BACKGROUND: Biological pacemakers based on the HCN2 channel isoform respond to beta-adrenergic and muscarinic stimulation, suggesting a capacity to respond to autonomic input. OBJECTIVE: The purpose of this study was to investigate autonomic response to emotional arousal in canines implanted with murine HCN2-based biological pacemakers using gene therapy. METHODS: An electronic pacemaker was implanted with its lead in the right ventricular apical endocardium (VVI 35 bpm). An adenoviral HCN2/GFP construct (Ad-HCN2, n = 7) or saline (control, n = 5) was injected into the left bundle branch on day 2 after radiofrequency ablation of the atrioventricular node to induce complete atrioventricular block. Emotional arousal was achieved by presenting food following an overnight fast. Autonomic control was evaluated with Poincare plots of R-R(N) against R-R(N+1) intervals to characterize heart rate variability (HRV) and with continuous RR interval assessment via 24-hour ambulatory ECG. The 24-hour ECG and Poincare plot shape were analyzed. RESULTS: During day 1 after biological pacemaker implantation, Poincare HRV parameters and RR intervals were unchanged with food presentation. However, on day 7, food presentation was accompanied by an increase in HRV (SD1, p < 0.07, and SD2, p < 0.05) and shortening of RR interval (P < .05) in dogs with Ad-HCN2 but not in controls. CONCLUSION: This is the first demonstration that biological pacemakers are capable of responding to natural arousal stimuli to elicit appropriate chronotropic responses, a potential advantage over electronic pacemakers.