| JOURNAL HOME | CME HOME | THIS MONTH | PAST ISSUES | ETOC | COLLECTIONS |
| AUTHORS | REVIEWERS | EDITORIAL BOARD | FEEDBACK | RSS | HELP |
| ||||||||||||||
| ||||||||||||||
|
|
|
|
||||||||||||
|
||||||||||||||
From the Departments of *Anesthesiology, and Internal Medicine, Osaka Dental University, Osaka, Japan; and Institute for Heart and Vascular Health, Albert Einstein Medical Center, Philadelphia, Pennsylvania.
Address correspondence and reprint requests to Masami Miyamae, MD, PhD, Department of Internal Medicine, Osaka Dental University, 8-1 Kuzuha hanazono-cho Hirakata, Osaka 573-1121, Japan. Address e-mail to kyotomiya{at}hotmail.com.
Abstract
BACKGROUND: Volatile anesthetics and regular ethanol consumption induce cardioprotection mimicking ischemic preconditioning. We investigated whether sevoflurane enhances ethanol preconditioning and whether inhibition of protein kinase C (PKC) and mitochondrial KATP channels attenuated this enhanced cardioprotection. The effects of regular ethanol consumption on expression of inducible (iNOS) and endothelial (eNOS) nitric oxide synthase were determined.
METHODS: Isolated perfused guinea pig hearts underwent 30-min global ischemia and 120-min reperfusion (Control: CTL). The ethanol group (EtOH) received 2.5% ethanol in their drinking water for 6 wk. Anesthetic preconditioning was elicited by 10-min exposure to sevoflurane (1 minimum alveolar anesthetic concentration; 2%) in ethanol (EtOH + SEVO) or nonethanol (SEVO) hearts. PKC and mitochondrial KATP channels were inhibited with chelerythrine and 5-hydroxydecanoate pretreatment, respectively. Contractile recovery was assessed by monitoring of left ventricular developed and end-diastolic pressures. Infarct size was determined by triphenyltetrazolium chloride staining. Expression of iNOS and eNOS were determined by Western blot analysis.
RESULTS: After ischemia-reperfusion, hearts from the EtOH, sevoflurane (SEVO), and EtOH + SEVO groups had higher left ventricular developed pressure and lower left ventricular end-diastolic pressure compared with CTL. Infarct size was reduced in EtOH and SEVO hearts compared with CTL (27% and 23% vs 45%, respectively, P < 0.001). Sevoflurane further reduced infarct size in EtOH hearts (27% vs 15%, P < 0.001). Chelerythrine and 5-hydroxydecanoate abolished cardioprotection in both SEVO and EtOH cardioprotected hearts. iNOS expression was reduced and eNOS expression was increased in EtOH hearts.
CONCLUSIONS: Sevoflurane enhances cardiac preconditioning induced by regular EtOH consumption. This effect is mediated in part by modulation of PKC and mitochondrial KATP channels, and possibly by altered modulation of NOS expression.
Volatile anesthetics induce cardiac preconditioning in a manner similar to ischemic preconditioning.1 Since Kersten et al.2 first documented anesthetic preconditioning with isoflurane, investigators have sought to elucidate the involved mechanisms mediating this cardioprotection. Sevoflurane has been shown to confer additional cardioprotection to late ischemic preconditioning, mediated via mitochondrial KATP channels.3 These findings suggest that sevoflurane may enhance cardioprotection in myocardium, which is already preconditioned.
Epidemiologic studies have shown that mortality rates for people who regularly drink alcohol in moderation are lower than in abstainers, primarily due to decreased fatal ischemic heart disease.4–7 Further, moderate alcohol consumers have lower mortality from myocardial infarction compared with abstainers.8 These beneficial cardiac effects may be due to the pleiotropic effects of ethanol on lipids,9 platelets, and fibrinolytic activity.10,11 We have demonstrated that regular ethanol consumption renders hearts more tolerant to ischemia-reperfusion injury, to a degree similar to acute ischemic preconditioning.12–14 We found this ethanol-induced preconditioning is mediated through myocyte adenosine A1 receptor12,13 and protein kinase C (PKC) activation.14 Ethanol and volatile anesthetic-induced cardiac preconditioning, thus, appear to share some common signaling pathways. Guiraud et al.15 confirmed that the combination of ethanol and ischemic preconditioning were synergistic in reducing infarct size.
We hypothesized that anesthetic preconditioning with sevoflurane would enhance ethanol-induced myocardial preconditioning, and that this effect is potentially mediated by PKC activation and modulation of mitochondrial KATP channels.16 Both sevoflurane and acute ethanol exposure increase nitric oxide (NO) release.17,18 Thus, we examined the effect of chronic ethanol exposure on the expression of inducible and endothelial NO synthases (iNOS and eNOS) expression.
METHODS
This study was conducted in accordance with the Osaka Dental University Guidelines for Animals Research at Osaka Dental University, and with the approval of the its Animal Experiment Committee, Osaka, Japan. Male Hartley guinea pigs were fed Lab Diet (RC4 TM, Oriental Yeast, Tokyo, Japan) and given water ad libitum.
Isolated Heart Perfusion and Measurement of Function
Animals weighing 650–700 g were given heparin (1000 U IP) and then anesthetized with pentobarbital (60 mg/kg IP). The hearts were excised and immediately arrested in cold isosmotic saline containing 20 mmol/L KCl. The aorta was cannulated and the isolated hearts perfused at 70 mm Hg on a nonrecirculating isovolumic perfused heart apparatus, using a Krebs–Henseleit (KH) perfusate: 118 mmol/L NaCl, 4.0 mmol/L KCl, 2.52 mmol/L CaCl2, 24.8 mmol/L NaHCO3, 1.7 mmol/L MgSO4, 1.2 mmol/L KH2PO4, 11.0 mmol/L glucose, 0.5 mmol/L EDTA, and 8 U/L insulin. The perfusate was insufflated continuously with 95%O2/5%CO2. The hearts were paced at 240 bpm using platinum-tipped electrodes connected to a Grass Instruments (Quincy, MA) SD-5 stimulus generator. Left ventricular (LV) developed pressure (LVDP) was measured from a 2.5F, high-fidelity micromanometer (Nihon-Kohden, Tokyo, Japan) passed into a compliant latex balloon inserted into the LV, and recorded on a PowerLab 2/20 Data Recording System (ADInstruments, Hayward, Australia). The balloon was connected to a Y-adapter with one end used to advance the micromanometer and the other used to fill the LV balloon with bubble-free water to an end-diastolic pressure (LVEDP) of 10 mm Hg. The maximum rate of increase of LV pressure (+dP/dtmax) and the minimum rate of decrease of LV pressure (–dP/dtmin) were calculated using ChartTM5 (ADInstruments). Coronary flow (CF) was measured by collecting effluent. Global ischemia was achieved by clamping the aortic inflow line. The temperature of the heart was continuously monitored by a digital thermometer (PTW-100A, Unique Medical, Japan). During ischemia, hearts were maintained at 37°C by enclosure in a water-jacketed air chamber. Warmed perfusate kept in the lower part of the chamber saturated the air with humidity and prevented cooling by evaporation.
Experimental Protocol
Animals were assigned to 1 of 8 groups (n = 10 each; Fig. 1). Animals were initially given 1.25% ethanol in their drinking water for 1 wk and then 2.5% ethanol for 6 wk. Serum ethanol levels at time of death were measured by gas chromatography (Shimadzu, Kyoto, Japan). Liver enzymes were measured to exclude the possible liver tissue damage.
|
After 20 min for equilibration, baseline LVDP, LVEDP, and CF were recorded. All hearts were subjected to 30 min of ischemia followed by 120 min of reperfusion. Anesthetic preconditioning was elicited by administration of sevoflurane (2%) for 10 min, followed by 10-min washout before ischemia. Sevoflurane was insufflated by passing the 95%O2/5%CO2 gas mixture through a calibrated vaporizer (ACOMA, Tokyo, Japan). Samples of coronary perfusate were collected anaerobically from the aortic cannula for measurement of sevoflurane concentration by an organic vapor sensor (OSP, Saitama, Japan). To investigate the role of PKC and mitochondrial KATP channels activation, their inhibitors, chelerythrine (CHE) and 5-hydroxydecanoate (5-HD), were administered for 20 min, starting 10 min before sevoflurane (EtOH + SEVO + CHE, EtOH + SEVO + 5HD) or saline vehicle (CTL + CHE, CTL + 5HD) administration. CHE (Alexis-Calbiochemicals, Tokyo, Japan) and 5-HD (Sigma Chemicals, St. Louis, MO) were dissolved in distilled water, and added to the KH perfusate to a final concentration of 10 and 200 µM, respectively. We performed experiments in the following order: first a CTL, followed by ETOH, SEVO, ETOH + SEVO, CTL + CHE, and so forth, until the first series of experiments was completed. This sequence was repeated 10 times.
Determination of Myocardial Infarct Size
At the end of experiments, the hearts were quickly frozen at –80°C for 15 min, then sliced into 2-mm thick transverse sections from apex to base (6 slices/heart). After removing the right ventricle and defrosting, each slice was weighed and incubated at 37°C with 1% triphenyltetrazolium chloride (Sigma Chemicals) in phosphate buffer (pH 7.4) for 10 min and then fixed in 10% formalin for at least 5 h to distinguish red stained viable tissue from pale unstained necrotic tissue.19 Each slice was photographed and the necrotic area was determined using Adobe Photoshop® CS (Adobe, CA) and multiplied by the weight of the slice, then expressed as a fraction of LV.
Western Blot Analysis
Separate experiments were performed (n = 4 in each group) to examine iNOS and eNOS expression. Myocardial tissue samples were collected before ischemia, and homogenized in ice-cold homogenizing buffer containing: 250 mM sucrose, 20 mM HEPES (pH 7.5), 10 mM KCl, 2 mM EGTA, 2 mM MgCl2, 25 mM NaF, 50 mM β-glycerophosphate, 1 mM Na3VO4, 1 mM PMSF, 1% Triton X, and protease inhibitor leupeptin (10 µg/mL). The homogenate was centrifuged at 1000g and 4°C for 5 min. The supernatant was recentrifuged at 10,000g and 4°C for 15 min. The protein concentration was estimated with a Bradford assay. Equivalent amounts (50 µg) of protein samples were loaded and separated on a 5%–10% SDS-PAGE gradient gel, then electrically transferred overnight to a PVDF membrane (Millipore CO, Billerica, USA). After blocking with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T), the membranes were incubated for 2 h at 4°C in TBS-T containing 5% milk and 1:200 dilution of rabbit primary antibody for iNOS and eNOS (H-174 and H-159, Santa Cruz Biotechnology, USA). Membranes were incubated with a 1:1000 dilution of horseradish peroxidase–labeled anti-rabbit immunoglobulin G (NA 934V, GE Healthcare, UK). The same blot was stripped and reblotted with antibodies to antitubulin to confirm equal protein loading. Bound antibody signals were detected with enhanced chemiluminescence (Pierce Biotechnology, IL) and visualized using VersaDoc 5000® Imaging System (Bio-Rad). Quantitative analysis of the band densities was performed by Quantity One® software (Bio-Rad).
Statistical Analysis
All data are expressed as mean ± sd. Two-factor repeated-measures analysis of variance was used to evaluate differences over time among groups. If differences were observed, a Tukey post hoc test was used to confirm the significance of differences among groups. The differences in expressions of iNOS and eNOS were determined by unpaired Student’s t-test. Power analysis demonstrated a sample size of n = 10 hearts per group was sufficient. A value of P < 0.05 was considered statistically significant.
RESULTS
Ten of 92 hearts were not used secondary to intractable ventricular fibrillation after reperfusion (4 in CTL, 3 in CTL + CHE, 2 in CTL + 5HD and 1 EtOH + SEVO + CHE) and two hearts were not used because of aortic rupture. Additional hearts were studied until each group had n = 10 successful experiments. There was no significant difference in body weight among groups. Serum ethanol levels at time of death was 2.7 ± 0.5 mmol/L. Ethanol was not detected in the effluent of the 10 EtOH hearts sampled after 50-min washout but before ischemia or sevoflurane exposure. There was no difference in liver enzymes levels between EtOH and CTL animals. The concentration of sevoflurane in the coronary perfusate after 10 min of exposure was 0.36 ± 0.09 mM. Sevoflurane was not detected in the effluent during the baseline, ischemic, and reperfusion periods.
Hemodynamics
Baseline LVDP, +dP/dtmax, –dP/dtmin, and CF were similar among the eight groups (Table 1). Administration of sevoflurane or treatment with CHE and 5-HD did not significantly affect LVDP or CF. Recovery of LVDP was greater in EtOH, SEVO, and EtOH + SEVO groups compared with CTL throughout the reperfusion period. Recovery of LVDP in EtOH + SEVO hearts was abolished by administration of CHE and 5-HD. Treatment with CHE and 5-HD alone did not affect the recovery of LVDP. LVEDP increased to 600% of baseline in CTL hearts after ischemia-reperfusion. The increase in LVEDP was significantly less in EtOH, SEVO, and EtOH + SEVO compared with CTL hearts during the reperfusion period. LVEDP in CHE and 5-HD groups was similar to that of CTL hearts. Treatment with CHE and 5-HD alone did not affect the increased LVEDP.
|
All groups had reduced +dP/dtmax after reperfusion compared with baseline. Recovery of +dP/dtmax was significantly greater in SEVO, EtOH, and SEVO + EtOH compared with CTL hearts, but not in hearts pretreated with CHE and 5-HD. Changes of –dP/dtmin during the reperfusion period had a similar pattern to those of +dP/dtmax.
There were no significant differences in CF among all groups throughout the experiment. This suggests that changes in CF could not account for the improved contractile recovery of SEVO or ETOH hearts (Table 1).
Myocardial Infarct Size
Myocardial infarct size (Fig. 2) in EtOH and SEVO groups was significantly reduced by approximately 50% compared with control hearts (EtOH: 27% ± 6%, SEVO: 23% ± 7% vs. CTL: 45% ± 11%, P < 0.001). The administration of sevoflurane with in EtOH hearts decreased infarct size compared with EtOH alone (EtOH + SEVO: 15% ± 4% vs. EtOH: 27% ± 6%, P < 0.001). Myocardial infarct size in EtOH and SEVO hearts pretreated with CHE and 5-HD was not different compared with CTL hearts (EtOH + SEVO + CHE: 44% ± 12%, P = 0.777; EtOH + SEVO + 5-HD: 45% ± 9%, P = 0.852 vs. CTL). Treatment with CHE and 5-HD alone did not affect the infarct size compared with the CTL group (P = 0.884; 0.355, respectively vs. CTL) (Fig. 2).
|
Western Blot Analysis
The densities of iNOS and eNOS were normalized against that of CTL. Chronic EtOH consumption increased expression of eNOS whereas it reduced expression of iNOS compared with the CTL groups (Fig. 3). Ethanol-treated and CTL hearts showed similar iNOS and eNOS expression in the presence of CHE and 5-HD with and without sevoflurane exposure (data not shown).
|
DISCUSSION
We found that pretreatment with sevoflurane and regular ethanol consumption equally preconditioned hearts against ischemia-reperfusion injury. Furthermore, sevoflurane enhanced the cardioprotective effect of regular ethanol consumption, resulting in smaller infarct size. This protection was abolished by inhibiting PKC or mitochondrial KATP channels, suggesting their role in mediating the observed cardiac preconditioning. Finally, we found that regular ethanol consumption increased eNOS expression and reduced iNOS expression. Our finding that hearts treated with sevoflurane or ethanol showed similar improved functional recovery, demonstrated by increased LVDP and lower LVEDP, as well as a nearly 50% decrease in infarct size, after ischemia-reperfusion, is consistent with previous reports.12,20,21 We observed that, despite a significant reduction of myocardial infarct size induced by the combination of ethanol and sevoflurane, compared with either preconditioning therapy alone, recovery of LVDP during reperfusion was comparable among SEVO, EtOH, and SEVO + EtOH hearts. This disparity between the functional recovery and infarct size might be explained by myocardial stunning from global ischemia that affected a larger area of the LV than that susceptible to infarction.22,23
A new finding of this study was that ethanol-induced preconditioning is enhanced by anesthetic-induced preconditioning. A possible mechanism for this effect may be that ethanol does not completely activate the signal transduction pathways eliciting cardioprotection. We previously demonstrated that exposure to 2.5% ethanol for 6 wk reduces ischemia-reperfusion injury to the same degree as 5%, 10%, and 20% ethanol for 6–12 wk.12 Thus, the ethanol dose used in the present study should induce the maximum ethanol-induced cardiac protection. Similarly, the sevoflurane dose (2%) used in the present study has been shown to produce the maximum cardiac protection.24 The additive preconditioning effect by the combination of sevoflurane and ethanol suggests that there are some differences in either their mechanisms of action or degree of signaling activation.
Opening of KATP channels is an important step in the signal transduction cascade of cardiac protection from both volatile anesthetics21 and chronic ethanol exposure.25 For example, Zaugg et al.26 demonstrated that sevoflurane increased mitochondrial flavoprotein oxidation, an index of mitochondrial KATP channel activity, and that this effect was completely abolished by CHE (2 µM) in rat cardiomyocytes. Mitochondrial KATP channels are activated by NO,27 a key mediator in late ischemic preconditioning.28 Others have shown that preconditioning with sevoflurane enhances the increased NO effluent induced by bradykinin and nitroprusside in isolated guinea pig hearts.17 Moreover, in humans, ethanol consumption increases serum NO production 30 min after drinking.29 Our data and prior reports suggest that increased eNOS expression due to chronic ethanol consumption could result in increased NO production. This is consistent with a previous study demonstrating that ethanol increases NO production through modulation of eNOS expression.18 The importance of eNOS in myocardial ischemic protection was demonstrated in an eNOS knockout mice model.30 Thus, augmentation of mitochondrial KATP channel activity by increased NO with the combination of sevoflurane and chronic ethanol consumption might explain the additive cardioprotection we observed in SEVO + EtOH hearts.
Previous experiments suggest that inflammation plays a role ischemia-reperfusion injury.31 NO derived from iNOS mediates cardiac dysfunction by inducing production of proinflammatory cytokines.32 Cytokines, such as interleukin-1β, interleukin-6, and tumor necrosis factor-
, are up-regulated rapidly in response to myocardial ischemia.33 Sevoflurane has been shown to reduce the production of tumor necrosis factor- in patients undergoing coronary bypass surgery.34 Interestingly, one study found that the late phase of ethanol’s myocardial preconditioning correlated with reduced leukocyte-endothelial cell adhesive interactions, suggesting an antiinflammatory effect.35 However, in the present study, with a crystalloid-perfused heart, leukocyte-endothelial cell adhesive interactions is not a factor. Nevertheless, EtOH hearts demonstrated reduced iNOS expression before ischemia compared with CTL. Thus, regular ethanol consumption could attenuate the inflammatory response to myocardial ischemia resulting from increased NO production via iNOS. Although iNOS is thought to be absent in myocytes under physiologic conditions, there is evidence that it is present in small vessel endothelium, vascular smooth muscle cells, and immune cells that infiltrate the heart.36
Studies demonstrate that acute (as opposed to chronic) ethanol exposure fails to exert cardioprotection when ethanol is present during ischemia-reperfusion.37 Although not withdrawn from the drinking water before death in this study, serum ethanol levels were low (2.7 ± 0.5 mmol/L) at the time of death. Ethanol was washed-out before ischemia using a crystalloid ethanol-free buffer. Ethanol was not detected after 50-min perfusion in the effluent. Ethanol freely diffuses in myocytes. Thus, tissue levels of ethanol were likely negligible at the time of study.
A limitation of the present study is that we did not directly measure PKC levels and mitochondrial KATP channel activity before and during ischemia-reperfusion. The doses of CHE and 5-HD used in the present study have been found to reduce PKC levels and inhibit mitochondrial KATP channel activity, respectively.14,38 Furthermore, we and other investigators have shown that the same concentrations of CHE and 5-HD inhibit the preconditioning-like effects of ethanol or sevoflurane alone.14,25,38 Prolonged ingestion of ethanol may lead to changes in heart rate and arterial blood pressure. Although we did not measure heart rate or arterial blood pressure before death in this study, a previous study demonstrated that feeding 10% ethanol (a much higher dose) for 40 wk to guinea pigs did not alter hemodynamics compared with control.39 Finally, animals were not randomized to each study group, although the order of study was standardized.
In conclusion, sevoflurane enhances cardiac preconditioning induced by regular ethanol consumption. Our findings suggest that PKC and mitochondrial KATP channels play a role in mediating this cardioprotection. NOS expression is altered by regular ethanol consumption.
Footnotes
Accepted for publication September 19, 2007.
Supported, in part, by Grant-in-Aid for Scientific Research (C) 16592032 from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MM), the Osaka Dental University Joint Research Founds (MM, ND), and the Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan (ND).
REFERENCES
This article has been cited by other articles:
|
|
 |
|
  X. Lu, H. Liu, L. Wang, and S. Schaefer Activation of NF-{kappa}B is a critical element in the antiapoptotic effect of anesthetic preconditioning Am J Physiol Heart Circ Physiol, May 1, 2009; 296(5): H1296 - H1304. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. S. Pagel Additive Cardioprotection by Ethanol and Sevoflurane: Are Sarcolemmal KATP Channels Also Involved? Anesth. Analg., June 1, 2008; 106(6): 1926 - 1926. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
