Phosphodiesterase-5 Inhibitor Sildenafil Preconditions Adult Cardiac Myocytes against Necrosis and Apoptosis
2005; Elsevier BV; Volume: 280; Issue: 13 Linguagem: Inglês
10.1074/jbc.m404706200
ISSN1083-351X
AutoresAnindita Das, Lei Xi, Rakesh C. Kukreja,
Tópico(s)Ion channel regulation and function
ResumoWe investigated the effect of sildenafil in protection against necrosis or apoptosis in cardiomyocytes. Adult mouse ventricular myocytes were treated with sildenafil (1 or 10 μm) for 1 h before 40 min of simulated ischemia (SI). Necrosis was determined by trypan blue exclusion and lactate dehydrogenase release following SI alone or plus 1 or 18 h of reoxygenation (RO). Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated nick end labeling assay and mitochondrial membrane potential measured using a fluorescent probe 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). Sildenafil reduced necrosis as indicated by decrease in trypan blue-positive myocytes and leakage of lactate dehydrogenase compared with untreated cells after either SI or SI-RO. The number of terminal deoxynucleotidyl transferase-mediated nick end labeling-positive myocytes or loss of JC-1 fluorescence following SI and 18 h of RO was attenuated in the sildenafil-treated group with concomitant inhibition of caspase 3 activity. An early increase in Bcl-2 to Bax ratio with sildenafil treatment was also observed in myocytes after SI-RO. The increase of Bcl-2 expression by sildenafil was inhibited by nitric-oxide synthase (NOS) inhibitor, l-nitro-amino-methyl-ester. The drug also enhanced mRNA and protein content of inducible NOS (iNOS) and endothelial NOS (eNOS) in the myocytes. Sildenafil-induced protection against necrosis and apoptosis was absent in the myocytes derived from iNOS knock-out mice and was attenuated in eNOS knock-out myocytes. The up-regulation of Bcl-2 expression by sildenafil was also absent in iNOS-deficient myocytes. Reverse transcription-PCR, Western blots, and immunohistochemical assay confirmed the expression of phosphodiesterase-5 in mouse cardiomyocytes. These data provide strong evidence for a direct protective effect of sildenafil against necrosis and apoptosis through NO signaling pathway. The results may have possible therapeutic potential in preventing myocyte cell death following ischemia/reperfusion. We investigated the effect of sildenafil in protection against necrosis or apoptosis in cardiomyocytes. Adult mouse ventricular myocytes were treated with sildenafil (1 or 10 μm) for 1 h before 40 min of simulated ischemia (SI). Necrosis was determined by trypan blue exclusion and lactate dehydrogenase release following SI alone or plus 1 or 18 h of reoxygenation (RO). Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated nick end labeling assay and mitochondrial membrane potential measured using a fluorescent probe 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). Sildenafil reduced necrosis as indicated by decrease in trypan blue-positive myocytes and leakage of lactate dehydrogenase compared with untreated cells after either SI or SI-RO. The number of terminal deoxynucleotidyl transferase-mediated nick end labeling-positive myocytes or loss of JC-1 fluorescence following SI and 18 h of RO was attenuated in the sildenafil-treated group with concomitant inhibition of caspase 3 activity. An early increase in Bcl-2 to Bax ratio with sildenafil treatment was also observed in myocytes after SI-RO. The increase of Bcl-2 expression by sildenafil was inhibited by nitric-oxide synthase (NOS) inhibitor, l-nitro-amino-methyl-ester. The drug also enhanced mRNA and protein content of inducible NOS (iNOS) and endothelial NOS (eNOS) in the myocytes. Sildenafil-induced protection against necrosis and apoptosis was absent in the myocytes derived from iNOS knock-out mice and was attenuated in eNOS knock-out myocytes. The up-regulation of Bcl-2 expression by sildenafil was also absent in iNOS-deficient myocytes. Reverse transcription-PCR, Western blots, and immunohistochemical assay confirmed the expression of phosphodiesterase-5 in mouse cardiomyocytes. These data provide strong evidence for a direct protective effect of sildenafil against necrosis and apoptosis through NO signaling pathway. The results may have possible therapeutic potential in preventing myocyte cell death following ischemia/reperfusion. Sildenafil citrate is a selective inhibitor of phosphodiesterase-5 (PDE5) 1The abbreviations used are: PDE5, phosphodiesterase-5; SI, simulated ischemia; RO, reoxygenation; LDH, lactate dehydrogenase; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; NOS, nitric-oxide synthase; iNOS, inducible NOS; eNOS, endothelial NOS; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; l-NAME, N-nitro-l-arginine methyl ester; RT, reverse transcription.1The abbreviations used are: PDE5, phosphodiesterase-5; SI, simulated ischemia; RO, reoxygenation; LDH, lactate dehydrogenase; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; NOS, nitric-oxide synthase; iNOS, inducible NOS; eNOS, endothelial NOS; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; l-NAME, N-nitro-l-arginine methyl ester; RT, reverse transcription. that catalyzes the breakdown of cGMP, one of the primary factors causing smooth muscle relaxation. By means of its potent action of enhancing NO-driven cGMP accumulation and ensuing vasodilatation in corpus cavernosum, sildenafil has become the most widely used drug for treating erectile dysfunction in men since its market debut under the trade name Viagra® in 1998 (1.Boolell M. Allen M.J. Ballard S.A. Gepi-Attee S. Muirhead G.J. Naylor A.M. Osterloh I.H. Gingell C. Int. J. Impot. Res. 1996; 8: 47-52PubMed Google Scholar, 2.Boolell M. Gepi-Attee S. Gingell J.C. Allen M.J. Br. J. Urol. 1996; 78: 257-261Crossref PubMed Scopus (522) Google Scholar, 3.Gibson A. Eur. J. Pharmacol. 2001; 411: 1-10Crossref PubMed Scopus (80) Google Scholar). In recent years, there are several studies on sildenafil for its therapeutic applications in diseases other than erectile dysfunction (4.Katz S.D. Balidemaj K. Homma S. Wu H. Wang J. Maybaum S. J. Am. Coll. Cardiol. 2000; 36: 845-851Crossref PubMed Scopus (237) Google Scholar, 5.Abrams D. Schulze-Neick I. Magee A.G. 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For example, sildenafil has been shown to enhance flow-mediated vasodilatation in chronic heart failure patients (4.Katz S.D. Balidemaj K. Homma S. Wu H. Wang J. Maybaum S. J. Am. Coll. Cardiol. 2000; 36: 845-851Crossref PubMed Scopus (237) Google Scholar). Because of its potent vasodilatory action, sildenafil has also been extensively studied for treating primary or hypoxia-induced pulmonary hypertension in adults and children (5.Abrams D. Schulze-Neick I. Magee A.G. Heart. 2000; 84: e4Crossref PubMed Scopus (206) Google Scholar, 6.Prasad S. Wilkinson J. Gatzoulis M.A. N. Engl. J. Med. 2000; 343: 1342Crossref PubMed Scopus (306) Google Scholar, 7.Wilkens H. Guth A. König J. Forestier N. Cremers B. Hennen B. Böhm M. Sybrecht G.W. Circulation. 2001; 104: 1218-1222Crossref PubMed Scopus (367) Google Scholar, 9.Michelakis E. Tymchak W. Lien D. Webster L. Hashimoto K. Archer S. Circulation. 2002; 105: 2398-2403Crossref PubMed Scopus (508) Google Scholar, 10.Sebkhi A. Strange J.W. Phillips S.C. Wharton J. Wilkins M.R. Circulation. 2003; 107: 3230-3235Crossref PubMed Scopus (217) Google Scholar). An epidemiological study (12.Shakir S.A.W. Wilton L.V. Boshier A. Layton D. Heeley E. Br. Med. J. 2001; 322: 651-652Crossref PubMed Scopus (123) Google Scholar) suggested that patients receiving sildenafil therapy for erectile dysfunction had reduced incidence of myocardial infarction than the general population matched with age, gender, and other risk factors. More interestingly, our laboratory recently discovered a powerful preconditioning-like effect of sildenafil in rabbit hearts (13.Ockaili R. Salloum F. Hawkins J. Kukreja R.C. Am. J. Physiol. 2002; 283: H1263-H1269Crossref PubMed Scopus (257) Google Scholar). Either intravenous or oral administration of sildenafil caused significant reduction of infarct size following ischemia/reperfusion in the myocardium. This protection was blocked by 5-hydroxydecanoate, a selective blocker of mitochondrial ATP-sensitive potassium channels. Sildenafil-induced cardioprotection has also been demonstrated in a model of global ischemia and reperfusion in mouse (14.Salloum F. Yin C. Xi L. Kukrej R.C. Circ. Res. 2003; 92: 595-597Crossref PubMed Scopus (209) Google Scholar) as well as rat (15.Das S. Maulik N. Das D.K. Kadowitz P.J. Bivalacqua T.J. Drugs Exp. Clin. Res. 2002; 28: 213-219PubMed Google Scholar). In addition, a recent study in rabbits has shown that early translocation of protein kinase C, especially the α, θ, and δ isoforms to the membranous fractions, may play an essential role in sildenafil-induced cardioprotection (16.Das A. Ockaili R. Salloum F. Kukreja R.C. Am. J. Physiol. 2004; 286: H1455-H1460Crossref PubMed Scopus (77) Google Scholar). Based on these studies (13.Ockaili R. Salloum F. Hawkins J. Kukreja R.C. Am. J. Physiol. 2002; 283: H1263-H1269Crossref PubMed Scopus (257) Google Scholar, 14.Salloum F. Yin C. Xi L. Kukrej R.C. Circ. Res. 2003; 92: 595-597Crossref PubMed Scopus (209) Google Scholar, 15.Das S. Maulik N. Das D.K. Kadowitz P.J. Bivalacqua T.J. Drugs Exp. Clin. Res. 2002; 28: 213-219PubMed Google Scholar, 16.Das A. Ockaili R. Salloum F. Kukreja R.C. Am. J. Physiol. 2004; 286: H1455-H1460Crossref PubMed Scopus (77) Google Scholar), we postulated that sildenafil triggers signaling cascade that involves the activation of protein kinase C, the generation of NO, and the accumulation of cGMP in the myocardium through inducible and endothelial nitric-oxide synthase (iNOS and eNOS), thereby leading to cardioprotection via opening of mitochondrial ATP-sensitive potassium channels (17.Kukreja R.C. Ockaili R. Salloum F. Yin C. Hawkins J. Das A. Xi L. J. Mol. Cell. Cardiol. 2004; 36: 165-173Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 18.Sasaki N. Sato T. Ohler A. O'Rourke B. Marban E. Circulation. 2000; 101: 439-445Crossref PubMed Scopus (317) Google Scholar, 19.Gross G.J. Basic Res. Cardiol. 2000; 95: 280-284Crossref PubMed Scopus (81) Google Scholar).Despite these novel and exciting observations, several fundamental issues concerning the mechanisms of sildenafil-induced cardioprotection still need to be addressed. First, it is possible that the profound vasodilatation caused by sildenafil may potentially trigger a preconditioning response in the heart. However, it remains unknown whether the drug also exerts a direct protective effect in the cardiomyocytes independent of its vascular/hypotensive effect. Second, although studies in intact hearts have demonstrated a significant protective effect of sildenafil against necrosis (infarction), there is absolutely no information in the literature suggesting that the drug also inhibits apoptosis or whether the mitochondria are the targets of protection in the cardiomyocytes. Third, despite the fact that NO signaling plays an important role in sildenafil-induced delayed preconditioning in the intact mouse heart, it is not known whether this pathway also regulates necrosis or apoptosis in the cardiomyocytes following simulated ischemia (SI) and reoxygenation (RO). Fourth, there is controversy over the presence of PDE5 in the mammalian ventricular cardiomyocytes (20.Ito M. Nishikawa M. Fujioka M. Miyahara M. Isaka N. Shiku H. Nakano T. Cell. Signal. 1996; 8: 575-581Crossref PubMed Scopus (39) Google Scholar, 21.Wallis R.M. Corbin J.D. Francis S.H. Ellis P. Am. J. Cardiol. 1999; 83: 3C-12CAbstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar, 22.Loughney K. Hill T.R. Florio V.A. Uher L. Rosman G.J. Wolda S.L. Jones B.A. Howard M.L. McAllister-Lucas L.M. Sonnenburg W.K. Francis S.H. Corbin J.D. Beavo J.A. Ferguson K. Gene (Amst.). 1998; 216: 139-147Crossref PubMed Scopus (195) Google Scholar, 23.Senzaki H. Smith C.J. Juang G.J. Isoda T. Mayer S.P. Ohler A. Paolocci N. Tomaselli G.F. Hare J.M. Kass D.A. FASEB J. 2001; 15: 1718-1726Crossref PubMed Scopus (198) Google Scholar), which may serve as the primary target for the action of sildenafil. To address these critical issues, we designed the current investigation to demonstrate the direct protective effect of sildenafil in a cardiomyocytes model of SI-RO injury. This model was particularly useful in studying the protective effect of sildenafil independent of any vascular effects or other cell types. We determined the effect of eNOS/iNOS in necrosis and apoptosis using cardiomyocytes derived from gene knock-out mice. Moreover, this study allowed us to directly examine the PDE5 expression in ventricular cardiomyocytes.EXPERIMENTAL PROCEDURESIsolation of Ventricular Cardiomyocytes—Adult male outbred ICR mice were purchased from Harlan (Indianapolis, IN). The adult male iNOS and eNOS knock-out mice (stock numbers 002596 and 002684) and their corresponding wild-type controls (B6,129 and C57BL/6J) were supplied by The Jackson Laboratory (Bar Harbor, ME). The animal experimental protocols were approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University. The ventricular cardiomyocytes were isolated using an enzymatic technique modified from the previously reported method (24.Xiao R.-P. Avdonin P. Zhou Y.-Y. Cheng H. Akhter S.A. Eschenhagen T. Lefkowitz R.J. Koch W.J. Lakatta E.G. Circ. Res. 1999; 84: 43-52Crossref PubMed Scopus (334) Google Scholar, 25.Zhou Y.-Y. Wang S.-Q. Zhu W. Chruscinski A. Kobilka B. Ziman B. Wang S. Lakatta E.G. Cheng H. Xiao R.-P. Am. J. Physiol. 2000; 279: H429-H436Crossref PubMed Google Scholar). In brief, the mouse was anesthetized with pentobarbital sodium (100 mg/kg intraperitoneally), and the heart was quickly removed from the chest. Within 3 min, the aortic opening was cannulated onto a Langendorff perfusion system (26.Xi L. Hess M.L. Kukreja R.C. Mol. Cell. Biochem. 1998; 186: 69-77Crossref PubMed Scopus (57) Google Scholar), and the heart was retrogradely perfused (37 °C) at a constant pressure of 55 mm Hg for ∼5 min with a Ca2+-free bicarbonate-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-butanedione monoxime, and 5 mm taurine that was continuously gassed with 95% O2 + 5% CO2. The enzymatic digestion was commenced by adding collagenase type II (Worthington; 0.5 mg/ml each) and protease type XIV (0.02 mg/ml) to the perfusion buffer and continued for ∼15 min. 50 μm Ca2+ was then added in to the enzyme solution for perfusing the heart for another 10–15 min. The digested ventricular tissue was cut into chunks and gently aspirated with a transfer pipette for facilitating the cell dissociation. The cell pellet was resuspended for a three-step Ca2+ restoration procedure (i.e. 125, 250, and 500 μm Ca2+). The freshly isolated cardiomyocytes were then suspended in minimal essential medium (catalogue number M1018, pH 7.35–7.45; Sigma) containing 1.2 mm Ca2+, 12 mm NaHCO3, 2.5% fetal bovine serum, and 1% penicillin-streptomycin. The cells were then plated onto 35-mm cell culture dishes that were precoated with 20 μg/ml mouse laminin in phosphate-buffered saline with 1% penicillin-streptomycin for 1 h. The cardiomyocytes were cultured in the presence of 5% CO2 for 1 h in a humidified incubator at 37 °C, which allowed cardiomyocytes to attach to the dish surface prior to the experimental protocol.Experiment Protocol—The cultured cardiomyocytes were incubated under 37 °C and 5% CO2, for 1 h with or without 1 or 10 μm sildenafil citrate, which was prepared by grinding Viagra tablets (Pfizer Inc.) into powder and dissolved in distilled water. The drug solution was filtered (0.45-μm pore size) before adding into cell medium. Cardiomyocytes were 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 MgCl2,20mm sodium lactate, 16 mm KCl, 10 mm 2-deoxyglucose (pH adjusted to 6.2) similar to those previously published (27.Rakhit R.D. Edwards R.J. Mockridge J.W. Baydoun A.R. Wyatt A.W. Mann G.E. Marber M.S. Am. J. Physiol. 2000; 278: H1211-H1217PubMed Google Scholar). In addition, the cells were incubated under hypoxic conditions at 37 °C during the entire SI period by adjusting the tri-gas incubator to 1–2% O2 and 5% CO2. RO was accomplished by replacing the ischemic buffer with normal medium under normoxic conditions. Assessment of cell necrosis and apoptosis was performed at two time points of RO, i.e. 1 and 18 h.Evaluation of Cell Viability—Cell viability was assessed by trypan blue exclusion assay and LDH release in the medium. At the end of protocol, 20 μl of 0.4% trypan blue (Sigma-Aldrich) was added into the culture dish. After ∼5 min of equilibration, the cells were counted under microscope. For LDH measurements, the cellular medium was collected, and the enzyme activity was monitored spectrophotometrically using an assay kit (Sigma-Aldrich).TUNEL Staining and Measurement of Mitochondrial Membrane Potential—Cardiomyocyte apoptosis was analyzed by TUNEL staining, using a kit purchased from BD Biosciences that detects nuclear DNA fragmentation via a fluorescence assay as previously reported (28.Das A. Chendil D. Dey S. Mohiuddin M. Mohiuddin M. Milbrandt J. Rangnekar V.M. Ahmed M.M. J. Biol. Chem. 2001; 276: 3279-3286Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). In brief, after SI and 18 h of RO, the cells in two chamber slides were fixed by 4% formaldehyde/phosphate-buffered saline at 4 °C for 25 min and subjected to TUNEL assay according to the manufacturer's protocol. The slides were then counterstained with Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (a DNA intercalating dye for visualizing nuclei in fixed cells; catalogue number H-1200, Vector Laboratories). Additionally, cardiomyocyte apoptosis was detected with a mitochondrial membrane potential (ΔΨm) detection kit provided by Biocarta. 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 15 minina5%CO2 incubator and rinsed with assay buffer according to the manufacturer's protocol. The stained cells were examined under an Olympus IX70 fluorescence microscope.Activated Caspase 3 Detection—Active caspase was detected using the CaspaTag™ Caspase 3,7 in situ assay kit (Chemicon, Temecula, CA) according to the manufacturer's instructions. In this assay, the cell-permeable noncytotoxic fluorochrome inhibitors of caspases binds covalently to a reactive cysteine residue on the large subunit of the active caspase heterodimer, thereby inhibiting further enzymatic activity. This kit uses a carboxyfluorescein labeled fluoromethyl ketone peptide inhibitor of caspases-3 and -7 (SR-DEVD-FMK), which emits a red fluorescence. The red fluorescent signal is a direct measure of the amount of active caspase 3 in the cell at the time the reagent was added. The stained cells were immediately examined under a Nikon epifluorescent microscope using a band pass filter (excitation, 550 nm; emission, >580 nm) to view the red fluorescence of active caspase-positive cells. Hoechst stain was detected using a UV filter with excitation at 365 and emission at 480 nm.RT-PCR of iNOS, eNOS, and PDE5—The total RNA was isolated from untreated and sildenafil-treated cardiomyocytes using TRI reagent (Molecular Research Center) as described previously (14.Salloum F. Yin C. Xi L. Kukrej R.C. Circ. Res. 2003; 92: 595-597Crossref PubMed Scopus (209) Google Scholar). The reverse transcription and PCR amplification were performed using a OneStep RT-PCR kit from Qiagen. The oligonucleotide primers were synthesized by integrated DNA technology, according to the published sequences for mouse iNOS (29.Wang Y. Guo Y. Zhang S.X. Wu W.-J. Wang J. Bao W. Bolli R. J. Mol. Cell. Cardiol. 2002; 34: 5-15Abstract Full Text PDF PubMed Scopus (83) Google Scholar), eNOS (30.Fagan K.A. Morrissey B. Fouty B.W. Sato K. Harral J.W. Morris Jr., K.G. Hoedt-Miller M. Vidmar S. McMurtry I.F. Rodman D.M. Respir. Res. 2001; 2: 306-313Crossref PubMed Scopus (82) Google Scholar), and β-actin (28.Das A. Chendil D. Dey S. Mohiuddin M. Mohiuddin M. Milbrandt J. Rangnekar V.M. Ahmed M.M. J. Biol. Chem. 2001; 276: 3279-3286Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), which served as internal control. The transcript levels of iNOS and eNOS were quantified by real time PCR performed in the ABI prism 7900HT sequence detector system (Applied Biosystems, Foster City, CA) using the TaqMan One Step PCR Master Mix reagent kit (Product 4309169). All of the samples were processed in triplicate according to the manufacturer's recommended conditions. The cycling conditions were: 48 °C for 30 min, 95 °C for 10 min, and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The cycle threshold was determined to provide the optimal standard curve values (0.98–1.0). The probes and primers (Table I) were designed by the Nucleic Acid Research Facilities of Virginia Commonwealth University, using Primer Express 2.0. The probes were labeled in the 5′ end with 6-carboxyfluoresceine and in the 3′ end with 6-carboxytetramethylrhodamine. Eukaryotic 18 S rRNA TaqMan endogenous control (Product 4310893E; Applied Biosystems) was used to normalize the transcript levels. The following amounts of RNA was used for real time PCR: 20 ng for iNOS, 4 ng for eNOS, and 1 ng for 18 S rRNA.Table IPrimer/probe sequences used for determination of iNOS, eNOS, and PDE5A expressionPrimer/probe sequencesiNOSForward: CAGGAGGAGAGAGATCCGATTTAGReverse: TGACCCGTGAAGCCATGACTagMan Probe: TGGAAGCAAAGAACACCACTTTCACCAAGAeNOSForward: CACCAGGAAGAAGACCTTTAAGGAReverse: CACACGCTTCGCCATCACTaqMan Probe: TAGCCAATGCAGTGAAGGTCTCTGCCTCPDE5AForward: AATACCACCCCTGGAGCACCReverse: TTCAAGGGCTCGCCAAAAGC Open table in a new tab PDE5 was amplified by using the primers based on the mouse PDE5A cDNA sequence (GenBank™ 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 soluble protein was extracted from the cells with reporter lysis buffer (Promega). The homogenate was centrifuged at 10,000 × g for 5 min under 4 °C, and the supernatant was recovered. 25 μg of protein from each sample 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. The membrane was then incubated with rabbit polyclonal primary antibody at a dilution of 1:1000 for each of the respective proteins, i.e. Bax, Bcl-2, iNOS, eNOS (Santa Cruz), or PDE5 (Calbiochem) for 2 h before being washed and incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:2000; Amersham Biosciences) for 1 h. The blots were developed using a chemiluminescent system. The optical density for each band was scanned and quantified using the identical densitometric system described above.Immunohistochemistry of PDE5—Localization of PDE5A protein in cardiomyocytes was performed according to the method of Senzaki et al. (23.Senzaki H. Smith C.J. Juang G.J. Isoda T. Mayer S.P. Ohler A. Paolocci N. Tomaselli G.F. Hare J.M. Kass D.A. FASEB J. 2001; 15: 1718-1726Crossref PubMed Scopus (198) Google Scholar). The cardiomyocytes were fixed in 4% paraformaldehyde and then treated with 0.1% sodium citrate and 0.1% Triton X-100. The cells were then preincubated with normal donkey antiserum for 30 min and then incubated overnight at 4 °C with polyclonal rabbit PDE5A antibody (Calbiochem) at 1:10,000 dilution. Incubation with secondary antibody was performed at room temperature for 1 h with anti-rabbit Alexa 488 (Molecular Probes). Imaging was performed on a Zeiss LSM 510 META confocal laser scanning microscope.Data Analysis and Statistics—The data are presented as the means ± S.E. The difference between control and sildenafil-treated groups or among the multiple treatment groups was analyzed respectively with unpaired t test or one-way analysis of variance followed by Student-Newman-Keuls post-hoc test. p < 0.05 was considered to be statistically significant.RESULTSEffect of Sildenafil on Cardiomyocyte Necrosis—Our method for cell preparations yielded at least 70% of the cardiomyocytes with rod shape morphology, which was similar to previously reported studies (24.Xiao R.-P. Avdonin P. Zhou Y.-Y. Cheng H. Akhter S.A. Eschenhagen T. Lefkowitz R.J. Koch W.J. Lakatta E.G. Circ. Res. 1999; 84: 43-52Crossref PubMed Scopus (334) Google Scholar, 25.Zhou Y.-Y. Wang S.-Q. Zhu W. Chruscinski A. Kobilka B. Ziman B. Wang S. Lakatta E.G. Cheng H. Xiao R.-P. Am. J. Physiol. 2000; 279: H429-H436Crossref PubMed Google Scholar). Fig. 1A shows a typical preparation of isolated adult mouse cardiomyocytes used in the present studies. After 40 min of SI, the percentage of trypan blue positive cardiomyocytes 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 h resulted in the decrease in trypan blue positive cardiomyocytes, i.e. from 35 ± 1% of the total counted cells in the untreated SI group to 17 ± 0.5 and 18 ± 1% 1 and 10 μm sildenafil-treated groups, respectively (n = 3; p < 0.05; Fig. 1D). After 40 min of SI and 1 h or 18 h of RO, the trypan blue positive cardiomyocytes further increased to 40 ± 2 and 54 + 1%, respectively. Again, prior treatment with sildenafil reduced the trypan blue-positive cells as compared with the untreated SI-RO group (p < 0.001, n = 3; Fig. 1, B–D). Similar results were obtained by measurement of LDH release in the medium. As shown in Fig. 1E, LDH increased in the medium following SI and SI-RO (1 or 18 h), which is attenuated by prior treatment with sildenafil. Also, the attenuation in LDH release was independent of the dose of sildenafil used in these experiments.Effect of Sildenafil on Cardiomyocyte Apoptosis—Despite significant necrosis following 40 min of SI and 1 h of RO (Fig. 1, D and E), apoptosis was not detectable under these conditions (data not shown). However, apoptotic cell death became clearly evident 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 to 3.5 ± 0.6% in cardiomyocytes treated with 10 μm sildenafil (p < 0.001 versus SI-RO and p < 0.05 versus untreated nonischemic control cardiomyocytes, n = 4; Fig. 2, A–I). The intense red fluorescence of active caspase was clearly observed in myocytes following 40 min of SI and 18 h of RO (Fig. 3B). But the red fluorescence decreased significantly in myocytes treated with 1 or 10 μm sildenafil before 40 min of SI and 18 h of RO (Fig. 3, C and D).Fig. 2Effect of sildenafil on inhibition of apoptosis in cardiomyocytes. Cells were treated with 10 μm sildenafil for 1 h followed by 40 min 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 μm 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 of cardiomyocytes 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Effect of sildenafil on caspase 3 activity in cardiomyocytes. Isolated myocytes were treated with 1 and 10 μm sildenafil for 1 h followed by 40 min of SI and 18 h of RO, and caspase 3 activity was detected by CaspaTag reagent under fluorescence microscope. Activated caspase 3 in adult myocytes (red, left column) and nuclei stained by Hoechst (blue, right column). A, nonischemic control; B, cardiomyocytes subjected to 40 min SI and 18 h of RO; C, cardiomyocytes pretreated with 1 μm sildenafil for 1 h before SI-RO; D, cardiomyocytes pretreated with 10 μm sildenafil for 1 h before SI-RO. It is apparent that a significant number of cardiomyocytes displayed red fluorescence staining after SI-RO (Fig. 3B). The cardiomyocytes pretreated with 1 or 10 μm sildenafil (C and D) showed a negligible amount of red fluorescence as compare with the untreated SI-RO cells.View Large Image Figure ViewerDownload Hi-res image
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