anesthesiology 1 miocitos protocolo

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Antilg, V XX N XX 1 XXX 2013

ABSTRACT

Background: Cardiovascular resuscitation upon intoxica-tion with lipophilic ion channelblocking agents has provenmost difficult. Recently, favorable results have been reportedwhen lipid rescue therapy is performed, i.e., the infusion ofa triglyceride-rich lipid emulsion during resuscitation. How-ever, the mechanism of action is poorly understood.Methods:Te authors investigate the effects of a clinicallyused lipid emulsion (Lipovens MC 20%; Fresenius KabiAG, Bad Homburg, Germany) on the block of the fastNa+current (I

Na) induced by the lipophilic local anesthetic

bupivacaine in adult rat left ventricular myocytes by usingthe whole cell patch clamp technique.Results: Bupivacaine at 10 M decreased I

Na by 54%

(19.3 1.9 pApF1 vs. 42.3 4.3 pApF1; n = 17;P< 0.001; V

Pip= 40 mV, 1 Hz). Addition of 10% lipid

emulsion in the presence of bupivacaine produced a 37%increase in I

Na (26.4 2.8 pApF1; n = 17; P< 0.001 vs.

bupivacaine alone). o test whether these results could beexplained by a reduction in the free bupivacaine concentra-tion by the lipid (lipid-sink effect), the authors removedthe lipid phase from the bupivacainelipid mixture byultracentrifugation. Also, the resulting water phase led toan increase in I

Na (+19%; n = 17; P < 0.001 vs.bupiva-

caine), demonstrating that part of the bupivacaine had

What We Already Know about This Topic

Cardiovascular resuscitation secondary to intoxication withlipophilic ion channelblocking agents has proven to be a sig-nicant therapeutic challenge. Recently, favorable results havebeen reported when lipid rescue therapy is performed.

This study determined whether the application of a clinicallyused lipid emulsion might reverse the bupivacaine-inducedchanges in the action potential and the fast Na+current (I

Na)

of left ventricular cardiomyocytes and compared the resultswith those obtained with the hydrophilic local anestheticmepivacaine

What This Article Tells Us That Is New

The study demonstrates that a lipid emulsion partially reversesthe effects of bupivacaine on the action potential and the fastNa+ current of left ventricular cardiomyocytes and providesevidence for a lipid-sink mechanism

Supplemental digital content is available for this article. DirectURL citations appear in the printed text and are available inboth the HTML and PDF versions of this article. Links to thedigital les are provided in the HTML text of this article on theJournals Web site (www.anesthesiology.org).

Copyright 2013, the American Society of Anesthesiologists, Inc. LippincottWilliams & Wilkins.Anesthesiology 2013; XX:00-00

* Research Associate, Medical Student, # Professor, Institutfr Zellulre und Molekulare Physiologie, Friedrich-Alexander-Universitt Erlangen-Nrnberg, Erlangen, Germany. Consultant, Research Associate, Professor, Klinik fr Ansthesiologie, Uni-

versittsklinikum Regensburg, Regensburg, Germany.

Received from the Institut fr Zellulre und Molekulare Physio-logie, Friedrich-Alexander Universitt Erlangen-Nrnberg, Erlan-gen, Germany. Submitted for publication April 30, 2013. Acceptedfor publication July 17, 2013. Support was provided by Friedrich-

Alexander-Universitt Erlangen-Nrnberg, Erlangen, Germany;Universittsklinikum Regensburg, Regensburg, Germany; and the

Johannes und Frieda Marohn-Stiftung, Erlangen, Germany. Thiswork has been presented in part at the 91st Meeting of the Ger-man Physiological Society, Dresden, Germany, March 23, 2012; the59th Annual Meeting of the German Society for Anesthesiology

and Intensive Care Medicine, Leipzig, Germany, May 5, 2012; the92nd Annual Meeting of the German Physiological Society, Heidel-berg, Germany, March 3, 2013; and the 79th Annual Meeting of theGerman Cardiac Society, Mannheim, Germany, April 5, 2013. Theauthors declare no competing interests. Drs. Wagner and Zausigcontributed equally to this work. Drs. Graf and Volk contributedequally to this work.

Address correspondence to Dr. Wagner or Dr. Volk: Institut frZellulre und Molekulare Physiologie, Friedrich-Alexander-Univer-sitt Erlangen-Nrnberg, Waldstrae 6, 91054 Erlangen, [email protected] or [email protected]. Information onpurchasing reprints may be found atwww.anesthesiology.orgor onthe masthead page at the beginning of this issue. ANESTHESIOLOGYsarticles are made freely accessible to all readers, for personal useonly, 6 months from the cover date of the issue.

Lipid Rescue Reverses the Bupivacaine-induced Blockof the Fast Na+Current (I

Na) in Cardiomyocytes of the

Rat Left Ventricle

Michael Wagner, M.D., Ph.D.,* York A. Zausig, D.E.A.A., M.D., Ph.D., Stefan Ruf, M.S.,

Elena Rudakova, Ph.D.,* Michael Gruber, Ph.D., Bernhard M. Graf, M.D., Ph.D.,Tilmann Volk, M.D., Ph.D.#

PAIN MEDICINE

Copyright by the American Society of Anesthesiologists. Unauthorized reproduction of this article is prohibited.
http://www.anesthesiology.org/mailto:[email protected]:[email protected]://www.anesthesiology.org/http://www.anesthesiology.org/mailto:[email protected]:[email protected]://www.anesthesiology.org/


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Anesthesiology 2013; XX:00-00 2 Wagnr et al.

Lipid Rescue and Cardiac INa

been removed during ultracentrifugation. Te substantiallyless lipophilic mepivacaine (40 M) reduced I

Na by 27%

(n = 24; P< 0.001). Te mepivacainelipid mixture causeda significant increase in I

Na(+17%; n = 24; P< 0.001). For

mepivacaine, only a small lipid-sink effect could be dem-

onstrated (+8%; n = 23; P< 0.01), reflecting its poor lipidsolubility.Conclusion: Te authors demonstrate lipid rescue onthe single-cell level and provide evidence for a lipid-sinkmechanism.

ACCIDENAL systemic toxicity of local anesthetics(LA) is a rare but potentially life-threatening com-plication of regional anesthesia.1,2Te symptoms usuallyinvolve the central nervous system and the heart, rangingfrom rather harmless signs such as mild discomfort, nausea,dizziness, or minor electrocardiogram changes up to sei-

zures, coma, ventricular fibrillation, and asystole.1

Terapyof severe systemic LA intoxication consisted of advancedcardiac live support and has been a challenge because nospecific therapy was available. Tis situation started tochange some 15 yr ago, when infusion of a commerciallyavailable lipid emulsion was shown to effectively treatintoxications with the lipophilic LA bupivacaine in rats3and later in dogs also.4 Only a few years later, the con-cept was transferred to the clinic, when patients with LAintoxication refractory to conventional treatment were suc-cessfully treated with infusion of a lipid emulsion.5,6Sincethen, numerous case reports have been published reporting

the successful use of lipid emulsion therapy in intoxicationswith LAs and, more recently, other lipophilic drugs7and,moreover, lipid infusion has meanwhile been included inresuscitation guidelines.8

o date, the mechanism of action of lipid resuscita-tion is not well understood. Among different concepts,the so-called lipid-sink hypothesis suggests an accumula-tion of the lipophilic LA in the lipid phase of the lipidemulsion, thereby decreasing the concentration of the LAin the water phase and hence in the tissue. 9According tothis concept, there are experimental observations that lipidemulsion infusion is effective in the treatment of intoxi-cations with the lipophilic LA bupivacaine, but not withthe less lipophilic LAs mepivacaine10and ropivacaine.11,12Moreover, with increasing concentrations of the lipid, adecrease in the myocardial concentration of bupivacainewas observed.13However, lipid resuscitation has also beensuccessfully applied to intoxications with the substantiallyless lipophilic LA lidocaine,14suggesting additional modesof action.

Although the exact mechanism of action is not clearlyunderstood, a common clinical observation reported duringthe application of the lipid emulsion in patients is a rapidimprovement in electrocardiogram alterations, i.e., short-ening of the QRS complexes, decrease in Qc-interval, or

termination of ventricular arrhythmias.15,16Tis suggests a

rather rapid action of the lipid emulsion on the effects ofthe LA on the cardiac action potential (AP) and hence, theunderlying ion channels.

In the current study, we therefore asked whether theapplication of a clinically used lipid emulsion might

reverse the bupivacaine-induced changes in the AP andthe fast Na+ current (INa

) of left ventricular cardiomyo-cytes and compared the results with those obtained withthe hydrophilic LA mepivacaine. We show that a lipidemulsion partially reverses the effects of bupivacaine onthe AP and the Na+current at the cellular level. Moreover,we provide evidence for a lipid-sink mechanism. Finally,by investigating the effect of lipid emulsion on the LAaction on heterologously expressed hKv4.2 + hKChIP2bchannels inXenopus laevisoocytes, we show that the effectof the lipid emulsion is not limited to the AP and the Na+current in cardiomyocytes, but appears as to be a generalmechanism on ion channels.

Materials and Methods

Isolation of Myocytes

Te investigation conforms to the Guide for the Care andUse of Laboratory Animalspublished by the U.S. NationalInstitutes of Health (National Institutes of Health Publica-tion No. 85-23, revised 1996) and was approved by localgovernment authorities (Government of Middle Franconia,Ansbach, Germany). Myocytes were isolated from the car-diac left ventricular free wall of female Wistar rats (weighingapproximately 220 g) as described previously.17,18 Briefly,

after induction of deep anesthesia by intraperitoneallyinjecting thiopental-sodium (100 mg/kg body mass; InresaArzneimittel GmbH, Freiburg, Germany), the heart wasquickly excised and placed into cold (4C) yrode solutionwhere it stopped beating immediately. Subsequently, theheart was perfused with modified yrode solution contain-ing 4.5 mMCa2+and 5 mMEGA (approximately 1 Mfree Ca2+) viathe ascending aorta at 37C for 5 min. Teperfusion was continued for 19 min, recirculating 25 mlof the same solution containing collagenase (CLS type II,160 U/ml; Biochrom KG, Berlin, Germany) and pro-tease (type XIV, 0.6 U/ml; Sigma-Aldrich Corporation,St. Louis, MO). Finally, the heart was perfused with storagesolution19containing 100 M Ca2+for 5 min. Using fineforceps, myocytes were carefully dissected from the subepi-cardial and the subendocardial layer of the left ventricularfree wall and placed in cell culture dishes containing thesame solution. issue pieces were minced and gently agi-tated to obtain single cardiomyocytes. After adaption tophysiological Ca2+levels, cells were transferred to cell cul-ture dishes containing storage solution supplemented with100 U/ml penicillin and 0.1 mg/ml streptomycin, stored at37C in a water saturated atmosphere containing 5% CO

2

and used for experiments for up to 36 h. Only quiescentsingle rod-shaped cells with clear cross striations were used

for experiments.



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Patch Clamp Technique

Te ruptured-patch whole cell configuration was used asdescribed previously.18,20 Currents were recorded using anEPC-10 amplifier (HEKA Elektronik Dr. Schulze GmbH,Lambrecht, Germany), controlled by the PULSE-Software

(HEKA). Membrane capacitance (Cm) and series resistance(Rs) were calculated using the automated capacitance com-

pensation procedure of the EPC-10 amplifier. During APmeasurements, R

s averaged 6.8 2.9 M (mean SD,

n = 95) and Cm

was 130.2 35.0 pF (mean SD, n = 95).During I

Nameasurements, R

saveraged 5.2 1.7 M(mean

SD, n = 88) and was compensated by 80%. Tis resultedin an average effective R

s of 1.0 M, leading to an aver-

age voltage error of 5.5 mV (average current 5.3 2.4 nA[mean SD], n = 88) for the native current, 2.5 mV (averagecurrent 2.4 1.0 nA [mean SD], n = 41) when blockedwith bupivacaine and 4.0 mV (average current 3.8 1.8 nA

[mean SD], n = 47) when blocked by mepivacaine (see alsothe section on limitations in the Discussion). Cm

averaged115.8 24.3 pF (mean SD, n = 88). Effective R

sand C

m

resulted in an average time constant of 120 s for chargingof the membrane capacitance. ogether with the relativelyslow kinetics of I

Na at 40 mV at room temperature, this

allowed the peak of the capacitive artifact to be clearly sepa-rated from the peak of the Na current. Te capacitive artifactwas subtracted using a P/4 leak subtraction protocol. Pipettepotentials were corrected for the liquid junction potential of13 or 9 mV for the solution with reduced Na+concentra-tion, respectively. All experiments were performed at roomtemperature (2224C). For each set of experiments, myo-cytes obtained from two to four rats were used. Some controlexperiments were performed on cells from one rat only. Allreported potentials are pipette potentials.

rains of 150 APs were elicited at 1 Hz in cells with a rest-ing membrane potential negative to 80 mV (a V

mpositive

to 80 mV was considered to be secondary to a leaky electri-cal access to the myocyte) by depolarizing current pulses of5 ms duration. After 50 APs, the solution was switched to thesolution containing the LA (bupivacaine or mepivacaine).After another 50 APs, the solution was switched to the mix-ture of lipid emulsion and the LA. Te last AP under eachcondition was evaluated.

o elicit INa

, cells were clamped to 40 mV for 20 ms andthen for 80 ms to 0 mV. Holding potential was 90 mV andcycle length was 1 s. I

Nawas measured at the pulse to 40

mV to assess the Na+current during the upstroke of the APwhile the pulse to 0 mV served to simulate the plateau phaseof the AP. Te standard protocol consisted of 200 pulses:50 under control conditions, 50 with LA, 50 with LA pluslipid emulsion, and another 50 under control conditions.Te last current under each condition was evaluated. Leaksubtraction was applied using a P/4 protocol, and R

s and

Cm

were automatically readjusted before each pulse. Teextracellular Na+ concentration was reduced to 20 mM by

replacing 118 mMNa+with Cs+. Moreover, this inhibited

the inward-rectifying K+current,21 thus rendering the leaksubtraction possible. All experiments were conducted undercontinuous perfusion of 7 ml/min. Current and voltagerecordings were low-pass filtered at 5 kHz and sampled at25 kHz. o compensate for variability in cell size, currents

were divided by the cell capacitance and are thus given ascurrent densities in pApF-1.

Isolation, Injection, and Maintenance of

X. laevisOocytes

Female X. laevis were anesthetized by immersing in tapwater containing 0.2% MS-222 for 10 min. Ovarian lobeswere surgically removed, and oocytes were isolated by enzy-matic digestion using collagenase (CLS type II, 260 U/ml;Biochrom KG, Berlin, Germany) in Ca2+-free OR2 solu-tion at 10C for 34 h. We used full-length complimentaryDNA transcripts encoding human Kv4.2 (hKv4.2) inserted

in pGEM and human KChIP2b (hKChIP2b) included inpGEM-HJ. Linearized plasmids were used as templatesfor coding RNA synthesis using the mMessage mMachineranscription Kit 7 (Life echnologies, Grand Island, NY).Defolliculated stage V and VI oocytes were injected with0.1 ng hKv4.2 + 0.5 ng hKChIP2b coding RNA. CodingRNAs were dissolved in RNAse-free water, and the total vol-ume injected was 50 nl per oocyte. After injection, oocyteswere maintained in ND96 solution and were studied 2 daysafter injection.

Two-electrode Voltage Clamp Experiments

Oocytes were transferred to a perfusion chamber, whichis continuously superfused with NaCl-95 solution, andimpaled with electrodes (0.11.5 M) filled with 3 M KCl.o increase the Lipovens (Fresenius Kabi AG, Bad Hom-burg, Germany) content to more than 10% in the bath solu-tion, a stock of a modified bath solution was designed whichwas diluted as needed with Lipovens or control to assurea constant K+ concentration of 4 mMand Ca2+ and Mg2+concentrations of 1 mMeach. Whole cell currents were mea-sured at room temperature (1922C) with the two-elec-trode voltage clamp technique using an OC-725C amplifier(Warner Instruments LLC, Hamden, C) controlled by thePulse-software (HEKA) viaan LIH-1600 interface (HEKA).An AgAgCl pellet placed directly in the bath solutionserved as a reference electrode for the current injection cir-cuit, whereas an additional AgAgCl pellet located close tothe oocyte was used to sense the bath potential to minimizeseries resistance errors. Currents were elicited by voltagesteps to +40 mV from a holding potential of 90 mV. Pulsedcurrent data were filtered at 1 kHz and sampled at 5 kHz.

Measurement of LA Concentrations

Bupivacaine and mepivacaine concentrations were mea-sured by gas chromatographymass spectrometry using anAgilent model 6890plus gas chromatograph and an MSD

5973 in the electron impact selected ion monitoring mode



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Lipid Rescue and Cardiac INa

(Agilent echnologies Inc., Santa Clara, CA). Extraction ofthe samples was adopted from the study by Colin et al.22Shortly, samples (50 l) were extracted by fluidfluid extrac-tion using hexane:ethyl acetate (70:30, 4 ml) plus 0.05%Lipofundin (20%; B. Braun Melsungen AG, Melsungen,

Germany) and glycine buffer at pH 9.0 (250 l), dried,solved in toluol:methanol (20:1, 250 l) and injected intothe gas chromatograph (0.2 l, split mode 20:1 at 285C).Ropivacaine (m/z = 126.1 and retention time = 3.97 min)was used as internal standard for bupivacaine (m/z = 140.1and retention time = 4.18 min, r2= 0.99) and mepivacaine(m/z = 98.1 and retention time = 3.65 min, r2 = 1.00)quantification. Separation was achieved on a PhenomenexZebron ZB-1ms column (30 m 0.25 mM 0.25 M; Phe-nomenex Inc., Aschaffenburg, Germany) at a starting tem-perature and period of 200C and 2 min followed by a lineartemperature gradient (60 K/min) up to 300C. Linearity of

the detector was given over the whole concentration rangewith relative recovery rates of 95.7 3.7% (mepivacaine,n = 17) and 100.0 6.3% (bupivacaine, n = 17) and lowerlimits of quantification of 1.43 M (mepivacaine) and1.03 M (bupivacaine).

Solutions and Drugs

Modified yrode solution was used for cell isolation and asbath solution and contained NaCl, 138 mM; KCl, 4 mM;MgCl

2, 1 mM; NaH

2PO

4, 0.33 mM; CaCl

2, 2 mM; glu-

cose, 10 mM; and HEPES, 10 mM (pH 7.30 with NaOH).Te Na+concentration was reduced to 20 mM by replacing118 mMNa+with Cs+ for measuring I

Na

. Myocyte storagesolution contained NaCl, 130 mM; NaH

2PO

4, 0.4 mM;

NaHCO3, 5.8 mM; MgCl

2, 0.5 mM; CaCl

2, 1 mM; KCl,

5.4 mM; glucose, 22 mM; and HEPES, 25 mM(pH 7.40with NaOH in the presence of 5% CO

2) and supplemented

with 1 mg/ml bovine serum albumin (albumin fraction V;Merck KGaA, Darmstadt, Germany), 100 U/ml of penicillin,and 0.1 mg/ml of streptomycin. Te pipette solution con-tained glutamic acid, 120 mM; KCl, 10 mM; MgCl

2, 4 mM;

EGA, 10 mM; HEPES, 10 mM; and Na2AP, 2 mM(pH

7.20 with KOH). ND96 solution contained NaCl, 96 mM;KCl, 2 mM; MgCl

2, 1 mM; CaCl

2, 1.8 mM; and HEPES, 5

mM(pH 7.40 with NaOH), supplemented with 100 U/mlof penicillin and 0.1 mg/ml of streptomycin (Sigma-AldrichCorporation). NaCl-95 solution contained NaCl, 95 mM;KCl, 4 mM; MgCl

2, 1 mM; CaCl

2, 1 mM; and HEPES, 10 mM

(pH 7.40 with ris). Te 5 stock of the modified bath solutioncontained NaCl, 60 mM; KCl, 18.25 mM; MgCl

2, 5 mM;

CaCl2, 5 mM; and HEPES, 20 mM(pH 7.4 with NaOH).

Bupivacaine and mepivacaine were obtained from Sigmaand were freshly dissolved in the bath solution on each dayof experiments. Bupivacaine was used at a concentration of10 M in most experiments, a plasma concentration thatcan be reached in patients.16 Mepivacaine was used at aconcentration of 40 M, a concentration that was reported

to cause similar cardiotoxicity as 10 M bupivacaine.10

Experiments in which the lipid emulsion (Lipovens MC20%; Fresenius Kabi AG) was applied were started with abath solution containing 10% of a control solution adaptedto the Na+content (approximately 2 mM) and osmolarity(290 mOsm, adapted with mannitol in the control solu-

tion) of Lipovens. Eventually, the solution was exchangedto the same solution containing 10% of the lipid emulsioninstead of control. Ultracentrifugation was performed at40,000 rpm (110,000g) in an Optima L60 ultracentrifuge(Beckman Coulter Inc., Brea, CA) using a i70.1 rotor for2 h at 4C.

Data Evaluation and Statistical Analysis

Data were analyzed using the PULSE-FI software (HEKA),IGOR Pro (WaveMetrics Inc., Lake Oswego, OR), andMicrosoft Excel (Microsoft Corporation, Redmond, WA) asdescribed previously.18o indicate the precision of the meanvalues, average data are given together with the SEM (mean SEM) and the number of experiments if not stated oth-erwise. o report the variability of the data, means togetherwith their respective SDs are given in the table, Supplemen-tal Digital Content 1, http://links.lww.com/ALN/A968,and 95% CIs are detailed in the table, Supplemental DigitalContent 2, http://links.lww.com/ALN/A969.23o estimatethe reduction in the bupivacaine concentration by the lipidemulsion, concentrationresponse analysis was performed inthe relevant range of concentrations assuming the previouslydescribed complete block of I

Na at high concentrations of

bupivacaine.24Concentrationresponse curves of Kv chan-nels were calculated at +40 mV from charge rather than

current amplitude because bupivacaine and mepivacaine areopen-channel blockers of Kv4 channels.25,26In both cases, aHill function with variable slope was fitted to the data. Sta-tistical significance was evaluated by paired or unpaired Stu-dent ttest or paired or unpaired one-way ANOVA followedby a NewmanKeuls multiple comparison test when morethan two groups were compared using Prism 5 (GraphPadSoftware Inc., La Jolla, CA). A two-tailed Pvalue of less than0.05 was considered statistically significant.

Results

Lipid Emulsion Reverses the Effects of Bupivacaine on

the Upstroke of the AP

Action potentials were recorded to assess the overall effect oflipid rescue on the cardiac cellular electrophysiology. Becauseof the heterogeneity of AP duration (APD) within the rat leftventricular free wall,27myocytes from epicardial and endo-cardial regions were analyzed separately. Figure 1 depicts theeffect of lipid rescue on the epicardial AP; 10 M of thelipophilic bupivacaine, a concentration reportedly reachedduring intoxication,16 significantly reduced the overshoot(fig. 1D; P< 0.001) and maximal upstroke velocity (by 56%;fig. 1E; P< 0.001) of the AP (table 1). Lipid rescue with 10%Lipovens partially reversed the effect on the overshoot (P

anesthesiology 1 miocitos protocolo

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