC attenuates calpain activation [13,14] and have related this effect to the protection afforded by IPC against apoptosis [13]. Neither the potential influence of calpain inhibition on structural protein degradation and sarcolemmal fragility in preconditioned myocardium nor the mechanism of the inhibitory effect of IPC on calpain have been elucidated.

A possible mechanism by which IPC may attenuate calpain activation is protein kinase A (PKA)-dependent phosphorylation of the enzyme. Previous studies have demonstrated norepinephrine release during ischemia in the isolated rat heart model [15,16]. Depletion of norepinephrine stores by pretreatment with reserpine abolishes the protection induced by IPC [16,17]. It has also been shown that preconditioning ischemia results in transient increases in cAMP and PKA activity, and that abolition of these increases by beta-blockade blunts IPC effects [17–19]. On the other hand, recent studies have described modulation of calpain activity by PKA phosphorylation [20,21].

This study tests the hypothesis that transient PKA activation during preconditioning ischemia results in calpain inhibition, and that this results in reduced hydrolysis of structural proteins reduced sarcolemmal fragility and less cell death during subsequent ischemia/reperfusion.


   1. Methods
Top
 Abstract
 1. Methods
2. Results
 3. Discussion
 4. Conclusion
 References
 
1.1. 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 intraperitoneal injection of pentobarbital (100 mg/kg), and the hearts were excised 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) at 10 ml/min. Left ventricular (LV) pressure was monitored as previously described [22].

1.2. Ischemia–reperfusion and preconditioning
Control hearts (n=14) were perfused normoxically for 40 min and then subjected to 60 min ischemia followed by 30 min of reperfusion. IPC (n=14) was induced by two cycles of 5 min of ischemia and 5 min of reperfusion (Fig. 1). Hearts from each group were frozen immediately before the onset of prolonged ischemia for measurement of calpain activity (n=4), after 10 min of ischemia for determination of ATP content (n=4), and after 30 min of reperfusion for determination of ATP content, calpain activity and Western blot analysis (n=6).


Figure 1
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  		Fig. 1 Ischemia–reperfusion protocols. Alp: alprenolol, IPC: ischemic preconditioning, ISO: isoproterenol.

 
1.2.1. Role of calpain inhibition in cardioprotection
In 15 hearts, the membrane-permeant calpain inhibitor MDL-28170 (Calbiochem) at 1 or 10 µM or its vehicle (DMSO at final concentration of 0.01% and 0.1%, respectively) was added to the perfusion media during the 15 min prior to ischemia and the first 10 min of reperfusion (Fig. 1), after which calpain activity and calpain-induced proteolysis were measured.

1.2.2. Effect of PKA on calpain activation
To test whether the inhibition of calpain in IPC was mediated by a transient activation of PKA during the trigger period, either the β-adrenergic receptor blocker alprenolol at 50 µM (Sigma; n=6) or the selective PKA inhibitor H89 (Calbiochem) at 10 µM dissolved in DMSO (final concentration <0.1%, n=6) was added to the KHB 5 min before the onset of the first preconditioning ischemia and during the following preconditioning reperfusion.

The drugs were washed out before the onset of sustained ischemia (Fig. 1). Control groups for those treatments consisted of non-IPC hearts subjected to two episodes of 5 min of perfusion with alprenolol or H89 (n=4 per group) interspersed by 5 min of perfusion without any drug. Drugs were washed out before sustained ischemia.

In a second set of experiments, PKA was activated by either β-adrenergic stimulation with 0.1 µM isoproterenol (Sigma; n=6) or by the cell-permeant 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 initiation of ischemia (Fig. 1).

In order to determine whether treatments widely accepted to blunt preconditioning protection also reduced the inhibitory effect of IPC on calpain, the mitochondrial KATP channel blocker 5-hydroxydecanoic acid (5HD) at 300 µM (n=6) or the opioid antagonist naloxone (100 µM, n=4) was added to the KHB 10 min before the onset of the first preconditioning ischemia and during the following two preconditioning reperfusions in additional experiments.

1.2.3. Contribution of caspase inhibition
The possible contribution of attenuated caspase activation to the reduced proteolysis in preconditioning hearts was tested in experiments (n=4) in which the broad-spectrum caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-fmk, Sigma) at 10 µM was added to the perfusion media together with 1 µM MDL-28170 during the 15 min previous to ischemia and the first 10 min of reperfusion.

1.3. Determinations
Lactate dehydrogenase (LDH) activity in the coronary effluent was measured by spectrophotometry [22]. Myocardial ATP content was determined by quantitative bioluminescence (Sigma). Myocardial water content was measured by desiccation as described [5].

1.3.1. Calpain activity assay
Frozen hearts were homogenized with ice-cold Tris-buffered saline (100 mM Tris–HCl, 145 mM NaCl, 10 mM EDTA, pH 7.3) and centrifuged (15 min at 15,000 x g). The supernatant was further centrifuged for 1 h at 100,000 x g, and the extracted cytosol was stored at –80 °C. Calpain activity was measured by fluorometry as previously described [23]. MDL-28170 at 10 µM was used to determine the specificity of the assay.

1.3.2. PKA activity assay
PKA activity was measured before the onset of the second preconditioning reperfusion in nontreated hearts and in hearts treated with either 50 µM alprenolol or 10 µM H89, as well as in normoxic hearts using a nonradioactive protein kinase assay kit (Calbiochem) according to the manufacturer's instructions.

1.3.3. Western blot
Frozen hearts were homogenized in lysis buffer (50 mM Tris–HCl, pH 7.3, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM dithiothretol, and 1% protease inhibitor cocktail; Sigma). The homogenate was centrifuged (15 min at 15,000 x g), and the supernatant was diluted with equal volume of Laemmli sample buffer (Sigma). Proteins were separated by electrophoresis on a 7.5% or 12% SDS gel and transferred onto nitrocellulose membrane (Hibond ECL, Amersham). Membranes were incubated with mouse monoclonal antibody to {alpha}-fodrin (Affiniti Research Products), which recognizes intact {alpha}-fodrin and the 145- to 150-kDa and 120-kDa fragments resulting from calpain- and caspase-mediated {alpha}-fodrin proteolysis, respectively. The other monoclonal antibodies used were against ankyrin (Ab-1, Oncogene Research Products), desmin (clone DE-U-10, Sigma), and troponin I (clone 8I-7, Spectral Diagnostics). An antimouse IgG peroxidase conjugate (Sigma) was used as secondary antibody. Protein bands were detected by chemiluminescence (SuperSignal West Dura Extended Duration Substrate, Pierce). Equal protein loading was confirmed by Ponceau Red staining (Sigma) of membranes.

1.4. Effect of IPC in hearts submitted to transient exposure to Na+-free perfusion
Because IPC reduces reperfusion-induced myocardial edema, and the contribution of this effect to reduced necrosis can be difficult to separate from that of attenuated sarcolemmal fragility, we sought to investigate the effect of IPC on cell death in hearts (n=18) submitted to acute cell swelling induced by readmission of Na+-containing buffer after 60 min exposure to Na+-free buffer (in mM: HEPES 6, mannitol 226, KCl 2.7, MgSO4 1, CaCl2 20, and glucose 11, pH 7.4). Nine of these hearts were preconditioned as in the ischemia/reperfusion protocol immediately before the Na+-free perfusion period. In 12 additional hearts, 1 µM MDL-28170 or vehicle was added to the Na+-free buffer perfusion and during the first 5 min of reintroduction of Na+, and in 6 hearts, the osmotic pressure of the Na+ readmission buffer was increased to 410 mOsm with mannitol.

1.5. Statistical analysis
Differences among groups were assessed by means of one-way ANOVA followed when necessary by the Bonferroni test for individual comparisons. Comparisons between two groups were evaluated by the t-test for independent samples. 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.


   2. Results
Top
 Abstract
 1. Methods
 2. Results
3. Discussion
 4. Conclusion
 References
 
2.1. Ischemic preconditioning and calpain activity
At the onset of sustained ischemia, there were no differences between IPC and control hearts regarding LV function, heart rate, or perfusion pressure. IPC accelerated the onset of rigor contracture (defined as the time required after onset of ischemia to reach an LVEDP value 5 mm Hg greater than its preischemic value) and reduced myocardial ATP content after 10 min of ischemia (Table 1), but reduced hypercontracture (as assessed by peak LV diastolic pressure after onset of reperfusion), LDH release, and contractile failure during reperfusion (Table 1; Figs. 2 and 3Go). Myocardial water content was markedly reduced in IPC hearts (494±5.6 ml/100 g dry weight vs. 586±19 ml/100 g dry weight in controls, P<0.001).


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  		Table 1 Ischemia (60 min) and reperfusion (30 min) in isolated rat hearts with and without ischemic preconditioning (IPC)

 

Figure 2
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  		Fig. 2 Protective effect of ischemic preconditioning in isolated rat hearts. Panel (A): representative LV pressure corresponding to control and IPC hearts. Panel (B): time course of LDH release during reperfusion. Data are mean±S.E.M.

 

Figure 3
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  		Fig. 3 Transient PKA stimulation and calpain inhibition mimic the protective effects of IPC. LV developed pressure (LVdevP, percentage of baseline values) after 30 min of reperfusion (panel A) and total LDH release during the 30 min of reperfusion (panel B) in the different groups of treatment: alprenolol (Alp), control (C), CPT-cAMP (cAMP), H89 (H89), isoproterenol (ISO), MDL-28170 (MDL), preconditioned (IPC). Data are mean±S.E.M. *P<0.05 respect to control group. $P<0.05 respect to IPC group.

 
Calpain activity was increased after ischemia/reperfusion, but IPC significantly attenuated this increase (Fig. 4). Calpain activity measured after completion of the two preconditioning cycles was reduced as compared to that measured in control hearts after 30 min of equilibration (5.67±0.73 pg AMC/min/mg protein vs. 8.14±0.56 pg AMC/min/mg protein, P=0.027). Western blot analysis showed a clear attenuation of {alpha}-fodrin proteolysis (Fig. 4) in IPC (145/150 kDa band: 156±12 au in control hearts vs. 48±18 au in IPC hearts, n=4). Perfusion of nonpreconditioned hearts with the calpain inhibitor MDL-28170 at 1 µM during 15 min before ischemia and the first 10 min of reperfusion mimicked the effects of IPC in that it attenuated calpain activation, reduced cleavage of {alpha}-fodrin (145/150 kDa band: 36±9 au, n=4; Fig. 4), and decreased hypercontracture, LDH release, and contractile dysfunction (Fig. 3). In addition, IPC attenuated degradation of ankyrin (62±11 au in control hearts vs. 168±23 au in IPC hearts, n=3) and desmin (43±19 au in control hearts vs. 186±18 au in IPC hearts, n=3; Fig. 5A) induced by reperfusion, and these effects were also mimicked by MDL-28170 (159±19 au for ankyrin and 177±23 au for desmin, n=3). IPC had also a marked protective effect against troponin I degradation (22 kDa band: 92±15 au in control hearts vs. 8±4 au in IPC hearts, n=3), but this effect was only partially reproduced by treatment with MDL-28170 (22 kDa band: 43±16 au; Fig. 5B). MDL-28170 at 10 µM did not result in greater protection against damage induced by ischemia/reperfusion.


Figure 4
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  		Fig. 4 Effect of IPC and other interventions on calpain activity measured after 30 min of reperfusion (panel A) and immunoblot analysis of {alpha}-fodrin and calpain-specific {alpha}-fodrin breakdown products (145/150 kDa; panel B). Nx: continuous perfusion without ischemia. The rest of abbreviations as in Fig. 3. Data are mean±S.E.M. *P<0.05 respect to Nx group.

 

Figure 5
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  		Fig. 5 Representative blots for ankyrin, desmin (panel A) and troponin I (panel B). Nx: perfusion without ischemia; C: control; IPC: preconditioned; MDL: MDL-28170; M+V: MDL-28170 plus Z-VAD-FMK.

 
Treatment with 300 µM 5-hydroxydecanoic acid (5HD) failed to attenuate the protection achieved with IPC. Hearts receiving 5HD during preconditioning had similar functional recovery than corresponding control IPC hearts not receiving the drug (38.3±4.5% of basal value and 42.6±4.3%, respectively, P=NS), as well as similar LDH release (15.5±2.3 U/30 min/gdw and 18.4±3.2 U/30 min/gdw, respectively, P=NS), calpain activity (8.6±0.8 pg AMC/min/mg protein and 9.2±0.6 pg AMC/min/mg protein, P=NS), and alpha-fodrin degradation than IPC hearts (145/150 kDa band: 54±15 au in control hearts vs. 45±12 au in IPC hearts, P=NS).

Inhibition of opioid receptors with naloxone significantly attenuated (P=0.003) the reduction in LDH release afforded by IPC but did not abolish it (43.5±4.6 U/min/gdw, 20.3±2.5 U/min/gdw, and 65.9±3.4 U/min/gdw, respectively, in naloxone, IPC, and control groups). In the naloxone group, calpain activity measured after 30 min of reperfusion was 10.9±0.4 pmol AMC/min/mg prot. [P=0.003 with respect to control hearts (12.8±0.4 pmol AMC/min/mg prot.) and P=0.110 with respect to IPC hearts (9.7±0.8 pmol/min/mg prot.)].

2.1.1. Effect of PKA on calpain activation
Preconditioning induced PKA activation (2745±280 pmol/mg prot/min vs. 1470±261 pmol/mg prot/min in control hearts, P=0.016). Perfusion with either alprenolol or H89 prevented PKA activation (1151±318 pmol/mg prot/min and 1020±149 pmol/mg prot/min, respectively), abolished the inhibitory effect of IPC on calpain activation and {alpha}-fodrin breakdown (145/150 kDa band: 133±22 au in alprenolol hearts and 119±18 au in H89 hearts, n=3; Fig. 4), and these effects were paralleled by a significant reduction of the beneficial effect of IPC on LDH release and functional recovery (Fig. 3). Neither alprenolol nor H89 had significant effects on calpain activity, LDH release or functional recovery during reperfusion of non-IPC hearts. Activation of PKA with either isoproterenol or CPT-cAMP before the onset of ischemia simulated the beneficial effects of IPC on calpain activation, {alpha}-fodrin cleavage (145/150 kDa band: 53±17 au in isoproterenol hearts and 61±15 au in CPT-cAMP hearts, n=3), LDH release, and functional recovery (Figs. 3 and 4)Go.

2.1.2. Contribution of calpain-independent caspase activation
Western blot analysis of {alpha}-fodrin did not show differences between control and ischemia/reperfusion group in the 120-kDa band resulting from specific cleavage by caspase-3. Simultaneous treatment with the caspase inhibitor Z-VAD-FMK and MDL-28170 did not significantly increase the protection afforded by MDL-28170 alone (functional recovery: 31.0±2.9% vs. 28.3±3.1% with respect to basal values; LDH release: 34.4±1.5 U/30 min/gdw vs. 38.4±3.5 U/30 min/gdw) and did not prevent the troponin I degradation observed in the presence of MDL-28170 (22-kDa band: 37±12 au; Fig. 5B).

2.1.3. IPC in hearts subjected to transient Na+-free perfusion
Perfusion with Na+-free buffer rapidly arrested contraction without inducing myocardial edema or LDH release. Reintroduction of Na+ caused severe myocardial edema, abrupt LDH release, and partial recovery of LV-developed pressure (33.3±5.4%; Fig. 6). When the osmolarity of the sodium readmission buffer was increased to 418 mOsm, myocardial edema and LDH release (28.4±2.3 U/30 min/gdw, P<0.001) were attenuated, and recovery of LV-developed pressure improved to 68.4±6.5% (P<0.001). Calpain activity measured after 30 min of Na+ reintroduction was significantly increased (Fig. 6).


Figure 6
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  		Fig. 6 Panel (A): myocardial water content 30 min after readmission of Na+ following 60 min of perfusion with Na+-free buffer (–Na+) was not modified by previous application of two cycles of preconditioning ischemia (–Na+/IPC), but was markedly reduced by increasing the osmolarity in the readmission Na+ buffer (Hyper). C: control group continually perfused with Na+-containing buffer. Panel (B): effect of these protocols and addition of the calpain inhibitor MDL-28170 during Na+ removal and readmission (MDL) on calpain activity after the Na+-readmission period and on LDH release during the Na+-readmission period. Data are mean±S.E.M. *P<0.05 respect to control group. $P<0.05 respect to Na+ depleted control group.

 
Application of the IPC protocol before Na+ withdrawal had no effect on myocardial edema occurring upon Na+-readmission. However, IPC markedly attenuated calpain activation and LDH release (Fig. 6), and improved functional recovery to 71.0±6.3% (P<0.001). Addition of 1 µM MDL-28170 to the Na+-free HEPES buffer mimicked the effects on LDH release (Fig. 6) and improved functional recovery to 51.0±4.2% (P=0.009).


   3. Discussion
Top
 Abstract
 1. Methods
 2. Results
 3. Discussion
4. Conclusion
 References
 
This study shows that IPC attenuates calpain activation and degradation of structural proteins during reperfusion, and that calpain inhibitors mimic the protective effect of IPC against cell death. Transient stimulation of PKA before index ischemia reproduces the effects of IPC on calpain activation, {alpha}-fodrin degradation, and cell death, while inhibition of PKA during the preconditioning stimulus has the opposite effects. The association between IPC, attenuated calpain activation, and reduced cell death was also observed in a model of transient Na+ removal in which cell death was induced by abrupt cell swelling after a period of Ca2+ overload. Altogether, the data obtained in this study suggest that reduced calpain-dependent sarcolemmal fragility is an end-effector mechanism of IPC, and that transient PKA activation is involved in the inhibitory effect of IPC on calpain.

3.1. Sarcolemmal fragility in IPC
Ischemia induces sarcolemmal fragility that may be detected as a reduced tolerance to osmotic stress [6], and that may persist or even be aggravated during the initial minutes of reperfusion [4,6]. Sarcolemmal fragility may allow that acute cell swelling and excessive contractile activation (hypercontracture) occurring during reperfusion result in sarcolemmal rupture and cell death in metabolically competent cardiomyocytes [3,4]. The mechanisms responsible for the development of sarcolemmal fragility during ischemia–reperfusion are not well known. Alterations in the lipidic composition [24] and hydrolysis of structural proteins have been proposed to play a role [25]. Armstrong et al. [10] recently described loss of sarcolemmal {alpha}-fodrin as an important determinant of ischemia-induced cell fragility. These authors observed in isolated cells that cardioprotective maneuvers as IPC or phosphatase inhibition with caliculin delay the onset of sarcolemmal fragility during ischemia [6].

The observation that reperfusion is associated with calpain activation and {alpha}-fodrin degradation agrees with previous observations [8,13]. As in previous studies [8,26], inhibition of calpain significantly attenuated {alpha}-fodrin degradation and LDH release during reperfusion and improved functional recovery. This is consistent with the notion that calpain activation contributes to cell death during myocardial reperfusion, that this effect is mediated, at least in part through degradation of {alpha}-fodrin and fragilization of cell membranes, and that IPC attenuates it by mechanisms that involve transient PKA activation.

Chen et al. [13] described activation of calpain in hearts submitted to 30 min of ischemia and 15 of reperfusion and observed that preconditioning suppressed this activation. They failed to detect caspase activation in reperfused myocardium, although release of cytochrome c was increased. Authors linked attenuation of calpain activation by preconditioning to cardioprotection through a reduced cleavage and activation of the protein Bid, a member of the Bcl-2 family [11]. This observation is coincident with the present results in pointing to attenuated calpain activation as an important mechanism of the protection afforded by preconditioning. Both studies appear as complementary and indicate that attenuated calpain activation can be protective through different effector systems.

The fact that prevention of {alpha}-fodrin proteolysis by calpain inhibition does not fully reproduce the protective effect of IPC suggests that the mechanisms of the protection induced by IPC are multiple.

In this study, the effects of IPC on functional recovery and calpain activity were not parallel. In an attempt to elucidate this discrepancy, the effects of IPC on degradation of additional cytoskeletal proteins (ankyrin and desmin) and of the contractile protein troponin I were analyzed and compared with those of MDL-28170. As in previous studies [8,13,27], ankyrin, desmin, and troponin I were degraded during reperfusion. Calpain inhibition fully reproduced the protective effects of IPC against proteolysis of ankyrin and desmin but only partially reproduced the effects of IPC on the hydrolysis of troponin I, indicating that other proteolytic systems different from calpains could be activated during ischemia/reperfusion. The more effective protection afforded by calpain inhibition against degradation of structural proteins as compared to troponin I was paralleled by more beneficial effect on cell death than on contractile failure (69% vs. 48% of the protection induced by IPC, respectively).

Because caspases are on one hand activated during reperfusion, and on the other hand are themselves substrate for calpain, the possibility exists that suppression of caspase activity contributes to the reduced protein degradation observed in IPC. However, our data do not point into this direction. First, Western blot analysis of {alpha}-fodrin did not detect any effect of ischemia/reperfusion, with or without IPC or MDL-28170, on the 120-kDa fragment resulting from caspase-3-mediated hydrolysis [28]. Second, inhibition of caspases with Z-VAD-FMK did not provide a significant additional protection against troponin I degradation, LDH release or contractile failure in hearts treated with the calpain inhibitor MDL-28170. Although these results do not exclude a certain contribution of caspases to protein degradation during initial reperfusion, they strongly suggest that this contribution is not major. The lack of evidence of a significant contribution of caspase activation to immediate reperfusion injury is not in disagreement with the contribution of caspases to cell injury after prolonged periods of reperfusion previously described [29]. However, this study does not rule out the possibility that calpain-independent hydrolysis of proteins of the contractile machinery other than Tn I, as Tn T [30], could contribute to postischemic contractile dysfunction.

3.2. Role of transient PKA activation
In this study, alprenolol and H89 were used to inhibit PKA activation during preconditioning ischemia. The concentration of alprenolol used, although lower than that used in other studies [17], was effective in inhibiting PKA activation. The concentration of H89 used was also effective. Although at the concentration in this study, H89 has been shown to inhibit kinases other than PKA, it does not significantly inhibit PKC [31], by far the most relevant of the inhibited kinases for classic IPC.

Our results suggest that the inhibitory effect of IPC on calpain activation is dependent on transient PKA activation during the preconditioning phase. Previous studies have shown that preconditioning protocols result in cyclic increases in cAMP and PKA, and that abolition of these increases by β-adrenergic blockade blunts the protective effect of IPC [17]. There is also evidence indicating that transient PKA activation has a cardioprotective effect that mimics that of IPC [17,18]. However, the mechanism proposed for this beneficial effect of PKA activation in previous studies was independent of any effect on calpain activation and was related to activation of p38MAPK. Several studies have identified transient activation of p38MAPK as an important element of the preconditioning cascade in pharmacological preconditioning or IPC [32,33]. One of the downstream effectors proposed for p38MAPK is HSP27, which phosphorylation induces its translocation and accumulation in the Z bands, increasing the resistance of the cytoskeleton to conformational changes and fragmentation [34]. It is thus possible that PKA reduces cell fragility in preconditioned myocardium by calpain independent mechanisms. However, in this study, the effect of IPC on calpain activity was simulated by transient PKA activation and was attenuated by treatments that prevented PKA activation, while calpain inhibition simulated the effects of IPC on {alpha}-fodrin proteolysis and cell death. Altogether, these results strongly suggest a role for reduced calpain activation in the protective effect of IPC. Previous studies have demonstrated the presence of several conserved PKA consensus sites in domain III of m-calpain [35] and that isolated calpains are highly phosphorylated [36]. Recently, Shiraha et al. [20], observed that phosphorylation at serine 369 by PKA maintains calpain in an inactive state.

3.3. Inhibition of IPC with 5HD or naloxone
In this study, 5HD at the concentrations of 100 and 300 µM failed to attenuate the protection achieved with IPC. Despite the large number of studies in which 5HD has been shown to antagonize IPC, other studies using Langendorff-perfused rat hearts have also failed to reverse the protective effects of IPC [37,38] and have identified KATP channels as independent targets of 5HD [38–40]. Indeed, most of the studies in which 5HD has been effective in blocking the protection obtained with IPC used either (1) models of regional ischemia or (2) models of global ischemia of relatively short duration in Langendorff-perfused hearts in which the main endpoint is functional recovery instead of cell death.

Our results with naloxone are consistent with other studies that showed only partial attenuation of the protective effect of IPC with this opioid antagonist [41,42]. While an in-depth analysis of the reasons of the failure of 5HD to abolish protection in our model is out of the scope of this study, the results obtained with this drug, in combination with those obtained with naloxone, show a clear parallelism between the degree of inhibition of calpain-dependent proteolysis and the magnitude of protection with different interventions; 5HD failed to both revert protection and to modify calpain inhibition, while naloxone partially blocked protection and induced milder inhibition of calpain activity.

3.4. Calpain and fragility during transient removal of extracellular Na+
Previous studies have shown that IPC reduces reperfusion-induced myocardial edema. This could obscure the interpretation of the present results, because it is difficult to dissociate the effects of attenuated fragility on sarcolemmal rupture from those of reduced cell swelling. To circumvent this problem, we used a protocol of transient Na+ withdrawal in which reperfusion-induced edema is not modified by IPC. In this model, exposure to Na+-free media induces Ca2+ influx through sarcolemmal Na+/Ca2+ exchange [43]. Reexposure to Na+-containing perfusate results in Ca2+ extrusion and massive Na+ gain through reverse-mode Na+/Ca2+ exchange and cell swelling. In contrast to what happens during reperfusion following ischemia, no hypercontracture was detected during Na+ readmission. This is consistent with a preponderant role of massive cell swelling in the observed cell death. The fact that IPC had a marked protective effect against calpain activation and cell death in this model in the absence of any effect on myocardial edema, and that this protective effect can be reproduced by inhibitors of calpain activity, strongly support the hypothesis that IPC has a direct effect on cell death secondary to calpain-dependent cell fragility.


   4. Conclusion
Top
 Abstract
 1. Methods
 2. Results
 3. Discussion
 4. Conclusion
References
 
This study is consistent with reduced sarcolemmal fragility secondary to PKA-dependent calpain inhibition being one of the end-effector mechanisms of the protective effect of IPC against necrotic cell death (Fig. 7). The results indicate that this mechanism acts in combination with others to afford the protective effect of IPC. The selective weight of attenuated fragility in comparison with other end-effectors yet to be identified remains to be determined.


Figure 7
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  		Fig. 7 Reduced sarcolemmal fragility as an end-effector mechanism of IPC. Calpain inhibition induced by transient PKA activation results in less degradation of {alpha}-fodrin, protein that confers elasticity and resistance to the cell membrane. The pharmacological interventions used in this study are also shown. βAR: β-adrenergic receptor.

 


   Acknowledgements
 
This study was partially supported by grants CICYT SAF 2002-0559 and FIS-RECAVA. We appreciate the excellent technical work of Mónica García.


   Notes
 
* Karin R. Sipido, University of Leuven, Belgium, served as Guest Editor for this article. Back

Time for primary review 43 days


   References
Top
 Abstract
 1. Methods
 2. Results
 3. Discussion
 4. Conclusion
 References
 

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