© 1998 by European Society of Cardiology
Copyright © 1998, European Society of Cardiology
Nuclear factor NF-
B in myocardium: developmental expression of subunits and activation by interleukin-1β in cardiac myocytes in vitro
Cardiothoracic Surgery, Imperial College School of Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, UK
* Corresponding author. Tel.: +44 (0)171 351 8184; Fax: +44 (0)171 376 3442; E-mail: p.barton@ic.ac.uk
Objective: The aims of the study were to investigate the pattern of expression of the major subunits of the NF-
B transcription factor complex in human and rat heart development, and to characterise the timing of NF-B activation by interleukin-1β (IL-1β) in rat neonatal cardiac myocytes. Methods: The expression of NF-B subunits p65 and p50 and the inhibitory subunits IB-
and IB-β in human and rat myocardial samples was measured by immunoblotting, using antibodies specific to each subunit. The activation of NF-B was measured in neonatal rat cardiac myocytes that were treated with IL-1β for different times (0–60 min). Depletion of the inhibitory factors IB- and IB-β was assessed by immunoblotting. The presence of NF-B DNA binding activity was measured directly in nuclear extracts by electrophoretic mobility shift assay (EMSA). Results: p65, p50, IB- and IB-β are expressed at all stages of development analysed. In human myocardial samples, expression of p50, p65 and IB- show an apparent gradual decline relative to total protein. In contrast, the level of IB-β remained relatively constant, suggesting a significant shift in the ratio of β and subunits with development. In rat myocardium, p65, p50, IB- and IB-β showed a gradual decline during development, with a particularly pronounced decrease between the ten day post-natal and adult samples. Treatment of neonatal rat cardiac myocytes with IL-1β (5 ng/ml) caused a rapid and transient depletion of IB- (reducing to 16±1.6% of initial levels within 5 min, returning to 82±10% within 60 min). A slower, less marked depletion is observed for IB-β (24±6% by 30 min, returning to only 49±5% by 60 min). Rapid and transitory accumulation of NF-B DNA binding activity was detected in the nucleus, with a pattern that correlated with the depletion of IB-. Conclusions: The principal NF-B subunits p65, p50, IB- and IB-β are present throughout development, suggesting that this transcription complex may participate in myocardial gene regulation throughout development and in the adult. Activation by IL-1β demonstrates that NF-B probably plays a direct role in the regulation of gene transcription in response to cytokine activation in cardiac myocytes.
KEYWORDS Development; Cardiac myocyte; NF-B; Interleukin-1β; Rat; Human