Activation of NFATc1 is directly mediated by IP3 in adult cardiac myocytes.

The Ca(2+)-sensitive nuclear factor of activated T cell (NFAT) transcription factors are implicated in cardiac development and cellular remodeling associated with cardiac disease. In adult myocytes it is not resolved what specific Ca(2+) signals control the activity of different NFAT isoforms in an environment that undergoes large changes of intracellular Ca(2+) concentration with every heart beat. Cardiac myocytes possess the complete inositol 1,4,5-trisphosphate (IP(3))/Ca(2+)-signaling cassette; however, its physiological and pathological significance has been a matter of ongoing debate. Therefore, we tested the hypothesis whether IP(3) receptor activation regulates NFAT activity in cardiac myocytes. We used confocal microscopy to quantify the nuclear localization of NFATc1-green fluorescent protein (GFP) and NFATc3-GFP fusion proteins (quantified as the ratio of nuclear NFAT to cytoplasmic NFAT) in response to stimulation with neurohumoral agonists. In rabbit atrial myocytes, an overnight stimulation with endothelin-1, angiotensin II, and phenylephrine induced nuclear accumulation of NFATc1 that was sensitive to calcineurin inhibitors (cyclosporin A or inhibitor of NFAT-calcineurin association-6) and prevented by the IP(3) receptor inhibitor 2-aminoethoxydiphenyl borate. Furthermore, a direct elevation of intracellular IP(3) with a cell-permeable IP(3) acetoxymethyl ester (10 μM) induced nuclear localization of NFATc1. With a fluorescence-based in vivo assay, we showed that endothelin-1 also enhanced the transcriptional activity of NFATc1 in atrial cells. The agonists failed to activate NFATc1 in rabbit ventricular cells, which express IP(3) receptors at a lower density than atrial cells. They also did not activate NFATc3, an isoform that is highly influenced by nuclear export processes, in both cell types. Our data show that the second messenger IP(3) is directly involved in the activation of NFATc1 in adult atrial cardiomyocytes.