Bishopric, Piero Anversa and Keith A. WebsterKazuhito Yamashita, Jan Kajstura, Daryl J. Discher, Bernard J. Wasserlauf, Nanette H.

Overexpressing Insulin-Like Growth Factor-1Reperfusion-Activated Akt Kinase Prevents Apoptosis in Transgenic Mouse Hearts

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 2001 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

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Reperfusion-Activated Akt Kinase Prevents Apoptosis inTransgenic Mouse Hearts Overexpressing Insulin-Like

Growth Factor-1Kazuhito Yamashita, Jan Kajstura, Daryl J. Discher, Bernard J. Wasserlauf, Nanette H. Bishopric,

Piero Anversa, Keith A. Webster

Abstract—Hearts of wild-type and insulin-like growth factor-1 overexpressing (Igf-11/2) transgenic mice were subjectedto Langendorff perfusions and progressive periods of ischemia followed by reperfusion. Apoptosis was measured byDNA nucleosomal cleavage and a hairpin probe labeling assay to detect single-base overhang. Transgenic heartssubjected to 20 minutes of ischemia and 4 hours of reperfusion (I/R) sustained a rate of apoptosis of 1.860.3% comparedwith 4.661.1% for wild-type controls (n54; P,0.03). Phosphorylation of the protein kinase Akt/protein kinase B waselevated 6.2-fold in transgenic hearts at baseline and increased another 4.4-fold within 10 minutes of reperfusion,remaining elevated for up to 2 hours. I/R activated Akt in wild-type hearts but to a lesser extent (1.660.3-fold).Pretreatment of transgenic hearts with wortmannin immediately before and during ischemia eliminated reperfusion-mediated activation of Akt and neutralized the resistance to apoptosis. The stress-activated kinase p38 was also activatedduring ischemia and reperfusion in both wild-type and transgenic hearts. Perfusion with the p38 inhibitor SB203580 (10mmol/L) blocked both p38 activation and phosphorylation of Akt and differentially modulated apoptosis in wild-typeand transgenic hearts. Pretreatment with SB203580 reduced apoptosis in wild-type hearts but increased apoptosis intransgenic hearts. These results demonstrate that Akt phosphorylation during I/R is modulated by IGF-1 and preventsapoptosis in hearts that overexpress the IGF-1 transgene.(Circ Res. 2001;88:609-614.)

Key Words: ischemian hypoxia n phosphoinositol-39-kinasen p38 mitogen-activated protein kinasen SB203580

Protective and antiapoptotic properties of insulin-likegrowth factor-1 (IGF-1) have been demonstrated in

different models of myocardial ischemia and infarction1–4 aswell as in isolated cardiac myocytes subjected to ischemic oroxidative stress.5,6 IGF-1 stimulates the phosphoinositol-39(PI3)-kinase pathway, producing phosphoinositides that pro-mote activation of the kinase Akt.7,8 Activated Akt kinaseplays a central role in suppressing apoptosis by modulatingthe activities of Bcl-2 family proteins,9 caspase 9,10 and Fasligand.11 Transfer of mutationally activated PI3-kinase andAkt genes has been shown to prevent apoptosis of cardiacmyocytes in vitro,6 and activated Akt delivered by an adeno-virus vector reduced apoptosis in the intact heart subjected toischemia and reperfusion (I/R).4

We previously reported that Igf-11/2 transgenic mice over-expressing IGF-1 had reduced rates of necrosis and apoptosisafter myocardial infarction caused by coronary artery liga-tion.3 The same hearts were also resistant to necrosis but notapoptosis caused by nonocclusive coronary artery constric-tion.12 Other work has shown that the endogenous PI3-kinase

pathway is activated in the postinfarcted myocardium, whereit may contribute to cell survival and hypertrophy.13–15

Regulation of the endogenous PI3-kinase pathway during I/Rhas not been described. We report here that Igf-11/2 trans-genic mouse hearts are resistant to I/R-mediated apoptosisthrough a novel pathway involving enhanced basal activationof Akt and superinduction by reperfusion. In these hearts,elevated basal IGF-1 supports an amplified response of thePI3-kinase pathway that shifts the balance between antiapo-ptotic Akt and proapoptotic p38 after reperfusion.

Materials and MethodsIGF-1 Transgenic MiceThe generation of transgenic mice expressing the human insulin-likegrowth factor-1 gene under the direction of the rata-myosin heavychain gene promoter has been described previously.16 In all studies,mice of both sexes heterozygous1/2 for the IGF-1 transgene between8 to 12 weeks of age were compared with age-matched controls.

Langendorff PerfusionsOur methods for isolating and perfusing mouse hearts by theLangendorff method have been described previously.17 Briefly,

Original received September 26, 2000; revision received January 25, 2001; accepted January 25, 2001.From the Department of Molecular and Cellular Pharmacology (K.Y., D.J.D., B.J.W., N.H.B., K.A.W.), University of Miami Medical Center, Miami,

Fla, and the Department of Medicine (J.K., P.A.), New York Medical College, Valhalla, NY.Presented in part at the 73rd Scientific Sessions of the American Heart Association, New Orleans, La, November 12–15, 2000, and published in abstract

form (Circulation. 2000;102[suppl II]:II-214).Correspondence to K.A. Webster, Department of Molecular and Cellular Pharmacology, University of Miami Medical Center, RMSB 6038, Miami,

FL 33136. E-mail Kwebster@chroma.med.miami.edu© 2001 American Heart Association, Inc.

Circulation Researchis available at http://www.circresaha.org

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Cellular Biology

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hearts were retrograde-perfused with a phosphate-free Krebs-Henseleit buffer equilibrated at 37°C with 5% CO2/95% O2, pH 7.4,and the Langendorff apparatus was housed in a temperature-controlled (37°C) and humidified incubator. Before treatments,hearts were perfused at constant pressure (80 mm Hg) with a flowrate of 2 to 4 mL/min for a 30-minute stabilization period. Globalischemia was applied by eliminating flow for the period indicated;hearts ceased to beat after 3 to 5 minutes of ischemia, and all heartsresumed contractions during reperfusion. Where indicated, drugsincluding wortmannin (Sigma Chemical Co) and SB203580 (Boeh-ringer) were added to the perfusion buffer 15 minutes beforeischemia and remained in the perfusion for 15 minutes afterreperfusion. At the end of the perfusion period, hearts were eitherfrozen rapidly on dry ice and stored at295°C or immersed in 1.5%paraformaldehyde.

Analysis of DNA Fragmentation (Laddering)and In Situ Ligation of Hairpin With Single-Base3* OverhangOur procedures for analyzing DNA fragmentation and in situ ligationwith hairpin probes have been described previously.12,17–19 Forfragmentation assays, DNA samples (5 to 8mg DNA) were subjectedto electrophoresis in 2% agarose gels and imaged by ethidiumbromide staining and digital photography. In some cases, the banddensity of the ladders was measured using an NIH Image programwith Adobe Photoshop. For in situ ligations, hearts were fixed in1.5% paraformaldehyde and tissue sections were labeled in a buffercontaining T4 ligase and 35 ng/mL hairpin probe with single base 39overhang (Synthetic Genetics Corp) followed by FITC-extravidinstaining (Sigma). Myocyte cytoplasm was detected bya-sarcomericactin labeling; nuclei were stained by propidium iodide. Sectionswere analyzed with a confocal microscope (MRC-1000, BioRadLaboratories). Apoptotic index was obtained by counting an averageof 15 000 myocyte nuclei in each heart.

Western Blot and Kinase AnalysisOur procedures for Western blots have been described in detailelsewhere.17 Blots were probed with specific antibodies against Akt,phospho-Akt, p38, and phospho-p38 (New England Biotechnology)and visualized using enhanced chemiluminescence (Pierce). Kinaseassays were used as described previously, with anti-p38 antibody toimmunoprecipitate p38 from solubilized heart tissues and myelinbasic protein as substrate.20

ResultsDNA Fragmentation During I/RIgf-11/2 and wild-type hearts were perfused for 30 minutesand subjected to increasing periods of ischemia followed by4 hours of reperfusion. DNA was extracted from the leftventricles and analyzed by gel electrophoresis as described inMaterials and Methods. Representative results are shown inFigure 1A. DNA nucleosomal cleavage was observed after 20minutes of ischemia and 4 hours of reperfusion and increasedwith the duration of ischemia up to 50 minutes (Figure 1, top,lanes 1 through 4). DNA cleavage in the Igf-11/2 hearts onlyappeared after 40 and 50 minutes of ischemia (n54). Toconfirm this finding, 4 hearts from each strain were subjectedto 20 minutes of ischemia and 4 hours of reperfusion, andapoptotic DNA fragmentation was measured in situ bydetection of single-base overhang using a hairpin probe assay,as described in Materials and Methods. Figure 1 (bottom)shows examples of myocyte fields from wild-type and Igf-11/2 hearts, both selected for high density of apoptotic nuclei.In these fields, 60% of the wild-type nuclei and 34% of theIgf-11/2 nuclei were identified as apoptotic. Analyses of more

than 15 000 nuclei in each of 4 hearts from both strainsrevealed a reduction in apoptosis; 1.860.3% versus4.661.1% (P,0.03) for transgenic and wild-type hearts,respectively, subjected to 20 minutes of ischemia and 4 hoursof reperfusion.

Activation of Akt and Reversal of Protectionby WortmanninWestern blots of wild-type and Igf-11/2 hearts probed withantibodies against phospho-Akt and total Akt are shown inFigure 2A, and quantitation of the results is shown in Figure2B. There was no difference in the total Akt protein betweenwild-type and Igf-11/2 samples (n53). Wild-type hearts wereweakly positive for phospho-Akt at baseline and showed asmall but significant 1.660.3-fold (n53) increase immedi-ately after reperfusion that remained elevated for 1 to 2 hours.In the Igf-11/2 hearts, phospho-Akt was 6.261.1-fold higherthan the wild-type at baseline (n54), and this was induced anadditional 4.460.9-fold (n53) within 10 minutes of reperfu-sion. Overall, there was.10-fold more phospho-Akt intransgenic hearts than in wild-type hearts 10 minutes afterreperfusion, and the activation of Akt was sustained for atleast 2 hours. Ischemia up to 50 minutes by itself was not

Figure 1. Apoptosis of wild-type and Igf-11/2 hearts induced byI/R. Excised hearts were perfused by the Langendorff system,as described in Materials and Methods. Top, Global ischemiawas sustained for intervals between 20 and 50 minutes, fol-lowed by 4 hours of reperfusion. Arrows indicate DNA frag-ments at '300, 500, 700, 900, and 1000 nucleotide base pairs.Bottom, Hairpin probe labeling of apoptotic myocytes from wild-type (A through C) and Igf-11/2 (D through F) hearts subjectedto 20 minutes of ischemia and 4 hours of reperfusion. Blue fluo-rescence (A and D) indicates total nuclei stained with propidiumiodide, green fluorescence (B and E) is the hairpin-FITC-extravidin stain, and red stain (C and F) is a-sarcomeric actin.

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sufficient to activate Akt, indicating a critical role for reper-fusion in the induction of this kinase; indeed, Akt kinaseactivation was also inhibited when 10 mmol/LN-acetylcysteine was included during I/R (n52, data not shown).

To determine whether Akt activation contributes to thecardioprotection observed in IGF-1–overexpressing hearts,wild-type and Igf-11/2 hearts were perfused with 200 nmol/Lwortmannin before and immediately after ischemia. Resultsfrom replicate hearts are shown in Figure 2C (left panel).Wortmannin had only minimal effect on I/R-induced DNAcleavage in wild-type hearts. In contrast, wortmannin treat-ment resulted in a marked increase in DNA cleavage in IGF-1transgenic hearts, equivalent to that seen in wild-type hearts.In experiments not shown here, we found that 200 nmol/Lwortmannin also inhibited extracellular signal–regulated ki-nase (ERK) activity in reperfused hearts; therefore, transgenicmouse hearts were treated with 20 nmol/L wortmannin beforeI/R, a concentration that does not effect ERK. Representativeresults are shown in the right panel of Figure 2C. The lowerwortmannin concentration also stimulated I/R-mediated apo-ptosis, supporting the predominant role of PI3-kinase inpreventing apoptosis in the transgenic hearts. Wortmannin

treatment alone did not cause increased DNA fragmentation.Densitometric quantitation of the bottom 3 bands in theladders, as described in Materials and Methods, indicated thatthe increased fragmentation in transgenic hearts by wortman-nin treatment was very significant (P,0.001, n54), whereaswortmannin treatment had no significant effect on fragmen-tation in the wild-type hearts. These results indicate thatsuperinduction of endogenous Akt is a key determinant of thecytoprotection observed in IGF-1–overexpressing mice.

Activation of p38 and Differential Responsesto SB203580The stress-activated protein kinase (SAPK) p38 has beenassigned a proapoptotic role in various models of I/R invitro21,22 and in vivo,23,24 an activity that is probably associ-ated with the p38a isoform.23 SAPK/p38 has also beenimplicated as an upstream kinase for mitogen-activated pro-tein kinase–activated protein kinase-2, which can activate Aktunder some conditions.25,26 Therefore, we analyzed the ex-pression of p38 in wild-type and Igf-11/2 mouse heartssubjected to I/R. The kinetics and magnitude of p38 phos-phorylation in response to I/R were similar (Figures 3A and3B). Phosphorylation was activated during the ischemicphase in both sets of hearts and was sustained through 1 hourof reperfusion, and there was a second later phase ofactivation starting at 2 to 4 hours of reperfusion. These 2phases probably correspond to stimuli initiated by ischemiaand reperfusion, respectively.27,28 Quantitation of the resultsindicated a possible slight lag in the responses of phopsho-p38 in the transgenic compared with the wild-type hearts(Figure 3B).

To determine the contribution of p38 activation to apopto-sis, hearts were perfused with the p38 inhibitor SB203580during ischemia, as described in Materials and Methods.These results are shown in Figure 3C. In wild-type hearts,pretreatment with SB203580 decreased but did not eliminateI/R-induced DNA cleavage, as would be predicted fromprevious studies.18,22,29 Unexpectedly, SB203580 treatmentincreased DNA fragmentation in the Igf-11/2 hearts. Densi-tometric quantitation of the ladders, as described for Figure 2,indicated that the effects of SB203580 were significant(P,0.001 for both wild-type and transgenic hearts, n54).There was no significant difference between wild-type andSB 203580–treated transgenic hearts subjected to I/R.

SB203580 has recently been shown to inhibit both p38 andAkt kinase within a narrow concentration range.30 As shownin Figure 3D, perfusion with wortmannin blocked Akt acti-vation selectively, but 10mmol/L SB203580, which is aconcentration frequently used for this kind of analysis,22,24,29

prevented the induction of both p38 and Akt by I/R. Theseresults present an interesting anomaly; the net effect ofsimultaneously inhibiting both p38 and Akt is protective inwild-type cells but promotes apoptosis in the transgenichearts. This suggests that p38 activation during I/R is domi-nant in wild-type hearts, whereas in Igf-11/2 hearts under thesame conditions, antiapoptotic Akt is dominant.

DiscussionOur results support previous studies that have describedcardioprotective and antiapoptotic roles of Akt in different

Figure 2. Expression of Akt and effects of wortmannin. A and B,Wild-type and Igf-11/2 hearts were subjected to I/R, andextracted proteins were analyzed by Western blot with anti-Aktantibodies as indicated. Western blots were quantitated by den-sitometric analyses, as described in Materials and Methods, andquantitation includes results from 3 separate experiments(6SEM). Phospho-Akt was significantly increased in the trans-genic hearts compared with wild-type hearts at all time points(P,0.01). C, Replicate hearts were perfused as in Figure 1 orwith 200 nmol/L wortmannin (left) or 20 nmol/L wortmannin(right) for 15 minutes before and immediately after ischemia orfor 4 hours without ischemia as indicated. After 4 hours ofreperfusion, DNA was analyzed by gel electrophoresis as in Fig-ure 1 (top).

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models of ischemia and hypoxia in vitro and in vivo.1,4,6 Inaddition, we show that the endogenous PI3-kinase pathway isactivated by reperfusion in a manner that reflects the avail-ability of IGF-1. Igf-11/2 transgenic myocytes from 75-day-old mice secrete 4-fold more IGF-1 and maintain almosttwice the serum levels of IGF-1 as their littermates.16 In thesehearts, we found that the basal level of phosphorylated Aktwas increased 6-fold above the equivalent wild-type level,presumably because of the continuous overproduction ofIGF-1 in these hearts. The additional stimulation of phospho-Akt by I/R, most pronounced in the Igf-11/2 hearts but alsoobserved in wild-type hearts, places Akt kinase among thelengthening list of kinases that respond to the reperfusionstimulus.

Both the induction of phospho-Akt by I/R and the resis-tance of the Igf-11/2 hearts to apoptosis were blocked bywortmannin. This confirms the role of PI3-kinase in bothresponses. The relatively greater induction of Akt in theIgf-11/2 hearts presumably reflects the primed state of PI3-kinase in these hearts. The stimulus for I/R-mediated activa-tion of Akt is not clear. A previous study using isolatedneonatal cardiac myocytes demonstrated that Akt was in-duced when the culture medium was switched from a low-pH,high-lactate medium containing sodium dithionite and deoxy-glucose to normal buffered medium.31 Akt is also activated bypreconditioning in the rat heart.32 Therefore, activation of Aktin response to reperfusion/reoxygenation occurs in vitro andin vivo. The stimulus may be directly linked to the redoxchanges that accompany I/R, as seems to be the case for c-JunN-terminal kinase and ERK activation.20,33,34 In results notshown here, we found that I/R in the presence of 10 mmol/LN-acetyl cysteine prevented Akt activation, supporting a rolefor reactive oxygen. The minimal impact of wortmannin onapoptosis in the wild-type hearts reflects the low basal levelof phospo-Akt and the weak response of Akt to I/R in thesehearts. This in turn presumably reflects the low basal activityof the PI3-kinase pathway in wild-type hearts.

The differential responses to SB203580 underscore theimportance of the balance between the PI3-kinase and p38pathways in determining cell fate in the ischemic reperfusedheart. Wild-type hearts have low basal phospho-Akt that isonly weakly activated by I/R. Therefore, endogenous Aktoffers only minimal protection in these hearts, and theprinciple action of SB203580 is to inhibit p38 and blockapoptosis. This result is consistent with several previousstudies that have described partial protection of normalmyocardium by treatments with SB 203580.22,29,35,36In con-trast, the PI3-kinase-Akt kinase pathway in IGF-11/2 hearts isextremely robust, generating.10-fold more phospho-Aktthan wild-type hearts during I/R and suppressing apoptosiseven though p38 is activated in parallel. Under these condi-tions, SB 203580 has both proapoptotic and antiapoptoticeffects because of the dual block of Akt and p38, and theoutcome is the net effect of these. Because inhibition of p38only partially prevents apoptosis,29 the contribution ofphospho-Akt seems to be dominant in the transgenic hearts,and the net effect of SB203580 treatment is to stimulateapoptosis.

Figure 3. Expression of p38 and effects of SB203580. A, Wild-type (top 2 panels) and Igf-11/2 transgenic (bottom 2 panels)hearts were subjected to I/R as indicated. Proteins wereextracted and analyzed by Western blot with anti-p38 antibodiesas indicated. Quantitation (B) includes results from 3 separateexperiments; treatment points from left to right are the same asshown on the top row of panel A without anisomycin controls.C, Replicate hearts, as indicated, were perfused as in Figure 1or with 10 mmol/L SB203580 for 15 minutes before and immedi-ately after ischemia. After 4 hours of reperfusion, DNA was ana-lyzed by gel electrophoresis as in Figure 1 (top). D, proteinswere extracted from Igf-11/2 hearts treated as indicated. Top,Western blots were probed with anti-phospho-Akt antibody.Bottom, Extracted proteins (100 mg) were immunoprecipitatedwith anti-p38 antibody and used in kinase assays with 5 mgmyelin basic protein/assay as substrate, as described in Materi-als and Methods. For kinase assays, hearts were exposed to 20nmol/L wortmannin or 10 mmol/L SB203580 as indicated.SB203580 inhibited Akt activation by .90% (n52).

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These results confirm the cardioprotective role of IGF-1and the PI3-kinase-Akt pathway. By extrapolation, augmen-tation of this pathway by either gene or protein transfer mayalso provide a promising clinical strategy. Indeed, elevatedIGF-1 correlates with improved recovery of postischemichearts.1,13–15Our studies have shown that Igf-11/2 transgenicmouse hearts are resistant to apoptosis or necrosis in 3different models of ischemia, confirming the disease-resistantphenotype of these hearts. However, the Igf-11/2 mice alsohave side effects that include an age-dependent hypertrophyand hyperplasia of the heart and various other organs thatappears within the first year of life and may compromise theprotective effects of IGF-1 in older mice.16 There may also beincreased cancer risk for these mice. Clearly, these sideeffects are not compatible with chronic global IGF-1 therapyas a treatment strategy for ischemic heart disease.

A key feature of the studies reported here is that protectionfrom I/R in the Igf-11/2 mice may actually be enhanced by thestimulus of reperfusion itself. Although increased circulatingIGF-1 supports a global increase of PI3-kinase and phospho-Akt in the Igf-11/2 hearts, superinduction of Akt is confinedto the ischemic-reperfused myocardium, providing both aspatial and temporal containment of the therapy. If the basalIGF-1 transgene expression could be silenced in the healthy(nondiseased) heart while maintaining or preferably amplify-ing activation during I/R, it may be possible to eliminate theside effects while maintaining protection. Incorporation ofhypoxia-responsive, oxidative stress, and silencer elements inthe transgene promoter that specifically activate gene expres-sion during ischemia or reperfusion may help to accomplishthis and perhaps create a feasible, safe method for deliveringIGF-1 genes to diseased hearts.37–39

AcknowledgmentsThis work was supported by grants HL44578 (to K.A.W.) andAG15756 (to P.A.) from the National Institutes of Health and anEstablished Investigator Award from the American Heart Associa-tion (to N.H.B.).

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