Direct inhibition of cardiac hyperpolarization-activated cyclic nucleotide-gated pacemaker channels by clonidine

Background - Inhibition of cardiac sympathetic tone represents an important strategy for treatment of cardiovascular disease, including arrhythmia, coronary heart disease, and chronic heart failure. Activation of presynaptic alpha(2)-adrenoceptors is the most widely accepted mechanism of action of the antisympathetic drug clonidine; however, other target proteins have been postulated to contribute to the in vivo actions of clonidine. Methods and Results - To test whether clonidine elicits pharmacological effects independent of alpha(2)-adrenoceptors, we have generated mice with a targeted deletion of all 3 alpha(2)-adrenoceptor subtypes (alpha(2ABC) (-/-)). alpha(2ABC) (-/-) mice were completely unresponsive to the analgesic and hypnotic effects of clonidine; however, clonidine significantly lowered heart rate in alpha(2ABC) (-/-) mice by up to 150 bpm. Clonidine-induced bradycardia in conscious alpha(2ABC) (-/-) mice was 32.3% ( 10 mu g/ kg) and 26.6% ( 100 mu g/ kg) of the effect in wild-type mice. A similar bradycardic effect of clonidine was observed in isolated spontaneously beating right atria from alpha(2ABC)- knockout and wild-type mice. Clonidine inhibited the native pacemaker current ( I-f) in isolated sinoatrial node pacemaker cells and the I-f- generating hyperpolarization-activated cyclic nucleotide - gated ( HCN) 2 and HCN4 channels in transfected HEK293 cells. As a consequence of blocking If, clonidine reduced the slope of the diastolic depolarization and the frequency of pacemaker potentials in sinoatrial node cells from wild-type and alpha(2ABC)-knockout mice. Conclusions - Direct inhibition of cardiac HCN pacemaker channels contributes to the bradycardic effects of clonidine gene-targeted mice in vivo, and thus, clonidine- like drugs represent novel structures for future HCN channel inhibitors.