The T-box factor Tbx3 is important in the development of the sinoatrial node (SAN) by repressing the development from embryonic (nodal) primary myocardium to working myocardium.¹ When Tbx3 is artificially over-expressed in mouse atria from prenatal stages onwards, ectopic pacemaker regions are formed in the adult atria in vivo.² In this study, we characterized: 1) ECG parameters of such Tbx3 over-expressing mice, 2) whether Tbx3 over-expressing induced ectopic activity can be modulated by the chronotropic agents acetylcholine (ACh), and noradrenalin (NA), and 3) electrophysiological properties of single cells. 6-lead ECGs were recorded in isoflurane (~1.0%) anesthetized control mice and atrial Tbx3 over-expressing mice. Tbx3 over-expression atrial preparations devoid of SAN tissue and SAN preparations (with attached atria) of control mice were exposed to increasing NA (0.1-50 µM) or ACh (0.03-50 µM) concentrations. Sharp microelectrode impalements were used to analyse beating frequency. Finally, single cells were isolated by enzymatic dissociation.³ Basic electrophysiological properties of Tbx3 over-expressing atrial and control SAN cells were assessed using perforated patch-clamp technique. Tbx3 over-expressing mice revealed severe morphological changes in ECG characteristics, including short PQ intervals, double deflections in the P-wave and partially or complete atrioventricular-block, coinciding with shortened interbeat intervals. However, the average interbeat interval was comparable to those of control mice. Tbx3 over-expressing right atria were visibly hypertrophied. Intrinsic firing frequency was not significantly lower in Tbx3 over-expressing atria (4.1±0.6 (average±SEM, n=16) vs 5.2±0.4 Hz (control SAN; n=13)). ACh slowed, and NA accelerated spontaneous activity in a dose-dependent fashion in both Tbx3 over-expressing and control mice. Dose-frequency relationships of both ACh and NA in Tbx3 over-expressing and control mice were identical. 50% of the Tbx3 over-expressing atrial single cells showed spontaneous activity. Compared to control SAN cells (n=7), intrinsic firing frequency (5.2±0.9 (Tbx3) vs 6.5±0.7 Hz (control SAN)), and all other action potential parameters of Tbx3 over-expressing atrial cells (n=7) were not significantly different. The spontaneously active Tbx3 over-expressing atrial cells showed a hyperpolarization activated current, I_f, whose density was similar to that in control SAN cells. In the silent Tbx3 over-expressing atrial cells, I_f was virtually absent. Embryonic Tbx3 over-expressing atria have electrophysiological features of SAN cells.