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3'phosphoinositide-dependent kinase-1 is essential for ischemic preconditioning of the myocardium

Grant R. Budas^*, Andrey Sukhodub^*, Dario R. Alessi and Aleksandar Jovanovi^*,1

^* Maternal and Child Health Sciences, Ninewells Hospital and Medical School, and

MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee, UK

¹Correspondence: Maternal and Child Health Sciences, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, UK. E-mail: a.jovanovic{at}dundee.ac.uk

SPECIFIC AIMS

Brief periods of ischemia and reperfusion that precede sustained ischemia lead to a reduction in myocardial infarct size. This phenomenon, known as ischemic preconditioning, is mediated by signaling pathway(s) that have yet to be fully defined. 3'-phosphoinositide-dependent kinase-1 (PDK1) has been implicated in numerous cellular processes. However, the involvement of PDK1 in preconditioning has yet to be elucidated. Studying PDK1 is not as straightforward as it is for the majority of kinases, due to the lack of a specific inhibitor of PDK1. Therefore, we have taken advantage of PDK1 hypomorphic mutant (PDK1^fl/–) mice with reduced expression of PDK1 to study the role of PDK1 in preconditioning.

PRINCIPAL FINDINGS

1. Preconditioning fails to protect PDK1^fl/– mice against myocardial infarction
Ischemia-reperfusion induced myocardial infarction in mice with normal PDK1 activity (PDK1^fl/+ mice) that was 52.3 ± 8.2% of the area at risk zone (Fig. 1 ). Preconditioning (four cycles of 5 min ischemia and 5 min reperfusion) significantly decreased the size of myocardial infarction induced by ischemia-reperfusion in these mice (Fig. 1) . When hearts from PDK1^fl/– were exposed to ischemia-reperfusion, the size of myocardial infarction was similar to those in PDK1^fl/+ mice (Fig. 1) . However, as opposed to PDK1^fl/+ mice, ischemic preconditioning was ineffective in hearts from PDK1^fl/– mice (Fig. 1) .

View larger version (75K): [in this window] [in a new window]   Figure 1. PDK1fl/– hearts can not be preconditioned against ischemia. Typical photographs of myocardial slices and corresponding graphs from PDK1fl/+ and PDK1fl/– exposed to ishemia-reperfusion. Infarcted areas are pale/gray, while viable myocardium is dark/red. In graphs, myocardial infarct size is expressed as a percentage of area at risk zone. Each bar represents mean ± SEM. *P < 0.05.

2. Cardiomyocytes from PDK1^fl/– mice can not be preconditioned
In PDK1^fl/+ mice, only 1 cell out of 7 nonpreconditioned cells has survived 30 min-long hypoxia. Similar results were obtained with PDK1^fl/–cells. An episode of 5 min-long hypoxia/5 min-long reoxygenation (preconditioning) administered prior to sustained hypoxia significantly increased the survival of cardiomyocytes from PDK^fl/+ mice. In contrast, preconditioning failed to protect PDK1^fl/– cardiomyocytes against hypoxia.

3. PDK1-mediated cardioprotection is not associated with changes in sarcolemmal membrane potential
It has been shown that preconditioning induces activation of sarcolemmal K_ATP channels, which would shorten action membrane potential duration and decrease intracellular Ca²⁺ loading and lead to cardioprotection. Preconditioning activates sarcolemmal K_ATP channels after 4 min of hypoxia in single beating cardiomyocytes. Therefore, we have measured membrane potential under control conditions and after 4 min of hypoxia with and without preconditioning. In cardiomyocytes from both PDK1^fl/+ and PDK1^fl/– mice, hypoxia induced prolongation of action membrane potential. Preconditioning prevented hypoxia-induced prolongation of action membrane potential in both phenotypes.

4. PDK1-mediated cardioprotection in preconditioning is associated with regulation of mitochondrial membrane potential
It has been proposed that preconditioning may activate putative mitochondrial K_ATP channels and/or inhibit the mitochondrial permeability transition pore. In both cases, the mitochondrial membrane potential would change. Therefore, we have monitored mitochondrial membrane potential in vivo in PDK1^fl/+ and PDK1^fl/– mice using the fluorescent dye JC-1, a ratiometric dye that is a reliable indicator of mitochondrial membrane potential. During first 20 min of hypoxia, the JC-1 ratio did not significantly change in nonpreconditioned cardiomyocytes regardless of their phenotype. However, in preconditioned PDK1^fl/+ cardiomyocytes, the JC-1 ratio steadily rose during hypoxia to reach statistical significance after 20 min. This effect of preconditioning was not observed in PDK1^fl/– mice. In preconditioned PDK1^fl/+ cells the rate of mitochondrial membrane depolarization was slow and associated with prolonged cell survival (Fig. 2 ). This finding was in contrast to preconditioned PDK1^fl/– cells where mitochondrial membrane depolarization did not occur in the first 20 min, but when it did it was fast and associated with cell death (Fig. 2) .

View larger version (15K): [in this window] [in a new window]   Figure 2. Preconditioning inhibits late and dramatic mitochondrial membrane depolarization and cell death in PDK1fl/+, but not PDK1fl/–, mice. A) Original images of preconditioned PDK1fl/+ and PDK1fl/– cardiomyocytes loaded with JC-1 exposed to hypoxia. Horizontal white bar represents 30 µm. B) Time course corresponding to (A).

5. PDK1/PKB/GSK-3ßbeta; cascade mediates preconditioning
Western blotting with protein kinase B (PKB) total antibody (Ab) demonstrated that levels of PKB in the heart remained steady under any of the experimental conditions and in both types of mice. When phospho-PKB (Thr308) Ab was used it was revealed that preconditioning induces phosphorylation of PKB in PDK1^fl/+, but not in PDK1^fl/– mice. The main target of PKB is glycogen synthase kinase-3 (GSK-3), an enzyme that has also been involved in cardioprotection. In PDK1^fl/+ mice, preconditioning was successful to maintain the degree of phosphorylation of GSK-3ßbeta; seen in control hearts. In contrast, in PDK1^fl/– mice, preconditioning was associated with a significant decrease in levels of phosphorylated form of GSK-3ßbeta;. In both cell types insulin (25 mU/ml) has significantly increased cell survival in hypoxia (all cells tested has survived hypoxia >90 min), which was associated with gradual mitochondrial membrane depolarization. Conversely, wortmannin (5 µM), an inhibitor of phosphoinositide 3-kinase (PI3K), has abolished any difference in response of preconditioned PDK1^fl/+ and PDK1^fl/–cells to hypoxia.

CONCLUSIONS AND SIGNIFICANCE

In conclusion, the present study has shown that full PDK1 activity is essential for preconditioning-induced cardioprotection. Furthermore, the signaling cascade PDK1/PKB/GSK-3ßbeta; is involved in regulation of mitochondrial membrane potential which in turn results in increased cellular resistance to ischemia/hypoxia (Fig. 3 ).

View larger version (8K): [in this window] [in a new window]   Figure 3. Schematic diagram summarizing conclusions of the present study.

Taken together, the data presented in this study suggest that ischemic heart disease in humans could perhaps be treated by strategies that trigger the activity of PDK1.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6252fje

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				Z. Cai, H. Zhong, M. Bosch-Marce, K. Fox-Talbot, L. Wang, C. Wei, M. A. Trush, and G. L. Semenza Complete loss of ischaemic preconditioning-induced cardioprotection in mice with partial deficiency of HIF-1{alpha} Cardiovasc Res, February 1, 2008; 77(3): 463 - 470. [Abstract] [Full Text] [PDF]

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-6252fjev1 20/14/2556    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Budas, G. R. Articles by Jovanovic, A. Search for Related Content PubMed PubMed Citation Articles by Budas, G. R. Articles by Jovanovic, A.