Abstract

Background

Anesthetic preconditioning (APC) is a clinically important phenomenon in which volatile anesthetics (VAs) protect tissues such as heart against ischemic injury. The mechanism of APC is thought to involve K+ channels encoded by the Slo gene family, and the authors showed previously that slo-2 is required for APC in Caenorhabditis elegans. Thus, the authors hypothesized that a slo-2 ortholog may mediate APC-induced cardioprotection in mammals.

Methods

A perfused heart model of ischemia–reperfusion injury, a fluorescent assay for K+ flux, and mice lacking Slo2.1 (Slick), Slo2.2 (Slack), or both (double knockouts, Slo2.x dKO) were used to test whether these channels are required for APC-induced cardioprotection and for cardiomyocyte or mitochondrial K+ transport.

Results

In wild-type (WT) hearts, APC improved post-ischemia–reperfusion functional recovery (APC = 39.5 ± 3.7% of preischemic rate × pressure product vs. 20.3 ± 2.3% in controls, means ± SEM, P = 0.00051, unpaired two-tailed t test, n = 8) and lowered infarct size (APC = 29.0 ± 4.8% of LV area vs. 51.4 ± 4.5% in controls, P = 0.0043, n = 8). Protection by APC was absent in hearts from Slo2.1−/− mice (% recovery APC = 14.6 ± 2.6% vs. 16.5 ± 2.1% in controls, P = 0.569, n = 8 to 9, infarct APC = 52.2 ± 5.4% vs. 53.5 ± 4.7% in controls, P = 0.865, n = 8 to 9). APC protection was also absent in Slo2.x dKO hearts (% recovery APC = 11.0 ± 1.7% vs. 11.9 ± 2.2% in controls, P = 0.725, n = 8, infarct APC = 51.6 ± 4.4% vs. 50.5 ± 3.9% in controls, P = 0.855, n = 8). Meanwhile, Slo2.2−/− hearts responded similar to WT (% recovery APC = 41.9 ± 4.0% vs. 18.0 ± 2.5% in controls, P = 0.00016, n = 8, infarct APC = 25.2 ± 1.3% vs. 50.8 ± 3.3% in controls, P < 0.000005, n = 8). Furthermore, VA-stimulated K+ transport seen in cardiomyocytes or mitochondria from WT or Slo2.2−/− mice was absent in Slo2.1−/− or Slo2.x dKO.

Conclusion

Slick (Slo2.1) is required for both VA-stimulated K+ flux and for the APC-induced cardioprotection.

Abstract

The authors have used novel gene-deleted mice to demonstrate that K+ flux via the KNa Slick channel encoded by the Slo2.1 gene is required for anesthetic preconditioning in mice. The identification of the role for Slick in anesthetic preconditioning will drive further development of novel cardiac-protective strategies and drugs for the clinical setting.

Supplemental Digital Content is available in the text.

What We Already Know about This Topic
  • Volatile anesthetics can protect the heart from ischemia– reperfusion injury, a phenomenon known as anesthetic preconditioning (APC)

  • The clinical efficacy of APC remains to be determined, and further understanding of the mechanism for APC is critical for the development of drugs and strategies for cardiac protection in the clinic

  • Ion channels in the cardiac myocyte and mitochondria play important roles in the mechanism of APC though the full identity of the channels remains unknown

What This Article Tells Us That Is New
  • The authors have used novel gene-deleted mice to demonstrate that K+ flux via the KNa Slick channel encoded by the Slo2.1 gene is required for anesthetic preconditioning in mice

  • The identification of the role for Slick in anesthetic preconditioning will drive further development of novel cardiac-protective strategies and drugs for the clinical setting

CLINICALLY relevant doses of halogenated volatile anesthetics (VAs) can protect tissues such as the heart from ischemia–reperfusion (IR) injury,1–3  a phenomenon known as “anesthetic preconditioning” (APC). APC is evolutionarily conserved from Caenorhabditis elegans4  to humans,3  and current American Heart Association/American College of Cardiology (AHA/ACA) guidelines specifically recommend the use of VAs during cardiac surgery for their protective effects.5 

The mechanism of APC is complex and involves many of the same effector signaling pathways as ischemic preconditioning (IPC), including protein kinases C6  and A,7,8  inhibitory G-proteins,9  adenosine,10,11  nitric oxide,12  and mitochondrial reactive oxygen species (ROS) generation.13,14  These signals are thought to converge at the level of mitochondria through distinct K+ channels, but the molecular identity of these channels is a subject of debate.

Several K+ channel types have been proposed to exist in mitochondria, including adenosine triphosphate (ATP)-sensitive (KATP),15  voltage-sensitive (KV),16  and large-conductance (BK) channels of the Slo gene family17  as well as a K+/H+ exchanger18  (for review, see the studies reported by Szabo and Zoratti19  and Szewczyk et al.20 ). Current evidence favors an involvement of mitochondrial KATP channels in IPC,21  whereas a mitochondrial Slo channel is thought to underlie APC.7,22,23 

The mammalian Slo channel family comprises Slo1 (KCNMA1; BK), Slo2.1 (KCNT2, Slick), Slo2.2 (KCNT1, Slack), and Slo3 (KCNU1).24 Slo3 expression is germline restricted,24,25  but the others are widely expressed. Because pharmacologic Slo1 channel activators can protect the heart against IR injury,17,26  it was widely assumed that a mitochondrial Slo1 channel (also termed BK, KCa, or BKCa) was responsible for APC. However, we recently showed that both C. elegans and mice lacking Slo1 channels can still be protected by APC, and mitochondria from these organisms contain a K+ channel activated by VA.22 

We have also previously demonstrated that the KCa channel SLO-2 is required for VA-induced protection of C. elegans against IR injury, with slo-2-mutant nematodes also lacking VA- stimulated mitochondrial K+ flux.22  Thus, we hypothesized that one of the mammalian SLO-2 orthologs, Slick or Slack (which are KNa, not KCa channels24,27 ), may underlie APC in mammals. Herein, we used novel gene-deleted mice (Slo2.1−/−, Slo2.2−/−, and Slo2.x double knockout [dKO]) to conclusively demonstrate that Slo2.1 codes the channel required for APC and for VA-stimulated K+ flux in cardiomyocytes and mitochondria.

Materials and Methods

Animals

Mice were housed in an Association for Assessment & Accreditation of Laboratory Animal Care-accredited pathogen-free facility with food and water available ad libitum. All procedures were approved by the University of Rochester’s Committee on Animal Resources (protocol no. 2010–030) in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (2011 revision). A Slo2.x dKO strain containing both mutant Slo2.x alleles28  was provided by Dr. Chris Lingle, Ph.D. (Washington University, St. Louis, Missouri). Mice were outcrossed more than six times to a C57BL/6 background, and conventional breeding was used such that experimental wild-type (WT) and knockout mice (males, 8 to 10 weeks old) were littermates. All animals were genotyped by tail-clip polymerase chain reaction (Kapa Biosystems, USA)29  with the following primers: Slo2.1 (Slick), C523-24C 5′-AACTTTATGAGTTCCTCTTCCATG-3′, C320-24F 5′-GAGCATCATACTTTGCTTTTTGGG-3′, KO = 269 base pairs (bp), WT = 579 bp; Slo2.2 (Slack) C311-30 5′-CCCATTCCACACTGCAGCCCTGTCTCTTTC-3′, C315-30 5′-TGTTTACTAGGGTCCAGGGAGAACCT ATGA-3′, KO = 200 bp, WT = 607 bp. Due to personnel limitations (i.e., same person handling mice and doing experiments), it was not feasible to blind the experimenter to animal genotype. However, for all experiments, mice of varying genotypes were randomly assigned to experimental groups, with treatments in randomized order across experimental days.

Ex Vivo Perfused Heart

Mouse hearts were perfused as previously described.22,26,29  In brief, after tribromoethanol anesthesia (100 mg/kg ip), the aorta was rapidly cannulated and perfused without pacing at a constant flow of 4 ml/min per 100 mg with Krebs–Henseleit buffer (118 mM NaCl, 4.7 mM KCl, 25 mM NaHCO3, 10 mM d-glucose, 1.2 mM MgSO4, 1.2 mM KH2PO4, and 2.5 mM CaCl2, gassed with 95% O2 and 5% CO2 at 37°C). Left ventricular (LV) pressure was measured via a water-filled transducer-linked low density polyethylene balloon. LV and coronary root pressures were monitored and digitally recorded at 1 kHz (DATAQ, USA). Parameters calculated from the LV balloon throughout perfusion included heart rate, systolic and end-diastolic pressures, LV-developed pressure (systolic − diastolic), rate × pressure product, dP/dTMAX (contraction), and dP/dTMIN (relaxation).

Hearts were equilibrated for 20 min before initiating data acquisition. A total of 163 hearts were analyzed, and none were excluded from the study. A 30-min normoxic perfusion was followed by 30 min of no flow global ischemia and then 60 min of reperfusion (fig. 1). Treatment groups were as follows: (1) control IR injury; (2) IPC comprising three cycles of 5-min ischemia plus 5-min reperfusion to replace the 30-min perfusion; (3) 10-min isoflurane (Henry Schein Animal Health, USA) infusion (100 µM or approximately 0.35 minimum alveolar concentration [MAC] for a C57BL/6 mouse30,31 ); (4) 20-min 7-chloro-3-methyl-4H-1,2,4-benzothiadiazine 1,1-dioxide (diazoxide, a K+ channel activator32 ; Sigma, USA) infusion (30 µM); and (5) 10-min 2,2’-sulfanediylbis(4,6-dichlorophenol) (bithionol a Slo channel activator33 ; TCI America, USA) infusion (2.5 nM). Pharmacologic agents were delivered via syringe pump just above the perfusion cannula for the indicated times, followed by 30-s washout before index ischemia. At the end of IR protocols, hearts were sliced, stained in 1% (wt/vol) 2,3,5-triphenyltetrazolium chloride for 20 min and fixed in 10% neutral buffered formalin for 24 h. Heart slices were imaged and infarct size was analyzed by planimetry as previously described.22,26,29 

Fig. 1.

Experimental protocol. Schematic shows the five perfused heart experimental conditions tested. Gray shading represents ischemia; 30-s washout was performed after administration of pharmacologic agents before ischemia. APC = anesthetic preconditioning; Bt = bithionol; dKO = double knockouts; DZX = diazoxide; IPC = ischemic preconditioning; WT = wild type.

Fig. 1.

Experimental protocol. Schematic shows the five perfused heart experimental conditions tested. Gray shading represents ischemia; 30-s washout was performed after administration of pharmacologic agents before ischemia. APC = anesthetic preconditioning; Bt = bithionol; dKO = double knockouts; DZX = diazoxide; IPC = ischemic preconditioning; WT = wild type.

Plasma Membrane Thallium (Tl+) Flux Assay on Isolated Mouse Cardiomyocytes and Human Embryonic Kidney (HEK)293 Cells

Mouse adult ventricular cardiomyocytes were isolated by collagenase perfusion as previously described.26  In brief, after anesthesia (tribromoethanol, see Ex Vivo Perfused Heart, above), hearts were cannulated and perfused with isolation buffer (IB) at 37°C (IB: 120 mM NaCl, 15 mM KCl, 0.6 mM Na2HPO4, 0.6 mM KH2PO4, 1.2 mM MgSO4, 10 mM HEPES, 4.6 mM NaHCO3, 30 mM taurine, 5.5 mM d-glucose, and 10 mM 2,3-butanedione monoxime, pH = 7.4) for less than 2 min before switching to digestion buffer (35 ml IB plus CaCl2 12.5 μM final, trypsin 400 μl at 2.5% [w/v], collagenase A 6.525 units, and collagenase D 15.375 units), and 8 min later placed in 2 to 3 ml of stop buffer (IB plus CaCl2 12.5 μM, 10% heat-inactivated Fetal Bovine Serum). Cardiomyocytes were dissociated and filtered through 75 μm mesh, and then, gravity sedimentation and resuspension were used to bring [Ca2+] stepwise to 1.8 mM, and the final pellet was placed into 1 ml of Minimal Essential Media (GIBCO, USA). Cell viability and yield were determined using Trypan blue and a hemocytometer, and preparations with greater than 85% viable rod-shaped cells were used experimentally. Cells were seeded (2500 cells per well) in a Falcon 96-well plate and incubated at 37°C for 1 h and then loaded with Thallos reagent (2 µM; Teflabs, USA) in 65-μl Hanks buffered salt solution (GIBCO) and monitored for thallium (Tl+) uptake by addition of 4 mM final Tl2SO4 in stimulus buffer (SB: 276 mM Na+ gluconate, 2.6 mM CaSO4, 1.6 mM MgSO4, 11.2 mM d-glucose, and 40 mM HEPES, pH = 7.3) in a BioTek Synergy plate reader in kinetic mode measuring every 7 s. (λex 488 nm; λem 525 nm) in the presence or absence of channel modulators. N-benzyl-N-(3-isobutoxy-2-pyrrolidin-1-yl-propyl)aniline (Bepridil; Sigma) is a calcium channel blocker that also inhibits Slo2 channels.33  Eight measurements were taken before additions. Paired control wells contained Tl+-free SB, enabling determination of Tl+-specific fluorescence changes.

HEK293 cells were seeded on glass 12-mm coverslips and transfected using Lipofectamine 3000 (Invitrogen, USA) with the expression vector pKT122 expressing an mCherry:rat Slick complementary DNA fusion in a pcDNA5/TO vector backbone (Invitrogen). After 24 h, cells were incubated in 1 ml of Hanks buffered saline solution containing 2 µM Thallos reagent (TefLabs) for 30 min and then transferred to an open-flow perfusion rig attached to a Nikon TE2000 microscope (Nikon, USA) equipped with a monochromator (TILL Photonics, Germany), charge coupled device camera (PCO-TECH, USA), and appropriate filter sets. Transfected cells were identified via mCherry fluorescence and perfused with SB, with or without Slo2 channel modulators, then SB with 4 mM Tl2SO4, and images acquired every 5 s (λex 488 nm; λem 535 nm). Changes in fluorescent emission were quantified for all cells in the field of view, both transfected and untransfected, using TILLvisION software (TILL Photonics).

Mitochondrial Thallium (Tl+) Flux Assay on Isolated Mouse Heart Mitochondria

After anesthesia (tribromoethanol as above), mitochondria were isolated from three pooled mouse hearts, loaded with BTC-AM (Life Technologies, USA) and monitored for Tl+ uptake (a surrogate for K+ channel flux) as previously described.22,29,34 

Isoflurane Dosing

In perfused hearts, isoflurane infusion via syringe pump afforded no possibility for volatilization before entering the heart. For C57BL/6 mice, 100 µM isoflurane equates to approximately 0.35 MAC.30,31  For cell and mitochondrial experiments, incubations generally lasted less than 1 min such that isoflurane volatilization was considered negligible. As such, isoflurane concentrations are denoted as “initial.”

Statistical Analysis

Significance was determined by using a two-way ANOVA and when warranted post hoc unpaired t testing (two tailed) analysis using Bonferroni multiple comparisons correction. P values reported in the results are from t tests, with P value less than 0.05 (following multiple comparisons correction) considered statistically significant. Sample size was determined based on experience and previous studies.

Results

Cardiac APC Requires Slo2.1 (Slick)

In C. elegans, SLO-2 is encoded by a single gene and contributes to VA-stimulated mitochondrial K+ flux and APC.22  However, in mammals, slo-2 has diverged into two paralogs (Slo2.1 and Slo2.2) with differing ion sensitivity to the C. elegans channel. By using a recently developed mouse strain with the genes coding for Slick (Kcnt2 or Slo2.1) and Slack (Kcnt1 or Slo2.2) both deleted,28  our goal herein was to determine whether either or both of these mammalian gene products are orthologous to the worm SLO-2 channel and facilitate K+ flux across the mitochondrial inner membrane in response to VA. First, we verified using genomic polymerase chain reaction that the Slo2.x dKO contained the expected lesions in each of the two genes and that each allele was unambiguously detectable (fig. 2, A and B). Next, we used Western blot analysis to query whether the channels themselves were similarly ablated. Slick and Slack are abundantly expressed in neural tissue, and our results demonstrate that we can readily detect both paralogs in brain lysates as well as their absence in the dKO background (fig. 2C). Finally, we report that Slo2.x dKO mice were viable and had normal cardiac electrical function (table S1, Supplemental Digital Content 1, http://links.lww.com/ALN/B256).

Fig. 2.

Slo2.1 and Slo2.2 genotyping and protein expression analysis of Slick and Slack in Slo2.x double knockout (dKO) mice. (A) Tail-clip PCR showing wild-type (WT) or knockout Slo2.1 products at the indicated mass, for WT (+/+), knockout (−/−), or heterozygous (+/−) animals. Primers were used as described in the Materials and Methods. Left lane = DNA ladder. (B) Similar to that in A, but with Slo2.2 primers. (C) Western blot for Slick and Slack (Neuromab antibody; Neuromab, USA) on brain tissue lysates from WT and Slo2.x dKO mice.

Fig. 2.

Slo2.1 and Slo2.2 genotyping and protein expression analysis of Slick and Slack in Slo2.x double knockout (dKO) mice. (A) Tail-clip PCR showing wild-type (WT) or knockout Slo2.1 products at the indicated mass, for WT (+/+), knockout (−/−), or heterozygous (+/−) animals. Primers were used as described in the Materials and Methods. Left lane = DNA ladder. (B) Similar to that in A, but with Slo2.2 primers. (C) Western blot for Slick and Slack (Neuromab antibody; Neuromab, USA) on brain tissue lysates from WT and Slo2.x dKO mice.

To interrogate the individual contribution of each Slo2.x paralog to APC, the mutant alleles were genetically separated through extensive backcrossing to a C57BL/6 background. Ex vivo perfused hearts from WT and Slo2-ablated mice (both the Slo2.x dKO and single mutant alleles) were then subjected to IR injury with optional APC (isoflurane in perfusion media). Injury was assessed by measuring post-IR functional recovery (fig. 3A) and infarct size (fig. 3B). APC was protective in WT hearts, with a 95% improvement in the post-IR recovery of rate pressure product (RPP; heart rate × left ventricular–developed pressure; P = 0.0005) and a 44% reduction in infarct size (P = 0.0043) versus control hearts. Slo2.2−/− mice exhibited a similar degree of protection by APC (132% improvement in recovery of RPP, P = 0.00016, 50% reduction in infarct size, P = 0.000005) versus control hearts. However, APC in Slo2.1−/− mice resulted in no protection (12% worse recovery of RPP, P = 0.057, 2% reduction in infarct size, P = 0.817, compared with IR alone). As expected, APC in dKO mice was similarly ineffective (8% worse recovery of RPP, P = 0.725, 2% increase in infarct size, P = 0.854, compared with IR alone). None of the genotypes exhibited significant differences in baseline susceptibility to ischemia.

Fig. 3.

Anesthetic preconditioning (APC) in wild-type (WT) and Slo2.x knockout mouse hearts. Langendorff-perfused hearts from WT (circles), Slo2.x double knockouts (dKO) (triangles), Slo2.1−/− (squares), and Slo2.2−/− (diamonds) were subjected to ischemia–reperfusion (IR) injury alone (open symbols) or IR with APC comprising 100 μM isoflurane infusion for 10 min before ischemia (filled symbols). (A) Cardiac function data (rate pressure product [RPP], product of heart rate × left ventricular developed pressure) throughout perfusion. RPP is expressed as a percentage of the value immediately before ischemia. Data are means ± SEM, with 95% CIs for the 60-min reperfusion time point shown adjacent to error bars (numbers shaded where appropriate to indicate which data set they belong to—see inset to B). Insets: representative heart cross-sections stained with 2,3,5-triphenyltetrazolium chloride (upper images) and threshold pseudo-colored images used to quantify infarct size (lower images). (B) Infarct size for control IR and APC + IR–treated hearts from each genotype. Within each group, individual data points are on the left, thereby indicating the number of replicates (N). Means ± SEM are on the right, with 95% CIs shown adjacent. Inset key shows position of mean, SEM, and 95% CIs on the graphs. *Statistically significant difference between control IR and APC + IR groups at 60 min of reperfusion (ANOVA, with post hoc unpaired t test).

Fig. 3.

Anesthetic preconditioning (APC) in wild-type (WT) and Slo2.x knockout mouse hearts. Langendorff-perfused hearts from WT (circles), Slo2.x double knockouts (dKO) (triangles), Slo2.1−/− (squares), and Slo2.2−/− (diamonds) were subjected to ischemia–reperfusion (IR) injury alone (open symbols) or IR with APC comprising 100 μM isoflurane infusion for 10 min before ischemia (filled symbols). (A) Cardiac function data (rate pressure product [RPP], product of heart rate × left ventricular developed pressure) throughout perfusion. RPP is expressed as a percentage of the value immediately before ischemia. Data are means ± SEM, with 95% CIs for the 60-min reperfusion time point shown adjacent to error bars (numbers shaded where appropriate to indicate which data set they belong to—see inset to B). Insets: representative heart cross-sections stained with 2,3,5-triphenyltetrazolium chloride (upper images) and threshold pseudo-colored images used to quantify infarct size (lower images). (B) Infarct size for control IR and APC + IR–treated hearts from each genotype. Within each group, individual data points are on the left, thereby indicating the number of replicates (N). Means ± SEM are on the right, with 95% CIs shown adjacent. Inset key shows position of mean, SEM, and 95% CIs on the graphs. *Statistically significant difference between control IR and APC + IR groups at 60 min of reperfusion (ANOVA, with post hoc unpaired t test).

Cardioprotection by IPC or the mKATP Channel Opener Diazoxide Are Independent of Slo2.x Channel Genotype

To discount the possibility that lack of protection by APC in the Slo2.1−/− or dKO hearts was due to an inability of these hearts to be protected at all, we examined two additional cardioprotective treatments that are unrelated to VA: namely IPC (fig. 4) and the mitochondrial KATP channel opener diazoxide (fig. 5).

Fig. 4.

Cardioprotection by ischemic preconditioning (IPC) in wild-type (WT) and Slo2.x knockout mouse hearts. Langendorff-perfused hearts were subjected to control ischemia–reperfusion (IR) injury alone (open symbols) or IR + IPC (filled symbols). Symbols for genotypes are the same as in figure 3. Data for controls (IR alone, no treatment) are reproduced from figure 3 and shown for comparative purposes. (A) Cardiac function data (rate pressure product [RPP], product of heart rate × left ventricular developed pressure) throughout perfusion. RPP is expressed as a percentage of the value immediately before ischemia. Data are means ± SEM, with 95% CIs for the 60-min reperfusion time point shown adjacent to error bars (see inset to B). Insets: representative heart cross-sections stained with 2,3,5-triphenyltetrazolium chloride (upper images) and threshold pseudo-colored images used to quantify infarct size (lower images). (B) Infarct size for control IR and IPC + IR–treated hearts from each genotype. Within each group, individual data on the left, thereby indicating the number of replicates (N). Means ± SEM are on the right, with 95% CIs shown adjacent. Inset key shows position of mean, SEM, and 95% CIs on the graphs. *Statistically significant difference between control IR and IPC + IR groups at 60 min of reperfusion (ANOVA, with post hoc unpaired t test). dKO = double knockout.

Fig. 4.

Cardioprotection by ischemic preconditioning (IPC) in wild-type (WT) and Slo2.x knockout mouse hearts. Langendorff-perfused hearts were subjected to control ischemia–reperfusion (IR) injury alone (open symbols) or IR + IPC (filled symbols). Symbols for genotypes are the same as in figure 3. Data for controls (IR alone, no treatment) are reproduced from figure 3 and shown for comparative purposes. (A) Cardiac function data (rate pressure product [RPP], product of heart rate × left ventricular developed pressure) throughout perfusion. RPP is expressed as a percentage of the value immediately before ischemia. Data are means ± SEM, with 95% CIs for the 60-min reperfusion time point shown adjacent to error bars (see inset to B). Insets: representative heart cross-sections stained with 2,3,5-triphenyltetrazolium chloride (upper images) and threshold pseudo-colored images used to quantify infarct size (lower images). (B) Infarct size for control IR and IPC + IR–treated hearts from each genotype. Within each group, individual data on the left, thereby indicating the number of replicates (N). Means ± SEM are on the right, with 95% CIs shown adjacent. Inset key shows position of mean, SEM, and 95% CIs on the graphs. *Statistically significant difference between control IR and IPC + IR groups at 60 min of reperfusion (ANOVA, with post hoc unpaired t test). dKO = double knockout.

Fig. 5.

Cardioprotection by diazoxide (DZX) in wild-type (WT) and Slo2.x knockout mouse hearts. Langendorff-perfused hearts were subjected to control ischemia–reperfusion (IR) injury alone (open symbols) or DZX + IR (filled symbols). Symbols for genotypes are the same as in figure 3. Data for controls (IR alone, no treatment) are reproduced from figure 3 and are shown for comparative purposes. (A) Cardiac function data (rate pressure product [RPP], product of heart rate × left ventricular developed pressure) throughout perfusion. RPP is expressed as a percentage of the value immediately before ischemia. Data are means ± SEM, with 95% CIs for the 60-min reperfusion time point shown adjacent to error bars (see inset to B). Insets: representative heart cross-sections stained with 2,3,5-triphenyltetrazolium chloride (upper images) and resulting pseudocolored images calculated via threshold masks and used to quantify infarct size (lower images). (B) Infarct size for control IR and DZX + IR–treated hearts from each genotype. Within each group, individual data are shown on the left, thereby indicating the number of replicates (N). Means ± SEM are on the right, with 95% CIs shown adjacent. Inset key shows position of mean, SEM, and 95% CIs on the graphs. *Statistically significant difference between control IR and DZX + IR groups (ANOVA, with post hoc unpaired t test). dKO = double knockout.

Fig. 5.

Cardioprotection by diazoxide (DZX) in wild-type (WT) and Slo2.x knockout mouse hearts. Langendorff-perfused hearts were subjected to control ischemia–reperfusion (IR) injury alone (open symbols) or DZX + IR (filled symbols). Symbols for genotypes are the same as in figure 3. Data for controls (IR alone, no treatment) are reproduced from figure 3 and are shown for comparative purposes. (A) Cardiac function data (rate pressure product [RPP], product of heart rate × left ventricular developed pressure) throughout perfusion. RPP is expressed as a percentage of the value immediately before ischemia. Data are means ± SEM, with 95% CIs for the 60-min reperfusion time point shown adjacent to error bars (see inset to B). Insets: representative heart cross-sections stained with 2,3,5-triphenyltetrazolium chloride (upper images) and resulting pseudocolored images calculated via threshold masks and used to quantify infarct size (lower images). (B) Infarct size for control IR and DZX + IR–treated hearts from each genotype. Within each group, individual data are shown on the left, thereby indicating the number of replicates (N). Means ± SEM are on the right, with 95% CIs shown adjacent. Inset key shows position of mean, SEM, and 95% CIs on the graphs. *Statistically significant difference between control IR and DZX + IR groups (ANOVA, with post hoc unpaired t test). dKO = double knockout.

The improvement in functional recovery (RPP) induced by IPC for each genotype was as follows: WT 148%, dKO 184%, Slo2.1−/− 108%, Slo2.2−/− 193%, compared with IR alone, P < 0.025 for all genotypes (fig. 4A). The reduction in infarct size induced by IPC was as follows: WT 53%, dKO 34%, Slo2.1−/− 45%, Slo2.2−/− 49%, compared with IR alone, P < 0.0025 for all genotypes (fig. 4B). Similar to IPC, diazoxide delivered consistent cardioprotection across all genotypes. Namely, the improvement in functional recovery (RPP) induced by diazoxide: WT 98%, dKO 171%, Slo2.1−/− 125%, Slo2.2−/− 143%, compared with IR alone, P < 0.01 for all genotypes (fig. 5A). The reduction in infarct size induced by IPC was as follows: WT 37%, dKO 45%, Slo2.1−/− 55%, Slo2.2−/− 48%, compared with IR alone, P < 0.001 for all genotypes (fig. 5B). Thus, cardioprotection by either IPC or diazoxide was consistent regardless of genotype, suggesting Slo2.1−/− hearts are still capable of being protected by these interventions and that cardioprotection, even via mechanisms thought to involve mitochondria, does not invariably require Slick.

In addition to functional recovery measured as RPP, data for dP/dTMAX and dP/dTMIN, LV-developed pressure, and LV end-diastolic pressure, for all genotypes and treatment regimens, are provided in Supplemental Digital Content 1, http://links.lww.com/ALN/B256, tables 2–4, respectively.

VA-stimulated K+ Transport in Cardiomyocytes and Mitochondria Requires Slo2.1 (Slick)

Given the proposed role of a mitochondrial large-conductance K+ channel in APC,7,22,23  we examined VA stimulation of K+ flux in both primary cardiomyocytes and isolated cardiac mitochondria by using a thallium (Tl+)-based fluorescence assay.22,34  All four genotypes were studied at the isolated mitochondrial level, but due to our initial results (vide supra) indicating that Slick but not Slack is involved in APC, studies in cardiomyocytes were limited to WT and Slo2.1−/−.

The VA isoflurane was found to stimulate K+ flux in cardiomyocytes (fig. 6A) as well as mitochondria (fig. 6B) from WT mice, and this effect was blocked by the inhibitor bepridil. Bepridil is thought to target cardiac KNa35  and other channels36  and has recently been shown to function as an Ebola antiviral.37  Its use here was motivated by its ability to inhibit Slo2 channels,33  which we validated using recombinant Slick expressed in HEK293 cells (fig. S1, Supplemental Digital Content 1, http://links.lww.com/ALN/B256). Notably, VA stimulation of K+ flux was absent in both cardiomyocytes and mitochondria from Slo2.1−/− hearts (fig. 6, A and B). At the mitochondrial level, the Tl+ flux pattern in Slo2.2−/− was similar to WT, and the pattern in dKO was similar to Slo2.1−/−. These data indicate that isoflurane induces a Slo2.1-dependent K+ flux in both cardiomyocytes and mitochondria.

Fig. 6.

Thallium (Tl+) flux in cardiomyocytes and mitochondria from mice of varying Slo2 genotype. (A) Cardiomyocytes were isolated from wild-type (WT) (white bars) and Slo2.1−/− (black bars) mice, and surface K+ channel activity assayed via thallium (Tl+) uptake. Where indicated, isoflurane (Iso, 150 μM initial) and/or bepridil (Bep, 10 μM) were added. Data are normalized to control (no addition, dashed line) and are means ± SEM. 95% CIs are shown at top, number of replicates (N) at bottom, of each graph bar—see inset key in B. *Statistically significant difference versus control within a given genotype (ANOVA, with post hoc unpaired t test). (B) Mitochondria were isolated from WT (white bars), Slo2.x double knockout (dKO) (hashed), Slo2.1−/− (black), and Slo2.2−/− (gray) hearts, and K+ channel activity assayed via Tl+ uptake. Adenosine triphosphate (ATP) was present throughout to block mKATP channels. Where indicated, isoflurane (Iso, 300 μM initial) and/or bepridil (Bep, 10 μM) were added. Data are normalized to control (no addition) and are means ± SEM. 95% CIs are shown at top, number of replicates (N) at bottom, of each graph bar—see inset key. *Statistically significant difference versus ATP condition within a given genotype (ANOVA, with post hoc unpaired t test). (C) Cardiomyocyte Tl+ flux, as in A, but stimulated with bithionol (Bt, 0.25 μM) instead of isoflurane. Data are normalized to control (no addition, dashed line) and are means ± SEM. 95% CIs and N as per inset key in B. *Statistically significant difference versus control within a given genotype (ANOVA, with post hoc unpaired t test). (D) Mitochondrial Tl+ flux as in B but stimulated with bithionol (Bt, 2.5 μM) instead of isoflurane. Data are normalized to control (no addition) and are means ± SEM. 95% CIs and N as per inset key in B. *Statistically significant difference versus ATP condition within a given genotype (ANOVA, with post hoc unpaired t test). DZX = diazoxide.

Fig. 6.

Thallium (Tl+) flux in cardiomyocytes and mitochondria from mice of varying Slo2 genotype. (A) Cardiomyocytes were isolated from wild-type (WT) (white bars) and Slo2.1−/− (black bars) mice, and surface K+ channel activity assayed via thallium (Tl+) uptake. Where indicated, isoflurane (Iso, 150 μM initial) and/or bepridil (Bep, 10 μM) were added. Data are normalized to control (no addition, dashed line) and are means ± SEM. 95% CIs are shown at top, number of replicates (N) at bottom, of each graph bar—see inset key in B. *Statistically significant difference versus control within a given genotype (ANOVA, with post hoc unpaired t test). (B) Mitochondria were isolated from WT (white bars), Slo2.x double knockout (dKO) (hashed), Slo2.1−/− (black), and Slo2.2−/− (gray) hearts, and K+ channel activity assayed via Tl+ uptake. Adenosine triphosphate (ATP) was present throughout to block mKATP channels. Where indicated, isoflurane (Iso, 300 μM initial) and/or bepridil (Bep, 10 μM) were added. Data are normalized to control (no addition) and are means ± SEM. 95% CIs are shown at top, number of replicates (N) at bottom, of each graph bar—see inset key. *Statistically significant difference versus ATP condition within a given genotype (ANOVA, with post hoc unpaired t test). (C) Cardiomyocyte Tl+ flux, as in A, but stimulated with bithionol (Bt, 0.25 μM) instead of isoflurane. Data are normalized to control (no addition, dashed line) and are means ± SEM. 95% CIs and N as per inset key in B. *Statistically significant difference versus control within a given genotype (ANOVA, with post hoc unpaired t test). (D) Mitochondrial Tl+ flux as in B but stimulated with bithionol (Bt, 2.5 μM) instead of isoflurane. Data are normalized to control (no addition) and are means ± SEM. 95% CIs and N as per inset key in B. *Statistically significant difference versus ATP condition within a given genotype (ANOVA, with post hoc unpaired t test). DZX = diazoxide.

To substantiate the hypothesis that K+ flux and cardioprotection occur through Slick activation, we turned to a second distinct reagent. Bithionol has been shown to block Slo-type channels33  and inhibits recombinant Slick expressed in HEK293 cells (fig. S1, Supplemental Digital Content 1, http://links.lww.com/ALN/B256). At the cardiomyocyte level, bithionol induced a bepridil-sensitive K+ flux in WT cells that was absent in Slo2.1−/− cells (fig. 6C). In mitochondria, bithionol induced a bepridil-sensitive K+ flux in WT that was absent in the dKO mitochondria and reduced in both the Slo2.1−/− and Slo2.2−/− mitochondria (fig. 6D). The cell data fully support our hypothesis. However, the mitochondria data suggest that both Slick and Slack partially contribute to bithionol-stimulated Tl+ uptake, which is potentially interesting (see Discussion). However, it is likely that bithionol stimulates other channel activities or even mitochondrial function through unknown mechanisms of action, and very low levels of Slo2.2 are expressed in the heart.28 

Finally, additional support for our hypothesis came from the observation that bithionol was cardioprotective in WT hearts (fig. 7, A and B: 129% improvement in RPP recovery, P = 0.0098, 57% reduction in infarct size, P = 0.00008, compared with IR alone). This protection was partially lost in Slo2.1−/− hearts (bithionol induced 97% improvement in RPP recovery, P = 0.028, 14% reduction in infarct size, P = 0.15). Overall, these data support a K+ transport pathway with the genetic and pharmacologic characteristics of Slick, being required for the cardioprotective effects of APC.

Fig. 7.

Effect of bithionol on ischemia–reperfusion (IR) injury outcomes in wild-type (WT) and Slo2.1 knockout mouse hearts. (A) Langendorff-perfused hearts from WT (circles) and Slo2.1−/− (squares) were subjected to IR injury alone (open symbols) or IR with bithionol (Bt, 2.5 nM for 20 min before the onset of ischemia, filled symbols), as detailed in the Materials and Methods and in figures 3 to 5. The control groups are reproduced from figure 3 and are shown here for comparative purposes. RPP is expressed as a percentage of the value immediately before ischemia. Data are means ± SEM, with 95% CIs for the 60-min reperfusion time point shown adjacent to error bars (see inset, B). Insets: representative heart cross-sections stained with 2,3,5-triphenyltetrazolium chloride (upper images) and resulting pseudo-colored images calculated via threshold masks and used to quantify infarct size (lower images). (B) Infarct size for control IR and Bt + IR–treated hearts from each genotype. Within each group, individual data points are shown on the left, thereby indicating the number of replicates (N). Inset key shows position of mean, SEM, and 95% CIs on the graphs. *Statistically significant difference between control IR and Bt + IR groups at 60 min of reperfusion (ANOVA, with post hoc unpaired t test).

Fig. 7.

Effect of bithionol on ischemia–reperfusion (IR) injury outcomes in wild-type (WT) and Slo2.1 knockout mouse hearts. (A) Langendorff-perfused hearts from WT (circles) and Slo2.1−/− (squares) were subjected to IR injury alone (open symbols) or IR with bithionol (Bt, 2.5 nM for 20 min before the onset of ischemia, filled symbols), as detailed in the Materials and Methods and in figures 3 to 5. The control groups are reproduced from figure 3 and are shown here for comparative purposes. RPP is expressed as a percentage of the value immediately before ischemia. Data are means ± SEM, with 95% CIs for the 60-min reperfusion time point shown adjacent to error bars (see inset, B). Insets: representative heart cross-sections stained with 2,3,5-triphenyltetrazolium chloride (upper images) and resulting pseudo-colored images calculated via threshold masks and used to quantify infarct size (lower images). (B) Infarct size for control IR and Bt + IR–treated hearts from each genotype. Within each group, individual data points are shown on the left, thereby indicating the number of replicates (N). Inset key shows position of mean, SEM, and 95% CIs on the graphs. *Statistically significant difference between control IR and Bt + IR groups at 60 min of reperfusion (ANOVA, with post hoc unpaired t test).

Discussion

Herein, we demonstrated that the KNa channel Slick, encoded by the Slo2.1 gene, is required for cardioprotection by APC in mice. We also showed that Slick is responsible for VA-stimulated K+ flux at both the cardiomyocyte plasma and mitochondrial membranes and that pharmacologic Slick openers are cardioprotective.

Although SLO-2 is a KCa channel in C. elegans, in mammals it has diverged into two paralogs, Slick and Slack, which are KNa channels.24,27  These channels may contribute to various physiologic functions attributed to KNa channels, including metabolic sensing and neuronal plasticity,24,27,38  and although the major focus of KNa channel studies to date has been neuronal, KNa channels are found in cardiac myocytes.39  Notably, they have not been reported in mitochondria. Although these results cement a cardioprotective role for Slick, the relative contribution of plasma membrane versus mitochondrial Slick channels to this effect remains unclear and is a topic of ongoing research.

Many signaling pathways such as APC and IPC are evolutionarily conserved,22  and there are many redundancies in their signaling cascades. For example, the mechanism of APC and IPC share commonalities such as protein kinase C activation,40  ROS generation,14,41–43  and mitochondrial K+ channels.44–46  Although the precise mechanism of APC is not well understood, evidence suggests that APC involves both altered mitochondrial function and K+ channel activity (reviewed in the study by Agarwal et al.47 ).

Studies have implicated the mitochondrial ATP-sensitive K+ channel (mKATP) in APC largely through the use of nonspecific measures of channel activity (e.g., flavoprotein fluorescence) and drugs that are known to have off-target mitochondrial effects (e.g., diazoxide or 5-hydroxydecanoate).44–46,48,49 Although these pharmacologic approaches suggest the involvement of the mKATP, the effects could have also been due to another K+ channel such as Slick. The current lack of a molecular identity for the mKATP channel precludes a definitive answer to this dilemma.

Moreover, the data presented herein do not rule out a potential cross talk between the mKATP and Slick. For example, because both diazoxide- and IPC-mediated cardioprotection were preserved in Slo2.1−/− mice, Slick activation may be upstream of mKATP channel activation. Overall, although there are many similarities between APC and IPC, there is also evidence for distinct signaling pathways,50  and herein we show that Slick appears to be involved only in APC, not in IPC.

In the field of APC, the canonical BK channel Slo1, which may be present in cardiac mitochondria,51  emerged as an early candidate effector of APC protection.7,23  However, the importance of this channel was recently questioned by the evidence that both IPC and APC protection are robust in Slo1-ablated organisms.22  Although the role of Slo1 in the heart remains to be fully elucidated, it is important to emphasize that our data do not preclude coexistence of Slo1 and Slick in cardiomyocytes or cardiac mitochondria. Rather, they conclude that only Slick is required for APC in the heart.

Our results are consistent with the findings in C. elegans, which showed that SLO-2 was required for APC.22  Despite the large sequence similarity (approximately 74% identical)38  between Slick and Slack, we found no role for Slack in cardiac APC or IPC. This is in agreement with the low expression levels of Slo2.2 in the heart.27  However, this does not rule out a role for Slack in protecting other tissues from ischemia. Both Slo2.1 and Slo2.2 are highly expressed in the nervous system,24  and there is evidence that Slick and Slack may coassemble into channels.52  In this respect, it is intriguing that both Slick and Slack appeared to contribute to bithionol-induced K+ flux in purified mitochondria (fig. 6D). However, this was the only condition where both Slick and Slack were additive in our hands. Moreover, the mechanism of action of bithionol is unknown, but its activity as an antihelminthic may rely on suppressing succinate oxidation.53  Hence, it is premature to conclude that Slack contributes to mitochondrial function. Nevertheless, it is possible that in other tissues such as the brain, Slo2.2 may play a role protecting against stress.

It is also interesting to speculate that Slick did not evolve to be opened by VAs, and given our results, we speculate that it may have a physiologic role in the mitochondrion. Although some mitochondrial K+ channels are demonstrated to play roles in pathophysiology (e.g., protection from IR injury),19  most are only reported at the phenomenological level as mitochondrial swelling or K+ flux sensitive to activators/inhibitors, with very little known about the normal function of these channels. The activation of mitochondrial K+ channels is hypothesized to modulate mitochondrial matrix volume, Ca2+ uptake capacity, and ROS production,54  and although these events are involved in the pathophysiology of IR injury, they also have effects on regular mitochondrial function and metabolism. For example, Ca2+ stimulates oxidative phosphorylation, and matrix volume can determine the efficiency of high-energy phosphate transport between the mitochondrion and cytosol. Ongoing research in our laboratory is aimed at determining the role of Slick in regulating physiologic mitochondrial function.

A potential limitation of this study is the low dose of isoflurane (approximately 0.35 MAC) used to elicit cardioprotection. It is unclear if higher doses, or other VAs (e.g., desflurane, sevoflurane), will exhibit similar Slo2.1-dependent cardioprotective effects. Nevertheless, we note that the current widespread clinical use of VAs, and the ACC/AHA recommendations for their use during cardiac surgery,5  suggests that the identification of Slick as a mediator of APC may drive development of novel cardioprotective drugs targeting this channel.

Acknowledgments

The authors thank Chris Lingle, Ph.D. (Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri), for providing Slo2.x knockout mice.

Supported by the National Institutes of Health (Bethesda, Maryland; grant no. R01-GM087483 to Drs. Brookes and Nehrke).

Competing Interests

The authors declare no competing interests.

References

1.
Cope
DK
,
Impastato
WK
,
Cohen
MV
,
Downey
JM
:
Volatile anesthetics protect the ischemic rabbit myocardium from infarction.
Anesthesiology
1997
;
86
:
699
709
2.
Stadnicka
A
,
Marinovic
J
,
Ljubkovic
M
,
Bienengraeber
MW
,
Bosnjak
ZJ
:
Volatile anesthetic-induced cardiac preconditioning.
J Anesth
2007
;
21
:
212
9
3.
Landoni
G
,
Biondi-Zoccai
GG
,
Zangrillo
A
,
Bignami
E
,
D’Avolio
S
,
Marchetti
C
,
Calabrò
MG
,
Fochi
O
,
Guarracino
F
,
Tritapepe
L
,
De Hert
S
,
Torri
G
:
Desflurane and sevoflurane in cardiac surgery: A meta-analysis of randomized clinical trials.
J Cardiothorac Vasc Anesth
2007
;
21
:
502
11
4.
Jia
B
,
Crowder
CM
:
Volatile anesthetic preconditioning present in the invertebrate Caenorhabditis elegans.
Anesthesiology
2008
;
108
:
426
33
5.
Hillis
LD
,
Smith
PK
,
Anderson
JL
,
Bittl
JA
,
Bridges
CR
,
Byrne
JG
,
Cigarroa
JE
,
Disesa
VJ
,
Hiratzka
LF
,
Hutter
AM
Jr
,
Jessen
ME
,
Keeley
EC
,
Lahey
SJ
,
Lange
RA
,
London
MJ
,
Mack
MJ
,
Patel
MR
,
Puskas
JD
,
Sabik
JF
,
Selnes
O
,
Shahian
DM
,
Trost
JC
,
Winniford
MD
;
2011 ACCF/AHA Guideline for Coronary Artery Bypass Graft Surgery
:
A report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Developed in collaboration with the American Association for Thoracic Surgery, Society of Cardiovascular Anesthesiologists, and Society of Thoracic Surgeons.
J Am Coll Cardiol
2011
;
58
:
e123
e210
6.
Ludwig
LM
,
Weihrauch
D
,
Kersten
JR
,
Pagel
PS
,
Warltier
DC
:
Protein kinase C translocation and Src protein tyrosine kinase activation mediate isoflurane-induced preconditioning in vivo: Potential downstream targets of mitochondrial adenosine triphosphate-sensitive potassium channels and reactive oxygen species.
Anesthesiology
2004
;
100
:
532
9
7.
Redel
A
,
Lange
M
,
Jazbutyte
V
,
Lotz
C
,
Smul
TM
,
Roewer
N
,
Kehl
F
:
Activation of mitochondrial large-conductance calcium-activated K+ channels via protein kinase A mediates desflurane-induced preconditioning.
Anesth Analg
2008
;
106
:
384
91
8.
Yang
C
,
Talukder
MA
,
Varadharaj
S
,
Velayutham
M
,
Zweier
JL
:
Early ischaemic preconditioning requires Akt- and PKA-mediated activation of eNOS via serine1176 phosphorylation.
Cardiovasc Res
2013
;
97
:
33
43
9.
Toller
WG
,
Kersten
JR
,
Gross
ER
,
Pagel
PS
,
Warltier
DC
:
Isoflurane preconditions myocardium against infarction via activation of inhibitory guanine nucleotide binding proteins.
Anesthesiology
2000
;
92
:
1400
7
10.
Kersten
JR
,
Orth
KG
,
Pagel
PS
,
Mei
DA
,
Gross
GJ
,
Warltier
DC
:
Role of adenosine in isoflurane-induced cardioprotection.
Anesthesiology
1997
;
86
:
1128
39
11.
Ishida
T
,
Yarimizu
K
,
Gute
DC
,
Korthuis
RJ
:
Mechanisms of ischemic preconditioning.
Shock
1997
;
8
:
86
94
12.
Novalija
E
,
Fujita
S
,
Kampine
JP
,
Stowe
DF
:
Sevoflurane mimics ischemic preconditioning effects on coronary flow and nitric oxide release in isolated hearts.
Anesthesiology
1999
;
91
:
701
12
13.
Vanden Hoek
TL
,
Becker
LB
,
Shao
Z
,
Li
C
,
Schumacker
PT
:
Reactive oxygen species released from mitochondria during brief hypoxia induce preconditioning in cardiomyocytes.
J Biol Chem
1998
;
273
:
18092
8
14.
Müllenheim
J
,
Ebel
D
,
Frässdorf
J
,
Preckel
B
,
Thämer
V
,
Schlack
W
:
Isoflurane preconditions myocardium against infarction via release of free radicals.
Anesthesiology
2002
;
96
:
934
40
15.
Garlid
KD
,
Halestrap
AP
:
The mitochondrial K(ATP) channel—Fact or fiction?
J Mol Cell Cardiol
2012
;
52
:
578
83
16.
Szabò
I
,
Bock
J
,
Jekle
A
,
Soddemann
M
,
Adams
C
,
Lang
F
,
Zoratti
M
,
Gulbins
E
:
A novel potassium channel in lymphocyte mitochondria.
J Biol Chem
2005
;
280
:
12790
8
17.
Xu
W
,
Liu
Y
,
Wang
S
,
McDonald
T
,
Van Eyk
JE
,
Sidor
A
,
O’Rourke
B
:
Cytoprotective role of Ca2+-activated K+ channels in the cardiac inner mitochondrial membrane.
Science
2002
;
298
:
1029
33
18.
Shi
GY
,
Jung
DW
,
Garlid
KD
,
Brierley
GP
:
Induction of respiration-dependent net efflux of K+ from heart mitochondria by depletion of endogenous divalent cations.
J Biol Chem
1980
;
255
:
10306
11
19.
Szabo
I
,
Zoratti
M
:
Mitochondrial channels: Ion fluxes and more.
Physiol Rev
2014
;
94
:
519
608
20.
Szewczyk
A
,
Jarmuszkiewicz
W
,
Kunz
WS
:
Mitochondrial potassium channels.
IUBMB Life
2009
;
61
:
134
43
21.
Kohro
S
,
Hogan
QH
,
Nakae
Y
,
Yamakage
M
,
Bosnjak
ZJ
:
Anesthetic effects on mitochondrial ATP-sensitive K channel.
Anesthesiology
2001
;
95
:
1435
340
22.
Wojtovich
AP
,
Sherman
TA
,
Nadtochiy
SM
,
Urciuoli
WR
,
Brookes
PS
,
Nehrke
K
:
SLO-2 is cytoprotective and contributes to mitochondrial potassium transport.
PLoS One
2011
;
6
:
e28287
23.
Stumpner
J
,
Lange
M
,
Beck
A
,
Smul
TM
,
Lotz
CA
,
Kehl
F
,
Roewer
N
,
Redel
A
:
Desflurane-induced post-conditioning against myocardial infarction is mediated by calcium-activated potassium channels: Role of the mitochondrial permeability transition pore.
Br J Anaesth
2012
;
108
:
594
601
24.
Salkoff
L
,
Butler
A
,
Ferreira
G
,
Santi
C
,
Wei
A
:
High-conductance potassium channels of the SLO family.
Nat Rev Neurosci
2006
;
7
:
921
31
25.
Schreiber
M
,
Wei
A
,
Yuan
A
,
Gaut
J
,
Saito
M
,
Salkoff
L
:
Slo3, a novel pH-sensitive K+ channel from mammalian spermatocytes.
J Biol Chem
1998
;
273
:
3509
16
26.
Wojtovich
AP
,
Nadtochiy
SM
,
Urciuoli
WR
,
Smith
CO
,
Grunnet
M
,
Nehrke
K
,
Brookes
PS
:
A non-cardiomyocyte autonomous mechanism of cardioprotection involving the SLO1 BK channel.
PeerJ
2013
;
1
:
e48
27.
Bhattacharjee
A
,
Joiner
WJ
,
Wu
M
,
Yang
Y
,
Sigworth
FJ
,
Kaczmarek
LK
:
Slick (Slo2.1), a rapidly-gating sodium-activated potassium channel inhibited by ATP.
J Neurosci
2003
;
23
:
11681
91
28.
Martinez-Espinosa
PL
,
Wu
J
,
Yang
C
,
Gonzalez-Perez
V
,
Zhou
H
,
Liang
H
,
Xia
XM
,
Lingle
CJ
:
Knockout of Slo2.2 enhances itch, abolishes KNa current, and increases action potential firing frequency in DRG neurons.
Elife
2015
;
4
29.
Wojtovich
AP
,
Urciuoli
WR
,
Chatterjee
S
,
Fisher
AB
,
Nehrke
K
,
Brookes
PS
:
Kir6.2 is not the mitochondrial KATP channel but is required for cardioprotection by ischemic preconditioning.
Am J Physiol Heart Circ Physiol
2013
;
304
:
H1439
45
30.
Sonner
JM
,
Gong
D
,
Li
J
,
Eger
EI
II
,
Laster
MJ
:
Mouse strain modestly influences minimum alveolar anesthetic concentration and convulsivity of inhaled compounds.
Anesth Analg
1999
;
89
:
1030
4
31.
Franks
NP
,
Lieb
WR
:
Temperature dependence of the potency of volatile general anesthetics: Implications for in vitro experiments.
Anesthesiology
1996
;
84
:
716
20
32.
Garlid
KD
,
Paucek
P
,
Yarov-Yarovoy
V
,
Murray
HN
,
Darbenzio
RB
,
D’Alonzo
AJ
,
Lodge
NJ
,
Smith
MA
,
Grover
GJ
:
Cardioprotective effect of diazoxide and its interaction with mitochondrial ATP-sensitive K+ channels. Possible mechanism of cardioprotection.
Circ Res
1997
;
81
:
1072
82
33.
Yang
B
,
Gribkoff
VK
,
Pan
J
,
Damagnez
V
,
Dworetzky
SI
,
Boissard
CG
,
Bhattacharjee
A
,
Yan
Y
,
Sigworth
FJ
,
Kaczmarek
LK
:
Pharmacological activation and inhibition of Slack (Slo2.2) channels.
Neuropharmacology
2006
;
51
:
896
906
34.
Wojtovich
AP
,
Williams
DM
,
Karcz
MK
,
Lopes
CM
,
Gray
DA
,
Nehrke
KW
,
Brookes
PS
:
A novel mitochondrial K(ATP) channel assay.
Circ Res
2010
;
106
:
1190
6
35.
Mori
K
,
Saito
T
,
Masuda
Y
,
Nakaya
H
:
Effects of class III antiarrhythmic drugs on the Na(+)-activated K+ channels in guinea-pig ventricular cells.
Br J Pharmacol
1996
;
119
:
133
41
36.
Yatani
A
,
Brown
AM
,
Schwartz
A
:
Bepridil block of cardiac calcium and sodium channels.
J Pharmacol Exp Ther
1986
;
237
:
9
17
37.
Johansen
LM
,
DeWald
LE
,
Shoemaker
CJ
,
Hoffstrom
BG
,
Lear-Rooney
CM
,
Stossel
A
,
Nelson
E
,
Delos
SE
,
Simmons
JA
,
Grenier
JM
,
Pierce
LT
,
Pajouhesh
H
,
Lehár
J
,
Hensley
LE
,
Glass
PJ
,
White
JM
,
Olinger
GG
:
A screen of approved drugs and molecular probes identifies therapeutics with anti-Ebola virus activity.
Sci Transl Med
2015
;
7
:
290ra89
38.
Kaczmarek
LK
:
Slack, slick and sodium-activated potassium channels.
ISRN Neurosci
2013
;
2013:pii: 354262
39.
Kameyama
M
,
Kakei
M
,
Sato
R
,
Shibasaki
T
,
Matsuda
H
,
Irisawa
H
:
Intracellular Na+ activates a K+ channel in mammalian cardiac cells.
Nature
1984
;
309
:
354
6
40.
Novalija
E
,
Kevin
LG
,
Camara
AK
,
Bosnjak
ZJ
,
Kampine
JP
,
Stowe
DF
:
Reactive oxygen species precede the epsilon isoform of protein kinase C in the anesthetic preconditioning signaling cascade.
Anesthesiology
2003
;
99
:
421
8
41.
Kevin
LG
,
Novalija
E
,
Riess
ML
,
Camara
AK
,
Rhodes
SS
,
Stowe
DF
:
Sevoflurane exposure generates superoxide but leads to decreased superoxide during ischemia and reperfusion in isolated hearts.
Anesth Analg
2003
;
96
:
949
55
42.
Novalija
E
,
Kevin
LG
,
Eells
JT
,
Henry
MM
,
Stowe
DF
:
Anesthetic preconditioning improves adenosine triphosphate synthesis and reduces reactive oxygen species formation in mitochondria after ischemia by a redox dependent mechanism.
Anesthesiology
2003
;
98
:
1155
63
43.
Tanaka
K
,
Weihrauch
D
,
Kehl
F
,
Ludwig
LM
,
LaDisa
JF
Jr
,
Kersten
JR
,
Pagel
PS
,
Warltier
DC
:
Mechanism of preconditioning by isoflurane in rabbits: A direct role for reactive oxygen species.
Anesthesiology
2002
;
97
:
1485
90
44.
Kersten
JR
,
Schmeling
TJ
,
Pagel
PS
,
Gross
GJ
,
Warltier
DC
:
Isoflurane mimics ischemic preconditioning via activation of K(ATP) channels: Reduction of myocardial infarct size with an acute memory phase.
Anesthesiology
1997
;
87
:
361
70
45.
Tanaka
K
,
Weihrauch
D
,
Ludwig
LM
,
Kersten
JR
,
Pagel
PS
,
Warltier
DC
:
Mitochondrial adenosine triphosphate-regulated potassium channel opening acts as a trigger for isoflurane-induced preconditioning by generating reactive oxygen species.
Anesthesiology
2003
;
98
:
935
43
46.
Kapinya
KJ
,
Löwl
D
,
Fütterer
C
,
Maurer
M
,
Waschke
KF
,
Isaev
NK
,
Dirnagl
U
:
Tolerance against ischemic neuronal injury can be induced by volatile anesthetics and is inducible NO synthase dependent.
Stroke
2002
;
33
:
1889
98
47.
Agarwal
B
,
Stowe
DF
,
Dash
RK
,
Bosnjak
ZJ
,
Camara
AK
:
Mitochondrial targets for volatile anesthetics against cardiac ischemia-reperfusion injury.
Front Physiol
2014
;
5
:
341
48.
Hanley
PJ
,
Mickel
M
,
Löffler
M
,
Brandt
U
,
Daut
J
:
K(ATP) channel-independent targets of diazoxide and 5-hydroxydecanoate in the heart.
J Physiol
2002
;
542
(
Pt 3
):
735
41
49.
Bantel
C
,
Maze
M
,
Trapp
S
:
Neuronal preconditioning by inhalational anesthetics: Evidence for the role of plasmalemmal adenosine triphosphate-sensitive potassium channels.
Anesthesiology
2009
;
110
:
986
95
50.
Sergeev
P
,
da Silva
R
,
Lucchinetti
E
,
Zaugg
K
,
Pasch
T
,
Schaub
MC
,
Zaugg
M
:
Trigger-dependent gene expression profiles in cardiac preconditioning: Evidence for distinct genetic programs in ischemic and anesthetic preconditioning.
Anesthesiology
2004
;
100
:
474
88
51.
Singh
H
,
Lu
R
,
Bopassa
JC
,
Meredith
AL
,
Stefani
E
,
Toro
L
:
MitoBK(Ca) is encoded by the Kcnma1 gene, and a splicing sequence defines its mitochondrial location.
Proc Natl Acad Sci U S A
2013
;
110
:
10836
41
52.
Chen
H
,
Kronengold
J
,
Yan
Y
,
Gazula
VR
,
Brown
MR
,
Ma
L
,
Ferreira
G
,
Yang
Y
,
Bhattacharjee
A
,
Sigworth
FJ
,
Salkoff
L
,
Kaczmarek
LK
:
The N-terminal domain of Slack determines the formation and trafficking of Slick/Slack heteromeric sodium-activated potassium channels.
J Neurosci
2009
;
29
:
5654
65
53.
Hamajima
F
:
Studies on metabolism of lung fluke genus Paragonimus. VII. Action of bithionol on glycolytic and oxidative metabolism of adult worms.
Exp Parasitol
1973
;
34
:
1
11
54.
Facundo
HT
,
Fornazari
M
,
Kowaltowski
AJ
:
Tissue protection mediated by mitochondrial K+ channels.
Biochim Biophys Acta
2006
;
1762
:
202
12