Avaliação da expressão genica atraves de tecnica de mRNA differential display em coraçoes de ratos submetidos a sobrecarga pressora aguda

AUTOR(ES)

Claudia Raquel Cantarelli Costa

DATA DE PUBLICAÇÃO

2002

RESUMO

To examine genes that are early differentially expressed in response to pressure overload we performed differential display reverse transcriptase-polymerase chain reaction on samples of rat left ventricle (LV) subjected to 2 and 4 hours of transverse aortic constriction. Specifically, we identified increased expression of five genes out of 43 candidate-cDNA ftagments. One such a gene, TBKI raised our interest because of its potential role on the regulation of pathways that control the activity of the transcription factor NF-kB, known to coordinate fundamental cellular processes in response to stressful stimuli. RT-PCR analysis with TBKI-specific primers in LV samples confirmed the enhanced expression of transcripts of TBKI in overloaded myocardium. To verify the functional significance of such finding we first examined TBKI expression in LV by immunoblot and immunohistochemical analysis. TBKI protein levels increased at 2 and remained elevated up to 24 hs of pressure overload. The immunostaining analysis showed TBKI expressed predominantly in cardiac myocytes and in the media of coronary vessels. Co-immunoprecipitation experiments indicated a load-induced increase in the association of TBKI with p50NF-kB that was paralleled by enhanced anti-TBKI immunocomplex kinase activity toward p50NF-kB. Experiments with myocardial nuclear and cytosolic extracts indicated a load-induced p50NF-kB translocation from cytosolic to nuclear fraction and increases in DNA-binding activity demonstrated by electrophoretic mobility shift assay, concurrent with increased TBKI expression and p50NF-kB phosphorylation by anti- TBKI immunoprecipitates. These results suggest that TBKI is rapidly activated and might regulate NF-kB in overloaded myocardium with potential implications in load-induced cardiac hypertrophy and remodeling.

ASSUNTO(S)

coração hipertrofia

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