Abstract 421: Mechanism of Rate-dependent Remodeling of Transient Outward Potassium Current in Canine Ventricular Myocytes
Background: Transient outward K+ current (Ito) downregulation is seen with in vivo tachycardia models and is usually attributed to heart failure; however, we have shown that tachycardia perse-binding protein almodulin regulates downregulates Ito in cultured cardiomyocytes. The Ca2+ - binding protein calmodulin regulates a kinase (CaMKII) and a phosphatase (calcineurin, CLN) that mediate activity-dependent regulation in neural systems. This study assessed mechanisms underlying rate-regulation of cardiac Ito expression.
Methods: Isolated left ventricular cardiomyocytes were cultured for 24hrs at 37°C in 2 groups/dog: cells paced continuously at 1 Hz (1HP) or 3 Hz (3HP). Ito was recorded with whole cell patch clamp.
Results: 3HP cells showed an ~44% reduction in Ito (eg at +40 mV: 10.8±1.0 pA/pF, vs 19.4±2.1 pA/pF for 1HP cells, P<0.01). Kv4.3 protein expression was reduced by 42% at 3 Hz, from 0.060±.02 to 0.03±0.01 ODU (Western blot, normalized to GAPDH), but KChIP2 was unchanged (0.46±0.08, 0.42±0.09 ODU for 3HP, 1HP respectively). Incubation with the CaMKII inhibitor KN93 (10 μM) prevented Ito downregulation in 3HP cells (Ito at 40 mV 17.3±0.9 pA/pF vs 18.32.4 pA/pF in 1HP). Incubation with the inactive KN93 analogue, KN92, did not affect rate-dependent Ito downregulation (19.2±1.4, 12.1±0.8 pA/pF, P<0.01 in 1HP, 3HP cells respectively). CaMKII activity assessed by CaMKII-phospholamban phosphorylation (P-PLB-T17) was increased ~35% in 3HP cells compared to 1HP. Total phospholamban expression was unchanged. Consistent with increased P-PLB-T17 (which should increase SERCA activity), Ca2+ re-uptake was accelerated: the decay time of the Ca2+I transient (Indo 1 AM microfluorescence) was 369±20 ms in 3HP cells vs 458±24 ms in 1HP, P<0.05. Incubation with the CLN blocker cyclosporin-A (CyA, 1 μg/ml) also prevented Ito downregulation (+40 mV: 17.9±2.5 pA/pF in 3HP cells vs 19.3±1.5 pA/pF in 1HP, P=NS; 9.2±0.9 pA/pF in parallel 3HP cells without CyA, P<0.01). Nuclear/cytosolic staining ratios for CLN-regulated NFATc3 and NFATc4 were increased by 22% and 26% respectively in 3HP cells compared to 1HP.
Conclusion: The calmodulin-dependent CaMKII and calcineurin/NFAT systems play important roles in rate-dependent control of Ito expression andcardiac electrophysiological remodeling.