NUCLEAR FACTOR NF-KAPPA-B IN MYOCARDIUM - DEVELOPMENTAL EXPRESSION OFSUBUNITS AND ACTIVATION BY INTERLEUKIN-1-BETA IN CARDIAC MYOCYTES IN-VITRO

Objective: The aims of the study were to investigate the pattern of ex pression of the major subunits of the NF-kappa B transcription factor complex in human and rat heart development, and to characterise the ti ming of NF-kappa B activation by interleukin-1 beta (IL-1 beta) in rat neonatal cardiac myocytes. Methods: The expression of NF-kappa B subu nits p65 and p50 and the inhibitory subunits I kappa B-alpha and I kap pa B-beta in human and rat myocardial samples was measured by immunobl otting, using antibodies specific to each subunit. The activation of N F-kappa B was measured in neonatal rat cardiac myocytes that were trea ted with IL-1 beta for different times (0-60 min). Depletion of the in hibitory factors I kappa B-alpha and IKB-beta was assessed by immunobl otting. The presence of NF-kappa B DNA binding activity was measured d irectly in nuclear extracts by electrophoretic mobility shift assay (E MSA). Results: p65, p50, I kappa B-alpha and IKB-beta are expressed at all stages of development analysed. In human myocardial samples, expr ession of p50, p65 and I kappa B-alpha show an apparent gradual declin e relative to total protein. In contrast, the level of I kappa B-beta remained relatively constant, suggesting a significant shift in the ra tio of beta and alpha subunits with development. In rat myocardium, p6 5, p50, I kappa B-alpha and I kappa B-beta showed a gradual decline du ring development, with a particularly pronounced decrease between the ten day post-natal and adult samples. Treatment of neonatal rat cardia c myocytes with IL-1 beta (5 ng/ml) caused a rapid and transient deple tion of I kappa B-alpha (reducing to 16+/-1.6% of initial levels withi n 5 min, returning to 82+/-10% within 60 min). A slower, less marked d epletion is observed for I kappa B-beta (24+/-6% by 30 min, returning to only 49+/-5% by 60 min). Rapid and transitory accumulation of NF-ka ppa B DNA binding activity was detected in the nucleus, with a pattern that correlated with the depletion of I kappa B-alpha. Conclusions: T he principal NF-kappa B subunits p65, p50, I kappa B-alpha and I kappa B-beta are present throughout development, suggesting that this trans cription complex may participate in myocardial gene regulation through out development and in the adult. Activation by IL-1 beta demonstrates that NF-kappa B probably plays a direct role in the regulation of gen e transcription in response to cytokine activation in cardiac myocytes . (C) 1998 Elsevier Science B.V. All rights reserved.