Abstract 18724: Stable and Synchronous Pacing Generated From the Tbx18-Induced Pacemaker Cells by Epithelial-to-Mesenchymal Transition Inhibition
The pacemaker cells of the sinoatrial node (SAN) exhibit highly variable individual beating rates, and yet pace the heart in synchrony. We have previously demonstrated conversion of ventricular cardiomyocytes to induced pacemaker cells (iPCs) by Tbx18. However, the iPCs ceased to pace the heart in a short while. In the embryonic heart, Tbx18 not only directs the SAN development, but also mobilizes epithelial-to-mesenchymal transition (EMT) in epicardial progenitors. We hypothesized that, during the conversion to iPCs, Tbx18 initiates both automaticity and EMT, and the EMT program runs counter to iPCs’ pacing function.
The iPCs were created by adenoviral gene transfer of human TBX18 to neonatal rat ventricular myocytes, while control myocytes were transduced with GFP alone (GFP-NRVMs). Cells that undergo EMT are known to migrate. Within 48hrs post-TBX18 gene transfer, significantly more iPCs exhibited a migratory behavior compared to GFP-NRVMs (216±12 vs. 28±4, p<0.05). A small molecule inhibitor of EMT, A83 (0.5μM), stopped the migration of iPCs to the level of GFP-NRVMs (30±7 cells in iPCs+A83). Normalized cell adherence between the iPCs and culture substrate decreased dramatically from day-1 to 7 (4.5±0.2 vs. 0. 8±0.1, p<0.05). EMT inhibition maintained iPC adherence from day-1 to 14 (4.7±0.3 vs. 5.0±0.7). Molecular drivers of EMT, Twist1, Rgs2 and Tgfβr1, were expressed higher in the iPCs compared to GFP-NRVMs (5.4-, 4.6-, and 3.3-fold, respectively, p<0.05). Measurements of electrical activity revealed that iPCs beat spontaneously, but fail to pace synchronously in <1-week. In contrast, iPCs+A83 demonstrated durable and synchronous pacing (139±12 bpm, n=3) for at least 2 weeks. Real-time Ca2+ imaging confirmed synchronous Ca2+ cycling in iPCs+A83, but not in iPCs, which correlated with increased Hcn4 expression at the transcript and protein levels (2.8- and 1.6-fold, respectively, p<0.05) in iPCs+A83 compared to iPCs. EMT inhibition partly restored gap-junctional coupling by translocating Cx43 from perinuclear to the sarcolemma in iPCs+A83.
Taken together, Tbx18-induced pacemaker cells generate durable and synchronous pacing upon inhibiting EMT. Our findings may give rise to biological pacemakers that are capable of long-term pacing.
Author Disclosures: J. Li: None. S. Gonzalez: None. J. Lehman: None. H. Cho: None.
- © 2016 by American Heart Association, Inc.