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

Abstract

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 are 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 mice with reduced expression of PDK1 to study the role of PDK1 in preconditioning. Whole heart and single cell models of preconditioning demonstrated that the hearts and cardiac cells from PDK1 hypomorphic mice could not be preconditioned. The cardioprotective effect of PDK1 was not related to the effect that preconditioning has on sarcolemmal membrane action potential as revealed by di-8-ANEPPS, a sarcolemmal-potential sensitive dye, and laser confocal microscopy. In contrast, experiments with JC-1, a mitochondrial membrane potential-sensitive dye, has demonstrated that intact PDK1 levels were required for preconditioning-mediated regulation of mitochondrial membrane potential. Western blotting combined with functional experiments have shown that intact PDK1 levels were required for preconditioning-induced phosphorylation of protein kinase B (PKB), glycogen synthase kinase-3beta (GSK-3beta), and cardioprotection. We conclude that PDK1 mediates preconditioning in the heart by regulating activating PKB-GSK-3beta to regulate mitochondrial but not sarcolemmal membrane potential. 3'Phosphoinositide-dependent kinase-1 (PDK1) is essential for ischemic preconditioning of the myocardium.

Figure 1

PDK1^fl/− hearts cannot be preconditioned against ischemia. Typical photographs of myocardial slices and corresponding graphs from PDK1^fl/+ and PDK1^fl/− exposed to ishemia-reperfusion. Infarcted areas are pale/gray, whereas viable myocardium is dark/red. In graphs, myocardial infarct size is expressed as a percentage of area at risk zone. Each bar represent mean ± sem. *P < 0.05 (as determined by the t test).

Figure 2

PDK1^fl/− cells cannot be preconditioned against hypoxia. Laser confocal images and a corresponding graph of PDK1^fl/+ and PDK1^fl/− cells exposed to 30-min-long hypoxia. Rod and rounded shape indicates that cell is alive or dead, respectively. White bar represents 25 μm. Graph depicts number of cells that survived/died in hypoxia. *P < 0.05 (as determined by χ² test).

Figure 3

The response of cardiac action membrane potential to hypoxia with and without preconditioning does not differ between PDK1^fl/+ and PDK1^fl/− mice. Original line-scans of di-8-ANEPPS-loaded cardiomyocytes and corresponding time-courses and bar graphs for nonpreconditioned and preconditioned PDK^fl/+ and PDK1^fl/− cardiomyocytes under control conditions and hypoxia. AU = arbitrary units. Each bar represents mean ± sem. *P < 0.05 (as determined by t test). Doted lines visualize action potential duration under control conditions.

Figure 4

Preconditioning induces depolarization of cardiac mitochondrias in PDK1^fl/+, but not in PDK1^fl/− mice. Original images of cardiomyocytes loaded with JC-1 and corresponding graphs under depicted conditions. The disappearance of red color is indicative of mitochondrial membrane depolarization. Horizontal white bar represents 35 μm. Each bar represent mean ± sem. *P < 0.05 (as determined by ANOVA followed by t test).

Figure 5

Preconditioning inhibits late and dramatic mitochondrial membrane depolarization and cell death in PDK1^fl/+, but not PDK1^fl/−, mice. A) Original images of preconditioned PDK1^fl/+ and PDK1^fl/− cardiomyocytes loaded with JC-1 exposed to hypoxia. Horizontal white bar represents 30 μm. B) Time course corresponding to (A).

Figure 6

Preconditioning phosphorylates PKB in PDK1^fl/+ hearts and inhibits dephosphorylation of GSK-3β. The effect of ischemia on phosphorylation of PKB and GSK-3β. Original Western blots with total PKB (A), phospho-PKB (B), total GSK-3β (C), and phospho-GSK-3β (D) antibodies applied on extracts from hearts under control conditions (control), and those exposed to 20-min-long ischemia without preconditioning (ischemia), to four cycles of ischemia/reperfusion (time 0 in preconditioning), 20-min-long ischemia after the preconditioning (ischemia after preconditioning) and treated with insulin (insulin), and corresponding graphs. Each bar represents mean ± sem. *P < 0.05 (as determined by ANOVA followed by t test).

Figure 7

Insulin protects PDK1^fl/+ and PDK1^fl/− cells against hypoxia, while wortmannin abolishes any difference between PDK1^fl/+ and PDK1^fl/− cells in the effect of preconditioning. Original images of cardiomyocytes loaded with JC-1 under depicted conditions. Each frame in each row corresponds to 0 min, 30 min, 60 min, and 90 min, respectively. Graphs on the right show time course of diameter ratio or JC-1 ratio (essentially the same result have been obtained in two more independent experiments).