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Chronic preconditioning: a novel approach for cardiac protection

Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio

Submitted 23 October 2006 ; accepted in final form 2 January 2007

	ABSTRACT

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Ischemic preconditioning is the most powerful protective mechanism known against lethal ischemia. Unfortunately, the protection lasts for only a few hours. Here we tested the hypothesis that the heart can be kept in a preconditioned state for constant protection against ischemia. In this study we chose BMS-191095 (BMS), a highly selective opener of mitochondrial ATP-sensitive K⁺ (mitoK_ATP) channels. BMS (1 mg/kg ip) was administered to rats every 24 h until 96 h. In other groups, BMS plus wortmannin (WTN, 15 µg/kg ip), an inhibitor of the phosphatidylinositol 3-kinase (PI3-K), or BMS plus 5-hydroxydecanoic acid (5-HD, 5 mg/kg ip), an inhibitor of mitoK_ATP, or BMS plus N-nitro-L-arginine methyl ester (L-NAME) (30 µg/kg ip), an inhibitor of nitric oxide (NO) synthase, were administered to rats. Rats were then subjected to 30-min left anterior descending coronary artery occlusion and 120-min reperfusion. Cardiac function, infarct size, pathological changes, and apoptosis were assessed at the end of treatments. Saline-treated hearts displayed marked contractile dysfunction and underwent pathological changes. BMS-treated rats showed significant improvement in cardiac function, and infarct size was significantly reduced in BMS-treated hearts. However, protection by BMS was abolished by 5-HD, WTN, or L-NAME. These data demonstrate that hearts can be chronically preconditioned and retain their ability to remain resistant against lethal ischemia and that this protection is mediated by activation of mitoK_ATP via NO and PI3-K/Akt signaling pathways.

mitochondrial ATP-sensitive K⁺ channels; BMS-191095; heart function

	MATERIALS AND METHODS

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All procedures on animals were approved by the University of Cincinnati Animal Care and Use Committee and were carried out in accordance with guidelines of National Institutes of Health.

Chemicals

BMS was a gift from Bristol-Myers Squibb. Diazoxide, wortmannin (WTN), and 5-hydroxydecanoic acid (5-HD) were purchased from Sigma Chemical. WTN was dissolved in DMSO before being added into the perfusion buffer. The final concentration of DMSO was <0.1%. Other reagents were dissolved in PBS. DMSO alone in this concentration had no effects on hemodynamics (31).

Surgical Preparation for Coronary Artery Ligation

The left anterior descending coronary artery (LAD) was ligated to produce infarction, as previously described (28). In brief, rats were anesthetized with pentobarbital sodium (40 mg/kg ip). A midline cervical skin incision was performed for intubation. The animals were mechanically ventilated with a Harvard model 683 respirator (Harvard Apparatus, South Natick, MA). The heart was exposed by left side thoracotomy, and LAD was dissected free above the first diagonal branch and ligated for 30 min, followed by 120 min of reperfusion. Body temperature was carefully monitored with a rectal probe (Cole-Parmer Instrument, Vernon Hill, IL) and maintained at 37°C throughout the surgical procedure.

Experimental Protocol

Eight-wk-old Fischer 344 adult male rats were used in these experiments. Rats were randomly divided into eight groups and underwent LAD occlusion for 30 min and 2 h of reperfusion, as shown in Fig. 1. In group 1, saline was administered (0.1 ml ip) at 0 h, 24 h, 48 h, 72 h, and 96 h, and hearts were then subjected to LAD occlusion and reperfusion. In group 2, animals received BMS (1 mg/kg ip) at 0 h and 24 h, followed by LAD occlusion and reperfusion. Group 3 was given BMS at 0 h, 24 h, and 48 h. Group 4 was given BMS at 0 h, 24 h, 48 h, and 72 h. Group 5 was given BMS at 0 h, 24 h, 48 h, 72 h, and 96 h. Group 6 was similar to group 5, except that 5-HD (5 mg/kg ip), a specific blocker of mitoK_ATP channel, was given with BMS at each interval. Group 7 was also similar to group 5, except that WTN (15 µg/kg ip), an inhibitor of Akt, was given together with BMS at different intervals. In group 8, N-nitro-L-arginine methyl ester (L-NAME; 30 µg/kg ip), a general inhibitor of nitric oxide (NO) synthase (NOS), was given together with BMS. The proper doses for BMS, WTN, and L-NAME have been published previously (1, 27). Effect of WTN or L-NAME alone on infarction was also determined (1, 27).

View larger version (17K): [in this window] [in a new window]   Fig. 1. Experimental groups and protocol. LAD, left anterior descending coronary artery; L-NAME, N-nitro-L-arginine methyl ester; BMS, BMS-191095; 5-HD, 5-hydroxydecanoic acid; WTN, wortmannin.

After anesthesia, the right carotid artery was cannulated with a microtip pressure transducer catheter (SPR-869, Millar Instruments) connected to a pressure-volume conductance unit (MPCU-200). Pressure-volume analysis provided analog outputs of the time-varying ventricular pressure and volume signals for data acquisition. The inferior vena cava was exposed, and inferior vena cava occlusion was performed by external compression, as described previously by Wang et al. (30). Hemodynamic parameter analysis was carried out using Millar's PVAN software (version 3.2).

Determination of Cell and Tissue Injury

Measurement of myocardial infarction. After left thoracotomy, animals were injected with 3 M potassium chloride directly into the left ventricle, and the heart was excised. The left ventricle was sliced parallel to the atrioventricular groove into 2- to 3-mm-thick sections, and the sections were incubated in 1% 2,3,5-triphenyltetrazolium chloride (TTC) solution prepared in phosphate buffer, pH 7.4, for 30 min at 37°C (25). In viable normal myocardium, TTC is converted by dehydrogenase enzyme to a red formazan pigment that stains tissue dark red. The infarcted myocardium that does not take up TTC stain due to loss of dehydrogenase enzyme remains pale in color. Slices were fixed in the 10% formalin and were digitally imaged by using an Olympus microscope, and infarct size was determined by using MetaMorph software (Molecular Devices, Downingtown, PA).

Terminal dUTP nick-end labeling assay for apoptosis. Terminal dUTP nick-end labeling (TUNEL) assay was performed on 6-µm-thick deparaffinized histological sections with a MEBSTAIN Apoptosis Kit II (Medical and Biological Laboratories). Sections were stained with 4-6-diamidino-2-phenylindole to visualize nuclei and later were photographed with an Olympus BX41 microscope (Olympus America, Melville, NY) equipped with a digital camera. Each apoptotic nucleus was carefully identified at high magnification (x1,000) and counted.

Statistical Analysis

Pressure-volume relationship was analyzed by one-way analysis of variance (ANOVA, StatView 4.0) for repeated measures. Individual variables, such as apoptotic myocytes, were tested by one-way ANOVA, followed by Bonferroni/Dunn test or unpaired t-test. P value <0.05 was considered significant. All data are presented as means ± SE.

	RESULTS

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Effect of PC on Infarct Size

The infarct size was found to be 48.7 ± 4.8% in ischemic control animals (Fig. 2, A and B) and was significantly decreased with the treatment of BMS to 30.5 ± 3.2%, 26.4 ± 2.8%, 33.8 ± 2.5%, and 21.7 ± 2.4% [in groups 2, 3, 4, and 5, respectively, as compared with control group (P < 0.05)]. Interestingly, hearts given BMS five times (group 5) at intermittent intervals were more tolerant to ischemia-reperfusion injury, resulting in marked reduction in infarct size compared with groups 2, 3, and 4 (P < 0.05) (Fig. 2B). However, administration of 5-HD, WTN, or L-NAME with BMS abolished the protection, and infarct was similar to control ischemic hearts (Fig. 2, A and B). Pretreatment of the hearts with these inhibitors did not cause any additional infarction (data not given).

View larger version (51K): [in this window] [in a new window]   Fig. 2. Effect of chronic preconditioning (PC) on infarct size. Shown are gross infarction, indicated by pale color (A), and quantitation of infarct size (B). As compared with ischemic control group, PC with BMS reduced the infarct size significantly, and the infarct size was the minimum after treatment with BMS for 96 h. Infarct-sparing ability of PC was abrogated when hearts were pretreated with 5-HD, WTN, or L-NAME, indicating that both mitochondrial ATP-sensitive K+ (mitoKATP) channels and phosphatidylinositol 3-kinase (PI3-K)/Akt as well as nitric oxide (NO) are involved in chronic PC. Values are expressed as means ± SE (n = 6–10). *P < 0.05 vs. ischemia-reperfusion control group (Con). P < 0.05 vs. groups 2–4 (G2–G4). 5-HD + BMS, BMS and 5-HD given together; WTN + BMS, wortmannin and BMS given together; L-NAME + BMS, L-NAME and BMS given together. LV, left ventricle.

Hemodynamic data are shown in Fig. 3. At the end of reperfusion, heart function was significantly decreased in the ischemic control group, as indicated by first derivative of left ventricular (LV) pressure increase (+dP/dt) (6,184.6 ± 326.4 mmHg), first derivative of LV pressure decrease (–dP/dt) (5,742.7 ± 371.5 mmHg), and LV ejection fraction (EF) (18.6 ± 4.4%), when compared with the BMS-treated group. A significant increase in LV +dP/dt, LV –dP/dt, and LV EF was observed in BMS treatment groups as compared with the control group. In the 96-h BMS treatment group, the cardiac function was better preserved than in other BMS-pretreated groups. However, the chronic protective effects of BMS were abolished by 5-HD, WTN, or L-NAME when given together with BMS (Fig. 3).

View larger version (14K): [in this window] [in a new window]   Fig. 3. Effect of various interventions on hemodynamic data. Hemodynamic parameters are significantly improved by chronic PC as indicated by positive and negative values of first derivative of LV pressure (+dP/dt and –dP/dt) (A and B) and percentage of ejection fraction (EF%; C). Optimal results were obtained in intermittently BMS-pretreated hearts for 96 h, as indicated by significant functional improvement. However, the beneficial effect of BMS was abolished by 5-HD, WTN, or L-NAME. Values are expressed as means ± SE (n = 6). *P < 0.05 vs. ischemia-reperfusion control group. P < 0.05 vs. G2–G4.

Apoptosis in BMS pretreatment groups at 24, 48, 72, or 96 h was significantly reduced compared with ischemia-reperfusion control group (Fig. 4). Among BMS-pretreated groups, the 96-h treatment group showed the lowest number of TUNEL-positive nuclei as compared with other BMS-treated groups (P < 0.05). However, the protective effect of BMS on apoptosis was abolished with 5-HD, WTN, or L-NAME given together with BMS.

View larger version (70K): [in this window] [in a new window]   Fig. 4. Representative photomicrographs of terminal dUTP nick-end labeling (TUNEL)-positive nuclei in various treatment groups. A: normal control; 4-6-diamidino-2-phenylindole (DAPI)-stained nuclei (blue; arrow). B: TUNEL-positive nuclei (green; arrow) in ischemia and reperfusion control group; sections stained with -sarcomeric actin, DAPI, and TUNEL. C: same area as B, except all nuclei were stained by DAPI. D: same section as B, except green fluorescence shows TUNEL-positive nuclei (arrow). E: intermittent PC after 96 h (G5). TUNEL-positive nuclei were significantly decreased in BMS-treated hearts as compared with ischemic control group. Rats treated with BMS at different intervals were also significantly protected from apoptosis (data not shown). F: occurrence of apoptosis after treatment with BMS + 5-HD. Treatment with WTN or L-NAME produced results similar to ischemic control (data not shown). Magnification, x400; n = 4 for each group; bars, 20 µm. G: semiquantitative estimate of TUNEL-positive nuclei in various groups. Values are means ± SE. *P < 0.05 vs. ischemia-reperfusion control. P < 0.05 vs. G2–G4.

	DISCUSSION

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MitoK_ATP channel plays a pivotal role in PC-induced protection. The importance of this channel is quite evident owing to the fact that it is localized in mitochondria, a lifeline cell organelle that occupies one-third of the cell volume. This channel is also believed to be the end effector of protection by PC (32). This study took advantage of the fact that pharmacological openers of this channel produce both immediate and late protection. BMS is a specific opener of mitoK_ATP channel, does not effect vasodilator or action potential shortening, and protects effectively against ischemic injury in the heart (8) and brain (13). We administered BMS intermittently at an interval of 24 h until 96 h. The heart was constantly preconditioned against the lethal ischemia. The cardiac function was well preserved at all time in these hearts. It could be argued that the prolonged protective effect of BMS is due to its accumulation within mitochondrial membranes for a longer duration, thus inducing PC effects. The half-life of BMS in rats is 3 h (21), and loss of effect after 12 h (in our unpublished data) strongly suggests that BMS is not trapped within the mitochondrial membranes.

Apoptosis was significantly reduced in BMS-pretreated hearts for 96 h as compared with the 24-h treated group, suggesting that the effect of chronic PC is more robust than that of acute PC. A specific inhibitor of mitoK_ATP channels, 5-HD, completely abrogated the effects of BMS-induced cardiac protection. These observation clearly depict that mitoK_ATP channel plays an important role in chronic PC.

Recent reports suggest that the heart can be put in preconditioned state with intermittent administration of opioids even after their withdrawal (18, 19). In the latter report (19), chronic morphine exposure gave stronger and sustained protection for 48 h after withdrawal of the drug. These observation are consistent with our results in which BMS was given intermittently at an interval of 24 h up to 96 h, resulting in significant protection as compared with one-time BMS-pretreated group (Figs. 2–4).

Signaling pathway for this unique phenomenon of constant protection is not completely known. The blockade of protection by mitoK_ATP channel antagonists and inhibitors of NOS and Akt suggests the involvement of mitoK_ATP channel, NO, and Akt signaling in this protection. However, it is not clear from this study whether mitoK_ATP channel remains open constantly or can be activated upon oxidative stimulus. It appears that the latter option is more likely because inducible NOS (iNOS) and Akt are known to be upregulated during the later phase of PC. The release of NO and Akt translocation to mitochondria could be important factors in the channel-mediated protection (1, 10, 20).

Infarct size is a standard marker of cell injury following permanent ischemia-reperfusion (9). A majority of cells die in the infarcted myocardium due to increased sarcolemmal permeability, ATP depletion, and loss of Ca²⁺ homeostasis (33), whereas many cells at the infarct border zone undergo apoptosis. PC effectively reduces both necrosis and apoptosis within the infarcted heart. The cardioprotective effects of Akt have been mainly attributed to the reduction of myocardial apoptosis and have a pivotal role in vascular homeostasis and angiogenesis (14, 24). The antiapoptotic activity of Akt is mediated through the activation of phosphatidylinositol 3-kinase (PI3-K) (6). There is strong support to the hypothesis that BMS activates PI3-K/Akt pathway during late PC (1). Previous studies have shown mitochondria as the downstream target of Akt activation, which causes phosphorylation of various proteins in the mitochondria (2). The eventual outcome is a reduced release of both apoptosis-inducing factor and cytochrome c, thus alleviating cell apoptosis (34).

The participation of PI3-K/Akt in opening of mitoK_ATP channels is highly attractive. Activation of PI3-K/Akt has multiple cardiovascular functions, including cell migration and growth, homeostasis, angiogenesis (24), and protection against ischemia-reperfusion injury (12). Akt is activated downstream of PI3-K during IPC and, in turn, phosphorylates a number of downstream targets relevant to cell survival, including the proapoptotic Bcl-2 family member Bad, NOS, and nuclear factor-B (24, 26). Phosphorylation of Bad by Akt inhibits its proapoptotic function and promotes cell survival (11). Akt, which is phosphorylated by BMS in the cytosol, is translocated to mitochondria (1). This is in agreement with the work of Sale et al. (22), who reported that Akt phosphorylation in mitochondria causes activation of various proteins, such as ATP synthase and GSK-3 within the mitochondria, which are eventually cardioprotective as they aid in the reduction of apoptosis. Thus we postulate that activation of the PI3-K signaling pathway upstream of mitoK_ATP channel results in decreased cardiac cell death and apoptosis.

NO regulates many physiological and pathological processes controlling the cardiovascular system (15). Modulation of mitoK_ATP channels occurs via enhanced levels of cGMP. The cGMP-dependent protein kinase in turn may phosphorylate mitoK_ATP channels and prime the channels to provide cardiac protection (4). In addition, there is a possibility for the involvement of NO in cardiac protection mediated by pharmacological PC with BMS. NO being upstream of PKC and the mitoK_ATP channels acts as a free radical, combining with superoxide to generate peroxynitrite, that could activate PKC, leading to opening of the mitoK_ATP channels (35). NO produced from cytosolic and mitochondrial NOS plays an important role as a trigger of both early and delayed PC (17) and is known to potentiate mitoK_ATP opening (3). Furthermore, L-NAME, the nonselective inhibitor of NOS, blocked the protective effect of BMS. The results of this study are consistent with other reports that NO enhances mitoK_ATP channel opening (23), and they are further supported by the observations that diazoxide did not provide any protection in iNOS knockout mice, thus suggesting that opening of mitoK_ATP channels was iNOS dependent (27). All these studies support our view that NO is an important trigger of mitoK_ATP channel activation during chronic PC.

Conclusion

Chronic or intermittent PC is a clinically relevant approach by which the heart could be protected against ischemia for a longer period via activation of mitoK_ATP channel, Akt translocation, and NO signaling pathways.

	GRANTS

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This study was supported in part by National Heart, Lung, and Blood Institute Grants HL-23597, HL-70062, HL-080686, R37-HL-074272 (to M. Ashraf), and HL-081859-01 (to Y. Wang).

	FOOTNOTES

 

Address for reprint requests and other correspondence: M. Ashraf, Dept. of Pathology and Laboratory Medicine, Univ. of Cincinnati Medical Center, 231 Albert Sabin Way, Cincinnati, OH 45267-0529 (e-mail: muhammad.ashraf{at}uc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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This Article Abstract Full Text (PDF) All Versions of this Article: 292/5/H2300    most recent 01163.2006v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science 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 ISI Web of Science (3) Citing Articles via Google Scholar Google Scholar Articles by Wang, Y. Articles by Ashraf, M. Search for Related Content PubMed PubMed Citation Articles by Wang, Y. Articles by Ashraf, M.