Lineage Reprogramming from Cardiomyocytes to Pacemaker Cells via a Single Transcription Factor

Abstract

The heartbeat originates in a small number of highly-specialized pacemaker cells found in the sinoatrial node (SAN). In an effort to create pacemaker cells from working cardiomyocytes, we expressed Tbx18, a transcription factor indispensible for embryonic development of the SAN. Within days, Tbx18-transduced neonatal rat cardiomyocytes (NRVMs) exhibited rhythmic spontaneous electrical firing. The mechanism of induced pacemaker activity resembled that of SAN cells, with enhanced voltage- and Ca2+-dependent “clock” pathways. The maximum diastolic potential was more positive in Tbx18-NRVMs in comparison to controls (−47±10 mV vs. −73±6 mV) with accompanying reduction in IK1. Line-scan confocal imaging of Tbx18-NRVMs loaded with Rhod-2 resolved localized Ca2+ release events (LCRs) that preceded each whole-cell Ca2+ transient resembling LCRs observed in native SAN cells. LCRs were not detected in control NRVMs. Spontaneous Ca2+-transients in Tbx18-NRVMs were suppressed on superfusion with ryanodine (10 μM) by 47±6% in comparison to 12±2% suppression in controls, indicative of increased relevance of [Ca2+]i stores. A 2.3-fold increase in [Ca2+]i stores was observed in Tbx18-NRVMs which can be attributed to increased sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) activity due to downregulation or phosphorylation of phospholamban (PLN), an inhibitor of SERCA. Tbx18-overexpression led to relative increase in the protein level of phosphorylated PLN (Ser16) similar to higher p-PLN level observed in the adult SAN relative to the left ventricle. In addition, Tbx18-NRVMs exhibited increased levels of cAMP and HCN4, shrank in size with disorganized myofibrils, and underwent epigenetic modifications consistent with durable reprogramming. Focal Tbx18 gene transfer in the guinea-pig ventricle consistently created ectopic pacemaker activity in vivo with a rhythm of 40±20 beats/min (n=5/7). Taken together, the data demonstrate faithful reprogramming of ventricular myocytes to pacemaker cells by a single transcription factor, Tbx18.