Tumor necrosis factor-alpha induces apoptosis via inducible nitric oxide synthase in neonatal mouse cardiomyocytes.
OBJECTIVE
It has been demonstrated that tumor necrosis factor-alpha (TNF alpha) induces apoptosis in cardiac myocytes. However, its mechanism of action is still not well understood. In the present study, we hypothesized that TNF alpha induces myocardial apoptosis by induction of inducible nitric oxide synthase (iNOS).


METHODS
Neonatal cardiac myocytes were isolated from iNOS (-/-) mutant and C57BL6 wild type mice. Cells were cultured for 3 days before treatment with an NO donor or TNF alpha. Following treatment with S-nitroso-N-acetyl-penicillamine (SNAP) or TNF-alpha, cells were tested for apoptosis by terminal deoxynucleotidyl transfer-mediated end labeling (TUNEL) staining and cell death detection ELISA. NO production was measured by nitrite concentration in the culture medium. Cardiomyocyte expression of iNOS and TNF type 1 receptor (TNFR1) mRNA was determined by reverse transcriptase-polymerase chain reaction (RT-PCR).


RESULTS
SNAP (0.01-100 microM) induced apoptosis of cardiac myocytes in a concentration-dependent manner in the wild type mice (n = 5, P < 0.01). TNFR1 mRNA was expressed in neonatal cardiomyocytes from both wild type and iNOS (-/-) mutant mice. TNF alpha induced a concentration-dependent increase in iNOS mRNA expression and nitrite production as well as significant apoptosis of cardiomyocytes in the wild type mice (n = 4, P < 0.01). However, without iNOS expression, the apoptotic effects of TNF-alpha were significantly attenuated in cardiomyocytes from iNOS (-/-) mutant mice (n = 4, P < 0.05).


CONCLUSIONS
TNF alpha induces apoptosis via iNOS expression and NO production in neonatal mouse cardiomyocytes.
Song
W
W
London Health Sciences Centre, Department of Medicine, Ontario, Canada.
Lu
X
X
Feng
Q
Q
D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects*; D002478:Cells, Cultured; D053938:DNA Fragmentation; D004790:Enzyme Induction; D020287:In Situ Nick-End Labeling; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D008854:Microscopy, Electron; D009206:Myocardium / Q000378:metabolism* / Q000648:ultrastructure; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse