Background Extensive research toward creating a biological pacemaker by enhancement of inward depolarizing current has been performed. However, studies have mainly focused on inducing spontaneous activity and have not adequately addressed ways to improve pacemaker function. In this study we attempted to improve pacemaker function by altering connexin expression in rat mesenchymal stem cells (MSCs) to a phenotype similar to native sinus node pacemaker cells.Methods To generate a biological pacemaker, MSCs were transduced with a cardiac pacemaker genehyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4), via transfection with a lentiviral vector. Funny current (If) in HCN4+ MSCs was recorded by voltage-clamp. Overexpression of connexin 45 (gene Gja7) in MSCs was achieved by transfection with the plasmid pDsRED2-N1-Gja7-RFP. Double-immunolabelling with anti-connexin 43 and anti-connexin 45 antibodies were used to identify the gap junction channels. The effects of the genetically modified MSCs on cardiomyocyte excitability were determined in MSCs cocultured with neonatal rat ventricular myocytes. Spontaneous action potentials of neonatal rat ventricular myocytes were recorded by current-clamp.Results High level time- and voltage-dependent inward hyperpoiarization current that was sensitive to 4 mmol/L Cs+ was detected in HCN4+ MSCs, confirming that HCN4 acted as If channels in MSCs. Connexin 43 and connexin 45 were simultaneously detected in CX45+ MSCs. Beating frequency was (82±8) beats per minute (n=5) in myocytes cocultured with nontransfected control MSCs, versus (129±11) beats per minute (n=5) in myocytes cocultured with HCN4+ MSCs.Myocytes cocultured with MSCs cotransfected with HCN4 and connexin 45 had the highest beatin g frequency at (147±9)beats per minute (n=5).Conclusion These findings demonstrate that overexpression of connexin 45 and subsequent formation of heteromeric connexin 45/connexin 43 gap junction channels in HCN4 expressing MSCs can improve their function as cardiac biological pacemakers in vitro.