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(Circulation Research. 2000;87:303.)
© 2000 American Heart Association, Inc.

Integrative Physiology

Protein Tyrosine Kinase Is Not Involved in the Infarct Size–Limiting Effect of Ischemic Preconditioning in Canine Hearts

Masafumi Kitakaze, Koichi Node, Hiroshi Asanuma, Seiji Takashima, Yasuhiko Sakata, Masanori Asakura, Shoji Sanada, Yoshiro Shinozaki, Hidezo Mori, Tsunehiko Kuzuya, Masatsugu Hori

From the Department of Internal Medicine and Therapeutics (M.K., K.M., H.A., S.T., Y. Sakata, M.A., S.S., T.K., M.H.), Osaka University Graduate School of Medicine, Suita, and Department of Physiology (Y. Shinozaki, H.M.), Tokai University School of Medicine, Isehara, Japan.

Correspondence to Masafumi Kitakaze, MD, PhD, Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita City, Osaka Pref 565-0871, Japan. E-mail kitakaze{at}medone.med.osaka-u.ac.jp

	Abstract

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Abstract—Protein kinase C (PKC) plays an important role in ischemic preconditioning (IP). Because (1) tyrosine kinase is located at the downstream of PKC for IP in the rabbit hearts and (2) we have reported that ecto–5'-nucleotidase is the substrate for PKC and plays a crucial role for the infarct size–limiting effect, we tested whether tyrosine kinase activation contributes to either activation of ecto–5'-nucleotidase or the infarct size–limiting effect of the early phase of IP in the canine heart. In dogs, the IP procedure (4 cycles of 5-minute occlusion of coronary artery) and exposure to 12,13-phorbol myristate acetate (PMA) each activated myocardial ecto–5'-nucleotidase and Lck tyrosine kinase. Genistein (10, 30, and 100 µg · kg^-1 · min^-1 IC), an inhibitor of tyrosine kinase, attenuated the activation of Lck tyrosine kinase but did not attenuate the activation of ecto–5'-nucleotidase due to either IP or PMA. In the other canine hearts, IP attenuated infarct size (49±5 versus 11±3 or 16±3%, P<0.01) due to 90 minutes of coronary occlusion followed by 6 hours of reperfusion, which was not blunted by 3 or 2 (30 and 100 µg · kg^-1 · min^-1) doses of genistein (infarct sizes, 15±4, 13±4, and 13±3%, respectively, and 17±3 and 15±4%, respectively) or lavendustin A. Tyrosine kinase does not activate ecto–5'-nucleotidase or trigger the infarct size–limiting effect of the early phase of IP in canine hearts.

Key Words: ischemic preconditioning • protein kinase C • genistein • lavendustin A

	Introduction

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Brief periods of ischemia that precede sustained ischemia limit infarct size markedly, a phenomenon known as ischemic preconditioning (IP).^{1 2 3} The mechanisms underlying this phenomenon have been studied extensively,^{4 5 6 7 8} and several lines of evidence support the idea that activation of protein kinase C plays an essential role in IP.^{9 10 11 12} We have previously reported that IP increases ecto–5'-nucleotidase activity,^{13 14} which seems to be consistent with the results of Liu et al,⁶ because adenosine production in the ischemic myocardium is attributable to ecto–5'-nucleotidase.^{15 16} The adenosine production via ecto–5'-nucleotidase may enhance the triggering mechanisms of IP or may contribute as an intermediate mediator to activate the final mediators such as the opening of ATP-sensitive K⁺ (K_ATP) channels. Interestingly, protein tyrosine kinase is reported to be located downstream of activation of protein kinase C in the rat and rabbit hearts.^{17 18 19 20 21 22} Indeed, protein kinase C activation phosphorylates and activates protein tyrosine kinase.^{21 22 23} Interestingly, Ping et al²⁴ reported that myocardial Lck and Src tyrosine kinases are phosphorylated and activated 5 and 30 minutes after the IP procedure, which may play an important role for the cardioprotection. However, it is not clarified what subtypes of Src protein tyrosine kinases are activated and whether the activated tyrosine kinase is involved in the infarct size–limiting effect of IP in canine hearts.

To clarify the role of Src tyrosine kinases and the linkage between tyrosine kinase and protein kinase C, we measured myocardial tyrosine kinase activity and infarct size in the control and preconditioned myocardium with or without administration of several doses of genistein or lavendustin A, each of which is an inhibitor of tyrosine kinase.

	Materials and Methods

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The procedure of the operation on the mongrel dogs (12 to 25 kg) for the experiments was described previously.^{9 10 11} All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH publication No. 85-23, revised 1985).

Experimental Protocols
Protocol I: Effect of Either Genistein or Lavendustin A on IP
Both coronary perfusion pressure and blood flow (CBF) were measured. Four cycles of 5 minutes of occlusion of the left anterior descending (LAD) coronary artery and a subsequent 5 minutes of reperfusion were performed (n=7; IP group). As a control, after 45 minutes of hemodynamic stabilization, the LAD artery was occluded for 90 minutes and reperfused for 6 hours (n=7; control group).

In 31 dogs, either genistein (10, 30, and 100 µg · kg^-1 · min^-1 [n=7 each]) or lavendustin A (2 and 20 µg · kg^-1 · min^-1 [n=5 each]) was administered into the LAD artery 5 minutes before and during IP. Genistein was diluted with saline, and lavendustin A was dissolved in 0.1% DMSO. Neither saline nor DMSO affected infarct size in the preliminary study. In 12 dogs, genistein (100 µg · kg^-1 · min^-1, n=7) or lavendustin A (20 µg · kg^-1 · min^-1, n=5) was infused into the LAD artery for 45 minutes before ischemia without IP.

Protocol II: Effect of Either Genistein or Lavendustin A on Pharmacological Preconditioning
Four cycles of administration of 12,13-phorbol myristate acetate (PMA, 0.5 pg · kg^-1 · min^-1) for 5 minutes with 5-minute intervals (n=7; PMA group) were performed.¹¹ PMA was dissolved with 0.1% DMSO.

In 19 dogs, either genistein (30 and 100 µg · kg^-1 · min^-1 IC [n=7 each]) or lavendustin A (20 µg · kg^-1 · min^-1 IC [n=5 each]) was administered 5 minutes before and during PMA administration.

Protocol III: Activities of Myocardial Tyrosine Kinase and Ecto–5'-Nucleotidase
With (n=10) and without (n=10) IP, or with (n=10) and without (n=10) PMA preconditioning, we measured tyrosine kinase and 5'-nucleotidase activities of the endocardial myocardium. In each condition, 5 dogs received 30 µg · kg^-1 · min^-1 of genistein, and the remaining 5 dogs received saline.

Measurements of the Physiological and Biochemical Parameters
We measured infarct size, risk area,^{9 10 11} collateral flow,²⁵ and endocardial ecto–5'-nucleotidase activity,^{9 10 11 12 13 14} as described previously. Tyrosine kinase activity was assessed by the enzymatic assay technique^{26 27} using the AUSA tyrosine kinase assay kit. We also performed the kinase-specific activity assays. Five Src tyrosine kinases (Src, Fyn, Lyn, Lck, and Blk) were expressed using reverse transcriptase–polymerase chain reaction, and the expression of 4 in 5 tyrosine kinases was detected by immunoblotting in the canine myocardium (Figure 1).

View larger version (33K): [in this window] [in a new window]   Figure 1. Immunoblots performed to identify the expression of Src tyrosine kinases in canine heart. Either canine placenta (P) or leukocyte (L) tissue was used as positive control for tyrosine kinase in canine heart (H).

The phosphorylation activity of all 4 members of the Src family was determined by immunoprecipitation followed by substrate-specific phosphorylation assay.

Statistical Analysis
Statistical analyses were performed using paired and unpaired t tests^{28 29} and were adjusted by a modified Bonferroni method. Analysis of covariance was used to account for the effect of collateral blood flow on infarct size. Each value was expressed as mean±SEM, with P<0.05 considered significant.

An expanded Materials and Methods section is available online at http://www.circresaha.org.

	Results

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Effects of Either Genistein or Lavendustin A in the Infarct Size–Limiting Effect of IP or PMA Preconditioning
Systolic (138 mm Hg) and diastolic (82 mm Hg) blood pressures and heart rate (139 bpm) before, during, and after 90 minutes of myocardial ischemia were not significantly changed either among the groups or throughout the protocol. In the IP group, coronary hyperemic flow during reperfusion after brief periods of ischemia was observed (89±2 to 298±13 mL/100 g · min^–1, P<0.001). Infusions of PMA slightly decreased CBF (90±2 to 81±3 mL/100 g · min^–1, P<0.005). Neither genistein nor lavendustin A during the IP procedure changed the extent of reactive hyperemic flow during reperfusion in the IP groups or the attenuation of CBF in the PMA groups.

The risk area (44%) and collateral flow (7.6 mL/100 g · min^–1) were comparable in all of the groups. Figures 2 and 3 show infarct size in these groups. IP markedly attenuated infarct size. No dose of genistein or lavendustin A abolished the infarct size–limiting effect of IP at every level of collateral flow. Figure 4 shows infarct size, and Figure 5 illustrates the regression plots of infarct size as a percentage of the area at risk against collateral flow in the PMA and control groups with and without either genistein or lavendustin A administration. Transient exposures of PMA markedly attenuated infarct size. No dose of genistein or lavendustin A abolished the infarct size–limiting effect of PMA preconditioning.

View larger version (22K): [in this window] [in a new window]   Figure 2. Infarct size in the following groups: control (n=7), IP (n=7), IP with 10 µg · kg-1 · min-1 IC genistein (n=7; IP+10Gen), IP with 30 µg · kg-1 · min-1 IC genistein (n=7; IP+30Gen), IP with 100 µg · kg-1 · min-1 IC genistein (n=7; IP+100Gen), IP with 2 µg · kg-1 · min-1 IC lavendustin A (n=5; IP+2Lav), IP with 20 µg · kg-1 · min-1 IC lavendustin A (n=5; IP+20Lav), 100 µg · kg-1 · min-1 IC genistein (n=7; 100Gen), and 20 µg · kg-1 · min-1 IC lavendustin A (n=5; 20Lav).

View larger version (30K): [in this window] [in a new window]   Figure 3. Plot of infarct size expressed as percentage of risk area and regional collateral flow during ischemia. In all groups, there are inverse relations between normalized infarct area and collateral flow. Infarct size in preconditioned hearts was smaller (P<0.001) with respect to any given value of collateral blood flow than that in control hearts. Infarct size was not affected in the genistein or the lavendustin A with IP group compared with the IP group. Abbreviations are as in Figure 2.

View larger version (19K): [in this window] [in a new window]   Figure 4. Infarct size in the following groups: PMA (n=7), PMA with 30 µg · kg-1 · min-1 IC genistein (n=7; PMA+30Gen), PMA with 100 µg · kg-1 · min-1 IC genistein (n=7; PMA+100Gen), and PMA with 20 µg · kg-1 · min-1 IC lavendustin A (n=5; PMA+20Lav). Infarct size was markedly decreased in the PMA group, and this effect was not blunted by either genistein or lavendustin A. Data from the control and IP groups, the 100 µg · kg-1 · min-1 IC genistein group (n=7, 100Gen group), and the 20 µg · kg-1 · min-1 IC lavendustin A group (n=5, the 20Lav group) were the same as in Figure 2, and we did not test the differences between these 4 groups and the data from protocol II.

View larger version (30K): [in this window] [in a new window]   Figure 5. Plot of infarct size expressed as percentage of the risk area and regional collateral flow during ischemia. In all groups, there were inverse relations between normalized infarct area and collateral flow. Infarct size in PMA-treated hearts was smaller (P<0.001) with respect to any given value in collateral blood flow than that in control hearts. Infarct size was not affected in the 3 doses of genistein with PMA groups compared with the PMA group. Data from the control and IP groups, the 100 µg · kg-1 · min-1 IC genistein group (n=7; 100Gen), and the 20 µg · kg-1 · min-1 IC lavendustin A group (n=5; 20Lav) were the same as in Figure 3, and we did not test the differences between these 4 groups and the data from protocol II. Abbreviations are as in Figures 2 and 4.

Changes in Myocardial Tyrosine Kinase and Ecto–5'-Nucleotidase Activities During IP With and Without Genistein
IP increased myocardial tyrosine kinase activity (71.8±6.2 to 143±19 pmol/mg protein · min^–1, P<0.05), which was blunted by genistein (33.6±6.7 to 30.6±8.6 pmol/mg protein · min^–1 in the genistein group and the genistein with IP group). PMA preconditioning increased myocardial tyrosine kinase activity (129±14 pmol/mg protein · min^–1, P<0.05), which was blunted by genistein (33.6±6.7 to 37.8.±5.9 pmol/mg protein · min^–1 in the genistein group and the genistein with PMA group).

Figure 1 shows the immunoblots of each kinase after immunoprecipitation using each antibody. The protein expression of 4 of 5 kinases (Src, Fyn, Lyn, and Lck) was confirmed in the particulate fraction of the canine heart. The expression of Blk was confirmed by reverse transcriptase–polymerase chain reaction using dog-specific primers; however, we were unable to detect the expression of Blk using available antibodies. Among these 4 tyrosine kinases, either IP or PMA increased Lck activity (Table). Activation of Lck after IP or PMA exposures was blunted by genistein. However, either IP or PMA preconditioning increased ecto–5'-nucleotidase activity in the myocardium (Figures 6 and 7). Genistein did not blunt the increases in ecto–5'-nucleotidase activity due to either ischemic or PMA preconditioning (Figures 6 and 7).

View this table: [in this window] [in a new window]   Table 1. The Src Tyrosine Kinase-Specific Activity (pmol · mg protein-1 · min-1) in the Particulate and Cytosolic Fractions of Nonpreconditioned and Reconditioned or PMA-Treated Myocardium

View larger version (39K): [in this window] [in a new window]   Figure 6. Comparison of canine ecto–5'-nucleotidase activity after 45 minutes of steady-state observation (control group; n=5), IP (n=5), IP with 3 doses of genistein (n=5 each), and IP with genistein (n=5).

View larger version (43K): [in this window] [in a new window]   Figure 7. Comparison of canine ecto–5'-nucleotidase activity after exposure to PMA (n=5), PMA with 3 doses of genistein (n=5 each), and genistein (n=5). PMA increased ecto–5'-nucleotidase activity, which was not blunted by genistein. Data from the control and IP groups were the same as in Figure 6.

	Discussion

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Infarct Size–Limiting Effect of IP: Role of Tyrosine Kinase
In the present study, we showed that neither genistein nor lavendustin A blunts the infarct size–limiting effect of IP and that genistein does not blunt the activation of ecto–5'-nucleotidase in the canine hearts. This result may contradict the previous studies showing that either genistein or another inhibitor of protein tyrosine kinase blunts the infarct size–limiting effect of IP in the rat or rabbit hearts.^{17 18 19 20 21}

There may be several possibilities to explain this disparity. The first possibility is the differences in the activation of Src tyrosine kinases in the dogs and rabbit hearts. IP phosphorylates and activates Src and Lck tyrosine kinases in the rabbit hearts,²⁴ but IP phosphorylates and activates Lck tyrosine kinases in the canine hearts in the present study, which may explain the differences. However, Src activation occurs 30 minutes after the IP procedure in the rabbit hearts,²⁴ suggesting that the activation of Src may not account for the cardioprotection of the early phase of IP. Therefore, Lck activation soon after the IP procedure is common in the rabbit and dog, and the differences in the role of tyrosine kinases in the previous^{17 18 19 20 21} and present studies may not be attributable to different activation of the subtypes of Src tyrosine kinases. The second idea is that there are several pathways to cause the cardioprotection due to IP, and the pathway to elicit cardioprotection may be different among the cycles of the IP procedure, the length of the sustained ischemia, and species. In rats, cardioprotection due to a single cycle of IP requires the activation of both protein kinase C and tyrosine kinase, but multiple-cycle IP requires the activation of either protein kinase C or tyrosine kinase.³⁰ On the other hand, in the pig hearts, synergistic activation of protein kinase C and tyrosine kinase is necessary to provoke full cardioprotection, proposed by the group of Vahlhaus et al,³¹ and the ratio of the role of these enzymes may be different among the species. To fully activate the end effector(s), different sites of phosphorylations of proteins or enzymes may be necessary via protein kinase C and tyrosine kinase. Furthermore, if K_ATP channels constitute the end effector of IP, the dual control of the activation of K_ATP channels via protein kinase C and tyrosine kinase may also be likely. This is because sarcolemmal K_ATP channels are activated by adenosine receptor activation³² or protein kinase C,³³ and mitochondrial K_ATP channels are activated by protein kinase C.³⁴

Limitations of the Present Study
We need to be careful of the doses and characteristics of the inhibitors of tyrosine kinase. We used 10 to 100 µg · kg^-1 · min^-1 of genistein, which corresponds to 10 to 100 mg/L or 37 to 370 µmol/L. Because the K_i value of genistein for deactivation of protein tyrosine kinase is 10 to 20 µmol/L,³⁵ the dose of 30 µg · kg^-1 · min^-1 of genistein seems to be enough to inhibit tyrosine kinase. We observed that genistein attenuates the activation of protein tyrosine kinase. Furthermore, 2 and 20 µg · kg^-1 · min^-1 of lavendustin A, which corresponds to 2 and 20 mg/L or 5 to 50 µmol/L, also seems to be enough to inhibit Src tyrosine kinases. The failure of the attenuation of the infarct size–limiting effect of IP using these 2 different tyrosine kinase inhibitors further supports the conclusion of this study.

Because we infused genistein or lavendustin A during the IP procedure into the coronary artery, genistein or lavendustin A was supposed to be washed out and should not have existed during the sustained ischemia and subsequent reperfusion. However, an effective amount of either genistein or lavendustin A infused into the coronary artery was spilled over and was diluted in the systemic blood, which may have affected the cardioprotection of IP. However, the recirculated amount of either genistein or lavendustin A may be less than the effective dose of genistein administered into the systemic vein. Even if either genistein or lavendustin A exists during sustained ischemia and reperfusion, the cardioprotection caused by IP is not blunted by the administration of either genistein or lavendustin A in the present study, suggesting the minimal role of tyrosine kinase as a trigger or even a mediator of IP in the canine hearts. In the rat or rabbit, genistein was infused into the systemic vein, and genistein may largely affect infarct size due to sustained ischemia and reperfusion. This difference may explain the different results using the inhibitors of tyrosine kinase in cardioprotection due to IP. We cannot deny this possibility. However, in the rabbit heart, Weinbrenner et al¹⁸ infused genistein into the coronary artery during the IP procedure in the Langendorff preparation and found that genistein inhibits the infarct size–limiting effect, suggesting that activation of protein tyrosine kinase can trigger the infarct size–limiting effect of IP in the rabbit hearts.

There is a possibility that the activation of Lck tyrosine kinase is involved in the late phase of IP.³⁶ Indeed, it is reported that tyrosine kinase activation is involved in the development of the late phase of IP in the rabbits. We need to test this issue in canine hearts.

Cellular Mechanisms of the Infarct Size–Limiting Effect of IP in Canine Hearts
If protein tyrosine kinase is not involved in the infarct size–limiting effect of IP, what is the trigger for IP in the canine hearts? In the present and previous studies, we have observed that IP translocates protein kinase C from cytosolic fractions to membrane fractions, indicating that IP activates protein kinase C in the canine myocardium.¹¹ There are several reports of the activation of the isoforms of protein kinase C,^{37 38} suggesting that total activity of protein kinase C may not be necessarily increased.

There are several ways to activate protein kinase C during IP. First, because adenosine is reported to trigger the infarct size–limiting effect of IP in the rabbit heart, adenosine may activate protein kinase C through G proteins. Second, in the rabbit heart, Goto et al³⁹ indicated that bradykinin is involved in the infarct size–limiting effect of IP through activation of protein kinase C. Furthermore, Schulz et al⁴⁰ reported that the combination of adenosine and bradykinin is important to cause IP by 10 minutes of ischemia in the swine. The third possibility is endogenous norepinephrine. We have previously reported that endogenous norepinephrine during the IP procedure is responsible for activation of ecto–5'-nucleotidase and mediates the infarct size–limiting effect.⁹ These observations indicate that endogenous norepinephrine⁴¹ can also trigger the infarct size–limiting effect of IP in the canine heart.

Taken together, the involvement of either protein tyrosine kinase or protein kinase C in IP may be important for cardioprotection, but its importance may vary among species.

	Acknowledgments

 
This work was supported by Grants-in-Aid for Scientific Research (12470153 and 12877107) from the Ministry of Education, Science, Sports and Culture of Japan and in part by grants from the Smoking Research Foundation in Japan.

Received April 3, 2000; revision received June 13, 2000; accepted June 22, 2000.

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