title: Nuclear factor NF-kappa B in myocardium: developmental expression of subunits and activation by interleukin-1 beta in cardiac myocytes in vitro.
abstract: 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 I kappa B-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 I kappa B-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.
authors.affiliation: Imperical College School of Medicine, National Heart and Lung Institute, London, UK.
authors.forename: D A
authors.initials: DA
authors.lastname: Norman
authors.forename: M H
authors.initials: MH
authors.lastname: Yacoub
authors.forename: P J
authors.initials: PJ
authors.lastname: Barton
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D004586:Electrophoresis; D005318:Fetal Heart / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins; D015151:Immunoblotting; D007375:Interleukin-1 / Q000494:pharmacology*; D007536:Isomerism; D009206:Myocardium / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
chemical_list: D004268:DNA-Binding Proteins; C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D007375:Interleukin-1; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606206:Nfkbia protein, rat; D000072000:NF-KappaB Inhibitor alpha