| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
a Department of Cardiothoracic Surgery, Faculty Division Ullevål University Hospital, Oslo, Norway
b Institute of Experimental Medical Research, Ullevål University Hospital, Oslo, Norway
c Department of Surgery, Faculty Division Ullevål University Hospital, Oslo, Norway
d Institute of Basic Medical Science, Department of Physiology, University of Oslo, Oslo, Norway
Received 19 September 2007; received in revised form 4 December 2007; accepted 10 December 2007.
* Corresponding author. Address: Department of Cardiothoracic Surgery, Ullevål University Hospital, 0407 Oslo, Norway. Tel.: +47 99727991; fax: +47 23015881. (Email: m.l.kaljusto{at}medisin.uio.no).
 |
Abstract |
|---|
 Top Abstract 1. Introduction2. Methods3. Results4. Discussion5. ConclusionsReferences |
|---|
Key Words: Preconditioning • Steroids • Hyperoxia • Ischemia–reperfusion injury
|
1. Introduction |
|---|
|
TopAbstract1. Introduction2. Methods3. Results4. Discussion5. ConclusionsReferences |
|---|
Recently, we demonstrated that hyperoxic preconditioning reduced infarct size and improved cardiac function in an isolated, perfused rat heart model and shifted the isolated vessel response towards a more relaxed state [3,4]. Hyperoxia generates reactive oxygen species and short exposure inducing a low grade oxidative stress may activate endogenous heart protection by induction of nuclear factor kappa B (NF
B)-dependent mechanisms [5]. However, in contrast to short exposure to hyperoxia, longer periods used in both clinical and experimental studies have shown mainly deleterious effects on most organ systems including the heart [6]. Another way of inducing myocardial protection is pretreatment with steroids. Such pretreatment reduces infarct size and improves postischemic function in isolated rat hearts, possibly through expression of heat shock protein (HSP) 72 [7].
The exact molecular mechanisms of preconditioning have not been clarified. Several and parallel pathways are apparently involved. Recent evidence indicates that survival kinases are important for cell survival in pre- and postconditioning [8]. These kinases include protein kinase B/AKT, extracellular signal-regulated kinases (ERK1/2), the stress-activated/c-Jun NH2 terminal kinases (SAPK/JNK), and p38 MAPK. Also, adenosine monophosphate dependent kinase (AMPK) has recently been linked to preconditioning [9].
We hypothesized that combined pretreatment with hyperoxia and steroids, which act through different molecular mechanisms, would potentiate the cardioprotection provided by each principle. Consequently, the purpose of the present investigation was to study the protection of dexamethasone and hyperoxia alone and in combination on ischemia–reperfusion injury in isolated, perfused rat hearts. Secondly, to investigate whether the survival kinases, AMPK or HSP72 were activated by and might explain the effect of steroids, hyperoxia or combined pretreatment. Thirdly, to investigate the effects on in vitro vascular reactivity, in particular to study whether combining steroids and hyperoxia would induce a shift towards relaxation.
|
2. Methods |
|---|
|
TopAbstract1. Introduction2. Methods3. Results4. Discussion5. ConclusionsReferences |
|---|
2.1 Isolated, perfused hearts
Rats were anesthetized with 5% sodium pentobarbital 60–80 mg/kg intraperitoneally, and 500 IU Heparin (Leo Pharma A/S, Denmark) was injected via the same route. After anesthesia rat hearts were rapidly excised, and placed in ice-cold, modified Krebs–Henseleit Buffer (KHB) containing (mmol/l): NaHCO3 25, KCl 4.7, KH2PO4 1.2, MgSO4/7H2O 1.2, glucose/1H2O 11.1, CaCl2 1.8 (Merck, Germany), NaCl 118.5. After aortic cannulation the hearts were mounted on a Langendorff system (AD Instruments Pty Ltd., Australia) and perfused with warm (37 °C), oxygenated (95% O2, 5% CO2, AGA Norway AS) KHB at constant pressure (70 mmHg). Heart temperature was kept constant during the experiment by the water-jacketed closed system. A fluid-filled latex balloon (Hugo Sachs Elektronik-Harvard Apparatus GmbH, Germany) was inserted into the left ventricle for measurements of pressures. Left ventricular end diastolic pressure (LVEDP) was set at 5–10 mmHg during stabilization by filling fluid in the balloon, and was not changed afterwards. Left ventricular developed pressure (LVDP) was calculated: left ventricular systolic pressure (LVSP) – LVEDP. Rate pressure product (RPP) was calculated: LVDP x heart rate (HR). Coronary flow (CF) was measured by timed collections of the coronary effluent. Arrhythmias were counted as an all or nothing response during the first 20 min of reperfusion and were evaluated from pressure curves as HR. Hearts with LVSP 
100 mmHg, CF 8 or 
20 ml/min or HR 220 beats per min (bpm) at the end of stabilization were excluded from the study (in total 30% of the hearts were excluded). The hearts were not paced.
All hearts were subjected to 20 min of stabilization, 30 min of global, normothermic ischemia by stopping the inflow of perfusate, and 120 min of reperfusion. Ten more hearts from each group were sampled after 20 min of stabilization and 10 min of reperfusion for immunoblotting, in addition to the hearts sampled after 120 min of reperfusion.
2.2 Infarct size
After 120 min of reperfusion, infarct size was measured by cutting the heart in 1 mm slices (hearts fixed in acrylic rat brain matrix by AgnThor's AB, Sweden), which were incubated for 15 min at 37° C in 1% triphenyltetrazoliumchloride. The heart sections were placed between two glass plates and digitally photographed (Nikon Coolpix 5400, Japan). Calculation of infarct area was computerized (Adobe Photoshop CS and Infarct Area Calculation Macro file, Copyright ©1998 Rob Bell, Hatter Institute, UCL, UK).
2.3 Groups
Rats were randomly assigned to the following groups, n
= 10 in each:
1. Controls: Twenty-four and 12 h before harvesting the heart, rats received an intramuscular (im) injection with phosphate-buffered saline (sham injection) and were exposed to a closed environment breathing air for 60 min immediately before isolating the heart (sham environment).
2. Dexamethasone group: Rats were injected with dexamethasone (4 mg/ml, MSD, Merck & Co., USA) 3 mg/kg im 24 and 12 h before heart isolation and were exposed to sham environment for 1 h immediately before harvesting the hearts. The dose of 3 mg/kg was found to give optimal protection in pilot experiments.
3. Hyperoxia group: Sham injection was performed 24 and 12 h before the heart isolation and rats were exposed to 60 min hyperoxic environment (90–95% O2) immediately before harvesting the hearts.
4. Combination of hyperoxia and dexamethasone: Rats were injected with dexamethasone im like in group 2 and exposed to hyperoxia like in group 3.
2.4 Isolated vessels
After the hearts were isolated in groups 1–4, the right femoral artery was harvested, placed in ice-cold KHB and the surrounding connective tissue removed. Two-millimeter arterial ring segments were cut and mounted on a four-chamber wire myograph (Multi Wire Myograph System Model 610 M, Danish Myo Technology A/S, Denmark) by two 40 µm tungsten wires inside the rings for measurements of isometric tension. One of the wires was attached to a force transducer, the other to a micrometer. KHB in the chambers was heated to 37 °C and continuously oxygenated with 95% O2 and 5% CO2. After equilibration for 60 min, the optimal vessel preparation and viability was confirmed by adding KHB with high potassium concentration (123.3 mM) twice, and the second precontraction was used as a reference contraction (100%). After washout and another 30 min of equilibration, the vessel rings were contracted with 10 µM phenylephrine and 2 µM acetylcholine was added to the baths to assess the viability of endothelium. Vessels without 50% relaxation of the precontraction value were excluded. Concentration–response cumulative curves (n
= 5) were assessed by adding vasoactive substances to the vessel chambers:
2.5 Immunoblots
The frozen heart samples were transferred to an ice-cold lysis buffer containing 210 mM sucrose, 40 mM NaCl, 30 mM Hepes, 5 mM EDTA, 100 µM sodium orthovanadate and 1% Tween-100. In addition one tablet of Complete EDTA-free protease inhibitor (Roche, Switzerland) and 250 µl phosphatase inhibitor cocktail 1 were added to 50 ml of extraction buffer. The tissue was homogenized using an Ultra-Turrax T 8 homogenizer (IKA, Germany) with 20 mg tissue/ml extraction buffer. Lysates were subsequently centrifuged at 12,000 x
g, 4 °C, for 10 min to remove insoluble material. One percent sodium dodecyl sulfate (SDS) was added to the supernatant and the samples were vortexed for 15 min at room temperature. The samples were again frozen in liquid nitrogen and stored for later analysis. Protein content was determined by Micro BCA protein assay kit (Pierce, USA). Proteins (10–20 µg/lane) were then subjected to 10% sodium SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and the fractionated products were electrophoretically transferred onto a hybond-P membrane (Amersham Biosciences Europe, Germany). The membrane was incubated for 1 h with 5% skimmed milk in Tris-buffered saline (TBS) (20 mM Trizma–base and 140 mM NaCl) containing 0.1% Tween 20 (TBST) in order to block nonspecific reactions. The membrane was then incubated in 5% BSA in TBST over night with primary antibody (rabbit polyclonal except AMPK which is rabbit monoclonal) (1:1000; phospho AKT (9271), AKT (9272), phospho ERK (9101), ERK (9102), phospho JNK (9251), phospho p38 (9211), phospho AMPK (2535) (Cell Signaling Technology, USA, product number in parenthesis), HSP72 (Stressgen) Sweden)). After washing with TBST, the membrane was incubated for 1 h in 5% skimmed milk and secondary antibody (goat anti-rabbit 1:2500, Southern Biotech, England) conjugated to horseradish peroxidase. After washing, the immunoreactions were visualized by chemiluminescence (ECL+, Amersham Biosciences) and pictures were taken with ImageReader LAS-1000 (Fujifilm). The densitometry of each band was investigated using ImageQuant (Amersham Biosciences). The membranes were stained with commasie blue (Bio-Rad Laboratories, USA), scanned (CanonScan Lide 35, Canon Europe NV, The Netherlands) and equal loading was investigated using ImageQuant on the scanned membranes. Membranes with uneven blotting were removed from the analysis. Negative controls were performed by removing the primary antibody from the protocol, and no unspecific binding was observed. Positive and negative control for phosphorylated AKT and ERK were also provided with the antibody kit and the immunoblot showed clear bands in the positive control, and none in the negative. When the results from the immunoblots revealed statistical trends, an additional immunoblot was performed with antibodies against total protein kinase.
2.5.1 Positive controls HSP
Twelve rats were anesthetized (ketamine/zylazine: 83/12 mg/kg), randomized in two groups and placed in a heating chamber. Core body temperature was measured rectally. The heat shock group was subjected to 1 h body temperature of 41.5 °C, while the control group was kept at 37.5 °C. After 1 h, the rats were removed from the chamber and left for 2 h fully anesthetized. The hearts was excised and proteins were extracted as described above.
2.5.2 Positive controls survival kinases
Twelve rats were randomized in two groups, anesthetized and the hearts were harvested and perfused as described in isolated, perfused hearts. After stabilization for 20 min, one group was subjected to a preconditioning protocol (two cycles of 5 min of global ischemia and 10 min of reperfusion) before sampling after 30 min of ischemia. The control group was only perfused for the same length of time (50 min). Tissue from both groups was treated as described above.
2.6 Chemicals
All chemicals, unless specified in text, were purchased from Sigma–Aldrich GmbH, Germany.
2.7 Statistical analysis
Continuous function data and infarct size in text are expressed as mean ± SD, while figures with infarct size show individual data and median value. All data regarding the isolated hearts and vessels were analyzed using the same software package (Graph Pad Prism Software Inc., USA), except incidence of arrhythmias analyzed by Fisher's exact test on Windows SPSS (SPSS Inc., USA). One-way analysis of variance was used to test differences in infarct size with a Bonferroni correction for multiple comparisons. Cardiac function parameters and Concentration–response curves for vessel rings were analyzed by ANOVA for repeated measures with Bonferroni correction for multiple comparisons.
The Western blots were analyzed in two ways. In two-group comparison, each gel was loaded with 14 heart samples (7 from the control group and 7 from one of the experimental groups) and signal intensity of the bands was quantified using ImageQuant. The average intensity of the control hearts were set to 0 and differences were calculated for each sample as percent change in comparison with the mean from the control hearts. If the analysis revealed change in relative phosphorylation between groups, an additional blot was performed using total kinase antibodies. A ratio between the nonphosphorylated and total protein kinase was created by dividing the individual measurements and normalized as described above. In four-group comparison, gels were loaded with three hearts from each group (control, dexamethasone, hyperoxia and combination) and normalized to the control group as in two-group comparison. The change in intensity for each sample was then calculated in percent compared to the mean of the control group. The setup and calculations were done twice giving a total of six biological replicas per group. Two group comparisons were evaluated by Welch corrected t-test. Four-group comparisons were analyzed by Kruskal–Wallis test.
A difference was considered significant when the corresponding p-value was 0.05, except for the cardiac function and vessel reactivity comparisons where p-value 0.0083 was regarded as significant due to the Bonferroni correction. Differences with p-values between 0.05 and 0.10 were considered as tendencies.
|
3. Results |
|---|
|
TopAbstract1. Introduction2. Methods3. Results4. Discussion5. ConclusionsReferences |
|---|
|
|
3.2.3 HR (Fig. 2)
No intergroup differences were present either in the baseline values or under reperfusion.
3.2.4 CF (Fig. 2)
Dexamethasone and combined pretreatment had higher CF at baseline (14 ± 1.5 ml/min, p
< 0.01 and 14 ± 1 ml/min, p
< 0.05, respectively) compared to controls and hyperoxia (12 ± 2 ml/min and 11.5 ± 1.5 ml/min, p
< 0.001 and p
< 0.05, respectively). Only the combined group increased CF during reperfusion compared to controls and hyperoxia.
3.2.5 RPP (Fig. 2)
There was no difference in the baseline values of RPP between the groups. Only combined pretreatment increased RPP compared to controls.
3.2.6 Arrhythmias (Fig. 2)
All pretreatments protected the heart against reperfusion arrhythmias without any difference between the groups.
3.3 Reactivity of isolated vessels
There were no differences in baseline values between the four groups (Fig. 3
). Differences between groups were observed regarding endothelium-independent relaxation by sodium nitroprusside, where the combined group enhanced relaxation compared to controls. Endothelium-dependent relaxation did not differ between groups.
|
|
|
|
|
|
4. Discussion |
|---|
|
TopAbstract1. Introduction2. Methods3. Results4. Discussion5. ConclusionsReferences |
|---|
Activation of survival kinases has recently been found to be an important mechanism of the endogenous cell defense including pre- and postconditioning [8]. However, apparently they are not important in cardioprotection by hyperoxia and steroids. Other molecular pathways are probably activated in the present study. We can only speculate what these pathways are.
4.1 Pretreatment with steroids
When dexamethasone binds to the glucocorticoid receptor, the receptor translocates from the cytoplasm to the nucleus, where it activates or suppresses gene transcription. Recent findings suggest the presence of rapid, nontranscriptional effects of steroids both in the heart and vasculature, possibly via activation of phosphatidylinositol 3-kinase and AKT [10]. However, we found no clear activation of any survival kinase including AKT. As different protocols for protein extraction and immunoblotting may give varying results, we performed positive controls, both supplied by the antibody companies and by performing supplementary experiments with ischemic preconditioning and heat shock. Ischemic preconditioning caused phosphorylation of ERK1/2 and P38, while the rats exposed to heat shock demonstrated significant upregulation of HSP72 and phosphorylation of ERK1/2 and AKT, showing that our protocols for immunoblotting and protein extraction worked. Interestingly, it has been shown that the AKT/phosphatidylinositol 3-kinase system is necessary to activate HSP72 in hyperthermic rat hearts [11]. However, we are not aware of any known connection between hyperthermia and ERK1/2 activation, and these findings need further attention.
Dexamethasone did not activate any survival kinases at all (Fig. 4). Previously dexamethasone was found to upregulate HSP72 [7]. This could not be verified in the present study. One explanation for this discrepancy might be the much higher dose of dexamethasone pretreatment used in our experiments: 3 mg/kg x 2 compared to 0.29 mg/kg [7]. Dexamethasone has a dose-dependent effect on HSP72 expression with a bell-shaped response curve [12].
Direct vasodilatory and membrane stabilizing action of glucocorticoids have been suggested [13,14] and this may partly explain the improved hemodynamic function, especially increased coronary flow in the combined group and antiarrhythmic effects in both groups with steroids (Fig. 2). Dexamethasone eliminated arrhythmias in our study. Clinical studies on steroids and postoperative arrhythmias have conflicting results [15,16]. However, these studies focus on atrial fibrillation after cardiac surgery and cannot directly be compared to the present study.
In the present study, endothelium-independent vasodilatation was enhanced in the combined group whereas there was no effect on the endothelium-dependent vasodilation. We have no definite explanation for this difference. The effects of corticosteroids on vascular tone are complex and may also vary between different vascular beds [17]. Steroids act both on the endothelium and directly on the vascular smooth muscle. Endothelium-dependent vasodilation may be inhibited by corticosteroids while there are both constrictory and dilatory effects on the vascular smooth muscle [17]. Retrospectively, coronary vessels might have been more relevant for the studies on vascular reactivity.
4.2 Pretreatment with hyperoxia
Hyperoxic pretreatment reduced LVEDP during reperfusion. It also reduced the occurrence of reperfusion arrhythmias (Fig. 2). Hyperoxic pretreatment reduced infarct size by 23% (Fig. 1). These results are in agreement with previous investigations [4]. The majority of studies on preconditioning find phosphorylation of AKT and ERK1/2 [8]. However, there are exceptions [8], e.g. preconditioning by adenosine does not cause phosphorylation of AKT and ERK1/2 [18] and other pathways may be responsible for heart protection by hyperoxia and steroids. A possible mediator could be NFB, already known as a mediator of ischemic preconditioning, and also activated by both 1 h of hyperoxia [19] and short cyclic episodes of hyperoxia [20]. Evaluation of NFB might have added interesting information to the present investigation.
4.3 Combined pretreatment with steroids and hyperoxia
Combined pretreatment with steroids and hyperoxia improved postischemic cardiac function compared to hyperoxia as single pretreatment. Combined pretreatment was also superior to dexamethasone regarding LVEDP (Fig. 2). However, the combined pretreatment did not reduce infarct size or occurrence of arrhythmias when compared to the single pretreatments (Figs. 1 and 2). These findings suggest that factors other than cell death might be influenced by the combined pretreatment during reperfusion. As hyperoxia [21] and steroids [22] (methylprednisolone) have previously shown beneficial effects on stunned myocardium, reduced stunning is probably the most likely explanation.
Interaction of glucocorticoids and NFB is thought to be partially responsible for glucocorticoid effects although the exact mechanisms are not resolved [23]. Moreover, cardiac protection by steroids might involve inhibition of cytochrome c release [24]. Inhibition of cytochrome c release has also been observed after hyperoxic treatment [20] and this is a possible mechanism where preconditioning stimuli converge. Release of cytochrome c may have a central role in ischemia–reperfusion injury as it is released during opening of the mitochondrial permeability transition pore [25], which again is central in ischemic preconditioning [26]. It is possible that several cardioprotective pathways unite at the mitochondrial permeability transition pore.
|
5. Conclusions |
|---|
|
TopAbstract1. Introduction2. Methods3. Results4. Discussion5. ConclusionsReferences |
|---|
|
Acknowledgments |
|---|
|
Footnotes |
|---|
This study has been supported by the University of Oslo (G.V. and J.V.), Ulleval University Hospital (K.O.S. and J.V.), the Norwegian Research Council (G.V.), and Eastern Norway Regional Health Authority and Norwegian National Health Association (J.V.).
1 Present address: Grand Self Defense Force 12th Brigade Headquarters, 1017-2 Arai, Shintoumura, Kitagunnmagun, Gunma 370-3594, Japan. 
2 Department of Pathophysiology, I.P. Pavlov Federal Medical University, St. Petersburg, Russia.
|
References |
|---|
|
TopAbstract1. Introduction2. Methods3. Results4. Discussion5. ConclusionsReferences |
|---|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ANN THORAC SURG | ASIAN CARDIOVASC THORAC ANN | EUR J CARDIOTHORAC SURG |
| J THORAC CARDIOVASC SURG | ICVTS | ALL CTSNet JOURNALS |
