Abstract 2815: Calcineurin/Nuclear Factor of Activated T-cells as a Final Common Signaling Pathway for Downregulation of Atrial Transient Outward Potassium Current
A variety of conditions downregulate transient outward K+ current (Ito) in atrium. Here we studied the mechanisms of atrial Ito downregulation in 3 systems:
atrial tachypacing (ATP, 400 bpm × 1 wk) dogs (n=12);
ventricular tachypacing (240 bpm × 2 wks) induced CHF (n=12 dogs); vs 20 CTL dogs;
24 hr in vitro tachypaced (3 Hz) atrial cardiomyocytes vs 1 Hz paced cells, from 34 dogs.
Methods: Ito was measured by patch clamp, proteins by immunoblot and immunofluorescence.
Results: CHF and ATP reduced Ito by ~50% versus CTL at +40 mV*** (***p<0.001) and reduced Kv4.3 protein expression by ~45% (p<0.01). Kv1.4 and KChIP2 protein were unchanged. Calcineurin activity increased from 0.87±0.07 nmol PO4 in control to 2.35±0.08*** in ATP and 2.56±0.18*** in CHF. Nuclear localization of NFAT c3/c4 was increased on both immunocytochemistry (Figure⇓) and selective nuclear/cytoplasmic immunoblot (nuclear/cyosolic ratio c3: 1.23±0.13 CTL, 3.50±0.14 ATP, 4.14±0.17 CHF; c4: 0.78±0.07 CTL, 1.51±0.06 ATP, 1.14±0.11 CHF). Ito was reduced by 53%*** in 3 Hz vs 1 Hz in-vitro paced cells, paralleling a ~50% decrease in Kv4.3 mRNA (qPCR) and protein. Ca2+-loading 1 Hz cells by incubating with 2×Ca2+o reduced Ito by 52%*** and suppression of Ca2+ loading (nimodipine or BAPTA-AM) prevented 3 Hz pacing effects. Calcineurin activity and nuclear localization of calcineurin-regulated nuclear factor of activated T cells (NFAT) c3/c4 were increased in 3 Hz cells. Inhibitors of calmodulin (W7), calcineurin (FK-506) and NFAT (INCA6) prevented Ito downregulation by 3 Hz pacing.
Conclusion: Ca2+ dependent calcineurin/NFAT signaling underlies atrial Ito downregulation for a wide range of experimental paradigms.