PKC-{epsilon} is upstream and PKC-{alpha} is downstream of mitoKATP channels in the signal transduction pathway of ischemic preconditioning of human myocardium

Ashraf Hassouna, Bashir M. Matata, and Manuel Galiñanes

Integrative Human Cardiovascular Physiology and Cardiac Surgery Unit, Department of Cardiovascular Sciences, University of Leicester, Glenfield Hospital, Leicester LE3 9QP, United Kingdom

Submitted 16 March 2004 ; accepted in final form 8 July 2004


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Protein kinase C (PKC) is involved in the process of ischemic preconditioning (IPC), although the precise mechanism is still a subject of debate. Using specific PKC inhibitors, we investigated which PKC isoforms were involved in IPC of the human atrial myocardium sections and to determine their temporal relationship to the opening of mitochondrial potassium-sensitive ATP (mitoKATP) channels. Right atrial muscles obtained from patients undergoing elective cardiac surgery were equilibrated and then randomized to receive any of the following protocols: aerobic control, 90-min simulated ischemia/120-min reoxygenation, IPC using 5-min simulated ischemia/5-min reoxygenation followed by 90-min simulated ischemia/120-min reoxygenation and finally, PKC inhibitors were added 10 min before and 10 min during IPC followed by 90-min simulated ischemia/120-min reoxygenation. The PKC isoforms inhibitors investigated were V1–2 peptide, GO-6976, rottlerin, and LY-333531 for PKC-{epsilon}, -{alpha}, -{delta} and -{beta}, respectively. To investigate the relation of PKC isoforms to mitoKATP channels, PKC inhibitors found to be involved in IPC were added 10 min before and 10 min during preconditioning by diazoxide followed by 90-min simulated ischemia/120-min reoxygenation in a second experiment. Creatine kinase leakage and methylthiazoletetrazolium cell viability were measured. Phosphorylation of PKC isoforms after activation of the sample by either diazoxide or IPC was detected by using Western blot analysis and then analyzed by using Scion image software. PKC-{alpha} and -{epsilon} inhibitors blocked IPC, whereas PKC-{delta} and -{beta} inhibitors did not. The protection elicited by diazoxide, believed to be via mitoKATP channels opening, was blocked by the inhibition of PKC-{alpha} but not -{epsilon} isoforms. In addition, diazoxide caused increased phosphorylation of PKC-{alpha} to the same extent as IPC but did not affect the phosphorylation of PKC-{epsilon}, a process believed to be critical in PKC activation. The results demonstrate that PKC-{alpha} and -{epsilon} are involved in IPC of the human myocardium with PKC-{epsilon} being upstream and PKC-{alpha} being downstream of mitoKATP channels.

cardioprotection; protein kinase C isoforms


PROTEIN KINASE C (PKC) plays an important role in the signaling pathway of ischemic preconditioning (IPC) in human (10, 27, 37) and animal (25, 29, 38, 51) studies, although this concept has been disputed by some investigators (41, 44). The reason for these discrepancies may be related at least, in part, to the complexity of PKC biology and pharmacology. PKC represent a family of at least 12 isoforms of closely related serine and threonine protein kinase (32, 40). Different PKC isoforms are known to be important in IPC; however, the precise role of specific isoforms appears to vary between animal species. For example PKC-{epsilon} has been shown to be an important component of IPC in rabbits (32), whereas in rats, both PKC-{epsilon} (20) and -{delta} (29) have been reported to be essential for cardioprotection by IPC. In contrast, in the canine heart model, it has been shown that PKC-{alpha} is important in provoking IPC (24). However, with the exception of the study by Julier et al. (23), there is little information in the literature regarding studies on human hearts.

The putative mitochondrial potassium-sensitive ATP (mitoKATP) channels also play a key role in the signal transduction mechanism of IPC (9, 14, 33, 34, 37). It has been reported that opening of the mitoKATP channels can activate PKC by generation of radical oxygen species (33), although it has also been suggested that opening of these channels can be potentiated by PKC (34). This may suggest that PKC isoforms may exist upstream and downstream of mitoKATP channels, but direct evidence of this does not exist. The present study was designed to investigate the role of different PKC isoforms in IPC of the human myocardium and to determine how they are related to the mitoKATP channels.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Study Subjects

The study was approved by the local ethical committee and was conducted with the consent of patients in accordance with the Helsinki Declaration. Atrial biopsies were collected from patients undergoing elective coronary artery bypass surgery and/or aortic valve surgery during cannulation of the right atrium before initiation of cardiopulmonary bypass. Patients with enlarged atriums, arrhythmias, poor left ventricular function (ejection fraction <30%), and right ventricular failure were excluded. Other criteria for exclusion were diabetes mellitus and the treatment with oral opioid analgesia, KATP channel openers, and catecholamines.

Processing of Samples and Experimental Preparation

This study was conducted by using an established model for quantification of the effect of ischemia and reperfusion on human cardiac muscle by using thin slices of atrial appendages (52). Briefly, sections ~40 mg wet weight and 300- to 500-µm thick were prepared from human right atrial appendages by using a surgical skin-graft knife. Sections were immediately equilibrated for 30 min in Krebs-Henseleit HEPES buffer (pH 7.4) oxygenated by bubbling with 95% oxygen-5% carbon dioxide and maintained at 37°C throughout the experiments. Simulated ischemia was induced by bubbling the medium with 95% N2-5% CO2 (pH 6.8–7.0) and replacing D-glucose with 2-deoxy-D-glucose as described previously (52). IPC was induced by 5-min ischemia followed by 5-min reoxygenation, a protocol that has been shown in our laboratory to be the most protective in this preparation (10).

Measurement of Tissue Injury and Viability

Tissue injury was determined by measuring the leakage of creatine kinase (CK) into the incubation medium during the reoxygenation period, and this was assayed by a kinetic method on the basis of the reduction of NADP to NADPH. The absorbance was read at 340 nm using a Bench plate reader (Bio-Rad, Hercules, CA). The rate of change in absorbance is directly proportional to CK activity, and the results were expressed as U/g wet wt. Tissue viability was assessed by the reduction of methylthiazoletetrazolium (MTT) to a blue formazan product at the end of the experimental time (43). The absorbance of the blue formazan precipitate formed was measured at 550 nm and the results were expressed as mmol/g wet wt.

Study Protocols

Identification of PKC isoforms involved in IPC. To identify the PKC isoforms involved in IPC, dose response studies were performed by using specific PKC inhibitors. After equilibration at 37°C in aerobic medium for 30 min, myocardial sections (n = 6 per group) were randomized to receive one of the protocols summarized in Fig. 1A. To serve as controls, some of the myocardial sections were subjected to the highest dose of the various PKC inhibitors used for 10 min followed by the induction of 90 min of simulated ischemia/120 min of reoxygenation (SI/R). The rest of the sections were treated with the various PKC inhibitors added 10 min before and 10 min during IPC followed by SI/R at various concentrations as follows: myristoylated V1–2 peptide (0.1, 1, 10 µM) a PKC-{epsilon} inhibitor; rottlerin (1, 10, 100 µM) a PKC-{delta} inhibitor; LY-333531 (1, 10, 100 nM) a PKC-({beta}1 + {beta}2) inhibitor; GO-6976 (1, 10, 100 nM), and Ro 32–0432 (0.1, 1, 10 µM) both PKC ({alpha} + {beta}) inhibitors. In view of the ongoing controversy on the use of rottlerin as a specific PKC-{delta} inhibitor, the selective PKC-{delta} activator bistratene A (1, 10, 100 nM) was administered for 10 min before simulated ischemia/reoxygenation.



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Fig. 1. Study protocol to identify the protein kinase C (PKC) isoforms involved in ischemic preconditioning (IPC) using specific inhibitors (A) and to investigate the sequence of involvement of PKC-{epsilon} and PKC-{alpha} in relation to mitoKATP channels (B).

 
Sequence of involvement of PKC isoforms in the mechanism of preconditioning and their relation to the activation of mitoKATP channels. To determine the sequence of events leading to the induction of IPC via activation of PKC isoforms, myocardial sections (n = 6 per group) were equilibrated for 30 min before they were randomized into any of the protocols summarized in Fig. 1B. The mitoKATP channel opener diazoxide was used alone or in combination with specific PKC isoform inhibitors. The concentration of 100 µM of diazoxide was used, because it was shown in our laboratory (10) to be optimally protective.

Phosphorylation of PKC isoforms. In this study, the phosphorylation of the PKC isoforms found to participate in IPC in the above studies was measured in myocardial sections (n = 4 per group) that were randomized after 30 min of equilibration to receive any one of the following protocols: 1) time-matched aerobic control for an additional 10 min, 2) IPC alone using 5 min of simulated ischemia followed by 5 min reoxygenation, and 3) 10 min preconditioning with diazoxide (100 µM). At the end of each protocol, the tissue sections were quickly frozen in liquid nitrogen and stored at –80°C until analysis. Samples were homogenized in RIPA buffer containing protease inhibitor, PMSF (1 mM), DDT (0.5 mM), glycerophosphate (25 µM), and sodium orthovanadate (1 mM). The homogenate was then centrifuged at 10,000 g for 30 min. The supernatant obtained was analyzed for protein concentration using the Bio-Rad protein assay kit.

Then 20 µg of the tissue supernatant were elecrophoresed on 10% SDS/PAGE and blotted onto nitrocellulose membrane. Membranes were blocked in TBS buffer containing 5% fat-free dry milk, followed by incubation with the appropriate primary antibodies at 1:1,000 dilution overnight at 4°C. Membranes were then detected by the appropriate horseradish peroxidase-conjugated secondary antibody (anti-rabbit goat antibody and anti-mouse goat antibody at 1:5,000 dilution). The signals were then developed by using an enhanced chemiluminescense Western blot analysis detection kit (Amersham, Little Chalfont, UK). Densitometry analysis of bands was performed by Scion image (Scion, Frederick, MD).

Solutions and Chemicals

Samples were incubated in Krebs medium composed of (in mM) 118 NaCl2, 4.8 KCl, 27.2 NaHCO3, 1.2 MgCl2, 1.0 KH2PO4, 1.25 CaCl2, 20 HEPES, and 10 D-glucose or 10 2-deoxy-D-glucose to induce simulated ischemia, and prepared in deionized distilled water. All of these chemicals, CK assay kit, MTT, diazoxide, and bistratene A were purchased from Sigma (St. Louis, MO). The specific PKC isoforms inhibitors GO-6976, myristoylated V1–2 peptide (to allow cell permeability), and rottlerin were obtained from BioMol Research Laboratories (Plymouth Meeting, PA), and LY-333531 was supplied by AG Scientific (San Diego, CA). The antibodies for PKC-{alpha} and -{epsilon}, phospho PKC-{alpha} (serine 657), and phospho PKC-{epsilon} (serine 729) were obtained from Upstate Cell Signaling Solution (Charlottesville, VA).

Statistical Analysis

All data are presented as means ± SE. Mean values were analyzed by ANOVA with a post hoc Tukey's test. Statistical significance was at P < 0.05.


    RESULTS
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 MATERIALS AND METHODS
 RESULTS
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Identity of PKC Isoforms Involved in IPC

The results shown in Figs. 2 to 5 indicate that IPC significantly reduced CK leakage and increased the MTT reduction of muscles subjected to ischemia/reoxygenation. None of the PKC inhibitors (at the highest doses used) in the absence of IPC had any significant effect on tissue damage or tissue viability. They also show that the PKC-({alpha} + {beta}) inhibitor GO-6976 at 100 nM blocked preconditioning, whereas PKC-{beta} inhibitor LY-333531 failed to block preconditioning. The effect of PKC-({alpha} + {beta}) inhibition was also confirmed with the use of Ro 32–0432 (data not shown). This would suggest that PKC-{alpha}, but not PKC-{beta}, is involved in signaling pathway of IPC. Furthermore, the PKC-{epsilon} inhibitor V1–2 peptide completely blocked protection by IPC at a concentration of 10 µM, whereas the PKC-{delta} inhibitor rottlerin had no effect at any of the study concentrations. Activation PKC-{delta} isoforms with bistratene A failed to induce protection in our model (Fig. 6). These results indicate that PKC-{epsilon} participate in the protection by IPC of the human myocardium with no involvement of PKC-{delta}.



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Fig. 2. Dose-response to PKC-({alpha} + {beta}) inhibition of IPC using GO-6976 on creatine kinase (CK) leakage (A) and methylthiazoletetrazolium (MTT) reduction (B). Data are expressed as means ± SE of n = 6 tissue sections/group. *P < 0.05 vs. IPC alone. A/C, aerobic/control; SI/R, simulated ischemia/reoxygenation.

 


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Fig. 5. Dose response to PKC-{delta} inhibition of IPC with rottlerin on CK leakage (A) and MTT reduction (B). Data are expressed as means ± SE of n = 6 tissue sections/group.

 


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Fig. 6. Dose response to PKC-{delta} activation with bistratene A on CK leakage (A) and MTT reduction (B). Data are expressed as means ± SE of n = 6 tissue sections/group. *P < 0.05 vs. SI/R.

 
Sequence of Involvement of PKC-{alpha} and -{epsilon} in Relation to mitoKATP Channel

Figure 7, A and B demonstrates that, as expected, IPC and diazoxide conferred a similar degree of cardioprotection as shown by the reduction of CK leakage and increased MTT viability. As seen previously, PKC-{epsilon} inhibitor V1–2 peptide (10 µM) blocked protection by IPC, but interestingly, it did not affect the protection induced by diazoxide. This would suggest that PKC-{epsilon} is upstream of mitoKATP channels. Figure 8, A and B demonstrate that the PKC-({alpha} + {beta}) GO-6976 (100 nM) blocked the protection seen with both IPC and diazoxide, which indicates that PKC-{alpha} is downstream of mitoKATP channels (note that the studies presented above suggested the absence of a role for the PKC-{beta} isoforms in IPC).



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Fig. 7. Effect of the PKC-{epsilon} inhibitor V1–2 peptide (10 µM) on the protection of IPC and diazoxide (DXZ) on CK leakage (A) and MTT reduction (B). Data are expressed as means ± SE of n = 6 tissue sections/group. *P < 0.05 vs. IPC without PKC-{epsilon} inhibition (PKC-{epsilon}I).

 


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Fig. 8. Effect of the PKC-({alpha} + {beta}) inhibitor GO-6976 (100 nM) on the protection of IPC and diazoxide on CK leakage (A) and MTT reduction (B). Data are expressed as means ± SE of n = 6 tissue sections/group. *P < 0.05 vs. IPC without PKC-({alpha} + {beta}) inhibition [PKC-({alpha}+{beta})I].

 
Phosphorylation of PKC Isoforms

To demonstrate the phosphorylation of PKC-{epsilon} and -{alpha} in relation to the opening of mitoKATP channels, tissue extract were detected by Western immunoblotting. As shown in Fig. 9, the basal level of 30–35% phosphorylation of PKC-{epsilon} and -{alpha} were observed in aerobic controls. IPC increased the phosphorylation of both PKC-{alpha} and -{epsilon}; however, diazoxide caused significant increase in PKC-{alpha} phosphorylation but failed to induce a significant increase in the phosphorylation of PKC-{epsilon}, which confirms that PKC-{epsilon} lies upstream and PKC-{alpha} lies downstream of mitoKATP channels.



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Fig. 9. The ratio of phosphorylated to nonphosphorylated PKC-{epsilon} (A) and PKC-{alpha} (B) in response to IPC and to DXZ by Western blot analysis and compared with A/C. Data are expressed as means ± SE of n = 4 tissue sections/group. *P < 0.05 vs. A/C. A and B top, representative blots.

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
The present studies provide novel information on the signal transduction pathway of IPC by demonstrating the involvement of PKC-{epsilon} and -{alpha} in the protection of the human myocardium and by showing that PKC-{epsilon} is upstream and PKC-{alpha} is downstream of mitoKATP channels.

These results in the human myocardium may find some support in reported animal studies. Thus for example, with the use of a cell culture model of hypoxic preconditioning, it was found that V1–2 peptide not only abolished cardioprotection but also inhibited the translocation of PKC-{epsilon} (13, 26). More recently, it has been shown that the disruption in the PKC-{epsilon} gene abolishes the infarct size reduction of preconditioning in the isolated buffer-perfused mouse heart (35). There is also evidence that the use of a PKC-{epsilon} selective activator protects the isolated perfused rat heart from ischemia/reperfusion injury (20). The above findings are consistent with the concept that activation of PKC-{epsilon} is necessary for cardioprotection by IPC.

The demonstration in our studies that PKC-{alpha} also participates in the cardioprotection by IPC in the human myocardium is in agreement with the demonstration that GO-6976, a potent inhibitor of PKC-{alpha}, significantly attenuates the protection of hypoxic preconditioning on cell death (42), and blocks the reduction of necrosis and apoptosis afforded by three 1-min cycles of simulated ischemia (49) in embryonic chick cardiomyocytes. Because GO-6976 is a potent inhibitor for {alpha}-isoforms and to a lesser extent of the {beta}-isoforms, it may be postulated that the results of the above studies may be due, at least in part, to PKC-{beta} inhibition (28). However, in our studies, we have demonstrated that in the human myocardium PKC-{beta} inhibition by LY-333531 had no effect on preconditioning, suggesting that the effect of GO-6976 is largely attributed to the inhibition of PKC-{alpha} rather than the PKC-{beta} isoforms.

The lack of a role for the PKC-{beta} and PKC-{delta} isoforms in IPC of the human myocardium is also supported by similar findings in the adult rabbit cardiomyocytes (50) although conflicting results have been published regarding the role of PKC-{delta}. In one study (6), the PKC-{delta} inhibitor rottlerin was found to block the cardioprotection induced by the activation of opioid receptors in the in vivo rat heart model. Another study by the same investigators (5) reported that rottlerin did not abolish the reduction in infarct size induced by IPC in the same species and experimental model. The most plausible explanation for the different results is that the protection elicited by activation of opioid receptors may use different PKC isoforms than the ones used in IPC. Certainly the elucidation of this issue would require further investigations.

The identification of the sequence of the involvement of the two PKC isoforms with PKC-{epsilon} being upstream and PKC-{alpha} being downstream of mitoKATP channels, demonstrated by pharmacological manipulation and by biochemical means, is of biological relevance. In previous studies (27, 30, 51), the use of nonselective PKC inhibitors such as chelerythrine, staurosporin, and calphostin C were insufficient to dissect this relationship, and any conclusion on the location of PKC isoforms in relation to mitoKATP channels using these tools should be interpreted with caution. Our laboratory has previously demonstrated (27) that protection of the human myocardium can be elicited by the PKC activator PMA in the presence of the mitoKATP channel closer 5-hydroxydecanoate, suggesting the PKC is downstream of mitoKATP, but the PKC responsible isoforms and whether other isoforms were involved upstream of mitoKATP channels was not investigated. In the literature, there is also evidence that mitoKATP channels are modulated by PKC (34), supporting the theory that PKC can be located both upstream and downstream of mitoKATP channels. Our finding that PKC-{alpha} is phosphorylated on the serine 657 by IPC and diazoxide and that PKC-{epsilon} is phosphorylated on serine 729 by IPC and not by diazoxide, supports the results obtained with the PKC isoform inhibitors. This argument is reinforced by the demonstration that in isolated, perfused rat hearts that diazoxide causes PKC-{alpha} translocation (46) but fails to induce translocation of PKC-{epsilon} (32). It is possible to speculate that the degree of protection from diazoxide was higher than that of IPC, such that diazoxide can overcome the PKC-{epsilon} block, whereas IPC cannot; however, this is unlikely, because both IPC and diazoxide afforded similar cardioprotection in terms of CK leakage and MTT reduction (Figs. 7 and 8).

The above findings support the view that PKC-{alpha} and PKC-{epsilon} isoforms act independently. As shown in the diagram depicted in the Fig. 10, the activation of sarcolemmal receptors triggers G protein-mediated signaling that, via the activation of PKC-{epsilon}, leads to the opening of mitoKATP channels and that, in turn, possibly through the release of reactive oxygen species, would activate PKC-{alpha}, this leading to the phosphorylation of p38 MAPK, which then effects a single or multiple end-effectors of IPC. The proposal by Garlid et al. (7) that PKC may be proximal and distal to the mitoKATP channels and the demonstration that PKC-{epsilon} interacts with and inhibis the permeability transition pore in the cardiac mitochondria and contributes to PKC-{epsilon}-induced cardioprotection (1) would support the present findings. However, the exact place and action of these PKC isoforms would require further investigations.



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Fig. 10. Proposed schematic representation of the signal transduction mechanism leading to cardioprotection by DXZ and IPC of the human myocardium. On activation of sarcolemmal receptors mitoKATP channels are activated via PKC-{epsilon}. The opening of mitoKATP channels will activate PKC-{alpha} possibly via the production of reactive oxygen species (ROS). PKC-{alpha} may then translocate to various cellular sites and activate p38MAPK. In turn, p38MAPK may activate a single or multiple end-effectors directly or via MAPK intermediates. PLC, phospholipase C; PLD, phospholipase D.

 
The concept of the existence of mitoKATP remains controversial with some authors (2) suggesting that the sulfonylurea-inhibitable mitoKATP channel may not exist, whereas others (19, 36) have reported the existence of native cardiac mitoKATP channels on the basis of pharmacological and histochemical studies. The specificity of diazoxide has also been questioned with diazoxide being inferred to activate the sarcolemmal, not the mitoKATP in mice and provides cardioprotection in this way (39). However, others have shown that diazoxide selectively opens and 5-hydroxydecanoate selectively blocks mitoKATP channels in isolated mitochondrial preparations (9) and intact cardiac myocytes. It has been suggested that diazoxide induces protection of the mouse heart and in the guinea pig heart by the opening of the sarcolemmal KATP channel and decreasing succinate oxidation in a dose-dependent manner without affecting NADH oxidation and that even at a concentration of 100 µm diazoxide does not decrease mitochondrial membrane potential as assessed by tetramethylrhodamine ethylester (16). By contrast, in our laboratory, we (10) have shown that mitochondrial and not sarcolemmal KATP channels are responsible for the cardioprotection of preconditioning in the human myocardium. In studies also conducted in our laboratory, we (18) have demonstrated that diazoxide causes partial depolarization of the mitochondria membrane potential as assessed by JC1 stains, which would support the argument that diazoxide induces preconditioning through its action on the mitoKATP channels and adds to the controversy of the role of sarcolemmal and mitoKATP channels in preconditioning. It is possible that species differences and variations in experimental conditions may account for the differing results;, however, it is clear that further investigation is required to fully elucidate the role played by sarcolemmal and mitoKATP channels.

Potential Limitations

The selectivity of some of the agents used to block the PKC isoforms may be questioned. V1–2 peptide is known to be a PKC-{epsilon} specific inhibitor (22); however, the selectivity of rottlerin to block PKC-{delta} has been controversial, and, whereas some investigators (15) have reported selective PKC-{delta} inhibition, others (3) have not. To overcome this problem we used the selective PKC-{delta} activator bistratene A (4, 47), which failed to precondition the tissue, thus confirming the lack of a role of PKC-{delta} in preconditioning. Although GO-6976 (28) and RO 32–0432 (48) are selective inhibitors of both PKC-{alpha} and -{beta}, the separation of the action of these two isoforms was carried out with the use of the PKC-({beta}1 + {beta}2) specific inhibitor LY-333531 (21).

Another potential limitation of our studies was the use of atrial tissue as opposed to ventricular myocardium, and therefore, any extrapolation must be conducted with caution. However, data from Yellon's laboratory (45) and from our own group (unpublished data) have demonstrated that the response to ischemia-reperfusion of ventricle and atrial tissue is similar.

Evidence in the literature has shown that the activation of PKC leads to translocation of different isoforms into various subcellular sites but has not shown that this is an important mechanism of cardioprotection by IPC. Our study, aimed to determine in heart tissue sections whether the activation of different isoforms (by determining phosphorylation-dephosphorylation states) imparts different properties on IPC, but did not investigate the subcellular locations, and this is a potential pitfall. The heart tissue model used in this study was not appropriate to establish whether or not subcellular translocation of phosphorylated PKC isoforms occurs.

Clinical Implications

The present findings may also have important clinical implications, because they indicate that it may be possible to target specific PKC isoforms to induce cardioprotection without activating other isoforms that may be involved in other cellular processes. In particular, it may be useful for eliciting cardioprotection in the diabetic heart that cannot be preconditioned by IPC or by diazoxide (12), suggesting a dysfunction at the level of mitoKATP channels. In contrast, diabetic myocardium could be preconditioned by PMA (17), which is a general PKC activator. Because evidence from this study has shown that PKC-{epsilon} is upstream of mitoKATP channels and because of the dysfunction at the level of mitoKATP channels in the diabetic myocardium, it is reasonable to conclude that PMA acts on a downstream PKC isoform, which in this case is PKC-{alpha}. Clearly, this has potential clinical/therapeutic implications for protecting the human diabetic myocardium by targeting the activation of specific PKC isoforms that potentiate preconditioning.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The work was partly funded by a grant from Diabetes United Kingdom (RD01/0002329) and by a personal contribution from Professor Manuel Galiñanes.



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Fig. 3. Dose response to PKC-{beta} inhibition of IPC with LY-333531 on CK leakage (A) and MTT reduction (B). Data are expressed as means ± SE of n = 6 tissue sections/group.

 


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Fig. 4. Dose response to PKC-{epsilon} inhibition of IPC with V1–2 peptide on CK leakage (A) and MTT reduction (B). Data are expressed as means ± SE of n = 6 tissue sections/group. *P < 0.05 vs. IPC alone.

 

    ACKNOWLEDGMENTS
 
We acknowledged the technical support and the advice of Dr. Alan Fowler and the comments and suggestions of Dr. James Downey.


    FOOTNOTES
 

Address for reprint requests and other correspondence: Manuel Galiñanes, Integrative Human Cardiovascular Physiology and Cardiac Surgery Unit, Dept. of Cardiovascular Sciences, Univ. of Leicester, Glenfield Hospital, Groby Rd., Leicester LE3 9QP, UK (E-mail: mg50{at}le.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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