Reprogramming Cardiac Progenitor Cells into Pacemaker Cells for Heart Repair

Sick sinus syndrome (SSS) is associated with the dysfunction of the sino atrial node (SAN), which is also the primary pacemaker of the heart. Dysfunction of the pacemaker prevents normal electrical conductivity, contributing to the recurrent development of the arrhythmias. Currently, electronic pacemaker implantation is the standard therapy for SSS and other cardiac rhythm disturbances. However, despite their clinical efficacy, they exhibit complications of infection, limited battery life, and lead fracturing. Hence, a therapy directed towards resorting normal electrical conductivity is of utmost clinical significance. This necessitates the need to generate biological pacemakers, which can integrate with the existing myocardium in a syncytium to enable synchronous beating of the heart without arrhythmias. In support of this concept, our objective was to use a unique cocktail of lentivirus- mediated transcription factors, that are critical to the embryonic development of the SAN for reprogramming cardiac progenitor cells (CPCs) into conducting Pacemaker-like cells. The CPCs treated with transcription factor combinations were initially screened for Pacemaker-like cells by FACS sorting for a reporter gene specific for pacemaker cells. Following which we assayed these cells for pacemaker marker genes. The results demonstrated a robust induction of Pacemaker marker genes in the SHOX2-HCN2-TBX5 (SHT5) activated CPCs by qPCR gene expression assays. Interestingly, the patch clamp recording of the Pacemaker-like cells exhibited funny currents (If) with a current density (pA/pF) of -5.03±1.36 (n=17). Further, we observed that the SHT5 cells (n=14) showed V1/2 of half of the maximal current (I/Imax at 0.5) as 125.28±3.83 mV, demonstrating the functional characteristic of pacemaker cells. Additionally, to expand our understanding of the transcriptome of SHT5 cells, we performed RNA sequencing and single cell sequencing of SHT5 mCherry+ cells. We observed that the transcriptome of SHT5 cells exhibited enrichment of pacemaker specific genes. In conclusion, SHOX2, HCN2 and TBX5 cocktail of transcription factors reprogrammed the CPCs into pacemaker-like cells. The SHT5 factors resulted in upregulation of pacemaker specific gene expression and the transcriptome, attributing pacemaker phenotype to the cells. These studies will facilitate the development of an optimal Pacemaker-like cell-based therapy within failing hearts through the recovery of lost contractile and electrical function between cardiomyocytes.