Embed Size (px)
Citation preview
2004;127:1262-1269 J Thorac Cardiovasc SurgGrand
T. Bish, Jeffrey Burdick, Timothy J. Pirolli, Mark F. Berry, Vivian Hsu and Todd Y. Joseph Woo, Matthew D. Taylor, Jeffrey E. Cohen, Vasant Jayasankar, Lawrence
prolonged myocardial ischemiaEthyl pyruvate preserves cardiac function and attenuates oxidative injury after
http://jtcs.ctsnetjournals.org/cgi/content/full/127/5/1262located on the World Wide Web at:
The online version of this article, along with updated information and services, is
2004 American Association for Thoracic Surgery Association for Thoracic Surgery and the Western Thoracic Surgical Association. Copyright ©
is the official publication of the AmericanThe Journal of Thoracic and Cardiovascular Surgery
on June 7, 2013 jtcs.ctsnetjournals.orgDownloaded from
CardiopulmonarySupport and
PhysiologyEthyl pyruvate preserves cardiac function and attenuatesoxidative injury after prolonged myocardial ischemiaY. Joseph Woo, MDa
Matthew D. Taylor, BSa
Jeffrey E. Cohena
Vasant Jayasankar, MDa
Lawrence T. Bish, BSb
Jeffrey Burdick, BSa
Timothy J. Pirollib
Mark F. Berry, MDa
Vivian Hsua
a
Todd Grand, BSFrom the Departments of Surgerya andPhysiology,b University of PennsylvaniaSchool of Medicine, Philadelphia, Pa.
Read at the Eighty-third Annual Meeting ofThe American Association for ThoracicSurgery, Boston, Mass, May 4-7, 2003.
Received for publication April 8, 2003; re-visions requested Oct 28, 2003; acceptedfor publication Nov 4, 2003.
Address for reprints: Y. Joseph Woo, MD,Assistant Professor of Surgery, Director,Minimally Invasive and Robotic CardiacSurgery Program, Division of Cardiotho-racic Surgery, Department of Surgery, Uni-versity of Pennsylvania, Silverstein 4, 3400Spruce St, Philadelphia, PA 19104 (E-mail:[email protected]).
J Thorac Cardiovasc Surg 2004;127:1262-9
0022-5223/$30.00
Copyright © 2004 by The American Asso-ciation for Thoracic Surgery
doi:10.1016/j.jtcvs.2003.11.032
1262 The Journal of Thoracic and CardDow
Objective: Myocardial injury and dysfunction following ischemia are mediatedin part by reactive oxygen species. Pyruvate, a key glycolytic intermediary, is aneffective free radical scavenger but unfortunately is limited by aqueous insta-bility. The ester derivative, ethyl pyruvate, is stable in solution and shouldfunction as an antioxidant and energy precursor. This study sought to evaluateethyl pyruvate as a myocardial protective agent in a rat model of ischemia-reperfusion injury.
Methods: Rats underwent 30-minute ischemia and 30-minute reperfusion of the leftanterior descending coronary artery territory. Immediately prior to both ischemiaand reperfusion, animals received an intravenous bolus of either ethyl pyruvate (n� 26) or vehicle control (n � 26). Myocardial high-energy phosphate levels weredetermined by adenosine triphosphate assay, oxidative injury was measured by lipidperoxidation assay, infarct size was quantified by triphenyltetrazolium chloridestaining, and cardiac function was assessed in vivo.
Results: Ethyl pyruvate administration significantly increased myocardial adenosinetriphosphate levels compared with control (87.6 � 29.2 nmol/g vs 10.0 � 2.4nmol/g, P � .03). In ischemic myocardium, ethyl pyruvate reduced oxidative injurycompared with control (63.8 � 3.3 nmol/g vs 89.5 � 3.0 nmol/g, P � .001). Ethylpyruvate diminished infarct size as a percentage of area at risk (25.3% � 1.5% vs33.6% � 2.1%, P � .005). Ethyl pyruvate improved myocardial function comparedwith control (maximum pressure: 86.6 � 2.9 mm Hg vs 73.5 � 2.5 mm Hg, P �.001; maximum rate of pressure rise: 3518 � 243 mm Hg/s vs 2703 � 175 mmHg/s, P � .005; maximal rate of ventricular systolic volume ejection: 3097 � 479�L/s vs 2120 � 287 �L/s, P � .04; ejection fraction: 41.9% � 3.8% vs 31.4% �4.1%, P � .03; cardiac output: 26.7 � 0.9 mL/min vs 22.7 � 1.3 mL/min, P � .01;and end-systolic pressure-volume relationship slope: 1.09 � 0.22 vs 0.59 � 0.2, P� .02).
Conclusions: In this study of myocardial ischemia-reperfusion injury, ethyl pyruvateenhanced myocardial adenosine triphosphate levels, attenuated myocardial oxida-
tive injury, decreased infarct size, and preserved cardiac function.iovascular Surgery ● May 2004 on June 7, 2013 jtcs.ctsnetjournals.orgnloaded from
Woo et al Cardiopulmonary Support and Physiology
CSP
Myocardial dysfunction and injury fol-lowing ischemia are attributed tomultiple factors, of which metabolicdepletion of high-energy phosphatesand formation of reactive oxygen spe-cies (ROS) are perhaps most predom-
inant. During ischemia, without citric acid cycle contribu-tion, energy requirements are poorly met by anaerobicglycolysis. Finely controlling glycolysis are multiple regu-lators including substrate limitation, oxidized nicotinamideadenine dinucleotide (NAD�), and allosterically governedenzymes. Researchers have attempted various approachestoward glycolytic substrate enhancement to augment aden-osine triphosphate (ATP) production. Pyruvate, one suchextensively studied efficacious glycolytic intermediate,yields increased cytosolic NAD�, the obligate electron ac-ceptor for glyceraldehyde-3-phosphate dehydrogenase,which oxidizes glyceraldehyde-3-phosphate to 1,3 bisphos-phoglycerate.1-4 In turn, phosphoglycerate kinase can thencatalyze 1,3 bisphosphoglycerate to 3-phosphoglycerategenerating ATP, augmenting cardiomyocyte energetics.1
The second primary mechanism of injury involves thegeneration of ROS during reperfusion via multiple reac-tions, most notably:
xanthine oxidase: 2O2 � NADPH 3 2O2�� � NADP� �
H�
superoxide dismutase: 2O2�� � 2H� 3 H2O2
nitric oxide: O2�� � NO� 3 ONOO�
ionized iron: H2O2 � Fe�2 3 OH� � OH� � Fe�3
Superoxide anion (O2��) and hydrogen peroxide (H2O2) are
moderately reactive, whereas peroxynitrite (ONOO�) andhydroxyl radical (OH�) are highly toxic and produce aspectrum of cellular injury via multiple mechanisms includ-ing membrane destabilization, mitochondrial disruption,and metabolic derangement. There exists a battery of nativeantioxidant enzyme systems that sequentially convert ROSto water and oxygen:
superoxide dismutase: 2O2� � 2H� 3 H2O2 � O2
catalase: 2H2O2 3 2H2O � O2
glutathione peroxidase: H2O2 � 2GSH 3 2H2O � GSSG
where GSH is reduced glutathione and GSSG is oxidizedglutathione. Multiple strategies for augmenting these nativesystems have likewise been investigated.5-8
Interestingly pyruvate, in the presence of hydrogen per-oxide, will decarboxylate to yield acetate, water, and carbondioxide.9 Pyruvate is also capable of scavenging hydroxylradical.10 Studies have shown that exogenous pyruvate ad-ministration diminishes ischemic myocardial oxidative in-jury,11-14 apparently in a dose-dependent manner.15 Further-more, independent of its metabolic and antioxidant features,
pyruvate may possess myocardial inotropic properties.The Journal of Thoracicjtcs.ctsnetjouDownloaded from
Pyruvate directly increases sarcoplasmic reticular ATPaseactivity and hence calcium cycling efficiency.15 In addition,pyruvate also indirectly augments beta agonist–mediatedinotropy.16 Unfortunately, the therapeutic potential of ex-ogenously administered pyruvate is significantly limited byextreme aqueous instability. Pyruvate can undergo sponta-neous condensation to parapyruvate, which can further un-dergo cyclization and either dehydration or reduction toyield a purported mitochondrial inhibitor.17,18
Ethyl pyruvate, a commercial food additive, is a li-pophilic ester derivative of pyruvate. Although poorly sol-uble in water or saline, ethyl pyruvate is highly soluble incalcium solutions, forming a dimolecular complex of theanionic enolate form stabilized with divalent cationic calci-um.19 In this form, ethyl pyruvate possesses far greaterstability than pyruvate and hence may serve as a practicalpyruvate precursor.
We therefore hypothesized that in a model of myocardialischemia-reperfusion injury, ethyl pyruvate administrationwould provide a cardioprotective effect.
MethodsAnimal Care and BiosafetyMale Wistar rats weighing 250 to 350 g were obtained fromCharles River (Boston, Mass). Food and water were provided adlibitum. This study was performed in accordance with the standardhumane care guidelines of the Institutional Animal Care and UseCommittee of the University of Pennsylvania, which conform tocurrent federal guidelines.
Preparation of Treatment SolutionsExperimental treatment groups were divided into animals receiv-ing Ringer’s solution as a control or Ringer’s solution containing28 mmol/L ethyl pyruvate obtained from Sigma Chemicals (StLouis, Mo). Ringer’s solution contained 130 mmol/L Na�, 4.0mmol/L K�, 2.7 mmol/L Ca�2, and 109 mmol/L Cl� at pH 7.0.
Acute in Vivo Ischemia-Reperfusion ModelAnimals (n � 52) underwent induction of general anesthesia withketamine (75 mg/kg) and xylazine (7.5 mg/kg), endotracheal in-tubation with a 14-gauge angiocatheter, and mechanical ventilationwith 2% isofluorane maintenance anesthesia with a respirator(Hallowell EMC, Pittsfield, Mass). Venous access for administra-tion of treatment solutions was obtained via exposure of the rightfemoral vein and insertion of a 24-gauge intravenous catheter. Aleft thoracotomy was performed through the fourth intercostalspace and the pericardium was reflected, exposing the heart. A 7-0polypropylene suture was then placed around the left anteriordescending (LAD) coronary artery and briefly snared to visuallyverify the territory of myocardial ischemia. Animals were thenrandomized to either the control (n � 26) or ethyl pyruvate group(n � 26) and received a 1.5 mL/kg intravenous bolus of eitherRinger’s solution or ethyl pyruvate. Two minutes later, ischemiawas initiated. The LAD was then occluded for 30 minutes. In asmall subset of animals (control n � 5, ethyl pyruvate n � 5),
hearts were harvested 10 minutes into the ischemia period forand Cardiovascular Surgery ● Volume 127, Number 5 1263 on June 7, 2013 rnals.org
Cardiopulmonary Support and Physiology Woo et al
CSP
analysis of myocardial energetic state via ATP assay. In the other42 animals, after the 30-minute ischemic period, just prior toreperfusion, the treatment solution was again intravenously bo-lused (3.0 mL/kg). Hearts were then reperfused for 30 minutes. Atthe end of this period, the animals were either put to death andhearts were harvested for lipid peroxidation analysis (control n �6, ethyl pyruvate n � 6) or animals were prepared for cardiacfunctional assessment and subsequent heart explantation for infarctsize determination (control n � 15, ethyl pyruvate n � 15).
Myocardial ATP LevelsATP levels were quantified using the commercially available EN-LITEN ATP luciferin/luciferase bioluminescence assay system(Promega, Madison, Wis). Myocardial tissue specimens from theischemic region were excised, immediately frozen in liquid nitro-gen, and individually pulverized into a fine powder by handgrinding with a dry ice–chilled steel mortar and pestle.20 Tenmilligrams of myocardium were homogenized with 1 mL of pre-cooled extractant (0.1% trichloroacetic acid) and centrifuged at4500 revolutions per minute (rpm) for 10 minutes.21 Supernatant(100 �L) was diluted 10-fold with 50 mmol/L Tris-acetate buffercontaining 2 mmol/L ethylenediaminetetraacetic acid (pH 7.75).Then 100 �L of sample extract or reference standard solution wasplaced in a tube luminometer (Turner Designs Luminometer TD-20/20, Promega), followed by the auto-injection of 100 �L of ATPluciferin/luciferase assay mix for ATP quantification. Lumines-cence was measured at a set lag time of 1 second and integrationtime of 10 seconds.
Myocardial Lipid Peroxidation MeasurementsMyocardial lipid peroxidation was quantified using the commer-cially available PeroxiDetect KIT (Sigma Chemicals). Myocardialtissue specimens were obtained from the ischemic anterolateral leftventricular wall and from the nonischemic remote basal postero-septal left ventricle and immediately frozen in liquid nitrogen.Lipid soluble components were extracted from the myocardial
22
Figure 1. ATP levels measured bioluminescently in ischemicmyocardium 10 minutes after the initiation of ischemia. Signifi-cantly higher levels of ATP were observed in ethyl pyruvate–treated hearts (n � 5) compared with controls (n � 5).
tissue specimens using a CHCl3/methanol extraction protocol.
1264 The Journal of Thoracic and Cardiovascular Surgery ● Majtcs.ctsnetjouDownloaded from
Briefly, 100 mg of tissue specimen was homogenized in a 2:1volume mixture of CHCl3 and 100% methanol at a ratio of 1 gtissue to 15 mL of the CHCl3/methanol mixture. The homogenizedmaterial was centrifuged at 10,000 rpm for 10 minutes, after whichthe supernatant was clarified with 0.3 mL of 0.9% NaCl per 1 g oftissue and then removed. The CHCl3 layer was then evaporatedwith nitrogen gas to leave the lipid peroxides from the specimenscontained in the methanol solvent. Specimen samples (100 �L)were added to a 1-mL mixture of Fe�2 ion and xylenol orange.Peroxides convert Fe�2 to Fe�3, which forms a color adduct withxylenol orange spectrophotometrically detectable at 560 nm. Lipidperoxide levels were then calculated using a reference standardcurve generated with defined quantities of tert-butyl hydroperox-ide.
Cardiac Functional Assessment Following Ischemiaand ReperfusionFollowing the 30-minute reperfusion period, a median sternotomywas performed and myocardial performance was assessed in vivo(control n � 15, ethyl pyruvate n � 15). A fully calibratedminiature pressure/volume conductance catheter (MIKRO-TIPcatheter and ARIA Pressure Volume Conductance System, MillarInstruments, Houston, Tex) was inserted into the left ventricularcavity through the apex. Calibration consisted of the cuvette2-point linear interpolation process and parallel conductance sub-traction via the hypertonic saline method. The digitized pressureand volume signals were displayed and recorded using Chartv4.1.2 software (AD Instruments, Colorado Springs, Colo). Mul-tiple cardiac functional parameters were measured in the 2 groupsusing Cardiac Pressure Volume Analysis Software-PVAN 2.9(Millar Instruments). Additionally, a flow probe monitor (Tran-sonic Systems, Ithaca, NY) was placed around the ascending aortato measure cardiac output. To provide a reference of baseline ratmyocardial function, a separate group of 15 noninfarcted nativeanimals underwent pressure-volume and cardiac output analysis.
Planimetric Determination of Left VentricularInfarction SizeFollowing the cardiac functional analysis, control and ethyl pyru-vate hearts were excised and rinsed in normal saline solution (pH7.4). The LAD was again snared and Evans blue dye was infusedinto the aortic root to facilitate determination of area at risk. Thespecimens were cut perpendicular to the long axis into 5 sectionsand incubated in 1% triphenyltetrazolium chloride (TTC) (SigmaChemicals) in phosphate-buffered saline solution (pH 7.4) at 37°Cfor 20 minutes. Following the incubation period, the TTC wasrinsed from the sections and a 10% formalin solution was addedfor tissue fixation. Sections were photographed using a digitalcamera and were downloaded onto a desktop computer containingdigital planimetry software (OpenLab, Lexington, Mass). Infarctsize as a percentage of area at risk was then measured.
Statistical MethodsStatistical analyses were performed using unpaired, 1-tailed Stu-dent t tests. All results were expressed as mean � standard error of
the mean (SEM).y 2004 on June 7, 2013 rnals.org
Woo et al Cardiopulmonary Support and Physiology
CSP
ResultsATP QuantificationAnalysis of ATP levels in ischemic hearts revealed amarked increase in animals receiving ethyl pyruvate com-pared with control animals (Figure 1). The ischemic regionof control hearts contained 10.0 � 2.4 nmol/g, whereas theischemic region of ethyl pyruvate hearts contained 87.6 �29.2 nmol/g (P � .03).
Lipid Peroxidation AnalysisSpectrophotometric quantification of lipid peroxides inmyocardium exposed to ischemia and reperfusion and innonischemic myocardium, which served as an internal con-trol, is represented in Figure 2. Lipid peroxide levels in thenonischemic myocardium did not differ significantly be-tween animals treated with ethyl pyruvate (42.3 � 4.8nmol/g; n � 6) and the control, Ringer’s solution (37.5 �5.1 nmol/g; n � 6), implying equivalent assay conditionsbetween control and ethyl pyruvate hearts. Ischemia signif-icantly increased the level of lipid peroxidation from 37.5 �5.1 nmol/g to 89.5 � 3.0 nmol/g in control hearts (P �.001). When comparing ischemic myocardium in ethylpyruvate hearts with control hearts, there was a statisticallysignificant decrement in lipid peroxidation with ethyl pyru-vate administration (63.8 � 3.3 vs 89.5 � 3.0 nmol/g, P �
Figure 2. Lipid peroxidation in myocardium following ischemiaand reperfusion. Lipid peroxidation in remote nonischemic myo-cardium was equivalent between control (n � 6) and ethylpyruvate (n � 6) hearts. Ischemia increased the level of lipidperoxidation in the control hearts. The administration of ethylpyruvate decreased the level of lipid peroxidation in the ischemicmyocardium when compared with control hearts.
.001).
The Journal of Thoracicjtcs.ctsnetjouDownloaded from
Infarction AnalysisMacroscopic analysis of TTC-stained cross sections follow-ing 30 minutes of ischemia and 30 minutes of reperfusiondemonstrated attenuated left ventricular infarction sizes inanimals treated with ethyl pyruvate (n � 15) as comparedwith the control group (n � 15; Figure 3). Ethyl pyruvateanimals had 25.3% � 1.5% of the left ventricular area atrisk infarcted as compared with 33.6% � 2.1% in controlanimals (P � .005).
Cardiac FunctionTable 1 displays heart rate and multiple pressure and vol-
TABLE 1. Hemodynamic data
ParameterNative
(n � 15)Control
(n � 15)
Ethylpyruvate(n � 15)
P value(controlvs ethyl
pyruvate)
Heart rate (bpm) 210 � 7.6 176 � 14.4 183 � 9.5 NSMaximum
pressure (mmHg)
98.7 � 2.9 73.5 � 2.5 86.6 � 2.9 �.001
Maximum dP/dt(mm Hg/s)
4974 � 199 2703 � 175 3518 � 243 .005
Minimum dP/dt(mm Hg/s)
�3940 � 258�1745 � 170 �2841 � 329 .003
Maximum dV/dt(�L/s)
3919 � 321 2120 � 287 3097 � 479 .04
Ejection fraction(%)
47.1 � 3.3 31.4 � 4.1 41.9 � 3.8 .03
Cardiac output(mL/min)
33.0 � 1.8 22.7 � 1.3 26.7 � 0.9 .01
bpm, Beats per minute; dP/dt, rate of pressure rise; dV/dt, rate of ventric-ular systolic volume ejection.
Figure 3. Myocardial infarction percentage measured by infarc-tion area (IA)/area at risk (AAR) following ischemia and reperfu-sion. Significant differences in left ventricular infarction wereobserved between animals receiving ethyl pyruvate (n � 15) andthe control group (n � 15).
ume parameters for native animals (n � 15) as a reference,
and Cardiovascular Surgery ● Volume 127, Number 5 1265 on June 7, 2013 rnals.org
Cardiopulmonary Support and Physiology Woo et al
CSP
Ringer’s control animals (n � 15), and ethyl pyruvateanimals (n � 15). Statistical comparisons between controland ethyl pyruvate animals are displayed. Heart rates wereequivalent. In all pressure and volume parameters mea-sured, there was a statistically significant increase in cardiacfunction among ethyl pyruvate animals compared with con-trols. The administration of ethyl pyruvate to infarcted an-imals brought hemodynamic parameters partially back tothose levels observed among noninfarcted native animals.End-systolic pressure-volume relationships were assessedand represented as a regression composite for each of the 3groups (Figure 4). Ethyl pyruvate animals exhibited signif-icantly improved myocardial function compared with con-trol animals, approaching native baseline values.
DiscussionIschemia-reperfusion injury of myocardium is a significantentity in many clinical situations, including coronary throm-bolysis, percutaneous coronary interventions, cardiac sur-
Figure 4. Composite representation of end-systolic pressure-vol-ume relationships (ESPVR) in native animals (n � 15) and incontrol (n � 15) and ethyl pyruvate–treated (n � 15) animalsfollowing ischemia and reperfusion. For native animals, mean �SEM slope, x-axis intercept (x-int), and correlation coefficient (r)were: 1.27 � 0.12, 10.4 � 2.0, and 0.97 � 0.07, respectively. Forcontrol animals, the values were: slope � 0.59 � 0.2, x-int � 9.9� 24.0, r � 0.68 � 0.13; for ethyl pyruvate–treated animals, thesevalues were: slope � 1.09 � 0.22, x-int � �4.1 � 19.9, r � 0.89� 0.03. Comparing the slopes of the control and ethyl pyruvatehearts demonstrates a statistically significant improvement incontractility with the administration of ethyl pyruvate (P � .02).Composite ESPVR for the native animals (n � 15) was 1.27(end-systolic volume [ESV]-10.39), control (n � 15) was 0.59(ESV-9.88), and ethyl pyruvate (n � 15) was 1.09 (ESV-4.06).Significant differences in the slope of the ESPVR were foundbetween ethyl pyruvate and control animals.
gery, and heart transplantation. Myocardial dysfunction and
1266 The Journal of Thoracic and Cardiovascular Surgery ● Majtcs.ctsnetjouDownloaded from
cellular injury occurs in part due to metabolic depletionduring ischemia followed by ROS formation during reper-fusion. Significant research efforts have investigated meansof protecting myocardium against ischemia-reperfusion in-jury. This article describes our utilization of ethyl pyruvateas an intravenous myocardial protection agent. Rats re-ceived bolus ethyl pyruvate before temporary coronary li-gation and just before reperfusion and demonstrated statis-tically significantly increased myocardial ATP levels,decreased tissue oxidative injury, decreased infarct sizes,and preserved myocardial function. To our knowledge, thisis the first report of a cardiovascular application of thisagent.
Ethyl pyruvate’s parent compound, the glycolytic prod-uct pyruvate, has been shown to attenuate myocardial isch-emic injury through both metabolic augmentation and anti-oxidant mechanisms but is limited in potential therapeuticefficacy by extreme aqueous instability.1,12,17 Specific stud-ies into the metabolic activity of the related pyruvate ester,methyl pyruvate, in pancreatic islet cells have demonstratedcellular and mitochondrial membrane permeation, deesteri-fication liberating free pyruvate for glycolytic substrate uti-lization, and subsequent augmentation of ATP generation.23
These studies, which were performed on nonischemic cells,also found a disproportionate increase in ATP generationcompared with glucose utilization. This enhanced produc-tion of ATP is felt to result from rapid intracellular dees-terification of methyl pyruvate to pyruvate and thus thegeneration of a significant transcellular gradient favoringfurther intracellular influx of methyl pyruvate. This resultsin the rapid accumulation of an intracellular excess pool ofpyruvate generated free of glycolytic regulators and withoutthe obligate initial ATP investment at the glucokinase andphosphofructokinase reactions. Pyruvate is then availablefor pyruvate dehydrogenase conversion to acetyl CoA andcitric acid cycle substrate provision.2 Such pyruvate storesmay be particularly useful in the myocardial reperfusionphase when the citric acid cycle becomes available again,yet ATP levels are very low and require rapid repletion.
Biologic investigation of ethyl pyruvate was first de-scribed in ophthalmologic research aimed at the preventionof free radical–induced lens injury, which normally leads tocataract formation.24,25 Ethyl pyruvate has subsequentlybeen studied in the trauma/critical care literature with mod-els of mesenteric ischemia, hemorrhagic shock, and endo-toxemic sepsis, all demonstrating a cytoprotective ef-fect.19,26-29 Another postulated role of ethyl pyruvate isrelated to its anti-inflammatory properties. In a murine hem-orrhagic shock model, ethyl pyruvate decreased expressionof tumor necrosis factor and interleukin-6.26 This could beexplained as an indirect finding related to ethyl pyruvate–mediated attenuation of multiorgan ischemic injury from the
induced state of hemorrhagic shock. In vitro, ethyl pyruvatey 2004 on June 7, 2013 rnals.org
Woo et al Cardiopulmonary Support and Physiology
CSP
does appear to directly inhibit the nuclear factor �B– andp38 mitogen–activated protein kinase pathways of inflam-matory cytokine activation. To date, there has not been areport of the application of ethyl pyruvate to the cardiovas-cular system.
This study sought to investigate the potential effect ofexogenous ethyl pyruvate administration on reducing myo-cardial injury and contractile dysfunction after ischemia.
The overall design of this study evaluated the effects ofethyl pyruvate at biochemical, cellular, and physiologiclevels. Myocardial high-energy phosphate levels, free radi-cal injury, myocardial infarction, and cardiac mechanicswere examined in a model of myocardial ischemia-reperfu-sion injury. The proportionate degree of myocardial injuryinduced is more pronounced than what occurs in a typicalcardiac surgical procedure. Nevertheless, the model wasselected for its predictability and functionality.
In the control group, Ringer’s solution was utilized topreclude any attribution of observed differences to electro-lyte composition of solutions. Also, in the unlikely eventthat some acid-base buffering capacity of ethyl pyruvatewas the only cause of observed differences, we also per-formed a small pilot series of control animals with lactatedRinger’s solution. These animals would have obtained anybuffering benefit, yet myocardial function and infarct sizewere equivalent to Ringer’s solution controls. The dose ofethyl pyruvate selected for this study was based upon aconcentration of ethyl pyruvate previously optimized in anintestinal ischemia model.19 The timing of administration ofthe ethyl pyruvate was designed to enhance the two pur-ported mechanisms of action, glycolytic substrate augmen-tation and antioxidation.
An initial left thoracotomy was employed during theischemic period for several reasons. This permitted ease ofaccess to the LAD, ease of confirmation of a large antero-lateral region of ischemic discoloration, and absence ofsternal bleeding during this period. A sternotomy was thenutilized for hemodynamic assessment because this alloweda more favorable angle of entry of the pressure-volumeconductance microcatheter into the left ventricle. In the rat,catheter insertion from a thoracotomy approach causes left-ward displacement of the cardiac apex with resultant torsionon the heart and hemodynamic compromise similar to thatseen clinically when displacing the heart during off-pumpgrafting of lateral coronary arteries. Furthermore, a sternot-omy facilitated easier access to the inferior vena cava forload variation to assess pressure-volume relationships.
To confirm the proposed mechanism of action of ethylpyruvate attributed to glycolytic substrate augmentation,tissue ATP levels were assayed in the ischemic myocardialterritory 10 minutes after LAD snaring. This period of timewas chosen to permit depletion of potentially confounding
myocardial high-energy phosphate reserves as well as toThe Journal of Thoracicjtcs.ctsnetjouDownloaded from
ensure dependence upon anaerobic glycolysis. As describedabove, excess exogenous pyruvate could liberate NAD� formore proximal glycolytic pathway generation of ATP.
Myocardial oxidative injury was diminished with ethylpyruvate. Compared with other inferential assays of freeradical injury, such as measuring GSH/GSSG levels, thelipid peroxidation assay is a direct measure of free radicaltissue injury. In this study, both an internal control withineach heart of ischemic versus nonischemic tissue and anexternal control of ethyl pyruvate–treated hearts versusRinger’s control hearts were employed. The equivalent re-sults in nonischemic tissue between ethyl pyruvate andcontrol groups further validated both the model and theassay. The reduction in lipid peroxidation of the ethyl pyru-vate group as compared with the control group is an indi-cation of reduced free radical injury in the animals treatedwith ethyl pyruvate.
Clearly, further studies are warranted. Future investiga-tions should evaluate ethyl pyruvate’s myocardial protectivecapacity in other models of myocardial ischemia as a meansof broadening the spectrum of clinical utility. To moreclosely mimic the typical ischemia occurring during on-pump cardiac surgery, the duration or proximity of ligationcould be altered to decrease the extent of myocardial injury.Shorter, transient periods of localized ischemia could beproduced to better model off-pump coronary surgery orpercutaneous coronary interventions.
Future directions should determine whether using higherdoses of ethyl pyruvate yields even greater myocardialprotective effects. Presently, only a limited dose-responsecurve of 3 concentrations of ethyl pyruvate spanning 3 logshas been studied.15 Alternate routes of administration, par-ticularly intracoronary, should also be evaluated to searchfor increased efficacy. A dual intravenous bolus techniquewas utilized with the assumption that a continuous infusionduring the period of coronary occlusion may not be useful.Alternate timing strategies should also be evaluated, such asprolonged pretreatment, continuous treatment, and postre-perfusion continuous infusion. Finally, ethyl pyruvateshould also be evaluated as an additive to transplant organpreservation solutions.
In this study, ethyl pyruvate—a nontoxic, inexpensive,intravenously administered antioxidant and potential glyco-lytic substrate—significantly preserved cardiac function,enhanced tissue ATP levels, attenuated myocardial oxida-tive injury, and markedly reduced infarct size after pro-longed myocardial ischemia.
References
1. Ochiai K, Zhang J, Gong G, Zhang Y, Liu J, Ye Y, et al. Effects ofaugmented delivery of pyruvate on myocardial high-energy phosphatemetabolism at high workstate. Am J Physiol Heart Circ Physiol.2001;281:H1823-32.
2. Valenti D, deBari L, Atlante A, Passarella S. L-lactate transport into
and Cardiovascular Surgery ● Volume 127, Number 5 1267 on June 7, 2013 rnals.org
Cardiopulmonary Support and Physiology Woo et al
CSP
rat heart mitochondria and reconstruction of the L-lactate/pyruvateshuttle. Biochem J. 2002;364:101-4.
3. Smolenski RT, Amrani M, Jayakumar J, Jagodzinski P, Gray CC,Goodwin AT, et al. Pyruvate/dichloroacetate supply during reperfu-sion accelerates recovery of cardiac energetics and improves mechan-ical function following cardioplegic arrest. Eur J Cardiothorac Surg.2001;19:865-72.
4. Saiki Y, Lopaschuk GD, Dodge K, Yamaya K, Morgan C, RebeykaIM. Pyruvate augments mechanical function via activation of thepyruvate dehydrogenase complex in reperfused ischemic immaturerabbit hearts. J Surg Res. 1998;79:164-9.
5. Woo YJ, Zhang JCL, Vijayasarathy C, Zwacka RM, Englehardt JF,Gardner TJ, et al. Recombinant adenovirus-mediated cardiac genetransfer of superoxide dismutase attenuates postischemic contractiledysfunction. Circulation. 1998;98:II255-261.
6. Johnson DL, Horneffer PJ, Dinatale JM Jr, Gott VL, Gardner TJ. Freeradical scavengers improve functional recovery of stunned myocar-dium in a model of surgical coronary revascularization. Surgery.1987;102:334-40.
7. Cheung PY, Wang W, Schulz R. Glutathione protects against myo-cardial ischemia reperfusion injury by detoxifying peroxynitrite. J MolCell Cardiol. 2000;32:1669-78.
8. Yoshida T, Watanabe M, Engelman DT, Engelman RM, Schley JA,Maulik N, et al. Transgenic mice overexpressing glutathione peroxi-dase are resistant to myocardial ischemia reperfusion injury. J MolCell Cardiol. 1996;28:1759-67.
9. Constantopoulos G, Barranger GA. Nonenzymatic decarboxylation ofpyruvate. Anal Biochem. 1984;139:353-8.
10. DeBoer LW, Bekx PA, Han L, Steinke L. Pyruvate enhances recoveryof rat hearts after ischemia and reperfusion by preventing free radicalgeneration. Am J Physiol Heart Circ Physiol. 1993;265:H1571-6.
11. Dobsak P, Courdertot-Masuyer C, Zeller M, Vergely C, Laubriet A,Assem M, et al. Antioxidative properties of pyruvate and protection ofthe ischemic rat heart during cardioplegia. J Cardiovasc Pharmacol.1999;34:651-9.
12. Mallet RT. Pyruvate: metabolic protector of cardiac performance. ProcSoc Exp Biol Med. 2000;223:136-48.
13. Mallet RT, Squires JE, Bhatia S, Sun J. Pyruvate restores contractilefunction and antioxidant defenses of hydrogen peroxide-challengedmyocardium. J Mol Cell Cardiol. 2002;34:1173-84.
14. Bassenge E, Sommer O, Schwemmer M, Bunger R. Antioxidantpyruvate inhibits cardiac formation of reactive oxygen species throughchanges in redox state. Am J Physiol Heart Circ Physiol. 2000;279:H2431-8.
15. Martin BJ, Valdivia HH, Bunger R, Lasley RD, Mentzer RM Jr.Pyruvate augments calcium transients and cell shortening in rat ven-tricular myocytes. Am J Physiol Heart Circ Physiol. 1998;274:H8-17.
16. Hermann HP, Zeitz O, Lehnart SE, Keweloh B, Datz N, Hasenfuss G,et al. Potentiation of beta-adrenergic inotropic response by pyruvate infailing human myocardium. Cardiovasc Res. 2002;53:116-23.
17. von Korff RW. Pyruvate-C14, purity and stability. Anal Biochem.1964;8:171-8.
18. Montgomery CM, Webb JL. Metabolic studies on heart mitochondria:II. The inhibitory action of parapyruvate on the tricarboxylic acidcycle. J Biol Chem. 1956;221:359-68.
19. Sims CA, Wattanasirichaigoon S, Menconi MJ, Ajami AM, Fink MP.Ringer’s ethyl pyruvate solution ameliorates ischemia/reperfusion-induced intestinal mucosal injury in rats. Crit Care Med. 2001;29:1513-8.
20. Manthorpe M, Cornefert-Jensen F, Hartikka J, Felgner J, Rundell A,Margalith M, et al. Gene therapy by intramuscular injection of plasmidDNA: studies on firefly luciferase gene expression in mice. Hum GeneTher. 1993;4:419-31.
21. Stanley P. Extraction of adenosine triphosphate from microbial andsomatic cells. Methods Enzymol. 1986;133:14-22.
22. Yagi K, Kiuchi K, Saito Y, Miike A, Kayahara N, Tatano T, et al. Useof a new methylene blue derivative for determination of lipid perox-ides in foods. Biochem Int. 1986;12:367-71.
23. Mertz RJ, Worley JFI, Spencer B, Johnson JH, Dukes ID. Activationof stimulus-secretion coupling in pancreatic beta-cells by specific
products of glucose metabolism. J Biol Chem. 1996;271:4838-45.1268 The Journal of Thoracic and Cardiovascular Surgery ● Majtcs.ctsnetjouDownloaded from
24. Varma SD, Devamanoharan PS, Ali AH. Prevention of intracellularoxidative stress to lens by pyruvate and its ester. Free Radic Res.1998;28:131-5.
25. Devamanoharan PS, Henein M, Ali AH, Varma SD. Attenuation ofsugar cataract by ethyl pyruvate. Mol Cell Biochem. 1999;200:103-9.
26. Yang R, Gallo DJ, Baust JJ, Uchiyama T, Watkins SK, Delude RL, etal. Ethyl pyruvate modulates inflammatory gene expression in micesubjected to hemorrhagic shock. Am J Physiol Gastrointest LiverPhysiol. 2002;283:G212-21.
27. Ulloa L, Ochani M, Yang H, Tanovic M, Halperin D, Yang R, et al. Ethylpyruvate prevents lethality in mice with established lethal sepsis andsystemic inflammation. Proc Natl Acad Sci U S A. 2002;99:12351-6.
28. Tawadrous ZS, Delude RL, Fink MP. Resuscitation from hemorrhagicshock with Ringer’s ethyl pyruvate solution improves survival andameliorates intestinal mucosal hyperpermeability in rats. Shock. 2002;17:473-7.
29. Venkataraman R, Kellum JA, Song M, Fink MP. Resuscitation withRinger’s ethyl pyruvate solution prolongs survival and modulatesplasma cytokine and nitrite/nitrate concentrations in a rat model oflipopolysaccharide-induced shock. Shock. 2002;18:507-12.
DiscussionDr W. Randolph Chitwood, Jr (Greenville, NC). What othermechanisms might be being involved here besides peroxidation?
Dr Woo. The data that we show relates primarily to theantioxidant aspects and that’s been well shown with pyruvate. Weare very interested in evaluating the potential ability to augmentglycolysis. I just bought a luminometer, and we’re going to look atATP levels through a luciferin-luciferase assay to see if there isany augmentation of the total cellular bioenergetic state.
Dr Jakob Vinten-Johansen (Atlanta, Ga). Can you tease outthe functional recovery aspects that you demonstrated? Becauseyou have an agent that exhibits positive inotropic effects, antiox-idant effects, and also metabolic support effects; any 1 of those 3could be responsible for the functional recovery you observed. Thereduction in the infarct size by itself perhaps could be responsiblefor that improvement in function.
In addition, when you looked at the ejection fraction, did youhave a control group to which you could compare that ejectionfraction? In other words, was it actually an increase in the treatedgroup relative to baseline, or does ejection fraction return to abaseline level?
Dr Woo. In terms of your first question, that’s sort of what Ithink Dr Chitwood was alluding to, whether or not there wassomething else going on. You, of course, have accurately definedwhat we have proposed in our background as the 3 primarymechanisms. And again, we have the data for the antioxidant effectand we’re starting the bioenergetics aspect.
I haven’t really thought about how to get at the direct inotropyaspect. There is, I think, a lot more support for pyruvate’s antiox-idant and metabolic aspects and less so for its direct inotropy, soI think that that is perhaps a little less likely to yield something.
In terms of how to distinguish all 3, that’s obviously going tobe of interest. We could, of course, try to block out 1 of thosepathways, perhaps by augmenting free radical production to try tooverwhelm the antioxidant effects and then see, so on and so forth.
In terms of your second question, we did not measure baselinelevels of ejection fraction in these animals. But in our separatepilot studies where we were basically working out the details of theMillar catheter, also as part of also our heart failure studies, we do
have a sense of where ejection fraction lies and it’s generally in they 2004 on June 7, 2013 rnals.org
Woo et al Cardiopulmonary Support and Physiology
CSP
40% to 50% range, so there is a decrement even in the treatedanimals. They do have an 8% infarct and they are just statuspost–significant ischemic injury. So we don’t necessarily expectthem to get back to a baseline level, but it’s mostly the differencebetween the 2 groups that we’re looking at.
Dr Vinten-Johansen. You gave the injections or the adminis-trations at ischemia and reperfusion to take advantage of both themetabolic effects as well as the antioxidant effects, is that therationale?
The Journal of Thoracicjtcs.ctsnetjouDownloaded from
Dr Vinten-Johansen. How did you decide on the dosage, theconcentration that you gave?
Dr Woo. We were guided somewhat by the intestinal ischemic-reperfusion work, although that’s just a starting point. There is nodose-dependent evaluation of ethyl pyruvate in the literature forany of the other trauma critical care work. There are only maybe5 published studies, period, looking at anything physiologic.
In terms of pyruvate, there is a dose-dependent benefit. So wecould potentially shoot for an equimolar concentration of pre-
Dr Woo. That’s right. sumed pyruvate release as another target point.
and Cardiovascular Surgery ● Volume 127, Number 5 1269 on June 7, 2013 rnals.org
2004;127:1262-1269 J Thorac Cardiovasc SurgGrand
T. Bish, Jeffrey Burdick, Timothy J. Pirolli, Mark F. Berry, Vivian Hsu and Todd Y. Joseph Woo, Matthew D. Taylor, Jeffrey E. Cohen, Vasant Jayasankar, Lawrence
prolonged myocardial ischemiaEthyl pyruvate preserves cardiac function and attenuates oxidative injury after
Continuing Medical Education Activities
http://cme.ctsnetjournals.org/cgi/hierarchy/ctsnetcme_node;JTCSSubscribers to the Journal can earn continuing medical education credits via the Web at
Subscription Information
http://jtcs.ctsnetjournals.org/cgi/content/full/127/5/1262#BIBLThis article cites 27 articles, 11 of which you can access for free at:
Citations
http://jtcs.ctsnetjournals.org/cgi/content/full/127/5/1262#otherarticlesThis article has been cited by 7 HighWire-hosted articles:
Subspecialty Collections
http://jtcs.ctsnetjournals.org/cgi/collection/myocardial_protection Myocardial protection
This article, along with others on similar topics, appears in the following collection(s):
Permissions and Licensing
http://www.elsevier.com/wps/find/obtainpermissionform.cws_home/obtainpermissionformreceipt, is available at: An on-line permission request form, which should be fulfilled within 10 working days of
. http://www.elsevier.com/wps/find/supportfaq.cws_home/permissionusematerialcan be found online at: General information about reproducing this article in parts (figures, tables) or in its entirety
on June 7, 2013 jtcs.ctsnetjournals.orgDownloaded from
![2-[2-(2-Pyridyl)ethyl]isoindolinium perchlorate](https://img.pdfslide.net/doc/110x75/6352bf780f35c933db00cfcb/2-2-2-pyridylethylisoindolinium-perchlorate.jpg)
