title: Phosphodiesterase-5 inhibitor sildenafil preconditions adult cardiac myocytes against necrosis and apoptosis. Essential role of nitric oxide signaling.
abstract: 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 microM) 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.
authors.affiliation: Division of Cardiology, Department of Internal Medicine, Virginia Commonwealth University Medical Center, Richmond, Virginia 23298-0281, USA.
authors.forename: Anindita
authors.initials: A
authors.lastname: Das
authors.forename: Lei
authors.initials: L
authors.lastname: Xi
authors.forename: Rakesh C
authors.initials: RC
authors.lastname: Kukreja
mesh_terms: D015106:3',5'-Cyclic-GMP Phosphodiesterases; D000818:Animals; D017209:Apoptosis; D001562:Benzimidazoles / Q000494:pharmacology; D015153:Blotting, Western; D002232:Carbocyanines / Q000494:pharmacology; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D008564:Membrane Potentials; D051379:Mice; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D018345:Mice, Knockout; D008928:Mitochondria / Q000378:metabolism; D032342:Muscle Cells / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology*; D019331:NG-Nitroarginine Methyl Ester / Q000378:metabolism; D009336:Necrosis; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D010100:Oxygen / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011687:Purines; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D000068677:Sildenafil Citrate; D013450:Sulfones; D013997:Time Factors; D014158:Transcription, Genetic; D014343:Trypan Blue / Q000378:metabolism / Q000494:pharmacology; D051020:bcl-X Protein
chemical_list: C494812:Bcl2l1 protein, mouse; D001562:Benzimidazoles; D002232:Carbocyanines; D017931:DNA Primers; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D010726:Phosphodiesterase Inhibitors; D010879:Piperazines; D019253:Proto-Oncogene Proteins c-bcl-2; D011687:Purines; D013450:Sulfones; D051020:bcl-X Protein; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D009569:Nitric Oxide; D000068677:Sildenafil Citrate; D007770:L-Lactate Dehydrogenase; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496317:Nos2 protein, mouse; C496315:Nos3 protein, mouse; D010727:Phosphoric Diester Hydrolases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C517382:Pde5a protein, mouse; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D014343:Trypan Blue; D010100:Oxygen; D019331:NG-Nitroarginine Methyl Ester