Abstract 16575: Synthetic TBX18 mRNA Induces Durable Reprogramming of Cardiac Myocytes to Pacemaker Cells

Abstract

Introduction: Adenoviral gene delivery of transcription factor, TBX18, has been proven to reprogram ordinary cardiomyocytes to pacemaker cells, and provide ventricular pacing in the rodent and porcine heart. Yet host innate immune response to recombinant viral therapies remains a hurdle to overcome for successful translation. Somatic cell reprogramming does not require persistent expression of the reprogramming factors after arriving at the final cell fate. We hypothesized that brief expression of synthetic TBX18 mRNA suffices to convert chamber cardiomyocytes to induced pacemaker cells, thus obviating issues inherent to adenoviral gene therapy. Methods: Synthetic mRNAs were in vitro transcribed as previously described. Neonatal rat ventricular myocytes (NRVMs) were used as an in vitro platform to test pacemaker reprogramming by TBX18 mRNA. GFP or fLuc encoded mRNA was used as controls in all experiments. Results: Three days after synthetic TBX18 mRNA transfection, Hcn4 transcript level increased >6-fold compared to control. After two weeks, genes enriched in the pacemaker tissue, e.g., Shox2, Hcn4, and Tbx3, continued to be expressed higher in TBX18-NRVMs compared to control by >4.2-fold. The level of endogenously expressed, rat Tbx18 increased by 50% in NRVMs transfected with synthetic human TBX18 mRNA, which point to long-term reprogramming. To identify and lineage-trace reprogrammed pacemaker cells in real time, we cultured ventricular cardiomyoytes from transgenic mouse line Hcn4 ( GFP/+ ) , in which de novo pacemaker cells expressing Hcn4 contain eGFP fluorescence. Twelve days after synthetic mRNA or adenoviral gene transfer of TBX18 or GFP control, a significantly higher number of Hcn4 ( GFP/+ ) cells were observed in both mRNA and adenoviral TBX18-treated wells compared to their respective controls. TBX18 mRNA-transfected NRVM monolayers exhibited >2-fold slower conduction velocities compared to fLuc-transfected myocytes. In line with slow conduction velocity in the induced pacemaker myocytes, expression of ventricular gap junction protein, Cx43, decreased significantly compared to control. Conclusions: Together, the data indicate durable nodal pacemaker cell reprogramming with transient expression of synthetic TBX18 mRNA.