INTRODUCTION

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WHEN THE HEART is exposed to brief periods of ischemia and reperfusion, resistance to a prolonged ischemic insult is conferred (14). This phenomenon has been termed ischemic preconditioning (IPC) and has been shown to occur in dogs (14), rabbits (10), pigs (19), rats (11), and possibly humans (25). IPC occurs in two phases: an early phase in which the window of cardioprotection lasts from 0 to 90 min post-IPC, and a second phase in which the window of protection reappears 24-72 h post-IPC. Many mediators and effectors have been proposed to be essential for IPC and include the ATP-sensitive K⁺ (K_ATP) channel (21, 23), protein kinase C (PKC) (13, 24), A₁-adenosine receptor (12), and the ₁-opioid (20) receptor.

More recently, evidence has been obtained that supports a role for the involvement of tyrosine kinase (TK) in early or classic IPC. Maulik et al. (13) found that IPC resulted in the stimulation of phospholipase D, mitogen-activated protein (MAP) kinase, and MAP-kinase-activated protein (KAP) kinase-2 in the isolated rat heart, and that this activation could be inhibited by genistein, a TK antagonist. These data suggested that TK activation is an early step in classic IPC. Conversely, recent evidence presented by Baines et al. (3) suggested that TK activation in the rabbit myocardium occurs during the prolonged ischemic period after IPC is conferred.

The few studies that have investigated the role of TK in IPC have utilized the TK inhibitor genistein. Genistein, originally thought to be selective for TK (1), has been found to exhibit extensive nonselective effects. Genistein inhibits tyrosine phosphorylation via TK inhibition at relatively low concentrations; however, it has also been shown to minimally inhibit protein serine/threonine kinases, such as PKC, at higher concentrations (1). Genistein has also been shown to inhibit voltage-sensitive sodium channels (17) and adenosine receptors (15). Because of the nonselective effects of genistein, lavendustin A (a more selective TK inhibitor) and daidzein (a structural analog of genistein), which lacks TK inhibitory activity (2) but shares several of the nonselective effects of genistein, were employed in our study.

Therefore, based on these previous results, the present study was designed to answer two major questions: 1) Does TK act during the initiation of IPC or the maintenance of IPC; and 2) Is genistein acting via TK inhibition to attenuate IPC? Our data suggest that TK is partially involved in the initiation of classic IPC in the intact rat heart and that genistein attenuates IPC via inhibition of TK.

	METHODS

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This study was performed in accordance with the guidelines of the Animal Care Committee of the Medical College of Wisconsin, which is accredited by the American Association for Accreditation of Laboratory Animal Care.

General surgical preparation. Male Wistar rats, weighing 350-450 g, were used for all phases of this study. The rats were anesthetized via intraperitoneal administration of inactin (100 mg/kg), a long-acting barbiturate. A tracheotomy was performed, and the trachea was intubated with a cannula connected to a rodent ventilator (model CIV-101, Columbus Instruments, Columbus, OH; or model 683, Harvard Apparatus, South Natick, MA). The rats were ventilated with room air supplemented with O₂ at 60-65 breaths/min. Atelectasis was prevented by maintaining a positive end-expiratory pressure of 5-10 mmH₂O. Arterial pH, PCO₂, and PO₂ were monitored at control, 15 min of occlusion, and 60 and 120 min of reperfusion by a blood gas system (AVL 995 pH/Blood Gas Analyzer) and maintained within a normal physiological range (7.35-7.45 pH; 25-40 mmHg PCO₂; and 80-110 mmHg PO₂) by adjusting the respiratory rate and/or tidal volume. Body temperature was maintained at 38°C with a heating pad, and bicarbonate was administered intravenously as needed to maintain arterial blood pH within normal physiological levels.

The right carotid artery was cannulated to measure mean arterial blood pressure (MABP) and heart rate (HR) via a Gould PE50 or Gould PE23 pressure transducer connected to a Grass (model 7) polygraph. The right jugular vein was cannulated for saline, bicarbonate, and drug infusion. A left thoracotomy was performed at the fifth intercostal space followed by a pericardiotomy and adjustment of the left atrial appendage to reveal the location of the left coronary artery. A ligature (6-0 prolene) was passed below the left descending vein and coronary artery from the area immediately below the left atrial appendage to the right portion of the left ventricle. The ends of the suture were threaded through a propylene tube to form a snare. The coronary artery was occluded by pulling the ends of the suture taut and clamping the snare with a hemostat onto the epicardial surface. Coronary artery occlusion was verified by epicardial cyanosis and subsequent decrease in MABP. Reperfusion of the heart was initiated via unclamping the hemostat and loosening the snare and was confirmed by visualizing an epicardial hyperemic response. HR and MABP were allowed to stabilize for 15 min before the following protocols were initiated.

Drugs. Inactin, a thiobutabarbital sodium salt, was purchased from Research Biochemical International (Natick, MA). 2,3,5-Triphenyltetrazolium chloride (TTC) was purchased from Sigma Chemical (St. Louis, MO). Genistein was purchased from Research Biochemicals. Daidzein and lavendustin A were purchased from BIOMOL (Biomolecules for Research, Plymouth Meeting, PA). Inactin was dissolved in distilled water. Genistein was dissolved in Alkamuls EL-620 (Rhone-Poulenc), 95% EtOH, and saline. Daidzein was dissolved in polyethylene glycol, 1 N NaOH, and Dulbecco's phosphate-buffered saline. Lavendustin A was dissolved in 95% EtOH and Dulbecco's phosphate-buffered saline.

Study groups and experimental protocols. Rats were randomly assigned to 1 of 11 groups (Fig. 1). All groups underwent a 30-min coronary artery occlusion and a 2-h reperfusion period after we administered the drug and/or IPC. Group A, constituting the control group, underwent a 30-min coronary artery occlusion and subsequent 2 h of reperfusion. Group B was subjected to IPC established via a 5-min coronary artery occlusion period and a 5-min reperfusion period repeated three times (IPC). Groups C-G were administered the nonselective TK inhibitor genistein (Gen). Group C was administered genistein (5 mg/kg) 30 min before a 30-min occlusion period and 2 h of reperfusion (Gen Con). Groups D (5 mg/kg) and E (10 mg/kg) were given genistein 30 min before IPC (Gen + IPC). Groups F and G were given genistein during the final 2 min of the first 5-min occlusion period (IPC + Gen-occ 1) or third 5-min occlusion period (IPC + Gen-occ 3) during IPC. Group H was given daidzein (Dzn, 5 mg/kg), an inactive structural analog of genistein, 30 min before a 30-min occlusion period and 2 h of reperfusion (Dzn Con). Group I was administered daidzein 30 min before IPC (Dzn + IPC). Groups J and K were administered the selective TK inhibitor lavendustin A (1.0 mg/kg). Group J was given lavendustin A 30 min before a 30-min occlusion and 2 h reperfusion period (Lav Con). Group K was subjected to lavendustin A treatment 30 min before IPC (Lav + IPC).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Protocol bar depicting experiments used to study effects of genistein (Gen), daidzein, and lavendustin A on ischemic preconditioning (IPC) in intact rat heart. All groups underwent a 30-min coronary artery occlusion and 2-h reperfusion period. Control (group A): 30-min occlusion and 2-h reperfusion; IPC (group B): 5-min occlusion period (I) followed by a 5-min reperfusion period (R) repeated 3 times; Drug Control (groups C, H, and J): genistein (5 mg/kg), daidzein (5 mg/kg), or lavendustin A (1 mg/kg), respectively, given 30 min before a 30-min occlusion and 2-h reperfusion period; Drug + IPC (groups D, E, I, and K): Gen (5 and 10 mg/kg), daidzein, or lavendustin A, respectively, administered 30 min before IPC; IPC + Gen, occlusion 1 (group F): Gen administered during first 5-min occlusion period; IPC + Gen, occlusion 3 (group G): Gen administered during final 5-min occlusion period.

Determination of infarct size. On completion of the above protocols, the coronary artery was reoccluded and the area at risk (AAR) was determined by negative staining. Patent blue dye was administered via the jugular vein to effectively stain the nonoccluded area of the left ventricle. The rat was euthanized with a 15% potassium chloride solution. The heart was excised, and the left ventricle was removed from the remaining tissue and subsequently cut into six thin cross-sectional pieces. This allowed for the delineation of the normal area, which stained blue, versus the AAR, which subsequently remained pink. The AAR was excised from the nonischemic area, and the tissues were placed in separate vials and incubated for 15 min with a 1% TTC stain in 100 mM phosphate buffer (pH = 7.4) at 37°C. TTC is an indicator of viable and nonviable tissue. TTC is reduced by dehydrogenase enzymes present in viable myocardium resulting in a formazan precipitate and inducing a deep red color in the viable tissue while the infarcted area remains gray (8). Tissues were stored in vials of 10% formaldehyde overnight, and the infarcted myocardium was dissected from the AAR under the illumination of a dissecting microscope (Cambridge Instruments). Infarct size (IS) and AAR were determined by gravimetry. IS was expressed as a percentage of the AAR (IS/AAR).

Exclusion criteria. We sucessfully completed the above protocols using 72 rats. We excluded rats from data analysis if they exhibited severe hypotension (<30 mmHg systolic blood pressure) or if we were unable to maintain adequate blood gas values within a normal physiological range due to metabolic acidosis.

Statistical analysis of data. All values are expressed as means ± SE. Analysis of variance with Bonferroni's test was used to determine whether any significant differences existed among groups for hemodynamics, IS, and AAR. Significant differences were determined at P < 0.05.

	RESULTS

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Hemodynamics. Table 1 summarizes HR, MABP, and rate-pressure product in all groups determined at baseline, 15 min postcoronary artery occlusion, and at 120 min of reperfusion. Blood pressures in the drug protocols were maintained at baseline values after either genistein, daidzein, or lavendustin A administration. No significant differences in baseline, 15 min postcoronary artery occlusion, or 2 h of reperfusion in hemodynamic parameters existed between groups.

IS and ARR. Table 2 summarizes the IS data for all groups. Left ventricular (LV) weight (g) did not differ significantly in all protocols vs. control or IPC. AAR and AAR expressed as a percentage of LV weight (AAR/LV) was not significantly different in the 11 groups.

Figures 2-4 show the IS as a percentage of the AAR (IS/AAR) for each group. The average IS/AAR in control rats was 56.3 ± 2.8%. IPC produced a marked reduction in IS/AAR (7.1 ± 2.0%) compared with control. Genistein, daidzein, and lavendustin A administered to nonpreconditioned rats had no significant effect on IS/AAR (52.3 ± 1.2%, 47.6 ± 6.5%, and 56.2 ± 3.0%, respectively); however, pretreatment with either genistein at 5 or 10 mg/kg or lavendustin A at 1 mg/kg significantly attenuated the cardioprotective effect of IPC (34.7 ± 2.2%, 33.5 ± 5.9%, and 30.1 ± 2.2%, respectively), although IS/AAR was still significantly reduced compared with the control group. The IS reduction produced by IPC was not affected by pretreatment with daidzein (7.9 ± 2.4%). Similarly, when genistein was administered after IPC had been conferred, either during the first or third occlusion period, it had no effect on IS/AAR (7.1 ± 2.0 vs. 6.4 ± 2.0% and 10.3 ± 2.9%, respectively).

View larger version (48K): [in this window] [in a new window]   Fig. 2.   Infarct size expressed as a percentage of area at risk (AAR) in rat hearts subjected to control and control + drug protocol. All groups were given Gen, daidzein (Dzn), or lavendustin A (Lav) 30 min before a 30-min occlusion and 2-h reperfusion period. Control: 30-min occlusion and 2-h reperfusion. Gen Con: genistein, a nonselective tyrosine kinase (TK) antagonist. Dzn Con: daidzein, an analog of genistein inactive in TK inhibitory activity. Lav Con: lavendustin A, a selective TK antagonist.

View larger version (38K): [in this window] [in a new window]   Fig. 3.   Graph demonstrating a role for TK in IPC. Infarct size expressed as a percentage of AAR in control rats and those subjected to IPC in absence and presence of TK inhibitors, genistein (Gen + IPC, 5 and 10 mg/kg) and lavendustin A (Lav + IPC), and inactive analog of genistein, daidzein (Dzn + IPC), given 30 min before initiation of IPC.

View larger version (36K): [in this window] [in a new window]   Fig. 4.   Graph showing that TK is involved in initiation of IPC. Infarct size expressed as a percentage of AAR in rats. All groups underwent a 30-min occlusion and 2-h reperfusion period; Gen was administered at a dose of 5 mg/kg. IPC was elicited via a 5-min occlusion and 5-min reperfusion period repeated 3 times either in absence of Gen (IPC) or before [IPC + Gen, during first (occ 1) or third occlusion (occ 3)] or after (Gen + IPC) Gen was administered.

	DISCUSSION

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In the present study we were able to demonstrate the involvement of a TK in the initiation of classic IPC. Second, our data suggest that genistein attenuates IPC in the intact rat heart via the inhibition of TK. The former question is of importance because it has been shown using genistein (5 mg/kg) that TK is involved in initiating IPC during the second window of protection (7), and it has been suggested that TK is also involved in early IPC (9); however, the point at which TK is involved in early IPC is controversial. The latter question is important to our study, because it has been determined by numerous investigators that genistein, originally thought to be selective for TK (1), has many unrelated targets. These targets include receptors (15), ion channels (17), and other kinases (2). Because most investigators who have examined the role of TK in IPC utilized only genistein in their studies, it was therefore important to determine whether genistein attenuated the cardioprotective effects of IPC via TK inhibition. In our study, we also utilized daidzein and lavendustin A. Lavendustin A, like genistein, is a TK inhibitor; however, it is structurally distinct and has been shown to be more selective than genistein as an inhibitor of TK. Daidzein, on the other hand, is a structural analog of genistein that lacks TK inhibitory activity (2).

Previous work in our laboratory has suggested the involvement of the opioid-₁ receptor (22), G_i/o proteins (20), and the K_ATP channel (6, 23) in IPC. The present data add another piece of the signaling pathway and suggest the involvement of a TK in the initiation of IPC. Currently, there is support for the involvement of TK in both early and late IPC. Imagawa et al. (7) showed that genistein, administered before the IPC stimulus, effectively blocked IPC 48 h after IPC was elicited during the second window of protection in the rabbit myocardium. Similarly, Kukreja and Qian (9) have presented preliminary data suggesting that genistein can abolish IPC in the intact rat heart, and they suggested that tyrosine phosphorylation is an important component of the signal transduction pathway leading to classic IPC. Maulik et al. (13) examined the signaling pathway in IPC in the isolated rat heart. Although they did not examine the effects of genistein on IS, they showed that genistein attenuated the increase in phospholipase D activity, which occurred in the preconditioned heart. They were also able to show that pretreatment with genistein attenuated the increase in both MAP kinase and MAPKAP kinase 2 activities, which occurred in the preconditioned heart. These observations led to the suggestion that TK acts proximal in the signal transduction pathway to phospholipase D and both MAP kinase and MAPKAP kinase 2. In our study, we utilized the intact rat heart model and demonstrated that genistein can also attenuate IPC, however, only when given before the IPC stimulus. In agreement with the results of Kukreja and Qian (9) and Maulik et al. (13), the present observation also suggests that TK is a proximal step in IPC rather than a more distal step in the signaling pathway. In contrast, recent work by Baines et al. (3) suggested that TK acts at a later step in classical IPC in the rabbit myocardium. The reason for the differences in data obtained by Baines et al. (3) in the rabbit heart compared with the present study and the data of Kukreja and Qian (9) and Maulik et al. (13) obtained in the isolated rat heart is most likely due to a species difference, because the same inhibitor genistein was used in all three studies, and we are unaware of any species differences concerning the inhibitory effects of genistein. Other factors such as differences in concentration and timing of genistein administration may also be involved; however, genistein at either 5 or 10 mg/kg attenuated IPC to values not significantly different from each other, and genistein given either during the first occlusion period or the last occlusion period did not affect IPC. Species differences have been found in IPC concerning the role of other effectors as well; therefore, a unique signaling pathway for IPC in the rabbit heart concerning TK is a distinct possibility.

The second aim of our study examined whether genistein attenuated IPC via inhibition of TK or via nonselective effects. Genistein was originally thought to be a selective TK inhibitor (1). This idea, however, has been refuted since genistein has been shown to exhibit extensive nonselective effects. Paillart et al. (17) found that genistein, at 250 µM, completely inhibited toxin-induced ²²Na⁺ influx through voltage-sensitive Na⁺ channels in cultured rat brain neurons. Similarly, daidzein, at 250 µM, was also able to block ²²Na⁺ uptake; however, lavendustin A, at 10 µM, had no significant effect on neurotoxin-induced ²²Na⁺ influx. We are not aware of studies concerning the inhibition of Na⁺ channels in the myocardium by genistein; however, if this were the case, genistein would not be expected to attenuate IPC, rather it might be expected to be cardioprotective. The observation that genistein, but not lavendustin A, inhibits Na⁺ channels in combination with our data suggest that even if genistein inhibits Na⁺ channels in the myocardium, this may not be of importance in IPC, because, in our study, similar attenuation of IS/AAR was shown when we compared genistein- and lavendustin A-treated hearts. The effects of genistein may also be attributed to inhibition of serine/threonine kinases such as PKC. This effect may be significant because there exists substantial evidence for the involvement of PKC in IPC (13, 24). Recent evidence points to the involvement of PKC- in IPC. Bogoyevitch and colleagues (4) suggested that PKC- is the major PKC isoform expressed in adult rat cardiac myocytes. Gray et al. (5) were able to abolish both the protective effects of hypoxic preconditioning and phorbol-mediated protection with the use of the PKC- selective antagonist V1-2 peptide. The involvement of PKC- in IPC was recently shown by Ping and colleagues (18), who demonstrated that both PKC- and -isozymes were translocated in IPC. However, Akiyama et al. (1) have shown that genistein exhibited inhibitory activity against the TKs EGF receptors pp60^v-src and pp110^gag-fes with half-maximal inhibitory concentration (IC₅₀) values of 22.2, 25.9, and 24.1 µM, respectively, but showed only a weak inhibitory effect against serine/threonine kinases such as cAMP-dependent protein kinase, phosphorylase kinase, and most importantly, PKC (2). We were unable to find any information concerning the specific inhibition of PKC- or PKA by genistein, and similarly, we were unable to find any publications examining the possible inhibition of PKC or PKA by daidzein. It was, however, reported that although genistein can inhibit protein kinases with an IC₅₀ of 0.7 µg/ml, daidzein exhibited an IC₅₀ > 100 µg/ml (2). Similar to genistein, it has been shown that lavendustin A inhibits the TK epidermal growth factor (EGF) receptor kinase with an IC₅₀ of 4.4 ng/ml but only weakly inhibits PKC and PKA with an IC₅₀ > 100 µg/ml (16). It has been shown, however, that lavendustin A can also inhibit phosphatidylinositol kinase with an IC₅₀ of 6.4 µg/ml (16). These data, in conjunction with the similar IS/AAR data obtained in rats pretreated with either genistein or lavendustin A, suggest that the effects of genistein on IPC cannot be attributed to inhibition of the serine/threonine kinase PKC.

On the other hand, recent results by Vahlhaus et al. (26) in pigs suggested that blockade of both TK and PKC is necessary to abolish IPC. On the basis of the present results which suggest that blockade of TK only partially attenuates IPC in the rat, it is also possible that inhibiting other kinases such as PKC may be necessary to completely abolish IPC in the intact rat heart. Future studies are necessary to address this interesting possibility.

It has been demonstrated that stimulation of the A₁-adenosine receptor can mimic IPC, and inhibition of this receptor can attenuate the cardioprotective effects of IPC in most species studied; however, the role of the adenosine receptor in IPC is still controversial in the rat model (11). It was suggested by Okajima et al. (15) that genistein, in thyroid cells, is a competitive antagonist for P₁-purinergic (adenosine) receptors, and they showed that genistein had a higher affinity for A₁-adenosine receptors compared with the EGF receptor TK. If this same phenomenon occurs in myocardial cells, it could be hypothesized that genistein was acting via A₁-adenosine receptor inhibition to attenuate IPC. However, it has been shown by Imagawa et al. (7) that genistein did not act as an A₁-adenosine receptor antagonist in the rabbit myocardium to block IPC. In control drug protocols, TK inhibition via genistein or lavendustin A had no significant effect on IS/AAR in the nonpreconditioned myocardium. Similarly, the inactive analog of genistein, daidzein, exhibited no effect in the nonpreconditioned myocardium. These data indicate that genistein, daidzein, and lavendustin A have no cardioprotective or negative effect in the nonpreconditioned rat heart. However, genistein or lavendustin A given before the initiation of IPC significantly attenuated the cardioprotective effects of IPC, and pretreatment with these structurally dissimilar chemicals produced a similar inhibition of IPC. Lavendustin A exhibited a dose-response relationship with respect to IPC attenuation as IS/AAR rose from 17.6 ± 3.0% (lavendustin A: 0.1 mg/kg, n = 5 rats, data not shown) to 30.1 ± 2.2% (lavendustin A: 1.0 mg/kg, Fig. 3). Conversely, daidzein was unable to alter IS/AAR in IPC-treated hearts. These data suggest that genistein most likely attenuates IPC via inhibition of a TK. These data also suggest that TK is involved in the initiation of IPC. An important finding consistent with this conclusion was that genistein administered after the first or third preconditioning cycle (groups F and G) was not able to significantly attenuate the cardioprotective effects of IPC compared with IPC controls.

In summary, the present results suggest that a TK is involved in the initiation of IPC in the intact rat heart, and genistein can attenuate the cardioprotective effects of IPC via inhibition of TK. Our conclusions that TK is inhibited by genistein during IPC and that TK is crucial in the initiation of IPC are based on the results of experiments in which both lavendustin A and genistein partially attenuated the cardioprotective effects of IPC and administration of genistein after IPC was conferred had no significant effect. Current studies did not examine the direct increase in TK activity in IPC; therefore, we cannot state unequivocally that the effects of IPC are due directly to a subsequent increase in TK activity. However, the involvement of TK in the initiation of IPC can be indirectly inferred. It will be important to further investigate the direct effects of IPC on TK activity in rat hearts to corroborate our results.