Nuclear factor NF-κB in myocardium:: developmental expression of subunits and activation by interleukin-1β in cardiac myocytes in vitro

Objective: The aims of the study were to investigate the pattern of expression of the major subunits of the NF-kappa B transcription factor complex in human and rat heart development, and to characterise the timing of NF-kappa B activation by interleukin-1 beta (IL-1 beta) in rat neonatal cardiac myocytes. Methods: The expression of NF-kappa B subunits p65 and p50 and the inhibitory subunits I kappa B-alpha and I kappa B-beta in human and rat myocardial samples was measured by immunoblotting, using antibodies specific to each subunit. The activation of NF-kappa B was measured in neonatal rat cardiac myocytes that were treated with IL-1 beta for different times (0-60 min). Depletion of the inhibitory factors I kappa B-alpha and IKB-beta was assessed by immunoblotting. The presence of NF-kappa B DNA binding activity was measured directly in nuclear extracts by electrophoretic mobility shift assay (EMSA). Results: p65, p50, I kappa B-alpha and IKB-beta are expressed at all stages of development analysed. In human myocardial samples, expression of p50, p65 and I kappa B-alpha show an apparent gradual decline relative to total protein. In contrast, the level of I kappa B-beta remained relatively constant, suggesting a significant shift in the ratio of beta and alpha subunits with development. In rat myocardium, p65, p50, I kappa B-alpha and I kappa B-beta 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-1 beta (5 ng/ml) caused a rapid and transient depletion of I kappa B-alpha (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 I kappa B-beta (24+/-6% by 30 min, returning to only 49+/-5% by 60 min). Rapid and transitory accumulation of NF-kappa B DNA binding activity was detected in the nucleus, with a pattern that correlated with the depletion of I kappa B-alpha. Conclusions: The principal NF-kappa B subunits p65, p50, I kappa B-alpha and I kappa B-beta are present throughout development, suggesting that this transcription complex may participate in myocardial gene regulation throughout development and in the adult. Activation by IL-1 beta demonstrates that NF-kappa B probably plays a direct role in the regulation of gene transcription in response to cytokine activation in cardiac myocytes. (C) 1998 Elsevier Science B.V. All rights reserved.