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Insulin-like growth factor-1 protects ischemic murine myocardium from ischemia/reperfusion associated… Davani, Ehsan Y; Brumme, Zabrina; Singhera, Gurpreet K; Côté, Hélène C; Harrigan, P R; Dorscheid, Delbert R Oct 10, 2003

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R176Critical Care    December 2003 Vol 7 No 6 Davani et al.ResearchInsulin-like growth factor-1 protects ischemic murinemyocardium from ischemia/reperfusion associated injuryEhsan Y Davani1, Zabrina Brumme2, Gurpreet K Singhera3, Hélène CF Côté4, P Richard Harrigan5 and Delbert R Dorscheid61Graduate Student, University of British Columbia, McDonald Research Laboratories/iCAPTURE Center, St Paul’s Hospital, Vancouver, BritishColumbia, Canada2Graduate Student, University of British Columbia, BC Centre for Excellence in HIV/AIDS, St Paul’s Hospital, Vancouver, British Columbia, Canada3Post-Doctoral Fellow, University of British Columbia, McDonald Research Laboratories/iCAPTURE Center, St Paul’s Hospital, Vancouver, BritishColumbia, Canada4Post-Doctoral Fellow, University of British Columbia, BC Centre for Excellence in HIV/AIDS, St Paul’s Hospital, Vancouver, British Columbia, Canada5Clinical Assistant, Professor of Medicine, University of British Columbia, BC Centre for Excellence in HIV/AIDS, St Paul’s Hospital, Vancouver, BritishColumbia, Canada6Assistant Professor of Medicine, University of British Columbia, McDonald Research Laboratories/iCAPTURE Center, St Paul’s Hospital, Vancouver,British Columbia, CanadaCorresponding author: Delbert R Dorscheid, ddorscheid@mrl.ubc.caANOVA = analysis of variance; CPK = creatine phosphokinase; IGF = insulin-like growth factor; MK = modified Kreb’s Henseleit working solution;mtDNA = mitochondrial DNA; nDNA = nuclear DNA; PCR = polymerase chain reaction; PI3 = phosphatidylinositol-3; ∆Psys/dia = differencebetween ex vivo systolic and diastolic pressure; ROS = reactive oxygen species; TNF = tumor necrosis factor; VEGF = vascular endothelial growthfactor.AbstractIntroduction Ischemia/reperfusion occurs in myocardial infarction, cardiac dysfunction during sepsis,cardiac transplantation and coronary artery bypass grafting, and results in injury to the myocardium.Although reperfusion injury is related to the nature and duration of ischemia, it is also a separate entitythat may jeopardize viable cells and ultimately may impair cardiac performance once ischemia isresolved and the organ heals.Method The present study was conducted in an ex vivo murine model of myocardialischemia/reperfusion injury. After 20 min of ischemia, isolated hearts were perfused for up to 2 hourswith solution (modified Kreb’s) only, solution plus insulin-like growth factor (IGF)-1, or solution plustumor necrosis factor (TNF)-α. Cardiac contractility was monitored continuously during this period ofreperfusion.Results On the basis of histologic evidence, IGF-1 prevented reperfusion injury as compared withTNF-α; TNF-α increased perivascular interstitial edema and disrupted tissue lattice integrity, whereasIGF-1 maintained myocardial cellular integrity and did not increase edema. Also, there was a significantreduction in detectable creatine phosphokinase in the perfusate from IGF-1 treated hearts. Byrecording transduced pressures generated during the cardiac cycle, reperfusion with IGF-1 wasaccompanied by markedly improved cardiac performance as compared with reperfusion with TNF-α ormodified Kreb’s solution only. The histologic and functional improvement generated by IGF-1 wascharacterized by maintenance of the ratio of mitochondrial to nuclear DNA within heart tissue.Conclusion We conclude that IGF-1 protects ischemic myocardium from further reperfusion injury,and that this may involve mitochondria-dependent mechanisms.Keywords apoptosis, mitochondrial DNA, myocardium, reperfusion injury, sepsisReceived: 26 April 2003Revisions requested: 7 July 2003Revisions received: 4 August 2003Accepted: 18 August 2003Published: 10 October 2003Critical Care 2003, 7:R176-R183 (DOI 10.1186/cc2375)This article is online at http://ccforum.com/content/7/6/R176© 2003 Davani et al., licensee BioMed Central Ltd(Print ISSN 1364-8535; Online ISSN 1466-609X). This is an OpenAccess article: verbatim copying and redistribution of this article arepermitted in all media for any purpose, provided this notice ispreserved along with the article's original URL.Open AccessR177Available online http://ccforum.com/content/7/6/R176IntroductionCardiovascular diseases are among the leading causes ofdeath in North America. The most important presentation ofcardiovascular disease is ischemia, which leads to tissuehypoxia, cellular necrosis, apoptosis and, in severe situations,organ dysfunction. The main treatment for acute ischemicheart disease is early vascular reperfusion to restore balanceto cardiac metabolic demands. Although reperfusion is thefoundation of therapy, it may actually initiate further injury tothe myocardium. Although the phenomenon of reperfusioninjury is related to the duration of ischemia, it is a separateentity and may be more severe than ischemic injury alone[1,2]. Ischemia/reperfusion injury can be generated in variouscardiovascular diseases/events or therapies, includingmyocardial infarction, cardiopulmonary bypass, coronarybypass grafting, heart transplantation, and coronary throm-bolytic therapy. It has also been speculated that the mecha-nism of myocardial dysfunction during septic shock is relatedto segmental ischemia and reperfusion in the left ventricularwall, because the involvement of persistent global ischemiahas been disproved [3,4].Ischemia results from the absence of or sluggish blood flowin coronary vessels. This leads to a mismatch betweencardiac metabolic supply and demand. Ischemia of shortduration may contribute to ‘stunned myocardium’ withouttissue injury, but prolonged ischemia results in a deficiency inenergy supplies and waste removal, with eventual initiation ofcellular necrosis and ‘priming’ of the myocytes for apoptosis[5]. Early restoration of blood flow or reperfusion reduces theextent of myocardium at risk for death from necrosis.However, in the presence of prolonged ischemia, reperfusionitself initiates mechanisms of injury that are fundamentally dif-ferent and potentially more severe than those of ischemia.Reperfusion injury is mediated by inflammation and character-ized by the production of reactive oxygen species (ROS).Production of ROS may be initiated during the ischemicphase, generating ‘primed’ myocardium. ROS activate tran-scription factors, such as nuclear factor-κB, both in cardiacmyocytes and the endothelium; in turn, this initiates transcrip-tion of genes including those encoding adhesion molecules,cytokines, coagulation mediators, and proteolytic enzymes[6]. In coordination with the complement cascade, ROS candisrupt the integrity of both cardiac myocyte and endothelialcell membranes [7]. These events can change intracellularion homeostasis, resulting in the accumulation of calcium andmetabolic byproducts. These changes increase the activationof enzymes that are utilized in the processes of necrosis andapoptosis, and that alter mitochondrial function [8]. At thetissue level, this is manifested by interstitial edema and dis-ruption of the tissue lattice. Concomitantly, neutrophils andother inflammatory cells migrate into the injured zone usingadhesion molecules such as intercellular adhesionmolecule-1 under the stimulation of secreted cytokines andchemotactic factors. Recruitment and infiltration of neu-trophils into the injured tissue is accompanied by neutrophildegranulation and further injury to the border zone of viablecells. These late cellular events in the myocardium only occurafter reperfusion [2,5,9,10].Sustainable functioning of the myocardium is the centralobjective of therapeutic intervention in myocardial infarction.Cardiac function and contractility are closely related tocardiac metabolism and energy production. In cardiomyo-cytes energy production is related to the number of mitochon-dria, with these organelles occupying up to 40% of thecardiomyocyte cytoplasm. Hence, the total number of mito-chondria in the myocardial tissue can be used as a measureof cardiomyocyte activity and health [11,12]. In HIV-infectedpatients with symptomatic hyperlactatemia receiving anti-retroviral therapy, Cote and coworkers [13] showed that theratio of mitchondrial DNA (mtDNA) to nuclear DNA (nDNA)can be used as a marker of drug-induced mitochondrial toxic-ity. During the past century, there have been major improve-ments in the strategies used to protect myocardial tissue fromischemia/reperfusion injury [14–18]. However, the mecha-nism of ischemia/reperfusion injury remains unknown, and ourabilities to treat and prevent it are therefore limited. Usingmethods similar to those used by Cote and coworkers [13],we investigated changes in heart mtDNA : nDNA ratio duringmyocardial ischemia and reperfusion phases, and comparedthese levels with additional measures of tissue injury.New markers of myocardial injury may provide mechanisticinsights and reveal therapeutic possibilities in reperfusioninjury. Here, we propose a new method, using insulin-likegrowth factor (IGF)-1, of protecting cardiac tissue againstischemia/reperfusion injury in an ex vivo murine model. Themechanism underlying this protective effect remains unclear.However, the integrity of the myocardial tissue lattice was pre-served and the development of interstitial edema in myocardialtissue was inhibited. These effects were correlated withimproved perfusion pressure and left ventricular compliance.We also demonstrated that this IGF-1 mediated protectionwas accompanied by preservation of mtDNA content. The rel-evance of this finding to tissue function is discussed.MethodsIschemia/reperfusion modelC57B6 mice (Jackson Laboratories, Bar Harbor, ME, USA),weighing 25–30 g, were anesthetized using 3% isoflurane(Baxter, Toronto, Ontario, Canada) for 1 min and maintainedwith 1% isoflurane for 3–5 min during cardiac excision. Toprevent coagulation in coronary vessels, 500 Uheparin–sodium (Organon Teknika Inc., Toronto, Ontario,Canada) was injected intraperitoneally 10 min before induc-tion of anesthesia. The heart was excised and assembled ona Langendorff apparatus and perfused with oxygenated (95%oxygen, 5% carbon dioxide), modified Kreb’s Henseleitworking solution (MK) at 37°C for 3–5 min [19–21] until mon-itoring revealed that the organ was stable. Transduced leftR178Critical Care    December 2003 Vol 7 No 6 Davani et al.ventricular and aortic pressures, and heart rate were moni-tored continuously using Power lab/8sp detectors (AD Instru-ments Pty Ltd., Castle Hill, Australia). Retrograde perfusionwas then stopped for 20 min to model global ischemia (aperiod of 20 min of ischemia was found to be optimal for theischemic phase in this model). The ischemic hearts were thenreperfused with MK solution alone, MK solution plus IGF-1(10 ng/ml), or MK solution plus tumor necrosis factor (TNF)-α(10 ng/ml) for 1 or 2 hours. After completion of the reperfu-sion period, the hearts were divided into halves. One half wasfrozen using 2-methylbutane (Isopentane; MERK KgaA,Darmstadt, Germany) in liquid nitrogen for 80 s for eventualsectioning and/or DNA isolation. Paraffin embedding of theother half followed fixation of the sample in 10% formalin.Histologic evaluation of different groupsSlides of paraffin embedded tissues from the apex to thebasal portion of the hearts were prepared and stained withhematoxylin and eosin. Serial 400× magnified images werecaptured using a Nikon E600 microscope and SpotAdvanced software (version 3.4.2; S. Leffler & Silicon Graph-ics Inc., Mountain View, CA, USA). Image Pro-Plus software(MediaCybernetics, Carlsbad, CA, USA) was used to evalu-ate the severity of interstitial edema around the perivascularspaces of coronary arteries and veins. Measures were takenin 10 sections from each of four hearts for all conditions andtime points. The extent of interstitial edema was measured byselecting a circular area with a radius two times greater thanthe vascular space contained within the drawn circle. Thetotal vascular and perivascular areas were measured. ‘Nontis-sue’ area was determined by color segmentation images con-structed by Image Pro-Plus. Total interstitial edema wasdetermined by subtracting the vascular area from the nontis-sue area and expressing this as a percentage of total perivas-cular area: percentage edema = ([nontissue area – totalvascular area]/total perivascular area) × 100%.Ventricular function assessmentPressure generated during the cardiac cycle was obtained bytransduction of the aortic cannula and recorded continuouslyduring ischemia and reperfusion using a Power lab/8spdetector. Using Powerlab software, the difference betweenex vivo systolic and diastolic pressure (∆Psys/dia) at differenttime points was calculated to assess ventricular performance.Pressure measured during systole reflected contractility, anddiastolic pressure drops reflected relaxation of the ventricle.Thus, greater ∆Psys/dia values indicate better overall perfor-mance of the left ventricle.Detection of creatine phosphokinaseA 1 ml sample of myocardial perfusate was collected every15 min during the reperfusion phase. The samples werefrozen in a mixture of ethanol and dry ice [22,23]. The level ofcreatine phosphokinase (CPK) was measured using VitrosCK slides (Ortho-Clinical Diagnostics, Rochester, NY, USA).Briefly, 11 µl perfusate was deposited on the slide and evenlydistributed. Samples were incubated for 5 min at 37°C. Afterfinal interaction, leuco-dye is oxidized by hydrogen peroxide inthe presence of peroxidase to form an insoluble dye. Reflec-tion densities are monitored during incubation, and the rate ofchange in reflection density is then converted to enzymeactivity by using 670 nm wavelength in the Vitros Chemistry250 System.Mitochondrial/nuclear DNA assayFrozen hearts embedded in opaque tissue fixation materialwere thawed, cut into small pieces (approximately 3 mg), andthen placed into lysis buffer. DNA was extracted using theQiagen DNA isolation kit (Qiagen Canada, Qiagen Inc., Mis-sissauga, Ontario, Canada), in accordance with the manufac-turer’s protocol. Extracts were then diluted 1:80 with bufferAE before performing the mtDNA assay, as reported previ-ously [11,13,24] but modified for application in murinetissues as described below.For each DNA extract, one murine nuclear gene (accessorysubunit of the murine mitochondrial DNA polymerase γ[ASPG]; Genbank accession number AF177202) and onemurine mitochondrial gene (cytochrome oxidase subunit 1[COX], Genbank accession number AB042432) were quan-tified separately with real-time, quantitative PCR, using theRoche LightCycler (Roche Diagnostics, Indianapolis, IN,USA). For the mitochondrial (COX) gene, the forward primermCOX1F (5′-TCGTTGATTATTCTCAACCAATCA-3′) and thereverse primer mCOX2R (5′-GCCTCCAATTATTATTGGTAT-TACTATGA-3′) were used. The oligonucleotides 3′-fluores-cein-mCOXPR1 (5′-AACCAGGTGCACTTTTAGGAGATGACC-F3′) and 5′-LC Red 640 3′-phosphate-blocked-mCOXPR2(5′L-AATTTACAATGTTATCGTAACTGCCCATGC-P3′) wereused as hybridization probes. For the nuclear (ASPG) gene,the forward primer mASPG1F (5′-GGAGGAGGCACTTTC-TCAGC-3′) and the reverse primer mASPG2R (5′-GAAGAC-CTGCTCCCTGAACAC-3′) were used. The oligonucleotides5′-flourescein-mASPGPR1 (GCGCTTTGGACCTTTGGG-TGTAG-F3′) and mASPGPR2 (5′L-GTTACGAAAGAACCT-AGCCTCACAGTGGT-P3′) were used as hybridizationprobes. PCR reactions and amplification cycles were per-formed as described elsewhere [13].A standard curve consisting of serially diluted mouse DNA(30 000, 6000, 1200, 240 and 48 nuclear genome equiva-lents) were included in each run. The same standard curvewas used to quantify both the nuclear (ASPG) and the mito-chondrial (COX) genes. mtDNA and nDNA genes wereassayed in duplicate. Results of the quantitative PCR assaywere expressed as the ratio of the mean value of the dupli-cate mtDNA measurements to the mean value of duplicatenDNA measurements. As a further quality control, a mouseDNA extract with a mtDNA : nDNA ratio known to be high,and an extract with a mtDNA : nDNA ratio known to be lowwere included in every run. Repeat sample and intrasamplevariations were under 5%.R179Statistical analysisValues are expressed as mean ± standard error. P < 0.05 wasconsidered statistically significant.ResultsPerivasular interstitial edema and tissue latticeintegrityThe cellular integrity of the myocardium was well preserved inthe tissue of hearts reperfused with IGF-1 (Fig. 1). The area ofinterstitial edema in hearts treated with IGF-1 plus MK was21 ± 4%, as compared with 34 ± 6% and 49 ± 5% for reper-fusions with MK only and MK plus TNF-α, respectively. Rep-resentative tissue histology images are presented in Fig. 1and were similar throughout the four hearts and in all condi-tions. Additional histology observations included an increasednumber of shrunken, contracted myocytes with dense pyc-notic nuclei with MK plus TNF-α reperfusion as comparedwith perfusate containing IGF-1. Using single factor analysisof variance (ANOVA), the differences in percentage edemabetween groups were statistically significant (P < 0.05).Insulin-like growth factor-1: improvement inmyocardial performance during reperfusionCardiac performance was determined by calculating thepressure difference between systole and diastole (i.e.∆Psys/dia) at set time points. The systolic and diastolic pres-sures were determined by taking the average values from awindow around the respective time points. Performance isthen a measure of both contractility (stroke volume andforce of left ventricular contraction, manifesting as systolicpressure) and diastolic function, or relaxation of the left ven-tricle (a reduction in diastolic pressures). Improved perfor-mance is manifested by a widening in ∆Psys/dia. Pressuremonitoring demonstrated that cardiac performanceincreased from 0 to 40 min of reperfusion for all conditions(Fig. 2). After 40 min of reperfusion, cardiac performancearrived at a plateau and became negative for the remainingminutes for the MK alone and the MK plus TNF-α reperfu-sions. With reperfusion with MK plus TNF-α the differencebetween systolic and diastolic pressure (∆Psys/dia) initiallyincreased to 6.8 ± 0.7 mmHg as compared with reperfusionwith MK alone (5.1 ± 0.6 mmHg). However, reperfusion withIGF-1 generated a ∆Psys/dia that was significantly greater(13.8 ± 1.2 mmHg) than that with TNF-α (6.8 ± 0.7 mmHg)by 20 min. This gain in cardiac performance was maintainedup to 120 min of reperfusion with IGF-1. The enhanced per-formance was reflected in improvements in both systolicand diastolic pressures. The late descent in slope at120 min of reperfusion with IGF-1 was similar to that occur-ring with reperfusion with MK alone and with MK plus TNF-α, but may relate to ex vivo conditions other than theischemia time and the reperfusion solution. A paired, twosample t-test for means between groups demonstrated astatistically significant difference between IGF-1 and MKalone and MK plus TNF-α (P < 0.005).Low creatine phosphokinase level in insulin-likegrowth factor-1 treated heartsCollected perfusate from hearts treated with IGF-1, at allreperfusion time points, contained significantly lower quanti-ties of detectable CPK (34.6U/l) than did perfusate from TNF-α treated hearts (113.6U/l). This is shown as an average forall time points in Fig.3. Single factor ANOVA revealed a statis-tically significant difference between groups (P<0.005).Ratio of mitochondrial to nuclear DNAIGF-1 maintained or improved the mtDNA : nDNA ratio duringreperfusion of ischemic myocardium as compared withcontrol reperfusion with MK alone. There was a significant dif-ference between all test groups (baseline, ischemia, reperfu-sion with MK alone, and reperfusion with MK plus IGF-1) inthe determined mtDNA : nDNA ratio (P < 0.05, by ANOVA).Based on previous work, it was thought useful to test theutility of mtDNA : nDNA ratio to assess the ‘cellular health’ ofischemic and reperfused myocardial tissues [11,12]. HowIGF-1 preserves mtDNA : nDNA ratio and if this also meansintact oxidative mitochondrial function that promotes cellularviability remains to be investigated.We found that IGF-1 appeared to protect heart tissue againsta reduction in the mtDNA : nDNA ratio, which was accompa-nied by improved histologic grading and improved organfunction in terms of contractility. A reduction in this ratio mayrepresent either necrosis of at-risk tissue or a reduction inmitochondrial number (mitoptosis) after the initial stimulusAvailable online http://ccforum.com/content/7/6/R176Figure 1Representative images of hematoxylin and eosin stained sections frommurine hearts subjected to ischemia/reperfusion. Images are of control,ischemia without reperfusion, and reperfusion with modified Kreb’sHenseleit working solution (MK) alone, MK plus tumour necrosis factor(TNF)-α, and MK plus insulin-like growth factor (IGF)-1, both at 1 and2 hours. Note the preservation of cellular and structural elements andthe lack of interstitial edema in the IGF-1 reperfused heart.Magnification for all images: 400×.Control                        IschemiaModified Krebs                     TNF-α                            IGF-11 h reperfusion2 h reperfusionR180Critical Care    December 2003 Vol 7 No 6 Davani et al.(ischemia) followed by subsequent reperfusion (Fig. 4). Sucha reduction was noted with reperfusion with MK alone afterthe initial increase in mtDNA : nDNA ratio that occurred afterischemia alone without reperfusion. Because this model uti-lized a cell-free perfusate, the mtDNA : nDNA ratio is not con-founded by potential contributions from immune cells – apoint that has been raised as a possible explanation forchanges in mtDNA : nDNA ratio.DiscussionDespite a range of clinical interventions, our ability to preventreperfusion injury after disruption of blood flow to vascularFigure 2Determination of cardiac performance. (a) Tracings from continuous monitor recordings obtained during ischemia and reperfusion. Each tracingdemonstrates the aortic and left ventricular transduced pressure over time. The mid-point in the tracing is at 20 min of reperfusion. The conditionsare modified modified Kreb’s Henseleit working solution (MK) alone, MK plus tumour necrosis factor (TNF)-α, and MK plus insulin-like growth factor(IGF)-1. (b) Determination of cardiac performance is as described in the Methods section (see text) and includes calculation of the pressuregradient between the systolic and diastolic pressure transductions from the aorta and left ventricle. As demonstrated, reperfusion with IGF-1generates a significant improvement in cardiac performance at all time points. Analysis demonstrated a significant difference between reperfusionwith IGF-1 plus MK as compared with MK alone and MK plus TNF-α (P < 0.005).25051015200 20 40 60 80 100 120Reperfusion time (min)Pressure (mmHg)MKTNFIGF-1-α∆(a)                                                                                           (b)Figure 3Measured creatine phosphokinase in perfusate from reperfused murinehearts. Hearts were prepared for Langendorf ex vivo reperfusion andperfused until the monitored heart rate and pressure were stable(approximately 3–5 min); then they were subjected to 20 min ofischemia followed by reperfusion. Perfusate solution was collected(approximately 1 ml/4 min) around each time point for determination ofCPK activity. For all time points, tumour necrosis factor (TNF)-αreperfusion generated a significant elevation in the detectable amountof CPK activity relative to that detected with insulin-like growth factor(IGF)-1 reperfusion (P < 0.005, by analysis of variance).TNF-αIGF-10501001502002503000 min 15 min 30 min 45 min 60 minCPK activity (Units/l)Figure 4Determination of mitochondrial DNA (mtDNA) : nuclear DNA (nDNA)ratio. The mtDNA : nDNA ratio was determined for the followingconditions: control; ischemia without reperfusion; modified Kreb’sHenseleit working solution (MK) alone for 1 hour; MK alone for 2 hours;MK plus insulin-like growth factor (IGF)-1 for 1 hour; and MK withinsulin-like growth factor (IGF)-1 for 2 hours. The number within eachhistogram represents the number of hearts processed for thatcondition. The values for mtDNA : nDNA ratio in the controls, ischemicmyocardial tissue, and either reperfusion group (MK or IGF-1) weresignificantly different from each other (P < 0.05).4666840.00.51.01.52.02.53.03.54.04.5Controls Ischemia MK1h MK2h IGF1h IGF2hmtDNA /nDNAR181beds remains disappointing. An appreciation of the mecha-nism of ischemia/reperfusion injury is central to developmentof better treatments. In the present study we demonstratedthat IGF-1 can lessen reperfusion injury following an initialischemic insult.This effect of IGF-1 on the ischemic myocardium was sup-ported by histologic evidence of improved tissue and cellularintegrity, including markedly less interstitial edema around theperivascular spaces. In this model, ischemic myocardiumtreated with IGF-1 had significantly lesser amounts ofdetectable CPK than did myocardium treated with TNF-α,suggesting reduced cellular injury. This is also consistent withthe cardiac performance and left ventricular contractility ofIGF-1 treated hearts, that exhibited a greater ∆Psys/dia. Itshould be noted that the detectable CPK levels would not beabove the normal range as determined for human wholeblood samples. However, there was considerable histologicevidence of tissue damage in the TNF-α treated hearts, sug-gesting the relative insensitivity of CPK in detecting lessermyocardial injuries. A more sensitive marker would be valu-able not only for studying the mechanism that underlies reper-fusion injury but also for evaluating the efficacy of therapeuticinterventions. This is particularly true when one considers thesegmental and intermittent ischemic/reperfusion zones thatcharacterize dysfunctional myocardium in sepsis. The initialimprovement in contractility, observed under all conditions ofreperfusion, was probably the result of a new supply of nutri-ents after the ischemic period, including oxygen. The additionof IGF-1 significantly augmented this improvement in left ven-tricular pressure generation and relaxation (thus increasing∆Psys/dia). This improvement was maintained throughout theperiod of reperfusion.Myocardial performance at the cellular level is associatedwith the number or functional capacity of mitochondria. Toinvestigate indirectly whether mitochondrial function may rep-resent a marker of this beneficial effect of IGF-1, we deter-mined the mtDNA : nDNA ratio in relation to myocardialfunction. Although not appreciated clinically, ischemia andreperfusion are two distinct periods [10,14,16,25,26]. Theischemic period has been described as ‘priming’ cardiacmyocytes for either necrotic or apoptotic death. A markedincrease in mtDNA : nDNA ratio was detected in the ischemicmyocardium relative to baseline control levels. Apoptosis hasbeen found to be an event that requires energy [27,28].Whether this increased mtDNA : nDNA ratio indicates anincrease in the number of mitochondria per cell or anincrease in the genome copy number per mitochondriaremains to be determined.Myocardial reperfusion injury, as a separate event, canincrease the extent of injury beyond that caused by ischemiaalone. It has been shown that modification of solutions orother conditions during the reperfusion phase can alter theextent of cellular and functional damage to the myocardium.We determined that the nature of the reperfusate can affectmtDNA : nDNA ratio. Reperfusion with MK alone resulted in areduction in mtDNA : nDNA ratio toward baseline values. Thismay reflect either mitochondrial mitoptosis in damaged and‘primed’ tissues, or necrotic loss of similar cells that were‘primed’, resulting in elevated mtDNA : nDNA ratio afterischemia. The net effect would be that the remaining tissue isspared and should reflect baseline tissue. However, amtDNA : nDNA ratio that does not differ from baseline doesnot indicate that the tissue is working normally. In fact, histol-ogy and contractility determinations demonstrated that theheart had sustained significant tissue damage and was dys-functional after MK reperfusion. With IGF-1 reperfusion thisreduction in mtDNA : nDNA ratio was prevented, suggestingthat the extent of injury is not associated with elevatedmtDNA : nDNA ratio alone. In fact, after ischemia/reperfusion,it was found that a normal mtDNA : nDNA ratio early afterreperfusion predicted significant tissue injury. The patterns ofmtDNA : nDNA ratio, as seen in this model, may prove usefulin future investigations of possible mitochondria-relatedmechanisms of reperfusion injury.IGF-1 can affect cardiomyocyte contractility through itsreceptor – a heterotetrameric protein with intracellular tyro-sine kinase activity [29]. Downstream signals after receptoractivation include Shc, Crk and phospholipase C, and activa-tion of phosphatidylinositol-3 (PI3) kinase. Guse and cowork-ers [30] demonstrated that IGF-1 can increase PI3 levels inrat cardiomyocytes. Through its action on PI3 kinase, IGF-1can affect both contractility [31] and apoptosis [32]. Theaction of IGF-1, as demonstrated in our myocardialischemia/reperfusion model, may occur via PI3 kinase and/oreffects on mitochondria. Increases in cardiomyocyte calciumlevels and cardiomyocyte sensitivity to calcium [33] havebeen demonstrated to effect cardiac performance. Alterationin calcium metabolism may interfere with the action ofcalcium because the filamentous network of cardiomyocytesand their contractile properties are extremely sensitive toeven small fluctuations in calcium ion concentration [34].A similar result to that presented here for IGF-1 in myocardialischemia/reperfusion has been demonstrated for vascularendothelial growth factor (VEGF), suggesting that a finalcommon ‘protective’ pathway may exist [35]. Anwar andcoworkers [36] showed that TNF-α decreased IGF-1 mRNAand increased IGF-1 binding protein-3 mRNA expression invascular smooth muscle cells. These actions of TNF-α effec-tively reduced free IGF-1 levels and activity, and promotedendothelial instability. Infusion of a modified IGF-1 reducedthe TNF-α induced apoptosis. An interaction between VEGFand IGF-1 was characterized in retinal neovascularization indiabetic patients [37]. The authors of that report describedcommon mitogen-activated protein kinase 44/42 pathwaysthat may be related to the mitogenic effect of those two mole-cules. However, the short time to effect for both IGF-1 andVEGF in myocardial ischemia/reperfusion models is mostAvailable online http://ccforum.com/content/7/6/R176R182probable through the Akt pathway [38,39]. Akt activation canimprove contractility through PI3 kinase signaling, and is alsoan initiator of protein kinase C activation upstream. Proteinkinase C plays an important role in cardiac function, calciummetabolism, and contractility. Michell and coworkers [38]showed that IGF-1 and VEGF both stimulate nitric oxide pro-duction from endothelial cells and that inhibition of PI3 kinaseby wortmannin and LY29004 decreases nitric oxide produc-tion and reduces cardiac function. Akt signaling has alsobeen demonstrated to prevent apoptosis. Whether thispathway alters the expression of Bcl-2 family members byIGF-1 exposure remains unknown.It has been shown that IGF-1 can protect myocardium andother tissues against apoptosis in various animal models[40–42]. IGF-1 may also improve cardiac function in diabeticpatients [41–45] and rat models of myocardial infarction andreperfusion [26]. It has been shown that IGF-1 can protectmyocardium by regulating changes in proapoptotic and/orantiapoptotic molecules such as Bcl-2, Bcl-XL and Bax.These are all related to the mitochondrial apoptotic pathwayand mitochondrial energetics [26]. This may explain, in part,how IGF-1 protects myocardium even in the later phase ofreperfusion injury.In an ex vivo model of myocardial ischemia and reperfusionwe demonstrated that IGF-1 protects against reperfusionassociated injury. We found this protective effect of IGF-1 tobe correlated with elevated mtDNA : nDNA relative to base-line, and this may represent a marker of preservation of mito-chondrial function. This study provides new insights intoischemia/reperfusion, and suggests possible mechanismsand treatments for the tissue injury and organ dysfunctionassociated with this process. The eventual benefit of this toour understanding of myocardial dysfunction in sepsis awaitsfurther study.Competing interestsNone declared.AcknowledgementsGrant support for this project was provided by the Heart & StrokeFoundation of British Columbia and Yukon. DRD is a recipient of aParker B Francis Fellowship in Pulmonary Research and a MichaelSmith Foundation for Health Research Scholar Award. The authorsthank Yijin Wang and Katherine Craig, MD, for their technical expertiseand contributions in the preparation of this manuscript.References1. Ganz W: Direct demonstration in dogs of the absence of lethalreperfusion injury. 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