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

Abstract

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.

Fig. 1.

Subcellular distribution of Ca²⁺-sensitive nuclear factor of activated T cell isoforms c1 and c3 (NFATc1 and NFATc3) in adult myocytes from rabbit. The isoform NFATc1 displayed nuclear localization in resting atrial (A,a) and ventricular (B,a) myocytes. A,b and B,b: corresponding cell nuclei were identified by staining with the cell-permeable DNA dye SYTO-59. A,c and B,c: fluorescence intensity profiles of NFATc1-green fluorescent protein (GFP) and SYTO-59 fluorescence across the nucleus and adjacent cytoplasmic regions, normalized to maximum fluorescence. In contrast to NFATc1, the isoform NFATc3 was distributed to the cytoplasm of atrial (A,d) and ventricular (B,d) myocytes. A,e and B,e: SYTO-59 staining of the cell nuclei. Corresponding NFATc3-GFP and SYTO-59 fluorescence intensity profiles for atrial (A,f) and ventricular (B,f) myocytes are shown. For images, scale bar = 10 μm. For line profiles, scale bar = 5 μm.

Fig. 2.

Neurohumoral agonists induce nuclear accumulation and transcriptional activity of NFATc1 in atrial (A–D) and ventricular (E) cells. Overnight stimulation with 100 nM endothelin-1 (ET-1) resulted in an increase in nuclear-localized NFATc1 (A,b compared with nonstimulated cells in A,a) by 50% [ratio of nuclear NFAT to cytoplasmic NFAT (NFAT_Nuc/NFAT_Cyt); A,c]. This effect was prevented by the inositol 1,4,5-trisphosphate (IP₃) receptor blocker 2-aminoethoxydiphenyl borate (2-APB; 2 μM) and was sensitive to the calcineurin (CaN) inhibitor of NFAT-CaN association-6 (INCA-6; 1 μM). B: In atrial myocytes expressing NFATc1-GFP, stimulation with ET-1 resulted in higher transcriptional activity, measured with NFAT-sensitive expression of the red fluorescent protein (RFP) (NFAT-RFP reporter; see text for details). F_RFP, fluorescence of RFP [in arbitrary units (AU)]. C and D: increases in nuclear localization of NFATc1 were also induced by ANG II (2 μM) and phenylephrine (Phe; 10 μM) and were prevented by 2-APB and CaN inhibition. CsA, cyclosporin A. E: all agonists failed to induce activation of NFATc1 in ventricular cells. Numbers in parentheses indicate the number of individual cells tested. NFAT_Nuc/NFAT_Cyt ratios are normalized (norm) to control. *P < 0.05, significantly different from control. Scale bar = 30 μm.

Fig. 3.

Activation of NFATc1 is directly mediated by IP₃ in atrial myocytes. A: short-term incubation with the membrane-permeable IP₃ acetoxymethyl ester (IP₃-AM; 10 μM, 2 h) resulted in nuclear accumulation of NFATc1, which was sensitive to 2-APB (2 μM). B: IP₃ induced activation of NFAT in overnight incubations. Inhibition of GSK3 with 1 μM alsterpaullone (Alsterp) resulted in robust nuclear accumulation of NFATc1, which was further enhanced by ET-1 in a 2-APB-sensitive manner (middle). B, right: nuclear accumulation of NFATc1 after full inhibition of nuclear export with 40 nM leptomycin B (LB). Numbers in parentheses indicate the number of individual cells tested. NFAT_Nuc/NFAT_Cyt ratios are normalized to control. *P < 0.05, significantly different from control.

Fig. 4.

NFATc3-GFP is not activated by neurohumoral agonists in rabbit myocytes. Incubation with the agonists ET-1 (100 nM), ANG II (2 μM), Phe (10 μM), or with IP₃-AM (10 μM) overnight did not activate NFATc3 in rabbit atrial (A) or ventricular (B) myocytes. Full inhibition of nuclear export with LB (40 nM) did result in nuclear accumulation of NFATc3. Numbers in parentheses indicate the number of individual cells tested. NFAT_Nuc/NFAT_Cyt ratios are normalized to control. *P < 0.05, significantly different from control.