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Cardiovascular Research Advance Access first published online on October 4, 2007
This version [Corrected Proof] published online on October 31, 2007
Cardiovascular Research, doi:10.1093/cvr/cvm028
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Novel functional role of heat shock protein 90 in ATP-sensitive K+ channel-mediated hypoxic preconditioning

Jun-Dong Jiao1,{dagger}, Vivek Garg1,{dagger}, Baofeng Yang2 and Keli Hu1,3,*

1 Division of Pharmacology, College of Pharmacy, The Ohio State University, 530 Parks Hall, 500 West 12th Avenue, Columbus, OH 43210, USA
2 Department of Pharmacology, Harbin Medical University, Harbin, Heilongjiang 150086, China
3 Institute of Mitochondrial Biology, The Ohio State University, Columbus, OH 43210, USA

* Corresponding author. Tel: +1 614 292 5433; fax: +1 614 292 9083. E-mail address: hu.175{at}osu.edu

Time for primary review: 31 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Aims: ATP-sensitive K+ (KATP) channels are implicated in the protective effect of ischaemic preconditioning (IPC). Kir6.2 has been shown to be involved in the cardioprotection of IPC. However, the mechanism by which Kir6.2-containing KATP channels protect the heart is still largely unknown. The present study was designed to explore the potential mechanism involved in KATP channel-mediated cardioprotection.

Methods and results: Cellular models of hypoxic preconditioning (HP) from rat heart-derived H9c2 cells and adult rat cardiomyocytes were employed. Dominant negative and small interfering RNA (siRNA) technology were utilized in combination with biochemical, immunofluorescent, and cell viability assays. The cell viability study revealed that HP significantly increased the viable cells after prolonged hypoxia and reoxygenation. This protective effect was prevented by expression of dominant negative Kir6.2AAA, siRNA targeting Kir6.2, or the KATP channel inhibitor 5-hydroxydecanoate. Further, our data showed that inhibiting heat shock protein 90 (HSP90) function with the HSP90 inhibitor geldanamycin or HSP90 expression with siRNA completely inhibited the protection of HP. We found that HSP90 was associated with Kir6.2 and its activity was linked to mitochondrial targeting of Kir6.2.

Conclusion: We demonstrate that Kir6.2 is critical in HP of cardiomyocytes. Importantly, we show that HSP90 is involved in KATP-mediated cytoprotection, possibly by promoting mitochondrial targeting of Kir6.2.

KEYWORDS Preconditioning; K-ATP channel; Mitochondria; Heat shock protein

Received April 23, 2007; revised September 1, 2007; accepted September 27, 2007


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
ATP-sensitive K+ (KATP) channels are implicated in the cardioprotection of ischaemic preconditioning (IPC).1–3 Although the cardiac KATP channels at both sarcolemmal (sarcKATP)4,5 and mitochondrial membrane (mitoKATP)6,7 have been suggested to play an important role in cardioprotection, the precise mechanism involved in KATP channel-mediated cardioprotection is largely unknown.

KATP channels on the sarcolemmal membrane are heterooctamers consisting of two structurally unrelated proteins: four pore-lining subunits that belong to the Kir6 subfamily of inwardly rectifying potassium channels and four regulatory subunits of the ATP-binding cassette superfamily.8–11 Although the structure of the sarcKATP channel has been clearly delineated and contains Kir6.2 and SUR2A, the molecular identity of the mitoKATP remains elusive.

The pore-forming subunits of KATP channels, Kir6.2 and Kir6.1, contribute to the diversity of KATP channels and are expressed in the heart. In addition, both subunits have been shown to be present in heart mitochondria.12–18 Interestingly, recent studies have shown that IPC provides no cardioprotection of Kir6.2 knockout mice. These animals did not respond to IPC with reduced infarct size or increased rates for ATP turnover and ATP synthesis, a mitochondrial function.19 However, the importance of Kir6.2 in IPC may have been exaggerated by the rapid heart rate in the murine model as the authors indicated.5 Nevertheless, the mechanism involved in KATP channel-mediated cardioprotection remains elusive. To define the molecular mechanism underlying KATP channel-mediated preconditioning, we employed the cellular model of hypoxic preconditioning (HP) and combined dominant negative and small interfering RNA (siRNA) technology with biochemical analysis, immunofluorecence images, and cell viability assay. Our data provided the first evidence in a cardiac myocyte model that Kir6.2 but not Kir6.1 is involved in the protection of HP. Importantly, our observations revealed a novel functional role of HSP90 involved in KATP channel-mediated HP.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Funding
 References
 
2.1 DNA constructs
Dominant negative constructs Kir6.2AAA and Kir6.1AAA, which were generated by mutating the pore region Gly-Phe-Gly to Ala-Ala-Ala and subcloned into a pEGFP vector, were kindly provided by Dr William A. Coetzee (New York University, New York, NY, USA). Adenoviral vector expressing Kir6.2AAA, Kir6.1AAA or GFP only was generated by Vector BioLabs (Philadelphia, PA, USA).

2.2 Small interfering RNA and transfection
The siRNA oligonucleotides targeting Kir6.2, Kir6.1 or HSP90 ({alpha} and ß isoforms) were purchased from Ambion Inc. (Austin, TX, USA). A negative control siRNA (scrambled) was included to monitor non-specific effects. Forty-eight to 72 h after transfection, Western blot was carried out to examine the knockdown of targeted proteins.

2.3 Cell culture of H9c2 cells and transfection
Rat heart-derived H9c2 cells were cultured in Dulbecco’s modified Eagle’s medium DMEM/F12 supplemented with 10% fetal bovine serum, 2 mM glutamine, and penicillin–streptomycin. Cells were transfected with cDNA using FuGENE6 or with siRNA using X-Tremegene siRNA transfection reagent.

2.4 Cardiomyocytes isolation and culture
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). Adult rat ventricular myocytes were isolated from adult Wistar rats (250–300 g) by enzymatic dissociation.20–22 The digested cardiomyocytes were plated in 35 mm Petri dishes precoated with laminin (1 µg/cm2) and maintained in culture medium M199 with 2% fetal bovine serum and 1% penicillin/streptomycin at 37°C.

2.5 Hypoxic preconditioning
Hypoxia was induced by incubating the cells in an airtight chamber in which O2 was replaced by N2 with glucose-free Tyrode’s solution that contains (in mmol/L) 139 NaCl, 4.7 KCl, 0.5 MgCl2, 1.0 CaCl2, and 5 HEPES, pH 7.4, at 37°C. The general experimental protocols employed are described below:

Group I (Control): The cells were incubated in Tyrode’s solution during the entire experimental period.

Group II (H/R): Cells were subjected to 120 min hypoxia and 160 min reoxygenation for H9c2 cells, and 90 min hypoxia and 120 min reoxygenation for rat adult cardiomyocytes.

Group III (HP + H/R): Cells were exposed to HP prior to H/R. HP was induced by exposing adult rat cardiomyocytes or H9c2 cells to 10 min of hypoxia and 30 min of reoxygenation.

2.6 Cell viability assessment
Trypan blue staining was used to distinguish viable cells from dead cells by haemocytometer cell counts. Cells that are able to exclude the stain were considered viable, and the percentages of non-blue cells over total cells were used as an index of viability. Cells were counted <15 min to minimize variability associated with changes in the ratio of stained/unstained cells over time.

2.7 Preparation of mitochondria
Intact mitochondria were prepared by differential centrifugation.18 Isolated cardiomyocytes or H9c2 cells were fractionated after treatment with or without HP. For immunofluorescence staining, cells were stained with mitochondrial-specific stain MitoTracker (250 nM) before fractionation. For co-immunoprecipitation and Western blot, isolated mitochondria were further purified by ultracentrifugation with 30% percoll.18

2.8 Immunofluorescent microscopy
As described previously,18 isolated mitochondria were fixed, blocked, permeablized, and then labelled with anti-Kir6.2 for 2 h and fluorescence-conjugated secondary antibody for 1 h. Immunofluorescence was visualized with a conventional fluorescence microscope (Nikon).

2.9 Co-immunoprecipitation and Western blot
Percoll-purified mitochondrial suspension was incubated with primary or control IgG overnight at 4°C. Antigen–antibody complexes were captured with r-protein-G agarose (4°C, 2 h). Agarose beads were washed four times with solubilization buffer before removal of bound proteins by boiling in SDS sample buffer. Samples were resolved by SDS–PAGE, transferred onto nitrocellulose membrane, and analysed by probing with various antibodies. Immunoblot analysis was carried out for the total cell homogenate and percoll-purified mitochondrial fraction as described previously.18

2.10 Data analysis
Group data were presented as means ± SE. Unpaired t-test was used to compare between groups. Multiple group means were compared by ANOVA followed by LSD post hoc test. Differences with a two-tailed P < 0.05 were considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Funding
 References
 
3.1 Involvement of Kir6.2 in the protection of hypoxic preconditioning
We first examined KATP subunit expression profile in the rat heart-derived H9c2 cells and found significant expression of Kir6.2, Kir6.1, and SUR2A, but not SUR1 and SUR2B (data not shown). To determine whether KATP channel pore-forming subunits are critical in a cellular model of HP, we studied the effect of Kir6.2 or Kir6.1 on the cytoprotection of HP by reducing expression of Kir6.2 or Kir6.1 with siRNA. Western blot analysis of cell lysates harvested 48–72 h after transfection with siRNA directed against Kir6.2 or Kir6.1 revealed that both subunits were dramatically decreased (Figure 1A). A negative control siRNA (C-siRNA) did not have any effect on Kir6.2 or Kir6.1 protein level. The cell viability assay with Trypan blue staining revealed that the percentage of viable cells after H/R with 120 min of hypoxia and 160 min of reoxygenation was significantly higher in the group treated with HP compared with that in the group treated without preconditioning (Figure 1B, 82.2% ± 7.7 vs. 45.5% ± 5.9, P < 0.01). The effect was largely eliminated in cells transfected with siRNA against Kir6.2 (45.7% ± 9.4 vs. 56.2% ± 6.6, P > 0.1) but not in cells transfected with siRNA against Kir6.1 (75.7% ± 7.6 vs. 48.5% ± 8.8, P < 0.05). Transfection with negative control siRNA had no effect on the cell viability in both HP + H/R and H/R groups (data not shown).


Figure 1
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  		Figure 1 Role of Kir6.2 in hypoxic preconditioning. (A) Western blots of cell lysate from H9c2 cells transfected with nothing (C), negative control siRNA (C-siRNA) or siRNA targeting Kir6.2 or Kir6.1. (B) Quantification of cell viability in H9c2 cells transfected with or without siRNA. n = 6. (C) Fluorescence image of rat adult cardiomyocytes infected with adenoviral vector expressing GFP. (D) Quantification of cell viability in adult rat cardiomyocytes infected with or without Kir6.1AAA or 6.2AAA. n = 6, **P < 0.01, *P < 0.05 vs. H/R.

 
To determine whether Kir6.2 has a similar role in rat adult cardiomyocytes, we infected adult rat cardiomyocytes with adenoviral vector expressing pore mutants of KATP channels—Kir6.2AAA (Ad-Kir6.2AAA) or Kir6.1AAA (Ad-Kir6.1AAA). These two mutants have been shown to suppress KATP current in a dominant negative fashion.23,24 Adenoviral vector expressing green fluorescent protein (Ad-CMV-GFP) was used as a viral control. Infection efficiency of Kir6.2AAA or Kir6.1AAA in cardiac myocytes was more than 90% based on our observation of GFP expression (Figure 1C). We noticed that adult cardiomyocytes preparations were more sensitive to hypoxic injury than H9c2 cells. We therefore decreased the duration of prolonged hypoxia and reoxygenation treatments for cardiomyocytes. The adult cardiomyocytes 2–3 days after adenoviral infection were subjected to 90 min of hypoxia and 120 min of reoxygenation with and without prior HP. Similar to the results in H9c2 cells, a single episode of 10 min of hypoxia and 30 min of reoxygenation preconditioned cells from H/R injury (Figure 1D, 70.3% ± 3.6 vs. 45.5% ± 5.9, P < 0.01). However, when cells were infected with Ad-Kir6.2AAA, the percentage of viable cells in the preconditioned group (HP + H/R) was not significantly different from that in the non-preconditioned group (51.3% ± 9.2 vs. 46.8% ± 7.6, P > 0.1). Kir6.1AAA viral infection did not affect the preconditioning effect (63.2% ± 8.2 vs. 40.2% ± 4.9, P < 0.05).

3.2 Role of HSP90 in mitochondrial targeting of Kir6.2
To determine the mechanism involved in Kir6.2-containing KATP-mediated protection, we first examined the role of HSP90 and HSP70 in HP by using a selective HSP90 inhibitor geldanamycin and HSP70 inhibitor quercetin. Geldanamycin (1 µM) or quercetin (50 µM) were added 15 min before and during HP. As shown in Figure 2A, HP significantly increased the cell viability (75.7% ± 10.5 vs. 41.7% ± 9.1, P < 0.01). Inhibition of HSP90 activity by geldanamycin treatment completely prevented the protective effect of preconditioning (45.8% ± 5.6 vs. 40.04% ± 5.6, P > 0.1). In contrast, quercetin, a selective HSP70 synthesis inhibitor,25 did not prevent the protective effect of preconditioning (80.7% ± 8.2 vs. 40.5% ± 7.7, P < 0.01). Western blot analysis revealed that HP caused a moderate increase in HSP90 but no change in HSP70 protein level (Figure 2B).


Figure 2
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  		Figure 2 Effect of inhibiting HSP90 activity on mitochondrial targeting of Kir6.2 in rat adult cardiomyocytes. (A) Quantification of cell viability with or without the treatment of geldanamycin (GA) or quercetin (Quer). GA and Quer were added 15 min before and during preconditioning. n = 6, **P < 0.01 vs. H/R. (B) Western blots of cell lysate with anti-HSP90 or anti-HSP70 antibodies following hypoxic preconditioning. (C) Representative immunofluorescence images of isolated mitochondria double-labelled with anti-Kir6.2 and MitoTracker. Scale bar: 10 µM. (D) Percentage of Kir6.2-positive mitochondria. Data were normalized to control. n = 3, **P < 0.01 vs. control.

 
We have previously shown that protein kinase C increases functional Kir6.2-containing KATP channels in mitochondria.18 Since protein kinase C is a central mediator of IPC, we tested whether HP has the same effect as protein kinase C in promoting Kir6.2 translocation to mitochondria18 and whether HSP90 activity modulates this effect. Figure 2C shows representative images of isolated mitochondria double-labelled with anti-Kir6.2 antibody and MitoTracker. Three independent experiments were conducted with five to 10 images in each group. At least five regions in each image were randomly selected (as long as mitochondria were not overlapping each other) and analysed. The number of Kir6.2-positive mitochondria as shown in yellow following HP was more than that in the control group. Geldanamycin prevented preconditioning-induced mitochondrial targeting of Kir6.2. The quantitative analysis revealed that the percentage of Kir6.2-positive mitochondria (normalized to control) following HP was significantly higher than that in control group (Figure 2D, 231.4% ± 35.7 vs. control, P < 0.01). Geldanamycin treatment (HP + GA) blocked most of the HP-induced increase in mitochondrial Kir6.2 (127.6% ± 24.3 vs. control, P > 0.1) with little effect on the mitochondrial localization of Kir6.2 under control condition (Figure 2D).

It is known that HSP90 or HSP70 inhibitor has many non-specific effects. To ensure the specific role of HSP90 in HP-mediated mitochondrial translocation of Kir6.2, we studied the effect of reduced HSP90 expression with siRNA targeting HSP90 {alpha} and ß isoforms in H9c2 cells. As shown in Figure 3, HSP90 protein level was significantly reduced by siRNA. Suppression of HSP90 expression with siRNA largely eliminated the protection of HP (data not shown). The HP induced increase in mitochondrial Kir6.2 was almost completely blocked by inhibiting HSP90 expression with siRNA (Figure 3B). Similar to cardiac myocytes, the basal level of mitochondrial Kir6.2 was not reduced by siRNA targeting HSP90. Further, HSP90 protein level was not significantly changed after HP in H9c2 cells. This result was different from that in adult rat cardiomyocytes where HSP90 was slightly increased following HP.


Figure 3
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  		Figure 3 Effect of inhibiting HSP90 expression on mitochondrial targeting of Kir6.2 in H9c2 cells. (A) Western blot of cell lysate from H9c2 cells transfected with control siRNA (C-siRNA) and siRNA targeting HSP90 ({alpha} and ß isoforms). (B) Quantification of Kir6.2-positive mitochondria. Data were normalized to the control. n = 3, **P < 0.01 vs. control. (C) Western blot of cell lysate with anti-HSP90 antibody.

 
3.3 Relationship between HSP90 and Kir6.2 in hypoxic preconditioning
To determine the effect of HP on Kir6.2 protein level, we performed Western blot analysis. Using the anti-Kir6.2 antibody, a single band at 40 kDa was detected in the prohibitin-enriched mitochondrial fraction (M) as well as in the total cell homogenate (T). The band in the mitochondrial fraction from cardiomyocytes treated with HP exhibited a more intense signal than that from cardiomyocytes without HP (Figure 4A). However, there was no difference in the Kir6.2 band from total cell homogenate with and without HP. With the anti-SUR2A antibody, we detected a very faint band in the mitochondrial fraction (Figure 4C).


Figure 4
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  		Figure 4 Association between HSP90 and Kir6.2. (A) Western blot of total cellular (T) and mitochondrial fraction (M) from cells treated with or without hypoxic preconditioning with anti-Kir6.2 antibody. (B) Immunoprecipitations of mitochondria fractions from cells treated with or without hypoxic preconditioning. Rabbit IgG was used as control immunoprecipitation. (C) Western blot of mitochondrial fraction with the anti-SUR2A antibody.

 
To explore whether HSP90 and Kir6.2 can physically associate with each other, immunoprecipitations of Kir6.2 were carried out in the percoll-purified mitochondrial fractions. Kir6.2 or HSP90 was immunoprecipitated from equivalent amounts of total protein in mitochondrial fraction. Kir6.2 was detected in the precipitate by anti-Kir6.2 antibody but not in the supernatant. Kir6.2 co-precipitated with HSP90 but not with COX IV. COX IV was detected in the supernatant of HSP90 or Kir6.2 (data not shown). The preconditioned cells exhibited relatively higher density bands of HSP90 and Kir6.2 in the Kir6.2 and HSP90 immunoprecipitates, respectively (Figure 4B). The use of anti-rabbit IgG for immunoprecipitation (antibodies against Kir6.2 or HSP90 were derived from rabbits) did not show a precipitation of either Kir6.2 or HSP90.

3.4 Effect of 5-HD on mitochondrial Kir6.2-containing KATP channels
To define whether Kir6.2-containing KATP channel in mitochondria are responsive to K+ channel activators or inhibitors, the effects of a putative mitochondrial KATP channel activator, diazoxide, and an inhibitor, 5-HD, on mitochondrial membrane potential (MMP) were assessed with tetramethylrhodamine (TMRE) fluorescence (Figure 5A). The fluorescence intensity of TMRE-loaded H9c2 cells gradually decreased during the application of diazoxide (100 µM) following HP, which reflects depolarization of MMP. Compared with the value before diazoxide, the TMRE fluorescence intensity was 78 ± 2.8% after 5 min of application of diazoxide. This effect was inhibited by not only 5-HD (500 µM) but also by siRNA targeting Kir6.2. Diazoxide had no effect on TMRE fluorescence intensity under normoxic condition (data not shown), indicating that K+ influx induced by diazoxide may not be sufficient enough to cause MMP changes. To explore the effect of 5-HD in the protection of HP and its relation to mitochondrial Kir6.2, we examined viable cells after H/R in adult rat cardiomyocytes treated with HP. 5-HD largely prevented the protection of HP and did not further reduce viable cells in the group transfected with dominant negative Kir6.2AAA.


Figure 5
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  		Figure 5 Effect of 5-HD on mitochondrial Kir6.2-containing KATP channels. (A) Representative immunofluorescence images of TMRE-loaded H9c2 cells (left panel) and relative TMRE fluorescence intensity over the value before hypoxia. 5-HD was added 5 min before addition of diazoxide (Diaz). Cells were treated with or without siRNA targeting Kir6.2. n = 5, *P < 0.05 vs. HP. (B) Quantification of cell viability from adult rat cardiomyocytes with or without infection of Kir6.2AAA (Ad-Kir6.2AAA). n = 6, *P < 0.05 vs. control.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Funding
 References
 
The key finding of the present study was that HP enhances translocation of Kir6.2 to mitochondria in a HSP90-dependent manner. We showed that the pore-forming subunit of KATP channels Kir6.2, but not Kir6.1, is involved in the protection of HP in a cardiac myocyte model of simulated ischaemia using both dominant negative and siRNA technology. We further demonstrated for the first time that HSP90 is critical in Kir6.2-mediated cytoprotection against hypoxia/reoxygenation-induced injury, possibly by promoting Kir6.2 targeting to mitochondria. Our data point out a novel mechanism involved in KATP channel-mediated cardioprotection.

Kir6.2 has been shown to be involved in the cardioprotection of IPC in mice.5,19 However, as noted by the authors,5 the relative importance of the sarcKATP channel may have been exaggerated due to the rapid heart rate in the murine model. The present study provides the first evidence in the quiescent (non-contracting) cardiomyocyte model that Kir6.2 subunit is critical in the cytoprotection against hypoxia. This cellular model, which is widely used for studying the mechanism of IPC, eliminated the potential influence of a faster heart rate in mice and excluded a contribution of sarcKATP channels. Our data thus indicate that Kir6.2-containing KATP channels can provide the protection independent of action potential duration (APD) shortening in cardiac myocytes. Budas et al.26 reported that an increase in the number of sarcKATP channels by forward trafficking is required in the protection of stimulated beating cardiomyocytes against hypoxia. However, we utilized non-contracting cardiomyocytes and did not find a significant increase in the number of functional sarcKATP channels following hypoxia and reoxygenation (unpublished observation). This discrepancy could be due to different cellular models between the two studies. It has been known that cardiac ventricular myocytes have an extremely high density of sarcKATP channels.27,28 Only a small fraction of sarcKATP channels needs to be open in order to cause appreciable shortening of the action potential.28 Excessive sarcKATP opening could actually be detrimental to the heart because significant shortening of action potentials by opening KATP channels is associated with cardiac arrhythmia.29–32

Although opening of sarcKATP channels may be important for cardioprotection, the mitochondrial site of action for Kir6.2-containing KATP channels has been suggested. Gumina RJ et al.19 demonstrated that the Kir6.2 null mutant heart failed to respond to IPC with an increase in ATP turnover or ATP synthesis rate, a primarily mitochondrial function. In addition, Kir6.2 has been localized in mitochondria both at physical and functional levels.12–17 The mechanism underlying Kir6.2-mediated protection is unknown. Our data showed that HSP90 was increased moderately following brief HP in adult cardiac myocytes but not in heart-derived H9c2 cells, implying that increased expression of HSP90 may not be critical in mediating Kir6.2 translocation and thus protection during ischaemia. We did not expect a significant increase in heat shock protein level since cells were harvested for Western blot right after brief HP, which lasts less than an hour. Posttranslational modification may likely play a role in HP-induced mitochondrial translocation of Kir6.2. Our co-immunoprecipitation experiments demonstrated that HSP90 was associated with Kir6.2 under normal conditions as well as following HP. This association was enhanced following HP, indicating that increased binding of Kir6.2 and HSP90 may likely contribute to the enhanced mitochondrial import of Kir6.2. Since the association of Kir6.2 with HSP90 was increased only moderately, an additional pathway or mechanism may also be involved in enhanced mitochondrial translocation of Kir6.2 by HP. Further, our data showed that reduced expression of HSP90 by siRNA only prevented the HP-induced increase in Kir6.2 but did not affect the basal level of Kir6.2 in mitochondria, indicating that HSP90 functions in a greater extent under stress condition than normal condition for Kir6.2 import into mitochondria. Incomplete inhibition of HSP90 function or expression with geldanamycin or siRNA could be one of the reasons for the basal Kir6.2 level in mitochondria. Taken together, our data are consistent with a recent discovery that HSP90 is required for mitochondrial protein import for hydrophobic membrane proteins, especially during stress conditions.33 Interestingly, connexin 43 in mitochondria, which has been shown to be involved in IPC, has been linked to HSP90.34,35

A previous study5 has shown that a flavoprotein oxidation response to diazoxide, a putative mitochondrial KATP channel opener, was preserved in cardiac cells of Kir6.2-deficient mice, indicating Kir6.2 may not be a part of putative mitochondrial KATP channels. Flavoprotein fluorescence measurements have been widely used to assay mitochondrial KATP channel activity in intact myocytes. However, this experimental approach has some limitations.36 It is known that diazoxide has some K+-independent effects,37–40 which may contribute to increase in flavoprotein oxidation. Although diazoxide, as a K+ channel opener, may activate mitochondrial KATP channels, the use of the diazoxide effect as an indicator of mitochondrial KATP activity requires more careful control experiments. Whether diazoxide protects the heart via its K+-independent or Kvplus;-dependent effects, KATP channels have been recorded in liver and heart mitochondria.17,41–44 In addition, Kir6.2 is localized in heart mitochondria both physically and functionally.12–18 Our study further showed that suppressing Kir6.2 expression with siRNA inhibited diazoxide-induced changes in MMP, implying diazoxide may activate mitochondrial Kir6.2-containing KATP channels. These studies were done in quiescent cardiomyocytes where the influence of sarcolemmal KATP channels by diazoxide on MMP should be minimal. Nevertheless, the present study was not aimed to define the molecular composition of putative mitochondrial KATP channels but rather to study the regulatory mechanism involved in Kir6.2-mediated cardioprotection. Even though Kir6.2-containing KATP channels in mitochondria are involved in preconditioning and responsive to diazoxide, our study does not exclude the possibility that a distinct mitochondrial KATP channel exists.

How does the mitochondrial Kir6.2 protein produce the preconditioning effect? We and others have shown that Kir6.2 is localized in mitochondria under normal condition at a relatively low level. These channels can be activated directly by stimulators in cells or in isolated mitochondria.17 Although they may constitutively present in mitochondria, they should not be active due to strong inhibition by relative high matrix ATP under normal condition. In the present study, we found that diazoxide caused Kir6.2-dependent depolarization of MMP only after HP, possibly via increase in mitochondrial Kir6.2. This result indicates that K+ influx induced by diazoxide-induced opening of mitochondrial Kir6.2-containing KATP channels may not be sufficient enough to cause changes in MMP under normal condition. However, this observation does not exclude the possibility that diazoxide may cause alterations in other mitochondrial functions under normal conditions such as mitochondrial volume.45 The exact mechanism by which increased Kir6.2-containing KATP in mitochondria protects cardiac myocytes against hypoxia/reoxygenation is currently unknown. While diazoxide-induced protection may involve K+-dependent and -independent mechanisms,6,40,46 Kir6.2-mediated preconditioning should definitely increase K+ influx in mitochondria. The mechanism in pharmacological preconditioning by diazoxide may be different from that in IPC or HP. Our data have shown that opening of KATP channels in mitochondria induces mild depolarization of MMP, indicating a possible contribution of decreased Ca2+ overload to cardioprotection.47,48

Isolated cardiomyocytes, obtained by enzymatic digestion of whole hearts, have multiple advantages. The most obvious advantage of studying cardiomyocytes compared with the whole heart is the elimination of other cell types, such as endothelial cells and fibroblasts, so that their influence is negligible. This is particularly important in studying the cell signalling in preconditioning involving subcellular components such as the cell membrane vs. mitochondria. It is desirable to have a pure collection of cardiomyocytes rather than the various cell types present in the whole heart. However, there are some limitations using cardiac myocytes. Isolated cardiac myocytes are quiescent (non-beating) whereas the heart is regularly beating in vivo. Thus, the cells we used in the present study cannot reproduce the same mechanical action. Mechanisms involved in those functions may not be revealed. Nevertheless, the protection of IPC has been consistently demonstrated in freshly isolated cardiomyocytes across multiple species, indicating that much of the innate protection of IPC resides in cardiomyocytes.

Giving the growing interest in mitochondria and cell death, our finding of an entirely new mechanism in KATP channel-mediated protection should be of general interest. It raises the intriguing possibility that mitochondrial protein import can be regulated differently under different physiological conditions.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
References
 
This study was supported by a research grant from American Heart Association Great Rivers Affiliate (K.H.).


   Acknowledgements
 
We would like to thank William Coetzee for the kind gifts of cDNA clones Kir6.1AAA and Kir6.2AAA. We are also grateful to Lane Wallace for critically reading the manuscript.

Conflict of interest: none declared.


   Notes
 
{dagger} These authors contributed equally to this work. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 

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  4. Patel HH, Gross ER, Peart JN, Hsu AK, Gross GJ. Sarcolemmal KATP channel triggers delayed ischemic preconditioning in rats. Am J Physiol Heart Circ Physiol (2005) 288:H445–H447.[Abstract/Free Full Text]
  5. Suzuki M, Sasaki N, Miki T, Sakamoto N, Ohmoto-Sekine Y, Tamagawa M, et al. Role of sarcolemmal K(ATP) channels in cardioprotection against ischemia/reperfusion injury in mice. J Clin Invest (2002) 109:509–516.[CrossRef][Web of Science][Medline]
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V. Garg, J. Jiao, and K. Hu
Regulation of ATP-sensitive K+ channels by caveolin-enriched microdomains in cardiac myocytes
Cardiovasc Res, April 1, 2009; 82(1): 51 - 58.
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