The Effect of Tumor Necrosis Factor-alpha in Cultured Neonatal Rat Cardiomyocytes

Kim, Su Jin; Lee, Dong Seok; Ko, Ok; Mun, Il Soo; Ko, Bok Hyun; Jung, Yong Wook

Abstract

PURPOSE: Tumor necrosis factor-alpha(TNF-alpha) is a pro-inflammatory cytokine that has been implicated in the pathogenesis of cardiovascular disease. Serum levels of TNF-alpha are elevated in many human cardiac related pathogenic conditions, including heart failure. It is well known that TNF-alpha inhibits myocardial contractility and induces apoptosis of adult rat cardiomyocytes via stimulation of TNF receptor 1. But pathophysiologically relevant low levels of TNF-alpha can not induce apoptosis of neonatal cardiomyocytes. So, we evaluated the effects of different concentrations of TNF-alpha in cultured rat neonatal cardiomyocytes : apoptosis or necrosis.
METHODS
Neonatal ventricular myocytes were isolated from 3-day-old rats by stepwise collagenase dissociation, and the cells were cultured for 3 days. After that, cardiomyocytes were treated with low(25 ng/mL) and high(250 ng/mL) concentration of TNF-alpha for 48 hours. Apoptosis was determined by terminal deoxynucleotidyl transfer-mediated end labelling(TUNEL) staining, and cell viability was evaluated by lactate dehydrogenase(LDH) measurements using cell culture supernatants.
RESULTS
Low dose TNF-alpha did not induce apoptosis compared with controls(10.5 +/- 3.5% : 10.4 +/- 4.3%). And high dose TNF-alpha also did not induce significant apoptosis(10.2 +/- 3.6% : 10.4 +/- 4.3%). There was no detectable morphological changes of cardiomyocytes after low and high concentration of TNF-alpha treatment. LDH levels after TNF-alpha treatment was not significant compared with control(control : low : high, 3.2 +/- 0.1% : 3.1 +/- 0.2% : 3.3 +/- 0.3%).
CONCLUSIONS
Our results suggest that high concentration of TNF-alpha alone can not induce apoptosis and significant cytotoxicity in neonatal rat cardiomyocytes.