Abstract: It is demonstrated that hyperpolarization-activated cyclic nucleotide-gated (HCN)-based biological pacing, especially that achieved by transduction of the HCN2 gene into cardiac cells in vivo, was significantly improved by co-transduction of the skeletal muscle sodium channel 1 (SkMI) gene. Expression of both genes hyperpolarized the action potential (AP) threshold. When viral biological pacemaker constructs carrying genes for HCN2 and SkMI were injected into the heart of dogs in vivo, the pacemaker function was facilitated by the slow depolarizing HCN2 current and the hyperpolarized AP threshold generated by SkMI. This dual gene therapy provided both highly efficient pacing and a brisk autonomic response that is superior to those of previously developed gene- or cell-based approaches.