Limiting Sarcolemmal Na+Entry during Resuscitation from Ventricular Fibrillation Prevents
Excess Mitochondrial Ca2+ Accumulation and Attenuates Myocardial Injury
Sufen Wang, PhD1; Jeejabai Radhakrishnan, PhD1; Iyad M. Ayoub, MS2; Julieta D. Kolarova, MD3;
Domenico M. Taglieri, MD1; and Raúl J. Gazmuri, MD, PhD, FCCM4
Department of Medicine, Division of Critical Care Medicine, and Department of Physiology &
Biophysics, Rosalind Franklin University of Medicine and Science
Medical Service, Section of Critical Care Medicine,
North Chicago VA Medical Center
North Chicago, Illinois 60064
1Post-doctoral Fellow, 2Research Instructor, 3Research Associate, 4Professor of Medicine and Associate
Professor of Physiology & Biophysics, Rosalind Franklin University of Medicine and Science and North
Chicago VA Medical Center.
Running Head: Limiting sarcolemmal Na+entry during ventricular fibrillation.
Please address correspondence to:
Raúl J. Gazmuri, MD, PhD, FCCM
Medical Service (111F)
North Chicago VA Medical Center
3001 Green Bay Road; North Chicago, Illinois, 60064
Phone: (224) 610-3681
Fax: (224) 610-3741
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Articles in PresS. J Appl Physiol (April 12, 2007). doi:10.1152/japplphysiol.01167.2006
Copyright © 2007 by the American Physiological Society.
Background: Intracellular Na+accumulation during ischemia and reperfusion leads to cytosolic Ca2+
overload through reverse mode operation of the sarcolemmal Na+-Ca2+ exchanger. Cytosolic Ca2+
accumulation promotes mitochondrial Ca2+ (Ca2+
m) overload leading to mitochondrial injury. We
investigated whether limiting sarcolemmal Na+entry during resuscitation from ventricular fibrillation (VF)
moverload and lessens myocardial dysfunction in a rat model of VF and closed-chest
resuscitation. Methods: Hearts were harvested from 10 groups of 6 rats each representing baseline, 15
minutes of untreated VF, 15 minutes of VF with chest compression given for the last 5 minutes (VF/CC),
and 60 minutes post-resuscitation (PR). VF/CC and PR included 4 groups each randomized to receive
before starting chest compression the new NHE-1 inhibitor AVE4454B (1.0 mg/kg), the Na+channel
blocker lidocaine (5.0 mg/kg), their combination, or vehicle control. The left ventricle was processed for
intracellular Na+and Ca2+
mmeasurements. Results: Limiting sarcolemmal Na+entry attenuated cytosolic
Na+increase during VF/CC and the PR phase and prevented Ca2+
moverload yielding levels that
corresponded to 77% and 71% of control hearts at VF/CC and PR, without differences among specific
Na+-limiting interventions. Limiting sarcolemmal Na+entry attenuated reductions in left ventricular
compliance during VF and prompted higher mean aortic pressure (110 ± 7 vs 95 ± 11 mmHg, p < 0.001)
and higher cardiac work index (159 ± 34 vs 126 ± 29 g·m/min/kg, p < 0.05) with lesser increases in
circulating cardiac troponin I at 60 minutes PR. Conclusions: Na+-limiting interventions prevented excess
maccumulation induced by ischemia and reperfusion and ameliorated myocardial injury and
Key words: Calcium, cardiopulmonary resuscitation, myocardial ischemia, sodium, ventricular
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Increased sarcolemmal Na+influx with consequent cytosolic Na+accumulation due to inability of the
Na+-K+ATPase to extrude Na+represents an important pathophysiological mechanism responsible for
cell injury during ischemia and reperfusion (3,28). Main routes for sarcolemmal Na+entry include the
sodium-hydrogen exchanger isoform-1 (NHE-1), Na+channels, and the Na+-HCO3
However, NHE-1 and Na+channels appear to be the preferred routes for Na+entry during ischemia and
reperfusion (21,38,54). NHE-1 is activated by the intense intracellular acidosis that accompanies ischemia,
initiating an electroneutral sarcolemmal Na+-H+exchange. Na+channels are activated following
sarcolemmal depolarization. However, they inactivate slowly during ischemia and contribute to cytosolic
Cytosolic Na+accumulation is believed to worsen ischemic injury mainly as a result of increased Ca2+
entry through the sarcolemmal Na+-Ca2+ exchanger isoform-1 (NCX-1) operating in reverse mode (1).
Cytosolic Ca2+ overload, in turn, leads to mitochondrial Ca2+ overload which can worsen cell injury by
disrupting mitochondrial function (32,61). Cytosolic Ca2+ overload can also favor reperfusion arrhythmias
through delayed afterdepolarizations causing ventricular arrhythmias (13,60). Despite the prevailing
belief that Ca2+ drives injury associated with cytosolic Na+overload, Iwai et al have reported that
cytosolic Na+overload may directly alter mitochondrial function by depolarizing its inner membrane and
reducing the rate of oxidative phosphorylation (30,31).
Most of the mechanistic knowledge gained on the beneficial effects of limiting cytosolic Na+overload has
resulted from work in isolated cardiac myocytes, isolated heart preparations, and intact animal models of
global or regional ischemia. We have focused our work on understanding the effects of NHE-1 inhibition
during resuscitation from cardiac arrest precipitated by VF. In this setting, the effects of ischemia (cardiac
arrest) and reperfusion (resuscitation) are compounded by VF, which intensifies ischemic injury and
prompts additional Na+entry through activation of Na+channels. We have previously reported that during
resuscitation from VF administration of NHE-1 inhibitors (i) ameliorates or prevents decreases in left
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ventricular compliance, (ii) attenuates reperfusion arrhythmias eliminating recurrent episodes of VF, and
(iii) lessens post-resuscitation myocardial dysfunction (4,5,16,18,37). We designed the current studies to
investigate during resuscitation from VF (i) whether the effects of NHE-1 inhibition are in fact associated
with lesser increases in cytosolic Na+, (ii) whether similar or additive effects can be elicited by blockade
of Na+channels, (iii) whether attenuation of cytosolic Na+overload limits mitochondrial Ca2+ increases,
and (iv) whether these cellular effects result in less myocardial injury and dysfunction in an intact rat
model of VF and closed-chest resuscitation.
Hearts were harvested at various time-events representative of baseline, untreated VF, closed-chest
resuscitation, and post-resuscitation while sarcolemmal Na+entry was limited by administration of the
new NHE-1 inhibitor AVE4454B, the Na+channels blocker lidocaine, or both. Left ventricular tissue was
processed to determine cytosolic Na+content (using Co-EDTA-as marker of the extracellular space) and
total mitochondrial Ca2+ content. These findings were related to myocardial function and circulating
levels of cardiac troponin I (cTnI). After observing that intracellular Na+remained elevated post-
resuscitation, we measured activity of the sarcolemmal Na+-K+ATPase in a separate series of
experiments. The studies showed that limiting sarcolemmal Na+-entry during resuscitation from VF
attenuates cytosolic Na+increases, prevents excess mitochondrial Ca2+ accumulation, attenuates increases
in cTnI, and ameliorates functional myocardial manifestations of ischemic injury.
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The studies were approved by our Institutional Animal Care and Use Committee and conducted in
accordance with institutional guidelines.
Adult male Sprague-Dawley rats (463 to 570 g) were anesthetized using sodium pentobarbital (45 mg/kg
intraperitoneal for induction and 10 mg/kg intravenous for maintenance every 30 minutes). A 5-F cannula
was orally advanced into the trachea and used for positive pressure ventilation during cardiac
resuscitation and the post-resuscitation interval. Proper placement was verified using an infrared CO2
analyzer (CO2SMO model 7100, Novametrix Medical Systems, Inc.). A conventional lead II ECG was
recorded through subcutaneous needles. PE50 catheters were advanced through the right femoral vein into
the right atrium and from the left femoral artery into the abdominal aorta for pressure measurement and
blood sampling. A thermocouple microprobe (IT-18, Physitemp) was advanced through the right femoral
artery into the thoracic aorta and used for measuring cardiac output and core temperature. A PE50
catheter was advanced through the left external jugular vein into the right atrium and used exclusively for
injection of thermal tracer. A 3-F catheter (C-PUM-301J, Cook, Inc.) was advanced through the right
external jugular vein into the right atrium. A pre-curved guide wire was then fed through its lumen,
advanced into the right ventricle, and used for electrical induction of VF. Core temperature was
maintained between 36.5°C and 37.5°C using an infrared heating lamp.
VF and Resuscitation Protocols
VF was induced by delivering a 60-Hz alternating current (0.1 to 0.6 mA) to the right ventricular
endocardium for an uninterrupted interval of three minutes after which the current was turned off and VF
allowed to continue until completion of a predetermined interval (described below). Chest compression
was then started using an electronically controlled and pneumatically driven (50 PSI) chest compressor
(CJ-80623, CJ Enterprises) set to deliver 200 compressions per minute with a 50% duty cycle. The depth
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of compression was adjusted within the first minute to attain an aortic diastolic pressure between 26 and
28 mmHg and ensure a coronary perfusion pressure above the resuscitability threshold of 20 mmHg in
rats (57). The depth of compression was adjusted during the remaining interval of chest compression to
maintain the aortic diastolic pressure within the target range. Positive pressure ventilation with 100%
oxygen was provided using a volume controlled ventilator (model 683, Harvard Apparatus) programmed
to deliver 6 ml/kg body weight at 25 breaths per minute unsynchronized to chest compression.
Defibrillation was attempted after 5 minutes of chest compression by delivering a maximum of two 3-J
transthoracic shocks using a biphasic waveform defibrillator (Smart Biphasic Heartstream XL M4735A,
Agilent Technologies). If VF persisted or an organized rhythm with a mean aortic pressure of ≤ 25 mmHg
ensued, chest compression was resumed for 30 seconds. The defibrillation-compression cycle was
repeated up to three additional times, increasing the energy of individual shocks if VF persisted to 5-J and
then to 7-J for the last two cycles. Successful resuscitation was defined as the return of an organized
cardiac rhythm with a mean aortic pressure ≥ 60 mmHg for ≥ 5 minutes. After return of spontaneous
circulation, the ventilation rate was increased to 60 breaths per minute. Resuscitated rats were ventilated
initially with 100% oxygen for 15 minutes and then continued with 50% oxygen for the remaining post-
resuscitation interval. Rats were monitored for a maximum of 60 minutes post-resuscitation. Successfully
resuscitated rats that died before 60 minutes post-resuscitation were excluded.
Rats were randomized after completion of surgical preparation to one of 10 groups of 6 rats each (Figure
1). In group 1, hearts were harvested at baseline (BL); in group 2, hearts were harvested after 15 minutes
of untreated VF (VF); in groups 3 to 6, hearts were harvested after 15 minutes of VF with chest
compression and ventilation provided during the last 5 minutes of VF (VF/CC); and in groups 7 to 10,
hearts were harvested at 60 minutes post-resuscitation (PR) following a VF and resuscitation protocol as
in VF/CC groups. Rats in VF/CC groups that spontaneously defibrillated and restored spontaneous
circulation before the designated harvest time were reassigned to the corresponding PR group. Rats in
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VF/CC and PR groups were randomized to one of four interventions. Three groups received Na+-limiting
interventions; namely, lidocaine vehicle followed by AVE4454B, lidocaine followed by AVE4454B
vehicle, and lidocaine followed by AVE4454B. One group served as control and received the vehicles of
lidocaine and AVE4454B. Randomization proceeded by blocks with the investigators blind to the
treatment assignment. Each block included one representative of each experimental group for a total of 6
Experimental Drugs and Vehicle Controls
AVE4454 hydrochloride (AVE4454B) − kindly donated by Sanofi-Aventis − was used for NHE-1
inhibition. AVE4454B is a selective NHE-1 inhibitor newly developed with the intent of circumventing
the adverse effects of cariporide reported in the EXPEDITION trial (41). Fluorometric image plate reader
assay performed by the manufacturer using human NHE subtypes demonstrated high potency and
selectivity for NHE-1 (IC50, 0.051 µM) compared with NHE-2 (IC50, 7.6 µM) and NHE-3 and -5 (no
inhibition at 10 µM). In a rat model of coronary occlusion and reperfusion, AVE4454B dose-dependently
(0.1-3 mg/kg intravenous and 3 mg/kg and 10 mg/kg oral) reduced infarct size (unpublished). The
AVE4454B dose and preparation followed the manufacturer’ s specifications. Accordingly AVE4454B
was dissolved in 1.8% glycine buffer (pH 4.00) to a concentration of 1.0 mg/ml and administered in bolus
dose of 1 mg/kg (1 ml/kg). For AVE4454B vehicle control, mannitol was dissolved in glycine buffer to a
final concentration of 1.3 mg/ml and given in bolus dose of 1 ml/kg. Lidocaine hydrochloride was
purchased from Sigma and used for Na+-channel blockade. Lidocaine was dissolved in 0.9% NaCl to a
concentration of 5 mg/ml and administered in bolus dose of 5 mg/kg (1 ml/kg). The dose was chosen
based on previous studies reporting myocardial protective effects in pig and rabbit models of ischemia
and reperfusion injury using 2 to 10 mg/kg (24,26,56). In preliminary studies, lidocaine administered in
bolus dose of 5 mg/kg to a 472 g and to a 490 g rat during spontaneous circulation elicited no
electrocardiographic or hemodynamic effects. For lidocaine vehicle control, 0.9% NaCl was administered
in bolus dose of 1 ml/kg.
Page 7 of 42
Cardiac output was measured after right atrial bolus injection of 200 µL of 0.9% NaCl at room
temperature. The dilution curves were analyzed using custom-developed LabVIEW-based software.
Cardiac index (CI) was calculated dividing cardiac output by body weight and reported as ml/min/kg.
Cardiac work index (CWI) was calculated multiplying CI by the difference between mean aortic and
mean right atrial pressures and reported as g·m/min/kg (after converting to work units multiplying by 1.36
×10-3 in kg/cm2/mmHg). The coronary perfusion pressure during closed-chest resuscitation was defined as
the pressure difference between the aorta and right atrium immediately before compression. Depth of
compression was measured using a displacement transducer (DSPL, World Precision Instruments)
enabling continuous recording throughout the interval of chest compression, and manually with a ruler at
the end of such interval. Both data were reported given a few instances in which the displacement
transducer failed. The ratio between coronary perfusion pressure and compression depth (CPP/Depth) was
used to assess changes in left ventricular compliance during chest compression as reported previously
Co-EDTA-Space: Co-EDTA-was used to measure the left ventricular extracellular space (ECS) based on
techniques previously described by Holman (27) and Goldberg (19). Co-EDTA-is presumed to cross cell
membranes in negligible quantities (50) such that concomitant measurement of Co3+ in plasma and in
tissue after intravenous administration of Co-EDTA-allows estimation of the ECS according to the
Co-EDTA-Space (%) = 100 × (MCot/[Co]p)/(Wt/1.053) (1)
where MCot = molar amount of Co-EDTA-in tissue (mmol), [Co]p= Co-EDTA-concentration in plasma
(mmol/L), Wt= tissue weight (kg), and 1.053 = density of rat heart tissue (kg/L) (55).
For this purpose, a bolus of Co-EDTA-100 mg/kg (prepared by dissolving 100 mg of Na[Co-
EDTA]ּ2H2O in 1.0 ml of 0.9% NaCl) was given intravenously (1.0 ml/kg) before harvesting. We
Page 8 of 42
confirmed previous studies (50) reporting no measurable physiologic effects elicited by such Co-EDTA-
dose during spontaneous circulation (n = 9 rats). To monitor for possible leak of Co-EDTA-to the
intracellular space, the interval between Co-EDTA-injection and heart harvesting was varied within
groups injecting Co-EDTA-at 10, 20, or 30 minutes before harvest in BL and PR groups and 25, 35, or 45
minutes in VF and VF/CC groups to account for the 15 minute interval of untreated VF. Intracellular leak
would manifest as time-dependent increases in the Co-EDTA-space.
Left Ventricular Tissue Analysis for Determination of Intracellular Na+and Mitochondrial Ca2+
Materials: Trace metal grade nitric acid (HNO3), perchloric acid (HClO4), sulfuric acid (H2SO4),
American Chemical Society (ACS) grade cobalt chloride hexahydrate (CoCl2ּ6H2O), sodium acetate
(CH3COONa), ethylenediamine tetraacetic acid (EDTA), 30% hydrogen peroxide (H2O2), and high
performance liquid chromatography (HPLC) grade alcohol were purchased from Fisher. Highest purity
(SigmaUltra) grade sodium chloride (NaCl), calcium carbonate (CaCO3), sucrose, 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), ethylene glycol-bis(2-aminoethylether)-
N,N,N′,N′-tetraacetic acid (EGTA), HPLC grade (+)-cis-diltiazem hydrochloride (diltiazem), and
technical grade ruthenium red (≈ 98%) were purchased from Sigma. Co-EDTA-sodium salt (Na[Co-
EDTA]ּ2H2O), which was prepared according to the method reported by Scheufler and Peters (50). A
mixture of CoCl2ּ6H2O (8 g), CH3COONa (20 g), and EDTA (10 g) in water (60 ml) was heated to near
boiling temperature. H2O23% (30 ml) was added gradually to obtain a deep red solution after which the
solution was allowed to equilibrate with room temperature and alcohol added to crystallize and wash.
Na[Co-EDTA]ּ2H2O crystals were obtained after four or five crystallization cycles.
Tissue Processing: Immediately before removing the heart, arterial blood (≈ 0.5 ml) was withdrawn into a
heparinized syringe, transferred to a 1.5-ml Eppendorff tube, centrifuged at 2300g for 10 minutes and the
plasma fraction (≈ 0.3 ml) stored at -20 °C until processing for Na+and Co-EDTA-. The heart was then
rapidly excised through a midline sternotomy and placed in an ice-chilled Petri dish. The right ventricle
and both atria were removed and ≈ 600 mg of the left ventricle apportioned for measuring intracellular
Page 9 of 42
Na+(posterolateral wall and posterior portion of the septum) and ≈ 100 mg for measuring mitochondrial
Ca2+ (anterolateral wall and anterior portion of septum). The fraction for intracellular Na+measurement
was kept at -20 °C until processing whereas the fraction for mitochondrial Ca2+ measurement was
processed immediately (see below under Mitochondrial Ca2+).
Co-EDTA-and Na+Measurements: The frozen left ventricular tissue was lyophilized for 24 hours at -50
°C and < 30 millibars (Lyph-Lock 4.5 Liter Freeze Dry System, Labconco) determining its water content
by differential weight measurement before and after complete lyophilization. The lyophilized tissue was
then powderized using a mixer mill (MM200, Retsch) set at 30 Hz for 5 minutes. Plasma (0.2 ml) and the
powderized tissue (≈ 30 mg dry weight) was acid digested using a 3:1:1 (vol:vol:vol) solution of HNO3,
HClO4, and H2SO4mixture (0.8 ml) at 80 °C overnight. Using deionized water, the plasma and tissue
digestates were diluted 400 and 200 times for Na+measurement and 100 and 20 times for Co-EDTA-
measurement, respectively. A Varian SpectrAA·640 system (Varian, Inc.) equipped with modules for
flame atomic absorption spectrometry (FAAS) and for high sensitive graphite furnace atomic absorption
spectrometry (GFAAS) was used with the aid of SpectrAA 5 PRO software (Varian, Inc.). Na+was
measured using FAAS at 589.6 nm whereas Co-EDTA-was measured using GFAAS detecting Co3+ at
242.5 nm. A Na+and Co3+ stock solution was prepared by dissolving 101.7 mg NaCl and 80.8 mg
CoCl2·6H2O in 2 L deionized water. The stock solution was diluted in deionized water to obtain 10 ml
aliquots of 0.1, 0.2, 0.5, 1.0, and 2.0 µg/ml of Na+standard solutions and 1.0 ml aliquots of 10, 20, 50,
and 100 ng/ml of Co3+ standard solutions and used to construct Na+and Co3+ standard curves. Samples
were measured in aliquots of 5.0 ml for Na+and 15 µL for Co3+ with standard Na+(0.5 µg/ml) and Co3+
(50 ng/ml) solutions measured every 10 samples for quality control.
Intracellular Na+[Na]iin mmol/L was calculated according to the following equation:
[Na]i= MNat/(Wt/1.053) - [Na]p× ECS (2)
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where MNat = molar amount of Na+in tissue (mmol), [Na]p= Na+concentration in plasma (mmol/L), and
ECS = extracellular space determined using Co-EDTA-[equation 1].
Mitochondrial Ca2+: The 100-mg sample of left ventricular tissue separated after heart harvesting was
quickly minced into small pieces and immersed into ice-cold inhibitor buffer as described by Pepe et al
(47). The inhibitor buffer was composed of 250 mmol/L sucrose (to maintain osmotic pressure and
preserve mitochondrial structural integrity), 3.2 mmol/L ruthenium red (to block the mitochondrial Ca2+
uniporter), 30 mmol/L diltiazem (to block mitochondrial Na+-Ca2+ exchange), 2 mmol/L EGTA (to
chelate free extra-mitochondrial Ca2+), and 10 mmol/L HEPES; the last two adjusted to pH 7.40 using
KOH. The sample was then hand homogenized using a Dounce homogenizer at a temperature between 0
and 4 °C to minimize Ca2+ redistribution. Mitochondria were then separated by differential centrifugation
as described by Hansford et al (23). Briefly, the left ventricular homogenate was diluted to 5 ml with
inhibitor buffer and centrifuged at 1,000g for 5 minutes to pellet nuclei and debris. The supernatant was
further centrifuged at 27,000g for 2.5 minutes and the pellet (mitochondrial fraction) re-suspended in 0.2-
ml of inhibitor buffer and frozen at -80°C until analysis. The frozen mitochondrial fraction was thawed on
ice and 0.05 ml of the suspension added to 0.2 ml of high purity HNO3for overnight digestion at 80°C.
The digestate was diluted 100 times with 0.5% HNO3(to prevent phosphate interference) before
measurement using GFAAS at 422.7 nm. Ca2+ standard stock solution (5.0 µg/ml) was prepared by
dissolving 25 mg CaCO3in 2.0 L 0.5% HNO3solution (instead of deionized water to completely dissolve
CaCO3). The stock solution was then diluted 5000, 2500, 1000, and 500 times using 0.5% HNO3solution
to obtain 1.0 ml aliquots of 1.0, 2.0, 5.0, and 10.0 ng/ml of Ca2+ standard solutions, respectively. Samples
were measured in aliquots of 1.0 ml with standard Ca2+ solution (5.0 ng/ml) measured every 10 samples
for quality control. Mitochondrial total protein was estimated using a protein assay kit (Micro BCA™
Protein Assay Kit, Pierce). Values of mitochondrial Ca2+ were expressed as nmol/mg protein.
Cardiac Troponin I
Plasma obtained at baseline and after resuscitation in the PR groups were assayed for cTnI using a
Page 11 of 42
commercially available one step “ sandwich” enzyme immunoassay method developed for human cTnI
(Dimension® clinical chemistry system using Cardiac Troponin-I Flex® reagent cartridge, Dade Behring
Inc.). The method had previously been shown to have excellent reactivity and specificity for rat cTnI
(92.8% homology) (6,12,45,46).
Na+-K+ATPase Activity in Left Ventricular Tissue
Activity of the Na+-K+ATPase was measured in a small subset of additional experiments after observing
that intracellular Na+remained elevated at 60 minutes post-resuscitation, using the same VF and
resuscitation protocol as in the main studies. The technique previously described by Schwinger et al. (52)
and Fuller et al. (14) was used with minor modifications. Hearts were harvested at 60 minutes post-
resuscitation and rinsed in ice-cold Tris-buffered saline buffer. The right ventricle and both atria were
removed and the left ventricle rapidly frozen by immersion in liquid N2. The frozen left ventricular tissue
was then weighed, diced, and agitated in 10 ml of a high salt solution (2 M NaCl, 20 mM HEPES, pH
7.40) for 30 minutes at 4°C to depolymerize myofilaments. The tissue was then rinsed and homogenized
using a Polytron homogenizer in a buffer containing 20 mM HEPES, 250 mM Sucrose, 2 mM EDTA, 1
mM MgCl2, 0.2 mM PMSF (pH 7.40) to a final concentration of 10 ml/g wet weight of left ventricular
tissue. Aliquots of ≈ 3 ml were stored at -80°C until analysis. Protein concentration was determined by
the BCA method. The Na+-K+ATPase activity was determined by measuring the ouabain-inhibitable
generation of inorganic phosphate (Pi) in the presence of excess ATP. All reagents and solutions were
prepared in phosphate-free glassware.
Assay Reaction: Two aliquots of total homogenate (500 µg protein, ≈ 65 µl) were mixed in 1.5 ml tubes
with 65 µl of reaction buffer (200 mM Tris– HCl, 30 mM MgCl2, 200 mM NaCl, 60 mM KCl, 10 mM
EGTA, 0.2 mM PMSF, and protease inhibitor cocktail, pH 7.50) with ouabain (2 mM) in one aliquot and
without ouabain in the other. The aliquots and buffer were mixed gently and kept on ice for 1 hour to
achieve maximal Na+-K+ATPase inhibition. The assay reaction was started by adding 14.4 µl of 0.1 M
ATP (pH 7.00) to a 10 mM final concentration followed by incubation at 37°C. The reaction was stopped
Page 12 of 42
after 10 minutes by adding 13 µl of 100% (w/v) ice-cold trichloroacetic acid. The samples were left on ice
for 1 hour to facilitate precipitation of proteins and then centrifuged at 20,000g for 30 minute. The
supernatant (60 µl) was assayed for inorganic phosphate by a colorimetric method described by King (34).
The supernatant (60 µl) was transferred to tubes containing 1.5 ml of 0.5% TCA and incubated at room
temperature for 10 minutes with 150 µl of 5% ammonium molybdate and 60 µl of ANS reagent (0.25%
aminonaphtholsulfonic acid [ANSA] in 15% NaHSO3and 6% Na2SO3). Absorbance was measured at 660
nm and converted to µmoles of phosphate using a standard curve prepared with KH2PO4. The Na+-K+-
ATPase activity was expressed as µmoles Pi/mg protein/hour.
A primary analysis was performed to determine whether limiting sarcolemmal Na+entry attenuated
cytosolic Na+increases and whether such effect influenced mitochondrial Ca2+ content and myocardial
injury and function. For this analysis, the data from all three Na+-limiting interventions were pooled
together. A secondary analysis was then performed to detect differences among the three Na+-limiting
For continuous variables, Student’ s t-test was used when comparing differences between Na+-limiting
interventions and control and one-way ANOVA when comparing differences among the three Na+-
limiting interventions and among the various time events (applying Dunnett's method for multiple
comparisons if overall differences were detected). In addition, two-way ANOVA was used in VF/CC and
PR groups to simultaneously test for treatment and time factors and to examine potential interactions
between these factors. The strength of association between variables was analyzed using Pearson’ s
product moment correlation test. Alternative non-parametric tests were used if the data failed tests for
normality or equal variance. The data were presented as mean ± SD unless otherwise stated. A two-tail
value of p < 0.05 was considered significant.
Page 13 of 42
Baseline measurements and cumulative dose of pentobarbital given before induction of VF were
comparable among all groups. During closed-chest resuscitation, adjustments in compression depth
successfully maintained the CPP above 20 mmHg in each rat in the VF/CC and PR groups. Yet, the
required depth of compression was less in the 36 rats treated with Na+-limiting interventions compared
with the 12 control rats (1.40 ± 0.11 vs 1.50 ± 0.19 cm using the manually measured depth; p = 0.028 by
Student’ s t-test). There were no statistically significant differences among the Na+-limiting interventions
despite a lower depth favoring the AVE4454B and lidocaine combination (AVE4454B, 1.42 ± 0.11 cm;
lidocaine, 1.41 ± 0.12 cm; and AVE4454B/lidocaine, 1.38 ± 0.11 cm). The time course of compression
depth and the CPP/depth ratio are shown in Figure 2.
Efforts to terminate VF and reestablish spontaneous circulation in the PR groups are shown in Figure 3.
Spontaneous defibrillation with return of spontaneous circulation − previously reported by us associated
with administration of the NHE-1 inhibitor cariporide in rats (16) − occurred before completion of the 5-
minute interval of chest compression or shortly thereafter in 1 control, 2 AVE4454B, 4 lidocaine, and 3
AVE4454B/lidocaine treated rats. As a result, fewer shocks and less cumulative energy were required to
terminate VF in Na+-limiting interventions. Recurrence of VF in the early post-resuscitation period (< 15
minutes) required delivery of additional electrical shocks in control and in AVE4454B groups
(unexpectedly) but not in the lidocaine and AVE4454B/lidocaine groups (Figure 3).
After return of spontaneous circulation, statistically significant reductions in cardiac index and cardiac
work index along with statistically insignificant decreases in mean aortic pressure occurred in all groups,
consistent with post-resuscitation myocardial dysfunction (Figures 4). However, Na+-limiting
interventions attenuated decreases in mean aortic pressure and cardiac work index although no
statistically significant differences were detected among the individual interventions.
Page 14 of 42
Left Ventricular Co-EDTA-Space
The left ventricular Co-EDTA-space at baseline and during VF corresponded to 21.1% and 20.7% of the
total left ventricular volume without dependency on the time of Co-EDTA-administration, and consistent
with values reported by other investigators using Co-EDTA-(40), TmDOTP5-, inulin, and mannitol (10).
During VF/CC, the left ventricular Co-EDTA-space decreased to 18.1% in control hearts and to 18.3% in
Na+-limiting interventions hearts (NS), without differences among interventions (Figure 5A). The Co-
EDTA-space, however, markedly increased in the PR group to 29.2% in control hearts and to 26.6% in
hearts subjected to Na+-limiting interventions, without statistically significant differences among specific
interventions. The increases in Co-EDTA-space occurred with minimal changes in left ventricular water
content, which varied between 75.9% at baseline and 77.9% at VF/CC (Figure 5B) suggesting that
increases in the Co-EDTA-space after resuscitation were unlikely the result of increases in the
extracellular space. Such effect would require concomitant and proportional reductions in the intracellular
space, and would be inconsistent with the reported increases − rather than decreases − in the intracellular
space during and after ischemia (2). We therefore elected to apply the baseline measurements of
extracellular space (21.1%) to the estimations of intracellular Na+for all groups.
Left Ventricular Intracellular Na+
Left ventricular intracellular Na+increased from 11.8 ± 3.0 mmol/L to 16.5 ± 1.5 mmol/L after 15
minutes of VF (NS). In control hearts, intracellular Na+further increased to 18.5 ± 1.8 mmol/L when VF
included chest compression and to 22.6 ± 11.1 mmol/L post-resuscitation (p < 0.05 for each group vs BL
by Kruskal-Wallis one-way ANOVA on ranks using Dunn's method for multiple comparisons). Na+-
limiting interventions prevented such increases in intracellular Na+yielding levels of 16.6 ± 3.7 mmol/L
in VF/CC groups and 15.7 ± 4.0 mmol/L in PR groups without statistically significant differences among
the specific interventions despite lower levels in the AVE4454B/lidocaine group (Figure 6A).
Left Ventricular Mitochondrial Ca2+
Page 15 of 42
Left ventricular mitochondrial Ca2+ remained relatively unchanged during the 15-minute interval of
untreated VF and during the interval of VF with chest compression. However, mitochondrial Ca2+
increased to approximately 140% of baseline during the post-resuscitation interval in control hearts. In
rats subjected to Na+-limiting interventions, mitochondrial Ca2+ corresponded to 77% and 71% of control
hearts at VF/CC and PR, without statistically significant differences among the specific Na+-limiting
interventions (Figure 6B). The changes in mitochondrial Ca2+ were correlated with changes in
intracellular Na+levels at PR (r = 0.44, n = 24, p < 0.04) but not at VF/CC (r = 0.11, n = 24, p = 0.63).
Similar analysis within individual groups at PR and VF/CC failed to disclose significant correlations (data
not shown). This was likely the result of small sample size and reduced variability within groups given
the controlled nature of the experiments.
Plasma Cardiac Troponin I
Plasma cTnI increased in control hearts from 0.7 ± 0.5 ng/mL at baseline to 48.6 ± 30.2 ng/mL at 60 post-
resuscitation. In rats treated with Na+-limiting interventions, plasma cTnI increased from 0.5 ± 0.3 ng/mL
at baseline to 27.5 ± 13.0 ng/mL at 60 minutes post-resuscitation (p < 0.05 vs control by Student’ s t-test),
without statistically significant differences among the specific interventions (Figure 7). Plasma cTnI
levels at 60 minutes post-resuscitation was positively correlated with left ventricular intracellular Na+and
left ventricular Co-EDTA-distribution space and inversely correlated with cardiac work index (Figure 7).
In separate studies − including 2 subjects per group − the Na+-K+ATPase activity measured at 60 minutes
post-resuscitation in control rats was reduced to ≈ 40% of baseline, from 2.82 ± 0.18 to 1.16 ± 0.39
µmoles Pi/mg protein/hr (p < 0.05 by Student’ s t-test). Compared to the control group, treatment with
AVE4454B was associated with further reduction in Na+-K+ATPase activity (0.69 ± 0.27 µmoles Pi/mg
protein/hr, p < 0.05), treatment with lidocaine was associated with increased Na+-K+ATPase activity
(4.43 ± 0.43 µmoles Pi/mg protein/hr, p < 0.05), and treatment with the AVE4454B/lidocaine
Page 16 of 42
combination preserved the Na+-K+ATPase activity at baseline levels (2.69 ± 0.32 µmoles Pi/mg
protein/hr, NS) by one-way ANOVA and Bonferroni t-test for multiple comparisons against control.
Page 17 of 42
The present studies demonstrate that limiting sarcolemmal Na+entry during resuscitation from VF (i)
prevents left ventricular intracellular Na+increases, (ii) maintains lower mitochondrial Ca2+ levels during
and after resuscitation, (iii) attenuates reductions in left ventricular compliance during chest compression,
(iv) lessens myocardial injury, and (v) ameliorates post-resuscitation myocardial dysfunction. No
statistically significant differences could be demonstrated among the various Na+limiting interventions
except for failure of AVE4454B to prevent recurrence of VF.
We have previously reported comparable increases in left ventricular intracellular Na+in an isolated rat
heart model of VF in which clinical resuscitation was simulated by providing 10 minutes of no-flow
ischemia followed by 15 minutes of low-flow reperfusion (18). In these studies increases in intracellular
Na+were halved if VF was absent despite identical periods of no-flow and low-flow reperfusion,
highlighting the importance of VF for promoting cytosolic Na+overload. VF may contribute to
intracellular Na+overload through Na+channel activation followed by slow inactivation during ischemia
(7). However, VF could also contribute by intensifying ischemic injury (17) and exacerbating
determinants of Na+overload such as energy depletion with further inhibition of the Na+-K+ATPase and
by intensifying intracellular acidosis increasing the rate of Na+-H+exchange.
The present studies points also to reperfusion as a factor that can intensify intracellular Na+overload. For
the same duration of VF (i.e., 15 minutes), intracellular Na+was higher when chest compression (i.e.,
reperfusion) was provided during the last 5 minutes of VF (Figure 6). Reperfusion has been shown to
increase the rate of Na+entry, and attributed to the wash out of protons accumulated in the extracellular
space during the period of no-flow ischemia (28). These observations are applicable to the setting herein
modeled in which ischemia and reperfusion were of short duration, global, and compounded by the
presence of VF. The findings, however, may not be directly extrapolated to other settings, such a regional
ischemia without VF.
Page 18 of 42
In addition to the (expected) aforementioned effects, we found (unexpectedly) that the intracellular Na+
levels were substantially higher at 60 minutes post-resuscitation than during chest compression,
challenging the presumption that restoration of aerobic metabolism could reverse the processes leading to
intracellular Na+accumulation. The possibility that determinants of intracellular Na+entry and overload
such as intracellular acidosis and decreased sarcolemmal Na+-K+ATPase activity persisted at 60 minutes
post-resuscitation was considered and additional exploratory experiments were conducted to assess the
Na+-K+ATPase activity after resuscitation. These studies demonstrated reductions in Na+-K+ATPase at
60 minutes post-resuscitation to ≈ 40% of baseline, suggesting that persistent intracellular Na+increases
could have been in part attributed to decreased removal of intracellular Na+excess. Na+-limiting
interventions had intriguing effects on Na+-K+ATPase activity. AVE4454B reduced, lidocaine increased,
and the AVE4454B/lidocaine combination maintained the Na+-K+ATPase activity at baseline levels.
These observations suggest the possibility of additional effects beyond those related to ion homeostasis.
Understanding these effects would require additional experiments beyond the scope of the present studies.
Another factor that may affect intracellular Na+is changes in NHE-1 activity. Studies in isolated rat hearts
have shown seven fold increases in NHE-1 messenger RNA early after ischemia and reperfusion (15).
NHE-1 activity has been shown to be increased by α1-adrenergic stimulation which can occur as part of
the neuroendocrine response to cardiac arrest (62,63).
Regardless of mechanisms, administration of Na+limiting interventions halted increases in left ventricular
intracellular Na+during the interval of chest compression and following return of spontaneous circulation
yielding levels comparable to those observed during VF in the absence of reperfusion. This effect was
associated with lower mitochondrial Ca2+ levels.
Changes in mitochondrial Ca2+ are directionally coupled with changes in cytosolic Ca2+ via the
mitochondrial Ca2+ uniporter (Ca2+ entry) and various antiporters, including the mitochondrial Na+-Ca2+
and H+-Ca2+ antiporters (Ca2+ efflux) such that increases in cytosolic Ca2+ would be expected to increase
mitochondrial Ca2+ and vice versa (9,29,35,42,48). Accordingly, attenuation in mitochondrial Ca2+
Page 19 of 42
increases by Na+limiting interventions in the present studies is best explained by reductions in Na+-
induced cytosolic Ca2+ entry (and overload). Mitochondrial Ca2+ overload during ischemia and
reperfusion has been shown to worsen mitochondrial injury compromising its capability to sustain
oxidative phosphorylation (61) and promoting the release of pro-apoptotic factors (8).
The functional benefits associated with Na+limiting interventions were as previously reported in various
animal models of VF and closed-chest resuscitation associated with NHE-1 inhibition (5,16,37). First,
they enabled hemodynamically more effective chest compression such that less depth of compression was
required to attain a predetermined coronary perfusion pressure. This effect is best explained by
amelioration (or prevention) of reductions in left ventricular compliance that leads to preload-dependent
reductions in forward blood flow (5,36,37). Second, Na+limiting interventions (with the exception of
AVE4454B as discussed below) prevented episodes of refibrillation eliminating the need for additional
electrical shocks during the early post-resuscitation interval. Finally, Na+limiting interventions reduced
myocardial cell injury, as evidenced by lower post-resuscitation plasma cTnI increases, and attenuated
post-resuscitation myocardial dysfunction yielding a higher cardiac work index with a higher mean aortic
Post-resuscitation myocardial injury and dysfunction was accompanied by a higher Co-EDTA-
distribution space. We interpreted these findings as indicative of disruption of sarcolemmal membrane
enabling Co-EDTA-to enter the intracellular space and cTnI to exit it and be measured in plasma.
Consistent with this explanation, the Co-EDTA-distribution space was lower in rats treated with Na+
limiting interventions, which also had lower plasma cTnI and higher cardiac work index. Likewise, the
Co-EDTA-distribution space was positively correlated with intracellular Na+and plasma cTnI and
negatively correlated with cardiac work index. We also observed lack of time-dependency between the
time of tracer injection and Co-EDTA-distribution space, favoring a mechanism of injury affecting a
small percentage of cells leading to rapid intracellular Co-EDTA-equilibration rather than a diffuse
alteration of plasma membrane permeability with slow Co-EDTA-intracellular “ leakage.”
Page 20 of 42
Differences among Interventions
There were no statistically significant differences among the three Na+limiting interventions except that
AVE4454B failed to prevent recurrent episodes of VF during the early post-resuscitation interval. This
contrasted with the prominent and consistent suppression or reperfusion arrhythmias reported using other
NHE-1 inhibitors including cariporide (5,16,20,59). Although the present data do not provide direct clues
on the mechanisms, previous observations using the more broadly known inhibitor cariporide might shed
some light. Cariporide given in bolus dose during closed-chest resuscitation in pigs produced plasma
levels that far exceeded the IC90 of NHE-1 (4) and reached levels which were previously shown to inhibit
slowly-inactivating Na+currents (51). These observations suggested that some of the benefits of
cariporide could be mediated through Na+channel blockade. Lidocaine, which characteristically inhibit
these channels, minimizes reperfusion arrhythmias (39,53). Accordingly, greater selectivity of
AVE4454B − lacking non-selective effects on slowly-inactivating Na+currents − could have been a
potential factor. Work beyond the scope of the present studies would be required to resolve this issue.
Lidocaine showed cardioprotective effects comparable to those of AVE4454B with the added benefits on
reperfusion arrhythmias. Lidocaine has been shown in several preclinical studies to be cardioprotective
(26,44,49) through mechanisms that involve attenuation of cytosolic Na+and Ca2+ overload (21,53).
These findings, however, contrast with the poor performance shown in clinical trials in patients suffering
out-of-hospital cardiac arrest (11). In these trials, however, lidocaine was administrated 25 minutes after
ambulance dispatch. Yet, one retrospective study showed higher resuscitability and hospital admission
rates in patient who had received lidocaine (25).
Whether an additive beneficial effect occurred between AVE4454B and lidocaine can not be established
based on the present data. Numerical differences favoring the combination were observe with regards to
depth of compression and intracellular Na+during chest compression, and intracellular Na+and cTnI at 60
minutes post-resuscitation. These differences, however, were not statistical significance. Larger sample
sizes per group would be required − corresponding to 141 for depth of compression, 35 for intracellular
Page 21 of 42
Na+, and 25 for cTnI − to demonstrate statistical significance for the reported numerical differences using
a power of 90%.
Limitations of Study
Model: Extrapolation of the present findings to clinical settings is constrained by several factors. VF was
induced electrically after prolonged electrical stimulation whereas clinically VF typically occurs in
patients with underlying coronary artery disease and often precipitated by coronary occlusion. In addition,
we cannot exclude that alterations in membrane composition occurred consequent to electrical stimulation.
However, in a previous study using a Langendorff rat heart preparation similar electrical stimulation at
normal perfusion flows elicited only transient myocardial depression after return of sinus rhythm without
changes in myocardial oxygen consumption (17). Inherent to animal models is the use of anesthesia
which exerts independent myocardial protective effects (33). However, the rat model herein used has been
highly effective in the development and testing of novel concepts with subsequent confirmation in larger
animal models, particularly when examining highly conserved mechanisms that transcend species
differences (i.e., Na+-H+exchange, Na+channel kinetics, and mitochondrial function).
Techniques: We examined changes in mitochondrial Ca2+ by measuring total instead of the
physiologically more relevant matrix free Ca2+. However, because previous studies had shown a good
linear correlation between total and matrix free mitochondrial Ca2+ in the isolated rat heart (22) such
compromise appeared reasonable given the limitations for measuring matrix free Ca2+ in intact animals.
For measuring intracellular Na+concentration, we concomitantly determined the Co-EDTA-space as
marker of the extracellular space based on previous studies indicating negligible plasma membrane
permeability to Co-EDTA-. However, we found post-resuscitation increases in Co-EDTA-space that
could not be explained by increases extracellular space (discussed in the Result section). We elected to
apply the baseline measurement of extracellular space for the calculations of intracellular space in all
groups. The possibility that prominent changes in extracellular space occurred and were not detected
seemed unlikely given the very minor changes in total left ventricular water. Even if small changes
Page 22 of 42
occurred, the effects of ischemia and reperfusion and the effects of Na+limiting interventions were
compared with proper controls.
Potential Clinical Significance
The effects of Na+limiting interventions reported here and in previous publications (5,37) are
physiologically relevant and would be expected to yield higher resuscitation and survival rates in patients
provided the effects could be replicated. Beyond the differences between animal models and clinical
resuscitation already mentioned, the timing of drug delivery is likely to be a critical determinant of
efficacy. It is widely accepted that the maximal efficacy of NHE-1 inhibitors is attained when given
before the onset of ischemia (58) with the efficacy diminishing as delivery is delayed during reperfusion
(43). We developed a protocol to simulate drug delivery at the earliest possible time in a clinical scenario
(i.e., after the onset of VF but before the start of cardiopulmonary resuscitation). Implementation of such
approach in humans would require a paradigm shift prompting drugs to be delivered before starting chest
compression. From this perspective, the intraosseous route for vascular access is available both in
children and adults without the limitations and delays imposed by cannulation of peripheral or central
veins in a crisis situation.
We have confirmed that limiting sarcolemmal Na+entry represents an important therapeutic goal during
resuscitation from VF that helps attenuate ischemia and reperfusion injury and preserve left ventricular
function. Given that NHE-1 inhibition and Na+channel blockade yielded comparable effects, and given
that NHE-1 inhibitors are not yet clinically available, use of lidocaine for the purpose of limiting
sarcolemmal Na+entry by early administration during resuscitation from VF should be examined.
Work supported by an NIH grant R01 HL71728-01 entitled “ Myocardial Protection by NHE-1 Inhibition”
and a VA Merit Review Grant entitled “ Myocardial Protection during Ventricular Fibrillation” .
Page 23 of 42
The authors wish to thank Linda C. Dowell, Laboratory Manager at the North Chicago VA Medical
Center, for conducting the determinations of plasma cardiac troponin I.
Page 24 of 42
1. An J, Varadarajan SG, Camara A, Chen Q, Novalija E, Gross GJ, Stowe DF. Blocking
Na(+)/H(+) exchange reduces [Na(+)](i) and [Ca(2+)](i) load after ischemia and improves function in
intact hearts. Am J Physiol 281: H2398-H2409, 2001.
2. Askenasy N, Tassini M, Vivi A, Navon G. Intracellular volume measurement and detection of
edema: multinuclear NMR studies of intact rat hearts during normothermic ischemia. Magn Reson
Med 33: 515-520, 1995.
3. Avkiran M, Ibuki C, Shimada Y, Haddock PS. Effects of acidic reperfusion on arrhythmias and
Na(+)-K(+)-ATPase activity in regionally ischemic rat hearts. Am J Physiol 270: H957-H964, 1996.
4. Ayoub IM, Kolarova J, Kantola RL, Sanders R, Gazmuri RJ. Cariporide minimizes adverse
myocardial effects of epinephrine during resuscitation from ventricular fibrillation. Crit Care Med 33:
5. Ayoub IM, Kolarova JD, Yi Z, Trevedi A, Deshmukh H, Lubell DL, Franz MR, Maldonado FA,
Gazmuri RJ. Sodium-hydrogen exchange inhibition during ventricular fibrillation: Beneficial
effects on ischemic contracture, action potential duration, reperfusion arrhythmias, myocardial
function, and resuscitability. Circulation 107: 1804-1809, 2003.
6. Bairoch AApweiler R. The SWISS-PROT protein sequence data bank and its new supplement
TREMBL. Nucleic Acids Res 24: 21-25, 1996.
7. Balser JR, Nuss HB, Romashko DN, Marban E, Tomaselli GF. Functional consequences of
lidocaine binding to slow-inactivated sodium channels. J Gen Physiol 107: 643-658, 1996.
8. Borutaite VBrown GC. Mitochondria in apoptosis of ischemic heart. FEBS Lett 541: 1-5, 2003.
9. Calderone VCavero I. [Ventricular arrhythmias. A potential risk associated with the use of non-
cardiovascular drugs prolonging the QT interval]. Minerva Med 93: 181-197, 2002.
Page 25 of 42
10. Dobson GPCieslar JH. Intracellular, interstitial and plasma spaces in the rat myocardium in vivo. J
Mol Cell Cardiol 29: 3357-3363, 1997.
11. Dorian P, Cass D, Schwartz B, Cooper R, Gelaznikas R, Barr A. Amiodarone as compared with
lidocaine for shock-resistant ventricular fibrillation. N Engl J Med 346: 884-890, 2002.
12. Fiorillo C, Pace S, Ponziani V, Nediani C, Perna AM, Liguori P, Cecchi C, Nassi N, Donzelli GP,
Formigli L, Nassi P. Poly(ADP-ribose) polymerase activation and cell injury in the course of rat
heart heterotopic transplantation. Free Radic Res 36: 79-87, 2002.
13. Fozzard HA. Afterdepolarizations and triggered activity. Basic Res Cardiol 87 Suppl 2: 105-113,
14. Fuller W, Parmar V, Eaton P, Bell JR, Shattock MJ. Cardiac ischemia causes inhibition of the
Na/K ATPase by a labile cytosolic compound whose production is linked to oxidant stress.
Cardiovasc Res 57: 1044-1051, 2003.
15. Gan XT, Chakrabarti S, Karmazyn M. Modulation of Na+/H+ exchange isoform 1 mRNA
expression in isolated rat hearts. Am J Physiol 277: H993-H998, 1999.
16. Gazmuri RJ, Ayoub IM, Hoffner E, Kolarova JD. Successful ventricular defibrillation by the
selective sodium-hydrogen exchanger isoform-1 inhibitor cariporide. Circulation 104: 234-239, 2001.
17. Gazmuri RJ, Berkowitz M, Cajigas H. Myocardial effects of ventricular fibrillation in the isolated
rat heart. Crit Care Med 27: 1542-1550, 1999.
18. Gazmuri RJ, Hoffner E, Kalcheim J, Ho H, Patel M, Ayoub IM, Epstein M, Kingston S, Han Y.
Myocardial protection during ventricular fibrillation by reduction of proton-driven sarcolemmal
sodium influx. J Lab Clin Med 137: 43-55, 2001.
Page 26 of 42
19. Goldberg SP, Digerness SB, Skinner JL, Killingsworth CR, Katholi CR, Holman WL. Ischemic
preconditioning and Na+/H+ exchange inhibition improve reperfusion ion homeostasis. Ann Thorac
Surg 73: 569-574, 2002.
20. Gumina RJ, Daemmgen J, Gross GJ. Inhibition of the Na(+)/H(+) exchanger attenuates phase 1b
ischemic arrhythmias and reperfusion-induced ventricular fibrillation. Eur J Pharmacol 396: 119-124,
21. Haigney MC, Lakatta EG, Stern MD, Silverman HS. Sodium channel blockade reduces hypoxic
sodium loading and sodium-dependent calcium loading. Circulation 90: 391-399, 1994.
22. Hansford RGCastro F. Effect of senescence on Ca2+-ion transport by heart mitochondria. Mech
Ageing Dev 19: 5-13, 1982.
23. Hansford RG, Hogue B, Prokopczuk A, Wasilewska E, Lewartowski B. Activation of pyruvate
dehydrogenase by electrical stimulation, and low-Na+ perfusion of guinea-pig heart. Biochim Biophys
Acta 1018: 282-286, 1990.
24. Hatori N, Roberts RL, Tadokoro H, Ryden L, Satomura K, Fishbein MC, Stiehm ER, Corday E,
Drury JK. Differences in infarct size with lidocaine as compared with bretylium tosylate in acute
myocardial ischemia and reperfusion in pigs. J Cardiovasc Pharmacol 18: 581-588, 1991.
25. Herlitz J, Ekstrom L, Wennerblom B, Axelsson A, Bang A, Lindkvist J, Persson NG, Holmberg
S. Lidocaine in out-of-hospital ventricular fibrillation. Does it improve survival? Resuscitation 33:
26. Hinokiyama K, Hatori N, Ochi M, Maehara T, Tanaka S. Myocardial protective effect of
lidocaine during experimental off-pump coronary artery bypass grafting. Ann Thorac Cardiovasc
Surg 9: 36-42, 2003.
Page 27 of 42
27. Holman WL, Skinner JL, Killingsworth CR, Rogers JM, Melnick S, Ideker RE, Digerness SB.
Controlled postcardioplegia reperfusion: mechanism for attenuation of reperfusion injury. J Thorac
Cardiovasc Surg 119: 1093-1101, 2000.
28. Imahashi K, Kusuoka H, Hashimoto K, Yoshioka J, Yamaguchi H, Nishimura T. Intracellular
sodium accumulation during ischemia as the substrate for reperfusion injury. Circ Res 84: 1401-1406,
29. Isenberg G, Han S, Schiefer A, Wendt-Gallitelli MF. Changes in mitochondrial calcium
concentration during the cardiac contraction cycle. Cardiovasc Res 27: 1800-1809, 1993.
30. Iwai T, Tanonaka K, Inoue R, Kasahara S, Kamo N, Takeo S. Mitochondrial damage during
ischemia determines post-ischemic contractile dysfunction in perfused rat heart. J Mol Cell Cardiol
34: 725-738, 2002.
31. Iwai T, Tanonaka K, Inoue R, Kasahara S, Motegi K, Nagaya S, Takeo S. Sodium accumulation
during ischemia induces mitochondrial damage in perfused rat hearts. Cardiovasc Res 55: 141-149,
32. Jesus Garcia-Rivas G, Guerrero-Hernandez A, Guerrero-Serna G, Rodriguez-Zavala JS,
Zazueta C. Inhibition of the mitochondrial calcium uniporter by the oxo-bridged dinuclear ruthenium
amine complex (Ru360) prevents from irreversible injury in postischemic rat heart. FEBS J 272:
33. Kato RFoex P. Myocardial protection by anesthetic agents against ischemia-reperfusion injury: an
update for anesthesiologists. Can J Anaesth 49: 777-791, 2002.
34. King EJ. The colorimetric determination of phosphorus. Biochem J 26: 292-297, 1932.
35. Kirichok Y, Krapivinsky G, Clapham DE. The mitochondrial calcium uniporter is a highly
selective ion channel. Nature 427: 360-364, 2004.
Page 28 of 42
36. Klouche K, Weil MH, Sun S, Tang W, Povoas HP, Kamohara T, Bisera J. Evolution of the stone
heart after prolonged cardiac arrest. Chest 122: 1006-1011, 2002.
37. Kolarova JD, Ayoub IM, Gazmuri RJ. Cariporide enables hemodynamically more effective chest
compression by leftward shift of its flow-depth relationship. Am J Physiol Heart Circ Physiol 288:
38. Lagadic-Gossmann D, Buckler KJ, Vaughan-Jones RD. Role of bicarbonate in pH recovery from
intracellular acidosis in the guinea-pig ventricular myocyte. J Physiol (Lond) 458: 361-384, 1992.
39. Li GRFerrier GR. Effects of lidocaine on reperfusion arrhythmias and electrophysiological
properties in an isolated ventricular muscle model of ischemia and reperfusion. J Pharmacol Exp
Ther 257: 997-1004, 1991.
40. Makos JD, Malloy CR, Sherry AD. Distribution of TmDOTP5- in rat tissues: TmDOTP5- vs.
CoEDTA- as markers of extracellular tissue space. J Appl Physiol 85: 1800-1805, 1998.
41. Mentzer RM, Jr. Effects of Na+/H+ exchange inhibition by cariporide on death and nonfatal
myocardial infarction in patients undergoing coronary artery bypass graft surgery: The EXPEDITION
study. Circulation 108, 3M. 2003.
Ref Type: Abstract
42. Miyata H, Silverman HS, Sollott SJ, Lakatta EG, Stern MD, Hansford RG. Measurement of
mitochondrial free Ca2+ concentration in living single rat cardiac myocytes. Am J Physiol 261:
43. Myers ML, Mathur S, Li GH, Karmazyn M. Sodium-hydrogen exchange inhibitors improve
postischemic recovery of function in the perfused rabbit heart. Cardiovasc Res 29: 209-214, 1995.
44. Nasser FN, Walls JT, Edwards WD, Harrison CE, Jr. Lidocaine-induced reduction in size of
experimental myocardial infarction. Am J Cardiol 46: 967-975, 1980.
Page 29 of 42
45. O'Brien PJ, Landt Y, Ladenson JH. Differential reactivity of cardiac and skeletal muscle from
various species in a cardiac troponin I immunoassay. Clin Chem 43: 2333-2338, 1997.
46. O'Brien PJ, Smith DE, Knechtel TJ, Marchak MA, Pruimboom-Brees I, Brees DJ, Spratt DP,
Archer FJ, Butler P, Potter AN, Provost JP, Richard J, Snyder PA, Reagan WJ. Cardiac
troponin I is a sensitive, specific biomarker of cardiac injury in laboratory animals. Lab Anim 40:
47. Pepe S, Tsuchiya N, Lakatta EG, Hansford RG. PUFA and aging modulate cardiac mitochondrial
membrane lipid composition and Ca2+ activation of PDH. Am J Physiol 276: H149-H158, 1999.
48. Pozzan TRizzuto R. The renaissance of mitochondrial calcium transport. Eur J Biochem 267: 5269-
49. Schaub RG, Stewart G, Strong M, Ruotolo R, Lemoie G. Reduction of ischemic myocardial
damage in the dog by lidocaine infusion. Am J Pathol 87: 399-414, 1977.
50. Scheufler EPeters T. Determination of the extracellular space with nonradioactive Co3+EDTA and
simultaneous estimation of Na, K, Ca, and Mg contents in isolated guinea-pig heart preparations by
atomic absorption spectrometry. Basic Res Cardiol 82: 341-347, 1987.
51. Scholz WAlbus U. Potential of selective sodium-hydrogen exchange inhibitors in cardiovascular
therapy. Cardiovasc Res 29: 184-188, 1995.
52. Schwinger RH, Wang J, Frank K, Muller-Ehmsen J, Brixius K, McDonough AA, Erdmann E.
Reduced sodium pump alpha1, alpha3, and beta1-isoform protein levels and Na+,K+-ATPase activity
but unchanged Na+-Ca2+ exchanger protein levels in human heart failure. Circulation 99: 2105-2112,
53. Tosaki A, Balint S, Szekeres L. Protective effect of lidocaine against ischemia and reperfusion-
induced arrhythmias and shifts of myocardial sodium, potassium, and calcium content. J Cardiovasc
Pharmacol 12: 621-628, 1988.
Page 30 of 42
54. Verdonck F, Bielen FV, Ver DL. Preferential block of the veratridine-induced, non-inactivating Na+
current by R56865 in single cardiac Purkinje cells. Eur J Pharmacol 203: 371-378, 1991.
55. Vinnakota KCBassingthwaighte JB. Myocardial density and composition: a basis for calculating
intracellular metabolite concentrations. Am J Physiol Heart Circ Physiol 286: H1742-H1749, 2004.
56. Vitola JV, Forman MB, Holsinger JP, Atkinson JB, Murray JJ. Reduction of myocardial infarct
size in rabbits and inhibition of activation of rabbit and human neutrophils by lidocaine. Am Heart J
133: 315-322, 1997.
57. von Planta I, Weil MH, von Planta M, Bisera J, Bruno S, Gazmuri RJ, Rackow EC.
Cardiopulmonary resuscitation in the rat. J Appl Physiol 65: 2641-2647, 1988.
58. Wann SR, Weil MH, Sun S, Tang W, Yu T. Cariporide for pharmacologic defibrillation after
prolonged cardiac arrest. J Cardiovasc Pharmacol Ther 7: 161-169, 2002.
59. Wirth KJ, Maier T, Busch AE. NHE1-inhibitor cariporide prevents the transient reperfusion-
induced shortening of the monophasic action potential after coronary ischemia in pigs. Basic Res
Cardiol 96: 192-197, 2001.
60. Wu YClusin WT. Calcium transient alternans in blood-perfused ischemic hearts: observations with
fluorescent indicator fura red. Am J Physiol 273: H2161-H2169, 1997.
61. Yamamoto S, Matsui K, Ohashi N. Protective effect of Na+ /H+ exchange inhibitor, SM-20550, on
impaired mitochondrial respiratory function and mitochondrial Ca2+ overload in ischemic/reperfused
rat hearts. J Cardiovasc Pharmacol 39: 569-575, 2002.
62. Yasutake MAvkiran M. Exacerbation of reperfusion arrhythmias by alpha 1 adrenergic stimulation:
a potential role for receptor mediated activation of sarcolemmal sodium-hydrogen exchange.
Cardiovasc Res 29: 222-230, 1995.
Page 31 of 42
63. Yokoyama H, Yasutake M, Avkiran M. Alpha1-adrenergic stimulation of sarcolemmal Na+-H+
exchanger activity in rat ventricular myocytes: evidence for selective mediation by the alpha1A-
adrenoceptor subtype. Circ Res 82: 1078-1085, 1998.
Page 32 of 42
Figure 1: Experimental groups depicting the various intervention and the time at which hearts were
harvested (a to d). BL = Baseline; VF = Ventricular fibrillation; VF/CC = VF with chest compression; PR
= Post-resuscitation. The interval between Co-EDTA-injection and harvesting was set at 10, 20, or 30
minutes in BL and PR group and 25, 35, or 45 minutes in VF and VF/CC groups to account for the 15
minute interval of VF. Rats in VF/CC and PR groups were randomized to AVE4454B and lidocaine
vehicle, AVE4454B vehicle and lidocaine, AVE4454B and lidocaine, and vehicles immediately before
starting chest compression (Drug in figure).
Figure 2: Depth of chest compression (Depth, A) and ratio between the coronary perfusion pressure (CPP)
and depth of compression (CPP/Depth, B) during closed-chest resuscitation in rats treated with Na+-
limiting interventions (closed circles, n = 36) or vehicle control (open circles, n = 12). Failure of the
displacement transducer for the initial 3 minutes of chest compression in experimental groups (1 rat) and
spontaneous defibrillation with return of circulation before completion of the 5-minute interval of chest
compression in 3 experimental and 1 control explain fewer observation (in brackets). Right panels depict
effects of individual Na+-limiting interventions at the fifth minute of chest compression. A = AVE4454B
and lidocaine vehicle; L = AVE4454B vehicle and lidocaine; A/L = AVE4454B and lidocaine. Mean ±
SEM. ∗p = 0.030 vs control by Student’ s t-test.
Figure 3: A: Shocks required in each rat to terminate ventricular fibrillation (VF) during closed-chest
resuscitation (R). B: Shocks required in each rat to terminate episodes of refibrillation during the post-
resuscitation interval (PR). C = vehicle control; A = AVE4454B and lidocaine vehicle; L = AVE4454B
vehicle and lidocaine; A/L = AVE4454B and lidocaine. The number in brackets denotes number of
shocks delivered. *p < 0.05 for differences among the 4 groups for Kruskal-Wallis one-way ANOVA on
ranks. Dunn’ s method for multiple comparisons failed to identify specific group differences.
Figure 4: A: Mean aortic pressure (MAP) at baseline (BL), at 15 minutes of untreated ventricular
fibrillation (VF), at 15 minutes of VF with chest compression for the last 5 minutes (VF/CC), and at 60
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minutes post-resuscitation (PR). B: Cardiac work index (CWI) and cardiac index (CI) at BL and PR.
Open bars denote control rats and closed bars rats treated with Na+-limiting interventions. Shaded bars
denote effects of individual Na+-limiting interventions. A = AVE4454B and lidocaine vehicle; L =
AVE4454B vehicle and lidocaine; A/L = AVE4454B and lidocaine. Mean ± SEM. *p < 0.05 vs BL by
Kruskal-Wallis one-way ANOVA on ranks using Dunn's Method for multiple comparisons; ‡ p < 0.001 vs
control by Student’ s t-test.; §p < 0.05 vs BL by one-way ANOVA using Dunnett's Method for multiple
comparisons; † p < 0.05 vs control by Student’ s t-test.
Figure 5: Left ventricular Co-EDTA-distribution space (A) and water content (B) at baseline (BL), at 15
minutes of untreated ventricular fibrillation (VF), at 15 minutes of VF with chest compression for the last
5 minutes (VF/CC), and at 60 minutes post-resuscitation (PR). Open bars denote control rats and closed
bars rats treated with Na+-limiting interventions. Shaded bars denote effects of individual Na+-limiting
interventions. A = AVE4454B and lidocaine vehicle; L = AVE4454B vehicle and lidocaine; A/L =
AVE4454B and lidocaine. Mean ± SEM. ∗p < 0.05 vs BL by Kruskall-Wallis one-way ANOVA on ranks
using Dunn's Method for multiple comparisons; #two-way ANOVA using time factor (VF/CC vs PR) and
treatment factor (control vs Na+-limiting interventions) was significant for time factor (p < 0.001).
Figure 6: Left ventricular intracellular Na+([Na+]i) (A) and mitochondrial Ca2+ ([Ca2+]m) (B) at baseline
(BL), at 15 minutes of untreated ventricular fibrillation (VF), at 15 minutes of VF with chest compression
for the last 5 minutes (VF/CC), and at 60 minutes post-resuscitation (PR). Open bars denote control rats
and closed bars rats treated with Na+-limiting interventions. Shaded bars denote effects of individual Na+-
limiting interventions. A = AVE4454B and lidocaine vehicle; L = AVE4454B vehicle and lidocaine; A/L
= AVE4454B and lidocaine. Mean ± SEM. ∗p < 0.05 vs BL by Kruskal-Wallis one-way ANOVA on
ranks using Dunn's Method for multiple comparisons; † p < 0.05 vs control by Student’ s t-test in PR
groups; #two-way ANOVA using time factor (VF/CC vs PR) and treatment factor (control vs Na+-limiting
interventions) was significant for treatment factor (p = 0.013) for [Na+]iand for both, time factor (p =
0.045) and treatment factor (p = 0.021) for ([Ca2+]m.
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Figure 7: Plasma cardiac troponin I (cTnI) at baseline (BL) and at 60 minutes post-resuscitation (PR) in
rats subjected to VF and resuscitation. Bar graphs: The open bar denotes control rats and the closed bar
rats treated with Na+-limiting interventions. Shaded bars denote effects of individual Na+-limiting
interventions. A = AVE4454B and lidocaine vehicle; L = AVE4454B vehicle and lidocaine; A/L =
AVE4454B and lidocaine. Mean ± SEM. *p < 0.05 vs control by Student’ s t-test. Scatterplots: Depict the
correlations and linear regressions between cTnI and cardiac work index (CWI), left ventricular
intracellular Na+([Na+]i), and left ventricular Co-EDTA-distribution space at 60 minutes PR. Closed
circles = AVE4454B and lidocaine vehicle; Down triangles = AVE4454B vehicle and lidocaine; Up
triangles = AVE4454B and lidocaine; Open circles = AVE4454B vehicle and lidocaine vehicle (control).
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VF 15-mins BL
VF 15-mins BLPR 60-mins
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Chest compression (mins)
A L A/L
A L A/L
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5 J 3 J 7 J
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66 1866 18
∗ ∗∗ ∗
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Co-EDTA-distribution space (%)
Water content (%)
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PR BL VFVF/CC
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10 Download full-text
r = 0.73
n = 24
p < 0.001
0 20 40 60 80100
r = 0.58
n = 24
p < 0.01
0 20 40 60 80100
r = 0.66
n = 24
p < 0.001
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