Abstract 236: Biphasic Reprogramming of Ventricular Cardiomyocytes to Pacemaker Cells by Tbx18

Abstract

Background: We have previously demonstrated that an embryonic transcription factor, TBX18, suffices to reprogram postnatal ventricular cardiomyocytes to induced pacemaker cells. Here, we sought to gain finer understanding of the reprogramming process by investigating genotype/phenotype of the de novo pacemaker cells in multiple temporal domains. Methods: TBX18-induced pacemaker cells (TBX18-iPMs) were created in vitro by somatic gene transfer of Adeno-TBX18 into neonatal rat ventricular myocytes (NRVMs). Control ventricular myocytes were transduced with Adeno- GFP. To investigate in vivo reprogramming, we delivered AAV9-TBX18 via tail vein of Hcn4(+/eGFP) heterozygous transgenic mice, which expresses eGFP under the Hcn4 promoter, so as to lineage-trace de novo pacemaker cells in the ventricular myocardium. Results: The automaticity of TBX18-iPMs was higher than that of GFP-NRVMs during D1-D3, decreased after D3 and rebound by D14. Monolayers of the iPMs revealed multiple foci of spontaneous field potentials with lower spike amplitude, spike slope, and conduction velocity, demonstrating phenotypic reprogramming to the iPMs by D14. RNAseq and qPCR revealed two distinct phases of reprogramming; an early phase that showed loss of expression of ventricular myocyte-related gene program and a late phase that showed gain of expression of nodal gene program which plateaued by D14. For example, Hcn4 gene expression began increasing during week 1 and continued the pattern during week 2. By week 3, Hcn4-positive TBX18-iPMs appeared in clusters of NRVM monolayers while GFP-NRVMs were spread evenly throughout the monolayers. We lineage-traced de novo, TBX18-reprogrammed pacemaker cells in vivo by delivering AAV9-TBX18 via tail-vein of Hcn4(+/eGFP) mice. In line with our in vitro data, in vivo pacemaker cell lineage tracing experiments indicated a progressive loss of ventricular gene program followed by gain of nodal gene program. Conclusions: Our data indicate that TBX18-induced reprogramming of ventricular myocytes to nodal pacemaker cells undergoes progressive changes in gene expression shedding ventricular gene/phenotype first, and then gaining nodal pacemaker genotype/phenotype, establishing fully functional nodal pacemaker by D14.