Hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) are expressed in the ventricle of fetal hearts, and are silenced during the development. These channels are up-regulated in the hypertrophied heart, and have been suggested to underlie arrhythmogenesis. To test this hypothesis, we generated a transgenic mice overexpressing HCN2 in the heart (HCN2-Tg) using alpha-MHC promoter, and analyzed the electrophysiological properties of ventricular myocytes. Mice (8-12 weeks old) were deeply anesthetized with 3% sevoflurane. Ventricular myocytes were isolated with collagenase digestion. Action potential and membrane currents were recorded with ruptured whole-cell patch clamp method using standard high K+ pipette solution. Values are means ± S.D. Statistical analysis was carried out using Student`s T-test. We first confirmed that amounts of mRNA and protein of HCN2 were significantly higher in HCN2-Tg than wild type (WT). However, contrary to our expectation, HCN2-Tg was not vulnerable to arrhythmia. In physiological bathing solution, the resting potential (RMP) of ventricular myocyte was not significantly different between WT (-81.5±1.0 mV) and HCN2-Tg (-81.6±1.6 mV, n=8, p=0.42). No spontaneous action potential (SAP) was recorded in HCN2-Tg myocytes, although robust HCN current (Ih) was recorded in the presence of 1 mM Ba2+. However, application of 0.3 μM isoproterenol (iso) to HCN2-Tg myocytes depolarized their RMP and subsequently induced SAP in 71%, coincident with the shifting of voltage-dependent activation curve of Ih; the voltage for the half maximal activation was -118.5±2.9 mV (control), and -90.2±1.5 mV (iso), respectively (n=4, p <0.01). In HCN2-Tg myocytes without SAP, RMP was significantly more depolarized (-77.2±2.1 mV) than that of WT (-82.1±1.0 mV, n=8, P<0.01). 3 μM ivabradine (iva), a specific HCN channel blocker, reversed iso-induced depolarization to the control level. We further compared the AP waveform elicited by electrical stimulation in WT and HCN2-Tg myocytes without SAP. In HCN2-Tg, action potential duration (APD) at -60 mV was significantly prolonged, and the rates of repolarization at -60 mV were significantly slower, both in the presence and absence of 0.3 μM iso. On the other hand, APD at 0 mV was significantly shorter in HCN2-Tg, most probably due to the outward tail current of Ih. When the myocytes were perfused with 3 mM K+ bathing solution, RMP was -95.6±1.5 mV in WT, and -91.2±3.3 mV in HCN2-Tg (n=6, p<0.01). Under this condition, SAP was induced in 57% of HCN2-Tg myocytes. Application of 3 μM iva hyperpolarized RMP in HCN2-Tg. These findings suggested overexpression of HCN2 potentially increased the vulnerability to arrhythmia, particularly in pathological conditions such as excessive β adrenergic stimulation or hypokalemia.