title: Angiotensin II and norepinephrine activate specific calcineurin-dependent NFAT transcription factor isoforms in cardiomyocytes.
abstract: Norepinephrine (NE) and angiotensin II (ANG II) are primary effectors of the sympathetic adrenergic and the renin-angiotensin-aldosterone systems, mediating hypertrophic, apoptotic, and fibrotic events in the myocardium. As NE and ANG II have been shown to affect intracellular calcium in cardiomyocytes, we hypothesized that they activate the calcium-sensitive, prohypertrophic calcineurin-nuclear factor of activated T-cell (NFATc) signaling pathway. More specifically, we have investigated isoform-specific activation of NFAT in NE- and ANG II-stimulated cardiomyocytes, as it is likely that each of the four calcineurin-dependent isoforms, c1-c4, play specific roles. We have stimulated neonatal ventriculocytes from C57/B6 and NFAT-luciferase reporter mice with ANG II or NE and quantified NFAT activity by luciferase activity and phospho-immunoblotting. ANG II and NE increased calcineurin-dependent NFAT activity 2.4- and 1.9-fold, measured as luciferase activity after 24 h of stimulation, and induced protein synthesis, measured by radioactive leucine incorporation after 24 and 72 h. To optimize measurements of NFAT isoforms, we examined the specificity of NFAT antibodies on peptide arrays and by immunoblotting with designed blocking peptides. Western analyses showed that both effectors activate NFATc1 and c4, while NFATc2 activity was regulated by NE only, as measured by phospho-NFAT levels. Neither ANG II nor NE activated NFATc3. As today's main therapies for heart failure aim at antagonizing the adrenergic and renin-angiotensin-aldosterone systems, understanding their intracellular actions is of importance, and our data, through validating a method for measuring myocardial NFATs, indicate that ANG II and NE activate specific NFATc isoforms in cardiomyocytes.
authors.affiliation: Institute for Experimental Medical Research, Oslo Univ. Hospital-Ullevaal, Bldg. 7, 4 floor, Kirkeveien 166, 0407 Oslo, Norway. i.g.lunde@medisin.uio.no
authors.forename: Ida G
authors.initials: IG
authors.lastname: Lunde
authors.forename: Heidi
authors.initials: H
authors.lastname: Kvaløy
authors.forename: Bjørg
authors.initials: B
authors.lastname: Austbø
authors.forename: Geir
authors.initials: G
authors.lastname: Christensen
authors.forename: Cathrine R
authors.initials: CR
authors.lastname: Carlson
mesh_terms: D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D019703:Calcineurin / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D018604:Epitope Mapping / Q000379:methods; D008215:Lymphocytes, Null; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D050778:NFATC Transcription Factors / Q000378:metabolism*; D009638:Norepinephrine / Q000378:metabolism / Q000494:pharmacology*; D014176:Protein Biosynthesis; D020033:Protein Isoforms; D015398:Signal Transduction
chemical_list: D004268:DNA-Binding Proteins; D050778:NFATC Transcription Factors; D020033:Protein Isoforms; D000804:Angiotensin II; D019703:Calcineurin; D002118:Calcium; D009638:Norepinephrine