Enhanced effects of isoflurane on the long QT syndrome 1-associated A341V mutant.

BACKGROUND: The impact of volatile anesthetics on patients with inherited long QT syndrome (LQTS) is not well understood. This is further complicated by the different genotypes underlying LQTS. No studies have reported on the direct effects of volatile anesthetics on specific LQTS-associated mutations. We investigated the effects of isoflurane on a common LQTS type 1 mutation, A341V, with an unusually severe phenotype. METHODS: Whole cell potassium currents (IKs) were recorded from HEK293 and HL-1 cells transiently expressing/coexpressing wild-type KCNQ1 (α-subunit), mutant KCNQ1, wild-type KCNE1 (ß-subunit), and fusion KCNQ1 + KCNE1. Current was monitored in the absence and presence of clinically relevant concentration of isoflurane (0.54 ± 0.05 mM, 1.14 vol %). Computer simulations determined the resulting impact on the cardiac action potential. RESULTS: Isoflurane had significantly greater inhibitory effect on A341V + KCNE1 (62.2 ± 3.4%, n = 8) than on wild-type KCNQ1 + KCNE1 (40.7 ± 4.5%; n = 9) in transfected HEK293 cells. Under heterozygous conditions, isoflurane inhibited A341V + KCNQ1 + KCNE1 by 65.2 ± 3.0% (n = 13) and wild-type KCNQ1 + KCNE1 (2:1 ratio) by 32.0 ± 4.5% (n = 11). A341V exerted a dominant negative effect on IKs. Similar differential effects of isoflurane were also observed in experiments using the cardiac HL-1 cells. Mutations of the neighboring F340 residue significantly attenuated the effects of isoflurane, and fusion proteins revealed the modulatory effect of KCNE1. Action potential simulations revealed a stimulation frequency-dependent effect of A341V. CONCLUSIONS: The LQTS-associated A341V mutation rendered the IKs channel more sensitive to the inhibitory effects of isoflurane compared to wild-type IKs in transfected cell lines; F340 is a key residue for anesthetic action.

Partial restoration of the long QT syndrome associated KCNQ1 A341V mutant by the KCNE1 ß-subunit.

BACKGROUND: The A341V mutation in the pore-forming KCNQ1 subunit of the slowly activating delayed-rectifier potassium current (IKs) underlies a common form of the long QT syndrome, and is associated with an unusually severe phenotype. However, there is controversy regarding the underlying mechanism responsible for the clinically observed phenotype. We investigated the biophysical characteristics of A341V in a cardiac environment by utilizing a cardiac cell line, and in particular the impact of the KCNE1 ß-subunit. METHODS: Whole-cell current were recorded from transiently transfected HL-1 cells, a cardiac cell line. Mutant KCNQ1 and KCNE1 were constructed by site-directed mutagenesis. RESULTS: The A341V mutant resulted in a non-functional channel when expressed alone. When co-expressed with wild type KCNE1, A341V produced a slowly activating current, with a smaller current density, slower rates of activation, and a depolarized shift in its activation curve compared to the wild type KCNQ1+KCNE1. Confocal microscopy confirmed the surface expression of GFP-tagged A341V, suggesting a functionally defective protein. A T58A mutation in KCNE1 abolished functional restoration of A341V. Under heterozygous conditions, the expression of A341V+KCNQ1+KCNE1 reduced but did not abolish the electrophysiological changes observed in A341V+KCNE1. A dominant negative effect of A341V was also observed. Action potential simulations revealed that the A341V mutation is arrhythmogenic. CONCLUSIONS: The KCNE1 ß-subunit partially rescued the non-functional A341V mutant, with electrophysiological properties distinct from the wild type IKs. GENERAL SIGNIFICANCE: The severity of the A341V phenotype may be due to a combination of a significant suppression of the IKs with altered biophysical characteristics.

Role of heat shock protein 90 and endothelial nitric oxide synthase during early anesthetic and ischemic preconditioning.

BACKGROUND: Nitric oxide is known to be essential for early anesthetic preconditioning (APC) and ischemic preconditioning (IPC) of myocardium. Heat shock protein 90 (Hsp90) regulates endothelial nitric oxide synthase (eNOS) activity. In this study, the authors tested the hypothesis that Hsp90-eNOS interactions modulate APC and IPC. METHODS: Myocardial infarct size was measured in rabbits after coronary occlusion and reperfusion in the absence or presence of preconditioning within 30 min of isoflurane (APC) or 5 min of coronary artery occlusion (IPC), and with or without pretreatment with geldanamycin or radicicol, two chemically distinct Hsp90 inhibitors, or N-nitro-L-arginine methyl ester, a nonspecific nitric oxide synthase NOS inhibitor. Isoflurane-dependent nitric oxide production was measured (ozone chemiluminescence) in human coronary artery endothelial cells or mouse cardiomyocytes, in the absence or presence of Hsp90 inhibitors or N-nitro-L-arginine methyl ester. Interactions between Hsp90 and eNOS, and eNOS activation, were assessed with immunoprecipitation, immunoblotting, and confocal microscopy. RESULTS: APC and IPC decreased infarct size (by 50% and 59%, respectively), and this action was abolished by Hsp90 inhibitors. N-nitro-L-arginine methyl ester blocked APC but not IPC. Isoflurane increased nitric oxide production in human coronary artery endothelial cells concomitantly with an increase in Hsp90-eNOS interaction (immunoprecipitation, immunoblotting, and immunohistochemistry). Pretreatment with Hsp90 inhibitors abolished isoflurane-dependent nitric oxide production and decreased Hsp90-eNOS interactions. Isoflurane did not increase nitric oxide production in mouse cardiomyocytes, and eNOS was below the level of detection. CONCLUSION: The results indicate that Hsp90 plays a critical role in mediating APC and IPC through protein-protein interactions, and suggest that endothelial cells are important contributors to nitric oxide-mediated signaling during APC.

Age-related attenuation of isoflurane preconditioning in human atrial cardiomyocytes: roles for mitochondrial respiration and sarcolemmal adenosine triphosphate-sensitive potassium channel activity.

BACKGROUND: Clinical trials suggest that anesthetic-induced preconditioning (APC) produces cardioprotection in humans, but the mechanisms of APC and significance of aging for APC in humans are not well understood. Here, the impact of age on the role of two major effectors of APC, mitochondria and sarcolemmal adenosine triphosphate-sensitive potassium (sarcKATP) channels, in preconditioning of the human atrial myocardium were investigated. METHODS: Right atrial appendages were obtained from adult patients undergoing cardiac surgery and assigned to mid-aged (MA) and old-aged (OA) groups. APC was induced by isoflurane in isolated myocardium and isolated cardiomyocytes. Mitochondrial oxygen consumption measurements, myocyte survival testing, and patch clamp techniques were used to investigate mitochondrial respiratory function and sarcKATP channel activity. RESULTS: After in vitro APC with isoflurane, the respiratory function of isolated mitochondria was better preserved after hypoxia-reoxygenation stress in MA than in OA. In isolated intact myocytes, APC significantly decreased oxidative stress-induced cell death in MA but not in OA, and isoflurane protection from cell death was attenuated by the sarcKATP channel inhibitor HMR-1098. Further, the properties of single sarcKATP channels were similar in MA and OA, and isoflurane sensitivity of pinacidil-activated whole cell KATP current was no different between MA and OA myocytes. CONCLUSION: Anesthetic-induced preconditioning with isoflurane decreases stress-induced cell death and preserves mitochondrial respiratory function to a greater degree in MA than in OA myocytes; however, sarcKATP channel activity is not differentially affected by isoflurane. Therefore, effectiveness of APC in humans may decrease with advancing age partly because of altered mitochondrial function of myocardial cells.

The A3 adenosine receptor agonist CP-532,903 [N6-(2,5-dichlorobenzyl)-3'-aminoadenosine-5'-N-methylcarboxamide] protects against myocardial ischemia/reperfusion injury via the sarcolemmal ATP-sensitive potassium channel.

We examined the cardioprotective profile of the new A(3) adenosine receptor (AR) agonist CP-532,903 [N(6)-(2,5-dichlorobenzyl)-3'-aminoadenosine-5'-N-methylcarboxamide] in an in vivo mouse model of infarction and an isolated heart model of global ischemia/reperfusion injury. In radioligand binding and cAMP accumulation assays using human embryonic kidney 293 cells expressing recombinant mouse ARs, CP-532,903 was found to bind with high affinity to mouse A(3)ARs (K(i) = 9.0 +/- 2.5 nM) and with high selectivity versus mouse A(1)AR (100-fold) and A(2A)ARs (1000-fold). In in vivo ischemia/reperfusion experiments, pretreating mice with 30 or 100 microg/kg CP-532,903 reduced infarct size from 59.2 +/- 2.1% of the risk region in vehicle-treated mice to 42.5 +/- 2.3 and 39.0 +/- 2.9%, respectively. Likewise, treating isolated mouse hearts with CP-532,903 (10, 30, or 100 nM) concentration dependently improved recovery of contractile function after 20 min of global ischemia and 45 min of reperfusion, including developed pressure and maximal rate of contraction/relaxation. In both models of ischemia/reperfusion injury, CP-532,903 provided no benefit in studies using mice with genetic disruption of the A(3)AR gene, A(3) knockout (KO) mice. In isolated heart studies, protection provided by CP-532,903 and ischemic preconditioning induced by three brief ischemia/reperfusion cycles were lost in Kir6.2 KO mice lacking expression of the pore-forming subunit of the sarcolemmal ATP-sensitive potassium (K(ATP)) channel. Whole-cell patch-clamp recordings provided evidence that the A(3)AR is functionally coupled to the sarcolemmal K(ATP) channel in murine cardiomyocytes. We conclude that CP-532,903 is a highly selective agonist of the mouse A(3)AR that protects against ischemia/reperfusion injury by activating sarcolemmal K(ATP) channels.

Volatile anesthetic-induced cardiac preconditioning.

Pharmacological preconditioning with volatile anesthetics, or anesthetic-induced preconditioning (APC), is a phenomenon whereby a brief exposure to volatile anesthetic agents protects the heart from the potentially fatal consequences of a subsequent prolonged period of myocardial ischemia and reperfusion. Although not completely elucidated, the cellular and molecular mechanisms of APC appear to mimic those of ischemic preconditioning, the most powerful endogenous cardioprotective mechanism. This article reviews recently accumulated evidence underscoring the importance of mitochondria, reactive oxygen species, and K(ATP) channels in cardioprotective signaling by volatile anesthetics. Moreover, the article addresses current concepts and controversies regarding the specific roles of the mitochondrial and the sarcolemmal K(ATP) channels in APC.

Cardioprotection by volatile anesthetics: new applications for old drugs?

PURPOSE OF REVIEW: Pharmacological interventions may play a prominent role in reducing organ damage in response to physiologic stress. A growing body of evidence indicates that volatile anesthetics exert protective effects against ischemia-reperfusion injury in vivo. Administration of volatile anesthetics before prolonged coronary artery occlusion and reperfusion has been shown to produce cardioprotection, a phenomenon termed anesthetic-induced preconditioning. Endogenous signal transduction proteins, reactive oxygen species, mitochondria, and ion channels have been implicated in anesthetic-induced preconditioning, and new data regarding the triggering and effector roles for these various components have been discovered that advance our understanding of the mechanisms responsible for anesthetic-induced preconditioning. This review will update and integrate these recent data into the current mechanistic model of anesthetic-induced preconditioning. RECENT FINDINGS: Despite a wealth of data from animal studies, the mechanism by which preconditioning with volatile anesthetics alleviates ischemic injury remains incompletely understood. Recent data have identified important interactions between reactive oxygen species and key intracellular signal transduction enzymes and proteins implicated in anesthetic-induced preconditioning. SUMMARY: This review highlights the major recent findings examining mechanisms of volatile anesthetic cardioprotection.

Isoflurane inhibits cardiac myocyte apoptosis during oxidative and inflammatory stress by activating Akt and enhancing Bcl-2 expression.

BACKGROUND: Volatile anesthetics attenuate apoptosis. The underlying mechanisms remain undefined. The authors tested whether isoflurane reduces apoptosis in cardiomyocytes subjected to oxidative or inflammatory stress by enhancing Akt and B-cell lymphoma-2 (Bcl-2). METHODS: Adult and neonatal rat ventricular myocytes and atrial HL-1 myocytes were exposed to hypoxia, hydrogen peroxide, or neutrophils with or without isoflurane pretreatment. The authors assessed cell damage and investigated apoptosis using mitochondrial cytochrome c release, caspase activity, and TUNEL assay. They determined expression of phospho-Akt and Bcl-2 and tested their involvement by blocking phospho-Akt with wortmannin and Bcl-2 with HA14-1. RESULTS: Isoflurane significantly reduced the cell damage and apoptosis induced by hypoxia, H2O2, and neutrophils. Isoflurane reduced hypoxia-induced mitochondrial cytochrome c release in HL-1 cells by 45 +/- 12% and caspase activity by 28 +/- 4%; in neonatal cells, it reduced caspase activity by 43 +/- 5% and TUNEL-positive cells by 50 +/- 2%. Isoflurane attenuated H2O2-induced caspase activity in HL-1 cells by 48 +/- 16% and TUNEL-positive cells by 78 +/- 3%; in neonatal cells, it reduced caspase activity by 30 +/- 3% and TUNEL-positive cells by 32 +/- 7%. In adult cardiomyocytes exposed to neutrophils, isoflurane decreased both mitochondrial cytochrome c and caspase activity by 47 +/- 3% and TUNEL-positive cells by 25 +/- 4%. Isoflurane enhanced phospho-Akt and Bcl-2 expression. Wortmannin and HA14-1 prevented the action of isoflurane (53 +/- 8% and 54 +/- 7% apoptotic cells vs. 18 +/- 1% without blockers). CONCLUSIONS: Isoflurane protects cardiomyocytes against apoptosis induced by hypoxia, H2O2, or activated neutrophils through Akt activation and increased Bcl-2 expression. This suggests that a reduction in apoptosis contributes to the cardioprotective effects of isoflurane.

Role of endothelial nitric oxide synthase as a trigger and mediator of isoflurane-induced delayed preconditioning in rabbit myocardium.

BACKGROUND: Isoflurane produces delayed preconditioning in vivo. The authors tested the hypothesis that endothelial, inducible, or neuronal nitric oxide synthase (NOS) is a trigger or mediator of this protective effect. METHODS: In the absence or presence of exposure to isoflurane (1.0 minimum alveolar concentration) 24 h before experimentation, pentobarbital-anesthetized rabbits (n = 128) instrumented for hemodynamic measurement received 0.9% saline (control), the nonselective NOS inhibitor N-nitro-l-arginine methyl ester (10 mg/kg), one of two of the selective inducible NOS antagonists aminoguanidine (300 mg/kg) or 1400W (0.5 mg/kg), or the selective neuronal NOS inhibitor 7-nitroindazole (50 mg/kg) administered before exposure to isoflurane (trigger; day 1) or left anterior descending coronary artery occlusion (mediator; day 2). All rabbits underwent 30 min of coronary occlusion followed by 3 h of reperfusion. Tissue samples for reverse-transcription polymerase chain reaction and immunohistochemistry were also obtained in the presence or absence of N-nitro-l-arginine methyl ester with or without isoflurane pretreatment. RESULTS: Isoflurane significantly (P < 0.05) reduced infarct size (23 +/- 5% [mean +/- SD] of the left ventricular area at risk; triphenyltetrazolium chloride staining) as compared with control (42 +/- 7%). N-nitro-l-arginine methyl ester administered before isoflurane or coronary occlusion abolished protection (49 +/- 7 and 43 +/- 10%, respectively). Aminoguanidine, 1400W, and 7-nitroindazole did not alter infarct size or affect isoflurane-induced delayed preconditioning. Isoflurane increased endothelial but not inducible NOS messenger RNA transcription and protein translation immediately and 24 h after administration of the volatile agent. Pretreatment with N-nitro-l-arginine methyl ester attenuated isoflurane-induced increases in endothelial NOS expression. CONCLUSIONS: The results suggest that endothelial NOS but not inducible or neuronal NOS is a trigger and mediator of delayed preconditioning by isoflurane in vivo.

Cardioprotection by volatile anesthetics.

Preconditioning describes a very powerful endogenous mechanism by which the heart may be protected against ischemia and reperfusion injury. Transient administration of a volatile anesthetic before a prolonged ischemic episode reduces myocardial infarct size to a degree comparable to that observed during ischemic preconditioning. Many components of the signal transduction pathways responsible for cardioprotection are shared by anesthetic and ischemic preconditioning. Exposure to volatile anesthetics generates small "triggering" quantities of reactive oxygen species (ROS) by directly interacting with the mitochondrial electron transport chain or indirectly through a signaling cascade in which G-protein-coupled receptors, protein kinases, and mitochondrial ATP-sensitive potassium (K(ATP)) channels play important roles. Several clinical studies also suggest that preconditioning by volatile anesthetics exerts beneficial effects in patients undergoing cardiac surgery. This review summarizes some of the recent major developments in the understanding of cardioprotection by volatile anesthetics.

Isoflurane protects against myocardial infarction during early reperfusion by activation of phosphatidylinositol-3-kinase signal transduction: evidence for anesthetic-induced postconditioning in rabbits.

BACKGROUND: Brief episodes of ischemia during early reperfusion after coronary occlusion reduce the extent of myocardial infarction. Phosphatidylinositol-3-kinase (PI3K) signaling has been implicated in this "postconditioning" phenomenon. The authors tested the hypothesis that isoflurane produces cardioprotection during early reperfusion after myocardial ischemia by a PI3K-dependent mechanism. METHODS: Pentobarbital-anesthetized rabbits (n = 80) subjected to a 30-min coronary occlusion followed by 3 h reperfusion were assigned to receive saline (control), three cycles of postconditioning ischemia (10 or 20 s each), isoflurane (0.5 or 1.0 minimum alveolar concentration), or the PI3K inhibitor wortmannin (0.6 mg/kg, intravenously) or its vehicle dimethyl sulfoxide. Additional groups of rabbits were exposed to combined postconditioning ischemia (10 s) and 0.5 minimum alveolar concentration isoflurane in the presence and absence of wortmannin. Phosphorylation of Akt, a downstream target of PI3K, was assessed by Western blotting. RESULTS: Postconditioning ischemia for 20 s, but not 10 s, reduced infarct size (P < 0.05) (triphenyltetrazolium staining; 20 +/- 3% and 34 +/- 3% of the left ventricular area at risk, respectively) as compared with control (41 +/- 2%). Exposure to 1.0, but not 0.5, minimum alveolar concentration isoflurane decreased infarct size (21 +/- 2% and 43 +/- 3%, respectively). Wortmannin abolished the protective effects of postconditioning (20 s) and 1.0 minimum alveolar concentration isoflurane. Combined postconditioning (10 s) and 0.5 minimum alveolar concentration isoflurane markedly reduced infarct size (17 +/- 5%). This action was also abolished by wortmannin (44 +/- 2%). Isoflurane (1.0 minimum alveolar concentration) increased Akt phosphorylation after ischemia (32 +/- 6%), and this action was blocked by wortmannin. CONCLUSIONS: Isoflurane acts during early reperfusion after prolonged ischemia to salvage myocardium from infarction and reduces the threshold of ischemic postconditioning by activating PI3K.