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Hypoxia-induced preconditioning in adult stimulated cardiomyocytes is mediated by the opening and trafficking of sarcolemmal K_ATP channels ¹

GRANT R. BUDAS, SOFIJA JOVANOVI, RUSSELL M. CRAWFORD and ALEKSANDAR JOVANOVI²

Maternal and Child Health Sciences, Tayside Institute of Child Health, Ninewells Hospital & Medical School, University of Dundee, Dundee, Scotland, UK

² Correspondence: Maternal and Child Health Sciences, Tayside Institute of Child Health, Ninewells Hospital & Medical School, University of Dundee, Dundee, DD1 9SY, Scotland, UK. E-mail: a.jovanovic{at}dundee.ac.uk

SPECIFIC AIMS

Sarcolemmal ATP-sensitive K⁺ (K_ATP) channels, composed of Kir6.2 and SUR2A subunits, couple the metabolic status of a cell with membrane excitability. Based on recent studies, we hypothesized that trafficking and activity of sarcolemmal K_ATP channels may be involved in mediating preconditioning (brief periods of ischemia/reoxygenation that precede sustained ischemia lead to a reduction in myocardial infarct size) in the heart. We took advantage of a model of preconditioning that uses adult cardiomyocytes stimulated to beat and examined whether activity and trafficking of K_ATP channels are involved in the intracellular signaling of preconditioning in the heart.

PRINCIPAL FINDINGS

1. Hypoxia induces death of cardiomyocytes without affecting levels of sarcolemmal K_ATP channel subunits
Single beating guinea pig cardiomyocytes responded to hypoxia with intracellular Ca²⁺ loading and irreversible hypercontracture, indicative of cell death. The average time of survival in cells exposed to hypoxia was 16.8 ± 2.8 min. To assess fluctuations in the number of sarcolemmal K_ATP channels during hypoxia, membrane fraction of cardiomyocytes was immunoprecipitated with anti-Kir6.2 antibody and probed with the anti-SUR2A antibody, and vice versa. These experiments revealed that levels of sarcolemmal K_ATP channel subunits remained steady during hypoxia.

2. A single episode of hypoxia/reoxygenation promotes survival of cardiomyocytes in hypoxia and increases the number of sarcolemmal K_ATP channels
When beating cardiomyocytes were exposed to 5 min hypoxia/5 min reoxygenation (preconditioning) before long-lasting hypoxia, the average time of survival was increased to 46.0 ± 8.3 min. Immunoprecipitation, followed by Western blot, demonstrated that levels of both subunits were increased specifically after a hypoxia/reoxygenation episode.

3. Preconditioning-induced increase in sarcolemmal K_ATP channels can be visualized and it is synchronized with the channels opening
To visualize the sarcolemmal K_ATP channel, we used laser confocal microscopy and anti-SUR2A antibody labeled with anti-sheep fluorescein. Measurement of the intensity of sarcolemma fluorescence revealed a pattern similar to that obtained with Western blot; the level of the SUR2A subunit was significantly increased after hypoxia/reoxygenation at the beginning of sustained hypoxia (Fig. 1 A, B). We applied perforated patch clamp electrophysiology to determine when, in relation to the observed increase in number of sarcolemmal K_ATP channels, K_ATP channels become active. During preconditioning (brief hypoxia and reoxygenation) there were no changes in steady-state current, and current density varied between 1 and 1.3 pA/pF (Fig. 1C ). At the beginning of long-lasting hypoxia, the current rose from 1.3 pA/pF to 10.1 pA/pF in 60 s, to 13.1 pA/pF in the next 60 s, and remained at this level until the end of the experiment (Fig. 1C ). On average, current density was 1.5 ± 0.5 pA/pF under control conditions and 9.1 ± 2.1 pA/pF at the beginning of sustained hypoxia (P=0.01, n=5, Fig. 1E ). When cells were exposed to hypoxia without preconditioning, current density was 3.6 ± 0.9 pA/pF after 15 min of hypoxia, significantly lower than in preconditioned cells (Fig. 1D, E , P=0.028, n=5).

View larger version (38K): [in this window] [in a new window]   Figure 1. Preconditioning induces trafficking and opening of sarcolemmal KATP channels in cardiomyocytes. A) Original images and corresponding graphs of cardiomyocytes stained with anti-SUR2A antibody labeled with fluorescein and collected at the depicted stages of experimental protocol. Graphs depict intensity of fluorescence drown against distance points in sarcolemma (each graph corresponds to the image above). B) Each bar represents mean ± SE (n=15–17). *P< 0.01. C) Line and scatter graph: time course of whole cell current density at membrane potential of +80 mV in a cardiomyocyte under control conditions, during preconditioning (5 min hypoxia/5 min reoxygenation, and in the first 5 min of long-lasting hypoxia). Insets: typical whole cell currents at membrane potential +80 mV during different stages of preconditioning. Membrane potential was held at –40 mV and the current was evoked by a 400 ms current step (to +80mV). Dotted line represents zero current line. D) Time course of whole cell current density at membrane potential of +80 mV in a cardiomyocyte under control conditions and hypoxia. Insets: typical whole cell currents at membrane potential +80 mV under control conditions and during hypoxia. Membrane potential was held at —40 mV and the current was evoked by a 400 ms current step (to +80mV). Dotted line represents zero current line. E) Average current density of non-preconditioned and preconditioned cardiomyocytes exposed to hypoxia. Bars represent mean ± SE (n=4–5); *P < 0.05 compared with preconditioned cells.

4. Inhibition of the opening of sarcolemmal K_ATP channels during sustained hypoxia abolishes preconditioning
Patch clamp electrophysiology suggested that opening of these channels occurs after an episode of brief hypoxia/reoxygenation. We tested whether inhibition of the activity of sarcolemmal K_ATP channels at this or any other stage of the experiment would abolish the effect of preconditioning. When glybenclamide (10 µM), a prototype antagonist of K_ATP channels, was present throughout the experiment, the beneficial effect of preconditioning was abolished. Survival time was significantly shorter when glybenclamide was present during long-lasting hypoxia but not at any other stage of experimental protocol. Similar results were obtained with a selective antagonist of sarcolemmal K_ATP channels, HMR 1098 (30 µM). When 5-HD (50 µM), an antagonist of putative mitochondrial K_ATP channels, was present throughout, the beneficial effect of preconditioning was abolished; 5-HD had no effect when it was applied at specific stages of experiments.

5. Inhibition of sarcolemmal K_ATP channels trafficking blocks preconditioning
Based on earlier studies, the increase in number of sarcolemmal K_ATP channels was expected to lead to a cellular phenotype more resistant to metabolic stress. To test this hypothesis, we performed experiments on cardiomyocytes treated with a cocktail of drugs known to inhibit protein trafficking: 5 µg/mL brefeldin A, 5 µM colchicines, and 20 µM nocodazole. This treatment did not affect the general sensitivity of cardiomyocytes. In treated cardiomyocytes, preconditioning did not increase the number of K_ATP channels in sarcolemma (Fig. 2 B, E). Under these conditions, an episode of brief hypoxia/reoxygenation did not promote the survival of cardiomyocytes exposed to hypoxia (Fig. 2C, D) .

View larger version (46K): [in this window] [in a new window]   Figure 2. Prevention of sarcolemmal KATP channels trafficking inhibits preconditioning. A) Epifluorescent images of Fura-2-loaded non-preconditioned cardiomyocytes pretreated with protein trafficking inhibitory cocktail (5 µg/mL brefeldin A, 5 µM colchicines, and 20 µM nocodazole) exposed to hypoxia. B) Original images and corresponding graphs of cardiomyocytes pretreated with protein trafficking inhibitory cocktail stained with anti-SUR2A antibody labeled with fluorescein and collected at depicted stages of experiments. Graphs depict intensity of fluorescence drawn against distance points in sarcolemma (each graph corresponds to the image above). C) Epifluorescent images of Fura-2-loaded preconditioned cardiomyocytes pretreated with protein trafficking inhibitory cocktail exposed to hypoxia. D) The average survival time ± SEM of non-preconditioned and preconditioned cardiomyocytes exposed to hypoxia under control conditions and when pretreated with protein trafficking inhibitory cocktail (n=4–10). *P < 0.01. E) Average intensity of fluorescence per unit of sarcolemma length ± SEM for cardiomyocytes pretreated with protein trafficking inhibitory cocktail collected at the depicted stages of experimental protocol (n=13–19).

CONCLUSIONS

In the present study we have demonstrated that stimulation of sarcolemmal K_ATP channel trafficking and channel activation by an episode of brief hypoxia/reoxygenation are essential for preconditioning in adult beating cardiomyocytes. This is the first account of an ion channel trafficking involved in preconditioning and cardioprotection.

Several lines of evidence suggest that sarcolemmal K_ATP channels are cardioprotective: coexpression of Kir6.2 with SUR2A confers resistance against metabolic stress in otherwise stress-sensitive cells; and ischemic preconditioning cannot be conferred in transgenic animals lacking sarcolemmal K_ATP channels. It was recently uncovered that trafficking of sarcolemmal K_ATP channels is regulated by PKC and adenosine, compounds involved in cardioprotective signaling. Using immunoprecipitation/Western blot and immunofluorescence/laser confocal microscopy, we have found that preconditioning induced an increase in the number of sarcolemmal K_ATP channels not observed when cardiomyocytes were exposed to hypoxia in the absence of preconditioning. As PKC is involved in preconditioning, it may at first sight appear that the results obtained disagree with earlier studies that reported PKC-mediated inhibition of K_ATP channels trafficking. However, it is known that activation and inhibition of certain subtypes of PKC may mediate cardioprotection and preconditioning. Thus, bearing in mind the complexity of signaling pathways in preconditioning that involve inhibition and activation of different subtypes of PKC, the results do agree with the suggestion that PKC may regulate trafficking of K_ATP channels and that recruitment of sarcolemmal K_ATP channels contributes to the cardioprotection afforded by preconditioning.

The preconditioning-induced increase in the number of sarcolemmal K_ATP channels had a time course similar to channel activation. Activation of sarcolemmal K_ATP channels was suggested to mediate preconditioning more than a decade ago, but this notion was challenged when it was discovered that some K_ATP channel-opening drugs may affect membrane potential of the mitochondria, which had been ascribed to the effect on putative mitochondrial K_ATP channels. Results obtained in the present study did not provide a definite answer regarding the involvement of mitochondrial K_ATP channels in preconditioning. In contrast, glybenclamide and HMR 1098, "nonselective" and selective antagonists of sarcolemmal K_ATP channels, respectively, abolished preconditioning when applied throughout the experiment or only during sustained hypoxia. These results suggest that activation of the channels at the beginning of sustained hypoxia is crucial for preconditioning-induced cardioprotection. This confirms the notion that the opening of these channels is essential for preconditioning; the timing of the channel opening would suggest that these channels serve primarily as end effectors rather then triggers of preconditioning.

It was recently reported that the number of expressed sarcolemmal K_ATP channels regulates the resistance to metabolic stress in cardiomyocytes. It is reasonable to assume that an acute increase in the number of sarcolemmal K_ATP channels would increase the cellular resistance to hypoxia. In support of this is our result that the inhibition of increase in number of sarcolemmal K_ATP channels abolished preconditioning. This shows that recruitment of K_ATP channels to sarcolemma is essential for preconditioning-induced cardioprotection.

In conclusion, we have demonstrated that sarcolemmal K_ATP channel activation and channel trafficking are essential for preconditioning-induced cardioprotection (Fig. 3 ). The importance of the channel trafficking in preconditioning had not been observed before, and this study may provide a basis for research that could lead to a better understanding of endogenous cardioprotective mechanisms and their exploitation in therapy for ischemic heart diseases.

View larger version (12K): [in this window] [in a new window]   Figure 3. Schematic diagram summarizing showing conclusions of the present study.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1602fje;

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This Article Full Text (PDF) All Versions of this Article: 18/9/1046 04-1602fjev1    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.