AMP-activated protein kinase mediates preconditioning in cardiomyocytes by regulating activity and trafficking of sarcolemmal ATP-sensitive K(+) channels

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 is complex and yet to be fully defined. AMP-activated kinase (AMPK) is activated in cells under conditions associated with ATP depletion and increased AMP/ATP ratio. In the present study, we have taken advantage of a cardiac phenotype overexpressing a dominant negative form of the alpha2 subunit of AMPK to analyze the role, if any, that AMPK plays in preconditioning the heart. We have found that myocardial preconditioning activates AMPK in wild type, but not transgenic mice. Cardiac cells from transgenic mice could not be preconditioned, as opposed to cells from the wild type. The cytoprotective effect of AMPK was not related to the effect that preconditioning has on mitochondrial membrane potential as revealed by JC-1, a mitochondrial membrane potential-sensitive dye, and laser confocal microscopy. In contrast, experiments with di-8-ANEPPS, a sarcolemmal-potential sensitive dye, has demonstrated that intact AMPK activity is required for preconditioning-induced shortening of the action membrane potential. The preconditioning-induced activation of sarcolemmal K(ATP) channels was observed in wild type, but not in transgenic mice. HMR 1098, a selective inhibitor of sarcolemmal K(ATP) channels opening, inhibited preconditioning-induced shortening of action membrane potential as well as cardioprotection afforded by AMPK. Immunoprecipitation followed by Western blotting has shown that AMPK is essential for preconditioning-induced recruitment of sarcolemmal K(ATP) channels. Based on the obtained results, we conclude that AMPK mediates preconditioning in cardiac cells by regulating the activity and recruitment of sarcolemmal K(ATP) channels without being a part of signaling pathway that regulates mitochondrial membrane potential.

Fig. 1

Intact AMPK is essential for cardiac preconditioning. Bar graphs. The activity of α1 and α2 isoforms of AMPK under depicted conditions in wild type and transgenic mice. Each bar represents mean±SEM (n=3 for each bar). *P<0.05. Line and scatter plot graph. Time-course of cellular survival under hypoxia for depicted experimental groups. Each point represent mean±SEM (n=3 for each point).

Fig. 2

The response of mitochondrial membrane potential to hypoxia does not differ between mice expressing wild type and dominant negative α2 AMPK. Original images of cardiomyocytes loaded with JC-1 (A) and corresponding graphs (B) under depicted conditions. The disappearance of red colour is indicative of mitochondrial membrane depolarization. Magnification 40×. Each bar represent mean±SEM (n=5-7). *P<0.05.

Fig. 3

The response of mitochondrial membrane potential to hypoxia after preconditioning does not differ between mice expressing wild type and dominant negative α2 AMPK. A: Original images of cardiomyocytes loaded with JC-1 before (control) and after preconditioning (IPC), and corresponding graphs. Magnification 40×. Each bar represent mean±SEM (n=4). *P<0.05. B: Original images of preconditioned cardiomyocytes loaded with JC-1 exposed to hypoxia and corresponding graphs. Magnification 40×. Each bar represent mean±SEM (n=4). *P<0.05. C: A typical example of JC-1 ratio dynamics in cell death. Original images of a cardiomyocyte loaded with JC-1 under hypoxia and corresponding time-course.

Fig. 4

Preconditioning shortens action membrane potential duration during hypoxia in wild type. Original line-scanes of di-8-ANEPPS-loaded cardiomyocytes, corresponding time-courses, and bar graphs under depicted conditions. AU, arbitrary units. Each bar represents mean±SEM (n=4). *P<0.05.

Fig. 5

Preconditioning fails to shorten action membrane potential duration during hypoxia in mice expressing dominant negative α2 AMPK. Original line-scanes of di-8-ANEPPS-loaded cardiomyocytes, corresponding time-courses, and bar graphs under depicted conditions. AU, arbitrary units. Each bar represents mean±SEM (n=4).

Fig. 6

Preconditioning activates sarcolemmal K_ATP channels in wild type, but not transgenic, cardiomyocytes. A: Typical superimposed whole cell currents at membrane potential +60 mV before and after preconditioning in wild type and transgenic cardiomyocytes. The membrane potential was held at −40 mV and the current was evoked by a 400 msec current step (to +80mV). Dotted line represents zero current line. B: Original line-scanes of di-8-ANEPPS-loaded cardiomyocytes and corresponding time-courses under depicted conditions in the presence of HMR 1098 (30 μM). AU, arbitrary units. Each bar represents mean±SEM.

Fig. 7

HMR 1098, a selective inhibitor of sarcolemmal K_ATP channels opening, inhibits preconditioning in cardiomyocytes from mice expressing wild type AMPK without having any effect on cells expressing dominant negative α2 AMPK. Time-course of cellular survival under hypoxia for depicted experimental group. Each point represent mean±SEM (n=3-4).

Fig. 8

The levels of sarcolemmal K_ATP channels are not affected by AMPK. A: Representative progress curves for the real-time PCR amplification of Kir 6.2 and SUR2A, and corresponding graphs showing cycle threshold for the real-time PCR amplification of Kir 6.2 and SUR2A. Each bar represents mean±SEM (n=3). B: Western blot with anti-Kir6.2 antibody of anti-SUR2 immunoprecipitate from cardiac membrane fractions obtained from wild type (WT) and transgenic (TG) mice and the corresponding graph. Each bar represent mean±SEM (n=3). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 9

Intact α2 AMPK is required for preconditioning-induced recruitment of sarcolemmal K_ATP channels. Western blot with anti-Kir 6.2 antibody of anti-SUR2 immunoprecipitate from cardiac membrane and cytosolic fractions obtained from wild type (WT) and transgenic (TG) mice under control conditions (A) and after preconditioning (B), and corresponding graphs depicting membrane/cytosol ratios. Each bar represent mean±SEM (n=3). *P<0.05.