AJP - Heart AJP: Regulatory, Integrative and Comparative Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 289: H761-H767, 2005. First published April 1, 2005; doi:10.1152/ajpheart.00012.2005
0363-6135/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
289/2/H761    most recent
00012.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Okada, T.
Right arrow Articles by Imamura, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Okada, T.
Right arrow Articles by Imamura, H.

Integrated pharmacological preconditioning and memory of cardioprotection: role of protein kinase C and phosphatidylinositol 3-kinase

Takayuki Okada, Hajime Otani, Yue Wu, Takamichi Uchiyama, Shiori Kyoi, Reiji Hattori, Tomohiko Sumida, Hiroyoshi Fujiwara, and Hiroji Imamura

Cardiovascular Center, Kansai Medical University, Moriguchi City, Japan

Submitted 6 January 2005 ; accepted in final form 29 March 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although protein kinase C (PKC) and phosphatidylinositol 3 (PI3)-kinase are implicated in cardioprotective signal transduction mediated by ischemic preconditioning, their role in pharmacological preconditioning (PPC) has not been determined. Cultured neonatal rat cardiomyocytes (CMCs) were subjected to simulated ischemia for 2 h followed by 15 min of reoxygenation. PPC of CMCs consisted of administration of 50 µM adenosine, 50 µM diazoxide, and 50 µM S-nitroso-N-acetylpenicillamine (SNAP), each alone or in combination, for 15 min followed by 30 min of washout before simulated ischemia. Although PKC-{epsilon} and PI3-kinase were significantly activated during treatment with adenosine, activation of these kinases dissipated after washout. In contrast, PPC combined with adenosine, diazoxide, and SNAP elicited sustained activation of PKC-{epsilon} and PI-3 kinase after washout. The combined-PPC, but not the single-PPC, protocol conferred antiapoptotic and antinecrotic effects after reoxygenation. The PKC inhibitor chelerythrine (5 µM) or the PI3-kinase inhibitor LY-294002 (10 µM) given during the washout period partially blocked the activation of PKC-{epsilon} and PI3-kinase mediated by the combined-PPC protocol, whereas combined addition of chelerythrine and LY-294002 completely inhibited activation of PKC-{epsilon} and PI3-kinase. Chelerythrine or LY-294002 partially blocked antiapoptotic and antinecrotic effects mediated by the combined-PPC protocol, whereas combined addition of chelerythrine and LY-294002 completely abrogated antiapoptotic and antinecrotic effects. These results suggest that the combined-PPC protocol confers cardioprotective memory through sustained and interdependent activation of PKC and PI3-kinase.

adenosine; apoptosis

MANY YEARS AGO, Murry et al. (28) demonstrated that repeated brief periods of ischemia significantly reduced infarct size after a prolonged period of coronary artery occlusion. This phenomenon has been termed ischemic preconditioning (IPC). Now, it is apparent that IPC is a powerful endogenous tool for myocardial protection against ischemia-reperfusion injury. However, clinical applications of IPC have been limited by ethical concerns. Thus pharmacological preconditioning (PPC) has emerged as an ideal alternative to IPC. PPC employs putative triggers of IPC, such as G protein-coupled receptor agonists, mitochondrial ATP-dependent K+ (KATP) channel openers, and nitric oxide (NO) donors. However, the efficacy of early single-drug PPC is variable. Several investigators reported that single-agent PPC was as effective as IPC (10, 26, 29, 33, 39); however, other studies, including our own, showed only marginal cardioprotection by the single-PPC strategy and require a combined-PPC technique in which multiple agents are used to elicit optimal cardioprotection (7, 9, 43, 49). Such conflicting observations may be attributed to differences in species and experimental models but, more importantly, may be related to the strength and duration of cardioprotective signaling activity.

The unique feature of cardioprotection conferred by IPC is the cardioprotective memory, which lasts for up to 1 h after the discontinuation of the preconditioning stimulus (50). Although the exact mechanism whereby IPC acquires the memory of cardioprotection has not been fully elucidated, we previously demonstrated that the memory of cardioprotection that mimics IPC cannot be achieved by simply increasing the dose of drugs that have been employed as an IPC mimetic, i.e., a G protein-coupled receptor agonist, a mitochondrial KATP channel opener, and an NO donor, but is successfully reproduced by combining these drugs (32). Because cardioprotective memory through combined PPC was thought to be mediated by coordinated interaction of cardioprotective signaling, we termed this technique the integrated PPC (43).

Cardioprotective signal transduction responsible for early IPC has been extensively investigated in the last decade. A growing body of evidence indicates that protein kinase C (PKC) and phosphatidylinositol 3 (PI3)-kinase play a crucial role in cardioprotective signal transduction mediated by IPC (3). Thus it is anticipated that PKC and PI3-kinase are also essential signaling elements in cardioprotection mediated by the integrated PPC. Accordingly, we have investigated the effect of single and combined PPC on cardioprotective memory by evaluating PKC and PI3-kinase activity during and after the PPC challenge and the relative contribution of these kinases to cardioprotection.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation and cultivation of neonatal rat cardiomyocytes. All experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (NIH Publ. No. 85-23, Revised 1996) and approved by Kansai Medical University Institutional Committee of Animal Care and Use. Neonatal rat cardiomyocytes (CMCs) were isolated and cultured as described previously (42). Briefly, neonatal rats were killed by decapitation, and their hearts were rapidly removed and placed in ice-cold Dulbecco's modified Eagle's medium (DMEM; GIBCO-BRL, Rockville, MD) containing penicillin G (300 U/ml), streptomycin (300 µg/ml), and amphotericin B (0.75 µg/ml; GIBCO-BRL). The ventricles were minced and incubated at 37°C for 10 min in Joklik's modified Eagle's medium (GIBCO-BRL) with 0.01% collagenase (Wako, Osaka, Japan). After incubation, minced pieces were stirred by pipette, and digested cell suspensions were transferred to DMEM with 10% FCS. Precipitate was removed and incubated in buffer with 0.01% collagenase at 37°C for 10 min. These procedures were repeated four to five times. The supernatants were centrifuged at 600 g and resuspended in DMEM with 10% FCS. Cells were plated for 30 min to reduce the number of contaminating non-CMCs. CMCs were pooled, counted, and incubated in DMEM with serum. CMCs (2 x 106 cells) were plated on a 60-mm culture dish and incubated for 48 h at 37°C under 5% CO2 in a tissue culture incubator.

Experimental protocols. Experimental protocols are shown in Fig. 1. CMCs were cultured overnight in Krebs-Henseleit bicarbonate (KHB) buffer solution containing (in mM) 118 NaCl, 4.7 KCl, 1.7 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, and 10 glucose, pH 7.4 with incubation in 5% CO2. CMCs were then treated with 50 µM adenosine (Sigma, Tokyo, Japan), 50 µM diazoxide (Sigma), and 50 µM S-nitroso-N-acetylpenicillamine (SNAP; Wako), each alone or in combination for 15 min; then these drugs were washed out for 30 min before 2 h of incubation in GasPak (Becton Dickinson) in modified KHB buffer solution in which 10 mM glucose was replaced by 2-deoxy-D-glucose. In some experiments where the dose-response effect on PKC and PI3-kinase activities was examined, the dose of adenosine, diazoxide, and SNAP was increased fourfold. The PKC inhibitor chelerythrine (5 µM; Sigma) and the PI3-kinase inhibitor LY-294002 (10 µM; Calbiochem) were administered during the washout period and simulated ischemia. Control CMCs were treated with the vehicle (0.05% dimethylsulfoxide) for 45 min before hypoxia and for 2 h during simulated ischemia. CMCs were reoxygenated for 15 min in the isosmotic KHB or the hyposmotic modified KHB buffer solution (140 mosM with reduced NaCl) to produce physical stress.



View larger version (24K):
[in this window]
[in a new window]
  		Fig. 1. Experimental protocol. Cultured neonatal rat cardiomyocytes (CMCs) were subjected to 2 h of simulated ischemia followed by reoxygenation (Reoxy) for 15 min in the presence or absence of osmotic stress. Time-matched control (Cont) CMCs were incubated under normoxic condition throughout the experiments. Pharmacological preconditioning (PPC) consisted of administration of adenosine (Ad), diazoxide (Dz), or S-nitroso-N-acetylpenicillamine (SNAP), each alone or in combination (CP), for 15 min followed by washout for 30 min before simulated ischemia. Chelerythrine (Che) or LY-294002 (LY), alone or in combination, was administered during washout and simulated ischemia. Hypo, hyposmotic.

 
PKC-{epsilon} activity assay. CMCs were treated with cell lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM {beta}-glycerophosphate, 1 mM Na3VO4, 1 mM PMSF, and a protease inhibitor cocktail (Complete, Roche Molecular Biochemicals, Manheim, Germany). The cell lysates were centrifuged at 37°C for 5 min at 14,000 g, and the supernatants were applied to the phosphorylation activity assay of PKC-{epsilon} as described previously (19).

Immunoblot assay for phosphorylated Akt. We measured phosphorylation of Akt as an index for PI3-kinase activity (37). The cell lysates obtained above were resolved on 7.5% SDS-polyacrylamide gels. Electroblotting procedures were used to transfer proteins to nitrocellulose membranes (Bio-Rad, Hercules, CA). Nitrocellulose membranes were blocked with a buffer containing 50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween 20, and 5% nonfat dry milk for 1 h at room temperature and incubated overnight at 4°C with the antibodies against rabbit polyclonal Akt or rabbit polyclonal phosphorylated Akt (Ser473) obtained from Cell Signaling (Beverly, MA). Immunolabeling was detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer's instructions and quantified with a densitometric analysis using the image analyzing software system Win Roof (Mitani, Fukui, Japan).

Caspase-3 activity assay. Caspase-3 activity in CMCs was evaluated by a colorimetric assay as described previously (42). Briefly, CMCs (2 x 106) were suspended in 50 µl of cold cell lysis buffer, incubated on ice for 10 min, centrifuged for 1 min at 10,000 g, diluted to 50 µg of protein, 50 µl of reaction buffer and 5 µl of the 4 mM DEVD-p-nitroanilide substrate (200 µM final concentration) were added, and the cells were incubated at 37°C for 1 h. p-Nitroanilide fluorescence was analyzed using a microtiter plate reader (Bio-Rad) at an emission wavelength of 405 nm.

DNA fragmentation assay. DNA fragmentation in CMCs was quantitatively measured with a Cell Death Detection ELISA kit (Roche) as described previously (42). Briefly, CMCs (2 x 106) were centrifuged for 10 min at 200 g. The supernatant of the medium was stored at 4°C. The pellet was then incubated in the cell lysis buffer for 30 min and centrifuged for 10 min at 200 g. The immunoreagent (biotin-labeled mouse monoclonal antibodies against histone and peroxidase-conjugated mouse monoclonal antibodies against DNA) was added to 20 µl of the cell lysate or the supernatant and incubated for 2 h. Then 2,2'-azino-di(3-ethylbenzthiazolinsulfonate) substrate was added, and the fluorescence was analyzed using a microtiter plate reader (Bio-Rad) at an emission wavelength of 405 nm.

Lactate dehydrogenase release. CMC necrosis was determined by lactate dehydrogenase (LDH) release in the culture medium. LDH was measured using an LDH assay kit (Sigma).

Statistical analysis. Values are means ± SE. Statistical analysis was performed by one-way ANOVA followed by Bonferroni's post hoc test. The differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Combined treatment with adenosine, diazoxide, and SNAP, but not each alone, provokes sustained activation of PKC-{epsilon} and PI3-kinase. Because PKC-{epsilon} and PI3-kinase have consistently been demonstrated as cardioprotective kinases against acute ischemia-reperfusion injury (15, 20, 24), we measured these protein kinase activities as an index for cardioprotective signal transduction. PKC-{epsilon} was significantly activated by treatment with adenosine, diazoxide, and SNAP, each alone (Fig. 2A). Similarly, Akt phosphorylation, measured to evaluate PI3-kinase activation, was significantly increased by treatment with adenosine (Fig. 2B). Treatment with diazoxide and SNAP also tended to increase Akt phosphorylation. PKC-{epsilon} activity and Akt phosphorylation were additively increased by combined treatment with adenosine, diazoxide, and SNAP. The activation of PKC-{epsilon} and the increase in Akt phosphorylation mediated by treatment with adenosine, diazoxide, and SNAP, each alone, dissipated 30 min after washout. In contrast, the combined addition of adenosine, diazoxide, and SNAP produced a sustained increase in PKC-{epsilon} activity and Akt phosphorylation after withdrawal of these drugs. Addition of chelerythrine or LY-294002 after withdrawal of adenosine, diazoxide, and SNAP partially inhibited the increase in PKC-{epsilon} activity and Akt phosphorylation, whereas combined addition of chelerythrine and LY-294002 completely inhibited the increase in PKC-{epsilon} activity and Akt phosphorylation mediated by combined PPC.



View larger version (22K):
[in this window]
[in a new window]
  		Fig. 2. Effect of single and combined addition of PPC drugs on PKC-{epsilon} and phosphatidylinositol 3 (PI3)-kinase activities. A: PKC-{epsilon} activity assay. B: immunoblot analysis of phosphorylated Akt (phospho-Akt, p-Akt) and total Akt. PKC-{epsilon} activity and phospho-Akt were measured 15 min after addition of 50 µM adenosine, 50 µM diazoxide, 50 µM SNAP, or CP and 30 min after washout of adenosine (–Ad), diazoxide (–Dz), SNAP (–SNAP), or combined drugs (–CP). Chelerythrine and LY-294002 were administered alone or in combination during washout. Values are means ± SE of 5 experiments. {dagger}P < 0.05; {dagger}{dagger}P < 0.01 vs. control. *P < 0.05; **P < 0.01 vs. –CP.

 
Although we used a conventional dose of adenosine, diazoxide, and SNAP in preconditioning studies, we employed a fourfold higher concentration of each drug to examine the possibility that the failure of the single-PPC protocol to sustain PKC-{epsilon} activation and Akt phosphorylation after washout was due to insufficient dose of the drugs. PKC-{epsilon} was significantly activated during incubation with 200 µM adenosine, 200 µM diazoxide, and 200 µM SNAP (Fig. 3A). Akt phosphorylation was significantly increased by incubation with 200 µM adenosine and 200 µM SNAP (Fig. 3B). However, PKC-{epsilon} activity and Akt phosphorylation were attenuated during washout of these drugs.



View larger version (19K):
[in this window]
[in a new window]
  		Fig. 3. Effect of high dose of a single PPC drug on PKC-{epsilon} and PI3-kinase activities. A: PKC-{epsilon} activity assay. B: immunoblot analysis of phospho-Akt and total Akt. PKC-{epsilon} activity and phospho-Akt were measured 15 min after addition of 200 µM adenosine, 200 µM diazoxide, or 200 µM SNAP and 30 min after washout of adenosine, diazoxide, or SNAP. {dagger}P < 0.05; {dagger}{dagger}P < 0.01 vs. control.

 
Combined treatment with adenosine, diazoxide, and SNAP, but not each alone, inhibits caspase-3 activity during reoxygenation in a PKC- and PI3-kinase-dependent manner. Caspase-3 activity was not significantly increased during hypoxia but was increased during reoxygenation (Fig. 4). The single-PPC technique with adenosine, diazoxide, and SNAP, each alone, did not inhibit caspase-3 activation during reoxygenation. In contrast, the combined-PPC technique significantly inhibited caspase-3 activation during reoxygenation. Chelerythrine and LY-294002 administered alone or in combination did not significantly increase caspase-3 activity during reoxygenation (not shown). However, combined-PPC-mediated inhibition of caspase-3 activation was partially blocked by chelerythrine or LY-294002 alone but was completely abrogated by combined addition of chelerythrine and LY-294002.



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 4. Caspase-3 activity assay. Caspase-3 activity was measured 2 h after normoxic incubation and simulated ischemia (SI) and 15 min after reoxygenation under isosmotic condition. PPC consisted of administration of adenosine, diazoxide, and SNAP, alone or in combination, and addition of chelerythrine and LY-294002, individually or in combination, as described in Fig. 1 legend. Values are means ± SE of 5 experiments. {dagger}P < 0.05; {dagger}{dagger}P < 0.01 vs. control. #P < 0.05; ##P < 0.01 vs. reoxygenation. *P < 0.05; **P < 0.01 vs. CP.

 
Combined treatment with adenosine, diazoxide, and SNAP, but not each alone, prevents DNA fragmentation during reoxygenation in a PKC- and PI3-kinase-dependent manner. DNA fragmentation was not significantly increased during hypoxia but was increased during reoxygenation (Fig. 5 ). PPC with adenosine, diazoxide, and SNAP individually did not inhibit DNA fragmentation during reoxygenation. In contrast, the combined-PPC protocol significantly inhibited DNA fragmentation during reoxygenation. Chelerythrine and LY-294002 administered alone or in combination did not significantly increase DNA fragmentation during reoxygenation (not shown). However, combined-PPC-mediated inhibition of DNA fragmentation was partially blocked by chelerythrine or LY-294002 alone but was completely abrogated by combined addition of chelerythrine and LY-294002.



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 5. DNA fragmentation assay. DNA fragmentation was measured 2 h after normoxic incubation and simulated ischemia and 15 min after reoxygenation under isosmotic condition. PPC consisted of administration of adenosine, diazoxide, and SNAP, each alone or in combination, and addition of chelerythrine and LY-294002, individually or in combination, as described in Fig. 1 legend. Values are means ± SE of 5 experiments. {dagger}{dagger}P < 0.01 vs. control. #P < 0.05; ##P < 0.01 vs. reoxygenation. *P < 0.05; **P < 0.01 vs. CP.

 
Combined treatment with adenosine, diazoxide, and SNAP, but not each alone, prevents LDH release during reoxygenation under the hyposmotic condition in a PKC- and PI3-kinase-dependent manner. No significant LDH release was observed during reoxygenation under the isosmotic condition (Fig. 6). Next, the hyposmotic buffer was employed to induce LDH release by imposing physical stress on the sarcolemmal membrane (4). Indeed, significant LDH release occurred during reoxygenation under the hyposmotic condition, which did not cause significant LDH release in normoxic CMCs. PPC with adenosine, diazoxide, and SNAP individually did not inhibit LDH release under the hyposmotic condition during reoxygenation. In contrast, combined PPC significantly inhibited LDH release under the hyposmotic condition during reoxygenation. Chelerythrine and LY-294002 administered alone or in combination did not significantly increase LDH release during reoxygenation under the hyposmotic condition (not shown). However, combined-PPC-mediated inhibition of LDH release was partially blocked by chelerythrine or LY-294002 but was completely abrogated by combined addition of chelerythrine and LY-294002.



View larger version (14K):
[in this window]
[in a new window]
  		Fig. 6. Lactate dehydrogenase (LDH) release. LDH release was measured 15 min after reoxygenation or time-matched normoxic incubation under isosmotic and hyposmotic conditions. PPC consisted of administration of adenosine, diazoxide, and SNAP, alone or in combination, and addition of chelerythrine and LY-294002, individually or in combination, as described in Fig. 1 legend. Values are means ± SE of 5 experiments. {dagger}{dagger}P < 0.01 vs. control. #P < 0.05; ##P < 0.01 vs. reoxygenation. *P < 0.05; **P < 0.01 vs. CP.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We previously demonstrated that integrated PPC with three putative triggers of IPC, i.e., a G protein-coupled receptor agonist, a mitochondrial KATP channel opener, and an NO donor, at a dose that does not confer cardioprotection alone or in combination with two triggering agents, produced powerful cardioprotection comparable to that produced by IPC in the isolated rat heart model (32, 43). The present study confirmed the efficacy of this combined-PPC technique in the cultured CMC model and demonstrated that combined PPC with adenosine, diazoxide, and an NO donor confers the memory of cardioprotection by sustained activation of PKC-{epsilon} and PI3-kinase. The sustained increase in PKC-{epsilon} and PI3-kinase activity and cardioprotective memory could not be achieved by simply increasing the dose of each drug; however, it was successfully reproduced by combining these drugs. These results suggest that the sustained activation of PKC-{epsilon} and PI3-kinase and the memory of cardioprotection are mediated by combined PPC with adenosine, diazoxide, and SNAP.

The memory of cardioprotection conferred by preconditioning and the underlying mechanisms have been a subject of extensive research for many years. It has been suggested that the mitochondrial KATP channel and PKC create a positive-feedback loop during the memory phase of IPC (44). Recent studies (21, 38) raised the hypothesis that reactive oxygen species (ROS) generated through activation of mitochondrial KATP channels play a pivotal role in the memory of cardioprotection. Juhaszova and associates (21) suggested that characteristic memory of PPC is mediated by moderate, reversible, and sustained mitochondrial swelling, which causes increased generation of ROS and, consequently, redox activation of PKC-{epsilon}. The persistence of mitochondrial swelling appears to be promoted by feedback amplification of mitochondrial KATP channel activity by PKC-{epsilon}.

Although adenosine alone can activate mitochondrial KATP channels and the endothelial isoform of NO synthase through the activation of PKC and PI3-kinase, respectively (35, 48), the present study suggests that coordinated interaction of distinct and overlapping downstream signaling generated by diazoxide and SNAP is necessary to amplify PKC and PI3-kinase activities. Adenosine stimulates adenosine A1, A2, and A3 receptors, which are coupled with Gi/o proteins (31). Gi/o protein activation results in membrane translocation and phosphorylation of PKC in a Ca2+-dependent (in the case of the classical PKC isoforms) and Ca2+-independent (in the case of the novel PKC isoforms) manner by generating the lipid second messenger diacylglycerol (12). The heterotrimeric G proteins can also activate PI3-kinase via the transactivation of receptor tyrosine kinases through the generation of ROS in certain cell types (27, 45). Although several Gi/o-coupled receptor agonists can generate ROS through the activation of mitochondrial KATP channels in the heart (10), ROS generation by Gi/o-coupled receptor stimulation may be transient, and the redox signaling is attenuated over time. Persistent generation of ROS can be obtained by diazoxide administration through the opening of mitochondrial KATP channels (25). This mechanism is also essential for activation of PKC by diazoxide (22). Indeed, Sato and associates (35) demonstrated that the primary effect of PKC activation by adenosine on mitochondrial KATP channel activation was to prime and enhance the channel activation by diazoxide but not to open the channels. On the other hand, NO-induced activation of PKC-{epsilon} occurs through ROS-dependent tyrosine nitration of PKC-{epsilon} (6) or through cGMP-dependent activation of mitochondrial KATP channels (47). Moreover, cardioprotective signal transduction induced by NO proceeds via the formation of the PKC-{epsilon}-Src kinase signaling module (46), each element of which may interdependently upregulate the other's kinase activity (19). Src kinase then leads to PI3-kinase activation through protein-protein interaction (1). Thus it seems likely that ROS and NO signaling produced by adenosine is insufficient for sustained activation of PKC-{epsilon} and PI3-kinase after the withdrawal of adenosine, and additional ROS and NO are necessary to provoke positive feedback and feedforward amplification of PKC-{epsilon} and PI3-kinase activities. However, it has been shown that acetylcholine and adenosine provoke sustained activation of PI3-kinase after withdrawal of these drugs in the isolated rabbit heart (23). In that model, acetylcholine and adenosine, each alone, appear to be capable of producing the memory of cardioprotection. Therefore, in certain experimental models, but not in others, G protein-coupled receptor agonists alone can produce ROS and NO or alternative molecules in an amount sufficient to integrate the positive amplification loop for sustained activation of PKC-{epsilon} and PI3-kinase.

We investigated the relative position of PKC and PI3-kinase in the integrated PPC-mediated cardioprotective signal transduction by employing the PKC inhibitor chelerythrine and the PI3-kinase inhibitor LY-294002. Administration of chelerythrine and LY-294002, each alone, during the washout period partially inhibited PKC-{epsilon} and PI3-kinase activities mediated by integrated PPC, whereas combined addition of chelerythrine and LY-294002 completely inhibited activation of PKC-{epsilon} and PI3-kinase. These results suggest that the cross talk between PKC-{epsilon} and PI3-kinase exists in a parallel position, and the activities of each of these kinases are regulated, at least in part, by the other's kinase, as is the case for the PKC-{epsilon}-Src kinase signaling module (46). It has been demonstrated that PI3-kinase exists upstream of PKC in cardioprotection mediated by IPC in the isolated and perfused rat heart model (40). Such a disparate observation may arise from the different experimental models and the preconditioning protocol. However, caution must be taken to interpret our results, because chelerythrine and LY-294002 are not completely specific for PKC and PI3-kinase, respectively.

We also investigated the relative contribution of PKC and PI3-kinase to the integrated PPC-mediated cardioprotection against apoptosis and necrosis. The apoptotic cascade was not activated during hypoxia in CMCs, as evaluated by caspase-3 activity and DNA fragmentation, but was activated during reoxygenation. This finding agrees with the previous observation that mitochondrial permeability transition pores, the regulatory channels for mitochondria-mediated death pathways (16), remain closed during ischemia, even though they are primed for activation but open on reperfusion (17). However, reoxygenation did not induce necrosis in CMCs under the isosmotic condition, as evaluated by LDH release, but reoxygenation indeed induced necrosis under the hyposmotic condition, which did not cause significant LDH release in normoxic CMCs. This finding suggests that fragility of the sarcolemma underlies the mechanism of necrosis in CMCs during reoxygenation. It has long been known that sarcolemmal fragility is a characteristic feature of reperfusion injury in the heart (14). Treatment with chelerythrine and LY-294002, each alone, before simulated ischemia was partially effective in blocking the inhibitory effect of integrated PPC on caspase-3 activation, DNA fragmentation, and LDH release during reoxygenation, whereas coadministration of chelerythrine and LY-294002 completely abrogated the combined-PPC-mediated cardioprotection against apoptosis and necrosis. These results suggest that the distal effectors activated by PKC-{epsilon} and PI3-kinase are different but ultimately converge on protection of mitochondria, because apoptosis and necrosis occur primarily as a consequence of mitochondrial damage.

Although the discrimination of apoptosis and necrosis is critical in the pathophysiology of ischemia and reperfusion injury, the present study did not provide a definite answer for this issue. It should be noted that DNA fragmentation is not specific for apoptosis but is also induced by necrosis associated with random DNA fragmentation at a late stage by release of lysosomal DNases (36). Our study demonstrating that caspase-3 activation and DNA fragmentation occurred without significant LDH release during reoxygenation under the isosmotic condition suggests that necrosis made no major contribution to DNA fragmentation in this experimental model. Nevertheless, more specific methods for the detection of apoptosis and the use of small interfering RNA targeting caspase-3 mRNA or a dominant-negative caspase-3 overexpression would be necessary to unequivocally address the role of caspase-3 in CMC injury during reoxygenation. In addition, because activation of the mitochondria-mediated cell death pathway upstream from caspase-3 is determined by the quantitative ratio of antiapoptotic to proapoptotic Bcl-2 family proteins in mitochondria (34), it would be intriguing to elucidate the relation between mitochondrial association of Bcl-2 family proteins and cardioprotection of PPC.

Among the distal effectors of PKC-{epsilon}, mitochondrial KATP channels (30), sarcolemmal KATP channels (2), and mitochondrial voltage-dependent anion channels (5) are the targets of PKC-{epsilon} responsible for cardioprotection. PI3-kinase is also known to activate signal transduction that converges on mitochondrial protection. It has been demonstrated that PI3-kinase confers cardioprotection by phosphorylating Akt, the substrate of PI3-kinase, which then modulates the phosphorylation status of several downstream kinases, such as glycogen synthase kinase-3{beta} and the target of rapamycin (18). A growing body of evidence suggests that inactivation of glycogen synthase kinase-3{beta} by Akt-dependent phosphorylation plays a cardioprotective role by inhibiting mitochondrial permeability transition (21). Akt also phosphorylates apoptosis-regulatory molecules such as Bad, procaspase-9, and cAMP-responsive element-binding protein in certain cell types (8, 11, 13). Recently, Uchiyama and associates (41) demonstrated that dominant-negative Akt abolished an antiapoptotic effect mediated by hypoxic preconditioning, and this detrimental effect was associated with the abrogation of phosphorylation of Bad in conjunction with the release of cytochrome c and the activation of caspase-3 in adult rat ventricular myocytes.

In conclusion, integrated PPC in combination with adenosine, diazoxide, and SNAP, but not individually, confers cardioprotective memory against CMC apoptosis and necrosis during reoxygenation. This memory of cardioprotective signal transduction is likely to be mediated by coordinated interaction of distinct cellular events produced by the G protein-coupled receptor agonist, the mitochondrial KATP channel opener, and the NO donor, promoting interdependent amplification of PKC-{epsilon} and PI3-kinase signaling.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan and a research grant from the Ministry of Education, Science, and Culture of Japan.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Otani, Dept. of 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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Abram CL and Courtneidge SA. Src family tyrosine kinases and growth factor signaling. Exp Cell Res 254: 1–13, 2000.[CrossRef][Web of Science][Medline]
  2. Aizawa K, Turner LA, Weihrauch D, Bosnjak ZJ, and Kwok WM. Protein kinase C-{epsilon} primes the cardiac sarcolemmal adenosine triphosphate-sensitive potassium channel to modulation by isoflurane. Anesthesiology 101: 381–389, 2004.[Web of Science][Medline]
  3. Armstrong SC. Protein kinase activation and myocardial ischemia/reperfusion injury. Cardiovasc Res 61: 427–436, 2004.[Abstract/Free Full Text]
  4. Armstrong SC and Ganote CE. Effects of the phospholipase inhibitor mepacrine on injury in ischemic and metabolically inhibited adult isolated myocytes. Am J Pathol 138: 545–555, 1991.[Abstract]
  5. Baines CP, Song CX, Zheng YT, Wang GW, Zhang J, Wang OL, Guo Y, Bolli R, Cardwell EM, and Ping P. Protein kinase C-{epsilon} interacts with and inhibits the permeability transition pore in cardiac mitochondria. Circ Res 92: 873–880, 2003.[Abstract/Free Full Text]
  6. Balafanova Z, Bolli R, Zhang J, Zheng Y, Pass JM, Bhatnagar A, Tang XL, Wang O, Cardwell E, and Ping P. Nitric oxide (NO) induces nitration of protein kinase C{epsilon} (PKC{epsilon}), facilitating PKC{epsilon} translocation via enhanced PKC{epsilon}-RACK2 interactions: a novel mechanism of NO-triggered activation of PKC{epsilon}. J Biol Chem 277: 15021–15027, 2002.[Abstract/Free Full Text]
  7. Belhomme D, Peynet J, Florens E, Tibourtine O, Kitakaze M, and Menasche P. Is adenosine preconditioning truly cardioprotective in coronary artery bypass surgery? Ann Thorac Surg 70: 590–594, 2000.[Abstract/Free Full Text]
  8. Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E, Frisch S, and Reed JC. Regulation of cell death protease caspase-9 by phosphorylation. Science 282: 1318–1321, 1998.[Abstract/Free Full Text]
  9. Cave AC, Collis CS, Downey JM, and Hearse DJ. Improved functional recovery by ischaemic preconditioning is not mediated by adenosine in the globally ischaemic isolated rat heart. Cardiovasc Res 27: 663–668, 1993.[Abstract/Free Full Text]
  10. Cohen MV, Yang XM, Liu GS, Heusch G, and Downey JM. Acetylcholine, bradykinin, opioids, and phenylephrine, but not adenosine, trigger preconditioning by generating free radicals and opening mitochondrial KATP channels. Circ Res 89: 273–278, 2001.[Abstract/Free Full Text]
  11. Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, and Greenberg ME. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91: 231–241, 1997.[CrossRef][Web of Science][Medline]
  12. De Jonge HW, Van Heugten HA, and Lamers JM. Signal transduction by the phosphatidylinositol cycle in myocardium. J Mol Cell Cardiol 27: 93–106, 1995.[Web of Science][Medline]
  13. Du K and Montminy M. CREB is a regulatory target for the protein kinase Akt/PKB. J Biol Chem 273: 32377–32379, 1998.[Abstract/Free Full Text]
  14. Ganote C and Armstrong S. Ischaemia and the myocyte cytoskeleton: review and speculation. Cardiovasc Res 27: 1387–1403, 1993.[Free Full Text]
  15. Gray MO, Karliner JS, and Mochly-Rosen D. A selective {epsilon}-protein kinase C antagonist inhibits protection of cardiac myocytes from hypoxia-induced cell death. J Biol Chem 272: 30945–30951, 1997.[Abstract/Free Full Text]
  16. Green DR and Reed JC. Mitochondria and apoptosis. Science 281: 1309–1312, 1998.[Abstract/Free Full Text]
  17. Griffiths EJ and Halestrap AP. Mitochondrial non-specific pores remain closed during cardiac ischaemia, but open upon reperfusion. Biochem J 307: 93–98, 1995.
  18. Gross ER, Hsu AK, and Gross GJ. Opioid-induced cardioprotection occurs via glycogen synthase kinase-{beta} inhibition during reperfusion in intact rat hearts. Circ Res 94: 960–966, 2004.[Abstract/Free Full Text]
  19. Hattori R, Otani H, Uchiyama T, Imamura H, Cui J, Maulik N, Cordis GA, Zhu L, and Das DK. Src tyrosine kinase is the trigger but not the mediator of ischemic preconditioning. Am J Physiol Heart Circ Physiol 281: H1066–H1074, 2001.[Abstract/Free Full Text]
  20. Hausenloy DJ and Yellon DM. New directions for protecting the heart against ischaemia-reperfusion injury: targeting the reperfusion injury salvage kinase (RISK) pathway. Cardiovasc Res 61: 448–460, 2004.[Abstract/Free Full Text]
  21. Juhaszova M, Zorov DB, Kim SH, Pepe S, Fu Q, Fishbein KW, Ziman BD, Wang S, Ytrehus K, Antos CL, Olson EN, and Sollott SJ. Glycogen synthase kinase-3{beta} mediates convergence of protection signaling to inhibit the mitochondrial permeability transition pore. J Clin Invest 113: 1535–1549, 2004.[CrossRef][Web of Science][Medline]
  22. Kis B, Rajapakse NC, Snipes JA, Nagy K, Horiguchi T, and Busija DW. Diazoxide induces delayed pre-conditioning in cultured rat cortical neurons. J Neurochem 87: 969–980, 2003.[CrossRef][Web of Science][Medline]
  23. Krieg T, Qin Q, McIntosh EC, Cohen MV, and Downey JM. ACh and adenosine activate PI3-kinase in rabbit hearts through transactivation of receptor tyrosine kinases. Am J Physiol Heart Circ Physiol 283: H2322–H2330, 2002.[Abstract/Free Full Text]
  24. Liu GS, Cohen MV, Mochly-Rosen D, and Downey JM. Protein kinase C-{epsilon} is responsible for the protection of preconditioning in rabbit cardiomyocytes. J Mol Cell Cardiol 31: 1937–1948, 1999.[CrossRef][Web of Science][Medline]
  25. Liu Y and O'Rourke B. Opening of mitochondrial KATP channels triggers cardioprotection. Are reactive oxygen species involved? Circ Res 88: 750–752, 2001.[Free Full Text]
  26. Lochner A, Marais E, Genade S, and Moolman JA. Nitric oxide: a trigger for classic preconditioning? Am J Physiol Heart Circ Physiol 279: H2752–H2765, 2000.[Abstract/Free Full Text]
  27. Luttrell LM, Daaka Y, and Lefkowitz RJ. Regulation of tyrosine kinase cascades by G-protein-coupled receptors. Curr Opin Cell Biol 11: 177–183, 1999.[CrossRef][Web of Science][Medline]
  28. Murry CE, Jennings RB, and Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74: 1124–1136, 1986.[Abstract/Free Full Text]
  29. Nakano A, Liu GS, Heusch G, Downey JM, and Cohen MV. Exogenous nitric oxide can trigger a preconditioned state through a free radical mechanism, but endogenous nitric oxide is not a trigger of classical ischemic preconditioning. J Mol Cell Cardiol 32: 1159–1167, 2000.[CrossRef][Web of Science][Medline]
  30. Ohnuma Y, Miura T, Miki T, Tanno M, Kuno A, Tsuchida A, and Shimamoto K. Opening of mitochondrial KATP channel occurs downstream of PKC-{epsilon} activation in the mechanism of preconditioning. Am J Physiol Heart Circ Physiol 283: H440–H447, 2002.[Abstract/Free Full Text]
  31. Olsson RA and Pearson JD. Cardiovascular purinoceptors. Physiol Rev 70: 761–845, 1990.[Free Full Text]
  32. Otani H, Okada T, Fujiwara H, Uchiyama T, Sumida T, Kido M, and Imamura H. Combined pharmacological preconditioning with a G-protein-coupled receptor agonist, a mitochondrial KATP channel opener and a nitric oxide donor mimics ischaemic preconditioning. Clin Exp Pharmacol Physiol 30: 684–693, 2003.[CrossRef][Web of Science][Medline]
  33. Pain T, Yang XM, Critz SD, Yue Y, Nakano A, Liu GS, Heusch G, Cohen MV, and Downey JM. Opening of mitochondrial KATP channels triggers the preconditioned state by generating free radicals. Circ Res 87: 460–466, 2000.[Abstract/Free Full Text]
  34. Reed JC, Jurgensmeier JM, and Matsuyama S. Bcl-2 family proteins and mitochondria. Biochim Biophys Acta 1366: 127–137, 1998.[Medline]
  35. Sato T, Sasaki N, O'Rourke B, and Marban E. Adenosine primes the opening of mitochondrial ATP-sensitive potassium channels: a key step in ischemic preconditioning? Circulation 102: 800–805, 2000.[Abstract/Free Full Text]
  36. Stadelmann C and Lassmann H. Detection of apoptosis in tissue sections. Cell Tissue Res 301: 19–31, 2000.[CrossRef][Web of Science][Medline]
  37. Stephens L, Anderson K, Stokoe D, Erdjument-Bromage H, Painter GF, Holmes AB, Gaffney PR, Reese CB, McCormick F, Tempst P, Coadwell J, and Hawkins PT. Protein kinase B kinases that mediate phosphatidylinositol 3,4,5-trisphosphate-dependent activation of protein kinase B. Science 279: 710–714, 1998.[Abstract/Free Full Text]
  38. Stowe DF and Kevin LG. Cardiac preconditioning by volatile anesthetic agents: a defining role for altered mitochondrial bioenergetics. Antioxid Redox Signal 6: 439–448, 2004.[CrossRef][Web of Science][Medline]
  39. Thornton JD, Liu GS, Olsson RA, and Downey JM. Intravenous pretreatment with A1-selective adenosine analogues protects the heart against infarction. Circulation 85: 659–665, 1992.[Abstract/Free Full Text]
  40. Tong H, Chen W, Steenbergen C, and Murphy E. Ischemic preconditioning activates phosphatidylinositol-3-kinase upstream of protein kinase C. Circ Res 87: 309–315, 2000.[Abstract/Free Full Text]
  41. Uchiyama T, Engelman RM, Maulik N, and Das DK. Role of Akt signaling in mitochondrial survival pathway triggered by hypoxic preconditioning. Circulation 109: 3042–3049, 2004.[Abstract/Free Full Text]
  42. Uchiyama T, Otani H, Okada T, Ninomiya H, Kido M, Imamura H, Nogi S, and Kobayashi Y. Nitric oxide induces caspase-dependent apoptosis and necrosis in neonatal rat cardiomyocytes. J Mol Cell Cardiol 34: 1049–1061, 2002.[CrossRef][Web of Science][Medline]
  43. Uchiyama Y, Otani H, Okada T, Uchiyama T, Ninomiya H, Kido M, Imamura H, Nakao S, and Shingu K. Integrated pharmacological preconditioning in combination with adenosine, a mitochondrial KATP channel opener and a nitric oxide donor. J Thorac Cardiovasc Surg 126: 148–159, 2003.[Abstract/Free Full Text]
  44. Uchiyama Y, Otani H, Wakeno M, Okada T, Uchiyama T, Sumida T, Kido M, Imamura H, Nakao S, and Shingu K. Role of mitochondrial KATP channels and protein kinase C in ischaemic preconditioning. Clin Exp Pharmacol Physiol 30: 426–436, 2003.[CrossRef][Web of Science][Medline]
  45. Ushio-Fukai M, Alexander RW, Akers M, Yin Q, Fujio Y, Walsh K, and Griendling KK. Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells. J Biol Chem 274: 22699–22704, 1999.[Abstract/Free Full Text]
  46. Vondriska TM, Zhang J, Song C, Tang XL, Cao X, Baines CP, Pass JM, Wang S, Bolli R, and Ping P. Protein kinase C-{epsilon}-Src modules direct signal transduction in nitric oxide-induced cardioprotection: complex formation as a means for cardioprotective signaling. Circ Res 88: 1306–1313, 2001.[Abstract/Free Full Text]
  47. Xu Z, Ji X, and Boysen PG. Exogenous nitric oxide generates ROS and induces cardioprotection: involvement of PKG, mitochondrial KATP channels, and ERK. Am J Physiol Heart Circ Physiol 286: H1433–H1440, 2004.[Abstract/Free Full Text]
  48. Xu Z, Park SS, Mueller RA, Bagnell RC, Patterson C, and Boysen PG. Adenosine produces nitric oxide and prevents mitochondrial oxidant damage in rat cardiomyocytes. Cardiovasc Res 65: 803–812, 2005.[Abstract/Free Full Text]
  49. Yao Z, Mizumura T, Mei DA, and Gross GJ. KATP channels and memory of ischemic preconditioning in dogs: synergism between adenosine and KATP channels. Am J Physiol Heart Circ Physiol 272: H334–H342, 1997.[Abstract/Free Full Text]
  50. Yellon DM and Dana A. The preconditioning phenomenon: a tool for the scientist or a clinical reality? Circ Res 87: 543–550, 2000.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
B. Zhong and D. H. Wang
Protease-activated receptor 2-mediated protection of myocardial ischemia-reperfusion injury: role of transient receptor potential vanilloid receptors
Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2009; 297(6): R1681 - R1690.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
A. J. Zatta, H. Kin, G. Lee, N. Wang, R. Jiang, R. Lust, J. G. Reeves, J. Mykytenko, R. A. Guyton, Z.-Q. Zhao, et al.
Infarct-sparing effect of myocardial postconditioning is dependent on protein kinase C signalling
Cardiovasc Res, May 1, 2006; 70(2): 315 - 324.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
289/2/H761    most recent
00012.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Okada, T.
Right arrow Articles by Imamura, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Okada, T.
Right arrow Articles by Imamura, H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.