Phosphodiesterase-5 inhibitor sildenafil preconditions adult cardiac myocytes against necrosis and apoptosis. Essential role of nitric oxide signaling.
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.
Das
Anindita
A
Division of Cardiology, Department of Internal Medicine, Virginia Commonwealth University Medical Center, Richmond, Virginia 23298-0281, USA.
Xi
Lei
L
Kukreja
Rakesh C
RC
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
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