Mechanisms Underlying Rate-Dependent Remodeling of Transient Outward Potassium Current in Canine Ventricular Myocytes

Abstract

Transient outward K + current ( I to ) downregulation following sustained tachycardia in vivo is usually attributed to tachycardiomyopathy. This study assessed potential direct rate regulation of cardiac I to and underlying mechanisms. Cultured adult canine left ventricular cardiomyocytes (37°C) were paced continuously at 1 or 3 Hz for 24 hours. I to was recorded with whole-cell patch clamp. The 3-Hz pacing reduced I to by 44% ( P <0.01). Kv4.3 mRNA and protein expression were significantly reduced (by ≈30% and ≈40%, respectively) in 3-Hz paced cells relative to 1-Hz cells, but KChIP2 expression was unchanged. Prevention of Ca 2+ loading with nimodipine or calmodulin inhibition with W-7, A-7, or W-13 eliminated 3-Hz pacing-induced I to downregulation, whereas downregulation was preserved in the presence of valsartan. Inhibition of Ca 2+ /calmodulin-dependent protein kinase (CaMK)II with KN93, or calcineurin with cyclosporin A, also prevented I to downregulation. CaMKII-mediated phospholamban phosphorylation at threonine 17 was increased in 3-Hz paced cells, compatible with enhanced CaMKII activity, with functional significance suggested by acceleration of the Ca 2+ i transient decay time constant (Indo 1-acetoxymethyl ester microfluorescence). Total phospholamban expression was unchanged, as was expression of Na + /Ca 2+ exchange and sarcoplasmic reticulum Ca 2+ -ATPase proteins. Nuclear localization of the calcineurin-regulated nuclear factor of activated T cells (NFAT)c3 was increased in 3-Hz paced cells compared to 1-Hz (immunohistochemistry, immunoblot). INCA-6 inhibition of NFAT prevented I to reduction in 3-Hz paced cells. Calcineurin activity increased after 6 hours of 3-Hz pacing. CaMKII inhibition prevented calcineurin activation and NFATc3 nuclear translocation with 3-Hz pacing. We conclude that tachycardia downregulates I to expression, with the Ca 2+ /calmodulin-dependent CaMKII and calcineurin/NFAT systems playing key Ca 2+ -sensing and signal-transducing roles in rate-dependent I to control.