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From the Departments of *Anesthesiology and Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin.
Address correspondence and reprint requests to Jianzhong An, MD, Department of Anesthesiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Address e-mail to jzan{at}mcw.edu.
Abstract
INTRODUCTION: Anesthetic preconditioning (APC) with volatile anesthetics improves recovery of contractile function and reduces calcium overload after ischemia/reperfusion (I/R). Mitochondrial and sarcolemmal KATP channel openings have been implicated in APC-induced cardioprotection. In this study, we investigated the effect of APC on major calcium cycling proteins and its relation to KATP channels.
METHODS: Isolated perfused rat hearts were divided into seven groups: Time control (n = 10), ischemia control (n = 8), APC (n = 8), Mitochondrial KATP inhibitor 5-hydroxydecanoate (5-HD, 200 µM, n = 8), Sarcolemmal KATP inhibitor HMR1098 (HMR, 20 µM, n = 8), and APC plus 5-HD or APC plus HMR1098 (n = 8 each). APC was initiated by administering 1.5% isoflurane for 15 min, followed by a 15 min washout before 30 min of myocardial ischemia and 60 min of reperfusion. Ca2+-release channels (RyR2), Ca2+-adenosine triphosphatase (SERCA2a), phospholamban, plasma membrane Ca2+ ATPase, and sodium–calcium exchanger in the homogenate were determined by Western blot assay.
RESULTS: APC improved contractile recovery (left ventricular developed pressure, +dP/dt, –dP/dt) after I/R, which was blocked by 5-HD and HMR. I/R depressed the density of RyR2, SERCA2a, and phospholamban, with no changes in the density of plasma membrane Ca2+ ATPase and sodium–calcium exchanger. APC reversed I/R-induced degradation of RyR2 and SERCA2a in the presence or absence of 5HD and HMR.
CONCLUSIONS: I/R-induced depression in cardiac performance is associated with a down-regulation of the major sarcoplasmic reticulum Ca2+-cycling proteins. Anesthesia preconditioning with isoflurane prevents I/R-related degradation of the RyR2 and SERCA2a in the sarcoplasmic reticulum. However, this effect was independent of its activation of KATP channels.
In the mammalian cardiac myocyte, Ca2+ homeostasis is maintained on a beat-to-beat basis. During excitation contraction coupling, Ca2+ entry via the sarcolemmal L-type channels triggers opening of the Ca2+-release channels (RyR2). Ca2+ stored in the sarcoplasmic reticulum (SR) is then released into the cytosol where it interacts with the myofilaments to initiate contraction. Ca2+ is removed from the cytosol to the SR by Ca2+-adenosine triphosphatase (SERCA2a) and extruded to extracellular space via plasma membrane Ca2+-adenosine triphosphatase (PMCA) and the Na+/Ca2+ exchanger (NCX) (1). Both Ca2+ release and uptake processes are regulated by protein phosphorylation/ dephosphorylation. Serine 2809 (ser2809) of RyR2 is a phosphorylation site for calmodulin-dependent kinase II (CaMKII) (2) or protein kinase A (PKA) (2,3). It is constitutively phosphorylated and responds to ß-adrenergic stimulation in the intact heart (4). RyR2 phosphorylation by PKA increases the rate of Ca2+ release from the SR, mostly as a result of increased luminal SR Ca2+ load (3,5). Ca2+ uptake by SERCA2a is dynamically regulated by phospholamban (PLB) phosphorylation. Phosphorylation of PLB by PKA or CaMKII relieves its inhibition of SERCA2a and stimulates SR Ca2+ uptake (5).
During ischemia and reperfusion (I/R), cytosolic Ca2+ is overloaded. This is mainly due to Ca2+ release from SR and Ca2+ influx from extracellular space via Na+/Ca2+ exchanger, causing myocardial injury (6–8). Cytosolic Ca2+ overload plays a major role in the development of myocardial injury from I/R (7,8). Volatile anesthetics such as isoflurane or sevoflurane, given briefly before ischemia, have been shown to protect the heart against I/R injury, an effect known as anesthetic-induced cardiac preconditioning (APC) (6,9,10). APC is proposed to involve the activation of G-protein-coupled receptors, multiple protein kinases, and sarcolemmal and mitochondrial ATP-sensitive potassium channels (KATP) (11). Volatile anesthetics have effects on intracellular Ca2+ homeostasis, explaining in part their negative effects on myocardial contractility (12–14). It is likely that the myocardial protection induced by APC affects the major Ca2+ cycling proteins and their function. We have reported that APC improved cardiac function and reduced cytosolic Ca2+ overload, SR protein degradation, and myocardial infarct size in isolated guinea pig hearts (6,15). In this study, we hypothesized that APC with isoflurane protects the rat heart against I/R injury by preserving the major Ca2+ cycling proteins, and KATP blockers reverse this protective action.
METHODS
The investigation conformed to the Guide for the Care and Use of Laboratory Animals (US National Institutes of Health (NIH No. 85-23, revised 1996) and approval was obtained from the Medical College of Wisconsin Animal Studies Committee.
Langendorff Isolated Heart Preparation and Measurements
Our methods have been previously described in detail (6,15–18). Forty-two male Wistar rats (280–330 g) were anesthetized with inactin (30 mg) and then given heparin (1000 U). The hearts were immediately perfused retrograde through the aorta with cold (4°C), modified Krebs–Ringer (KR) solution (equilibrated with 95% O2 and 5% CO2) and then rapidly excised. The KR perfusate (pH 7.39 ± 0.01, Po2 560 ± 10 mm Hg) had the following composition (nonionized): Na+ 118 mM, K+ 4.7 mM, Mg2+ 1.2 mM, Ca2+ 1.6 mM, Cl– 134 mM, HCO3– 25 mM, H2PO4– 1.2 mM, glucose 11 mM, and HEPES 10 mM. Perfusate and bath temperatures were maintained at 37.2°C ± 0.1°C using a thermostatically controlled water circulator (1150S, VWR, Bristol, CT).
At constant temperature and perfusion pressure (100 mm Hg), left ventricular pressure (LVP) was measured isovolumetrically using a transducer connected to a thin, saline-filled latex balloon inserted into the left ventricle through the mitral valve from an incision in the left atrium. Coronary artery flow (CF) was measured by an ultrasonic flowmeter (Transonic T106X, Ithaca, NY) placed directly into the aortic inflow line. Coronary inflow and coronary venous Na+, K+, Ca2+, and pH were measured off-line with an intermittently self-calibrating analyzer system (Radiometer Copenhagen ABL 505, Copenhagen, Denmark). Coronary outflow (coronary sinus) O2 tension was also measured on-line continuously with a Clark-type O2 electrode (203B, Instech; Plymouth Meeting, PA). The percentage O2 extraction was calculated as 100 x (Po2a – Po2v)/Po2a (where p= partial pressure in mm Hg, a= coronary artery and v= coronary sinus); myocardial O2 consumption (MVO2) was calculated as (CF/g heart weight) x (arterial Po2 – venous Po2) x 24 µL O2/mL at 760 mm Hg; and cardiac work efficiency was calculated as (systolic LVP – diastolic LVP) x heart rate/MVO2. At the end of the experiments, the hearts were freeze-clamped and stored at –80°C until used in Western blot assays (see below).
Experimental Protocol
Animals were divided into seven groups. The untreated time control (nonischemia) group (n = 10) was not subjected to global ischemia. The ischemia control (ISC, n = 8) group underwent 45 min of coronary perfusion, followed by 30 min of global ischemia, and 60 min of reperfusion. Global ischemia was induced in all experiments by stopping coronary artery perfusion. The same time protocols were used in animals receiving the sarcolemmal KATP inhibitor HMR (1-[5-[2-(5-chloro-o-anisamido)ethyl]-2mythoxyphenyl]-3-methylthiourea), sodium salt; Aventis, Frankfurt, Germany; 20 µM, n = 8), the mitochondrial KATP inhibitor 5-HD (5-hydroxydecanoate, Sigma, St. Louis, MO, 200 µM, n = 8), APC (isoflurane 1.5%, Abbott Laboratories, North Chicago, IL, n = 8), and APC + HMR or 5-HD groups (n = 8 each) (see below). In the APC group, isoflurane was given for 15 min, followed by a 25 min washout before 30 min of ischemia. Isoflurane was bubbled into the KR solution using an agent-specific vaporizer placed in the O2–CO2 gas mixture line. HMR or 5-HD in KR solution was given for 35 min, followed by washout for 10 min before 30 min ischemia and 60 min reperfusion. In two other groups, HMR or 5-HD treatments were combined with APC. Samples of coronary perfusate were collected from a port in the aortic cannula to measure isoflurane concentration by chromatography (Shimadzu, Kyoto, Japan).
Western Blot Assays for Ca2+-Cycling Proteins
Western blot assays were conducted as previously described (4). Briefly, aliquots of homogenate samples of the left ventricle were solubilized in Laemmli sample buffer and fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using a 4%–20% slab gel. After the transfer to nitrocellulose membranes, they were blocked with 5% nonfat milk in phosphate-buffered saline and probed with primary antibodies against RyR2 (1:2000), SERCA2a (1:2000), PLB (1:1000), and PMCA (1:1000). The above-mentioned antibodies were purchased from Affinity Bioreagents Inc. Golden, CO. The antibody against NCX (1:1000) was obtained from Sigma (St. Louis, MO). The antibodies against RyR-ser2809 and PLB-thr17 were purchased from Badrilla Ltd. (Leeds, UK; 1:1000). Secondary antibodies were conjugated to horseradish peroxidase and the specific signals were measured with chemiluminescence. The density of proteins were determined by densitometry using Kodak 1D software and normalized to actin. Protein assays were done with Bradford methods using a BioRad assay kit (BioRad Laboratories, Hercules, CA).
Statistical Analysis
Data are expressed as means ± sd. Two-way analysis of variance was used to assess the differences among the groups for continuous data at the discrete study time points before ischemia and during reperfusion. Analysis of variance for repeated measures was used to assess within group differences over time. Student–Newman–Keuls multiple-comparison post hoc tests were used to differentiate the differences within or between groups. Differences among the means were considered significant when P < 0.05 (two-tailed).
RESULTS
Cardiac Performance
HR and other cardiac measurements before and after myocardial ischemia are listed in Table 1. HR was more rapid in all APC-treated groups (inflow isoflurane concentration was 0.29 ± 0.02 mM, which is equivalent to a minimal alveolar concentration of approximately 1.0 ± 0.2) when compared with the ISC Group 2 min after initiation of reperfusion. It was likely due to relative bradycardia in the ISC group compared with other groups. Throughout reperfusion, CF was lower than baseline in each ischemia group, but higher in all of the APC-treated groups (return to 68% in APC, 66% in APC + 5-HD, 65% in APC + HMR) compared with the ISC group (50%). Oxygen delivery and the percentage of O2 extraction were significantly increased in APC group. MVO2 was decreased throughout reperfusion in each group, but it was less during initial reperfusion in the APC-treated groups (75% in APC, 65% in APC + 5-HD, 64% in APC + HMR) than in the ISC group (59%). During reperfusion, cardiac efficiency was decreased in each group, but it was higher in the APC groups than the ISC group. After 60 min of reperfusion, MVO2 and cardiac efficiency were higher in the APC group (60%) than in the ISC group (45%), but it remained lower than baseline. In groups with 5-HD or HMR-1098 alone, no significant differences in recovery of HR, CF, or other cardiac measurements were observed when compared with ischemia control group (data not shown).
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Left ventricular developed pressure (LVDP) was decreased after I/R compared with baseline in each group, but it was higher in the APC group than in the ISC, APC + HMR, or APC + 5HD groups on reperfusion (Fig. 1, Panel A). Left ventricular end-diastolic pressure (LVEDP) in the ischemia groups showed a marked increase (Fig. 1, Panel B) compared with the control group. However, preischemic treatment with 1.5% isoflurane resulted in less increase in LVEDP compared with the ISC group, whereas sarcolemmal and mitochondrial KATP channel blockers partially inhibited this APC-induced attenuation in LVEDP increase. APC resulted in better recovery of +dP/dt and –dP/dt (Panel C, D) compared with the ISC group, an effect abolished by 5-HD and HMR-1098. In groups with 5-HD or HMR-1098 alone, no significant differences in recovery of LVDP, LVEDP, +dP/dt, or –dP/dt were observed when compared with the ischemia control group (data not shown).
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Effect of APC on the Ca2+ Regulatory Proteins
Figure 2 displays the protein density of the cardiac Ca2+ release channel, RyR2, and its phosphorylation level at ser-2809. There was a significant reduction from baseline (approximately 36%) in the density of RyR2 and in the phosphorylation level of RyR2 at ser-2809 (approximately 38%) in the ISC only group. The decline in the latter is, thus, largely a result of decreased RyR2 density, but not a change of phosphorylation status after I/R. APC significantly prevented the degradation of RyR2 and its phosphorylation at ser-2809 (87% ± 8% and 84% ± 10% of control) due to I/R. Inhibition of sarcolemmal KATP and mitochondrial KATP channel did not affect this preservation by APC.
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Figure 3 shows SERCA2a density, which was decreased significantly after I/R (60% ± 10% from baseline). This reduction was partially attenuated by APC treatment (83% ± 12% of control). Like that observed with RyR2, the protective effect of APC was not affected by 5-HD or HMR-1098. Figure 4 shows the density of PLB and its phosphorylation at thr-17, which were significantly decreased after 30 min of I/R (84% ± 6% and 76% ± 9% of baseline). However, APC failed to attenuate this decline in all other treatment groups.
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The density of PMCA and NCX are shown in Figure 5. There was no alteration of PMCA and NCX in any groups exposed to myocardial ischemia and reperfusion.
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DISCUSSION
I/R-induced myocardial injury is primarily due to cytosolic Ca2+ overload and oxidative stress (6–8). APC has been shown to reduce Ca2+ overload and oxidative stress and to improve contractile function during I/R (15,19). We recently showed that, in the guinea pig heart, APC with sevoflurane and blockade of the Na+/Ca2+ exchanger attenuated Ca2+ overload and reduced degradation of SR Ca2+ cycling protein (15). In this study, we have confirmed these findings in an isolated rat heart experimental preparation. We further show that inhibition of sarcolemmal KATP and mitochondrial KATP channels prevented isoflurane-induced improvement in contractile function after I/R, supporting the role of these ion channels in the mechanism of APC. Surprisingly, however, blockade of these KATP channels did not reverse the preservation of SR proteins RyR2 and SERCA2a by APC. This intriguing observation suggests that isoflurane-induced APC may activate alternative protective mechanisms that prevent protein proteolysis.
APC and SR Protein Preservation
Cytosolic Ca2+ overload during I/R causes proteolysis due to its activation of Ca2+-sensitive proteases (20,21). Inhibition of calpain prevented I/R-induced degradation of key SR Ca2+ cycling proteins (i.e., RyR2 and SERCA2a) and improved contractile function (20). Similarly, exercise training was shown to inhibit calpain activation during I/R and reduce SERCA degradation (22). Proteolysis of RyR2 by trypsin induced subconductance, increased RyR2 channel opening (4), and precipitated increased Ca2+ leak and cytosolic Ca2+ overload during I/R.
In the current study, APC-induced recovery of cardiac contractile function after I/R was associated with preservation of RyR2 and SERCA2a protein contents, similar to our observations in the guinea pig heart (15). These results suggest that APC inhibited proteolysis, probably by attenuation of Ca2+ overload and/or direct inhibition of protease activity. Numerous studies have shown that APC suppress Ca2+ overload, and thus subsequent protease activation. Since we have observed a dissociation between depressed contractile function and preservation of SR proteins after APC blockade with KATP blockers, we speculate that isoflurane-induced APC directly inhibits protease activity. It may inhibit protease calpain directly and/or via its endogenous antagonist calpastadin (22). Further studies are required to validate our hypothesis.
Despite the preservation of SERCA2a and RyR2 proteins after APC blockade, we have seen depressed contractile function, and by extension, cytosolic Ca2+ overload. This is likely a result of increased oxidative damage to the Ca2+ cycling proteins after APC blockade. Oxidation/nitrosative modifications modify the properties of RyR2-mediated Ca2+ release and SERCA2a-mediated Ca2+ uptake (23,24). Sulfhydryl oxidation or S-nitrosylation of the cardiac RyR has been reported to activate the channel (25). Nitrosylation of SERCA2a inhibits Ca2+ uptake (24,26). These malfunctions likely contribute to Ca2+ overload. The precise role of oxidation/ nitrosative modifications in APC needs to be further investigated.
APC and Sarcolemmal Ca2+ Cycling Proteins
During steady-state contractions in the heart, Ca2+ entry into the sarcolemma through membrane L-type Ca2+ channels (ICa) is balanced by an equal efflux of Ca2+ from the cell via the NCX (27) and the PMCA (28). Our results revealed no alteration of PMCA and NCX contents after I/R. Thus, it appears that these two sarcolemmal proteins are less sensitive to proteolysis than SERCA2a and RyR2 in I/R. This is not surprising, as sarcolemmal Na+/K+-ATPase was also found not to be degraded during I/R, but the cytoskeletal proteins 
-fodrin and ankyrn-B, which anchor the Na+-K+-ATPase, were cleaved by calpain after ischemia (21). It remains to be seen whether similar defects occur to PMCA and NCX during I/R. Oxidative damage likely also affects Ca2+ cycling by sarcolemmal proteins PMCA and NCX.
Although APC treatment does not alter the contents of PMCA and NCX after I/R, volatile anesthetics themselves may have some inhibitory effect on sarcolemmal Ca2+-adenosine triphosphatase and Na+/Ca2+ exchanger. Volatile anesthetics, such as isoflurane and sevoflurane, inhibit Ca2+ extrusion from the cell by affecting the sarcolemmal Ca2+-adenosine triphosphatase (29). In a whole-cell patch-clamp study, both sevoflurane and halothane significantly reduced the outward and inward Na+/Ca2+ exchanger current, which could limit the negative inotropic effects and help to maintain SR Ca2+ content (30). These results suggest that APC may have a memory effect that reduces contractile failure by modulating PMCA and NCX function during I/R.
In summary, we have shown that, in isolated rat hearts, isoflurane-induced APC protects the heart against I/R injury by preserving major Ca2+ cycling proteins. However, APC may induce an alternative protective mechanism that prevents proteolysis, independent of its activation of KATP channels. Further studies are warranted to validate this hypothesis.
Footnotes
Accepted for publication July 3, 2007.
Supported, in part, by grants from the American Heart Association, Dallas, Texas: 026518Z (to J.A.), and the National Institutes of Health, Bethesda, Maryland: HL-34708 and PO1GM 66730 (to Z.J.B.).
Presented, in part, at the annual meeting of the American Society of Anesthesiologist, Atlanta, Georgia, October 23–27, 2005, and published in an abstract form in Anesthesiology 2005;105:A-474.
The authors have no conflicts of interest to report.
REFERENCES
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