Ca(2+) loading occurs during myocardial reperfusion injury. Volatile anesthetics can reduce reperfusion injury. The authors tested whether sevoflurane administered before index ischemia in isolated hearts reduces myoplasmic diastolic and systolic [Ca(2+)] and improves function more so than when sevoflurane is administered on reperfusion.
Four groups of guinea pig hearts were perfused with crystalloid solution (55 mmHg, 37 degrees C): (1) no treatment before 30 min global ischemia and 60 min reperfusion (CON); (2) 3.5 vol% sevoflurane administered for 10 min before ischemia (SBI); (3) 3.5 vol% sevoflurane administered for 10 min after ischemia (SAI); and (4) 3.5 vol% sevoflurane administered for 10 min before and after ischemia (SBAI). Phasic myoplasmic diastolic and systolic [Ca(2+)] were measured in the left ventricular free wall with the fluorescence probe indo-1.
Ischemia increased diastolic [Ca(2+)] and diastolic left ventricular pressure (LVP). In CON hearts, initial reperfusion greatly increased diastolic [Ca2+] and systolic [Ca(2+)] and reduced contractility (systolic-diastolic LVP, dLVP/dt(max)), relaxation (diastolic LVP, dLVP/dt(min)), myocardial oxygen consumption (MvO(2)), and cardiac efficiency. SBI, SAI, and SBAI each reduced ventricular fibrillation, attenuated increases in systolic and systolic-diastolic [Ca(2+)], improved contractile and relaxation indices, and increased coronary flow, percent oxygen extraction, MvO(2), and cardiac efficiency during 60 min reperfusion compared with CON. SBI was more protective than SAI, and SBAI was generally more protective than SAI.
Sevoflurane improves postischemic cardiac function while reducing Ca(2+) loading when it is administered before or after ischemia, but protection is better when it is administered before ischemia. Reduced Ca(2+) loading on reperfusion is likely a result of the anesthetic protective effect.
REPERFUSION after cardiac ischemia can cause reversible mechanical dysfunction (stunning) or cell death (infarction) depending on the duration and magnitude of ischemia. 1Myoplasmic Ca2+overload during ischemia, and particularly on reperfusion, contributes to the injury. 2–6Ca2+loading is due largely to intracellular acidosis and a large transmembrane pH gradient that promotes Na+–H+exchange and, in turn, reverse-mode Na+–Ca+exchange at the onset of reperfusion. 2–4Impaired adenosine triphosphate synthesis on reperfusion results from cytoskeletal damage by reactive oxygen (O2) species and mitochondrial Ca2+overload. 7–9These effects can cause hypercontracture and incomplete relaxation of myofibrils or cell death. 1,9
It has long been known that administering a volatile anesthetic before ischemia, or before and after hypoxia or ischemia, improves cardiac function and reduces the occurrence of dysrhythmias on reperfusion. 10–14This was thought to arise from a metabolic sparing effect or from inhibition of Ca2+influx by the anesthetic. It was discovered earlier that hearts can be protected by “preconditioning” before ischemia, i.e. , by brief pulses of ischemia before prolonged ischemia. 15Protection was assessed by reduced infarct size, attenuated mechanical dysfunction, or limited ultrastructural abnormality. More recently, anesthetics have also been shown to precondition hearts against ischemia. 16–21We have shown that sevoflurane preconditioning mimics ischemic preconditioning by improving vascular, mechanical, and metabolic function and induced endothelial nitric oxide release in guinea pig hearts. 17,18Because these effects were blocked by glibenclamide, the ATP-sensitive potassium channel (KATP) opening has an important role in mediating the protective effect. 18We also reported recently that anesthetic preconditioning, like ischemic preconditioning, reduces Ca2+loading and improving function on reperfusion 21; this indicates an important link between cardiac function and Ca2+homeostasis during both ischemic and anesthetic preconditioning.
We questioned whether treatment with a volatile anesthetic before ischemia protects hearts better than treatment on initial reperfusion. We proposed that protection obtained by the former treatment was triggered or mediated by depressed cardiac metabolism and effected by reduced myoplasmic Ca2+loading on reperfusion; protection afforded by the latter treatment was due to a direct metabolic sparing effect mediated only on reperfusion that reduced Ca2+loading. To test this, we administered sevoflurane before and/or after global ischemia in isolated hearts and examined the changes in mechanical, metabolic, and vascular function associated with myoplasmic systolic and diastolic [Ca2+] on reperfusion.
Materials and Methods
Langendorff Heart Preparation
The investigation conformed to the Guide for the Care and Use of Laboratory Animals (US National Institutes of Health No. 85–23, revised 1996). Approval was obtained previously from the Medical College of Wisconsin animal studies committee (Milwaukee, WI). Portions of our methods have been described in detail previously. 5,6,21–25Sixty guinea pig hearts were isolated and perfused with a modified Krebs-Ringer's solution 6,21,24,25and perfused at an aortic root perfusion pressure of 55 mmHg at 37.3 ± 0.1°C. Left ventricular pressure (LVP) was measured isovolumetrically, and right atrial and ventricular electrograms were recorded to monitor spontaneous heart rate (HR) and atrioventricular conduction time. 21–25Coronary flow (CF) was measured at the aorta, and coronary sinus venous partial pressure of oxygen (Po2) tension was measured continuously on-line with an O2Clark-type electrode. Percent O2extraction was calculated as 100 · (Po2a − Po2v)/Po2a, myocardial O2consumption (Mvo2) was calculated as CF/g · (Po2a − Po2v) · 24 μl O2/ml at 760 mmHg, and cardiac work efficiency was calculated as systolic–diastolic LVP · HR/Mvo2.
Measurement of Cytosolic and Noncytosolic Free Ca2+in Intact Hearts
Loading Fluorescent Probe Indo-1 and Recording Ca2+Transients.
We have published this methodology in detail. 5,6,21,22,24,25Experiments were performed in a light-blocking Faraday cage. The heart was partially immobilized by hanging it from the aortic cannula, the pulmonary artery catheter, and the left ventricular balloon catheter. The heart was immersed in a bath. The distal end of a trifurcated fiber silica fiberoptic cable (optical surface area 3.85 mm2) was placed against the left ventricular epicardial surface through a hole in the bath. A rubber O ring was placed between the ferrule and the heart to reduce cardiac motion at the contact point of the fiberoptic tip. Background autofluorescence was determined for each heart after initial perfusion and equilibration at 37°C. Thereafter, hearts were loaded with indo-1 acetoxymethyl ester (AM) at room temperature (25 ± 0.6°C) for 20–30 min with 165 ml recirculated Krebs-Ringer's solution containing 6 μm indo-1 AM (Sigma Chemical, St. Louis, MO). Indo-1 AM was initially dissolved in 1 ml dimethyl sulfoxide containing 16% (weight/volume) Pluronic I-127 (Sigma Chemical) and was diluted to 165 ml with modified Krebs-Ringer's solution. Loading was stopped when the F385intensity was increased 10-fold. Residual indo-1 AM was washed out by perfusing the heart with standard perfusate for at least another 20 min, and then each heart was rewarmed to 37.2 ± 0.1°C before initiating the study. The perfusate contained probenecid (100 μm) to retard leakage of indo-1. Loading and washout of indo-1 reduces LVP approximately 25%, an effect due to the vehicle and to intracellular Ca2+buffering by indo-1 per se . 6,22,24
Fluorescence emissions at F385and F456were recorded using a modified luminescence spectrophotometer (SLM Aminco-Bowman II; Spectronic Instruments, Urbana, IL). The left ventricular region of the heart was excited with light from a xenon arc lamp, and the light was filtered through a 355-nm monochromator with a bandwidth of 8 nm. The beam was focused onto the in-going fibers of the optic bundle. The arc lamp shutter was only opened for 2.5-s recording intervals. Fluorescence emissions were collected by fibers of the remaining two limbs of the cable and filtered by square interference filters (Corion, Franklin, MA) at 385 (390 ± 5) and 456 (460 ± 5) nm.
An advantage of the indo-1 ratiometric procedure is that although both F385and F456decrease over time, the F385/F456ratio remains stable during the experimental period. 6In companion studies, tissue autofluorescence was measured throughout each experiment in the absence of ischemia (autofluorescence control, n = 8) and before, during, and after ischemia (ischemia control, n = 8). The mean F385and F456background values obtained were then subtracted from the corresponding F385and F456values from the indo-1–treated hearts at the same time point and for the same experimental condition.
Calculation of Cytosolic and Noncytosolic Ca2+Concentrations from F385/F456Transients.
The Ca2+transient obtained from the indo-1 fluorescence ratio F385/F456is proportional in a nonlinear manner to [Ca2+]. Calibration curves were derived according to previously published protocols by Brandes et al. 26,27using modifications of a standard equation for fluorescent indicators. 28Total (tot) intracellular [Ca2+] was calculated from the totF385/totF456ratio (totR), Rmax= Sr/bH (for > 100 μm Ca2+), Rmin= Rmax· S385/S456(for 0 Ca2+), S385= I385/I385(at min/max Ca2+) = 0.05, S456= I456/I456(at min/max Ca2+) = 2.4, and Kd, according to the equation:
where S = ratio of light intensities (I) at the same wavelength at min and max Ca2+, Sr= (1 − S456)/(1 − S385) =−1.48, and bH = the average slope (b) of totF385as a function of totF456=−0.25.
We calculated the dissociation constant (Kd) of indo-1 using homogenized guinea pig heart protein as 249 ± 8 nm at 37°C. 24Rmaxwas calculated as 5.986, and Rminwas calculated as 0.059.
Non cytosolic (primarily mitochondrial) mito[Ca2+] was calculated similarly:
where mitoR was calculated as the ratio of the noncytosolic fluorescence, mitoF385, and mitoF456, respectively. Noncytosolic fluorescence was measured at the end of each experiment (185 min) in control (CON; n = 8), sevoflurane before ischemia (SBI; n = 7), sevoflurane before and after ischemia (SBAI; n = 9), and sevoflurane after ischemia (SAI; n = 7) groups after perfusing hearts with 100 μm MnCl2for 10 min to quench fluorescence derived from the cytosolic (cyto) compartment. 6,27 mitoF385and mitoF456were calculated at each time point by multiplying the residual mitochondrial fluorescence fractions (f385and f456) by total end-diastolic fluorescence so that:
Similar to equations 1 and 2, cytosolic cyto[Ca2+] was calculated as:
where cytoR was derived from the ratio of the cytosolic fluorescence, cytoF385, and cytoF456, respectively, calculated at each time point by effectively subtracting mitochondrial compartment Ca2+(mito[Ca2+]) from tot[Ca2+] and multiplying the remainder by total end-diastolic fluorescence (as in equation 3) so that:
Nonstimulated endothelium does not contribute significantly to tot[Ca2+]. 6,22,27
There were four primary groups subjected to ischemia reperfusion; each experiment lasted 195 min beginning after 30 min equilibration. An untreated time control group (n = 9) was not subjected to ischemia; these data are not plotted. The four ischemia groups underwent 30 min of fluorescent dye loading, 20 min of washout, and 45 min of equilibration; this was followed by 30 min of global, no-flow ischemia, 60 min of reperfusion, and 10 min of Mn2+quenching. During ischemia, hearts were immersed in a bath of Krebs-Ringer's solution at 37.3 ± 0.3°C. The SBI group had 85 min of perfusion followed by a 10-min exposure to 3.5 vol% sevoflurane immediately before 30 min of ischemia. The SAI group had 95 min of perfusion, 30 min of ischemia, and 60 min reperfusion, with exposure to 3.5 vol% sevoflurane only during the first 10 min of reperfusion. Hearts of the SBAI group were exposed to 3.5 vol% sevoflurane for 10 min just before 30 min ischemia and also immediately during the first 10 min of reperfusion. If ventricular fibrillation occurred, a 0.25-ml bolus of lidocaine (250 μg) was administered immediately via the aortic cannula. All hearts reverted to sinus rhythm, and data were collected after stabilization of LVP. Sevoflurane was bubbled into the perfusate using an agent-specific vaporizer placed in the oxygen–carbon dioxide gas mixture line. Coronary effluent was collected anaerobically from the aortic cannula to measure sevoflurane concentration by gas chromatography. 12–14,17,18Inflow concentrations (in mm) were 0.62 ± 0.04 for SBI, 0.66 ± 0.04 for SAI, and 0.63 ± 0.05 and 0.67 ± 0.03 for SBAI, which combined was equivalent to 3.34 ± 0.28 vol% atmospheres for a minimal alveolar concentration (MAC of sevoflurane = 2.4%) of approximately 1.52 ± 0.2%. Sevoflurane was not detectable in the effluent during the initial equilibration period, the onset of the reperfusion period (CON and SBI groups), or from 15 to 60 min of reperfusion (all groups).
Data Presentation and Interpretation
Cytosolic Systolic and Diastolic Ca2+.
Initial measurements obtained throughout fluorescence dye loading and vehicle washout are not displayed; results shown in graphs begin at 50 min before ischemia. Mechanical, metabolic, and F385and F456measurements were made at 1- to 30-min intervals throughout the study. F385, F456, and F385/F456Ca2+transient signals, LVP, and dLVP/dt were displayed simultaneously on a computer screen and stored digitally using proprietary software (SLM Aminco-Bowman II, series 2, on OS/2 version 3, International Business Machines Corp., Thornwood, NY). After correcting for tissue autofluorescence over time, with or without ischemia and reperfusion, and quenching cytosolic (myoplasmic) compartment Ca2+to reveal noncytosolic compartment Ca2+, the signals were calibrated to nm [Ca2+] using algorithms developed by our group. LVP and raw metabolic data were recorded (MacLab; AD Instruments, Castle Hills, Australia) and, together with the Ca2+transient data, were later analyzed together (Microsoft Excel®; Microsoft Corp., Redmond, WA). Several characteristics of myoplasmic [Ca2+] were analyzed: peak systolic, peak diastolic, and phasic systolic–diastolic [Ca2+], i.e. , released or phasic [Ca2+]; mitochondrial (noncytosolic) [Ca2+], measured at the end of each experiment, is not phasic. Characteristics of LVP analyzed were systolic, diastolic, and systolic–diastolic LVP, and dLVP/dtmaxand dLVP/dtmin.
All data were expressed as mean ± standard error of the mean (SEM), and a P value (two-tailed) less than 0.05 was considered significant. Within-group data over time at specific times (88,124,128, and 181 min) for a given variable were compared with a pretreatment control period (at 78 min) by the Duncan comparison of means test whenever univariate analysis of variance fluorescence values for repeated measures were significant (P < 0.05; Super ANOVA 1.11®software for Macintosh®from Abacus Concepts, Inc., Berkeley, CA). Among-group data were analyzed at specific time points before (at 78 min) and after (at 88,124, 128, and 181 min) ischemia using multiple analysis of variance (Super ANOVA®). If fluorescence values were significant, post hoc comparisons of means were performed using the Student t test with Duncan adjustment for multiplicity to differentiate among the four analyzed treatment groups. The incidence of ventricular fibrillation versus sinus rhythm per group and the number of ventricular fibrillations per heart per group were determined by Fisher exact test. Differences in ventricular fibrillation duration were determined by unpaired t tests. Some among-group statistical notations that are not given in the figures are given in the text. Within-group comparisons on reperfusion versus before ischemia are given only in the text.
In the time control group, no measured mechanical or metabolic variable changed over 180 min perfusion; systolic [Ca2+] and diastolic [Ca2+] also did not change. Variables unchanged (not significant) at 60 min of reperfusion from 50 min before ischemia and averaged for all ischemia groups, were HR, 254 ± 3 to 251 ± 3 beats/min, and atrioventricular conduction time, 76 ± 2 to 77 ± 2 ms. Venous pH values for CON, SBI, SBAI, and SAI groups, respectively, were not different before ischemia, 7.16 ± 0.03, 7.12 ± 0.03, 7.12 ± 0.04, and 7.11 ± 0.05, and were not different at 60 min of reperfusion, 7.09 ± 0.05, 7.08 ± 0.06, 7.09 ± 0.04, and 7.08 ± 0.05. Ventricular fibrillation was the most notable dysrhythmia observed on reperfusion. The percentage of hearts that fibrillated during reperfusion was 100% for CON, 72% for SBI, 92% for SBAI, and 89% for SAI (P > 0.05). The median number of fibrillations per heart was 3.0 for CON, 1.0 for SBI, 1.0 for SBAI, and 1.0 for SAI; this number was lower (P < 0.05) in SBI, SAI, and SBAI groups than in the CON group.
Figures 1A–6Bdisplay the associated temporal changes in cytosolic [Ca2+] and cardiac function at 25 time points beginning 45 min before the onset of 30-min ischemia and 60-min reperfusion. Systolic [Ca2+] decreased slowly over 10 min during ischemia and then increased abruptly twofold during initial reperfusion in the CON group (fig. 1A). The increase in systolic [Ca2+] on 1 min reperfusion was much less in SBI, SAI, and SBAI groups and returned to control levels after 5 min in each treated group but only after 15 min of reperfusion in CON. Systolic LVP decreased faster than systolic [Ca2+] during early ischemia (fig. 1B) and began to increase after 25 min of ischemia. Systolic LVP increased on initial reperfusion to the control level in SBI and SBAI groups, but not in SAI and CON groups; moreover, systolic LVP remained higher in the SBI group than in the SAI group for the first 30 min of reperfusion. Diastolic [Ca2+] increased progressively twofold during ischemia and up to fourfold during initial reperfusion in each group (fig. 2A); diastolic [Ca2+] remained increased up to 30 min in CON but returned faster toward controls in sevoflurane-treated groups. Diastolic LVP (fig. 2B) increased in each group during the last 5 min of ischemia. During reperfusion, diastolic LVP increased less in SBI, SBAI, and SAI groups compared with CON and remained above the control level only in CON and SAI groups at 60 min of reperfusion.
Systolic–diastolic [Ca2+], i.e. , phasic [Ca2+], (fig. 3A) decreased slowly over 10 min in all groups during initial ischemia, after which the transients disappeared until reperfusion. Systolic–diastolic [Ca2+] increased abruptly approximately twofold during the first minute of reperfusion in CON but less so in sevoflurane-treated groups and remained higher during reperfusion in CON compared with the SBI and SAI groups. Unlike phasic [Ca2+], developed systolic–diastolic LVP (fig. 3B) decreased abruptly to near zero at the onset of ischemia. At 5 min of reperfusion, systolic–diastolic LVP was decreased by 66% in CON, whereas it was reduced approximately 41% averaged for all sevoflurane-treated groups. Between 20 and 60 min of reperfusion, systolic–diastolic LVP was higher in SBI and SBAI than in CON and SAI groups and higher in SAI than in CON.
Contractility, dLVP/dtmax(fig. 4A), was decreased moderately during SBI and SBAI treatments and approached zero in each group during ischemia; dLVP/dtmaxwas decreased by 70% on initial reperfusion in CON and by approximately 34% averaged for the sevoflurane-treated groups. dLVP/dtmaxremained depressed at 60 min reperfusion in the CON and SAI groups, but not in the SBI and SBAI groups. Relaxation, dLVP/dtmin(fig. 4B), exhibited trends for each group similar to those of dLVP/dtmax.
Coronary flow (fig. 5A) was not altered by sevoflurane in SBI and SBAI groups before ischemia. Coronary flow at 60 min of reperfusion remained lower than before ischemia in each group but was higher in each sevoflurane-treated group. Mvo2(fig. 5B) was indeterminate during ischemia and was lower in all groups but the SBI group on initial reperfusion and was higher in each sevoflurane group after 20 min of reperfusion compared with the CON group. Among the sevoflurane groups, Mvo2was highest on final reperfusion in the SBI and SBAI groups and lowest in the SAI group.
Cardiac work efficiency (fig. 6A) was not altered by sevoflurane in SBI and SBAI groups before ischemia among the groups. On reperfusion, cardiac efficiency was higher in all sevoflurane groups and especially in the SBI and SBAI group; it remained decreased at 60 min of reperfusion only in CON. Percent O2extraction (fig. 6B) was reduced by sevoflurane in SBI and SBAI groups before ischemia; on reperfusion, it was lower in all sevoflurane-treated groups compared with CON.
We examined cardioprotective effects of a volatile anesthetic, sevoflurane, administered for 10 min immediately before global ischemia, for 10 min on reperfusion immediately after ischemia, and both before and after ischemia to ascertain whether the timing of exposure to the anesthetic influences the extent of protection. At the same time, we measured whether diastolic and systolic myocyte [Ca2+] during ischemia and reperfusion are modulated by the protective effect of the anesthetic when administered before or after ischemia. As in previous studies using other volatile anesthetics, we observed that sevoflurane treatment afforded cardioprotection by reducing dysrhythmias and improving mechanical and metabolic function. Concomitantly, we observed that improved function with each sevoflurane treatment was associated with reduced systolic and phasic Ca2+loading on reperfusion.
It was interesting that sevoflurane administered before ischemia was more protective than sevoflurane administered on reperfusion. One might expect that because it is during the reperfusion period that the major damage to cells occurs, administering a depressant agent during reperfusion would be more protective that when administered before ischemia. Sevoflurane administered before ischemia exerted minimal cardiac effects except for small decreases in contractility (dLVP/dtmax), relaxation (dLVP/dtmin), and percent O2extraction. However, this small metabolic sparing effect before ischemia could trigger mechanisms that lead to cardioprotection on reperfusion. Indeed, the degree of protection was similar to that found previously for sevoflurane preconditioning. 17,18,21Although there was no memory phase after anesthetic exposure involved in these experiments, i.e. , no washout, as in our previous studies, 17,18,21it is likely that the anesthetic activates protective cellular pathways regardless of whether ischemia subsequently occurs. We speculate that the small sevoflurane-induced decrease in metabolic rate, without a change in O2delivery, metabolically triggers a protective mechanism mediated by a change in mitochondrial energetics, e.g. , a small decrease in mitochondrial Ca2+, an increase in reduced nicotinamide adenine dinucleotide (NADH), or a small release of O2-derived free radicals. 29Because we could not statistically differentiate effects of the three sevoflurane treatments on reducing Ca2+loading during ische-mia and reperfusion, we suggest that the reduction in Ca2+loading is a consequence rather than a cause of improved function after sevoflurane treatment.
The immediate period of reperfusion sets the stage for Ca2+overload 2–7and a large release of reactive O230,31species that leads to mitochondrial and cellular injury and demise. In a preliminary study 31using a protocol identical to the SBI group, we found that sevoflurane pretreatment reduced peroxynitrite fluorescence in the effluent by 77%. Because peroxynitrite is the product of two free radicals, superoxide and nitric oxide, the reduced release of one or both of these species after ischemia is likely associated with the reduced Ca2+overloading we observed. This suggests that sevoflurane pretreatment triggers a sequence of events to protect the heart by reducing free radical damage and Ca2+loading. It is noteworthy that not only was sevoflurane administered before ischemia more protective than when administered on initial reperfusion, but there was also no additive protective effect when sevoflurane was administered both before and after ischemia. Also, postischemic sevoflurane treatment did not seem to suppress metabolism or function compared with sevoflurane pretreatment. This suggests that sevoflurane initiates a maximal protective effect before ischemia that overrides a lesser protective effect elicited by sevoflurane on reperfusion. A major effector of both preischemic and postischemic sevoflurane was reduced Ca2+loading; combining these protocols also did not additively reduce Ca2+loading. As in anesthetic preconditioning, the more favorable outcome was garnered if the anesthetic was administered before ischemia.
Role of Cations in Ischemia and Reperfusion Injury
Postischemic reperfusion injury results in large part from mitochondrial 6–8and myoplasmic Ca2+overloading. 2–6,21,25If the increase in [Ca2+] is prolonged, a cascade of events is initiated, which ultimately results in lethal injury. 1,15We have recently detailed the time course of changes in contractility and relaxation associated with altered [Na+], myoplasmic and mitochondrial [Ca2+], and [NADH] during reperfusion injury in the intact heart. 6It was apparent that contractile performance becomes dissociated from myoplasmic Ca2+, particularly during early reperfusion, in that higher Ca2+concentrations are associated with reduced contractile force and impaired relaxation. Moreover, the increased [Na+] and mitochondrial [Ca2+] during and after ischemia probably underlies contractile dysfunction. Because Ca2+loading was temporally associated with Na+loading during early reperfusion 6,25and because [Na+] and [Ca2+] were reduced after blocking Na+–H+exchange, 25our studies strongly support the notion that Na+–H+exchange and reverse Na+–Ca+exchange are major cationic factors that underlie reperfusion injury.
Anesthetic-induced Cardiac Protection
It is not understood how volatile anesthetics protect the myocardium from reperfusion damage, but the overall mechanism is likely multifactorial. We reported previously that halothane and isoflurane improve function and metabolism on reperfusion and reduce dysrhythmia development when administered 10 min before and after hypoxia 12or graded or complete global ischemia 13,14in isolated guinea pig hearts. Many other studies have supported the cardiac protective effects of anesthetics. 10,11,16–21,23We showed that halothane protects against dysrhythmias and improves mechanical, metabolic, and vascular endothelial function when administered during low-flow perfusion for 1 day at 3°C. 23Isoflurane administered for 45 min before and during 15 min of coronary occlusion in dogs enhanced recovery of regional myocardial contractile function after 5 h of reperfusion. 32Because this effect was partially blocked by glibenclamide, a role for isoflurane to enhance KATPchannel activation during ischemia and reperfusion was suggested.
Preconditioning by isoflurane, 16,19,33enflurane, 16and halothane 16reduced infarct size in rabbit hearts; the protection was similar to that of ischemic preconditioning. In dogs, isoflurane administered alone or during four 5-min occlusions before 60 min of regional myocardial ischemia reduced infarct size. 20Because the protective effect was reversed by glibenclamide, this again supported a role for KATPchannel opening. Anesthetics may activate A1receptors or increase the sensitivity of adenosine A1receptors to attenuated adenosine release. 34In turn, anesthetic-induced increases in protein kinase C activity may underlie reduced infarct size 33and improved contractility 35after ischemia.
We reported that anesthetic preconditioning (APC) with sevoflurane is as effective as ischemic preconditioning (IPC) in improving not only mechanical function, but also cardiac rhythm, perfusion, metabolic function, and basal- and nitric oxide–mediated coronary flow. 18Moreover, the vascular and myocardial protective effects of APC by sevoflurane and IPC were antagonized by glibenclamide, suggesting a common final mechanism via activation of KATPchannels. In a recent study, we showed that equivalent improvements in metabolic and contractile functions after IPC and APC by sevoflurane were accompanied by equivalent reductions in Ca2+loading on reperfusion after global ischemia. 21If KATPchannel antagonism also reverses the reduced Ca2+loading effects of IPC and APC, this would suggest that cardioprotection is afforded, at least in part, by reduced Ca2+loading when KATPchannels are open. Interestingly, both APC and IPC reduce cytosolic Ca2+loading and protect hearts via KATPchannel opening. It is widely believed that IPC activates protein kinase C and tyrosine kinase pathways that in turn promote phosphorylation of sarcolemmal and mitochondrial KATPchannels. 36–38It seems clear that APC and IPC lead to protective effects via a common final mechanism that opens KATPchannels and reduces cytosolic Ca2+loading.
In contrast to APC or anesthetic pretreatment, anesthetic treatment on reperfusion was not quite as protective. It is possible that anesthetic pretreatment triggers a powerful protective effect, as good as IPC, 21and that anesthetic posttreatment is insufficient to reduce the metabolic rate with the onset of reperfusion. Indeed, sevoflurane administered immediately on reperfusion did not seem to reduce function or metabolism compared with the control group. However, because Ca2+loading during reperfusion was reduced by sevoflurane administered during reperfusion, the improvement in Ca 38homeostasis and function is likely linked through an anesthetic-induced effect.
Volatile anesthetics are often selected for patients with coronary artery disease who are at risk for ischemia and infarction during cardiac and noncardiac surgery. Temporary ischemia is often induced during cardiac surgery and angioplasty. 39A volatile anesthetic may be a safe and efficacious method to protect the heart during cardiac and noncardiac procedures in patients with coronary artery disease. 40In this study, we investigated possible differences in protective mechanisms when the anesthetic was administered before versus after ischemia and showed that similarly reduced Ca2+loading contributes to, or results from, both treatment protocols. Further research will seek to determine whether improved cardiac function and Ca2+homeostasis on reperfusion after anesthetic pretreatment results from factors mediated by intracellular signaling pathways that lead to early activation of KATPchannel opening during the treatment. Future studies will also be directed to determine whether improved function and Ca2+homeostasis after anesthetic posttreatment is a consequence of enhanced KATPchannel opening on reperfusion.
(1) Our studies in the guinea pig may not be easily compared with those of other species, especially the rat. Ischemia in rats causes a marked contracture beginning early during ischemia that is accompanied by a marked increase in diastolic [Ca2+] (Stowe et al. , laboratory observations, July 1999); on early reperfusion, diastolic LVP remains increased, and diastolic [Ca2+] does not increase further. (2) It is possible but unlikely that a portion of the 150-nm increase in diastolic [Ca2+] that we found in each group on initial reperfusion was derived from washout of indo-1–bound Ca2+rather than from cytosolic Ca2+. Subepicardial left ventricular tissue at the location of the fiberoptic probe was not infarcted but was likely stunned. Because it is phasic, systolic–diastolic [Ca2+] must represent cytosolic [Ca2+]. (3) It is not possible to separate mitochondrial compartment Ca2+from other compartments, e.g. , nuclear or Ca2+from other cells, i.e. , endothelial, vascular, or nerve. The flux of Ca2+through each of these compartments is likely very slow. These compartments likely contribute little to the total rapid phasic signal of the myoplasm. We estimated average noncytosolic Ca2+from MnCl2quenching at the end of each experiment, and Mn2+may leak into this compartment over time. We assumed that the residual fluorescence recorded after quenching cytosolic fluorescence arises predominantly from mitochondrial Ca2+, but this may be an overestimate. (4) Our study required a crystalloid perfusate devoid of cells with a high O2tension, so our findings may not exactly mimic those in blood-perfused hearts. (5) Our study was limited to one concentration of one anesthetic.
The authors thank Amadou Camara, Ph.D., Qun Chen, M.D., Ming-Tao Jiang, Ph.D., James Heisner, B.S., and Anita Tredeau (Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI), and Samhita Shahane Rhodes, M.S. (Department of Biomedical Engineering, Marquette University, Milwaukee, WI), for their valuable contributions to this study.