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Am J Physiol Regul Integr Comp Physiol 292: R920-R926, 2007. First published September 28, 2006; doi:10.1152/ajpregu.00520.2006
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NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION

Myocardial preconditioning against ischemia-reperfusion injury is abolished in Zucker obese rats with insulin resistance

Prasad V. G. Katakam,1 James E. Jordan,1,2 James A. Snipes,1 Christina D. Tulbert,1 Allison W. Miller,1 and David W. Busija1

1Department of Physiology and Pharmacology and 2Department of Cardiothoracic Surgery, Wake Forest University Health Sciences, Winston-Salem, North Carolina

Submitted 20 July 2006 ; accepted in final form 25 September 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Insulin resistance (IR) precedes the onset of Type 2 diabetes, but its impact on preconditioning against myocardial ischemia-reperfusion injury is unexplored. We examined the effects of diazoxide and ischemic preconditioning (IPC; 5-min ischemia and 5-min reperfusion) on ischemia (30 min)-reperfusion (240 min) injury in young IR Zucker obese (ZO) and lean (ZL) rats. ZO hearts developed larger infarcts than ZL hearts (infarct size: 57.3 ± 3% in ZO vs. 39.2 ± 3.2% in ZL; P < 0.05) and also failed to respond to cardioprotection by IPC or diazoxide (47.2 ± 4.3% and 52.5 ± 5.8%, respectively; P = not significant). In contrast, IPC and diazoxide treatment reduced the infarct size in ZL hearts (12.7 ± 2% and 16.3 ± 6.7%, respectively; P < 0.05). The mitochondrial ATP-activated potassium channel (KATP) antagonist 5-hydroxydecanoic acid inhibited IPC and diazoxide-induced preconditioning in ZL hearts, whereas it had no effect on ZO hearts. Diazoxide elicited reduced depolarization of isolated mitochondria from ZO hearts compared with ZL (73 ± 9% in ZL vs. 39 ± 9% in ZO; P < 0.05). Diazoxide also failed to enhance superoxide generation in isolated mitochondria from ZO compared with ZL hearts. Electron micrographs of ZO hearts revealed a decreased number of mitochondria accompanied by swelling, disorganized cristae, and vacuolation. Immunoblots of mitochondrial protein showed a modest increase in manganese superoxide dismutase in ZO hearts. Thus obesity accompanied by IR is associated with the inability to precondition against ischemic cardiac injury, which is mediated by enhanced mitochondrial oxidative stress and impaired activation of mitochondrial KATP.

mitochondrial KATP; obesity; Type 2 diabetes

INSULIN RESISTANCE (IR) is defined as a defect in the ability of insulin to stimulate glucose uptake and is characterized by impaired glucose tolerance and hyperinsulinemia (40). On the basis of the American Diabetes Association (12a) and Adult Treatment Panel III (19) criteria, approximately one-third of the healthy population (13) may be insulin resistant. Without necessary dietary and lifestyle modifications, the IR syndrome progresses to Type 2 diabetes mellitus (40). Both IR and Type 2 diabetes are known risk factors for the development of atherosclerosis and myocardial infarction (3, 20, 37). Several studies have shown that, in both humans and animals, those with diabetes have larger infarctions accompanied by decreased survival rates after myocardial infarction than their nondiabetic counterparts (5, 9, 20, 30, 32). However, the impact of IR that precedes diabetes on ischemic cardiac injury is not known.

Zucker obese (ZO) rats with leptin receptor mutation (fa/fa, homozygous for the mutation) have been widely used as models of IR and Type 2 diabetes, with Zucker lean (ZL) rats as controls (Fa/fa, heterozygous for the mutation) (3). Previous data from our laboratory and others have shown that ZO rats develop IR with a metabolic profile very similar to the human condition (11, 12). We and others have also reported abnormal coronary vasoreactivity and cardiac function in ZO rats compared with ZL controls (26, 36, 38, 47). Furthermore, our group (25) has observed greater myocardial damage in ZO rats after myocardial ischemia-reperfusion injury (MI/R) than in the ZL controls (25). On the basis of the underlying metabolic derangements, coronary vascular dysfunction, and impaired tolerance to myocardial ischemia present in IR, we hypothesized that ZO rats will be resistant to the protection afforded by preconditioning against MI/R injury. Therefore, we examined the effects of ischemic and pharmacological preconditioning on MI/R in ZO rats. To determine the effects of IR associated with obesity on cardiovascular function, we studied the ZO rats at the age of 10–12 wk when they are euglycemic and largely normotensive (14, 15, 33, 45).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The animal protocol was approved by the Institutional Animal Care and Use Committee of Wake Forest University Health Sciences. All experiments complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male ZO and ZL rats were obtained at 10 wk of age and housed in the animal care facility. The animals received standard rat chow and tap water.

MI/R studies. The rats aged 10–12 wk were randomized to receive one of the following treatments (Fig. 1) : 1) index ischemia of 30 min followed by 4-h reperfusion, 2) normal saline as placebo, 3) ischemic preconditioning (IPC; 5-min ischemia followed by 5-min reperfusion), 4) 5-hydroxydecanoic acid (5-HD; 10 mg/kg iv) + IPC, 5) diazoxide (10 mg/kg iv), or 6) diazoxide + 5-HD. Treatments 2–6 were followed by index ischemia-reperfusion. 5-HD has been proposed as a specific blocker of mitochondrial ATP-activated potassium (KATP) channels and has been used in numerous studies to block preconditioning against MI/R injury (10, 16, 41). The rats were anesthetized with pentobarbital sodium (induction: 60 mg/kg ip; maintenance: 25 mg/kg iv, as needed) and were ventilated with 100% oxygen by a rodent ventilator adjusted to maintain exhaled CO2 between 3.5 and 4.5%. A left thoracotomy was performed, and a suture was placed around the left coronary artery. After appropriate treatments, index ischemia was initiated by tightening the suture around the coronary artery; reperfusion was then achieved by loosening the suture. Subsequently, infarct size was determined by differential staining and triphenyltetrazolium chloride, as described previously, with minor modifications (25), and is expressed as follows: area of necrosis/area at risk x 100.


Figure 1
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  		Fig. 1. Infarct size expressed as a percentage of the area at risk. *P < 0.05 compared with placebo-treated Zucker lean (ZL) rats. IPC, ischemic preconditioning; 5-HD, 5-hydroxydecanoic acid.

 
Isolation of mitochondria. Heart mitochondria were isolated from animals that were not subjected to ischemia-reperfusion injury, using a previously described method (24). Briefly, hearts were homogenized in ice-cold isolation buffer containing (in mmol/l) 225 mannitol, 75 sucrose, 5 MOPS, 0.5 EGTA, and 2 taurine, with 0.2% BSA (pH 7.25). The homogenate was centrifuged twice at 1,000 g for 5 min (4°C), and the supernatant was then centrifuged at 10,000 g for 10 min (4°C). After the pellet was washed, it was resuspended in buffer containing (in mmol/l) 225 mannitol, 25 sucrose, 5 MOPS, 1 EGTA, 5 KH2PO4, and 2 taurine supplemented with 0.2% BSA (pH 7.4), placed on ice, and used within 3 h. Mitochondrial isolation was carried out simultaneously from a matched pair of ZO and ZL hearts.

Electron microscopy. As previously reported (39), an isolated mitochondrial pellet or a finely chopped heart tissue was fixed in 2.5% glutaraldehyde. It is noted that the hearts were not subjected to ischemia-reperfusion injury before electron microscopy studies. After they were washed in 0.1 M Millonig phosphate buffer, the samples were postfixed in 2% osmium tetroxide followed by dehydration in a graded series of ethanol. Next, the samples were infiltrated in a propylene oxide and spurr resin before embedding and curing. Ultrathin sections were cut, placed on copper grids, and stained with uranyl acetate and lead citrate. Electron microscopy was performed with a Philips TEM 400 transmission electron microscope.

Confocal microscopy. Sample mitochondria from ZO and ZL hearts were examined in blind studies (n = 5 each), one rat pair at a time and in an alternating sequence. Mitochondria placed in poly-D-lysine-coated glass-bottom culture dishes were studied with the use of an inverted microscope connected to a Zeiss LSM-510 laser-scanning confocal system with a Zeiss C-Apochromat x63/numerical aperture 1.2 water-immersion objective. All experiments were performed at room temperature, and imaging conditions such as gain levels, confocal aperture size, and laser power were held constant. The average pixel intensity of mitochondria in each field (n = 30–50) was determined with Zeiss software.

Mitochondrial membrane potential was monitored with tetramethylrhodamine ethyl ester (TMRE; 100 nmol/l), and reactive oxygen species (ROS) were assessed with dihydroethidium (HEt; 5 µmol/l) (Molecular Probes, Eugene, OR) as previously described (4, 39). TMRE fluorescent images (excitation wavelength = 543 nm, emission wavelength of >560 nm) were recorded after application of vehicle or diazoxide (100 or 500 µmol/l) or 1 µmol/l CCCP. CCCP is an ionophore that dissipates the mitochondrial membrane potential by a non-ion channel mechanism leading to depolarization of the mitochondria. In contrast, K+ channel activators such as NS-1619 (calcium-activated potassium channel activator) or diazoxide (mitochondrial KATP channel activator) depolarize the mitochondria by opening K+ channels. Thus depolarization by CCCP was assessed in ZO and ZL mitochondria to compare the mitochondrial membrane potential and to evaluate the specificity of diazoxide activation of mitochondrial KATP channels. The mitochondrial depolarization was expressed as the percent decrease in average TMRE fluorescence from the average steady-state fluorescence before application of drugs. For measurement of ROS, mitochondria were treated with vehicle or diazoxide (100 or 500 µmol/l), and 5 min later, HEt was administered in the dark. HEt fluorescence images were obtained (excitation wavelength = 488 nm and emission wavelength of > 560 nm), and average peak intensity of HEt fluorescence was determined.

Fluorescent detection of mitochondrial ROS. To validate the HEt fluorescence measurements from confocal microscopy, additional experiments were performed with the use of a fluorescence spectrophotometer (BMG Labtechnologies, Durham, NC) at 37°C, with filter wavelengths of 510-nm excitation and 590-nm emission. Isolated mitochondrial samples (0.05–0.1 mg/ml) with and without diazoxide (100 and 500 µmol/l) and 5 µmol/l HEt were prepared on a 96-well plate. HEt fluorescence of mitochondria from ZL and ZO rats (on the same plate) was measured in relative fluorescence units once every 60 s for 45 cycles. Data were expressed as a percentage of the average fluorescence of untreated ZL mitochondria.

Immunoblot quantification of manganese superoxide dismutase. Equal amounts of protein from mitochondrial lysate samples were separated by 4–20% SDS-PAGE, transferred onto a polyvinylidine difluoride membrane, and blocked with 5% skim milk powder, Tris-buffered saline, and 0.1% Tween 20. Blots were incubated with monoclonal anti-manganese superoxide dismutase (MnSOD; 1:2,500; BD Transduction Laboratories). The membranes were then washed and incubated with anti-mouse IgG (1:10,000; Jackson Immuno Research) conjugated to horseradish peroxidase. The bound antibodies were visualized by enhanced chemiluminescence, and the densities of the immunobands were quantitated.

Real-time PCR quantification of MnSOD. The tissue was homogenized, and total cellular RNA was extracted by an SVtotal RNA isolation system (Promega). Real-time RT-PCR was performed with the use of an ABI Prism 7700 sequence detection system (Applied Biosystems). PCR primers and probes were designed by the Primer Express program, with the forward and reverse primers located in different exons and the probe spanning an intron-exon boundary. PCR products were detected with probes labeled with fluorescent dye 6-carboxy-fluorescein at the 5' end and quencher dye 6-carboxy-tetramethyl-rhodamine at the 3' end. The 2–{Delta}{Delta}Ct method described by Livak and Schmittgen (29) was used to analyze the results. In brief, the threshold cycle (Ct) value of a gene was subtracted from the Ct value of a reference housekeeping gene (GAPDH) to standardize for the amounts of RNA template and efficiencies of reverse transcription. The resulting change in Ct values was then converted to a linear form using 2–{Delta}Ct and used in subsequent statistical analysis.

Data analysis. All values are expressed as means ± SE. ANOVA with a Newman-Keuls post hoc test was used to determine whether any significant differences existed among groups for area at risk or infarct size. Similarly, Tukey's post hoc test was used for TMRE and HEt fluorescence data. Significance was established at P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of IPC and diazoxide on infarct size. All groups studied had a similar area of the left ventricle made ischemic after occlusion of the left coronary artery (area at risk), with no significant differences among the groups [P = not significant (NS)] (Table 1). In the saline (placebo) groups, the area of necrosis was significantly greater in the ZO rats (57.3 ± 3%, n = 7) than in the ZL rats (39.2 ± 3.2%, n = 9; P < 0.05) (Fig. 1). IPC decreased the area of necrosis in ZL rats (12.7 ± 2%, n = 9; P < 0.05), whereas it had no significant effect on ZO rats (47.2 ± 4.3%, n = 8; P = NS). Administration of 5-HD before IPC partially inhibited the protective effect of IPC in ZL rats (27.4 ± 3.9%, n = 12; P < 0.05 vs. IPC alone), whereas it had no effect on IPC in ZO rats (51.9 ± 3.5%, n = 4; P < 0.05 vs. IPC alone). Administration of diazoxide before induction of the index ischemia reduced the area of necrosis in ZL rats (16.3 ± 6.7%, n = 5; P < 0.05 vs. untreated group). In contrast, diazoxide administration had no effect on area of necrosis in ZO rats (52.5 ± 5.8%, n = 7; P = NS vs. untreated). Administration of 5-HD to diazoxide-treated ZL rats before induction of the index ischemia reversed the protection afforded by diazoxide (41.9 ± 2.5%, n = 7; P < 0.05 vs. diazoxide alone). In contrast, 5-HD had no effect on the area of necrosis in diazoxide-treated ZO rats (45 ± 2.8%, n = 6; P = NS vs. diazoxide alone).


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  		Table 1. Area at risk (% of left ventricle)

 
Morphological changes of mitochondria. The yield of mitochondrial proteins (mg/g heart wet wt) was consistent within each group of animals, suggesting minimal variation in the preparations of the mitochondrial fraction (data not shown). Electron micrograph images of isolated mitochondria established the presence of mitochondria in the preparations and validated our isolation procedures (Fig. 2A). Isolated mitochondria tend to be fragile during sample processing for electron microscopy. Therefore, we studied the morphology of in situ mitochondria in sections of the hearts. Electron micrograph images of randomly selected heart sections from ZO hearts displayed fewer and more widely dispersed mitochondria than images from ZL hearts. Also, focal morphological changes suggestive of mitochondrial stress, such as disorganized cristae and vacuolation were observed in ZO hearts (Fig. 2, B and C).


Figure 2
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  		Fig. 2. Transmission electron micrographs showing the ultrastructural features of mitochondria. A: isolated heart mitochondria (x25,000). B and C: microsections of hearts from Zucker obese (ZO) and ZL rats. B: cross sections of hearts at low (x3,300) and high (x19,500; from boxed area) magnification. Mitochondria from ZO hearts exhibit vacuolation (V) and disorganized cristae (C). C: longitudinal sections of hearts (x5,500) from ZL and ZO rats. Focal areas of widely dispersed mitochondria were observed in ZO hearts compared with the tightly packed mitochondria in ZL hearts.

 
Effect of diazoxide on mitochondrial membrane potential. Diazoxide at 500 µmol/l decreased the TMRE fluorescence in isolated mitochondria from ZL hearts, indicating depolarization of mitochondria. However, the percent decrease in TMRE fluorescence after diazoxide treatment was significantly less in mitochondria from ZO hearts than from ZL hearts (73 ± 9% in ZL mitochondria vs. 39 ± 9% in ZO mitochondria, n = 5 rats each; P < 0.05) (Fig. 3A). CCCP elicited a similar decrease in TMRE fluorescence in ZL (50 ± 3%) and ZO (49 ± 4%; n = 5 each, P = NS) mitochondria.


Figure 3
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  		Fig. 3. Membrane potential measured by tetramethylrhodamine ethyl ester (TMRE) fluorescence and reactive oxygen species (ROS) measured by dihydroethidium (HEt) fluorescence. A: representative images of TMRE-loaded mitochondria from ZL and ZO hearts (top) and corresponding differential interference contrast (DIC) images merged with TMRE fluorescence (bottom). Diazoxide-induced depolarization of ZL mitochondria is indicated by a decrease in TMRE fluorescence. B: representative images of HEt-treated mitochondria from ZL and ZO hearts (top) and corresponding DIC images merged with HEt fluorescence (bottom). Diazoxide enhanced HEt fluorescence in ZL mitochondria, whereas ZO mitochondria were unaffected.

 
Effect of diazoxide on mitochondrial ROS production. After application of HEt, isolated mitochondria exhibited fluorescence, indicating ROS generation. This baseline HEt fluorescence (relative fluorescence units) was elevated modestly in mitochondria from ZO (154 ± 30) compared with ZL (101 ± 4, n = 3 rats each; P < 0.05) hearts. Diazoxide at 500 µmol/l increased the HEt fluorescence in ZL mitochondria (209 ± 25; P < 0.05) compared with HEt fluorescence in the absence of diazoxide. However, the ZO mitochondria did not show enhancement of HEt fluorescence after diazoxide application (145 ± 36; P = NS) (Fig. 3B). Similar observations were made by measuring HEt fluorescence in a fluorescence plate reader in which it was possible to simultaneously study several hundred mitochondria. HEt fluorescence was significantly elevated in ZO mitochondria at baseline compared with ZL mitochondria. Normalized HEt fluorescence was 170 ± 37% in ZO compared with 101 ± 1% in ZL mitochondria. Diazoxide (100 µmol/l) increased HEt fluorescence in mitochondria to 123 ± 5% in ZL hearts (n = 4; P < 0.05), whereas it failed to elicit any response in ZO hearts (170 ± 38%, n = 4; P = NS).

Expression of MnSOD. Immunoblots identifying the MnSOD showed increased immunoband intensity in ZO mitochondrial protein (168 ± 3.3 pixels, n = 6; P < 0.05) compared with ZL samples (133 ± 9.6 pixels, n = 6) (Fig. 4). However, the mRNA transcripts of MnSOD in the heart showed identical expression in ZO and ZL hearts [2–{Delta}{Delta}Ct in arbitrary units: 1.1 ± 0.3 (n = 5) in ZL vs. 1.1 ± 0.3 (n = 6) in ZO; P = NS].


Figure 4
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  		Fig. 4. Immunoblots of manganese superoxide dismutase (MnSOD) were performed with the lysates of isolated mitochondria from ZL (left) and ZO (right) hearts. ZO mitochondria showed a modest increase in MnSOD protein.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major findings of our study are as follows. 1) Hearts from ZO rats with IR sustain larger infarct sizes after MI/R injury than those from ZL rats. 2) IPC and diazoxide-induced preconditioning failed to confer cardioprotection in ZO rats compared with ZL rats. 3) Inhibition of mitochondrial KATP channels abolished both diazoxide-induced preconditioning and IPC in ZL hearts, whereas it had no effect on ZO hearts. 4) Diazoxide elicited significantly diminished membrane depolarization and ROS generation in isolated mitochondria from ZO hearts compared with ZL hearts. 5) Mitochondria from ZO hearts exhibit enhanced ROS production and morphological features suggestive of metabolic and oxidative stress.

Studies in our laboratory have demonstrated for the first time that hearts from IR ZO rats exhibited larger infarct area after MI/R injury than those from ZL rats (25). Recently, this observation was confirmed in Zucker diabetic fatty rats, which are genetically identical to the ZO rats used in our study, except that they develop severe hyperglycemia at an early age (27). Furthermore, these investigators showed that preconditioning does not occur in Zucker diabetic fatty rats (27). Because the ZO rats exhibit greater susceptibility to ischemic injury than ZL rats, we hypothesized that IR in the absence of hyperglycemia may also impair preconditioning against ischemic cardiac injury. IPC is a mechanism observed in almost all vital organs in which a short period of ischemia followed by reperfusion affords protection from subsequent ischemia-reperfusion injury. Several pharmacological agents such as diazoxide and anesthetics also induce preconditioning against ischemic injury. Because IPC and pharmacological preconditioning are known to share many mechanisms, we studied preconditioning by both approaches in ZO and ZL rats. Consistent with our hypothesis, we observed that IPC and diazoxide preconditioning reduced the infarct size in ZL hearts, whereas ZO rats failed to respond to preconditioning. Thus it appears that ZO hearts have lost their ability to precondition against ischemic injury.

It is widely accepted that mitochondria are the site of origin of critical signals that mediate IPC (6, 17, 18, 21, 23, 41). Mitochondrial depolarization via opening of mitochondrial KATP channels is one such important mechanism known to mediate preconditioning (6, 18). Inhibition of mitochondrial KATP channels in ZL hearts with 5-HD abolished the protection conferred by both IPC and diazoxide, suggesting that opening of mitochondrial KATP channels is critical to preconditioning. However, in ZO hearts, 5-HD had no effect on infarct size after IPC, suggesting impaired activation of mitochondrial KATP channels. Therefore, we evaluated the opening of mitochondrial KATP channels in isolated mitochondria in response to diazoxide, a prototype mitochondrial KATP channel opener.

Diazoxide has been used extensively to study pharmacological preconditioning where it appears to confer cardioprotection by activating mitochondrial KATP channels (6, 17). In isolated mitochondria from ZL hearts, diazoxide induced depolarization of mitochondria, indicating the opening of mitochondrial KATP channels. In contrast, diazoxide elicited diminished depolarization of mitochondria from ZO hearts. Thus, mitochondrial KATP channels from ZO hearts were resistant to activation by diazoxide. Interestingly, membrane depolarization in response to CCCP, an ionophore that dissipates the mitochondrial membrane potential and uncouples oxidative phosphorylation, was similar in mitochondria from both ZL and ZO hearts. This suggests that depolarization specifically mediated by mitochondrial KATP channels was abnormal in mitochondria from ZO hearts.

ROS have been implicated in adverse outcomes in cardiovascular disease. However, generation of ROS in the context of preconditioning has been shown to be beneficial. Enhanced generation of mitochondrial ROS in response to preconditioning has been proposed as a trigger for signaling pathways conferring cardioprotection (6, 16, 18, 21). In the present study, ROS production at baseline was elevated in mitochondria from ZO hearts, providing further evidence of a state of increased oxidative stress. Consistent with published reports (4, 22), diazoxide induced enhanced ROS generation in isolated mitochondria from ZL hearts. However, diazoxide failed to enhance ROS generation in mitochondria from ZO hearts. Thus it may be explained that increased oxidative stress at baseline causes oxidative modification of key mitochondrial enzymes and/or ion channels, leading to altered diazoxide binding and/or signaling in ZO hearts. Furthermore, the inability of diazoxide to increase ROS levels in mitochondria from ZO hearts may eliminate an essential preconditioning stimulus.

Because oxidative stress may also result from a deficiency of antioxidant mechanisms, we determined the expression of protein and mRNA of MnSOD, the primary antioxidant enzyme of mitochondria. Interestingly, we observed an increase in MnSOD protein in the isolated mitochondrial preparations from ZO obese hearts, although mRNA levels were unchanged compared with ZL hearts. Oxidative stress has been reported to increase expression of antioxidant enzymes in several pathological models (7, 31, 42). Thus increased expression of MnSOD in ZO hearts may be an adaptation in response to oxidative stress. However, the modest increase in MnSOD in ZO hearts may account for some but not all of the reduced ROS levels with diazoxide, and it is likely that other factors are more important.

Several reports have described an association between mitochondrial oxidative injury and alterations in mitochondrial morphology (28, 34, 43). In ZO hearts, we observed a decrease in numbers of mitochondria and biogenesis, features linked to decreased capacity to generate ATP and mitochondrial stress. However, only a few scattered mitochondria with morphological correlates of increased oxidative stress, such as swelling, vacuolation, and disorganized cristae (8) were seen in ZO hearts. Thus, morphologically, ZO hearts exhibit relatively mild but definite evidence of mitochondrial dysfunction indicative of oxidative damage.

Recent reports have questioned the specificity of 5-HD as a mitochondrial KATP channel antagonist (21). 5-HD is a well-accepted inhibitor of IPC and pharmacological preconditioning (10, 16, 41), and thus we believe that the preconditioning that we observed is a viable mechanism for myocardial protection. Moreover, there is no general agreement concerning the role of mitochondrial KATP channels in membrane depolarization and the specific ion channels mediating preconditioning (IPC and diazoxide). Similarly, conflicting reports exist regarding the molecular structure of mitochondrial KATP channels and the contribution of inward-rectifier potassium channels (44, 46). In the absence of ideal pharmacological probes of preconditioning, we used diazoxide, the most extensively studied agent (6, 10, 17, 34, 35) in preconditioning, although possible limitations are acknowledged. We are confident that impaired depolarization and ROS generation are two key mitochondrial defects that underlie the pathology associated with IR. Indeed, our findings in ZO rats are consistent with recent observations in patients with diabetes where isolated cardiac mitochondria did not depolarize or enhance ROS production in response to diazoxide. (22).

Based on our observations, we conclude that an increased mitochondrial oxidative stress is central to the impaired preconditioning in ZO hearts. Oxidative free radicals could potentially alter the activation of mitochondrial KATP channels by direct modification of channel proteins or alteration of membrane properties by lipid peroxidation. This could lead to diminished depolarization in response to diazoxide or ischemia and impaired preconditioning. Similarly, ROS can inhibit mitochondrial enzymes, including electron transport chain enzymes, leading to the inability to generate proton motive force across the inner mitochondrial membrane, decreased oxidative phosphorylation (ATP generation), and increased predisposition to ischemic injury.

The present study demonstrates for the first time that IR accompanying obesity impairs the ability of hearts to precondition against ischemic injury. It appears that increased mitochondrial oxidative stress in IR underlies the altered activation of mitochondrial KATP channels and critical protective signaling pathways, leading to the inability to precondition the heart against ischemia-reperfusion injury.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by National Institutes of Health Grants HL-30260, HL-50587, HL-46558, DK-62372, HL-77731, HL-65380, and HL-66074, American Heart Association Grant 0270114N, and Y. F. Wu Research and Education Fund, WFUSM Venture Fund, K. G. Phillips Fund for Prevention and Treatment of Heart Disease, and WFUSM Interim Funding.


   ACKNOWLEDGMENTS
 
The authors appreciate the technical guidance with electron microscopy provided by Kenneth Grant of the Department of Pathology, Wake Forest University Health Sciences. We thank Nancy Busija for editing the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. V. G. Katakam, Dept. of Physiology and Pharmacology, Wake Forest Univ. Health Sciences, Hanes 1050, Medical Center Blvd, Winston-Salem, NC 27157 (e-mail: pkatakam{at}wfubmc.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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Bray GA. The Zucker-fatty rat: a review. Fed Proc 36: 148–153, 1977.[Web of Science][Medline]
  2. Bressler P, Bailey SR, Matsuda M, DeFronzo RA. Insulin resistance and coronary artery disease. Diabetologia 39: 1345–1350, 1996.[CrossRef][Web of Science][Medline]
  3. Busija DW, Katakam P, Rajapakse NC, Kis B, Grover G, Domoki F, Bari F. Effects of ATP-sensitive potassium channel activators diazoxide and BMS-191095 on membrane potential and reactive oxygen species production in isolated piglet mitochondria. Brain Res Bull 66: 85–90, 2005.[CrossRef][Web of Science][Medline]
  4. Cho E, Rimm EB, Stampfer MJ, Willett WC, Hu FB. The impact of diabetes mellitus and prior myocardial infarction on mortality from all causes and from coronary heart disease in men. J Am Coll Cardiol 40: 954–960, 2002.[Abstract/Free Full Text]
  5. Costa AD, Quinlan CL, Andrukhiv A, West IC, Jaburek M, Garlid KD. The direct physiological effects of mitoKATP opening on heart mitochondria. Am J Physiol Heart Circ Physiol 290: H406–H415, 2006.[Abstract/Free Full Text]
  6. Csonka C, Pataki T, Kovacs P, Muller SL, Schroeter ML, Tosaki A, Blasig IE. Effects of oxidative stress on the expression of antioxidative defense enzymes in spontaneously hypertensive rat hearts. Free Radic Biol Med 29: 612–619, 2000.[CrossRef][Web of Science][Medline]
  7. Dong F, Zhang X, Yang X, Esberg LB, Yang H, Zhang Z, Culver B, Ren J. Impaired cardiac contractile function in ventricular myocytes from leptin-deficient ob/ob obese mice. J Endocrinol 188: 25–36, 2006.[Abstract/Free Full Text]
  8. Donnan PT, Boyle DI, Broomhall J, Hunter K, MacDonald TM, Newton RW, Morris AD. Prognosis following first acute myocardial infarction in Type 2 diabetes: a comparative population study. Diabet Med 19: 448–455, 2002.[CrossRef][Web of Science][Medline]
  9. Dos Santos P, Kowaltowski AJ, Laclau MN, Seetharaman S, Paucek P, Boudina S, Thambo JB, Tariosse L, Garlid KD. Mechanisms by which opening the mitochondrial ATP-sensitive K+ channel protects the ischemic heart. Am J Physiol Heart Circ Physiol 283: H284–H295, 2002.[Abstract/Free Full Text]
  10. Erdos B, Snipes JA, Miller AW, Busija DW. Cerebrovascular dysfunction in Zucker obese rats is mediated by oxidative stress and protein kinase C. Diabetes 53: 1352–1359, 2004.[Abstract/Free Full Text]
  11. Erdos B, Snipes JA, Tulbert CD, Katakam P, Miller AW, Busija DW. Rosuvastatin improves cerebrovascular function in Zucker obese rats by inhibiting NAD(P)H oxidase-dependent superoxide production. Am J Physiol Heart Circ Physiol 290: H1264–H1270, 2006.[Abstract/Free Full Text]
  12. Expert Committee on the Diagnosis and Classification of Diabetes Mellitus. Report of the expert committee on the diagnosis, and classification of diabetes mellitus. Diabetes Care 26, Suppl 1: S5–S20, 2003.
  13. Ford ES, Abbasi F, Reaven GM. Prevalence of insulin resistance and the metabolic syndrome with alternative definitions of impaired fasting glucose. Atherosclerosis 181: 143–148, 2005.[CrossRef][Web of Science][Medline]
  14. Frisbee JC. Hypertension-independent microvascular rarefaction in the obese Zucker rat model of the metabolic syndrome. Microcirculation 12: 383–392, 2005.[CrossRef][Web of Science][Medline]
  15. Frisbee JC, Stepp DW. Impaired NO-dependent dilation of skeletal muscle arterioles in hypertensive diabetic obese Zucker rats. Am J Physiol Heart Circ Physiol 281: H1304–H1311, 2001.[Abstract/Free Full Text]
  16. Fryer RM, Eells JT, Hsu AK, Henry MM, Gross GJ. Ischemic preconditioning in rats: role of mitochondrial KATP channel in preservation of mitochondrial function. Am J Physiol Heart Circ Physiol 278: H305–H312, 2000.[Abstract/Free Full Text]
  17. Garlid KD. Opening mitochondrial K(ATP) in the heart—what happens, and what does not happen. Basic Res Cardiol 95: 275–279, 2000.[CrossRef][Web of Science][Medline]
  18. Gross GJ, Peart JN. KATP channels and myocardial preconditioning: an update. Am J Physiol Heart Circ Physiol 285: H921–H930, 2003.[Abstract/Free Full Text]
  19. Grundy SM, Brewer HB Jr, Cleeman JI, Smith SC Jr, Lenfant C. Definition of metabolic syndrome: report of the National Heart, Lung, and Blood Institute/American Heart Association conference on scientific issues related to definition. Arterioscler Thromb Vasc Biol 24: e13–18, 2004.[Free Full Text]
  20. Haffner SM, Lehto S, Ronnemaa T, Pyorala K, Laakso M. Mortality from coronary heart disease in subjects with type 2 diabetes and in nondiabetic subjects with and without prior myocardial infarction. N Engl J Med 339: 229–234, 1998.[Abstract/Free Full Text]
  21. Hanley PJ, Mickel M, Loffler M, Brandt U, Daut J. K(ATP) channel-independent targets of diazoxide and 5-hydroxydecanoate in the heart. J Physiol 542: 735–741, 2002.[Abstract/Free Full Text]
  22. Hassouna A, Loubani M, Matata BM, Fowler A, Standen NB, Galinanes M. Mitochondrial dysfunction as the cause of the failure to precondition the diabetic human myocardium. Cardiovasc Res 69: 450–458, 2006.[Abstract/Free Full Text]
  23. Honda HM, Korge P, Weiss JN. Mitochondria and ischemia/reperfusion injury. Ann NY Acad Sci 1047: 248–258, 2005.[CrossRef][Web of Science][Medline]
  24. Imahashi K, Schneider MD, Steenbergen C, Murphy E. Transgenic expression of Bcl-2 modulates energy metabolism, prevents cytosolic acidification during ischemia, and reduces ischemia/reperfusion injury. Circ Res 95: 734–741, 2004.[Abstract/Free Full Text]
  25. Jordan JE, Simandle SA, Tulbert CD, Busija DW, Miller AW. Fructose-fed rats are protected against ischemia/reperfusion injury. J Pharmacol Exp Ther 307: 1007–1011, 2003.[Abstract/Free Full Text]
  26. Katakam PV, Tulbert CD, Snipes JA, Erdos B, Miller AW, Busija DW. Impaired insulin-induced vasodilation in small coronary arteries of Zucker obese rats is mediated by reactive oxygen species. Am J Physiol Heart Circ Physiol 288: H854–H860, 2005.[Abstract/Free Full Text]
  27. Kristiansen SB, Lofgren B, Stottrup NB, Khatir D, Nielsen-Kudsk JE, Nielsen TT, Botker HE, Flyvbjerg A. Ischaemic preconditioning does not protect the heart in obese and lean animal models of type 2 diabetes. Diabetologia 47: 1716–1721, 2004.[CrossRef][Web of Science][Medline]
  28. Lebovitz RM, Zhang H, Vogel H, Cartwright J Jr, Dionne L, Lu N, Huang S, and Matzuk MM. Neurodegeneration, myocardial injury, and perinatal death in mitochondrial superoxide dismutase-deficient mice. Proc Natl Acad Sci USA 93: 9782–9787, 1996.[Abstract/Free Full Text]
  29. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2–{Delta}{Delta}CT method. Methods 25: 402–408, 2001.[CrossRef][Web of Science][Medline]
  30. Marfella R, D'Amico M, Di Filippo C, Piegari E, Nappo F, Esposito K, Berrino L, Rossi F, Giugliano D. Myocardial infarction in diabetic rats: role of hyperglycaemia on infarct size and early expression of hypoxia-inducible factor 1. Diabetologia 45: 1172–1181, 2002.[CrossRef][Web of Science][Medline]
  31. Methy D, Bertrand N, Prigent-Tessier A, Stanimirovic D, Beley A, Marie C. Differential MnSOD and HO-1 expression in cerebral endothelial cells in response to sublethal oxidative stress. Brain Res 1003: 151–158, 2004.[CrossRef][Web of Science][Medline]
  32. Nadeau A, Tancrede G, Jobidon C, D'Amours C, and Rousseau-Migneron S. Increased mortality rate in diabetic rats submitted to acute experimental myocardial infarction. Cardiovasc Res 20: 171–175, 1986.[Web of Science][Medline]
  33. Oltman CL, Richou LL, Davidson EP, Coppey LJ, Lund DD, Yorek MA. Progression of coronary and mesenteric vascular dysfunction in Zucker obese and Zucker diabetic fatty rats. Am J Physiol Heart Circ Physiol 291: H1780–H1787, 2006.[Abstract/Free Full Text]
  34. Ozcan C, Bienengraeber M, Dzeja PP, Terzic A. Potassium channel openers protect cardiac mitochondria by attenuating oxidant stress at reoxygenation. Am J Physiol Heart Circ Physiol 282: H531–H539, 2002.[Abstract/Free Full Text]
  35. Ozcan C, Holmuhamedov EL, Jahangir A, Terzic A. Diazoxide protects mitochondria from anoxic injury: implications for myopreservation. J Thorac Cardiovasc Surg 121: 298–306, 2001.
  36. Picchi A, Gao X, Belmadani S, Potter BJ, Focardi M, Chilian WM, Zhang C. Tumor necrosis factor-alpha induces endothelial dysfunction in the prediabetic metabolic syndrome. Circ Res 99: 69–77, 2006.[Abstract/Free Full Text]
  37. Plutzky J, Viberti G, Haffner S. Atherosclerosis in type 2 diabetes mellitus and insulin resistance: mechanistic links and therapeutic targets. J Diabetes Complications 16: 401–415, 2002.[CrossRef][Web of Science][Medline]
  38. Prakash R, Mintz JD, Stepp DW. Impact of obesity on coronary microvascular function in the zucker rat. Microcirculation 13: 389–396, 2006.[CrossRef][Web of Science][Medline]
  39. Rajapakse N, Shimizu K, Payne M, Busija D. Isolation and characterization of intact mitochondria from neonatal rat brain. Brain Res Brain Res Protoc 8: 176–183, 2001.[CrossRef][Medline]
  40. Reaven GM. Insulin resistance/compensatory hyperinsulinemia, essential hypertension, and cardiovascular disease. J Clin Endocrinol Metab 88: 2399–2403, 2003.[Free Full Text]
  41. Riess ML, Camara AK, Novalija E, Chen Q, Rhodes SS, Stowe DF. Anesthetic preconditioning attenuates mitochondrial Ca2+ overload during ischemia in guinea pig intact hearts: reversal by 5-hydroxydecanoic acid. Anesth Analg 95: 1540–1546, 2002.[Abstract/Free Full Text]
  42. Sam F, Kerstetter DL, Pimental DR, Mulukutla S, Tabaee A, Bristow MR, Colucci WS, Sawyer DB. Increased reactive oxygen species production and functional alterations in antioxidant enzymes in human failing myocardium. J Card Fail 11: 473–480, 2005.[CrossRef][Web of Science][Medline]
  43. Santos DL, Palmeira CM, Seica R, Dias J, Mesquita J, Moreno AJ, Santos MS. Diabetes and mitochondrial oxidative stress: a study using heart mitochondria from the diabetic Goto-Kakizaki rat. Mol Cell Biochem 246: 163–170, 2003.[CrossRef][Web of Science][Medline]
  44. Seharaseyon J, Ohler A, Sasaki N, Fraser H, Sato T, Johns DC, O'Rourke B, Marban E. Molecular composition of mitochondrial ATP-sensitive potassium channels probed by viral Kir gene transfer. J Mol Cell Cardiol 32: 1923–1930, 2000.[CrossRef][Web of Science][Medline]
  45. Stepp DW. Impact of obesity and insulin resistance on vasomotor tone: nitric oxide and beyond. Clin Exp Pharmacol Physiol 33: 407–414, 2006.[CrossRef][Web of Science][Medline]
  46. Suzuki M, Kotake K, Fujikura K, Inagaki N, Suzuki T, Gonoi T, Seino S, Takata K. Kir6.1: a possible subunit of ATP-sensitive K+ channels in mitochondria. Biochem Biophys Res Commun 241: 693–697, 1997.[CrossRef][Web of Science][Medline]
  47. Toblli JE, DeRosa G, Rivas C, Cao G, Piorno P, Pagano P, Forcada P. Cardiovascular protective role of a low-dose antihypertensive combination in obese Zucker rats. J Hypertens 21: 611–620, 2003.[CrossRef][Web of Science][Medline]



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