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Am J Physiol Heart Circ Physiol 281: H1066-H1074, 2001;
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Vol. 281, Issue 3, H1066-H1074, September 2001

Src tyrosine kinase is the trigger but not the mediator of ischemic preconditioning

Reiji Hattori1, Hajime Otani2, Takamichi Uchiyama2, Hiroji Imamura2, Jianhua Cui1, Nilanjana Maulik1, Gerald A. Cordis1, Li Zhu1, and Dipak K. Das1

1 Cardiovascular Division, Department of Surgery, University of Connecticut School of Medicine, Farmington, Connecticut 06030; and 2 Department of Thoracic and Cardiovascular Surgery, Kansai Medical University, Moriguchi City 570-8507, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The signal cascade that triggers and mediates ischemic preconditioning (IPC) remains unclear. The present study investigated the role of the Src family of tyrosine kinases in IPC. Isolated and buffer-perfused rat hearts underwent IPC with three cycles of 5-min ischemia and 5-min reperfusion, followed by 30-min ischemia and 120-min reperfusion. The Src tyrosine kinase family-selective inhibitor PP1 was administered between 45 and 30 min before ischemia (early PP1 treatment) or for 15 min before IPC [early PP1-preconditioning (PC) treatment]. PP1 was also administered for 5 min before the sustained ischemia (late PP1 treatment) or after IPC (late PP1-PC treatment). Src kinase was activated after 30 min of ischemia in both the membrane and cytosolic fractions. Src kinase was also activated by IPC but was attenuated after the sustained ischemia. Early and late PP1 treatment inhibited Src activation after the sustained ischemia and reduced infarct size. Early PP1-PC inhibited Src activation after IPC but not after the sustained ischemia and blocked cardioprotection afforded by IPC. Late PP1-PC treatment abrogated IPC-induced activation of Src and protein kinase C (PKC)-epsilon in the membrane but not in the cytosolic fraction. This treatment modality abrogated Src activation after the sustained ischemia and failed to block cardioprotection afforded by IPC. These results suggest that Src kinase activation mediates ischemic injury but triggers IPC in the position either upstream of or parallel to membrane-associated PKC-epsilon .

protein kinase C-epsilon ; PP1


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ISCHEMIC PRECONDITIONING (IPC) is a receptor-mediated process and is realized via signal transduction pathways. Several investigators have proposed the unifying hypothesis that activation of protein kinase C (PKC) represents a link between cell surface receptor activation and putative end-effector sarcolemmal or mitochondrial ATP-sensitive K+ (KATP) channels (25, 26, 29, 37, 44), although arguments against the role of PKC in mediating IPC have also been provided (9, 30). On the other hand, the possible involvement of protein tyrosine kinases in IPC was proposed for the first time by Maulik and colleagues (27, 28). It is now increasingly clear that protein tyrosine kinases play a crucial role in mediating IPC in some animal species. Protein tyrosine kinases may act in parallel to (17, 43, 45), downstream of (5, 33), or upstream of (18) PKC in eliciting IPC. However, the exact member of protein tyrosine kinase involved in IPC remains unclear.

Among the large number of tyrosine kinases, we focused on the Src family of tyrosine kinases as a candidate for the protein tyrosine kinase member responsible for triggering or mediating IPC. Src tyrosine kinase acts as membrane-attached molecular switch that links a variety of cues to crucial intracellular signaling pathways. Src tyrosine kinase has been implicated in mechanisms of cell survival and death, which are regulated by complex signal transduction processes (22, 38). Thus Src tyrosine kinase activation may represent a key element in mediation of myocardial injury or protection associated with ischemia and reperfusion. Myocardial ischemia represents a powerful stimulus for activation of the Src family of tyrosine kinases. It has been shown that hypoxia causes rapid activation of the Src family of tyrosine kinases in cultured rat cardiac myocytes (39).

Rapid activation of the Src family of tyrosine kinases after ischemia has also been documented in the isolated guinea pig heart (42). Because the Src family of tyrosine kinases are activated by stimulation of G protein-coupled receptors (12, 36), an increase in intracellular Ca2+ (15), oxidative stress (2), and enhanced nitric oxide synthesis (3), all of which can be elicited by IPC challenges (4, 7, 20, 35), we anticipated that IPC could function as a trigger for the activation of the Src family of tyrosine kinases.

The involvement of the Src family of tyrosine kinases and PKC-epsilon , an isoform known to play a crucial role in mediating IPC, in late PC has been investigated by Ping and colleagues (33) using the conscious rabbit model. Their study demonstrated that PKC-epsilon and the Src family of tyrosine kinases are activated after IPC, although the Src family of tyrosine kinases appears to be activated downstream of PKC-epsilon . However, the time course of activation of the Src family of tyrosine kinases during IPC, sustained ischemia, and reperfusion remains unknown in the previous studies. Therefore, we investigated the temporal relationship between Src kinase activity and myocardial injury or protection during IPC and sustained ischemia using a Src family-selective tyrosine kinase inhibitor, 4-amino-5-(4-methylphenyl)-7-(t-butyl)-pyrazolo-3,4-D-pyrimidine (PP1) (19). We also studied the position of PKC-epsilon relative to Src kinase in triggering IPC. The results of our study suggest that Src kinase activation mediates ischemic injury but triggers IPC at the position either upstream of or parallel to membrane-associated PKC-epsilon .


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental animals. Male Sprague-Dawley rats weighing 275-300 g were used in the present study. These animals received humane care and were quarantined in quiet quarters for at least 1 wk before the study. The rats were anesthetized with pentobarbital sodium (80 mg/kg ip injection). After intravenous administration of heparin (500 IU/kg), the chest was opened, and the heart was rapidly excised and mounted on a nonrecirculating Langendorff perfusion apparatus. The perfusion buffer used in this study consisted of a modified Krebs-Henseleit bicarbonate buffer (KHB) [containing (in mol/l) 118 NaCl, 4.7 KCl, 1.7 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, and 10 glucose], pH 7.4, aerated with a 95% O2-5% CO2 gas mixture and filtered through a 5-µm filter to remove any particle contaminants. The buffer was maintained at a constant temperature of 37°C and was gassed continuously for the entire duration of the experiment. The working mode of the heart was introduced by switching the flow to the left atrium from the aortic root with a constant preload of 17 cmH2O and an afterload of 100 cmH2O. After steady-state cardiac function was attained, baseline measurements of heart rate, left ventricular (LV) developed pressure (LVDP), the first derivative of LVDP (LV dP/dtmax), aortic flow, and coronary flow were performed. LV pressure was measured using a Gould P23XL pressure transducer (Gould Instrument System; Valley View, OH). The signal was amplified using a Gould 6600 series signal conditioner and monitored on a CORDAT II real-time data acquisition and analysis system (Triton Technologies; San Diego, CA). Heart rate, LVDP, and LV dP/dtmax were all derived and calculated from the continuously obtained pressure signal. Aortic flow was measured using a calibrated flowmeter (Gilmont Instruments; Barrington, IL), and coronary flow was measured by timed collection of the coronary effluent.

Experimental protocol. The hearts were randomly divided into four groups, with six hearts in each group (Fig. 1). In the control group, isolated hearts were perfused for 45 min with KHB buffer. The hearts of the early PP1 group received 5 µM PP1 (Alexis Biochemicals; San Diego, CA), which was dissolved in DMSO at a final concentration of 0.01%, for 15 min and then perfused without PP1 for another 30 min. The hearts of the late PP1 group received PP1 for 5 min just before 30 min of ischemia. In the PC group, the hearts were preperfused with KHB buffer for 15 min and subjected to three cycles of 5-min global ischemia and 5-min reperfusion. The early PP1-PC group received PP1 for 15 min, followed by IPC. The late PP1-PC group received PP1 for 5 min after IPC. All the hearts were then subjected to 30 min of global ischemia, followed by 120 min of reperfusion. The hearts were in the retrograde mode to allow postischemic recovery for the first 10 min of reperfusion and were switched thereafter to the antegrade working mode.


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  		Fig. 1.   Experimental protocol. Isolated rat hearts were randomly divided into four groups, with six hearts in each group. In the control group, hearts were perfused for 45 min with Krebs-Henseleit bicarbonate buffer. The hearts of the early 4-amino-5-(4-methylphenyl)-7-(t-butyl)-pyrazolo-3,4-D-pyramidine (PP1) group received 5 µM PP1 for 15 min and were then perfused without PP1 for another 30 min. The hearts of the late PP1 group received PP1 for 5 min just before 30 min of ischemia. In the ischemic preconditioning (PC) group, isolated hearts were preperfused for 15 min and subjected to three cycles of 5-min global ischemia (shaded bars) and 5-min reperfusion. The hearts of the early PP1-PC group received PP1 for 15 min, followed by PC. The hearts of the late PP1-PC group received PP1 for 5 min after PC. All the hearts were then subjected to 30 min of global ischemia, followed by 120 min of reperfusion. The hearts were in the retrograde mode to allow postischemic recovery for the first 10 min of reperfusion and were switched thereafter to the antegrade working mode.

Evaluation of infarct size. At the end of 120 min of reperfusion, the hearts were perfused with phosphate-buffered saline containing 1% triphenyltetrazolium chloride for 15 min at 37°C. The hearts were trimmed to remove the atrium and connective tissues, frozen at 20°C, and sliced transversely in a plane perpendicular to the apical-basal axis into ~0.5-mm-thick sections. The sections were blotted dry, mounted on microscope slides, and scanned with a Hewlett-Packard Scanjet 5P single-pass flat-bed scanner (Hewlett-Packard; Palo Alto, CA). With the use of NIH 1.61 Image processing software, each digitized image was subjected to equivalent degrees of background subtraction, brightness, and contrast enhancement for improved clarity and distinctness. The areas at risk (equivalent to total ventricular mass) as well as the infarct zones of each slice were traced, and the respective areas were calculated in terms of pixels. The infarct volume was calculated, and the sum of all slices was used to compute a ratio of percent infarct to total LV mass.

Tissue sample preparation. In a separate series of experiments for the measurement of Src kinase and PKC-epsilon activity, the heart was perfused according to the protocol described earlier. The ventricular tissue was excised at the time point of 45 min after baseline and frozen in liquid nitrogen. The frozen myocardial tissue samples were powdered under liquid nitrogen and homogenized in five volumes of buffer [containing 320 mmol/l sucrose, 10 mmol/l Tris · HCl (pH 7.5), 1 mmol/l EDTA, 1 mmol/l EGTA, 10 mmol/l benzamidine, 50 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 0.3% beta - mercaptoethanol] with a Polytron homogenizer at the maximum speed, three times for 15 s each. The homogenates were mixed with an equal volume of the same buffer and centrifuged at 1,000 g for 10 min, and the supernatant was then centrifuged at 100,000 g for 60 min. The 100,000-g supernatant was referred to as the cytosolic fraction. The 100,000-g pellet was extracted with the homogenization buffer supplemented with 1% Triton X-100 for 30 min. The solubilized membrane fraction was collected by centrifugation at 50,000 g for 60 min. Protein concentrations were determined by the method of Bradford (8) using an assay kit (Bio-Rad Laboratories; Yokohama, Japan).

Src kinase assay. Src kinase activity in the rat myocardial tissue was determined by substrate-specific phosphorylation assay (13) using an assay kit purchased from Upstate Biotechnology (Lake Placid, NY). Briefly, equal concentrations of tissue proteins (50 µg) were immunoprecipitated overnight with 5 µg of rabbit polyclonal anti-Src antibodies (Santa Cruz Biotechnology; Santa Cruz, CA) and 10 µl of protein A/G agarose beads (Santa Cruz Biotechnology). The Src kinase-specific activity was then determined by subjecting the immunoprecipitates to a phosphorylation assay. The immunoprecipitates were incubated with 10 µg substrate peptide (KVEKIGEGTYGVVYK) in reaction buffer (total volume of 40 µl) containing 10 µCi of [gamma -32P]ATP and with (in mmol/l) 16.9 MnCl2, 0.11 ATP, 4.5 MOPS (pH 7.2), 5.6 beta -glycerol phosphate, 1.1 EGTA, 0.23 sodium orthovanadate, and 0.23 dithiothreitol for 15 min at 30°C. The reaction was terminated by the addition of 20 µl of 40% trichloroacetic acid. The phosphorylated substrate was transferred to P81 phosphocellulose paper as described by the manufacturer. The P81 binding papers were washed three times in 0.75% phosphoric acid and once in acetone, and the radioactivity was measured using a beta -scintillation counter. The Src kinase-specific activity was calculated from the specific counts (total counts minus nonspecific counts). The nonspecific counts were determined by performing parallel assays in the absence of tissue immunoprecipitates. Each assay was performed twice, and the results were averaged. The data was expressed as a percentage of baseline Src kinase activity.

PKC-epsilon -selective phosphorylation activity assay. The phosphorylation activity of the epsilon -isoform of PKC was determined as previously described (33). Briefly, 50 µg of protein from either the cytosolic or membrane fraction were immunoprecipitated overnight with PKC-epsilon antibodies (Santa Cruz Biotechnology). The immunoprecipitates were then subjected to a phosphorylation assay using a PKC assay kit (Upstate Biotechnology). Purified PKC from the rat brain (Upstate Biotechnology) was used as a standard to calculate specific activities.

Immunoblotting and quantification of phosphorylated PKC-epsilon . Activation of PKC-epsilon was also evaluated by measuring the phosphorylated form of PKC-epsilon (phospho-PKC-epsilon ) (31). For this, equal concentrations of cytosolic and membrane proteins were subjected to SDS-PAGE with 7.5% polyacrylamide gels and then immunoblotted according to Yoshida et al. (46). The blots were blocked with 5% skim milk in buffer containing 150 mmol/l NaCl, 10 mmol/l Tris · HCl (pH 7.4), and 0.05% Tween 20 for at least 1 h and then incubated with 500-fold diluted antibodies against phospho-PKC-epsilon (Upstate Biotechnology) for 1 h at room temperature, and the immunoblot bands were visualized with the use of an enhanced chemiluminescence Western blotting detection kit. The amount of PKC-epsilon on the immunoblots was measured with a densitometric analysis using NIH 1.61 Image processing soft ware. Because the total amounts of protein transferred from each lane to the nitrocellulose membranes during blotting were rarely identical despite a careful attempt to achieve equal protein loading in all lanes of the gel, the phospho-PKC-epsilon signal was normalized to the corresponding Coomassie blue stain signal determined by densitometric analysis as described by Ping et al. (32).

Statistical analysis. All numerical data are expressed as means ± SE. Statistical analysis was performed by one-way ANOVA and Scheffé's multiple-comparison test. The differences were considered significant at a P value of <0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Src kinase activity. In the control group of hearts, Src kinase activity in the membrane fraction was increased by fivefold after 30 min of ischemia but decreased to a nearly baseline level by 15 min after reperfusion (Fig. 2). A similar but less prominent increase in Src kinase activity was observed in the cytosolic fraction after 30 min of ischemia. Whereas no significant inhibition of Src kinase activity just before 30 min of ischemia was noted by early PP1 treatment, late PP1 treatment significantly inhibited the baseline activity of Src kinase in the membrane and cytosolic fractions. However, early and late PP1 treatment inhibited the increase in Src kinase activity after 30 min of ischemia. Whereas IPC increased Src kinase activity in the membrane and cytosolic fractions by 3.7- and 2.5-fold, respectively, IPC significantly inhibited Src kinase activation in these fractions after 30 min of ischemia. Although early and late PP1-PC treatment significantly inhibited IPC-induced activation of Src kinase, only late PP1-PC treatment inhibited sustained ischemia-induced Src kinase activation.


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  		Fig. 2.   Src kinase activity. Frozen ventricular tissues were separated into membrane (A) and cytosolic fractions (B) as described in MATERIALS AND METHODS. Src kinase activity in each fraction was determined by substrate-specific phosphorylation assay. The data were expressed as a percentage of baseline Src kinase activity. R, reperfusion. Each symbol represents the mean ± SE of six experiments. *P < 0.05 and **P < 0.01 compared with control; #P < 0.05 and ##P < 0.01 compared with PC.

PKC-epsilon activity. The PKC-epsilon -selective phosphorylation activity in both the membrane and cytosolic fractions was significantly increased after IPC (Fig. 3). Although early PP1 treatment had no significant effect on PKC-epsilon activity just before the sustained ischemia, late PP1 treatment exerted a marked inhibition on basal PKC-epsilon activity in the membrane fraction. This inhibition by PP1 treatment was not observed in the cytosolic fraction. The IPC-induced activation of PKC-epsilon that occurred in the membrane but not in the cytosolic fraction was significantly inhibited by early PP1-PC treatment and was abrogated by late PP1-PC treatment.


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  		Fig. 3.   Protein kinase C (PKC)-epsilon -selective phosphorylation activity assay. PKC-epsilon -selective phosphorylation activity after ischemic PC in the membrane and cytosolic fractions was assessed as described in MATERIALS AND METHODS. Open bars, cytosolic fraction; solid bars, membrane fraction. Each bar represents the mean ± SE of six experiments. *P < 0.05 and **P < 0.01 compared with baseline (BL); dagger P < 0.05 and dagger dagger P < 0.01 compared with PC.

Phospho-PKC-epsilon Western blotting. Because phospho-PKC-epsilon recognizes the active form of PKC-epsilon (31), activation of PKC-epsilon in the membrane and cytosolic fractions after IPC was evaluated by Western blot analysis using phospho-PKC-epsilon antibodies (Fig. 4). The amount of phospho-PKC-epsilon was significantly increased in the membrane and cytosolic fractions after IPC. Early PP1 treatment resulted in a significant reduction in the amount of phospho-PKC-epsilon in the membrane but not in the cytosolic fraction. Moreover, late PP1 treatment abolished the increase in immunodetectable phospho-PKC-epsilon only in the membrane fraction. Similarly, the IPC-induced increase in membrane-associated phospho-PKC-epsilon was inhibited by early PP1-PC treatment and more profoundly by late PP1-PC treatment.


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  		Fig. 4.   Phosphorylated PKC-epsilon (P-PKC-epsilon ) immunoblotting. A: Western blot analysis for P-PKC-epsilon after ischemic PC in the membrane and cytosolic fractions. B: quantification of P-PKC-epsilon immunoblot by densitometry analysis. Open bars, cytosolic fraction; solid bars, membrane fraction. Each bar represents the mean ± SE of six experiments. *P < 0.05 and **P < 0.01 compared with control; dagger P < 0.05 and dagger dagger P < 0.01 compared with PC.

Functional recovery. There were no differences in baseline function among all groups (Table 1). As was expected, upon reperfusion, the values of heart rate, LVDP, LV dP/dtmax, and aortic flow were decreased in all groups of hearts compared with baseline values, whereas coronary flow remained little changed throughout reperfusion. The hearts treated with IPC showed significantly better recovery of heart rate, LVDP, LV dP/dtmax, and aortic flow during reperfusion compared with control hearts. Early PP1 treatment conferred significantly better recovery of LVDP, LV dP/dtmax, and aortic flow during reperfusion compared with the control. On the other hand, late PP1 exerted a modest improvement of cardiac function during the late reperfusion period. Although early PP1-PC treatment significantly attenuated the IPC-induced improvement of cardiac function, especially with respect to LV dP/dtmax and aortic flow during reperfusion, late PP1-PC treatment failed to block the IPC-induced improvement of cardiac function.

                              
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  		Table 1.   Effect of PP1 and ischemic PC on the recovery of cardiac function

Infarct size. The normalized infarct size, calculated as the percent infarct area divided by the total ventricular mass in the control heart, was 33.5 ± 1.7% (Fig. 5). Infarct size was significantly reduced by IPC (21.3 ± 1.7%) compared with the control. Early PP1 (24.0 ± 2.2%) and late PP1 treatment (23 ± 4.5%) were also capable of reducing infarct size. Interestingly, early PP1-PC treatment abrogated the infarct size-limiting effect of IPC (32.2 ± 2.1%), whereas late PP1-PC treatment failed to block the infarct size-limiting effect of IPC (16.0 ± 3.0%).


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  		Fig. 5.   Infarct size. At the end of 120 min of reperfusion, the heart was perfused with 1% triphenyltetrazolium chloride for 15 min at 37°C . The hearts were sliced, and the infarct volume was calculated with image processing soft ware as described in MATERIALS AND METHODS. Each bar represents the mean ± SE of six experiments. *P < 0.05 and **P < 0.01 compared with control; dagger P < 0.05 compared with PC.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The signal cascade involved in IPC remains a matter of debate. There is little doubt that IPC generates, by stimulating G protein-coupled receptors, the signal that is eventually transmitted to effector machinery such as sarcolemmal or mitochondrial KATP channels. Although PKC is known to play an essential role in this signaling pathway, accumulating evidence suggest that protein tyrosine kinases may also be involved in transmission of the signal generated by IPC (5, 17, 33, 43, 45). However, the specific member of tyrosine kinase responsible for triggering or mediating IPC has not been elucidated because of the lack of selective inhibitors against certain families of tyrosine kinases.

A recently discovered Src family-selective tyrosine kinase inhibitor, PP1 (19), has been employed as a powerful tool in investigating the molecular and cellular mechanisms of physiological and pathophysiological events associated with the activation of the Src family of tyrosine kinases. PP1 is known to inhibit the Src family of tyrosine kinase at submicromolar concentrations in vitro and low micromolar concentrations in intact cells without affecting other protein tyrosine kinase and receptor tyrosine kinase activities except for platelet-derived growth factor receptors (6). At least nine members of the Src family of tyrosine kinases (Fyn, Yrk, Fgr, Yes, Src, Lyn, Hck, Lck, and Blk) have been identified in various mammalian tissues (10, 41). Although each Src family member exhibits some specific functions, they are also functionally overlapping and compensatory. Such redundant functions of the Src family of tyrosine kinases in a biological system has led investigators to employ PP1 or its analog PP2 as the most practical means to clarify the role of the Src family of tyrosine kinases in physiological and pathophysiological events. Indeed, it has been shown that the inhibition of extracellular signal-regulated kinase activation by angiotensin II in vascular smooth muscle cells is only partial in c-Src knockout mice or using retroviral transduction of dominant-negative c-Src but is complete using PP1 treatment (21). With the use of this Src family-selective tyrosine kinase inhibitor, we examined the role of the Src family of tyrosine kinases in IPC as well as in ischemia and reperfusion injury.

The present study demonstrated that Src kinase activity was increased in both the membrane and cytosolic fractions after 30 min of ischemia. Src kinase was also activated by IPC, but the activity was attenuated after 30 min of ischemia in this group of hearts. Early and late PP1 treatment inhibited Src kinase activation after 30 min of ischemia. Although early PP1-PC treatment inhibited the activation of Src kinase induced by IPC, the activity was regained after 30 min of ischemia in this group of hearts. The mechanism of relatively rapid loss of the effect of PP1 on Src kinase activity in the heart treated with IPC is currently unknown but may be related to different intracellular kinetics of PP1 between the IPC-treated and nontreated hearts. Late PP1-PC treatment, on the other hand, inhibited IPC-induced and sustained ischemia-induced Src kinase activation. Functional studies and infarct size measurements have demonstrated that early PP1 treatment improves the recovery of cardiac function during reperfusion and reduces infarct size. Late PP1 treatment also exerted a beneficial effect on infarct size but modest improvement of cardiac function. Preconditioned hearts showed significantly better recovery of cardiac function during reperfusion and smaller infarct size compared with control hearts. However, early PP1-PC treatment inhibited the beneficial effect of IPC on the recovery of cardiac function and infarct size. In contrast, late PP1-PC treatment failed to block the functional protection and infarct size limitation afforded by IPC. The temporal correlation between Src tyrosine kinase activation after 30 min of ischemia and aggravation of myocardial injury tends to suggest that activation of Src kinase itself mediates myocardial injury during a sustained ischemia, but it also plays a crucial role in triggering IPC in the isolated and perfused rat heart.

Although the Src family of tyrosine kinases has been shown to be activated by hypoxia, ischemia, and PC challenges (33, 39, 42), a causative role of activation of the Src family of tyrosine kinases in eliciting IPC has not been documented until recently. Bolli et al. (7) hypothesized that the Src family of tyrosine kinases activated after IPC challenges could trigger delayed protection in the rabbit heart. The present study is the first to show that the activation of the Src family of tyrosine kinase could also be a trigger for early PC. Although activation of the Src family of tyrosine kinases during sustained ischemia is deleterious, it is not harmful when induced by a brief period of ischemia but is capable of generating signals that eventually confer myocardial protection, a concept consistent with the triggering role of activation of the Src family of tyrosine kinases in IPC. In this respect, activation of PKC-epsilon may also be a trigger for the myocardial protection afforded by IPC because late PP1-PC treatment failed to block the myocardial protection afforded by IPC despite the fact that this treatment modality abrogated IPC-induced activation of PKC-epsilon in the membrane fraction. Because PKC activation appears to be a common mediator of IPC in a wide variety of species (9, 40), the controversial issue as to whether PKC acts as a trigger, a mediator, or both remains to be addressed.

We investigated the position of Src kinase relative to PKC-epsilon by administering PP1 before and after IPC. We were able to show that late PP1 treatment abrogated baseline PKC-epsilon activity and that late PP1-PC treatment abrogated IPC-induced activation of PKC-epsilon in the membrane but not in the cytosolic fraction. The temporal relationship between PP1 inhibition of Src kinase and PKC-epsilon activities suggests that Src kinase is not downstream of PKC-epsilon but is either upstream of or parallel to PKC-epsilon in the membrane fraction, where intimate interaction of each of the kinases may occur. However, there is also a Src kinase-independent pathway in activating PKC-epsilon in the cytosolic fraction. Such a differential regulation of PKC-epsilon activity may provide a clue in resolving the complex issues regarding the role of tyrosine kinase-dependent and -independent signal cascades in mediating IPC.

Although activation of both Src kinase-dependent and -independent signal transduction pathways may be a necessary step to elicit optimal IPC with respect to myocardial protection in some species, it should be emphasized that these cascades may not act in a coordinated fashion in modifying ischemia and reperfusion injury in other species. Kitakaze and colleagues (23) recently demonstrated that protein tyrosine kinases are not involved in the infarct size-limiting effect of early PC in the canine heart. Such a conflicting result may be related to the species difference in the activity of the Src family of tyrosine kinases, because no members of the Src family of tyrosine kinases except for Lck were activated by IPC in the canine heart.

The mechanism underlying myocardial injury induced by activation of the Src family of tyrosine kinase during ischemia remains elusive because of quite complicated nature of Src family tyrosine kinase-activated signaling pathways. It is well known that the Src family of tyrosine kinases forms a scaffold for the binding of three important signal transduction elements, i.e., phospholipase C-gamma , phosphatidylinositol-3-kinase, and the guanine-nucleotide exchange factor Grb2/Sos (34). Phospholipase C-gamma activation appears to be an essential step for PKC activation after stimulation of cytokine and growth factor receptors in a wide variety of cell types (11) and may also play a role in IPC-induced PKC activation in the heart, because phospholipase C-gamma is the most abundant phospholipase C isoform in the heart (14). Phosphatidylinositol-3-kinase, on the other hand, activates the serine/threonine protein kinase Akt/PKB, which in turn modulates the function of the death regulatory Bcl-2 family of proteins (16). Finally, Grb/Sos activation results in initiation of a mitogen-activated protein (MAP) kinase cascade by sequential phosphorylation and activation of the protooncogenes Ras and Raf (24). The MAP kinase family has been implicated in myocardial ischemia and reperfusion injury (1). Despite a tremendous research effort in this field, the exact role of those signaling systems in myocardial injury and protection remains obscure. Clarification of Src family tyrosine kinase-activated signal cascades and their physiological and pathophysiological roles in the heart await further investigation.

In conclusion, the present study suggests that activation of the Src family of tyrosine kinases mediates myocardial injury during ischemia but triggers IPC at the position either upstream of or parallel to membrane-associated PKC. The exact role of Src kinase-dependent and -independent activation of PKC in the signaling cascade provoked by IPC remains to be answered.


    ACKNOWLEDGEMENTS

We are grateful for technical assistance by Rie Yasuda.


    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grants HL-34360, HL-22559, and HL-33889 (to D. K. Das) and HL-56803 (to N. Maulik) and by Ministry of Education, Science, and Culture of Japan Research Grant 10671275 (to H. Otani).

Address for reprint requests and other correspondence: H. Otani, Thoracic and Cardiovascular Surgery, Kansai Medical Univ., 10-15 Fumizono-cho, Moriguchi City 570-8507, Japan (E-mail: otanih{at}takii.kmu.ac.jp).

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.

Received 11 December 2000; accepted in final form 4 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 281(3):H1066-H1074
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