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Am J Physiol Heart Circ Physiol 293: H1457-H1465, 2007. First published June 1, 2007; doi:10.1152/ajpheart.01282.2006
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Dynamic changes in nitric oxide and mitochondrial oxidative stress with site-dependent differential tissue response during anoxic preconditioning in rat heart

Dang Van Cuong, Mohamad Warda, Nari Kim, Won Sun Park, Jae Hong Ko, Euiyong Kim, and Jin Han

Mitochondrial Signaling Laboratory, Department of Physiology and Biophysics, College of Medicine, Mitochondrial Research Group-Frontier Inje Research Science and Technology Project, Cardiovascular and Metabolic Diseases Center, Inje University, Busan, Korea

Submitted 22 November 2006 ; accepted in final form 26 March 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In this study, dynamic changes in nitric oxide (NO) and mitochondrial superoxide (O2bullet–) were examined during anoxic preconditioning (AP) in rat heart model. AP and anoxia-reoxygenation (A/R) were performed on isolated hearts and single cardiomyocytes. The cellular insult in the form of infarct size and DNA damage were localized and correlated with NO synthases (endothelial and inducible) expression levels. The results showed that endocardium was the most affected region in AP groups, whereas the larger area of infarct was confined to mid- and epicardium in the A/R group. Interestingly, a high-level expression of immunofluorescent NO synthases was restricted to viable areas in the AP. In contrast to the gradual increase in O2bullet– level that occurred in the AP group, a sudden massive increase in its level was demonstrated at the onset of reoxygenation in the A/R group. The observed increase in NO production during reoxygenation in the AP group was attenuated by inducible NO synthase inhibitor. The study revealed, on a real-time basis, the role played by preconditioning for modulating NO and O2bullet– levels on behalf of cell survival. The results afford a better understanding of cardiac-adapting mechanism during AP and the role of inducible NO synthase in this important phenomenon.

mitochondrial superoxide; endocardium; inducible nitric oxide synthase

HEART FAILURE DUE TO ischemic-reperfusion injury is one of the most serious problems in heart disease. Ischemic preconditioning, in which short-term ischemia and reperfusion are followed by long-term ischemia, can protect the heart against ischemia and reperfusion-induced cardiac injuries (25). Although preconditioning by a nitric oxide (NO) donor can mimic the anoxic preconditioning (AP) phenomenon to protect the heart from anoxia-reoxygenation (A/R) injuries, the evidence that a change in NO concentration contributes to either pro- or antiapoptotic effects remains equivocal (13). NO stimulates soluble guanylate cyclase, leading to an activation of both the sarcolemmal and mitochondrial ATP-sensitive K+ (KATP) channels, thus reducing ischemia and reperfusion-induced heart damage (10, 18). However, this is a double-edged sword, since NO interacts with the reactive oxygen species (ROS) superoxide (O2bullet–) to form peroxynitrite (ONOO–), which is a reactive nitrogen species (RNS) that causes oxidative DNA damage (4, 16, 27). An early state of reoxygenation produces a massive increase in ROS levels, and the resulting apoptosis and/or necrosis impair cardiac function through arrhythmia or heart failure (26).

Recent studies have demonstrated that differences in O2bullet– dismutase distribution, action potential duration, and myosin-actin dynamics lead to regional differences in ischemia and reperfusion-induced heart injuries (5, 28). However, the process of regional injury localization during global ischemia is not completely understood. Using the rat heart as an experimental model, we propose that O2bullet– and NO contribute not only to infarction during global A/R injury but also to regional localization of the infarct. To date, no correlation has been drawn between cellular NO and mitochondrial ROS levels or their impacts on pro- or antiapoptotic outcomes during cardiac A/R. In the present study, we followed the dynamic changes in NO and O2bullet– levels during A/R using specific fluorescent probes. Regional expression differences between NO synthases [endothelial and inducible (eNOS and iNOS, respectively)] were compared for damaged and viable areas (including the endocardium, midcardium, and epicardium) by triphenyltetrazolium chloride (TTC) staining. A possible protective role against oxidative damage during these events, particularly for nuclear (n)DNA and mitochondrial (m)DNA, was also addressed.

We aimed to evaluate in vivo site-specific cardiac damage as a function of A/R-induced global injuries in a laboratory animal model. The degree of damage in the A/R-treated heart was correlated with the resulting NO and O2bullet– level changes. This study tested whether regional NO production and nuclear or mitochondrial damage could be superimposed on the recorded local gross damage during cellular oxidative stress.

Oxidative damage of nDNA and mDNA was evaluated using digestion with endonuclease III (Endo III), which is a glycosylase that hydrolyzes the N-glycosylic bond between deoxyribose and a damaged pyrimidine base (7). While digested mDNA was tested by standard agarose gel electrophoresis, nDNA fragmentation at Endo III-sensitive sites was detected by the more sensitive method of single-cell gel electrophoresis (the comet assay). Using these techniques, we sought to identify regional injury differences and to determine the protective effect of AP.

To the best of our knowledge, this might be the first study that localizes anatomic abnormalities and correlates them with the dynamic change in NO and mitochondrial O2bullet– during A/R. Therefore, we can understand better their contributions in the AP phenomenon.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Whole Heart and Single Cardiac Myocyte Preparations

Hearts were isolated from male Sprague-Dawley rats. The investigation conforms to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996) and was approved by the Institutional Animal Care and Use Committee of the College of Medicine, Inje University. The isolation of single ventricular cardiomyocytes was performed as previously described (9, 10). The experimental protocols are depicted in Fig. 1, A and B.


Figure 1
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  		Fig. 1. Experimental protocol and proposed hypothesis. A: experimental design for whole heart and single cardiomyocyte groups. For fluorescent imaging in vitro, single cells were stained with specific dyes and subsequently subjected to experimental protocols. B: excluding group 1 [control (CT)], all of the protocols involved anoxic perfusion with anoxic solution for 30 min followed by reoxygenation with Tyrode solution for 60 min. Whole heart perfusion or single-cell incubation were performed with anoxic solution for 30 min only [group 2, anoxia-reoxygenation (A/R)] or were pretreated 3 times with 2 min of exposure to anoxic solution [group 3, anoxic preconditioning (AP)]. In group 4, samples were treated as AP in the presence of 100 µM S-methylisothiourea (SMT). In addition, 24 h before heart and single-cell isolation in group 4, rats received injections of SMT (5 mg/kg body wt ip). Triphenyltetrazolium chloride (TTC) staining, Western blot analysis, fluorescent-based immunoassay, comet assay, and mitochondrial (mt)DNA fragmentation assay were performed after experimental protocols described above (arrowhead). To measure changes in mitochondrial superoxide (O2bullet–) and nitric oxide (NO), single cells were preincubated with specific dyes and then treated as described in MATERIALS AND METHODS. C: proposed signal transduction pathway. iNOS, inducible NO synthase; KATP, ATP-sensitive K+ channels; ROS, reactive oxygen species.

 
After a 20-min period of stabilization, hearts and single cardiomyocytes were sorted into the following four perfusion groups: group 1 [control (CT)], perfused with normal Tyrode solution for 120 min total; group 2 (A/R), perfused with normal Tyrode solution for 30 min followed by global anoxia for 30 min using anoxic solution and then reoxygenated for 60 min; the remaining groups were preconditioned twice with 5 min of global anoxia (group 3, AP) or underwent two single 5 min of AP in the presence of 100 µM S-methylisothiourea (SMT) as an iNOS-selective inhibitor (group 4, SMT). After being preconditioned, the hearts and cells were perfused with normal Tyrode solution for 60 min, followed by 30 min of anoxic perfusion. The animals in group 4 were additionally injected with SMT (5 mg/kg iv) 24 h before heart isolation.

TTC Staining and Regional Dissection

Regional myocardial viability was assessed by TTC staining (1). Following each treatment as predicted in the experimental protocol, the hearts were perfused for 10 min with 1% TTC containing normal Tyrode solution. The hearts were then cross-sectioned into 1.5-mm slices using a Stadie-Riggs microtome (Thomas Scientific). After being scanned (HP Scanjet 4300), the viable (red) and infarcted (white) areas were separated to evaluate the degree of damage in each slice. The data were analyzed with ImageJ (version 1.34s) software (NIH).

Quantification of DNA Damage

Comet assay. Oxidative DNA damage was evaluated in treated cardiomyocytes by the comet assay, as previously described (10). Briefly, cells were suspended in low-melting point agarose (1% in PBS) and plated onto standard agarose-coated slides. The plated cells were lysed overnight in lysis buffer. The cellular DNA was digested with Endo III at 37°C for 45 min and then denatured for 40 min in electrophoresis buffer. Electrophoresis was carried out at 25 V for 40 min. Following electrophoresis, the samples were washed in neutralizing solution and the DNA was stained with propidium iodide (5 µg/ml). Stained DNA samples were examined under a laser-scanning microscope (Axiovert200; Zeiss). Based on the comet head-to-tail ratio, DNA damage was categorized in the range from type I (undamaged, no discernible tail) to type V (highest level of DNA damage, negligible head). The comets were scored visually and imaged using the LSM510-META software (Zeiss). To avoid atypical comets, cells at the slide edges were excluded from the score. Between 200 and 400 comets were scored for each slide, and the assay was repeated five times (6, 14).

mDNA fragmentation. mDNA was isolated from separated viable and infarcted areas using the mtDNA extractor CT kit (Wako, Japan). To check for sites of oxidative damage, the mtDNA was incubated with Endo III at 37°C for 45 min to release damaged pyrimidines from the double-stranded DNA, thereby generating apurinic sites. The mtDNA was loaded onto 1% agarose gels and electrophoresed in Tris acetate-EDTA buffer. The agarose gel was stained with ethidium bromide, and images were captured (RAS3000; Fuji film).

Fluorescence-Based Immunoassay and Western Blot Analysis for Detecting eNOS and iNOS

Treated hearts were sliced into 10 µm-thick cross-sections using a microtome as previous described (10). The first antibodies were polyclonal rabbit anti-eNOS (amino acids 2–160 of the human eNOS NH2-terminal portion) and anti-iNOS (mouse iNOS COOH-terminal peptide) antibody, and the secondary antibody was anti-rabbit Alexa Fluor 488. The slices were visualized by confocal laser-scanning microscopy (Axiovert 200; Zeiss) using a He-Ne light source and the LSM510-META software. Alexa Fluor 488 was detected at 488-nm excitation and 510-nm emission wavelength. The endo-, mid-, and epicardium were imaged as shown in Fig. 3D.


Figure 3
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  		Fig. 3. Regional changes in NO synthase expression. To test the effect of TTC staining on the expression of endothelial NO synthase (eNOS) and iNOS, a whole heart was perfused for 20 min with normal Tyrode solution with or without 1% TTC, and infarcted and viable areas were then carefully separated. Whole tissue extractions were used for Western blotting with anti-beta-tubulin, anti-eNOS, and anti-iNOS antibodies (A). Following experimental protocols (see MATERIALS AND METHODS and Fig. 1), hearts were stained with TTC, infarcted vs. viable areas were separated, and protein samples were prepared for Western blot analysis. Polyclonal rabbit anti-eNOS and anti-iNOS were used as primary antibodies. Goat anti-rabbit were used as secondary antibody. Reactive bands were detected by enhanced chemiluminescence and imaged using the LAS3000 apparatus. Specific bands at 135 and 130 kDa correspond to eNOS and iNOS, respectively. Values are given as means ± SE. *P < 0.05 vs. CTi; {dagger}P < 0.05 vs. A/Rv group (n = 4–5 for each). i, infarcted areas; v, viable areas (B and C). E: immunofluorescence assay results. Primary and secondary antibodies were anti-iNOS antibody and goat anti-rabbit AlexaFluor 488, respectively. Samples were imaged by laser-scanning confocal microscopy (excitation/emission wavelengths, 488/505–530 nm). Upper images show expression of iNOS for each experimental group in endo-, mid-, and epicardial areas. Two-dimensional pseudocolor images are shown below. Endocardial area is defined as innermost portion of each heart section, whereas epicardial area is defined as outermost edge of each heart section (D). All images were captured in the left ventricular areas. Pseudocolor images were analyzed by LSM510-META software [intensity range, 0–4,000 arbitrary units (AU)].

 
The detection of eNOS and iNOS in viable and infarcted tissues was carried out by Western blot analysis as previously described (10). Reactive bands were detected using the LAS-3000 apparatus with the Image Reader LAS-3000 version 1.2 software (Fuji Photofilm) and analyzed by the Multi Gauge version 2.2 software (Fuji Photofilm). To exclude the possibility of false positives due to the TTC staining, a parallel Western blot analysis was performed with proteins extracted from a TTC-stained control heart.

Fluorescent NO, O2bullet– Measurement in Single Cardiomyocytes

Single cardiomyocytes were stained with the NO-specific fluorescent probe 4-amino-5-methylamino-2',7'-diflurofluorescein (DAF-FM) diacetate at 5 µM concentration (excitation and emission wavelengths of 495 and 515 nm, respectively) (21). To monitor mitochondrial O2bullet– production, cells were incubated with 5 µM MitoSox red (excitation/emission wavelengths of 510/580 nm). Fluorescence imaging was carried out using a confocal laser-scanning microscope (Inverted Axiovert, 200 M; Zeiss) at x200 and x400 magnifications, using the appropriate laser lines and filter sets. Images were analyzed using the LSM510-META software (Zeiss).

Statistical Analysis

The data were analyzed by a multifactorial ANOVA based on repeated measurements with substitution of missing values and using the Microcal Origin version 6.0 software. All the data are presented as means ± SE. A P value <0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Regional Differences Induced by A/R and the Effects of AP and iNOS Inhibition on Infarct Areas

There were 81.2% slices from the A/R group; 13 from a total of 16 hearts showed the spread of infarct from the endocardium to the epicardium, whereas the damaged areas in the AP groups seemed to be located in the endocardium areas (Fig. 2B, real and converted images). Based on these findings, we developed further approaches on the regional changes induced by the AP and A/R mechanism.


Figure 2
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  		Fig. 2. Regional changes in infarcted and viable areas. After experimental protocols were carried out, hearts were stained with TTC and cross-sectioned into 6 slices from S1 (base head) to S6 (apex head). A: each slice was scanned, and infarcted (pale color) and viable (red, pink color) areas were distinguished and analyzed by ImageJ software. Black and white rows indicate viable and infarcted areas, respectively, as percentages of total slice area. B: sum of infarct and viable areas from S1–S6 for each heart. Upper images represent experimental groups. Black and white negative images below enhance discrimination of infarct and viable areas (n = 5 for CT group and n = 16 for A/R and AP groups). Values are given as means ± SE. *P < 0.05 vs. CT group; {dagger}P < 0.05 vs. A/R group.

 
A/R-induced cardiac damage with an infarct size of 55.6 ± 6.9% of the total heart compared with the CT group (14.7 ± 6.4%). AP produced relatively smaller infarcts (33.8 ± 4.2% of the total heart). Blocking iNOS expression during AP episodes increased the infarct size by about 19.5% compared with that in the AP group (Fig. 2B). Cross-sectional analysis showed that there was more damage in the heart apex than in the basal areas (Fig. 2A). Converted image analysis revealed differential localization of the infarct, with centrally damaged endocardial areas in the AP groups and randomly diffuse damage in the A/R and SMT groups (Fig. 2B).

Since TTC staining was the first step in discriminating between viable and infarcted areas, it was important to confirm that TTC itself did not influence the results obtained for the various treatment groups. It is clear from Fig. 3A that TTC staining itself neither contributes to protein expression nor mtDNA fragmentation when tested in a normal Tyrode solution-perfused heart as a control.

Differential eNOS and iNOS Expression in Viable and Infarcted Areas of the Heart

Both eNOS and iNOS were generally expressed at comparatively higher levels in viable than in infarcted areas. From Western blot analysis, the relative band intensities of expressed eNOS in viable and infarcted areas of the AP-treated group were 1.31 ± 0.06 and 0.73 ± 0.13, respectively. These values correspond to 1.15 ± 0.1 and 0.6 ± 0.08 in the A/R group. The expression of iNOS in viable and infarcted areas for the AP group was 1.2 ± 0.08 and 0.47 ± 0.03, respectively. Interestingly, the iNOS expression levels in viable and infarcted areas for the A/R group were not significantly different (0.68 ± 0.05 and 0.59 ± 0.09, respectively). In the AP group, eNOS expression was 1.8 times greater in the viable area than in the infarcted area. iNOS expression, however, showed relative higher value (2.6-fold) in the viable area than in the infarcted one. With the use of equal loading of protein samples, the reactive band of iNOS in the AP-group viable area was almost 1.7-times more intense than that in the A/R group (Fig. 3, B and C).

Fluorescence-based immunohistology confirmed the higher expression levels of iNOS and eNOS in the AP and A/R groups compared with those in the control group for both the endocardium and epicardium. The level of iNOS expression in the myocardium was not significantly different between the A/R and AP groups (Fig. 3E).

Oxidative Stress-Induced DNA Differential Damage in the Nucleus and Mitochondria

Oxidative damage to nDNA was estimated using the comet assay with or without Endo III (Fig. 4, B and C). A/R significantly increased the DNA damage score compared with that in the control (P < 0.05). The inclusion of Endo III in the assays significantly decreased (P < 0.05) the type IV and type V DNA damage scores for both the A/R (42 ± 3% and 21 ± 3% of total comets) and AP (6 ± 2% and 3 ± 0.5% of total comets) groups. In general, Endo III digestion produced higher DNA damage scores, especially of types IV and V, for all the experimental groups.


Figure 4
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  		Fig. 4. Oxidative stress-induced nuclear and mtDNA damage. Comet assay for DNA damage. DNA was stained with propidium iodide and examined under a fluorescence microscope. A: DNA damage was assessed in 5 categories (types I–V) based on comet head and tail lengths (see MATERIALS AND METHODS). B: comet assay without enzymatic treatment. Upper images show propidium iodide-stained DNA for each of the groups depicted in chart below. Preconditioning of myocytes with anoxic solution decreases A/R-induced DNA damage. Inhibition of iNOS by SMT increases DNA damage compared with that in the AP group. C: comet assay performed with endonuclease III (Endo III) digestion for 45 min. Upper images show the propidium iodide-stained DNA for each of the experimental conditions presented in chart below. Values are shown as means ± SE. *P < 0.05 compared with type I of CT group; {dagger}P < 0.05 compared with type V of A/R group (n = 6 for each). D: results of mtDNA fragmentation assay. After experimental protocols, hearts were stained with TTC, and infarcted and viable areas were separated. mtDNA was isolated and incubated for 45 min at 37°C with or without Endo III. Samples were electrophoresed in 1% agarose gels, stained with ethidium bromide, and visualized using the LAS3000 apparatus (n = 4 for each experimental condition). MK, DNA molecular marker.

 
The mtDNA fragmentation assay showed higher levels of oxidative damage for all A/R, AP, and SMT groups compared with the CT group. Moreover, a higher level of DNA fragmentation in infarcted tissue compared with viable tissue was noted for A/R. Interestingly, cutting mtDNA with Endo III resulted in more fragments in the AP group than in the A/R or CT groups. (Fig. 4D).

Effect of Successive Reoxygenation Episodes on O2bullet– Levels and the Role of Mitochondrial KATP Channels

MitoSOX red is a specific dye that binds to mitochondrial O2bullet– (22). When compared with that in the CT group, the O2bullet– level tended to decrease during the period of anoxia in the A/R group (Fig. 5). Upon perfusion, this decrease was converted into an abrupt increase, with a slope of 1.98 ± 0.3 over a 20-min period. This was not the case for the AP group, in which two successive AP periods before reoxygenation resulted in an above-baseline increase in the O2bullet– level after a small decrease. This increase was relatively small (slope of 1.16 ± 0.1) compared with that for A/R, and it persisted until the end of the experiment. As shown in Fig. 5, the fluorescent intensity increased in the A/R group from 93.7 ± 6.3% to 145 ± 4%, and the MitoSOX red level changed within the same period from 106.7 ± 5.2% up to 129.4 ± 4.2%. (Fig. 5, A–E). Inhibition of iNOS by SMT showed a higher level of O2bullet– during both anoxic preconditioning and anoxia. Especially, O2bullet– increased sharply in some first minutes of reoxygenation as similar with A/R with slope of 1.62 ± 0.1. During reoxygenation, O2bullet– level proved to be higher in SMT group than both AP and A/R groups.


Figure 5
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  		Fig. 5. Dynamic changes in mitochondrial O2bullet– levels. Single cardiomyocytes were isolated and stained with mitochondrial O2bullet–-specific MitoSOX red. Cells were then placed in perfusion chamber. Fluorescence was detected every 10 s by laser-scanning confocal microscopy. Values are shown as normalized intensity (F x 100/F0). A: changes in fluorescence for CT group. B: images were captured from CT group, represented for transmitted (TM) image, MitoSOX red image, and converted to pseudocolor image. C, left: changes in fluorescence for the A/R group. a, transmitted image; b, c, and d, pseudocolor images representing fluorescent intensities at times indicated. D, left: changes in fluorescence for AP group. E, left: changes in fluorescence for SMT group. Light-red shading highlights changes in MitoSOX red staining. Within the first minutes of reoxygenation, intensity increases sharply in A/R group, whereas this change is slower in AP group. Slopes are compared for the change in intensity during same time period. Values are given as means ± SE (n = 5 for each). Pseudocolor images were analyzed by LSM510-META software (intensity range, 0–4,000 AU).

 
Application of diazoxide (100 µM), a specific activator of mtKATP channels, increased mitochondrial O2bullet– production following preconditioning episodes. Interestingly, diazoxide improved the balance of O2bullet– during anoxia and reduced the increase slope of this ROS shortly after reoxygenation (Fig. 6A). The slope of MitoSOX red intensity during the first 20 min of reoxygenation was 1.37 ± 0.3 versus 1.16 ± 0.1 in AP and 1.98 ± 0.3 in A/R. To demonstrate the role of mtKATP channels in H2O2/O2bullet– turnover, the MitoSOX red level was traced with or without the addition of diazoxide (Fig. 6B). Preincubation of cardiomyocytes with 100 µM diazoxide significantly reduced the O2bullet– production from its H2O2 precursor. The data from isolated mitochondria model showed that the activation of mtKATP channels by diazoxide depolarized mitochondrial membrane potential (Fig. 6C).


Figure 6
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  		Fig. 6. Preconditioning with mitochondrial ATP-sensitive K+ (KATP) channel activator reduced mitochondrial O2bullet– production during oxygenation. To test effect of transient changes in mitochondrial O2bullet– on mitochondrial KATP channels, cardiomyocytes were stained with 5 µM MitoSOX red and applied to 2x preconditioning with 100 µM diazoxide before 30 min of anoxia and 60 min of reoxygenation. Fluorescent intensity was monitored by a confocal laser-scanning microscope as described in MATERIALS AND METHODS (A). A, right: pseudocolor images are represented for a, b, and c times indicated in A, left. Diazoxide treatment decreases 100 µM H2O2-induced MitoSOX red intensity. B, left: changes in MitoSOX red fluorescence. B, right: pseudocolor images are presented for the respective time points indicated in B, left. To test changes of mitochondrial membrane potential induced by diazoxide, mitochondria was isolated by Percoll gradient methods as previously described (9), incubated in a energized high K+ solution, stained with 2 µM tetramethylrhodamine ethyl esters (TMRE) (excitation/emission wavelength, 488/513 nm). C, left and middle: changes in TMRE in isolated mitochondria sample as control and 100 µM diazoxide treated. Right panels are transmitted and TMRE-stained mitochondria images.

 
The Dynamic Changes in NO Level During A/R

As shown in Fig. 7, anoxic episodes triggered NO production. A significant rise in the NO level was initiated by anoxic perfusion in both the A/R and AP groups. In the AP group, NO was increased after two successive intervals of AP. This increase persisted during a long-term anoxic perfusion. Within 30 min of anoxic perfusion, the increase of NO in the AP group was significantly higher than in the A/R group (132.1 ± 3.5% for A/R vs. 196.2 ± 4.3% for AP). Interestingly, for both AP and A/R, a sudden drop in the NO level occurred upon reoxygenation (indicated by the small arrow in Fig. 7, C and F). This was followed by a continuous increase over the 60 min of reoxygenation. The highest levels of NO during the 60 min of reoxygenation were 138.3 ± 3% for the A/R group and 234.4 ± 4.4% for the AP group. The fluctuating NO levels that were observed during preconditioning in the SMT-treated group remained high during a continuous period of anoxia.


Figure 7
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  		Fig. 7. Dynamic changes in NO during A/R. Single cardiomyocytes were isolated and stained with NO-specific 4-amino-5-methylamino-2',7'-diflurofluorescein (DAF-FM) diacetate. Cells were then placed in a perfusion chamber. Fluorescence was detected every 10 s by laser-scanning confocal microscopy. Values are reported as normalized intensities (F x 100/F0). Changes in fluorescence levels and images captured of CT group (A and B), changes in fluorescence levels of A/R group (C–E), changes in fluorescence levels of AP group (F–H), changes in fluorescence levels of the SMT group (I–K) are shown. Fluorescence intensity values (D, G, and J) and pseudocolor images (E, H, and K, respectively) were represented at respective times (C, F, and I). Values are given as means ± SE; n = 6 for every experiment. *P < 0.05 compared with value measured at time a; {dagger}P < 0.05 compared with the value measured at time b. Pseudocolor images were analyzed by LSM510-META software (intensity range, 0–4,000 AU).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Adaptive changes during ischemic preconditioning are currently subjects of considerable debate. Many recent investigations have studied adaptive changes in cellular organelles, as well as alterations in NO metabolism and the formation of ROS, which include ONOO– (8). NO contributes to the activation of cGMP and protein kinase G (PKG), thus stimulating both sarcolemmal and mtKATP (10, 18).

More recently, we observed that the NO-cGMP-PKG-KATP pathway protects against A/R-induced injuries. The opening of these channels may play a role in shortening the action potential, thus saving energy during long-term anoxia and balancing the levels of ROS and RNS, which increase dramatically at the onset of reoxygenation (2). Our further attempt was made in the current study to localize anatomic abnormalities, as well as the final apoptotic outcome, and then to correlate them with the dynamic changes related to NO and O2bullet– production during A/R. In terms of localization, our results indicate that the endocardium was the most affected region in the AP-treated hearts, whereas the epicardium showed a relatively larger area of infarct in the A/R-treated heart. However, further study is required to disclose the real cause behind these discrepancies. Our results in AP-treated hearts agreed with other previous findings reported after ischemic myocardial damage in vivo (23), where the geometry of infarct scars in myocardial damage started from the subendocardium extending toward the epicardium. Moreover, it was recently confirmed that patients with ischemic cardiomyopathy tended to have a larger endocardial than epicardial scar (3). This observed similarity between our AP model alterations and that of in vivo ischemic myocardial damage suggests a possible common mechanism that copes in both cases with cellular insult. This calls our attention to the importance of a careful design in any further in vitro experimental model to simulate the in vivo reality of ischemic cellular insult.

The dynamic changes in both NO and O2bullet– levels in the AP group compared with those in the A/R group support the idea that adaptive measures taken during AP counteract the oxygen and nutrient deficiencies and compete with accumulated metabolic wastes during the anoxic period. In the AP experiment, short successive AP periods resulted in a marginal decrease in O2bullet– production. This decrease in O2bullet– did not continue during long periods of anoxia. However, reoxygenation induced a moderate increase in the O2bullet– levels. In contrast, the gradual decrease in O2bullet– production observed during long anoxic periods in the A/R experiment was followed by a considerable surge in the O2bullet– level at the onset of reoxygenation.

Unlike the A/R experiment, which showed a sharp increase in the NO level at the onset of anoxia, AP mediated a gradual increase in the NO level in the AP experiment.

Balancing oxygen supply and utilization is a major consideration for maintaining maximal muscle performance. NO, on the other hand, is an important regulator of tissue oxygen consumption in cardiac and skeletal muscle. It has been suggested that the inhibitory effect of NO on oxygen consumption is the result of reduced activities of mitochondrial enzymes, such as aconitase in the Krebs cycle, and complexes I and II of the mitochondrial electron transport chain due to the formation of a nitrosyl complex with the iron-sulfur center (12, 15) of the enzyme. NO binds reversibly at a low micromolar concentration to the oxygen-binding site of cytochrome oxidase, which is the terminal enzyme of the mitochondrial electron transport chain, to decrease oxygen consumption. This binding may be increased under conditions of lowered oxygen availability to match the tissue oxygen supply (29). In addition to modulating the impact of NO on energy abundance (11), it has recently been shown that decreased NO production negatively influences muscle performance by increasing oxygen consumption (20). Furthermore, NO-induced opening of both mtKATP and mitochondrial Ca2+-activated K+ channels protects the heart during long-term ischemia by attenuating mitochondrial anion channels, decreasing the mitochondrial membrane potential and decreasing intramitochondrial Ca2+ influx. This, in turn, influences the efflux of ROS to the cytoplasm (17, 30). It is of interest to note that mtKATP activation by the channel opener diazoxide affords an additional means of protection by decreasing the rate of H2O2 conversion to O2bullet–. However, as depicted in Fig. 6, although the long and gradual increase in NO performs an overall protective role in cardiac myocytes during AP, it may withhold the efflux of ROS to the cytoplasm, with concomitant elevation of the mitochondrial ROS level. This may contribute to greater oxidative stress on mtDNA, as observed in the case of AP. In contrast, the massive efflux of mitochondrial ROS in the A/R experiment at the onset of perfusion clearly had a greater impact on the nDNA than on the mtDNA. Therefore, the relative increase in NO clearly enhances cardiac performance in different ways during both periods of A/R in the AP model compared with the A/R model. These results are further supported by the Western blotting data, which revealed an upregulated expression of both eNOS and iNOS in the viable areas rather than the infarcted areas of both heart models.

Mitochondrial complex I O2bullet– production is promoted by inhibiting respiration at a site distal to the O2bullet–-generating redox center (e.g., NO-induced cytochrome oxidase inhibition with low electron flow) (27). Therefore, the essential state 4 condition is favored by the onset of reperfusion with a sudden increase in oxygen availability. The elevated level of O2bullet– during this short period, in turn, scavenges NO to form ONOO– (24), which is a shorter-lived and less-potent vasorelaxant than NO; therefore, the increase in O2bullet– production may cause a decrease in NO bioavailability (19). Consistent with this assumption, and as shown in RESULTS, the change in NO bioavailability may account for the short sharp drop in the NO level at the onset of perfusion in both the AP and A/R models, in contrast to the SMT model.

Since oxidative stress, which generates an excessive amount of reactive species, contributes to the vast majority of apoptotic/necrotic damage, we studied the differential influence of oxidative stress on nDNA and mtDNA in the rat heart AP and A/R models. Fragmentation assays for nDNA and mtDNA were performed to monitor the degrees of damage. Comet assays were used as the ultimate judge of eventual cellular outcome. Interestingly, profound mtDNA damage was observed in the AP model rather than in the A/R model. On the other hand, nDNA exhibited more fragmentation in the A/R model than in the AP model. It is well known that mtDNA is more susceptible than nDNA to ROS, and, consequently, mtDNA is more prone to oxidative injury than nDNA (31). This can partly explain our findings provided that, unlike the A/R model, preconditioning in the AP model introduces early O2– species that influence mainly the mtDNA in situ. The level of O2– species needed to induce remote DNA damage failed to cause significant fragmentation of mtDNA in the case of the A/R model. Regardless of our previous assumption depicted in Fig. 5 of elevated mitochondrial ROS level in the AP model, this discrepancy may attribute to a decreased yield of surviving isolated mitochondria after the drastic A/R treatment. The comet assay revealed the final cellular outcomes in our experiments. When compared with the control, AP-treated cells showed greater stability and survival rates than the A/R-treated cells. The cross-talk between ROS and both nDNA and mtDNA damage as well as the role of mitochondrial channels during A/R was shown in Fig. 8. Since this cardioprotection mechanism does not rely solely on iNOS-produced NO, the SMT-treated cells are more likely to exhibit comparable stability and lower cellular damage than the A/R-treated cells. Nevertheless, preconditioning appeared to be counteracted by an addition of SMT. This can explain the fluctuating level of NO during successive AP and its relatively stability at the onset of reoxygenation.


Figure 8
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  		Fig. 8. Possible mechanism by which O2bullet– and NO interact to protect the heart during AP. ONOO–, peroxynitrite; MPTP, mitochondrial permeable transition pore; IMAC, inner membrane anion channels; ETC, electron transport chain; mKATP, mitochondrial KATP channel; mKCa, mitochondrial Ca2+-activated K+ channel; MCU, mitochondrial Ca2+ uniporter. During long-term anoxia, the level of O2bullet– initially decreases and then increases sharply during the first minutes of reperfusion. This may disrupt the ROS balance in mitochondria (including ONOO– and H2O2), thereby damaging the mtDNA. The flux of O2bullet– out of the mitochondria through MPTP and/or IMAC leads to nuclear (n)DNA damage (Ref. 27). Oxidative stress and damage caused to mtDNA and nDNA may initiate both necrotic and apoptotic pathways (Ref. 31) (A). A brief episode of AP increases the production of NO. This NO activates mKATP channels via cGMP-PKG pathways or other K+ channels (Ref. 10). These channels attenuate overload of Ca2+, reducing membrane potential during later long-term anoxia, thereby shortening action potential and saving energy. Additional NO production during later anoxia contributes to further activation of protective channels. The opening of these channels attenuates influx of Ca2+ into mitochondria and inhibits efflux of O2bullet– to cytosol, thus reducing the cytosolic ROS level and decreasing oxidative stress-induced nDNA damage (B).

 
In conclusion, to the best of our knowledge, this study is the first to demonstrate the real-time dynamic changes in the NO and O2bullet– levels of AP- and A/R-treated rat ventricular cardiomyocytes. The attempt was made further to localize anatomic abnormalities, as well as the final apoptotic outcome, and to correlate them with the profound dynamic changes. The observed similarity between our AP model alterations and those of in vivo ischemic myocardial damage suggests a possible common anti-insult coping mechanism and highlights the importance of careful designing any further in vitro experimental model to simulate the in vivo ischemic insult.

In the AP model, NO production played a pivotal role in scavenging the massive increase in mitochondrial O2bullet– during post-A/R. This scavenging effect is clearly demonstrated in our model by the short-lived drop in the NO level at the onset of reoxygenation. Our results suggest that the continuous increase in O2bullet– species during preconditioning affords an accommodating mechanism to enable cell survival by protecting the cell from sudden O2bullet– shock at the onset of reoxygenation. The work is step toward a new prospective for a better management of ischemic cardiac insult.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by the Korea Science and Engineering Foundation grant funded by the Korean government Ministry of Science and Technology (ROA-2007-000-20085-0 and R13-2007-023-00000-0).


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Han, Mitochondrial Signaling Lab., Dept. of Physiology and Biophysics, College of Medicine, Mitochondrial Research Group-Frontier Inje Research Science & Technology Project, Cardiovascular and Metabolic Diseases Ctr., Inje Univ., 633-165 Gaegeum-Dong, Busanjin-Ku, Busan 614-735, Korea (e-mail: phyhanj{at}inje.ac.kr)

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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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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