In order to investigate the physiological role of HCN4, a major subunit of pacemaker current of sinoatrial node (SAN), we generated two lines of transgenic mice; HCN4+/ tetA_TRE overexpressing HCN4 at their physiological locus (tTA) ; HCN4Luc/tetA_TRE, a double knock-in mouse that enables complete knockdown of HCN4 expression with doxycycline (TET-off). In vivo, TET-off mouse showed significant bradycardia with intermittent sinus pause or irregular beat, whereas the heart rate of tTA was similar to that of wild type (WT). When we stimulated parasympathetic nerve, WT and tTA equally showed bradycardia. In contrast, complete sinus pause was induced in TET-off. We next recorded action potential of SAN, and found unstable spontaneous firing in TET-off. Application of acetylcholine completely stopped spontaneous firing, and set the membrane potential at stable level. In tTA, this level was more depolarized. These findings indicate that HCN4 may act as a limiter for parasympathetic hyperpolarization of SAN.
