Sage Journals: Discover world-class research

Abstract

Bupivacaine is frequently used for conducting regional anesthesia. When accidentally injected or excessively absorbed into circulation, bupivacaine can induce severe arrhythmia and potentially lead to cardiac arrest. The specific mechanisms underlying this cardiotoxicity, however, remain to be clarified. We transfected HEK-293 cells to express the small conductance calcium-activated potassium type-2 channel (SK₂), and used a whole-cell patch clamp method in order to explore how bupivacaine affected these channels. We subsequently used SK₂ knockout mice to explore the relevance of SK₂ channels in bupivacaine-induced cardiotoxicity in isolating mouse hearts, mounting them on a Langendorff apparatus, and perfusing them with bupivacaine. Using this system, arrhythmia, asystole, and cardiac functions were monitored. We observed dose-dependent inhibition of SK₂ channels by bupivacaine: half-maximal inhibitory concentration (IC50) value = 18.6 μM (95% CI 10.8–32.1). When SK₂ knockout (SK₂ ^−/−) or wild-type (WT) mice were perfused with Krebs-Henseleit buffer (KHB), we did not observe any instances of arrhythmia. When SK₂ ^−/− mice or WT were perfused with KHB containing bupivacaine (40 μM), the time to arrhythmia (T_arrhythmia) and time to asystole (T_asystole) were both significantly longer in SK₂ ^−/− mice relative to WT mice (P < 0.001). Similarly, SK₂ ^−/− mice exhibited a significantly longer time to 25%, 50%, and 75% reductions in heart rate (HR) and rate-pressure product (RPP) relative to WT mice following bupivacaine perfusion (P < 0.001). These results reveal that bupivacaine was able to mediate a dose-dependent inhibition of SK₂ channels in HEK-293 cells, and deletion of SK₂ channels can delay bupivacaine-induced cardiotoxicity in isolated mouse hearts.

Keywords

bupivacaine cardiotoxicity isolated heart mouse

Introduction

Regional anesthesia achieved through local administration of local anesthetics (LAs) is commonly employed for obstetric or orthopedic surgical operations, and for achieving postoperative analgesia.^1,2 Bupivacaine is an amide LA that is commonly used in the clinic owing to its desirable analgesic properties and extended duration of efficacy.³ Typically, bupivacaine is safe, but in some cases the accidental administration or excess absorption of this LA into circulation can lead to severe arrhythmia and cardiac arrest, leading to poor patient outcomes.^4
–6 As such, anesthesiologists need to be aware of the optimal strategies for both preventing and treating bupivacaine-induced cardiotoxicity.

As the mechanisms governing bupivacaine-induced cardiotoxicity are complex, they remain to be fully elucidated.⁴ This toxicity is known to be associated with the ability of bupivacaine to substantially inhibit ion channel and receptor activity on cardiomyocytes.⁴ In particular, research has suggested that this toxicity is a secondary consequence of the inhibition of sodium,⁷ L-calcium,⁸ and transient outward potassium current channels. The sodium current is the primary current important for mediating depolarization during action potential formation (phase 0), thereby controlling cardiomyocyte excitability and conductivity.⁹ As bupivacaine can interfere with this depolarization in the context of cardiomyocyte action potentials through a mechanism linked with impaired Na+ influx, this can result in subsequent arrhythmias and potentially cardiac arrest.¹⁰ These findings led to the conclusion that inhibition of sodium channels is the primary mechanism whereby bupivacaine induces cardiotoxicity.^7,11 However, several additional ion channels have more recently been found to play a role in controlling the excitation and conduction processes of cardiomyocytes.^12,13 As such, it is important that the possible role of these receptors in bupivacaine-induced cardiotoxicity be investigated.

Calcium-activated potassium channels are triggered in response to changes in levels of calcium within cells, and serve as key regulators of normal cardiomyocyte functionality.^13,14 There are three categories of calcium-activated potassium channels in humans: large, medium, and small conductance channels. Small conductance calcium-activated potassium type 2 channel (SK₂) is an important potassium channel that is involved in the hyperpolarization that occurs following an action potential.^12,13,15 These SK₂ channels are present in both the nervous and circulatory system, with distribution on atrial cells,¹⁶ ventricular cells,¹⁶ and Purkinje cells,¹² playing vital roles in regulating cardiac conduction. Indeed, these SK₂ channel have recently been shown to play a direct role in atrial fibrillation in humans,¹² with SK₂ channel upregulation contributing to heart failure.^17,18 Given their key role in the context of action potential propagation, the dysfunction of SK₂ channels has the potential to induce atrial or ventricular arrhythmia.¹⁹ Bupivacaine-induced cardiotoxicity is known to occur due to disruption in the activation of cardiac ion channels,²⁰ but the effects of bupivacaine on SK₂ channels is yet to be reported. We therefore hypothesized that bupivacaine may directly suppress SK₂ currents, thereby contributing to the cardiotoxicity associated with this LA agent.

In this report, we utilized HEK-293 cells expressing SK₂ channels for whole-cell patch clamp studies, and we further utilized isolated hearts from SK₂-knockout mice in order to explore the role of SK₂ channels in bupivacaine-induced cardiotoxicity.

Method

Chemicals

Human embryonic kidney 293 cells (HEK293 cells) were from the Institute of Life Sciences of the Chinese Academy of Sciences. Additionally, the following were purchased: Dulbecco’s modified Eagle’s medium (DMEM) from Hyclone (USA); fetal bovine serum (FBS), penicillin/streptomycin, and 0.25% trypsin from Gibco (USA); pCDNA3/rSK Ca²⁺ plasmids from OriGene (USA); Lipofectamine 2000 from Invitrogen (USA); and bupivacaine from Sigma Aldrich (USA). The pipette solution was composed of the following, in mM: MgCl₂ 1.15, Potassium gluconate 144, and CaCl₂ 1.0, pH was maintained at 7.3 with KOH.

Cell culture and transfection

HEK293 Cells were grown using DMEM containing 10% FBS at 37°C in a 5% CO₂ incubator with penicillin/streptomycin, and 0.25% trypsin was used for detecting cells. Prior to transfection cells were plated 2 × 105 cells/cm2, and cells were grown to 85% confluency at which time cells were transfected with the pCDNA3/rSK Ca2 plasmids using Lipofectamine 2000 based on provided directions. This allowed us to generate HEK-293 cells stably expressing SK₂ channels (SK2 cells), as described previously.²¹ Prior to patch clamp studies, SK₂ cells were played in a glass cover for 24 h.

SK₂ current recordings

A whole-cell patch clamp approach was used as described previously.²² The coverslip containing the SK₂ cells was placed into an inverted Olympus microscope (IX70, Japan) on the cell chamber. A superfusion system was then used for the assay, with appropriate solutions added to reservoirs. The system used was a DAD-VC system with a Micromanifold made up of eight polyamide-coated quarts tubes (100 um ID). This manifold allows for the simultaneous flow of up to eight different solutions into a single <1 uL, and can be readily maneuvered around cells to accurately direct it at target cells.

An EPC-10 amplifier was used for whole-cell patch clamping. Briefly, a glass electrode with a 1.2 mm outer diameter was pulled with a microelectrode puller, yielding a 1.5–3.0 MΩ resistance after the pipette solution was added. We then picked SK₂ cells with smooth cell membranes using microscopic guidance and recorded currents for these cells. After achieving a giga-seal, negative pressure was used to disrupt SK₂ cell membrane, and the Pulse 8.0 software (HEKA, Germany) was used for voltage stimulation and data recording, with all experiments being conducted at 36°C. The SK₂ currents stimulation program was as follows: a holding potential was maintained at −80 mV, the step clamp voltage was from −120 mV to +40 mV with a step of 10 mV and a 200 ms duration. Cells were able to produce stable currents at 0 mV; therefore, currents at 0 mV were used for comparisons in the following experiments.

Preparation of Langendorff-isolated heart

Age-, sex- and weight-matched adult SK₂ knockout and WT C57BL/6J mice (8–10 weeks old) were used for this study. The SK₂ ^−/− mice were generated, validated, and bred onto a C57BL/6J background at the Nanjing Model Institute of Zoology. All experiments were conducted in the laboratory of Wenzhou Medical University with approval from the Animal Care and Use Committee of Wenzhou Medical University. Animal care and treatments were carried out in accordance with the guidelines of the National Institutes of Health.

Mice were intraperitoneally administered 80 mg/kg ketamine hydrochloride and 12 mg/kg xylazine to achieve anesthesia, after which they were injected with 500U/kg heparin to prevent intracoronary microthrombi formation. We then rapidly excised the hearts from these animals and subjected them to retrograde perfusion with modified Krebs-Henseleit buffer (KHB; NaCl: 118 mmol/L, KCl: 4.7 mmol/L, pH: 7.40 ± 0.05) at 37°C and 80 mmHg. A latex balloon in the left ventricle was used for monitoring left ventricular pressure, with saline being intermittently injected into this balloon so as to keep left ventricular end-diastolic pressure between 4 mmHg and 6 mmHg. Electrocardiograph (ECG) electrodes were consistently placed in a “lead Ⅱ” position. The PowerLab biological signal processing and analysis system and the Chart 5.5.6 biological signal recording software were used for all data collection. The experimental protocol was initiated following 25 minutes of KHB perfusion, at which time steady-state conditions had been achieved.

Assessment of cardiotoxicity

Cardiotoxicity was first assessed via perfusing normal modified KHB through the isolated hearts of WT and SK₂ ^−/− mice (n = 8 per group) (Experimental Protocol was illustrated in Figure 1). In addition, to test the effects of bupivacaine on cardiotoxicity, WT and SK₂ ^−/− mice were perfused with KHB containing 40 uM bupivacaine (0.5%, Sigma-Aldrich Co., St. Louis, MO, P code: B5274-5G) (n = 8 per group). The times corresponding to the following parameters were measured for all groups: the time to first ventricular premature beat following bupivacaine administration (T_arrhythmia); the time to asystole following bupivacaine administration (T_asystole); and the time to 25%, 50%, and 75% reductions in heart rate (HR) and rate-pressure product (RPP = HR × (systolic pressure − diastolic pressure)) relative to baseline following bupivacaine administration. Baseline hemodynamic parameters were established prior to bupivacaine infusion.

Figure 1.

Experimental protocol. WT: wild type mice; SK₂ ^−/−: SK₂ knockout mice; B: bupivacaine; T_asystole: time to asystole.

Statistical analyses

All statistical analyses were conducted using SPSS (19.0, IL, USA). Shapiro-Wilk tests were used to assess the normality of measured data. The dose-dependent ability of bupivacaine to inhibit SK₂ currents was assessed via non-linear fitting of the data using GraphPad Prism 5.0 (CA, USA): Y = Bottom + (Top-Bottom)/(1 + 10^((LogIC50-X)*HillSlope)). Continuous data were given as means ± standard deviation. Weights, T_arrhythmia and T_asystole were assessed via one-way ANOVAs with LSD tests used for pairwise comparisons of significant results. Cardiac function parameters were compared via repeated-measured ANOVAs, with Scheffe tests being used to facilitate multiple comparisons. P < 0.05 was the significance threshold.

Results

Bupivacaine inhibits SK₂ currents in a dose-dependent fashion

We first used HEK-293 cells which were transfected to express SK₂ channels in order to assess the ability of bupivacaine to inhibit SK₂ currents. We found that increasing bupivacaine concentrations were associated with a reduction in SK₂ current. We were able to fit this dose-response data for bupivacaine inhibition of SK₂ currents (at 0 mV) using a non-linear model, yielding a bupivacaine half-maximal inhibitory concentration (IC₅₀) value of 18.6 μM (95% CI 10.8–32.1) (Figure 2).

Figure 2.

Bupivacaine inhibits SK₂ currents in a dose-dependent manner. The ability of bupivacaine to inhibit SK₂ currents (0 mV) in a dose-dependent fashion was assessed via non-linear fitting to yield the IC₅₀ value shown. The error bar indicates the standard deviation from the mean value. The equation was Y = Bottom + (Top-Bottom)/(1 + 10^((LogIC50-X)*HillSlope)). A total of 1 μM free calcium was present within the pipette.

Baseline parameters, T_arrhythmia and T_asystole for mouse studies

There were no differences in baseline weight (23.2 ± 3.1, 22.5 ± 2.5, 23.4 ± 2.3, and 23.3 ± 3.4 g), HR (394.1 ± 7.4, 396.6 ± 7.8, 401.5 ± 11.7 and 396.5 ± 9.5 beats/s) or RPP (3741.0 ± 1081.0, 38322.9 ± 1771.1, 37673.8 ± 3610.2, and 37577.0 ± 1831.3 mm Hg·beats/s) in the WT, SK2^−/−, WT + B, or SK2^−/− + B groups, respectively. Hearts in the SK2^−/− + B group had significantly longer T_arrhythmia and T_asystole values than did hearts in the WT + B group (both P < 0.001). There was no occurrence of arrhythmia in the WT or SK2^−/− group (Table 1).

Table 1.

Times to defined HR, RPP, and ECG endpoints after bupivacaine perfusion.^a

Times to event (second)	WT	SK2^−/−	WT + B	SK2^−/− + B
T_arrhythmia	—	—	169.4 ± 10.6	296.3 ± 18.1
T_asystole	—	—	354.8 ± 17.8	561.6 ± 18.0
HR, 25% reduction	—	—	132.9 ± 9.7	209.9 ± 22.7
HR, 50% reduction	—	—	247.9 ± 14.2	339.3 ± 20.0
HR, 75% reduction	—	—	303.8 ± 9.6	426.0 ± 15.3
RPP, 25% reduction	—	—	126.4 ± 9.8	208.5 ± 25.3
RPP, 50% reduction	—	—	222.4 ± 19.2	321.1 ± 15.3
RPP, 75% reduction	—	—	282.5 ± 12.4	404.9 ± 9.3

WT: wild type mice; SK₂ ^−/−: SK₂ knockout mice; B: bupivacaine. T_arrhythmia: the time to first ventricular premature beat after perfused with KHB containing 40 uM bupivacaine; T_asystole: the time to asystole after perfused with KHB containing 40 uM bupivacaine; HR: heart rate; RPP: rate-pressure product.

^a Data are given as the mean ± standard deviation.

Cardiac function variables

Following bupivacaine infusion initiation, the hearts in the SK₂ ^−/− + B group exhibited a significantly longer time to 25%, 50%, and 75% reduction in HR and RPP relative to those in the WT + B group (Figures 3 and 4). Within 1–7 minutes of bupivacaine perfusion, the HR and RPP values in the SK₂ ^−/− + B group had significantly increased than the values in WT + B group (Figures 3 and 4).

Figure 3.

Changes in HR (beats/s) among the four study groups (n = 8 for each group). HRs (Heart Rate) for the four treatment groups are shown. Data are means ± standard deviation. The error bar on each data point indicates the standard deviation from the mean value. *P < 0.05, between WT + B and SK₂ ^−/− + B groups. WT: wild type mice; SK₂ ^−/−: SK₂ knockout mice; B: bupivacaine.

Figure 4.

Changes in RPP (mm Hg·beats/s) among the four study groups (n = 8 for each group). RPPs (Rate-pressure product) for the four treatment groups are shown. Data are means ± standard deviation. The error bar on each data point indicates the standard deviation from the mean value. *P < 0.05, between WT + B and SK₂ ^−/− + B groups. WT: wild type mice; SK₂ ^−/−: SK₂ knockout mice; B: bupivacaine.

Discussion

The results of this study revealed that bupivacaine was capable of dose-dependent inhibition of SK₂ currents, with an IC₅₀ value of 18.6 µM. We further found that deleting SK₂ channels was associated with a significant increase in time to first arrhythmia, time to asystole, and time to 25%, 50%, and 75% reductions in HR and RPP following cardiac bupivacaine infusion.

The isolated cardiac perfusion model has certain advantages. First, it can exclude the systematic effect of bupivacaine on our observations of bupivacaine cardiotoxicity. In addition, the interference factor in this model is small, which can reduce the total requirement of knockout mice. The bupivacaine concentration (40 µM) used in the present study was derived from our previous work,²³ at which all isolated hearts stop beating. Due to ventricular premature was very easy to identify on the electrocardiogram, so we defined it as arrhythmia in the study.

There are several different types of ion channels that play essential roles in regulating cardiac function, including sodium channels,⁷ L-calcium channels,²⁴ transient outward potassium channels,²⁵ and beta adrenergic receptors.²⁶ Abnormal functioning of these channels or receptors can disrupt normal cardiac electrophysiology and systolic function, and its abnormalities can affect the normal functioning of the heart. Sodium channels are particularly important for action potential depolarization, whereas L-calcium channels are important for the repolarization plateau, and transient outward potassium channel currents are vital for normal repolarization. The disrupted function of these three types of ion channels has previously been proposed as the primary mechanism underlying bupivacaine-induced cardiotoxicity.^10,27,28 However, there are many other ion channels within the cardiac tissue, raising the possibility that other pathways are also involved in this toxicity.

Very few researchers have assessed the impact of bupivacaine on calcium-activated potassium channels. Martín et al.²⁹ assessed the ability of bupivacaine to inhibit large conductance calcium-activated potassium channels (BK) using human umbilical artery smooth muscle cells. In so doing he observed dose-dependent BK inhibition mediated by bupivacaine. However, no previous reports have explored the ability of bupivacaine to affect SK₂ channel activity. In the present study, we found that bupivacaine inhibited currents through SK₂ channels on HEK293 cells, with an IC₅₀ value of 18.6 μM. This suggests that SK₂ currents are sensitive to bupivacaine. In clinical instances where bupivacaine accidentally enters the bloodstream, circulating levels can readily exceed 10 μM. To further verify the role of SK₂ channels in bupivacaine-induced cardiotoxicity, we therefore next conducted in vivo studies using mice in which this ion channel had been knocked out.

Following knockout of certain genes, compensatory pathways are often engaged, leading to the lack of any apparent phenotype under baseline conditions following gene knockout.^30,31 Consistent with this, SK₂ knockout mice did not exhibit arrhythmia at baseline. This may be because a loss of SK₂ channel expression in the cardiomyocytes may have led to the induction of compensatory mechanisms that achieved normal cardiomyocyte homeostasis, although the specific pathways involved remain uncertain.

We hypothesized that SK₂ channel knockout may help to reduced bupivacaine-induced cardiotoxicity as these channels are targeted by bupivacaine. Consistent with this hypothesis, SK₂ knockout mice exhibited significantly longer times to arrhythmia and cardiac arrest following bupivacaine induction than did WT mice. This thus revealed that SK₂ gene knockout delayed the onset of bupivacaine-induced cardiotoxicity in isolated mouse hearts.

SK₂ currents can be blocked by apamin, with Chang et al.³² having shown that apamin induced early afterdepolarizations, premature ventricular beats, and torsades de pointes ventricular arrhythmia from area failing rabbit ventricles exhibiting secondary. In addition, SK₂ gene variants are linked to lethal ventricular arrhythmias in patients suffering from underlying heart diseases.¹⁸ This suggests that inhibiting SK₂ current can alter normal cardiac conductivity. Our present results further reveal that bupivacaine is capable of blocking SK₂ currents, which likely contributes to bupivacaine-induced cardiotoxicity. While SK₂-knockout mice did eventually suffer from cardiac arrest, there was a significant delay in arrhythmia development relative to WT mice, suggesting that the knock out of this gene improved bupivacaine resistance in mice isolated hearts.

Limitations

In this report we utilized HEK-293 cells expressing SK₂ channels for whole-cell patch clamp experiments. Such an approach was limited by the fact that this expression was not physiological, and so may not be representative of normal human or mouse cardiomyocytes. Furthermore, we have not explored potential compensatory mechanisms which may be active in mice following knockout of the SK₂ gene.

Conclusion

Bupivacaine was able to mediate a dose-dependent inhibition of SK₂ channels in HEK-293 cells. Knockout of SK₂ channel was associated with a delay in the ability of bupivacaine to induce cardiotoxicity in mouse isolated hearts. Our team will try to further verify our results in vivo.

Footnotes

Abbreviations

Acknowledgment

The authors would like to thank Thomas J Papadimos of University of Toledo College of Medicine and Life Sciences and Yun Xia of the Ohio State University Medical Center for revising the manuscript.

Author contributions

HC participated in conduct of the study, data collection, manuscript preparation, and data analysis; FX was responsible for study design, conduct of the study, data analysis, and manuscript preparation; XC and YC were responsible for study design, conduct of the study, data analysis, and study data collection; and ZJ was responsible for study design, conduct of the study, and manuscript preparation.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Natural Science Foundation of Zhejiang Province, China [Grant Number: LQ18H090006] and Wenzhou Municipal Scientific and Technological Program Projects [Grant Number: Y20190410].

ORCID iD

H Chen

References

Baliuliene

Macas

Rimaitis

. The optimal concentration of bupivacaine and levobupivacaine for labor pain management using patient-controlled epidural analgesia: a double-blind, randomized controlled trial. Int J Obstet Anesth 2018; 35: 17–25.

Dolin

Cashman

Bland

. Effectiveness of acute postoperative pain management: I. Evidence from published data. Br J Anaesth 2002; 89: 409–423.

Moore

Bridenbaugh

Thompson

, et al. Bupivacaine: a review of 11,080 cases. Anesth Analg 1978; 57: 42–53.

Slagt

Ketelaars

Swenne

, et al. Local anesthetic systemic toxicity (LAST), needs treatment. J Emerg Med 2019; 56: 107–108.

Hussain

McCartney

Neal

, et al. Local anaesthetic-induced myotoxicity in regional anaesthesia: a systematic review and empirical analysis. Br J Anaesth 2018; 121: 822–841.

Neal

Barrington

Fettiplace

, et al. The third American Society of Regional Anesthesia and Pain Medicine practice advisory on local anesthetic systemic toxicity: executive summary 2017. Reg Anesth Pain Med 2018; 43: 113–123.

Clarkson

Hondeghem

. Mechanism for bupivacaine depression of cardiac conduction: fast block of sodium channels during the action potential with slow recovery from block during diastole. Anesthesiology 1985; 62: 396–405.

Rossner

Freese

. Bupivacaine inhibition of L-type calcium current in ventricular cardiomyocytes of hamster. Anesthesiology 1997; 87: 926–934.

Lee

Weeks

Kao

, et al. Sodium current in single heart muscle cells. Nature 1979; 278: 269–271.

10.

Shibuya

Momose

Ito

. Effects of bupivacaine on contraction and membrane potential in isolated canine papillary muscles. Pharmacology 1993; 47: 158–166.

11.

Valenzuela

Snyders

Bennett

, et al. Stereoselective block of cardiac sodium channels by bupivacaine in guinea pig ventricular myocytes. Circulation 1995; 92: 3014–3024.

12.

Reher

Wang

Hsueh

, et al. Small-conductance calcium-activated potassium current in normal rabbit cardiac Purkinje cells. J Am Heart Assoc 2017; 6: e005471.

13.

Timofeyev

Tuteja

, et al. Ablation of a Ca²⁺-activated K⁺ channel (SK2 channel) results in action potential prolongation in atrial myocytes and atrial fibrillation. J Physiol 2009; 587: 1087–1100.

14.

Xia

Fakler

Rivard

, et al. Mechanism of calcium gating in small-conductance calcium-activated potassium channels. Nature 1998; 395: 503–507.

15.

Willis

Trieb

Leitner

, et al. Small-conductance calcium-activated potassium type 2 channels (SK2, KCa2.2) in human brain. Brain Struct Funct 2017; 222: 973–979.

16.

Tuteja

Timofeyev

, et al. Differential expression of small-conductance Ca²⁺-activated K⁺ channels SK1, SK2, and SK3 in mouse atrial and ventricular myocytes. Am J Physiol Heart Circ Physiol 2005; 289: H2714–2723.

17.

Skibsbye

Poulet

Diness

, et al. Small-conductance calcium-activated potassium (SK) channels contribute to action potential repolarization in human atria. Cardiovasc Res 2014; 103: 156–167.

18.

Hsieh

Chang

Hsueh

, et al. Apamin-sensitive potassium current modulates action potential duration restitution and arrhythmogenesis of failing rabbit ventricles. Circ Arrhythm Electrophysiol 2013; 6: 410–418.

19.

Chia-Ti

Chen

, et al. KCNN2 polymorphisms and cardiac tachyarrhythmias. Medicine (Abingdon) 2016; 95: e4312.

20.

Butterworth

. Models and mechanisms of local anesthetic cardiac toxicity: a review. Reg Anesth Pain Med 2010; 35: 167–176.

21.

Chen

Liu

Sun

, et al. The natural flavone acacetin blocks small conductance Ca²⁺-activated K⁺ channels stably expressed in HEK-293 cells. Front Pharmacol 2017; 8: 716.

22.

Liu

, et al. Epinephrine reversed high-concentration bupivacaine-induced inhibition of calcium channels and transient outward potassium current channels, but not on sodium channel in ventricular myocytes of rats. BMC Anesthesiol 2015; 15: 66.

23.

Chen

Xia

Liu

, et al. Lipid emulsion reverses bupivacaine induced asystole in isolated rat hearts: concentration response and time response relationships. Anesthesiology 2010; 113(6): 1320–1325.

24.

Schroüder

Handrock

Beuckelmann

, et al. Increased availability and open probability of single L-type calcium channels from failing compared with nonfailing human ventricle. Circulation 1998; 98: 969–976.

25.

Kuryshev

Brittenham

Fujioka

, et al. Decreased sodium and increased transient outward potassium currents in iron-loaded cardiac myocytes: implications for the arrhythmogenesis of human siderotic heart disease. Circulation 1999; 100: 675–683.

26.

Ungerer

Böhm

Elce

, et al. Altered expression of beta-adrenergic receptor kinase and beta 1-adrenergic receptors in the failing human heart. Circulation 1993; 87: 454–463.

27.

Zapata-Sudo

Trachez

Sudo

, et al.

Is comparative cardiotoxicity of S(−) and R(+) bupivacaine related to enantiomer-selective inhibition of L-type Ca²⁺ channels?

Anesth Analg 2001; 92: 496–501.

28.

Courtney

Kendig

. Bupivacaine is an effective potassium channel blocker in heart. Biochim Biophys Acta 1988; 939: 163–166.

29.

Martín

Enrique

Palomo

, et al. Bupivacaine inhibits large conductance, voltage- and Ca²⁺-activated K⁺ channels in human umbilical artery smooth muscle cells. Channels (Austin) 2012; 6: 174–180.

30.

Wilkinson

. Genetic paradox explained by nonsense. Nature 2019; 568: 179–180.

31.

Rossi

Kontarakis

Gerri

, et al. Genetic compensation induced by deleterious mutations but not gene knockdowns. Nature 2015; 524: 230–233.

32.

Chang

Hsieh

Hsueh

, et al. Apamin induces early afterdepolarizations and torsades de pointes ventricular arrhythmia from failing rabbit ventricles exhibiting secondary rises in intracellular calcium. Heart Rhythm 2013; 10: 1516–1524.

Ablation of small conductance calcium-activated potassium type-2 channel (SK 2 ) delays occurrence of bupivacaine-induced cardiotoxicity in isolated mouse hearts

Abstract

Keywords

Introduction

Method

Chemicals

Cell culture and transfection

SK2 current recordings

Preparation of Langendorff-isolated heart

Assessment of cardiotoxicity

Statistical analyses

Results

Bupivacaine inhibits SK2 currents in a dose-dependent fashion

Baseline parameters, Tarrhythmia and Tasystole for mouse studies

Cardiac function variables

Discussion

Limitations

Conclusion

Footnotes

Abbreviations

Acknowledgment

Author contributions

Declaration of conflicting interests

Funding

ORCID iD

References

SK₂ current recordings

Bupivacaine inhibits SK₂ currents in a dose-dependent fashion

Baseline parameters, T_arrhythmia and T_asystole for mouse studies