AbstractPacemaking dysfunction (PD) may result in heart rhythm disorders, syncope or even death. Current treatment of PD using implanted electronic pacemaker has some limitations, such as finite battery life and the risk of repeated surgery. As such, the biological pacemaker has been proposed as a potential alternative to the electronic pacemaker for PD treatment. Experimentally it has been shown that bio-engineered pacemaker cells can be generated from non-rhythmic ventricular myocytes (VMs) by knocking down genes related to the inward rectifier potassium channel current (IK1) or by overexpressing hyperpolarization-activated cyclic nucleotide gated channel genes responsible for the “funny” current (If). Such approaches can turn the VM cells into rhythmic pacemaker cells. However, it is unclear if a bio-engineered pacemaker based on the modification of IK1- and If-related channels simultaneously would enhance the ability and stability of bio-engineered pacemaking action potentials (APs). This study aimed to investigate by a computational approach the combined effects of modifying IK1and Ifdensity on the initiation of pacemaking activity in human ventricular cell models. First, the possible mechanism(s) responsible for VMs to generate spontaneous pacemaking APs by changing the density of IK1and Ifwere investigated. Then the integral action of targeting both IK1and Ifsimultaneously on the pacemaking APs was analysed. Our results showed a reciprocal interaction between IK1and Ifon generating stable and robust pacemaking APs in VMs. In addition, we thoroughly investigated the dynamical behaviours of automatic rhythms in VMs in the IK1and Ifparameter space, providing optimal parameter ranges for a robust pacemaker cell. In conclusion, to the best of our knowledge, this study provides a novel theoretical basis for generating stable and robust pacemaker cells from non-pacemaking VMs, which may be helpful in designing engineered biological pacemakers for application purposes.Author SummaryPacemaking dysfunction has become one of the most serious cardiac diseases, which may result in arrhythmia and even death. The treatment of pacemaking dysfunction by electronic pacemaker has saved millions of people in the past fifty years. But not every patient can benefit from it because of possible limitations, such as surgical implication and lack of response to autonomic stimulus. The development of bio-pacemaker based on gene engineering technology provides a promising alternative to electronic pacemaker by manipulating the gene expression of cardiac cells. However, it is still unclear how a stable and robust bio-pacemaker can be generated. The present study aims to elucidate possible mechanisms responsible for a bio-engineered pacemaker by using a computational electrophysiological model of pacemaking cells based on modifying ion channel properties of IK1and incorporating Ifin a human ventricular cell model, mimicking experimental approaches of gene engineering. Using the model, possible pacemaking mechanisms in non-pacemaking cells, as well as factors responsible for generating robust and stable biological pacemaker, were investigated. It was shown that the reciprocal interaction between reduction of IK1and incorporation of Ifplayed an important role for producing robust and stable pacemaking. This study provides a novel insight into understanding of the initiation of pacemaking behaviours in non-rhythmic cardiac myocytes, providing a theoretical basis for experimental designing of biological pacemakers.