Phosphodiesterase-5 Inhibitor Sildenafil Preconditions AdultCardiac Myocytes against Necrosis and ApoptosisESSENTIAL ROLE OF NITRIC OXIDE SIGNALING*

Received for publication, April 28, 2004, and in revised form, January 4, 2005Published, JBC Papers in Press, January 24, 2005, DOI 10.1074/jbc.M404706200

Anindita Das, Lei Xi‡, and Rakesh C. Kukreja§

From the Division of Cardiology, Department of Internal Medicine, Virginia Commonwealth University Medical Center,Richmond, Virginia 23298-0281

We investigated the effect of sildenafil in protectionagainst necrosis or apoptosis in cardiomyocytes. Adultmouse ventricular myocytes were treated with silde-nafil (1 or 10 �M) for 1 h before 40 min of simulatedischemia (SI). Necrosis was determined by trypan blueexclusion and lactate dehydrogenase release followingSI alone or plus 1 or 18 h of reoxygenation (RO). Apo-ptosis was assessed by terminal deoxynucleotidyl trans-ferase-mediated nick end labeling assay and mitochon-drial membrane potential measured using a fluorescentprobe 5,5�,6,6�-tetrachloro-1,1�,3,3�-tetraethylbenzimida-zolyl-carbocyanine iodide (JC-1). Sildenafil reduced ne-crosis as indicated by decrease in trypan blue-positivemyocytes and leakage of lactate dehydrogenase com-pared with untreated cells after either SI or SI-RO. Thenumber of terminal deoxynucleotidyl transferase-medi-ated nick end labeling-positive myocytes or loss of JC-1fluorescence following SI and 18 h of RO was attenuatedin the sildenafil-treated group with concomitant inhibi-tion of caspase 3 activity. An early increase in Bcl-2 toBax ratio with sildenafil treatment was also observed inmyocytes after SI-RO. The increase of Bcl-2 expressionby sildenafil was inhibited by nitric-oxide synthase(NOS) inhibitor, L-nitro-amino-methyl-ester. The drugalso enhanced mRNA and protein content of inducibleNOS (iNOS) and endothelial NOS (eNOS) in the myo-cytes. Sildenafil-induced protection against necrosisand apoptosis was absent in the myocytes derived fromiNOS knock-out mice and was attenuated in eNOSknock-out myocytes. The up-regulation of Bcl-2 expres-sion by sildenafil was also absent in iNOS-deficient myo-cytes. Reverse transcription-PCR, Western blots, andimmunohistochemical assay confirmed the expressionof phosphodiesterase-5 in mouse cardiomyocytes. Thesedata provide strong evidence for a direct protective ef-fect of sildenafil against necrosis and apoptosis throughNO signaling pathway. The results may have possibletherapeutic potential in preventing myocyte cell deathfollowing ischemia/reperfusion.

Sildenafil citrate is a selective inhibitor of phosphodiester-ase-5 (PDE5)1 that catalyzes the breakdown of cGMP, one ofthe primary factors causing smooth muscle relaxation. Bymeans of its potent action of enhancing NO-driven cGMP ac-cumulation and ensuing vasodilatation in corpus cavernosum,sildenafil has become the most widely used drug for treatingerectile dysfunction in men since its market debut under thetrade name Viagra® in 1998 (1–3). In recent years, there areseveral studies on sildenafil for its therapeutic applications indiseases other than erectile dysfunction (4–11). For example,sildenafil has been shown to enhance flow-mediated vasodila-tation in chronic heart failure patients (4). Because of its potentvasodilatory action, sildenafil has also been extensively studiedfor treating primary or hypoxia-induced pulmonary hyperten-sion in adults and children (5–7, 9, 10). An epidemiologicalstudy (12) suggested that patients receiving sildenafil therapyfor erectile dysfunction had reduced incidence of myocardialinfarction than the general population matched with age, gen-der, and other risk factors. More interestingly, our laboratoryrecently discovered a powerful preconditioning-like effect ofsildenafil in rabbit hearts (13). Either intravenous or oral ad-ministration of sildenafil caused significant reduction of infarctsize following ischemia/reperfusion in the myocardium. Thisprotection was blocked by 5-hydroxydecanoate, a selectiveblocker of mitochondrial ATP-sensitive potassium channels.Sildenafil-induced cardioprotection has also been demon-strated in a model of global ischemia and reperfusion in mouse(14) as well as rat (15). In addition, a recent study in rabbitshas shown that early translocation of protein kinase C, espe-cially the �, �, and � isoforms to the membranous fractions, mayplay an essential role in sildenafil-induced cardioprotection(16). Based on these studies (13–16), we postulated that silde-nafil triggers signaling cascade that involves the activation ofprotein kinase C, the generation of NO, and the accumulationof cGMP in the myocardium through inducible and endothelialnitric-oxide synthase (iNOS and eNOS), thereby leading tocardioprotection via opening of mitochondrial ATP-sensitivepotassium channels (17–19).

Despite these novel and exciting observations, several fun-damental issues concerning the mechanisms of sildenafil-induced cardioprotection still need to be addressed. First, it ispossible that the profound vasodilatation caused by sildenafilmay potentially trigger a preconditioning response in the

* This work was supported in part by National Institutes of HealthGrants HL51045, HL59469, and HL79424 (to R. C. K.) and GrantCA16059 (to the Imaging Cytometry Facility of the Massey CancerCenter) and by American Heart Association, Mid-Atlantic AffiliateGrant 0060289U (to L. X.). The costs of publication of this article weredefrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

‡ Recipient of Research Career Enhancement Award from The Amer-ican Physiological Society.

§ To whom correspondence should be addressed: Division of Cardiol-ogy, Box 980281, VA Commonwealth University Medical Center, Rich-mond, VA 23298. Tel.: 804-828-0389; Fax: 804-828-8700; E-mail:rakesh@hsc.vcu.edu.

1 The abbreviations used are: PDE5, phosphodiesterase-5; SI, simu-lated ischemia; RO, reoxygenation; LDH, lactate dehydrogenase; JC-1,5,5�,6,6�-tetrachloro-1,1�,3,3�-tetraethylbenzimidazolylcarbocyanine io-dide; NOS, nitric-oxide synthase; iNOS, inducible NOS; eNOS, endo-thelial NOS; TUNEL, terminal deoxynucleotidyl transferase-mediatednick end labeling; L- NAME, N-nitro-L-arginine methyl ester; RT, re-verse transcription.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 13, Issue of April 1, pp. 12944–12955, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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heart. However, it remains unknown whether the drug alsoexerts a direct protective effect in the cardiomyocytes inde-pendent of its vascular/hypotensive effect. Second, althoughstudies in intact hearts have demonstrated a significant pro-tective effect of sildenafil against necrosis (infarction), thereis absolutely no information in the literature suggesting thatthe drug also inhibits apoptosis or whether the mitochondriaare the targets of protection in the cardiomyocytes. Third,despite the fact that NO signaling plays an important role insildenafil-induced delayed preconditioning in the intactmouse heart, it is not known whether this pathway alsoregulates necrosis or apoptosis in the cardiomyocytes follow-ing simulated ischemia (SI) and reoxygenation (RO). Fourth,there is controversy over the presence of PDE5 in the mam-malian ventricular cardiomyocytes (20–23), which may serveas the primary target for the action of sildenafil. To addressthese critical issues, we designed the current investigation todemonstrate the direct protective effect of sildenafil in acardiomyocytes model of SI-RO injury. This model was par-ticularly useful in studying the protective effect of sildenafilindependent of any vascular effects or other cell types. Wedetermined the effect of eNOS/iNOS in necrosis and apo-ptosis using cardiomyocytes derived from gene knock-outmice. Moreover, this study allowed us to directly examine thePDE5 expression in ventricular cardiomyocytes.

EXPERIMENTAL PROCEDURES

Isolation of Ventricular Cardiomyocytes—Adult male outbred ICRmice were purchased from Harlan (Indianapolis, IN). The adult maleiNOS and eNOS knock-out mice (stock numbers 002596 and 002684)and their corresponding wild-type controls (B6,129 and C57BL/6J) weresupplied by The Jackson Laboratory (Bar Harbor, ME). The animalexperimental protocols were approved by the Institutional Animal Careand Use Committee of Virginia Commonwealth University. The ven-tricular cardiomyocytes were isolated using an enzymatic techniquemodified from the previously reported method (24, 25). In brief, themouse was anesthetized with pentobarbital sodium (100 mg/kg intra-peritoneally), and the heart was quickly removed from the chest. Within3 min, the aortic opening was cannulated onto a Langendorff perfusionsystem (26), and the heart was retrogradely perfused (37 °C) at aconstant pressure of 55 mm Hg for �5 min with a Ca2�-free bicarbon-ate-based buffer containing 120 mM NaCl, 5.4 mM KCl, 1.2 mM MgSO4,1.2 mM NaH2PO4, 5.6 mM glucose, 20 mM NaHCO3, 10 mM 2,3-butane-dione monoxime, and 5 mM taurine that was continuously gassed with95% O2 � 5% CO2. The enzymatic digestion was commenced by addingcollagenase type II (Worthington; 0.5 mg/ml each) and protease typeXIV (0.02 mg/ml) to the perfusion buffer and continued for �15 min. 50�M Ca2� was then added in to the enzyme solution for perfusing theheart for another 10–15 min. The digested ventricular tissue was cutinto chunks and gently aspirated with a transfer pipette for facilitatingthe cell dissociation. The cell pellet was resuspended for a three-stepCa2� restoration procedure (i.e. 125, 250, and 500 �M Ca2�). The freshlyisolated cardiomyocytes were then suspended in minimal essential me-dium (catalogue number M1018, pH 7.35–7.45; Sigma) containing 1.2mM Ca2�, 12 mM NaHCO3, 2.5% fetal bovine serum, and 1% penicillin-streptomycin. The cells were then plated onto 35-mm cell culture dishesthat were precoated with 20 �g/ml mouse laminin in phosphate-buff-ered saline with 1% penicillin-streptomycin for 1 h. The cardiomyocyteswere cultured in the presence of 5% CO2 for 1 h in a humidifiedincubator at 37 °C, which allowed cardiomyocytes to attach to the dishsurface prior to the experimental protocol.

Experiment Protocol—The cultured cardiomyocytes were incubatedunder 37 °C and 5% CO2, for 1 h with or without 1 or 10 �M sildenafilcitrate, which was prepared by grinding Viagra tablets (Pfizer Inc.) intopowder and dissolved in distilled water. The drug solution was filtered(0.45-�m pore size) before adding into cell medium. Cardiomyocyteswere subjected to SI for 40 min by replacing the cell medium with an“ischemia buffer” that contained 118 mM NaCl, 24 mM NaHCO3, 1.0 mM

NaH2PO4, 2.5 mM CaCl2-2H2O, 1.2 mM MgCl2, 20 mM sodium lactate, 16mM KCl, 10 mM 2-deoxyglucose (pH adjusted to 6.2) similar to thosepreviously published (27). In addition, the cells were incubated underhypoxic conditions at 37 °C during the entire SI period by adjusting thetri-gas incubator to 1–2% O2 and 5% CO2. RO was accomplished byreplacing the ischemic buffer with normal medium under normoxic

conditions. Assessment of cell necrosis and apoptosis was performed attwo time points of RO, i.e. 1 and 18 h.

Evaluation of Cell Viability—Cell viability was assessed by trypanblue exclusion assay and LDH release in the medium. At the end ofprotocol, 20 �l of 0.4% trypan blue (Sigma-Aldrich) was added into theculture dish. After �5 min of equilibration, the cells were counted undermicroscope. For LDH measurements, the cellular medium was col-lected, and the enzyme activity was monitored spectrophotometricallyusing an assay kit (Sigma-Aldrich).

TUNEL Staining and Measurement of Mitochondrial Membrane Po-tential—Cardiomyocyte apoptosis was analyzed by TUNEL staining,using a kit purchased from BD Biosciences that detects nuclear DNAfragmentation via a fluorescence assay as previously reported (28). Inbrief, after SI and 18 h of RO, the cells in two chamber slides were fixedby 4% formaldehyde/phosphate-buffered saline at 4 °C for 25 min andsubjected to TUNEL assay according to the manufacturer’s protocol.The slides were then counterstained with Vectashield mounting me-dium with 4�,6-diamidino-2-phenylindole (a DNA intercalating dye forvisualizing nuclei in fixed cells; catalogue number H-1200, Vector Lab-oratories). Additionally, cardiomyocyte apoptosis was detected with amitochondrial membrane potential (��m) detection kit provided by Bio-carta. The cells were stained with a dye, 5,5�,6,6�-tetrachloro-1,1�,3,3�-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1), at 37 °C for 15min in a 5% CO2 incubator and rinsed with assay buffer according to themanufacturer’s protocol. The stained cells were examined under anOlympus IX70 fluorescence microscope.

Activated Caspase 3 Detection—Active caspase was detected usingthe CaspaTagTM Caspase 3,7 in situ assay kit (Chemicon, Temecula,CA) according to the manufacturer’s instructions. In this assay, thecell-permeable noncytotoxic fluorochrome inhibitors of caspases bindscovalently to a reactive cysteine residue on the large subunit of theactive caspase heterodimer, thereby inhibiting further enzymatic activ-ity. This kit uses a carboxyfluorescein labeled fluoromethyl ketonepeptide inhibitor of caspases-3 and -7 (SR-DEVD-FMK), which emits ared fluorescence. The red fluorescent signal is a direct measure of theamount of active caspase 3 in the cell at the time the reagent was added.The stained cells were immediately examined under a Nikon epifluo-rescent microscope using a band pass filter (excitation, 550 nm; emis-sion, �580 nm) to view the red fluorescence of active caspase-positivecells. Hoechst stain was detected using a UV filter with excitation at365 and emission at 480 nm.

RT-PCR of iNOS, eNOS, and PDE5—The total RNA was isolatedfrom untreated and sildenafil-treated cardiomyocytes using TRI rea-gent (Molecular Research Center) as described previously (14). Thereverse transcription and PCR amplification were performed using aOneStep RT-PCR kit from Qiagen. The oligonucleotide primers weresynthesized by integrated DNA technology, according to the publishedsequences for mouse iNOS (29), eNOS (30), and �-actin (28), whichserved as internal control. The transcript levels of iNOS and eNOS werequantified by real time PCR performed in the ABI prism 7900HTsequence detector system (Applied Biosystems, Foster City, CA) usingthe TaqMan One Step PCR Master Mix reagent kit (Product 4309169).All of the samples were processed in triplicate according to the manu-facturer’s recommended conditions. The cycling conditions were: 48 °Cfor 30 min, 95 °C for 10 min, and 40 cycles of 95 °C for 15 s and 60 °C for1 min. The cycle threshold was determined to provide the optimalstandard curve values (0.98–1.0). The probes and primers (Table I)were designed by the Nucleic Acid Research Facilities of Virginia Com-monwealth University, using Primer Express 2.0. The probes werelabeled in the 5� end with 6-carboxyfluoresceine and in the 3� end with6-carboxytetramethylrhodamine. Eukaryotic 18 S rRNA TaqMan en-dogenous control (Product 4310893E; Applied Biosystems) was used tonormalize the transcript levels. The following amounts of RNA wasused for real time PCR: 20 ng for iNOS, 4 ng for eNOS, and 1 ng for 18S rRNA.

PDE5 was amplified by using the primers based on the mousePDE5A cDNA sequence (GenBankTM accession number NM_153422;also see Table I). The RT-PCR products were electrophoresed on 2%Tris-acetate-EDTA-agarose gel.

Western blots for Bax, Bcl-2, iNOS, eNOS, and PDE5—Total solubleprotein was extracted from the cells with reporter lysis buffer (Pro-mega). The homogenate was centrifuged at 10,000 � g for 5 min under4 °C, and the supernatant was recovered. 25 �g of protein from eachsample was separated by 12% acrylamide gels and transferred to nitro-cellulose membrane and then blocked with 5% nonfat dry milk in TBST(10 mM Tris-HCl, pH 7.4, 100 mM NaCl, and 0.1% Tween 20) for 1 h. Themembrane was then incubated with rabbit polyclonal primary antibodyat a dilution of 1:1000 for each of the respective proteins, i.e. Bax, Bcl-2,

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iNOS, eNOS (Santa Cruz), or PDE5 (Calbiochem) for 2 h before beingwashed and incubated with anti-rabbit horseradish peroxidase-conju-gated secondary antibody (1:2000; Amersham Biosciences) for 1 h. Theblots were developed using a chemiluminescent system. The opticaldensity for each band was scanned and quantified using the identicaldensitometric system described above.

Immunohistochemistry of PDE5—Localization of PDE5A protein incardiomyocytes was performed according to the method of Senzaki et al.(23). The cardiomyocytes were fixed in 4% paraformaldehyde and thentreated with 0.1% sodium citrate and 0.1% Triton X-100. The cells werethen preincubated with normal donkey antiserum for 30 min and thenincubated overnight at 4 °C with polyclonal rabbit PDE5A antibody(Calbiochem) at 1:10,000 dilution. Incubation with secondary antibodywas performed at room temperature for 1 h with anti-rabbit Alexa 488(Molecular Probes). Imaging was performed on a Zeiss LSM 510 METAconfocal laser scanning microscope.

Data Analysis and Statistics—The data are presented as themeans � S.E. The difference between control and sildenafil-treatedgroups or among the multiple treatment groups was analyzed respec-tively with unpaired t test or one-way analysis of variance followed byStudent-Newman-Keuls post-hoc test. p � 0.05 was considered to bestatistically significant.

RESULTS

Effect of Sildenafil on Cardiomyocyte Necrosis—Our methodfor cell preparations yielded at least 70% of the cardiomyocyteswith rod shape morphology, which was similar to previouslyreported studies (24, 25). Fig. 1A shows a typical preparation ofisolated adult mouse cardiomyocytes used in the present studies.After 40 min of SI, the percentage of trypan blue positive car-diomyocytes increased with respect to control cardiomyocytes, i.e.from 3 � 0.2% of total counted cells in the control group to 35 �1% in the SI group, p � 0.05. Pretreatment with sildenafil for 1 hresulted in the decrease in trypan blue positive cardiomyocytes,i.e. from 35 � 1% of the total counted cells in the untreated SIgroup to 17 � 0.5 and 18 � 1% 1 and 10 �M sildenafil-treatedgroups, respectively (n 3; p � 0.05; Fig. 1D). After 40 min of SIand 1 h or 18 h of RO, the trypan blue positive cardiomyocytesfurther increased to 40 � 2 and 54 � 1%, respectively. Again,prior treatment with sildenafil reduced the trypan blue-positivecells as compared with the untreated SI-RO group (p � 0.001,n 3; Fig. 1, B–D). Similar results were obtained by measure-ment of LDH release in the medium. As shown in Fig. 1E, LDHincreased in the medium following SI and SI-RO (1 or 18 h),which is attenuated by prior treatment with sildenafil. Also, theattenuation in LDH release was independent of the dose of sil-denafil used in these experiments.

Effect of Sildenafil on Cardiomyocyte Apoptosis—Despite sig-nificant necrosis following 40 min of SI and 1 h of RO (Fig. 1, Dand E), apoptosis was not detectable under these conditions(data not shown). However, apoptotic cell death became clearlyevident following 40 min of SI and 18 h of RO, i.e. 24.7 � 1.2%of TUNEL-positive cells (p � 0.001 versus nonischemic control,n 4). The number of TUNEL-positive cells were reduced to3.5 � 0.6% in cardiomyocytes treated with 10 �M sildenafil (p �0.001 versus SI-RO and p � 0.05 versus untreated nonischemiccontrol cardiomyocytes, n 4; Fig. 2, A–I). The intense redfluorescence of active caspase was clearly observed in myocytesfollowing 40 min of SI and 18 h of RO (Fig. 3B). But the red

fluorescence decreased significantly in myocytes treated with 1or 10 �M sildenafil before 40 min of SI and 18 h of RO (Fig. 3,C and D).

Effect of Sildenafil on Mitochondrial Membrane Poten-tial—It has been demonstrated that components of ischemiaactivate the mitochondrial death pathway in cardiac myocytes(31). In the present study, we used cationic lipophilic probeJC-1 (32–34) to assess the mitochondrial membrane potential(��m). In this assay, the mitochondria of nonapoptotic cellsappeared in red following the aggregation of the JC-1 reagent,which emits red fluorescence at 590 nm (Fig. 4A). In contrast,in the apoptotic or dead cells the JC-1 dye remained in itsmonomeric form, thereby emitting relatively more green fluo-rescence (Fig. 4C). For positive control, cardiomyocytes weretreated with an uncoupler of mitochondrial respiration, car-bonyl cyanide m-chlorophenylhydrazone (10 �M), for 15 minbefore the initiation of JC-1 dye loading. As shown in Fig. 4B,there was a dissociation of JC-1-aggregates in the mitochon-drion as a result of drop in ��m. Consequently, intense greenfluorescence was observed in the presence of carbonyl cyanidem-chlorophenylhydrazone in these cells. A reduction in theratio of red to green fluorescence indicates a fall in ��m. In thecardiomyocytes subjected to SI and RO for 18 h, the red aggre-gated fluorescence decreased, and the monomer green fluores-cence increased (Fig. 4C). Quantitative measurements showedthat SI-RO cardiomyocytes had a lower JC-1 aggregate/mono-mer ratio as compared with the control nonischemic cells (i.e.16.8 � 0.5 versus 110.7 � 0.7 p � 0.001, n 3; Fig. 4E). TheJC-1 aggregate/monomer ratio was increased significantly to34.9 � 0.5 in cardiomyocytes treated with 10 �M sildenafil (Fig.4, D and E).

Effect of Sildenafil on Bcl-2 Expression: Role of NO Signal-ing—We further examined the effect of sildenafil on expressionof anti-apoptotic protein Bcl-2 and pro-apoptotic protein, Bax inthe cardiomyocytes. As shown in Fig. 5 (A–C), sildenafil en-hanced the expression of Bcl-2 in a time-dependent manner.The robust induction of Bcl-2 occurred in 10 �M sildenafil-pretreated myocytes after 40 min of SI and 1 h of RO butdisappeared after 18 h of RO. When normalized with the ex-pression of �-actin, there was a significant increase in Bcl-2 toBax ratio following 1 h of RO in sildenafil-treated cardiomyo-cytes (Fig. 5C). This early induction of Bcl-2 by sildenafil couldbe responsible for its anti-apoptotic effect in the present study(Fig. 2).

To demonstrate a possible link between NO signaling andinduction of Bcl-2 by sildenafil, we coincubated cardiomyocyteswith 100 �M of nonselective NOS inhibitor L-NAME with 10 �M

sildenafil prior to SI-RO. The results show that sildenafil-induced Bcl-2 up-regulation was completely abolished by L-NAME, suggesting a NOS-dependent anti-apoptotic effect ofsildenafil in cardiomyocytes (Fig. 5, D and E).

Effect of Sildenafil on iNOS and eNOS Expression in Car-diomyocytes—An increase in mRNA of iNOS and eNOS (to alesser extent) was observed after 1 h of incubation with silde-nafil (Fig. 6, A and B). Quantitative real time RT-PCR showedthat the ratio of iNOS/18 S rRNA and eNOS/18 S rRNA wasincreased by 3.5- and 1.5-fold, respectively, after 1 h of treat-ment with sildenafil. The expression of iNOS and eNOS proteinwas also enhanced in a time-dependent fashion (Fig. 6, D–F).This increase in protein expression reached �2-fold for iNOSand �1.5-fold for eNOS by 30 min after treatment withsildenafil.

Effect of Sildenafil on Necrosis and Apoptosis in iNOS- oreNOS-deficient Cardiomyocytes—To further establish the di-rect cause-and-effect relationship between sildenafil-inducedcytoprotection and iNOS/eNOS, we used cardiomyocytes de-

TABLE IPrimer/probe sequences used for determination of iNOS,

eNOS, and PDE5A expression

Primer/probe sequences

iNOS Forward: CAGGAGGAGAGAGATCCGATTTAGReverse: TGACCCGTGAAGCCATGACTaqMan Probe: TGGAAGCAAAGAACACCACTTTCACCAAGA

eNOS Forward: CACCAGGAAGAAGACCTTTAAGGAReverse: CACACGCTTCGCCATCACTaqMan Probe: TAGCCAATGCAGTGAAGGTCTCTGCCTC

PDE5A Forward: AATACCACCCCTGGAGCACCReverse: TTCAAGGGCTCGCCAAAAGC

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rived from mice deficient in iNOS or eNOS (i.e. iNOS-KO oreNOS-KO). As shown in Fig. 7A, pretreatment of B6,129 wild-type mouse cardiomyocytes (the background strain of iNOS-KO) with 1 �mol/liter sildenafil resulted in a decrease in ne-crosis following SI-RO, i.e. from 24.5 � 2.2 to 14.7 � 1.5% oftotal counted cells (n 3; p � 0.05). However, the protectiveeffect of sildenafil was completely blunted in the iNOS-KO

cardiomyocytes (Fig. 7A). Sildenafil also reduced necrosis inC57BL/6J wild-type cardiomyocytes (the background strain ofeNOS-KO mice), as shown by a decrease in myocyte viabilityfrom 34.6 � 2.9 to 14.7 � 0.5% of total counted cells after SI-RO(n 3; p � 0.001; Fig. 7B). The sildenafil induced protectionwas not abolished in eNOS-KO cardiomyocytes as indicated byreduction of necrosis from 32.2 � 0.7 to 24.7 � 1.9% of total

FIG. 1. Representative images of cardiomyocytes and quantitative data showing the effect of sildenafil pretreatment on myocyteviability following SI or SI-RO. A, normal isolated mouse cardiomyocytes. B, myocytes subjected to a 40 min of SI and 1 h of RO. Cell necrosisis clearly evident by the increased number of trypan blue-positive myocytes. C, myocytes treated with 10 �M sildenafil (Sil) for 1 h prior to SI(showing clearly fewer trypan blue-positive myocytes compared with the untreated myocytes in B). D and E, quantitative results of anti-necroticprotection of sildenafil in the cardiomyocytes preincubated with 1 or 10 �M of sildenafil for 1 h prior to SI or SI-RO (n 3/group). Note that bothof the number of trypan blue positive cells (D) and the release of LDH (E) into the cell culture medium were significantly reduced insildenafil-treated myocytes. *, p � 0.01 versus all other groups; #, p � 0.05 versus SI; �, p � 0.01 versus SI; ‚, p � 0.001 versus SI-RO 1 h; E, p �0.05 versus sildenafil (1 �M) � SI-RO 1 h; f, p � 0.01 versus Control; �, p � 0.001 versus SI-RO 18 h.

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counted cells after SI-RO (n 3; p � 0.05; Fig. 7B). Neverthe-less, this protective effect of sildenafil in the eNOS-KO car-diomyocytes, although statistically significant, was not as re-markable as observed in wild-type cardiomyocytes. Similarly,sildenafil significantly reduced TUNEL-positive nuclei in wild-type and eNOS-KO mice (Fig. 7C). The drug had no effect oninhibition of apoptosis in cardiomyocytes from iNOS-KO mice(Fig. 7D).

Effect of Sildenafil on Bcl-2 expression in iNOS- or eNOS-deficient Cardiomyocytes—As shown in Fig. 7 (E–G), pretreat-ment of B6,129 and C57BL/6J wild-type mouse cardiomyocyteswith 1 �M sildenafil enhanced the expression of Bcl-2 after 40

min of SI and 1 h of RO (n 3; p � 0.05). The increase of Bcl-2expression by sildenafil was abolished in myocytes derivedfrom iNOS-KO mice (Fig. 7, E and F). Although pretreatmentwith sildenafil enhanced the Bcl-2 level after SI-RO comparedwith untreated myocytes derived from eNOS-KO mice, such anincrease was not statistically significant (Fig. 7, E and G).

Expression of PDE5 in Cardiomyocytes—The expression ofPDE5 in cardiomyocytes was evaluated with three distinctassays. First, using RT-PCR we detected mRNA of PDE5 in theintact heart as well as isolated cardiomyocytes (Fig. 7A). Sec-ond, using Western blots we detected the protein expression ofPDE5 in mice cardiomyocytes (Fig. 7B). Two splice variants of

FIG. 2. Effect of sildenafil on inhibition of apoptosis in cardiomyocytes. Cells were treated with 10 �M sildenafil for 1 h followed by 40min of SI and 18 h of RO. Apoptotic nuclei were observed using TUNEL assay. A–C, nonischemic control; D–F, after 40 min of SI and 18 h of RO;G–I, pretreatment with 10 �M sildenafil before 40 min of SI and 18 h of RO. A, D, and G, cell morphology; B, E, and H, total nuclei(4�,6-diamidino-2-phenylindole staining); C, F, and I, TUNEL-positive myocyte nuclei (stained in green fluorescent color). A significant number ofcardiomyocytes underwent apoptosis (i.e. TUNEL-positive; Fig. 2F), whereas sildenafil pretreatment reduced TUNEL-positive nuclei (Fig. 2I). J,bar diagram showing quantitative data from three independent experiments.

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PDE5 band (90–100 kDa) were observed, which is consistentwith the previously reported PDE5A in dog cardiomyocytes(23). Furthermore, using immunohistochemistry and laser con-focal microscopy, the green fluorescence for PDE5A in normalisolated myocyte was clearly evident, thereby confirming thelocalization of PDE5A in adult mouse cardiomyocytes (Fig. 7G).The green fluorescence color was not observed in the cardiom-yocytes without antibody incubation (Fig. 7C) or incubationwith blocking peptide (Fig. 7D), primary antibody PDE5A (Fig.7E), or Alexa 488-labeled anti-rabbit antibody (Fig. 7F).

DISCUSSION

We previously demonstrated a powerful preconditioning-likecardioprotective effect of sildenafil in the rabbit (13) and mousehearts (14). In the present study, we have shown for the firsttime that sildenafil directly protects adult cardiomyoctyesagainst necrosis and apoptosis following ischemia-reoxygen-ation injury. Sildenafil-induced inhibition of apoptosis was ev-ident by the significant decrease in the TUNEL-positive nucleiand preservation of ��m, which is essential for production ofATP and cellular homeostasis. These results suggest that va-sodilatation caused by sildenafil or the presence of other celltypes may not be a prerequisite for the potent preconditioning

effect of this drug, at least in the isolated cardiomyocyte modelof SI-RO. Sildenafil also induced early up-regulation of Bcl-2/Bax ratio, which may have played an important role in theantiapoptotic effect of the drug.

Sildenafil is a selective potent inhibitor of PDE5 (a cGMP-specific isoform of PDE), which promotes increase in cGMP invascular smooth muscle cells by preventing its breakdown withPDE5 (3). We recently demonstrated that sildenafil up-regu-lated iNOS and eNOS in the intact murine myocardium, anddelayed protection induced by this drug was completely abol-ished with 1400W, a selective inhibitor of iNOS (14). In addi-tion, it is now well appreciated that various stimuli such asbrief episodes of ischemia (35), endotoxin-derivatives (36), ago-nists of G protein-coupled receptors (37, 38), and systemichypoxia (39) induce delayed preconditioning in an iNOS-de-pendent manner. The present results suggest that the NOS-dependent mechanism in sildenafil-induced protection plays acrucial role in the inhibition of apoptosis and necrosis in thecardiomyocytes also. This is demonstrated by absence of theprotective effect of sildenafil both against necrosis and apo-ptosis in the cardiomyocytes derived from iNOS gene knock-outmice (Fig. 7). Although sildenafil-induced protection remained

FIG. 3. Effect of sildenafil oncaspase 3 activity in cardiomyocytes.Isolated myocytes were treated with 1and 10 �M sildenafil for 1 h followed by 40min of SI and 18 h of RO, and caspase 3activity was detected by CaspaTag rea-gent under fluorescence microscope. Acti-vated caspase 3 in adult myocytes (red,left column) and nuclei stained by Ho-echst (blue, right column). A, nonischemiccontrol; B, cardiomyocytes subjected to 40min SI and 18 h of RO; C, cardiomyocytespretreated with 1 �M sildenafil for 1 hbefore SI-RO; D, cardiomyocytes pre-treated with 10 �M sildenafil for 1 h be-fore SI-RO. It is apparent that a signifi-cant number of cardiomyocytes displayedred fluorescence staining after SI-RO(Fig. 3B). The cardiomyocytes pretreatedwith 1 or 10 �M sildenafil (C and D)showed a negligible amount of red fluo-rescence as compare with the untreatedSI-RO cells.

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statistically significant in eNOS knock-out cardiomyocytes, themagnitude of protection was less than the wild-type mice.These results suggest that NO derived from eNOS may havepartially contributed to the signaling mechanism leading tocytoprotection by sildenafil.

Our results (Fig. 6, A–D) also showed a significant increasein mRNA and protein expression of iNOS and eNOS (to a lesserextent). The increase in the levels of iNOS and eNOS proteinswas evident at the end of 1 h of preincubation with sildenafil,

suggesting that NO was available to trigger the precondition-ing effect in the cardiomyocytes. Although the role of NO in theimmediate cardioprotection with brief episodes of ischemia (is-chemic preconditioning) has been debatable, sildenafil clearlyinduced early protection in the cardiomyocytes through NO.NO is known to activate guanylate cyclase, resulting in en-hanced formation of cGMP. Subsequently, cGMP may activateprotein kinase G that in turn opens the KATP channel, resultingin the cardioprotective effects as reported earlier (40). Our

FIG. 4. Effect of sildenafil on preservation of mitochondrial membrane potential in cardiomyocytes. A, isolated murine cardiomyo-cytes loaded with cationic lipophilic probe JC-1. Note the predominant aggregate (nonapoptotic) mitochondria (in red/orange color). B, the nearlycomplete monomer (apoptotic) mitochondria (in green color) induced by 10 �M carbonyl cyanide m-chlorophenylhydrazone, an uncoupler ofmitochondrial respiration, treated for 15 min before the initiation of JC-1 dye loading. C, cardiomyocytes subjected to 40 min of SI and 18 h of RO.Note the majority of myocytes became JC-1 monomeric green cells (i.e. apoptotic cells). D, cardiomyocytes pretreated with 10 �M sildenafil for 1 hbefore SI-RO. Note that as compared with the untreated SI-RO cells (C), sildenafil-treated myocytes (D) showed more nonapoptotic mitochondria(in red/orange color). E, quantification of JC-1 aggregate/monomer ratio, which shows sildenafil (Sil) increased significantly the ratio (i.e. lesser lostin ��m) as compared with the untreated SI-RO group (p � 0.05).

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FIG. 5. Effect of sildenafil on expression of Bax and Bcl-2 in cardiomyocytes: role of NO signaling. A, representative Western blotshowing expression of Bax, Bcl-2, and �-actin (house keeping gene), during 40 min of SI and 1 h or 18 h of RO in the presence or absence of 10 �M

sildenafil. B–D, bar diagrams showing averaged expression of Bax, Bcl-2, and Bcl-2 to Bax ratio after SI and 1- or 18-h RO in the presence orabsence of 10 �M sildenafil (Sil) pretreatment. Note that the Bcl-2 to Bax ratio was significantly increased after 1 h of sildenafil treatment (Fig.4D). E, Western blot showing effect of sildenafil (10 �M) on expression of Bcl-2 and Bax in the presence and absence of an inhibitor of NO synthases,L-NAME (100 �M). F, bar diagram depicting the Bcl-2 to Bax ratio. Note that the increase in Bcl-2 or Bax expression as well as Bcl-2 to Bax ratiowith sildenafil was completely reversed by L-NAME (Fig. 4, E and F).

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FIG. 6. Effect of sildenafil on gene transcription and protein expression of iNOS and eNOS in cardiomyocytes. The total RNA wasextracted after treatment for 1 h with 10 �M sildenafil and subjected to RT-PCR as described under “Experimental Procedures.” A, representativegel showing iNOS, eNOS, and �-actin; B and C, bar diagram showing the quantitative change of iNOS and eNOS mRNA assessed by real time PCR.Note that the mRNA level of iNOS (Fig. 5B) as well as eNOS (to a lesser extent; Fig. 5C) was significantly increased in sildenafil-treated myocytes.D, iNOS and eNOS proteins were measured at the indicated time points following 1-h incubation with 10 �M sildenafil, using Western analysis.E and F, bar diagrams showing normalized density of iNOS and eNOS expression at each of the time points.

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FIG. 7. Effect of sildenafil on necrosis, apoptosis, and Bcl-2 expression in cardiomyocytes derived from iNOS and eNOS geneknock-out mice. Myocytes were prepared from either iNOS gene knock-out mice (iNOS-KO) and their B6,129 wild-type control mice (B6,129,WT), or eNOS gene knock-out (eNOS-KO) mice and their C57BL/6J wild-type control mice (C57BL/6J WT). The myocytes were subjected to 40 minof SI and 1 h of RO. A and B, cell viability determined by Trypan blue assay. Note that pretreatment with sildenafil (S) significantly decreased thecell necrosis caused by SI-RO in myocytes isolated from B6,129 wild-type mice, whereas this drug failed to protect the cells from iNOS-KO mice(A). On the other hand, sildenafil significantly preserved viability of myocytes from C57BL/6J wild-type mice as well as eNOS-KO mice ( B). C andD, cardiomyocyte apoptosis assessed with TUNEL assay following 40 min of SI and 18 h of RO. Note that sildenafil significantly reducedTUNEL-positive nuclei in the myocytes from B6,129 wild-type, C57BL/6J wild-type, and eNOS-KO mice (D), whereas the drug provided noinhibition on apoptosis in the myocytes from iNOS-KO mice (C). Sildenafil significantly increased Bcl-2 expression and Bcl-2/Bax ratio followingSI-RO in the cardiomyocytes isolated from both strains of wild-type mice (i.e. B6,129 and C57BL/6J (E–G). In contrast, such an increase in Bcl-2expression and Bcl-2/Bax ratio was not found in the myocytes from iNOS-KO mice (E and F) and also was attenuated in the eNOS-KO myocytes(E and G).

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preliminary results suggest definite involvement of proteinkinase G in mediating the anti-necrotic and anti-apoptotic ef-fects of sildenafil in isolated mouse cardiomyocytes.2

One of the salient findings in this study is that sildenafilsignificantly inhibited apoptosis in the cardiomyocytes (Figs. 2and 7). It was recently documented that sildenafil could induceapoptosis in B-chronic lymphocytic leukemia cells (41). How-ever, this pro-apoptotic effect of sildenafil appeared to be selec-tive for the leukemic B cells because it did not induce apoptosisin normal B lymphocytes even at a drug concentration as highas 100 �g/ml (41). It is notable that the protective doses ofsildenafil used in the present study are 1 and 10 �M (�0.69 and6.9 �g/ml, respectively), which are much lower than used todemonstrate the pro-apoptotic effect in the cancer cells (41).Apoptotic cell death has been appreciated as an importantcomponent of the pathogenesis of myocardial ischemia-reper-fusion injury (42–46). Our results on sildenafil-induced anti-apoptotic effect in cardiomyocytes may have potential thera-peutic ramification in chronic ischemic heart diseases andheart failure, where apoptosis serves as the primary drivingforce for the loss of cardiomyocytes. Furthermore, the signifi-cant increase in the Bcl-2 to Bax ratio (Fig. 5) is likely to be one

of the responsible factors for the inhibitory effect of sildenafilon apoptosis in the cardiomyocytes, because Bcl-2 has beenrecognized as a key anti-apoptotic protein (47). Interestingly,our results show that either coincubation with NOS inhibitor,L-NAME (Fig. 5) or genetic deletion of iNOS (Fig. 7) completelyabolished the sildenafil-induced Bcl-2 expression in cardiomyo-cytes. These results established a key link between NO signal-ing and the expression of cytoprotective antiapoptotic protein,Bcl-2. Studies in neuronal cells also suggested a role for NO ininduction and regulation of Bcl-2 (48, 49). In addition, themitochondrial ATP-sensitive potassium channel opening prop-erty of sildenafil (13) may also contribute to its role in theinhibition of apoptosis. A previous report by Akao et al. (50) hasshown that the openers of the KATP channel (i.e. diazoxide andpinacidil) were able to reduce apoptosis induced by oxidativestress in the neonatal rat cardiomyocytes.

In the present study, we have provided unequivocal proof ofthe presence of PDE5 in cardiomyocytes with three assays thatdetected mRNA, protein expression, and localization of PDE5.There has been a dominant view that PDE5 is not present inthe myocardium (11), mainly based on the earlier studies by Itoet al. (20) and Wallis et al. (21), who failed to find enzymeactivity and/or immunoreactive bands of PDE5 in human ven-tricular tissues. However, this concept was challenged by a2 A. Das and R. C. Kukreja, unpublished observations..

FIG. 8. Expression of PDE5 inmouse cardiomyocytes. A, RT-PCR ofPDE5 and �-actin mRNA extracted fromwhole heart tissue or isolated cardiomyo-cytes; B, Western blot of PDE5 proteinexpression in isolated cardiomyocytes;C–G, immunohistochemical staining forPDE5A in murine cardiomyocytes. Themagnified images of cardiomyocytes cap-tured with laser confocal microscopy(400�) represent fluorescence (left panel);differential interference contrast (middlepanel), and overlay (right panel). The re-agent staining conditions are: withoutany antibody incubation (C); incubatedwith blocking peptide only (D); with pri-mary antibody for PDE5A only (E); withsecondary antibody only (F, i.e. Alexa 488labeled anti-rabbit antibody); and incu-bated with both primary and secondaryantibody (G, i.e. antibody for PDE5A plusAlexa 488 labeled anti-rabbit antibody).The presence of PDE5A (in green fluores-cence) was clearly visible in a diffusedpattern in the cardiomyocytes (G).

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recent study where abundant expression of PDE5 was observedin isolated canine ventricular cardiomyocytes with immunohis-tochemistry (23). In agreement with this observation, our studydemonstrated for the first time the presence of PDE5 in murineventricular cardiomyocytes (Fig. 8). The discrepancy may beattributed to a possible species difference between human andother mammalian species like dog and mice. However, in thestudy by Loughney et al. (22), a strong band was also detectedfor PDE5 mRNA in human heart sample. The presence ofPDE5 in guinea pig cardiomyocytes was also suggested byenhanced accumulation of cGMP level without affecting cAMP(51) with zaprinast, a less potent inhibitor of PDE5 than silde-nafil (3). Thus it is possible that sildenafil could also enhancecGMP partially through direct inhibition of PDE5 in the mousecardiomyocytes.

In summary, for the first time, we have shown a directprotective action of sildenafil in isolated adult cardiomyocytesthat is independent of any vascular response or presence ofother cell types. Our data, based on the pharmacological inhi-bition of NOS and cardiomyocytes derived from iNOS/eNOSgene knock-out mice, also suggest an important role of NOsignaling in protective effect of sildenafil against apoptosis andnecrosis. Because of the presence of PDE5 in mouse ventricularcardiomyocytes, its direct inhibition by sildenafil may also con-tribute to the cytoprotective effect by maintaining cGMP accu-mulation. Overall, these results provide an important mecha-nistic basis of sildenafil-induced cardioprotection, which mayfurther give support in exploiting the new therapeutic applica-tions of sildenafil and other clinically approved PDE5 inhibi-tors for treatment of ischemic heart diseases (11, 17).

Acknowledgments—We are grateful to Dr. R.-P. Xiao and B. Ziman(Laboratory of Cardiovascular Science, NIA, National Institutes ofHealth) for kindly sharing their technical expertise in isolation of adultmouse cardiomyocytes. We also thank Dr. C. Yin for efforts in testingand optimizing the primer and RT-PCR protocol to detect PDE5 mRNAin mouse heart tissues.

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Anindita Das, Lei Xi and Rakesh C. KukrejaSIGNALING

against Necrosis and Apoptosis: ESSENTIAL ROLE OF NITRIC OXIDE Phosphodiesterase-5 Inhibitor Sildenafil Preconditions Adult Cardiac Myocytes

doi: 10.1074/jbc.M404706200 originally published online January 24, 20052005, 280:12944-12955.J. Biol. Chem. 

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