Ischemic preconditioning (IP) is a cardioprotective mechanism against myocellular death and cardiac dysfunction resulting from reperfusion of the ischemic heart. At present, the precise list of mediators involved in IP and the pathways of their mechanisms of action are not completely known. The aim of the present study was to investigate the role of platelet-activating factor (PAF), a phospholipid mediator that is known to be released by the ischemic-reperfused heart, as a possible endogenous agent involved in IP. Experiments were performed on Langendorff-perfused rat hearts undergoing 30 min of ischemia followed by 2 h of reperfusion. Treatment with a low concentration of PAF (2 × 10−11 M) before ischemia reduced the extension of infarct size and improved the recovery of left ventricular developed pressure during reperfusion. The cardioprotective effect of PAF was comparable to that observed in hearts in which IP was induced by three brief (3 min) periods of ischemia separated by 5-min reperfusion intervals. The PAF receptor antagonist WEB-2170 (1 × 10−9 M) abrogated the cardioprotective effect induced by both PAF and IP. The protein kinase C (PKC) inhibitor chelerythrine (5 × 10−6 M) or the phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 (5 × 10−5 M) also reduced the cardioprotective effect of PAF. Western blot analysis revealed that following IP treatment or PAF infusion, the phosphorylation of PKC-ε and Akt (the downstream target of PI3K) was higher than that in control hearts. The present data indicate that exogenous applications of low quantities of PAF induce a cardioprotective effect through PI3K and PKC activation, similar to that afforded by IP. Moreover, the study suggests that endogenous release of PAF, induced by brief periods of ischemia and reperfusion, may participate to the triggering of the IP of the heart.
ischemic preconditioning (IP) is the phenomenon whereby brief periods of ischemia and reperfusion increase the resistance to myocardial infarction and contractile dysfunction induced by a subsequent sustained episode of ischemia (9, 34–36). In all species tested, the beneficial effects of IP include protection against necrotic and apoptotic cell death. In some species, it has been shown that IP also induces the prevention of ischemia-reperfusion-induced arrhythmias, a faster recovery from reperfusion-induced myocardial stunning, and a protection of microvasculature function (for reviews, see Refs. 33 and 35). Because of its proven efficacy, understanding the mechanisms by which IP enhances the tolerance to ischemic injury may therefore be of considerable interest. Several mechanisms have been suggested to explain the protective effect of IP at the myocardial level. It has been shown that brief periods of ischemia induce the release of several agonists such as adenosine, bradykinin, and opioids. Acting on G protein-coupled receptors (GPCR), these mediators initiate a signaling cascade that involves the activation of protein kinase C (PKC), phosphoinositide 3-kinase (PI3K), endothelial nitric oxide (NO) synthase (NOS), and other signaling pathways (33). Among these pathways, mitochondrial ATP-sensitive K+ channels (mitoK) may play a central role in cardioprotection (33).
Currently, the precise list of triggers involved in IP and related intracellular cascades are not completely known. It has been suggested that there are several triggers and that they may all participate to reach a threshold for PKC activation (8).
The aim of the present study was to test the hypothesis that the platelet-activating factor (PAF) pathway may play a role in IP. PAF may be a candidate as a trigger of IP because specific PAF receptors are present on several cell types, including cardiomyocytes and endothelial and smooth muscle cells (17, 30, 40, 43); their stimulation leads to the production of diacylglycerol, which in turn activates PKC (30, 43). Recent findings suggest a further PAF-induced signal transduction mechanism, implicating a Gi-dependent activation of PI3K and a consequent phosphorylation of protein kinase B (PKB or Akt) and NOS3 (2).
Up to now, however, studies performed on the cardiac effects of PAF were mainly devoted to investigate the effects exerted by high concentrations of this mediator comparable to those released in severe pathophysiological conditions, such as after a long-lasting period of ischemia (19, 32) or acute anaphylactic shock (10, 24). Several in vivo and in vitro studies demonstrated that severe ischemia-reperfusion of the heart leads to the release of amounts of PAF, which are enough to activate inflammatory blood cells and to induce severe myocardial dysfunction, including profound electrophysiological alterations and a negative inotropic effect (40, 44).
We cannot exclude, however, that brief periods of ischemia and reperfusion induce the release of very small quantities of PAF, unable per se to alter myocardial contractility but enough to activate PKC and/or PI3K, leading to an IP of the heart. As a matter of fact, PAF is thought to be a mediator of cell-to-cell communication, and some of its above-mentioned actions are reported to be achieved at concentrations as low as 10−12 M. Such a concentration, however, in many species does not appreciably affect heart function (30, 43).
We hypothesized that the administration of a very low dose of PAF (10−12–10−11 M) is able to induce protective effects akin to IP and that the activation of PAF receptors may play a role in triggering IP. To test this hypothesis, we compared the protective effects of low doses of PAF infused just before ischemia-reperfusion with those induced by three cycles of IP. In separate groups, PAF perfusion or IP were performed in the presence of the PAF receptor blocker WEB-2170. The possible role of PKC and PI3K in the signaling pathway involved in PAF-induced protection was studied by coinfusion of PAF with the specific inhibitors of these kinases chelerythrine or LY-294002, respectively.
Experiments were performed on male Wistar rats (n = 76, 450–550 g body wt). Rats were housed in identical cages and were allowed access to tap water and a standard rodent diet ad libitum. The animals received humane care in accordance with Italian law (DL-116, Jan. 27, 1992), which is in compliance with the National Institutes of Health publication Guide for the Care and Use of Laboratory Animals, and the scientific project, including animal care, was supervised and approved by the local ethical committee. In brief, rats were heparinized (2.500 units im) and anesthetized with urethane (1 g/kg ip) 10 min later. The hearts were then rapidly excised, placed in ice-cold buffer solution, and weighed. Isolated hearts were attached to the perfusion apparatus and retrogradely perfused with oxygenated Krebs-Henseleit buffer containing (in mM) 127 NaCl, 17.7 NaHCO3, 5.1 KCl, 1.5 CaCl2, 1.26 MgCl2, and 11 d-glucose, supplemented with 5 μg/ml lidocaine and gassed with 95% O2-5% CO2 (38, 41). The hearts were instrumented as previously described (36, 38, 41). A constant flow (9 ± 2 ml·min−1·g wet wt−1) was adjusted to obtain a coronary perfusion pressure of 80–85 mmHg within the first 5–10 min of stabilization and kept constant thereon. A constant proportion of 10% of the flow rate was applied by means of one of two perfusion pumps (Terumo, Tokyo, Japan) by using a 50-ml syringe connected to the aortic inflow cannula. Drug applications (see Experimental Protocols) were performed by switching from the syringe containing buffer alone to the syringe of the other pump containing the ×10 drug stock solution. A small hole in the left ventricular wall allowed drainage of the thebesian flow, and a polyvinyl chloride balloon was placed into the left ventricle and connected to an electromanometer to record left ventricular pressure (LVP). The hearts were electrically paced at 280–300 beats/min and kept in a temperature-controlled chamber (37°C). Coronary perfusion pressure (CPP) and coronary flow were monitored with a second electromanometer and an electromagnetic flow probe, respectively, both placed along the perfusion line. Coronary flow, CPP, and LVP were recorded and analyzed offline with LabView software (National Instruments), which allowed quantification of the maximum rate of increase of LVP (+dP/dtmax). If not otherwise indicated, chemicals were purchased from Sigma (St. Louis, MO).
Each heart was allowed to stabilize for 30 min; at this time, baseline parameters were recorded. After stabilization, hearts were randomly assigned to one of the treatment groups described below and then subjected to 30 min of global, no-flow ischemia followed by 120 min of reperfusion (I/R) (see Fig. 1). Pacing was discontinued at the beginning of the ischemic period and restarted after the third minute of reperfusion. After stabilization, hearts of the control group (group 1; n = 7) were perfused with buffer for an additional 29-min period before ischemia. Group 2 hearts (n = 7) underwent a preconditioning protocol, which consisted of three brief periods of ischemia (3 min) separated by 5-min reperfusion intervals. After the third episode of ischemia, the hearts were reperfused with buffer for 10 min before induction of 30 min ischemia. To study the possibility that PAF may mimic the effects of IP, the hearts of group 3 (n = 7) were perfused with PAF (2 × 10−11 M) for 19 min and then with buffer alone for 10 min. In preliminary experiments performed to study the dose response to PAF in our experimental setting, we observed that the concentration of PAF used in this study (n = 5) had no significant effect on cardiac performance, whereas higher doses of PAF (2 × 10−10, n = 5, and 2 × 10−9 M, n = 4) exerted a dose-dependent negative inotropic effect (LVP = 81.5 ± 4.8% and 57.5 ± 3.6% of baseline level, respectively) comparable to those reported by other authors on the same preparation (1).
Hearts of group 4 (n = 5) underwent a preconditioning protocol like group 2 hearts, bracketed by a 29-min infusion of the PAF receptor antagonist WEB-2170 (1 × 10−9 M; Boheringer Ingelheim), a triazolobenzodiazepine derivative (4, 16, 27, 31). Groups 5, 6, and 7 hearts (n = 5 for each group) were perfused with PAF for 19 min like group 3 hearts, bracketed by a 29-min infusion of the receptor antagonist WEB-2170 (1 × 10−9 M), the PKC inhibitor chelerythrine (5 × 10−6 M; Ref. 14), and the PI3K inhibitor LY-294002 (5 × 10−5 M; Ref. 18), respectively, before ischemia.
To test the role of the used antagonists per se on I/R damages, in three additional groups of hearts (n = 5 for each group), the antagonists WEB-2170 (group 8), chelerythrine (group 9), or LY-294002 (group 10) were infused for 29 min without PAF and IP.
Assessment of ventricular function.
To obtain maximal developed LVP, the volume of the intraventricular balloon was adjusted to an end-diastolic LVP (LVEDP) of 5 mmHg during the stabilization period, as reported elsewhere (36). LVEDP, developed LVP, and +dP/dtmax were continuously monitored during all the experiments. Contracture development was defined as an increase in intrachamber pressure of 4 mmHg above preischemic LVEDP values. Developed LVP and +dP/dtmax values during reperfusion were expressed as the percentage of respective preischemic values. Maximal recovery of developed LVP and +dP/dtmax during reperfusion was compared with respective preischemic values.
Assessment of myocardial injury.
To obtain infarct areas, hearts were rapidly removed from the perfusion apparatus at the end of reperfusion, and the left ventricle was dissected into 2- to 3-mm circumferential slices. After 20 min of incubation at 37°C in 0.1% solution of nitro blue tetrazolium in phosphate buffer, unstained necrotic tissue was carefully separated from stained viable tissue by an independent observer who was not aware of the nature of the intervention. The weights of the necrotic and nonnecrotic tissues were then determined, and the necrotic mass was expressed as a percentage of total left ventricular mass (26, 36).
Assessment of kinases activation.
Six additional hearts were treated as group 1 (n = 2), group 2 (n = 2), or group 3 (n = 2) without I/R. They were then rapidly removed from the perfusion system, snap frozen, and used for Western blotting analysis to determine the level of phosphorylation of PKC and Akt.
For immunodetection of proteins, frozen heart tissue samples were dismembrated in 250 ml of 100 mM NaCl, 10 mM Tris (pH 7.6), 1 mM EDTA (pH 8), 0.1 mg/ml phenylmethylsulfonyl fluoride, 0.1 mg/ml aprotinin and leupeptin, and 1.54 mM Na orthovanadate. After 5 min of centrifugation at 12,000 rpm, pellets were discarded, and the protein content was measured in the supernatant. A total protein of 20 (for PKC and Akt analysis) or 50 mg (for phosphorylated PKC and Akt analysis) for each sample was separated on 12% (for PKC) or 10% (for Akt) SDS-PAGE gel and transferred to Hybond ECL nitrocellulose membranes (Amersham) (7). As positive controls, lysates from Jurkat (PKC and Akt), 3T3/A31 (phospho-PKC), or calyculin-stimulated Jurkatt cells were used. Blots were probed with primary rabbit polyclonal anti-PKCε (Upstate 06-991), anti-phospho-PKCε (Ser729, Upstate 06-821), anti-Akt (Cell Signaling 9272), and anti-phospho-Akt (Ser473, Cell Signaling 9271) diluted 1:1,000 according to manufacturer's instructions. Blots were then probed with horseradish peroxidase-conjugated anti-rabbit antibody. Protein were detected using an enhanced chemiluminescence ECL Western blotting detection reagent, and bands were visualized through Kodak Image Station 440 CF. To confirm equal protein loading, blots were stripped with 0.4 M NaOH and then reblotted with an anti-actin antibody (Sigma 2066). Image analysis were performed by the Kodak 1D 3.5 software.
All values are presented as means ± SE. Data were subjected to ANOVA followed by the Bonferroni correction for post hoc t-tests. Significance was accepted at a P level <0.05.
Effects of Treatment Before Ischemia on Ventricular Function
Baseline functional parameters did not differ significantly among the seven groups of hearts (Table 1). Group 1 hearts showed stable cardiac performance during the period (29 min) in which they were perfused with buffer before ischemia. Preconditioning protocol did not significantly modify LVEDP and LVP developed by group 2 hearts. In group 3 hearts, the infusion of PAF (2 × 10−11 M) caused a slight transient increase of developed LVP (116.2 ± 2.8%), followed by a subsequent recovery to baseline value during washout. In the hearts that underwent an IP protocol while perfused with WEB-2170 (10−9 M; group 4) cardiac performance was not affected. Perfusion of group 5 hearts with both WEB-2170 and PAF did not significantly modify cardiac performance. Similar results were observed in group 6 hearts: the perfusion with PAF in the presence of chelerythrine induced a slight (not significant) increase of developed LVP (121.3 ± 10.4%). In group 7, coinfusion of PAF and LY-294002 induced a transient reduction of developed LVP (87.5 ± 11.0%) followed by a recovery to baseline level during washout. No changes were observed in the cardiac performance of hearts of additional groups (groups 8-10) in which only WEB-2170, chelerythrine, or LY-294002 was infused.
Effects of 30 min Ischemia on Ventricular Function
In all experimental groups, a similar marked reduction of developed LVP was observed while LVEDP slowly increased. At the end of the period of ischemia, all hearts were not developing pressure, whereas the rise of LVEDP (contracture) caused by ischemia (about 12–20 mmHg over baseline value) was statistically similar in all the experimental groups (Fig. 2).
Ventricular Function During Reperfusion
In control (group 1) hearts, reperfusion caused a further marked worsening of diastolic contracture during ischemia, which reached its peak at 30–40 min (about 85–90 mmHg over preischemic values) and then tended to recovery slowly. The recovery, however, was largely incomplete, because LVEDP persisted near to 60–65 mmHg over baseline values after 120 min of reperfusion. Developed LVP, as well +dP/dtmax, slowly improved during reperfusion as diastolic pressure declined. Maximum recovery of developed LVP and contractility was usually attained within 90–100 min of reperfusion. The protective effect of IP was evident for group 2 hearts (Figs. 3A and 4A). In this group, the increase of LVEDP during reperfusion was significantly reduced (P < 0.01 vs. group 1) compared with group 1 hearts. In particular, in the preconditioned hearts the peak of LVEDP recorded at 30 min of reperfusion and at the end of reperfusion was 55–60 and 30–35 mmHg over preischemic values, respectively (Fig. 2A). In parallel to LVEDP, IP also improved the recovery of developed LVP and +dP/dtmax during reperfusion (Figs. 3A and 4A). Pretreatment of group 3 hearts with PAF mimicked the effects of IP, leading to a significant reduction (P < 0.01 vs. group 1) of the entity and duration of diastolic contracture during reperfusion (45–50 and 35 mmHg and over preischemic values at 30 and 120 min of reperfusion, respectively) and contractile dysfunction observed during reperfusion. The cardioprotective effect exerted by PAF was superimposable to that induced by IP in group 2 hearts (Figs. 3 and 4).
Pretreatment of hearts with PAF receptor antagonist (WEB-2170) completely blocked the cardioprotective effect induced by both IP (group 4) and PAF (group 5). Besides, in the hearts of group 6 or 7 the block of PKC or PI3K with chelerythrine or LY-294002, respectively, significantly reduced the cardioprotective effect induced by PAF (Figs. 2B, 3B, and 4B).
Coronary Perfusion Pressure During I/R
As expected, CPP dropped during ischemia and increased in all groups during reperfusion. The percent increase of CPP during reperfusion was similar in all treated groups. Although there was no statistical difference among groups, it is noteworthy that there are three different situations in the groups in which PAF was coinfused with inhibitors that blocked its protective effects. Compared with those of PAF alone (group 3), the percent variations of CPP were either superimposable (group 5, PAF + WEB-2170) (Fig. 5A), slightly higher (group 6, PAF + chelerythrine), or slightly lower (group 7, PAF + LY-294002) (Fig. 5B).
In group 1 hearts, the infarct size measured at the end of reperfusion was 63.3 ± 8.6% of the area at risk (Fig. 6). IP exerted a significant cardioprotective effect in group 2 hearts, in which infarct size was reduced to 32.3 ± 6.5% of the area at risk (P < 0.05 vs. group 1). The infusion of PAF (group 3) before ischemia induced a protective effect, which was superimposable to that caused by IP; in these hearts, the infarct size (33.7 ± 4.3% of the area at risk) was indeed reduced by 40% with respect to that of group 1 (P < 0.05 vs. group 1). The presence of WEB-2170 abolished the protection induced by both IP (group 4) and PAF (group 5); in these hearts, infarct size was comparable to that measured in control hearts of group 1 (infarct size = 72.6 ± 6.7% and 70.0 ± 6.0% of the area at risk, respectively). Similar results were observed in groups 6 and 7 hearts, which were treated with PAF in the presence of chelerythrine or LY-294002 before the induction of ischemia (infarct size = 71.2 ± 5.2 and 59.3 ± 7.8% of the area at risk, respectively). In groups 8–10, in which only WEB-2170, chelerythrine, or LY-294002 was infused, the infarct size was not statistically different from that observed in the control group (group 1), being 59 ± 7%, 52 ± 8%, and 53 ± 6.0%, respectively.
PKC and Akt activation/phosphorylation by PAF or IP was confirmed by Western blot analysis. Densitometrical analysis of the scanned blots, presented in Fig. 7, showed a well-evident increase of phosphorylated PKC and Akt in both PAF- and IP-treated hearts compared with the control hearts.
The present study confirms the hypotheses that very low concentrations of PAF are able to induce protective effects akin to IP and that the activation of PAF receptors play a role in the triggering of IP. Moreover, this study strongly indicates that the signaling pathways downstream the PAF receptor stimulation involve PI3K and PKC activation.
IP is a cardioprotective mechanism whereby hearts exposed to brief, sublethal ischemic insults are more resistant against subsequent prolonged ischemia, reducing myocellular death and postischemic cardiac dysfunction. Although several studies were devoted to investigate the role of different mediators involved in IP and their intracellular pathways, the mechanisms of IP are still to be fully elucidated. Several agonists, among which are adenosine, bradykinin, and opioids, are able to trigger preconditioning of the heart. Although these mediators exert a cardioprotective effect by modulating different signaling pathways, recent findings suggest that these diverse signals converge on a few final common effectors that improve myocardial function and cell viability. Most of the agonists involved in IP stimulate PI3K, leading to activation of downstream enzymes, such as protein kinase B/Akt and endothelial NOS. The activation of PKC also seems downstream to PI3K activation (33, 47). One of the effects of the activation of these pathways is the alteration of the activity of mitoK, leading to lowered cell metabolism and reduced cell death, thus resulting in cardioprotection (33, 35). It has been also proposed that PKC activation is potentiated by mitoK activation (37, 39). This reverberant activation of PKC makes this kinase one of the fundamental steps of cardioprotection.
Nevertheless, PKC activation can be achieved only if triggers released by preconditioned myocardium reach a threshold (8). It is conceivable that, besides adenosine, bradykinin, and opioids, which are more studied up to now, other substances released from ischemic-reperfused cardiac tissue may participate in the induction of IP via PKC activation. Among these, PAF may play an important role.
In ongoing experiments we have tested PAF effects in the presence of the opioid receptor antagonist naloxone (n = 5) or adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DCPX) (n = 5). The doses of the antagonists were 1 × 10−5 M and 5 × 10−6 M, respectively (22, 25). In these preliminary experiments, we found that PAF-induced protection was partially blunted by the adenosine receptor antagonist and was not affected by opioid receptors antagonist. The results suggest that the protection by PAF is independent of opioid receptors, whereas a cross talk between PAF and adenosine receptors may exist. Further studies are required to elucidate this interaction.
It has been shown that PAF can be released in significant amounts from ischemic-reperfused hearts (19, 27, 32). Besides cardiomyocytes, several cellular elements within the myocardium, such as vascular endothelial cells and resident mast cells, have been shown to be capable of producing PAF. Therefore, the heart itself may be a source of PAF independently from inflammatory cells (13, 30, 40).
When released after a long-lasting period of ischemia, PAF induces severe cardiac effects, which are comparable to those exerted by relatively high concentrations (up to 1 × 10−9 M) of this mediator (1, 32). When activated, the receptor for PAF, which is present on cardiomyocytes and belongs to the GPCR superfamily (17, 40, 43), relays signals causing a negative inotropic effect (3, 45). Actually, PAF is considered as one of the most important GPCR agonist depressing myocardial contractility, causing further distress to the ischemic organ in ischemic heart disease (44).
From the results of the present study, we can argue that brief periods of ischemia and reperfusion induce the release of very small quantities of PAF, which cannot alter myocardial contractility, but are enough to participate in the activation of PKC and/or PI3K, leading to an IP of the heart. This reasoning is supported by comparing the effects of IP induced by brief periods of ischemia and reperfusion to those caused by a brief treatment with a very low dose of PAF (2 × 10−11 M), as well as the effect of PAF receptors blockade in hearts undergoing IP. Although, at this low concentration, PAF had no significant effect on cardiac activity, it reduced the extension of infarct size and improved the recovery of developed LVP during reperfusion. The cardioprotective effect of PAF was comparable to that observed in hearts undergoing IP. The fact that pretreatment of the hearts with a PAF receptor antagonist abrogated the cardioprotective effect induced by IP further supports the hypothesis that the release of PAF induced by brief periods of ischemia is involved in IP.
Most notably the beneficial effects exerted by PAF involve both myocardial contractility and relaxation (i.e., reduction in contracture development) at reperfusion. This is an intriguing result that deserves further investigation, because it may indicate an improvement of myofilament responsiveness to Ca2+, which may be a feature of IP (5). Moreover, Gelpi et al. (15) suggested that prevention of contracture might be “a more robust indicator of protection” than contractility itself, especially in the rat heart. According to these authors, in this species, the improvement of contractility may be attributed to reduced formation of free radicals via a xanthine oxidase (XO) pathway due to the reduced release of purine by preconditioned heart. In fact, in XO-deficient rabbit hearts, recovery of function was not a function of IP despite marked reduction in infarct size in preconditioned hearts (15). On the contrary, contracture improvement was similar in both species, i.e., this parameter is not influenced by the presence of XO.
Regarding the mechanisms by which PAF may induce IP, it must be stressed that several lines of evidence indicate that in most cases IP is induced by PI3K stimulation, which in turn leads to activation of downstream kinases such as PKB/Akt, endothelial NOS, and PKC (33, 35). The intracellular signaling pathways activated by PAF receptors stimulation share the same kinases cascade. Indeed, stimulation of PAF receptors leads to the production of diacylglycerol, which in turn activates PKC (for reviews, see Refs. 13, 30, and 43). Moreover, recent findings demonstrated a further PAF-induced signal transduction mechanism in cardiac cells, implicating a Gi-dependent activation of PI3K and a consequent phosphorylation of PKB/Akt and NOS3 (2). In the present study, we confirmed by Western blot analysis the involvement of these kinases in IP- and PAF-induced cardioprotection. Moreover, we investigated the role of PKC and PI3K in intracellular signaling pathways involved in PAF-induced preconditioning by using the specific inhibitors chelerythrine and LY-294002. Our observation that both chelerythrine and LY-294002 abolished the protective effects of PAF strongly support a role for PKC and PI3K in PAF-induced cardioprotection. These two kinase blockers are reported to abolish IP-induced protection (47, 48, 50). Here, we show that chelerythrine and LY-294002 per se do not affect I/R damages, as previously reported (11, 29, 47). Moreover, for the first time, we show that also WEB-2170 given before ischemia does not affect I/R injuries.
In our model of I/R with constant flow during baseline and reperfusion periods, it is unlikely that disparities in tissue perfusion may contribute to the differences observed between groups. Increase of coronary pressure during reperfusion could have worsened the myocardial injuries (49). The fact that CPP increased similarly in all groups suggests that if present the worsening was similar in all groups. Therefore, our findings about PAF-induced cardioprotection are not dependent on the CPP variations.
Physiological Significance of the Findings in Cardioprotection
It is known that exercise can mimic the protective effect of IP (9, 12, 21, 51). Evidence for preconditioning in humans derives also from in vitro studies (42) and observations made during coronary angioplasty and cardiac surgery (12, 21, 51). Also, the warm-up phenomenon (i.e., enhanced resistance to further ischemia in patients suffering from angina a few minutes after a previous effort) has been attributed to a preconditioning-like effect (9, 46). In patients with stable angina, it has been recently demonstrated that exercise delays the appearance of ST segment depression during a subsequent effort in the early and late periods of protection after exercise-induced ischemia (9, 20, 28).
It has been also observed that a very low increase in PAF levels occurs during exercise in normoalbuminuric diabetic patients (6) as well as during atrial pacing (30). It may then be argued that low levels of PAF participate to exercise-induced cardioprotection. The fact that low levels of PAF may be released in certain conditions [e.g., noninfarcting ischemias (present study), atrial pacing (30), and during exercise (6)], and the fact that these low levels of PAF participate to IP (present study) may be taken into account when strategies of myocardial protection are considered.
In conclusion, taken together, our data indicate that 1) low concentrations of exogenous PAF are able to trigger preconditioning-like effects without evident cardiodepressant effect; and 2) the signaling events downstream PAF receptor stimulation involve PI3K and PKC activation. Our results also suggest that endogenous synthesis and release of low quantities of PAF, induced by brief periods of ischemia and reperfusion, might play a crucial role in the triggering of ischemic preconditioning.
This work was supported by Ministero dell’Istruzione, dell’Università e della Ricerca, Compagnia di S. Paolo, Torino, and Istituto Nazionale per la Ricerca Cardiovascolare.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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