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Cardiovascular Research 2004 61(3):610-619; doi:10.1016/j.cardiores.2003.10.022
© 2004 by European Society of Cardiology
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Copyright © 2003, European Society of Cardiology

Ischemic preconditioning activates AMPK in a PKC-dependent manner and induces GLUT4 up-regulation in the late phase of cardioprotection

Yasuhiro Nishinoa, Tetsuji Miura*,a, Takayuki Mikia, Jun Sakamotoa,b, Yuichi Nakamuraa, Yoshihiro Ikedaa, Hironori Kobayashia and Kazuaki Shimamotoa

aSecond Department of Internal Medicine, Sapporo Medical University School of Medicine, South-1 west-1, Chou-ku Sapparo 060-8543, Japan
bDepartment of Pharmacology Sapporo Medical University School of Medicine South-1 west-1, Chou-ku Sapparo 060-8543, Japan

* Corresponding author. Tel. :+81-11-611-2111; fax: +81-11-644-7958. miura{at}sapmed.ac.jp

Received 30 July 2003; revised 22 October 2003; accepted 23 October 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Experiment 1: effects...
 4. Experiment 2: effects...
 5. Experiment 3:...
 6. Results
 7. Discussion
 References
 
Objective: The aim of this study was to determine the role of AMP-activated protein kinase (AMPK) and its link to protein kinase C (PKC) in the late phase of cardioprotection afforded by ischemic preconditioning (PC) against myocardial stunning. Methods and results: Rabbits were instrumented with a balloon occluder around a coronary artery and with a Doppler sensor to monitor the thickening fraction (TF). Conscious rabbits underwent five cycles of 5-min ischemia/5-min reperfusion (I/R) on 2 consecutive days (days 1 and 2). Reduction of TF after I/R was significantly less and recovery of TF was faster on day 2, indicating a late PC effect. PC provoked translocation of PKC-{varepsilon} from the cytosol to the membrane and significantly increased AMPK activity by 100% immediately after PC. The mRNA level of GLUT4, a glucose transporter, was elevated by 150% at 3 h after PC, and the total protein level of GLUT4 was increased by 107% at 24 h after PC. The level of sarcolemmal GLUT4 protein after I/R on day 2 was 41% higher than its level after I/R on day 1. AMPK activation and up-regulation of GLUT4 by PC were abrogated by pre-treatment with PKC inhibitors. Conclusion: PC activated AMPK and up-regulated GLUT4 expression in a PKC-dependent manner. This GLUT4 up-regulation at 24 h after PC may contribute to attenuation of myocardial stunning.

KEYWORDS Ischemia; Preconditioning; Protein kinases; Stunning


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Experiment 1: effects...
 4. Experiment 2: effects...
 5. Experiment 3:...
 6. Results
 7. Discussion
 References
 
Brief periods of ischemia induce myocardial tolerance against stunning and infarction 24–48 h after a brief period of ischemia in experimental animals [1–6]. This phenomenon is termed late (or delayed) preconditioning (PC) or second window of protection (SWOP), to differentiate it from cardioprotection that develops immediately after PC (i.e., classic or early PC). Recent clinical studies have suggested that late PC underlies the protective effects of angina pectoris on exercise tolerance in patients with stable angina and the effect of protection afforded by pre-infarct angina on contractile function in patients with acute myocardial infarction [7,8]. Intensive investigations in the last decade have revealed that late PC is triggered by nitric oxide (NO), oxygen free radicals, and adenosine and that this triggering of late PC leads to activation of multiple signaling pathways, resulting in up-regulation of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX2), aldose reductase and Mn-superoxide dismutase (Mn-SOD) [1–6]. Enhanced production of NO and PGI2/PGE2 and removal of oxygen free radicals by Mn-SOD during ischemic insult and/or subsequent reperfusion have been suggested to be mechanisms of attenuation of myocardial injury by late PC. However, alteration in metabolic responses to ischemia by late PC has not been fully characterized.

It has been established that anaerobic glycolysis is important for ATP generation during ischemia and crucial for attenuation of ischemic injury in the heart [9–11]. Increased glucose uptake in ischemic cardiomyocytes is primarily achieved by translocation of a glucose transporter, GLUT4, from its intracellular compartments to the sarcolemma [12,13]. It has recently been found that the expression level of GLUT4 is increased by activation of AMP-activated protein kinase (AMPK). In a study by Holmes et al. [14], administration of 5-aminoimidazole-4-carboxamine ribonucleoside (AICAR), an AMPK activator, for 5 days increased GLUT4 protein level by approximately two-fold in the rat skeletal muscle in situ. AMPK is a sensor of cellular energy charge and its activity increases in response to various cellular stresses that increase cytosolic-free AMP [14–17]. Based on these earlier findings, we hypothesized that PC activates AMPK and thus induces GLUT4 up-regulation in the late phase of PC in the heart.

To test this hypothesis, we assessed the effects of PC on AMPK activity and on mRNA and protein levels of GLUT4 in a rabbit model of myocardial stunning. In addition, the possible link between protein kinase C-{varepsilon} (PKC-{varepsilon}) and AMPK in late PC was examined for the following two reasons. First, PKC-{varepsilon} plays an obligatory role in the anti-stunning effect of late PC in rabbits [1,18,19]. Second, AMPK is regulated by phosphorylation of its kinase domain [15,16], though direct phosphorylation of AMPK by PKC has not been reported to date. The results of the present study support the notion that PC activates AMPK by a PKC-dependent mechanism and induces GLUT4 up-regulation in the late phase of PC.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Experiment 1: effects...
 4. Experiment 2: effects...
 5. Experiment 3:...
 6. Results
 7. Discussion
 References
 
This study conformed to 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).


   3. Experiment 1: effects of PC on regional contractile function
Top
 Abstract
 1. Introduction
 2. Methods
 3. Experiment 1: effects...
4. Experiment 2: effects...
 5. Experiment 3:...
 6. Results
 7. Discussion
 References
 
3.1 Surgical preparation
Chronically instrumented rabbits were prepared according to methods used in earlier studies [2,3,19]. In brief, male albino rabbits (Japanese White), weighing 2.5±0.2 (S.E.) kg, were anesthetized with sodium pentobarbital, intubated and mechanically ventilated. Under sterile conditions, the chest was opened via a left thoracotomy and a balloon occluder was placed around the marginal branch of the left coronary artery. A 10-MHz Doppler ultrasonic crystal (PrimeTech, Japan) was secured in the center of the marginal branch territory, and two ECG electrodes were tied to the chest muscle layer. The distal end of the balloon occluder, ECG leads and wires of ultrasonic crystals were passed under the subcutaneous tissue and exteriorized from the interscapular area. Cefazolin (125 mg) was administered as a single intramuscular injection for infection prophylaxis.

Rabbits were brought into the laboratory two weeks after surgery, and leads from the ultrasonic crystal were connected to a Pulse Doppler Dimension System VF-1 (Crystal Biotec, USA) for measuring regional wall thickening. A phonocardiogram was recorded with a hand-held precordial microphone (TA-701T, Nihon-Kohden, Japan) during recording of hemodynamic parameters to identify systolic periods. Each systolic period was determined as the interval between onset of the first cardiac sound and onset of the second cardiac sound. Thickening fraction (TF) was calculated as the net systolic thickening expressed as a percentage of end-diastolic thickness.

3.2 Experimental protocol
Rabbits (n=12) underwent five cycles of 5-min ischemia/5-min reperfusion on 2 consecutive days (i.e., days 1 and 2) with a 24-h interval (Fig. 1A). TF and ECG were continuously measured before and during repetitive ischemia/reperfusion and 2 h after the last reperfusion. No anti-arrhythmic agents were administered throughout the experiments. At the end of the experiment, 2000 units of heparin were injected and then each rabbit was killed by pentobarbital overdose. The hearts were excised, and the positional relationship between the ultrasonic crystal and the area at risk was confirmed by injecting Evans Blue into the aorta after coronary ligation.


Figure 1
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  		Fig. 1 Protocols in Experiments 1 and 2. In the myocardial stunning experiments (panel A), conscious rabbits were subjected to five cycles of 5-min coronary occlusion (solid bars) separated by 5-min reperfusion (open bars) on 2 consecutive days. In the protocol for PKC analysis (panel B), rabbits received no pretreatment (control) or five cycles of 5-min coronary occlusion/5-min reperfusion (PC), and ventricular tissue was sampled immediately after the PC or its time control period. In the protocol for AMPK and GLUT4-mRNA analyses (panel C), rabbits were subjected to vehicle injection alone (Control), PC or injection of a PKC inhibitor (Che or GF) plus PC. Chelerythrine (5 mg/kg) and GF109203X (0.05 mg/kg) were injected intravenously 5 min before ischemia. Saline was used for the vehicle. Only chelerythrine was used in the series of GLUT4 experiments. Che=chelerythrine, GF=GF109203X.

 

   4. Experiment 2: effects of PC on PKC, AMPK and GLUT4 mRNA levels
Top
 Abstract
 1. Introduction
 2. Methods
 3. Experiment 1: effects...
 4. Experiment 2: effects...
5. Experiment 3:...
 6. Results
 7. Discussion
 References
 
4.1 Surgical preparations and protocols
Rabbits were instrumented with a coronary occluder as in Experiment 1. In a series of PKC experiments (Fig. 1B), rabbits were subjected to no treatment for 50 min (controls, n=5) or PC with five cycles of 5-min ischemia/5-min reperfusion (n=5), and then the hearts were excised. In a series of AMPK experiments (Fig. 1C), rabbits underwent no ischemia (controls, n=7), PC (n=7), chelerythrine injection (5 mg/kg) plus PC (n=7) or GF109203X injection (0.05 mg/kg) plus PC (n=5). Chelerythrine and GF109203X were administered intravenously at 5 min before PC, and the hearts were excised at 5 min after the last reperfusion. In a series of GLUT4 mRNA analysis (Fig. 1C), rabbits received no treatment (n=6), PC (n=7) or chelerythrine injection plus PC (n=6), and the hearts were removed for tissue sampling at 3 h after the last 5-min reperfusion or corresponding time control period. Excised hearts were immediately soaked in ice-cold saline, and tissues in the anterior reperfused region and in the posterior non-ischemic region were sampled and frozen in liquid nitrogen.

4.2 Western blotting for PKC
Tissue samples were processed to obtain cytosolic fractions and particulate fractions from the myocardial homogenate, and protein levels of PKC-{varepsilon} and PKC-{alpha} in each fraction were determined by Western blotting as previously reported [20,21]. SigmaGel (SPSS, USA) was used to quantify the PKC signals on immunoblots.

4.3 Determination of AMPK activity
AMPK extraction and assay of its activity were performed as previously reported [22]. In brief, frozen tissue was homogenized in ice-cold buffer containing 50 mM Tris–HCl (pH 7.5), 250 mM mannitol, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 4 µg/ml soybean trypsin inhibitor and 1 mM benzamidine. The homogenate was centrifuged at 14000xg for 20 min at 4 °C. The supernatant was brought to 2.5% (w/v) polyethylene glycol (PEG) with 25% PEG 6000 and was agitated for 10 min at 4 °C. Samples were centrifuged at 10000xg for 10 min at 4 °C. The supernatant was then made up to 6% PEG 6000 and was agitated once again at 4 °C. This fraction was centrifuged at 10000xg for 10 min, and the pellet was washed with homogenization buffer containing 6% PEG 6000 and finally centrifuged at 10000xg for 10 min at 4 °C. Then the pellet was re-suspended with suspending buffer that consisted of homogenizing buffer plus 10% glycerol, and the re-suspended enzyme samples were used for determination of AMPK activity by assessment of incorporation of 32P into a synthetic substrate (AMARAASAAALARRR peptide) [22].

4.4 Quantitative RT-PCR for GLUT4 mRNA
Total RNA was isolated from tissue samples using RNeasy Midi kits (Qiagen, USA) according to the manufacturer's protocol. Since the nucleotide sequence of rabbit GLUT4 was unknown, we first designed primers for rabbit GLUT4 based on mouse GLUT4 (GenBank association no. AB008453 [GenBank] ). We selected the following primers: GLUT4 mRNA sense (5'-CAGTATGTTGCGGATGCTATGG-3', positions 1322–1344) and GLUT4 mRNA antisense (5'-GGCACTTTTAGGAAGGTGAAGATG-3', positions 1390–1413). One-step RT-PCR was performed and we obtained a single clear band at the expected 92 bp in length, and this band was excised from the gel and purified by using QIAquick GelR Extraction Kits (Qiagen, USA). The cDNA fragment was cloned into the pGEM-T Easy vector (Promega, USA) and its sequence was determined. Sequence analysis showed the partial rabbit GLUT4 cDNA between the two primers to be 89.1%, 91.3% and 93.5% homologous to the mouse, rat and human GLUT4 transcripts, respectively. Since cloned nucleic acid sequences in these regions were highly conserved compared to those in the mouse, rat and human, we concluded that the isolated cDNA corresponded to a portion of the rabbit GLUT4 transcripts and designed a TaqMan probe for rabbit GLUT4 (5'-CTACGTCTTCCTCCTCTTTGC-3') between the two primers.

As an internal control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) transcripts in each sample were also determined. As primers and a TaqMan probe for rabbit GAPDH (GenBank association no. AB008453 [GenBank] ), we used 5'-TTCTTCTCGTGCAGTGCTAGCG-3' (GAPDH mRNA sense, positions 38–59), 5'-TAAAAGCAGCCCTGGTGACCA-3' (GAPDH mRNA antisense, positions 118–138) and 5'-AGTGAACGGATTTGGCCGCATTGG-3' (GAPDH TaqMan probe, positions 89–112).

Quantitative RT-PCR and analysis of mRNA expression were performed using an ABI PRISMR 7700 Sequence Detection System (Applied Biosystems, USA). The conditions of one-step RT-PCR were as follows: 30 min at 48 °C (stage 1, RT), 10 min at 95 °C (stage 2, RT inactivation and AmpliTaq Gold activation), and then 40 cycles of amplification for 60 s at 95 °C, 60 s at 55 °C and 30 s at 72 °C (stage 3, PCR).


   5. Experiment 3: immunohistochemistry of GLUT4
Top
 Abstract
 1. Introduction
 2. Methods
 3. Experiment 1: effects...
 4. Experiment 2: effects...
 5. Experiment 3:...
6. Results
 7. Discussion
 References
 
5.1 Surgical preparation and protocols
Rabbits were prepared and divided into a control group, PC group and chelerythrine plus PC group (Che+PC group) as in Experiment 2. As shown in Fig. 2, in the control group, tissue samples were taken under the baseline condition (n=7) or immediately after five cycles of 5-min ischemia/5-min reperfusion (n=7). In the PC group and Che+PC group, rabbits received PC (i.e., five cycles of 5-min ischemia separated by 5-min reperfusion) on day 1, and tissue sampling was performed under the baseline condition on day 2 (n=6 in the PC group, n=7 in the Che+PC group) or immediately after five cycles of 5-min ischemia/5-min reperfusion on day 2 (n=5 in the PC group, n=6 in the Che+PC group). Ventricular tissues were placed in 3% paraformaldehyde for 2 h and then transferred to 0.5% paraformaldehyde and stored overnight at 4 °C. After fixation, tissue samples were immersed overnight in phosphate-buffered saline containing 30% sucrose and 0.2% NaN3 at 4 °C. The tissue was then embedded in Tissue-Tek O.C.T. compound (Miles, USA), and standard techniques were used to prepare slides stained with monoclonal mouse anti-GLUT4 antibodies (R&D systems, Minneapolis, MN), FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, USA) and also with rhodamine-conjugated Lens Culinaris agglutinin (50 µg/ml) (Vector Laboratories, USA) to identify the sarcolemma.


Figure 2
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  		Fig. 2 Protocol in Experiment 3. The control group received vehicle injection and tissue was sampled without ischemia (control-baseline) or after five cycles of 5-min ischemia/5-min reperfusion (control-I/R). The PC group and Che+PC group received a vehicle and chelerythrine (5 mg/kg), respectively, 5 min before ischemia and then underwent five cycles of 5-min ischemia/5-min reperfusion. Tissue samples were taken under the baseline condition on day 2 (PC-baseline, Che+PC-baseline) or after five cycles of 5-min ischemia/5-min reperfusion on day 2 (PC-I/R, Che+PC-I/R).

 
5.2 Image analysis
The slides were examined by using a confocal laser scanning microscope (LSM 510, Zeiss, Germany). Specimens were scanned using a Plan-Neofluar 20x/0.3 objective at a 512x512 pixel resolution. We confirmed that there was no cross-talk between the two channels (green, 505–530-nm band-pass filter; red, 560-nm long pass filter). In each tissue sample, 12 randomly chosen areas were analyzed. Images (8-bit) were recorded at eight-times frame averaging, and then binary images were created automatically by using Adobe Photoshop 7.0 (Adobe Systems, Mountain View, CA, USA) as shown in Fig. 3. To create binary images of GLUT4, each pixel was either on or off, as determined by a pre-set threshold of 55 on a 255-point gray scale (Fig. 3C). For sarcolemmal images, we set an appropriate threshold to sharply outline the sarcolemma for each sample (Fig. 3B). Intracellular GLUT4 images were obtained by subtracting the sarcolemmal images from the total GLUT4 images (Fig. 3E). The level of GLUT4 expression was expressed as number of positive pixels.


Figure 3
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  		Fig. 3 Image analysis for determination of GLUT4 protein in the sarcolemma and intracellular compartments. Panels A and B are confocal images of GLUT4 distribution and sarcolemma that was stained with rhodamine-conjugated Lens Culinaris agglutinin, respectively. Binary images of GLUT4 (panel C) and the sarcolemma (panel D) were produced from panels A and B, and subtraction of panel D from panel C produced an image of intracellular GLUT4 (panel E). Total GLUT4 level and sarcolemmal GLUT4 level were determined as number of positive pixels in panel C and difference between positive pixels in panel C and those in panel E.

 
5.3 Statistical analysis
Data are presented as means±S.E. Inter-group differences except for those in the time courses of heart rates and TF were tested by one-way analysis of variance (ANOVA). Time courses of heart rates and TF throughout the experiments were compared between study groups by two-way repeated measures ANOVA. When ANOVA indicated statistically significant differences, multiple comparisons were performed by using the Student–Newman–Keuls post-hoc test. All statistical analyses were performed using SigmaStat (SPSS USA). The difference was considered significant if the p-value was less than 0.05.


   6. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Experiment 1: effects...
 4. Experiment 2: effects...
 5. Experiment 3:...
 6. Results
7. Discussion
 References
 
6.1 Experiment 1
Nineteen rabbits were used in this series of experiments, and three of them died after the surgery and additional four rabbits were excluded because of technical problems (i.e., malfunction of the balloon occluder, disruption of a lead from the Doppler crystal) or lethal ventricular fibrillation during coronary occlusion.

There was no significant difference between heart rates on days 1 and 2 (Table 1). Baseline TF on day 1 was 18.9±1.8%, and five episodes of 5-min ischemia/5-min reperfusion reduced TF to 37.3% of the baseline level and then it slowly recovered on day 1. On day 2, baseline TF was 18.1±1.6%, which was not statistically different from the baseline TF on day 1, but the same protocol of ischemia/reperfusion resulted in significantly better recovery than that on day 1 (Fig. 4). These results indicate that the five cycles of 5-min ischemia/5-min reperfusion on day 1 could precondition the myocardium against myocardial stunning on day 2, which is consistent with previous observations [1–3].


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  		Table 1 Heart rate in Experiment 1

 

Figure 4
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  		Fig. 4 Regional contractile function after repetitive ischemia in conscious rabbits. The time courses of thickening fraction (% baseline) after reperfusion were significantly different on days 1 and 2 (p<0.05 by two-way repeated measures ANOVA). *p<0.05 vs. thickening fraction on day 1 at the corresponding time point.

 
6.2 Experiment 2
In untreated controls, PKC-{varepsilon} levels in the cytosolic and particulate fractions were 48.8±2.6% and 51.2±2.6% of the total, respectively, in the anterior ventricle and 48.9±2.3 and 51.1±2.3 of the total, respectively, in the posterior ventricle. As shown in Fig. 5, in the preconditioned myocardium, PKC-{varepsilon} in the cytosolic fraction was reduced (39.1±3.3%) and PKC-{varepsilon} in the particulate fraction was increased (60.9±3.3%) compared with the levels in controls, indicating PC-induced translocation of PKC-{varepsilon} [20,21]. Interestingly, such PKC-{varepsilon} translocation was observed in the non-preconditioned region as well (PKC-{varepsilon} in the cytosolic and particulate fractions were 36.7±3.6% and 63.3±3.6%, respectively). PKC-{alpha} was not translocated after PC as in our previous studies [20,21] (data not shown).


Figure 5
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  		Fig. 5 Intracellular distribution of PKC-{varepsilon}. Representative Western blots and summary of protein levels of PKC-{varepsilon} in the cytosolic fraction and the particulate fraction as percentages of total. *p<0.05 vs. control group. Ant=anterior ventricular region, Post=posterior ventricular region, IZ=preconditioned ischemic region, NZ=non-ischemic region. Lane 6=positive control of PKC-{varepsilon} obtained from rat brain lysate.

 
AMPK activity in the preconditioned myocardium (895.5±89.2 pmol min–1 mg protein–1) was significantly higher than AMPK activities in the non-preconditioned region and the corresponding region of the control group (454.2±84.2 and 447.1±135.8 pmol min–1 mg protein–1, respectively) (Fig. 6). Pretreatment with chelerythrine and GF109203X abolished the PC-induced increase in AMPK activity. AMPK activity in the non-preconditioned region of the preconditioned heart did not differ from that in untreated controls. Three hours after PC, the GLUT4 mRNA level was increased, and the ratio of GLUT4 mRNA to GAPDH mRNA (GLUT4/GAPDH) was significantly larger in the preconditioned region (2.5±0.4) than in the non-preconditioned region and untreated control (1.6±0.4 and 1.0±0.2, respectively). This effect of PC was reduced by chelerythrine as shown in Fig. 6. GLUT4/GAPDH in the preconditioned region of the chelerythrine plus PC group (2.0±0.4) tended to be higher than that in the controls, but the difference was not statistically significant. There was no significant inter-group difference in GLUT4/GAPDH in the myocardium not rendered ischemic.


Figure 6
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  		Fig. 6 Levels of AMPK activity and GLUT4-mRNA. Solid bars=preconditioned region or its untreated control (i.e., anterior region), open bars=non-preconditioned region or its untreated control (i.e., posterior region). Che=chelerythrine, GF=GF109203X. *p<0.05 vs. control group, #p<0.05 vs. non-ischemic region.

 
6.3 Experiment 3
Data obtained from immunohistochemical analyses of GLUT4 protein are summarized in Fig. 7. On day 1, five cycles of 5-min ischemia/5-min reperfusion did not alter the level of total GLUT4 but increased sarcolemmal GLUT4, indicating translocation of GLUT4, in the ischemic region. On day 2, GLUT4 levels in the non-preconditioned regions did not differ from those on day 1. However, in the preconditioned region, baseline level of total GLUT4 protein on day 2 was higher by 107%. Furthermore, the level of sarcolemmal GLUT4 after ischemia/reperfusion on day 2 was significantly higher than that on day 1. Chelerythrine inhibited the PC-induced increase in total GLUT4 level and abolished the PC-induced augmentation of GLUT4 translocation after ischemia/reperfusion on day 2.


Figure 7
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  		Fig. 7 GLUT4 protein after PC. Representative samples of GLUT4 immunohistochemistry (panel A) and summary of total and sarcolemmal GLUT4 protein levels determined by image analysis (panel B). Solid bars=region subjected to ischemia or its control, open bars=non-ischemic region or its untreated control (i.e., posterior region), Base=baseline, I/R=after ischemia/reperfusion. *p<0.05 vs. control group, #p<0.05 vs. non-ischemic region.

 

   7. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Experiment 1: effects...
 4. Experiment 2: effects...
 5. Experiment 3:...
 6. Results
 7. Discussion
References
 
The present study confirmed that PC with five cycles of 5-min ischemia/5-min reperfusion could induce a late PC effect against myocardial stunning induced in rabbit hearts (Fig. 4) [2,3,19]. In this cardioprotective protocol of PC, PKC-{varepsilon} was translocated to the membrane fraction and AMPK activity increased immediately after PC, GLUT4 mRNA level was elevated 3 h after PC, and GLUT4 protein level was increased 24 h after PC. Two structurally different PKC inhibitors, chlerythrine and GF109203X, abolished the PC-induced activation of AMPK, and chelerythrine eliminated the up-regulation of both GLUT4 mRNA and protein levels. These findings indicate that PC activates AMPK and up-regulates GLUT4 expression in a PKC-dependent manner. Together with results of earlier studies [14,15] showing that AMPK activation increased GLUT4 expression, the results in the present study support the notion that PC up-regulates GLUT4 expression by activation of AMPK in PKC dependent manner. To the best of our knowledge, this is the first study demonstrating a link between PKC, AMPK and GLUT4 expression in cardiomyocytes.

AMPK is a hetero-trimer consisting of {alpha}, β and {gamma} subunits, and its activity is regulated by AMP, an allosteric activator, and by phosphorylation of the kinase domain in the {alpha}-subunit [15,16]. AMP increases the level of AMPK activity by preventing interaction of a kinase domain of the {alpha}-subunit with the autoinhibitory domain. AMPK phosphorylation is thought to be induced by AMPK kinase, but its molecular identity and regulation are unclear. The present study showed for the first time that PKC inhibitors abolished AMPK activation by ischemic episodes in the myocardium, indicating a crucial role of PKC in AMPK activation during PC. Whether PKC directly phosphorylates AMPK or AMPK kinase remains to be examined. Nevertheless, it is notable that PKC activation alone was not sufficient to activate AMPK, since the level of AMPK activity increased only in the preconditioned region despite the fact that similar PKC-{varepsilon} translocation occurred in preconditioned and non-preconditioned regions (Fig. 5). Presumably, elevation of cytosolic AMP level during PC, in addition to PKC activation, is necessary induction of substantial AMPK activation by PC.

Chelerythrine has been used as a specific inhibitor of PKC in a number of studies on the mechanism of PC [19,23,24]. However, some recent studies have raised a question about the specificity and efficacy of chelerythrine as a PKC inhibitor [25 26]. Thus, we also examined the effect of structurally different PKC inhibitor, GF109203X, on AMPK activation by PC. GF109203X completely inhibited elevation of AMPK activity by PC as did chelerythrine (Fig. 6), and these findings support the notion that activation of AMPK by PC is PKC-dependent.

The finding that PKC-{varepsilon} was translocated in both the ischemic and non-ischemic myocardium is not surprising, since "remote" PC, which refers to protection of the myocardium from infarction by transient ischemia in remote areas of the heart or in extracardiac organs, has been shown to be PKC-mediated [27–30]. Although the precise mechanism of PKC activation in the non-ischemic myocardium remains unclear, involvement of the sympathetic nervous system has been suggested, at least in some preparations [28,29].

The present findings regarding the effect of AMPK activation on GLUT4 expression in the myocardium are consistent with results of earlier studies [14,31] showing that an AMPK activator, AICAR, increased the level of MEF2, a transcriptional factor, and the level of GLUT4 protein in skeletal muscle. MEF2 reportedly undergoes post-translational regulation by MAP kinases, and binding of MEF2 to the GLUT4 promotor is essential for GLUT4 expression in skeletal and cardiac muscle [31,32]. In addition, Akt also participates in GLUT4 expression, at least in adipocytes [33]. Since PC has been shown to activate MAP kinases [1] and PI3-Akt pathways in the heart [34,35], there is the possibility that regulation of MEF2 by AMPK, MAP kinases and Akt contributed to the up-regulation of GLUT4 expression after PC.

The results of the present study do not allow us to draw a clear conclusion on the role of up-regulated GLUT4 in the anti-stunning effect of delayed PC. Since there is no selective inhibitor of GLUT4 that is applicable for rabbit hearts in situ, it was not possible to directly assess the role of GLUT4 in myocardial stunning in the present rabbit preparation. However, there are several lines of circumstantial evidence supporting the notion that up-regulation of GLUT4 contributes to attenuation of myocardial stunning. First, the rate of glycolysis during myocardial ischemia determines functional recovery after reperfusion [9,36]. Second, the rate of glycolysis in the ischemic myocardium depends on glucose delivery [37]. Third, infusion of insulin, which induces GLUT4 translocation, increases glucose uptake and attenuates ischemia/reperfusion injury [11,36]. Fourth, inhibition of GLUT4 translocation by cytochalasin B or cardiac-specific ablation of the GLUT4 gene aggravated ischemia-induced depletion of ATP and post-ischemic contractile dysfunction in isolated rodent hearts [38,39]. Finally, chelerythrine at the same dose as that used in the present study has been shown to abolish the anti-stunning effect of late PC in conscious rabbits [19]. Taken together, the findings argue for the possibility that enhancement of glycolysis by up-regulated GLUT4 is a part of the mechanism of delayed PC against stunning.

It is notable that AMPK activation by ischemia causes an untoward effect on myocardial function immediately after reperfusion [17,40]. Activated AMPK inhibits acetyl-CoA carboxylase and thus reduces the level of cytosolic malonyl-CoA, an endogenous inhibitor of carnitine palmitoyl-transferase 1, resulting in enhancement of fatty acid oxidation. Such an increase in fatty acid oxidation inhibits glucose oxidation at the level of pyruvate dehydrogenase, which perturbs functional recovery of the myocardium after reperfusion [17,40]. Therefore, AMPK-induced up-regulation of GLUT4 may be an adaptive mechanism that attenuates AMPK activation during subsequent ischemia by increasing ATP generation from glycolysis. Nevertheless, the results of the present study suggest that transient activation of AMPK is not entirely detrimental and actually triggers an adaptive response in the heart.

In conclusion, PC activates AMPK by a PKC-dependent mechanism and induces up-regulation of GLUT4 24 h after PC in rabbit hearts. This GLUT4 up-regulation may contribute to late PC against myocardial stunning.


   Notes
 
Time for primary review 00 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Experiment 1: effects...
 4. Experiment 2: effects...
 5. Experiment 3:...
 6. Results
 7. Discussion
 References
 

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