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Cardiovascular Research 2005 66(3):530-542; doi:10.1016/j.cardiores.2005.02.010
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Copyright © 2005, European Society of Cardiology

PI 3-kinase, protein kinase C, and protein kinase A are involved in the trigger phase of β1-adrenergic preconditioning

Arnaud Robinet*, Guillaume Hoizey and Hervé Millart

Department of Pharmacology, Reims University Hospital, 51, rue Cognacq-Jay, 51095, Reims cedex, France

* Corresponding author. Tel.: +33 3 26 91 80 31; fax: +33 3 26 91 35 30. Email address: arnaud.robinet{at}univ-reims.fr

Received 9 July 2004; revised 24 January 2005; accepted 10 February 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Using an isolated non-working rat heart model, this study investigated the mechanisms of pharmacological preconditioning (PC) induced by transient β1-adrenoreceptor (β1-AR) stimulation with xamoterol (XA).

Methods: After 6-hydroxydopamine (6-OHDA) pretreatment and a 20-min stabilization period, hearts were perfused at constant pressure for 20 min then subjected to 40 min of global ischemia and 30 min of reperfusion (I/R, Ctrl); exposed to 0.01 µM XA for 5 min with or without 10 µM atenolol (ATE), a specific antagonist of β1-AR, followed by a 15-min XA-free perfusion before I/R (PC, ATE-PC, respectively); treated during 20 min with either phosphoinositide (PI) 3-kinase inhibitors, LY-294002 (LY, 15 µM), or wortmaninn (WO, 0.1 µM); protein kinase C (PKC) inhibitor, GF-109203X (GF, 4 nM); or protein kinase A (PKA) inhibitor, H89 (H89, 1 µM), with an infusion starting 3 min before XA (LY-PC, WO-PC, GF-PC, and H89-PC, respectively). The main endpoints were the mean coronary flow (MCF), the left ventricular end-diastolic pressure (LVEDP), rate-pressure product (RPP), and creatine kinase (CK) release.

Results: XA induced an increase in the MCF after I/R (t 105 min) and a protective effect on the LVEDP, which were blocked by ATE and abolished with the different inhibitors. The transient increase in RPP following XA infusion was blocked by ATE and was not modified by the inhibitors except for H89. Recovery of RPP, measured 25 min after reperfusion, was improved by XA, blocked by ATE, and decreased with the different inhibitors. Fifteen minutes after the end of ischemia, CK release reached maximal values in all groups. XA provided significant protection whereas ATE and the four inhibitors suppressed XA-induced protection.

Conclusion: The transient preischemic exposure to nanomolar concentrations of a β1-AR agonist is protective against I/R. PI 3-kinase, PKC, and PKA are implicated in the trigger phase of PC. These observations were confirmed by Western blots.

KEYWORDS Ischemia; Preconditioning; Protein kinases; Beta1-adrenoreceptor; Signal transduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The term preconditioning (PC) was first introduced in 1986 by Murry [1] and refers to the mechanism that non-lethal short periods of ischemia, interspersed with reperfusion, protect the heart from injury during a prolonged subsequent period of ischemia. Apart from reduced infarct size, PC also reduces postischemic left ventricular dysfunction, arrhythmias, and apoptosis, depending on the model and species studied [2]. Beside ischemia-induced activation of cardioprotection by the prominent role of noradrenergic {alpha}1-receptors via a noradrenaline release [3], the β-adrenergic signaling pathway during PC should also be considered a trigger in eliciting PC [4,5]. Recently, we have suggested that the β-AR-induced PC (β-PC) was mediated less by myocardial β2-AR than by β1-AR activation [5]. Myocardial ischemia and I/R activate several protein kinase families that include the protein kinase C (PKC) isoforms, phosphoinositide (PI) 3-kinase/Akt, and cAMP-dependent protein kinase (PKA) [6]. Tong et al. [7] showed recently that ischemic preconditioning (I-PC) activated PI 3-kinase upstream of PKC. Lochner et al. [4] proposed that in addition to stimulation of G protein-coupled receptors (GPCRs) such as muscarinic, angiotensin II, or opioid receptors that carry to PKC activation, ischemia-induced activation of the β-adrenergic signaling pathway should be considered as a trigger or contributory factor in eliciting PC. The exact role of PKA still needs to be resolved. The purpose of our study was to investigate, in rat pretreated with 6-OHDA to attenuate the effect of endogenous catecholamines [8], the role of PI 3-kinase, PKC, and PKA in the triggering of β1-PC by using MCF, LVEDP, RPP, and creatine kinase (CK) release as main endpoints.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Compounds
6-Hydroxydopamine (6-OHDA) and wortmaninn (WO) were purchased from Sigma® (Saint Quentin Fallavier, France). PI 3-kinase p85, PKC{varepsilon}, PKA{alpha}, p-PI 3-kinase p85 (Tyr 508), p-PKC epsilon (PKC{varepsilon}) (Ser 729), and p-PKA alpha (PK{alpha}) (Ser 96) antibodies were purchased from Santa Cruz Biotechnology® (Heidelberg, Germany). Xamoterol hemifumarate, GF-109203X (GF), H89, and LY-294002 (LY) were obtained from Tocris® (Illkirch, France); atenolol (ATE) was a gift from Astra Zeneca (Rueil Malmaison, France). XA, LY, WO, GF, and H89 were first dissolved in 0.01% DMSO and sterile water, at a final concentration which had no effect either on the RPP or CK release, added to the Krebs–Henseleit buffer (KHB) just before oxygenation, warming, and use.

2.2. Isolated heart preparation
All experimentation procedures on animals adhered to the "Principles of Laboratory Animal Care" (National Institutes of Health, publication no. 23, revised 1985). Two-month-old male Wistar rats weighing 280–330 g were fed a standard diet and acclimatized in a quiet quarantine room for 5 days before the experiments. They were anesthetized with 50 mg/kg of sodium pentobarbital injected intraperitoneally (IP) and heparinized with a 500 IU injection. After deep anesthesia was reached, the chest was quickly opened. The hearts were promptly excised, placed first into a beaker of ice-cold perfusion solution to isolate an adequate length of aorta artery for cannulation. The hearts were perfused with an oxygenated KHB at 37 °C in accordance with the non-working Langendorff mode. We used the balloon method for recording of isovolumetric pressure in the isolated perfused hearts. Retrograde perfusion was started immediately after the aortic cannulation at a constant pressure (67 mm Hg) and a latex balloon (Hugo Sachs Elektronik) was inserted into the left ventricle through the left atrium and connected to a pressure transducer (Spectramed model P10EZ transducer, Gould 8000S recorder; Gould Electronics, Ballainvilliers, France). The balloon was filled with distilled water such that the initial diastolic pressure was kept constant at 10 mm Hg.

2.3. Experimental protocol
All experiments lasted a total of 110 min. All hearts were allowed to stabilize for 20 min with the standard KHB. The mM concentrations of constituents of the KHB were: NaCl 118, KCl 4.7, CaCl2 1.25, MgSO4 1.2, KH2PO4 1.2, glucose 11, NaHCO3 22.6, EDTA 0.027. The temperature of the perfusion buffer was adjusted to 37 °C by means of a Harvard thermocirculator and a Water thermostat type VTS 13 and the buffer was continuously bubbled with 95% O2, 5% CO2. Measurements recorded after a 20-min stabilization period were considered as the baseline values. Global ischemia was produced for 40 min by closing the inflow of physiologic solution. The hearts were maintained in a thermocontrolled chamber (37 °C) containing 0.9% physiologic serum. This ischemia period was followed by reperfusion for 30 min. The hearts were paced during 5 min from t 105 min to t 110 min. When paced, the beat rate was set to 360 beatsmin–1 and the hearts were defibrillated when necessary. All the rats were treated with 6-OHDA 4 and 3 days beforehand (50 mg/kg IP) [8].

2.3.1. Experiment 1: preconditioning experiment
This experiment was performed to confirm the ability of a low concentration of XA to mimic pharmacological PC induced by isoproterenol (ISO) [5]. We examined the CF, LVEDP, RPP, +LVdP/dtmax, and CK release of hearts pretreated with 0.01 µM XA with or without the specific antagonist of β1-AR, ATE. All the hearts were subjected to a 40-min global normothermic (37 °C) ischemia and then reperfused.

Group 1: Control group (Ctrl, n=5), isolated rat hearts were perfused with KHB during and after stabilization.
Group 2: 0.01 µM XA (PC, n=5), isolated rat hearts were exposed after stabilization (from t 20 min to t 25 min) to XA, then to a 15-min drug-free perfusion period.
Group 3: 10 µM ATE (ATE, n=5), isolated rat hearts were perfused with KHB containing ATE throughout the protocol.
Group 4: 10 µM ATE (ATE-PC, n=5), isolated rat hearts were perfused with KHB containing ATE throughout the protocol and to 0.01 µM XA from t 20 min to t 25 min.

2.3.2. Experiment 2: blockade of PC
Selective inhibitors of PI 3-kinase (LY, 15 µM and WO, 0.1 µM), PKC (GF, 4 nM), and PKA (H89, 1 µM) were applied before ischemia and 2 min before XA administration, to the XA-treated hearts. All the hearts were subjected to 40-min global normothermic (37 °C) ischemia and then reperfused for 30 min.

Group 5: LY (n=5), isolated rat hearts were exposed after stabilization (from t 18 min to 37 min) to 15 µM LY.
Group 6: WO (n=5), isolated rat hearts were perfused with KHB containing 0.1 µM WO (from t 18 min to t 37 min).
Group 7: GF (n=5), isolated rat hearts were exposed after stabilization (from t 18 min to t 37 min) to 4 nM GF.
Group 8: H89 (n=5) isolated rat heart with perfused with KB containing 1 µM H89 (from t 18 min to t 37 min).
Group 9: LY-PC (n=5), isolated rat hearts were exposed after stabilization (from t 18 min to 37 min) to 15 µM LY and to 0.01 µM XA from t 20 min to t 25 min.
Group 10: WO-PC (n=5), isolated rat hearts were perfused with KHB containing 0.1 µM WO (from t 18 min to t 37 min) and to 0.01 µM XA from t 20 min to t 25 min.
Group 11: GF-PC (n=5), isolated rat hearts were perfused with KHB containing 4 nM GF (from t 18 min to t 37 min) and to 0.01 µM XA from t 20 min to t 25 min.
Group 12: H89-PC (n=5) isolated rat heart with perfused with KB containing 1 µM H89 (from t 18 min to t 37 min) and to 0.01 µM XA from t 20 min to t 25 min.

A diagrammatic representation of the 2 experiments is shown in Fig. 1.


Figure 1
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  		Fig. 1 Schematic representation of the experimental protocol, Group 1 (Ctrl), Group 2 (PC), Group 3 (ATE), Group 4 (ATE-PC), Group 5 (LY), Group 6 (WO), Group 7 (GF), Group 8 (H89), Group 9 (LY-PC), Group 10 (WO-PC), Group 11 (GF-PC), and Group 12 (H89-PC).

 
2.4. Measurements
2.4.1. Contractile parameters
The contractile parameters were measured during 110 min. The systolic pressure (mm Hg) and the left ventricular end-diastolic pressure (LVEDP, mm Hg) were measured at t 20, t 25, t 40, t 80, t 85, t 90, t 95, t 105, and t 110 min and the difference between these two values represented the developed pressure (mm Hg). The heart rate (beatsmin–1) and +LVdP/dtmax (mm Hgsec–1) were measured in the same time. The rate-pressure product (RPP, mm Hgbeatmin–1) was calculated by multiplying the developed pressure and the heart rate. Reperfusion arrhythmias were not diagnosed.

2.4.2. Mean coronary flow (MCF)
The MCF (ml/min) was measured using the perfusate draining out of the right atrium for 1 min.

2.4.3. Biochemical determination
Effluents from the hearts were collected and CK activity (mIUmin–1g–1 of wet weight), which denotes myocardial cell cytolysis in a model of global ischemia, was determined at t 95 min, a time where enzymatic activity reached maximal values in all groups, and at t 105 min. Measurements were based on a fully enzymatic method using a commercially available reagent pack (Roche-Boehringer®, France).

2.5. Western blotting
After the different treatments and before ischemia (t 40 min), hearts were snap frozen for protein kinase and phospho-protein kinase analysis. Frozen hearts were powdered in a prechilled mortar and pestled with liquid nitrogen. Ice-cold lysis buffer (75 mM NaCl, 20 mM HEPES, 2.5 mM MgCl2, 0.1 mM EDTA, 0.1% Triton X-100, 10 µg/ml aprotinin, 20 mM glycerophosphate, 0.5 mM dithiothreitol, 0.1 mM sodium orthovanadate, 200 µg/ml phenylmethylsulfonyl fluoride, 1 mM sodium fluoride, pH 7.7) was added to powdered tissue. The homogenate was centrifuged at 45,000 x g at 4 °C for 10 min. Protein concentration was measured (BCA method, Interchim, Montluçon, France). Aliquots of supernatant containing equal amounts of protein (20 µg) were boiled in sample loading buffer for 5 min before loading on 10% SDS–polyacrylamide gel and electrophoretically transferred onto Immobilon-P membranes (Millipore, Saint Quentin en Yvelines, France). The non-specific binding sites on the membranes were blocked with 5% non-fat milk in Tris-buffered saline plus Tween-20 (TBST: 20 mM Tris–HCl, 150 mM NaCl, 0.1%Tween-20, pH 7.4). The membranes were incubated in 5% non-fat milk in TBST containing PI 3-kinase, phospho-PI 3-kinase, PKC{varepsilon}, phospho-PKC{varepsilon}, PKA{alpha}, or phospho-PKA{alpha} antibodies (1:1000 dilution) all night at 4 °C and then incubated in horseradish peroxydase-conjugated anti-rabbit IgG antibody in TBST for 1 h at room temperature. Finally, the immunoreactive bands were visualized by a chemiluminescence reagent.

2.6. Expression of results and statistical analysis
Statistical analyses were performed using SPSS® 8.0 for Windows®. Values we reported as means ± standard error of the mean (S.E.M.). Data were analyzed using simple one-way analysis of variance (ANOVA). If the F ratios were significant, post hoc tests (Dunnett's multiple comparison test) were applied to assess significance (p<0.05).


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. XA, a specific agonist of β1-ARs protects against I/R injury
At the end of the prolonged global ischemia, ATE blocked the increase in MCF induced by XA (Fig. 2) and the XA-induced protective effect on LVEDP (Fig. 3A). After stabilization, the transient increase in RPP (Fig. 3C) following XA infusion (121 ± 4% vs. 96 ± 5% in Ctrl, p<0.05) was blocked by ATE. Recovery of RPP after 25 min of reperfusion, was improved by XA (75 ± 6% vs. 37 ± 10% in Ctrl, p<0.05) and this beneficial effect was suppressed by ATE (53 ± 8% in ATE-PC vs. 49 ± 6% in ATE, NS) (Fig. 3C). These observations were strengthened by +LVdP/dtmax measurements (Table 1). Concerning the CK release (Table 2), XA provided significant protection 15 min after ischemia (706 ± 76 vs.1127 ± 116 in Ctrl, p<0.05). ATE blocked the protective effect of XA on CK release (1041 ± 87 vs. 1012 ± 39 in ATE, NS). Matched control groups did not differ from Ctrl groups for LVEDP, RPP, and CK values.


Figure 2
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  		Fig. 2 Effects of ATE, LY, WO, GF, and H89 on the improvement in MCF recovery (% of baseline values) induced by XA at t 105 min. PC group differed from all other groups. *p<0.05.

 

Figure 3
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  		Fig. 3 Graph showing the effect of ATE on postischemic recovery of LVEDP (mm Hg) (A), the heart rate data (B) (//, hearts were paced (360 beats.min–1) during 5 min from t 105 min to t 110 min), and the time course of changes in RPP (%) normalized to RPP after equilibration (C). *p<0.05, PC vs. Ctrl; #p<0.05, PC vs. ATE-PC; NS, ATE vs. Ctrl; NS, ATE-PC vs. ATE. –{triangleup}– control (n=5), –{square}– xamoterol 0.01 µM (n=5), –{circ}– xamoterol 0.01 µM+atenolol 10 µM (n=5), –{diamond}– atenolol 10 µM (n=5).

 

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  		Table 1 Time course of changes of the maximal rate pressure development (+LVdP/dtmax) (mm Hg.s–1)

 

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  		Table 2 Creatine kinase (CK) activity 15 and 25 min after global ischemia

 
3.2. PI 3-kinase activation triggers pharmacologically-induced β1-PC
LY and WO blocked the increase in MCF induced by XA during reperfusion (Fig. 2) and the XA-induced protective effect on LVEDP (Fig. 4A). As shown in Fig. 4C, there was no difference in RPP after transient XA stimulation (114 ± 3% in LY-PC and 117 ± 5% in WO-PC vs.121 ± 4% in PC). In contrast, after 25 min of reperfusion, LY and WO abolished the beneficial effect of XA on RPP recovery (47 ± 4% in LY-PC, 57 ± 7% in WO-PC vs. 75 ± 6% in PC, p<0.05) (Fig. 4C). When hearts were pretreated with LY or WO, after 15 min of reperfusion, the protective XA effect on CK release was abolished. With the two inhibitors, the CK release was higher than in PC group (1119 ± 121 in LY-PC and 987 ± 190 in WO-PC vs. 706 ± 76 in PC, p<0.05) and did not differ from Ctrl group (Table 2). The implication of PI 3-kinase in β1-PC was supported by Western blot analysis as shown in Fig. 4C; XA induced phosphorylation of the p85 subunit of PI 3-kinase which was suppressed by LY and by WO.


Figure 4
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  		Fig. 4 Graph showing the effect of LY and WO on postischemic recovery of LVEDP (mm Hg) (A), the heart rate data (B) (//, hearts were paced (360 beats.min–1) during 5 min from t 105 min to t 110 min), and the time course of changes in RPP (%) normalized to RPP after equilibration (C).*p<0.05, PC vs. Ctrl; +p<0.05, PC vs. LY-PC; °p<0.05, PC vs. WO-PC; # p<0.05, LY vs. Ctrl, and {infty}p<0.05, WO vs. Ctrl. –{triangleup}– control (n=5), –lhblk– xamoterol 0.01 µM (n=5), –{circ}– xamoterol+LY-294002 15 µM (n=5), –bullet– xamoterol+wortmaninn 0.1 µM (n=5), –{diamond}– LY-294002 15 µM (n=5), –{diamondsuit}– wortmaninn 0.1 µM (n=5). PI 3-kinase activation was assessed by Western blot (D).

 
3.3. PKC is involved in the signaling pathway of β1-PC
GF blocked the increase in MCF induced by XA (Fig. 2) and the XA-induced protective effect on LVEDP (Fig. 5A). As shown in Fig. 5C, there was no difference in RPP after transient β1-AR stimulation (113 ± 3% vs. 121 ± 4% in PC). In contrast after 25 min of reperfusion, GF abolished the protective effect of XA on RPP recovery (39 ± 6% vs. 75 ± 6% in PC, p<0.05) (Fig. 5C). After 15 min of reperfusion and when hearts were treated by GF, CK release was higher than in PC group and did not differ from Ctrl (Table 2). The implication of PKC{varepsilon} was supported by Western blot analysis. XA-induced PKC{varepsilon} phosphorylation was prevented by GF (Fig. 5D).


Figure 5
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  		Fig. 5 Graph showing the effect of GF on postischemic recovery of LVEDP (mm Hg) (A), the heart rate data (B) (//, hearts were paced (360 beats.min–1) during 5 min from t 105 min to t 110 min), and the time course of changes in RPP (%) normalized to RPP after equilibration (C). *p<0.05, PC vs. Ctrl; #p<0.05, PC vs. GF-PC; NS, GF vs. Ctrl and NS, GF-PC vs. GF. control (n=5), xamoterol 0.01 µM (n=5), xamoterol 0.01 µM+GF-109203X 4 nM (n=5), GF-109203X 4nM (n=5). PKC{varepsilon} activation was assessed by Western blot (D).

 
3.4. Blockade of PKA activity prevents β1-PC
H89 blocked the increase in MCF induced by XA (Fig. 2) and the XA-induced protective effect on LVEDP (Fig. 6A) As shown in Fig. 6C, administration of H89 decreased RPP value after transient β1-AR stimulation (110 ± 5% vs. 121 ± 4% in PC, p<0.05). After 25 min of reperfusion, H89 abolished the beneficial effect of XA on RPP recovery (50 ± 5% vs. 75 ± 6% in PC, p<0.05) (Fig. 6C). After 15 min of reperfusion, CK release was higher than in PC group and did not differ from Ctrl (Table 2). The implication of PKA was supported by Western blot analysis. Phosphorylation of PKA{alpha} induced by XA was blocked by H89 (Fig. 6D).


Figure 6
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  		Fig. 6 Graph showing the effect of H89 on postischemic recovery of LVEDP (A), the heart rate data (B) (//, hearts were paced (360 beats.min–1) during 5 min from t 105 min to t 110 min), and the time course of changes in RPP (%) normalized to RPP after equilibration (C). *p<0.05, PC vs. Ctrl; #p<0.05, PC vs. H89-PC; NS, H89 vs. Ctrl and NS, H89-PC vs. H89. –{triangleup}– control (n=5), –{square}– xamoterol 0.01 µM (n=5), –{circ}– xamoterol 0.01 µM+H89 1 µM (n=5), –{diamond}– H89 1 µM (n=5). PKA activation was assessed by Western blot (D).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The major new findings of this current study are as follows. In hearts from rats which had been catecholamine depleted with 6-OHDA [8], a 5-min perfusion with a concentration as low as 0.01 µM XA, followed by a 15-min drug-free perfusion before a prolonged I/R, restored postischemic contractile function and attenuated CK release, a marker of cell injury. The XA-induced protective effect was completely blocked by β1-AR blocker ATE. LY, WO, GF, and H-89, when administered before and during the XA protocol, abolished cardioprotection provided by β1-adrenergic stimulation, suggesting that PI 3-kinase, PKC, and PKA activation does act as a trigger of cardioprotection.

A previous study from our laboratory showed the beneficial role of β1-AR stimulation in pharmacological PC. In rat hearts treated with reserpine 24 h before experimentation, infusion of 0.02 µM isoproterenol (ISO) during 5 min, before a 40-min global ischemia followed by 30-min reperfusion, provided significant protection, causing a better functional recovery and a decrease of CK release. These beneficial effects were similar to those obtained by I-PC. The ISO-induced protective effect was completely blocked by β1-AR blocker ATE, suggesting that classic β-PC is mainly mediated by β1-ARs [5].

ISO has been shown to stimulate the phosphorylation of protein kinase B (PKB or Akt), a downstream target of PI 3-kinase, in neonatal rat ventricular cardiac myocytes [9]. We used LY and WO, two structurally unrelated inhibitors, to identify potential role of PI 3-kinase in acute pharmacological PC induced by β1-adrenergic stimulation (β1-PC). XA induced an increase in p85 subunit phosphorylation as demonstrated by Western analysis, effects inhibited by preincubation with WO or LY. These data indicate that XA activated PI 3-kinase. Since LY and WO fully inhibited the effects of β1-PC, activation of the PI 3-kinase pathway must lead to secondary signals that maintain contractile function and plasma membrane integrity in reperfused myocardium. In an in vitro perfused rabbit heart model, a 5-min exposure to insulin prior to a prolonged ischemic period led to a significant reduction in infarct size, similar to that seen with I-PC, which could be prevented by PI 3-kinase inhibition [10]. Recently, administration of insulin at the beginning of reperfusion has been reported to improve postischemic recovery of contractile function through PI 3-kinase in isolated working rat heart [11]. In ischemic-preconditioned rat hearts submitted to 20 min of global ischemia, inhibition of PI 3-kinase with either WO or LY, added before and throughout PC, blocked the recovery of left ventricular developed pressure [7]. In isolated perfused rat hearts subjected to 35-min regional ischemia, inhibition of PI 3-kinase, with the above-mentioned agents infused during the preconditioning protocol, partially abrogated the infarct sparing effect of I-PC [12]. The newly identified PI 3-kinase-dependent pathway of protection against reperfusion injury works primarily via a reduction of hypercontracture-induced cellular necrosis [13]. Considered together, these data show that activation of PI 3-kinase, during the trigger phase of PC, might be a common mediator for β1-PC and I-PC, via endogenous catecholamine release. All these observations show the important role of PI 3-kinase in cardioprotection induced by either β1-PC, insulin, or I-PC.

Our study suggests that PKC is implicated as a trigger in β1-PC. Treatment with polymyxin B, a PKC inhibitor, has been reported to annul the ISO-PC-induced improvement of RPP and suppression of CK release in an isolated non-working rat heart model [14]. Since the hearts were not catecholamine-depleted, the PC effects of ISO may have resulted partly from {alpha}1-stimulation. PKC was the first kinase examined in detail in I/R and I-PC. The role of PKC in I-PC was initially indicated by the loss of cardioprotection by pharmacological inhibition [15]. Recently, studies utilizing molecular biology techniques have clearly demonstrated the contribution of PKC{varepsilon} to I-PC in isolated perfused mouse hearts [16]. In Langendorff-perfused rat hearts, I-PC induces phosphorylation of PKB and translocation of PKC{varepsilon}, and it increases NO production. These effects which are blocked by WO suggest a role for PI 3-kinase in I-PC upstream of PKC and NO [7]. Recently, distinct functions of protein kinase isoforms have been reported during I/R. In isolated perfused rat hearts, pre-treatment with a selective PKC{varepsilon} activator, followed by a 10-min washout before I/R, improved cardiac function and decreased CK release whereas administration of a selective PKC{delta} inhibitor during reperfusion conferred cardioprotective effects against reperfusion injury [17]. GF is a specific PKC inhibitor which does not, however, distinguish between PKC{delta} and PKC{varepsilon}. Nevertheless, the suppression of the cardioprotective action of β1-PC when GF was administered around xamoterol is in favour of the implication of PKC activation as a trigger of PC, with PKC{varepsilon} being probably the isoform involved since we observed phosphorylation of PKC{varepsilon} using Western blot analysis. PKC{varepsilon} is activated by DAG, a product of PLC. Beside this well established signaling pathway, PKC{varepsilon} activation by PIP3, a product of PI 3-kinase has been reported to occur [18]. Recent evidence indicates that PLC-{gamma} can be activated by PIP3 in the absence of PLC-{gamma} tyrosine phosphorylation [19]. It has been shown that the binding of the PH domain of PLC-{gamma} to PIP3 present in the membrane as a result of PI 3-kinase activation leads to the activation of PLC-{gamma} [20].

In open-chest dogs, transient pretreatment with olprinone, a phosphodiesterase III inhibitor, or dibutyryl-cAMP (db-cAMP) injected intravenously 30 min before 90-min ischemia, followed by 6 h of reperfusion, has cardioprotective effects as assessed by reduced infarct size. The effect of olprinone was blunted by H-89, but not by GF suggesting that cardioprotective effect afforded by transient exposure to phosphodiesterase III inhibitors implies cAMP-PKA-dependent but PKC-independent mechanisms [21]. Trapidil is an antianginal compound which specifically activates PKAII by means of a cAMP sensitizing action in ischemic hearts. In Langendorff-hearts of New Zealand White rabbits, trapidil prevents I/R-induced reduction in phospholamban (PLB) phosphorylation, a major substrate of PKA in the heart, possibly explaining at least parts of the cardioprotective effects of the compound in I/R [22]. One of the prominent features of I/R injury in the heart is the disturbed Ca2+ homeostasis, causing a Ca2+ overload to cardiomyocytes. Potentially, the recovery in PLB phosphorylation allows for improved Ca2+ uptake into the SR, thereby reducing the cytosolic Ca2+ level. Functional consequences of this are a significant reduction in diastolic contracture. The phosphorylase kinase/glycogen phosphorylase cascade is another potential target for PKAII, eventually providing the ischemic heart with additional substrate for glycolytic ATP production [22]. Recently, Rho-kinase has been shown to negatively regulate Akt via inhibitory effects on PI 3-kinase [23] and transient preischemic activation of PKA has been shown to reduce infarct size through Rho-kinase inhibition [24]. The direct engagement of PI 3-kinase in the signaling of β1-AR has been reported recently with βARK1 as well as Gβ{gamma} signaling being involved in β1-AR-mediated PI 3-kinase activation [25]. Therefore, β1-AR transient stimulation elevates PI3K activity likely via both the Gs{alpha}- and the Gβ{gamma}-βARK1-directed signaling pathways.

β1- and β2-adrenergic receptors (β-ARs) co-exist in mammalian heart, and it is generally accepted that both activate adenylyl cyclase (AC), resulting in increased levels of cAMP and subsequent activation of L-type Ca2+ channels. The role of cAMP in β2-AR signaling and its functional relevance in adult rat heart have been the subject of controversy. β2-AR-stimulated increases in cAMP are not coupled to phosphorylation of cytosolic proteins, including phospholamban, troponin I, and glycogen phosphorylase kinase, in rat and canine heart cells [26]. In another study, β2-AR stimulation has been reported to induce PKA-dependent phospholamban phosphorylation in both adult cardiomyocytes and in adult hearts of rats and the β2-AR-mediated shortening of relaxation time correlated with Ser16 phosphorylation. Only β1-AR stimulation produced significant Ca2+/calmodulin kinase II (CaMKII)-related Thr17M phosphorylation of phospholamban, troponin I phosphorylation, and activation of phosphorylase a [27]. Phosphorylation of phospholamban (PLB) at Ser 16 (PKA site) and at Thr17 (CaMKII site) increases sarcoplasmic reticulum Ca2+ uptake and myocardial contractility and relaxation. Although both PLB phosphorylation sites are involved in the mechanical recovery after ischemia, Thr17 (CaMKII site) appears to play a major role [28]. Zhu et al. demonstrated that CaMKII constitutes a novel linkage of β1-AR stimulation to cardiomyocyte apoptosis independent of PKA signalling [29]. The low concentration of β1-AR agonist used in our study may have led to an increased PKA activity without increasing CaMKII activity [30]. However, whether CaMKII activation might be beneficial or detrimental in the trigger phase of β1-PC needs further investigations since pre-treatment for 6 days of rats with the CaMKII inhibitor, KN-93, has been shown to produce detrimental effects on recovery of cardiac function in the globally ischemic rat heart [31].

In rat hearts, I-PC leads to a parallel activation of both PKC and adenylyl cyclase. In global ischemia, adenylyl cyclase is sensitized after 5–15 min of ischemia and this sensitization can be prevented by an inhibition of PKC [32]. In our study, PKA and PKC pathways were activated by XA before global ischemia whereas in another study, PKC did not appear to be involved in cardioprotection afforded by β-adrenergic stimulation [4]. Transduction mechanisms following stimulation of β1-ARs in isolated perfused rat heart involve the activation of PKA, PI 3-kinase, and PKC{varepsilon} pathways and are necessary in the triggering of β1-PC to limit postischemic cell injury and contractile dysfunction. Although both PKA and PKC are simultaneously activated during sympathetic stimulation through β- and {alpha}1-adrenergic receptors, respectively [32], sequential activation of these two kinases is more likely to occur in our catecholamine-depleted isolated heart preparations. PKC activation downstream PKA stimulation is not a commonly accepted rule but paracrine mechanisms for PKC activation cannot be ruled out. However, activation of both PKA and PKC pathways in a single sequence of events with PKA prior to PKC activation has been reported in OK cells following stimulation of dopamine D1 receptors [33].

In conclusion, it appears that β1-PC is a powerful mechanism of anticipation of cardioprotection reflecting the role of catecholamines during stress. One possible interpretation of our results considers a single sequence of events, with PKA activation prior to PKC activation in the cascade downstream to stimulation of β1-AR. Fig. 7 shows a schematic representation of the putative interactions between the different signaling pathways activated by XA. Activation of PKC{varepsilon} following transient β1-AR stimulation during the β1-PC protocol suggests a protective feedback regulatory mechanism which could be vital in the settings of excessive release of catecholamines [34].


Figure 7
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  		Fig. 7 Suggested model for the signaling pathway involved in the trigger phase of early β1-PC. For explanation, see Discussion. AC: Adenylate Cyclase; PIP3: Phosphatidylinositol 3,4,5-triphosphate; PIP2: Phosphatidylinositol 4,5-bisphosphate; DAG: Diacylglycerol; PL{gamma}: Phospholipase C{gamma}.

 


   Acknowledgements
 
We thank Françoise Moreau and Jean Pisani from Pharmacological Laboratory for technical assistance.


   Notes
 
Time for primary review 25 days


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

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