Enis Novalija

Reactive oxygen species as mediators of cardiac injury and protection: the relevance to anesthesia practice.

Reactive oxygen species (ROS) are central to cardiac ischemic and reperfusion injury. They contribute to myocardial stunning, infarction and apoptosis, and possibly to the genesis of arrhythmias. Multiple laboratory studies and clinical trials have evaluated the use of scavengers of ROS to protect the heart from the effects of ischemia and reperfusion. Generally, studies in animal models have shown such effects. Clinical trials have also shown protective effects of scavengers, but whether this protection confers meaningful clinical benefits is uncertain. Several IV anesthetic drugs act as ROS scavengers. In contrast, volatile anesthetics have recently been demonstrated to generate ROS in the heart, most likely because of inhibitory effects on cardiac mitochondria. ROS are involved in the signaling cascade for cardioprotection induced by brief exposure to a volatile anesthetic (termed “anesthetic preconditioning”). ROS, therefore, although injurious in large quantities, can have a paradoxical protective effect within the heart. In this review we provide background information on ROS formation and elimination relevant to anesthetic and adjuvant drugs with particular reference to the heart. The sources of ROS, the means by which they induce cardiac injury or activate protective signaling pathways, the results of clinical studies evaluating ROS scavengers, and the effects of anesthetic drugs on ROS are each discussed.

sevoflurane Mimics Ischemic Preconditioning Effects on Coronary Flow and Nitric Oxide Release in Isolated Hearts

BACKGROUND Like ischemic preconditioning, certain volatile anesthetics have been shown to reduce the magnitude of ischemia/ reperfusion injury via activation of K+ adenosine triphosphate (ATP)-sensitive (K(ATP)) channels. The purpose of this study was (1) to determine if ischemic preconditioning (IPC) and sevoflurane preconditioning (SPC) increase nitric oxide release and improve coronary vascular function, as well as mechanical and electrical function, if given for only brief intervals before global ischemia of isolated hearts; and (2) to determine if K(ATP) channel antagonism by glibenclamide (GLB) blunts the cardioprotective effects of IPC and SPC. METHODS Guinea pig hearts were isolated and perfused with Krebs-Ringers solution at 55 mm Hg and randomly assigned to one of seven groups: (1) two 2-min total coronary occlusions (preconditioning, IPC) interspersed with 5 min of normal perfusion; (2) two 2-min occlusions interspersed with 5 min of perfusion while perfusing with GLB (IPC+GLB); (3) SPC (3.5%) for two 2-min periods; (4) SPC+GLB for two 2-min periods; (5) no treatment before ischemia (control [CON]); (6) CON+GLB; and (7) no ischemia (time control). Six minutes after ending IPC or SPC, hearts of ischemic groups were subjected to 30 min of global ischemia and 75 min of reperfusion. Left-ventricular pressure, coronary flow, and effluent NO concentration ([NO]) were measured. Flow and NO responses to bradykinin, and nitroprusside were tested 20-30 min before ischemia or drug treatment and 30-40 min after reperfusion. RESULTS After ischemia, compared with before (percentage change), left-ventricular pressure and coronary flow, respectively, recovered to a greater extent (P<0.05) after IPC (42%, 77%), and treatment with SPC (45%, 76%) than after CON (30%, 65%), IPC+GLB (24%, 64%), SPC+GLB (20%, 65%), and CON+GLB (28%, 64%). Bradykinin and nitroprusside increased [NO] by 30+/-5 (means +/- SEM) and 29+/-4 nM, respectively, averaged for all groups before ischemia. [NO] increased by 26+/-6 and 27+/-7 nM, respectively, in SPC and IPC groups after ischemia, compared with an average [NO] increase of 8+/-5 nM (P<0.01) after ischemia in CON and each of the three GLB groups. Flow increases to bradykinin and nitroprusside were also greater after SPC and IPC. CONCLUSIONS Preconditioning with sevoflurane, like IPC, improves not only postischemic contractility, but also basal flow, bradykinin and nitroprusside-induced increases in flow, and effluent [NO] in isolated hearts. The protective effects of both SPC and IPC are reversed by K(ATP) channel antagonism.

Sevoflurane exposure generates superoxide but leads to decreased superoxide during ischemia and reperfusion in isolated hearts.

Reactive oxygen species (ROS) are largely responsible for cardiac injury consequent to ischemia and reperfusion, but, paradoxically, there is evidence suggesting that anesthetics induce preconditioning (APC) by generating ROS. We hypothesized that sevoflurane generates the ROS superoxide (O2·−), that APC attenuates O2·− formation during ischemia, and that this attenuation is reversed by bracketing APC with the O2·− scavenger manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) or the putative mitochondrial adenosine triphosphate-sensitive potassium (mKATP) channel blocker 5-hydroxydecanoate (5-HD). O2·− was measured continuously in guinea pig hearts by using dihydroethidium. Sevoflurane was administered alone (APC), with MnTBAP, or with 5-HD before 30 min of ischemia and 120 min of reperfusion. Control hearts underwent no pretreatment. Sevoflurane directly increased O2·−; this was blocked by MnTBAP but not by 5-HD. O2·− increased during ischemia and during reperfusion. These increases in O2·− were attenuated in the APC group, but this was prevented by MnTBAP or 5-HD. We conclude that sevoflurane directly induces O2·− formation but that O2·− formation is decreased during subsequent ischemia and reperfusion. The former effect appears independent of mKATP channels, but not the latter. Our study indicates that APC is initiated by ROS that in turn cause mKATP channel opening. Although there appears to be a paradoxical role for ROS in triggering and mediating APC, a possible mechanism is offered.

Reactive Oxygen Species Precede the ε Isoform of Protein Kinase C in the Anesthetic Preconditioning Signaling Cascade

Background Protein kinase C (PKC) and reactive oxygen species (ROS) are known to have a role in anesthetic preconditioning (APC). Cardiac preconditioning by triggers other than volatile anesthetics, such as opioids or brief ischemia, is known to be isoform selective, but the isoform required for APC is not known. The authors aimed to identify the PKC isoform that is involved in APC and to elucidate the relative positions of PKC activation and ROS formation in the APC signaling cascade. Methods Isolated guinea pig hearts were subjected to 30 min of ischemia and 120 min of reperfusion. Before ischemia, hearts were either untreated or treated with sevoflurane (APC) in the absence or presence of the nonspecific PKC inhibitor chelerythrine, the PKC-&dgr; inhibitor PP101, or the PKC-&egr; inhibitor PP149. Spectrofluorometry and the fluorescent probes dihydroethidium were used to measure intracellular ROS, and effluent dityrosine as used to measure extracellular ROS release. Results Previous sevoflurane exposure protected the heart against ischemia–reperfusion injury, as previously described. Chelerythrine or PP149 abolished protection, but PP101 did not. ROS formation was observed during sevoflurane exposure and was not altered by any of the PKC inhibitors. Conclusions APC is mediated by PKC-&egr; but not by PKC-&dgr;. Furthermore, PKC activation probably occurs downstream of ROS generation in the APC signaling cascade.

Anesthetic preconditioning improves adenosine triphosphate synthesis and reduces reactive oxygen species formation in mitochondria after ischemia by a redox dependent mechanism.

Background Mitochondrial changes that characterize the heart after anesthetic preconditioning (APC) or the mechanisms by which mitochondrial triggering factors lead to protection are unknown. This study hypothesized that generation of reactive oxygen species (ROS) during APC is required to initiate the mitochondrial protective effects, and that APC leads to improved mitochondrial electron transport chain function and cardiac function during reperfusion. Methods Isolated guinea pig hearts were subject to 30 min ischemia and 120 min reperfusion. Prior to ischemia hearts were either untreated (I/R), or treated with sevoflurane (APC), in the presence or absence of the ROS scavenger tiron (TIR), or the superoxide dismutase mimetic MnTBAP (TBAP). Intracellular ROS were measured by spectrofluorometry using the fluorescent probe dihydroethidium (DHE). In another series of experiments, using the same protocol, hearts were reperfused for only 5 min and removed for measurement of adenosine triphosphate (ATP) synthesis by luciferin–luciferase luminometry and ROS generation by dichlorohydro-fluorescein (DCF) fluorescence in isolated mitochondria. Results The APC improved cardiac function and reduced infarction. Tiron or MnTBAP abrogated the protection afforded by APC. Mitochondrial ATP synthesis was decreased by 70 ± 3% after IR alone, by only 7 ± 3% after APC, by 69 ± 2% after APC+TIR, and by 71 ± 3% after APC + TBAP. Mitochondrial ROS formation (DCF) increased by 48 ± 3% after IR alone, by 0 ± 2% after APC, by 43 ± 4% after APC + TIR, and by 46 ± 3% after APC + TBAP. ROS generation (DHE) was increased in I/R group at 5 and 120 min reperfusion. This was attenuated by APC but this protective effect was abrogated in APC + TIR and APC + TBAP groups. Conclusions The results indicate that ROS are central both in triggering and mediating APC, and that the mitochondrion is the target for these changes.

Sevoflurane before or after Ischemia Improves Contractile and Metabolic Function while Reducing Myoplasmic Ca2+Loading in Intact Hearts

Background Ca2+ 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 [Ca2+] and improves function more so than when sevoflurane is administered on reperfusion. Methods Four groups of guinea pig hearts were perfused with crystalloid solution (55 mmHg, 37°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 [Ca2+] were measured in the left ventricular free wall with the fluorescence probe indo-1. Results Ischemia increased diastolic [Ca2+] and diastolic left ventricular pressure (LVP). In CON hearts, initial reperfusion greatly increased diastolic [Ca2+] and systolic [Ca2+] and reduced contractility (systolic–diastolic LVP, dLVP/dtmax), relaxation (diastolic LVP, dLVP/dtmin), myocardial oxygen consumption (Mvo2), and cardiac efficiency. SBI, SAI, and SBAI each reduced ventricular fibrillation, attenuated increases in systolic and systolic–diastolic [Ca2+], improved contractile and relaxation indices, and increased coronary flow, percent oxygen extraction, Mvo2, and cardiac efficiency during 60 min reperfusion compared with CON. SBI was more protective than SAI, and SBAI was generally more protective than SAI. Conclusions Sevoflurane improves postischemic cardiac function while reducing Ca2+ loading when it is administered before or after ischemia, but protection is better when it is administered before ischemia. Reduced Ca2+ loading on reperfusion is likely a result of the anesthetic protective effect.

Anesthetic preconditioning attenuates mitochondrial Ca2+ overload during ischemia in Guinea pig intact hearts: reversal by 5-hydroxydecanoic acid.

Cardiac ischemia/reperfusion (IR) injury is associated with mitochondrial (m)Ca2+ overload. Anesthetic preconditioning (APC) attenuates IR injury. We hypothesized that mCa2+ overload is decreased by APC in association with mitochondrial adenosine triphosphate-sensitive K+ (mKATP) channel opening. By use of indo-1 fluorescence, m[Ca2+] was measured in 40 guinea pig Langendorff-prepared hearts. Control (CON) hearts received no treatment for 50 min before IR; APC hearts were exposed to 1.2 mM (8.8 vol%) sevoflurane for 15 min; APC + 5-hydroxydecanoate (5-HD) hearts received 200 &mgr;M 5-HD from 5 min before to 15 min after sevoflurane exposure; and 5-HD hearts received 5-HD for 35 min. Sevoflurane was washed out for 30 min and 5-HD for 15 min before 30 min of global ischemia and 120 min of reperfusion. During ischemia, the peak m[Ca2+] accumulation was decreased by APC from 489 ± 37 nM (CON) to 355 ± 28 nM (P < 0.05); this was abolished by 5-HD (475 ± 38 nM m[Ca2+]). APC resulted in improved function and reduced infarct size on reperfusion, which also was blocked by 5-HD. 5-HD pretreatment alone did not affect m[Ca2+] (470 ± 34 nM) or IR injury. Thus, preservation of function and morphology on reperfusion is associated with attenuated mCa2+ accumulation during ischemia. Reversal by 5-HD suggests that APC may be triggered by opening mKATP channels.

Preconditioning with sevoflurane reduces changes in nicotinamide adenine dinucleotide during ischemia-reperfusion in isolated hearts: reversal by 5-hydroxydecanoic acid.

Background Ischemia causes an imbalance in mitochondrial metabolism and accumulation of nicotinamide adenine dinucleotide (NADH). We showed that anesthetic preconditioning (APC), like ischemic preconditioning, improved mitochondrial NADH energy balance during ischemia and improved function and reduced infarct size on reperfusion. Opening adenosine triphosphate–sensitive potassium (KATP) channels may be involved in triggering APC. The authors tested if effects of APC on NADH concentrations before, during, and after ischemia are reversible by 5-hydroxydecanoate (5-HD), a putative mitochondrial KATP channel blocker. Methods Nicotinamide adenine dinucleotide fluorescence was measured in 60 guinea pig Langendorff-prepared hearts assigned into five groups: (1) no treatment before ischemia; (2) APC by exposure to 1.3 mm sevoflurane for 15 min; (3) 200 &mgr;m 5-HD from 5 min before to 15 min after sevoflurane exposure; (4) 35 min 5-HD alone; and (5) no treatment and no ischemia. Sevoflurane was washed out for 30 min, and 5-HD for 15 min, before 30-min ischemia and 120-min reperfusion. Results Nicotinamide adenine dinucleotide was reversibly increased during sevoflurane exposure before ischemia, and the increase and rate of decline in NADH during ischemia were reduced after APC. 5-HD abolished these changes in NADH. On reperfusion, function was improved and infarct size reduced after APC compared with other groups. Conclusion Anesthetic preconditioning was evidenced by improved mitochondrial bioenergetics as assessed from NADH concentrations during ischemia and by attenuated reperfusion injury. Reversal of APC by bracketing sevoflurane exposure with 5-HD suggests that APC is triggered by mitochondrial KATP channel opening or, alternatively, by attenuated mitochondrial respiration without direct involvement of mitochondrial KATP channel opening.

Anesthetic preconditioning: Effects on latency to ischemic injury in isolated hearts

Background Anesthetic preconditioning (APC) is protective for several aspects of cardiac function and structure, including left ventricular pressure, coronary flow, and infarction. APC may be protective, however, only if the duration of ischemia is within a certain, as yet undefined range. Brief ischemia causes minimal injury, and APC would be expected to provide little benefit. Conversely, very prolonged ischemia would ultimately cause serious injury with or without APC. Previous investigations used a constant ischemic time as the independent variable to assess ischemia-induced changes in dependent functional and structural variables. The purpose of the study was to define the critical limits of efficacy of APC by varying ischemic time. Methods Guinea pig hearts (Langendorff preparation; n = 96) underwent pretreatment with sevoflurane (APC) or no treatment (control), before global ischemia and 120 min reperfusion. Ischemia durations were 20, 25, 30, 35, 40, and 45 min. Results At 120 min reperfusion, developed (systolic–diastolic) left ventricular pressure was increased by APC compared with control for ischemia durations of 25–40 min. Infarction was decreased by APC for ischemia durations of 25–40 min, but not 20 or 45 min. APC improved coronary flow and vasodilator responses for all ischemia durations longer than 25 min, and decreased ventricular fibrillation on reperfusion for ischemia durations longer than 30 min. Conclusions Although APC protects against vascular dysfunction and dysrhythmias after prolonged ischemia, protection against contractile dysfunction and infarction in this model is restricted to a range of ischemia durations of 25–40 min. These results suggest that APC may be effective in a subset of patients who have cardiac ischemia of intermediate duration.

Sevoflurane preconditioning before moderate hypothermic ischemia protects against cytosolic [Ca2+] loading and myocardial damage in part via mitochondrial KATP channels

Background Brief sevoflurane exposure and washout (sevoflurane preconditioning [SPC]) before 30-min global ischemia at 37°C is known to improve cardiac function, decrease cytosolic [Ca2+] loading, and reduce infarct size on reperfusion. It is not known if anesthetic preconditioning (APC) applies as well to hypothermic ischemia and reperfusion and if KATP channels are involved. The authors examined in guinea pig isolated hearts the effect of sevoflurane exposure before 4-h global ischemia at 17°C on cardiac function, cytosolic [Ca2+] loading, and infarct size. In addition they tested the potential role of the mitochondrial KATP channel in eliciting the cardioprotection by SPC. Methods Hearts were randomly assigned to (1) a nontreated hypothermic ischemia group (CON), (2) a group given 3.5 vol% sevoflurane for 15 min with a 15-min washout before hypothermic ischemia (SPC), and (3) an SPC group in which anesthetic exposure was bracketed with 200 &mgr;m 5-hydroxydecanoate (5-HD) from 5 min before until 5 min after sevoflurane (SPC + 5-HD). Cytosolic [Ca2+] was measured in the left ventricular (LV) free wall with the intracellularly loaded fluorescence probe indo-1. Results Initial reperfusion in CON hearts markedly increased systolic and diastolic [Ca2+] and reduced contractility (dLVP/dtmax), relaxation (diastolic LVP, dLVP/dtmin), myocardial oxygen consumption (Mvo2), and cardiac efficiency. In SPC hearts, cytosolic [Ca2+] overloading (especially diastolic [Ca2+]) was decreased with increased myocardial [Ca2+] influx (d[Ca2+]/dtmax) and efflux (d[Ca2+]/dtmin), improved contractility, relaxation, coronary flow, Mvo2, cardiac efficiency, and decreased infarct size. In SPC + 5HD hearts, the reduction in infarct size was antagonized by 5-HD, but functional return was less affected by 5-HD. Conclusions Anesthetic preconditioning occurs after long-term hypothermic ischemia, and the infarct size reduction is the result, in part, of mitochondrial KATP channel opening.