Mechanisms underlying rate-dependent remodeling of transient outward potassium current in canine ventricular myocytes

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 degrees 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 approximate to 30% and approximate to 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 Ito 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.