Abstract 14024: Tbx18-Reprogrammed Cardiomyocytes Exhibit Pacemaker Phenotype with Upregulated Calcium and Voltage-Sependent Clock Mechanisms
The heartbeat originates within the sinoatrial node (SAN), a diminutive, highly specialized structure containing a small number (<10,000) of genuine pacemaker cells. The remaining number of working cardiomyocytes in the atria and ventricles number in the billions in the typical mammalian heart. In an effort to create rare pacemaker cells, we transduced ordinary neonatal rat cardiomyocytes (NRCMs) with Tbx18, a transcription factor expressed in the nascent SAN during embryonic development. Within days, Tbx18-NRCMs 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 depolarized in Tbx18-NRCMs in comparison to GFP-transduced controls (-47±10 vs. -73±6 mV) with accompanying reductions in IK1 (p<0.05). Tbx18-NRCMs also expressed the pacemaker current If (-5.2±1.3 pA/pF at -140 mV, n=3). An index of automaticity (the % of autonomously-beating cells multiplied by the frequency of AP oscillations in those cells) was much greater in Tbx18-NRCMs compared to controls (70±16 vs. 13±4 % bpm, p<0.001). Line-scan confocal imaging of Tbx18-NRCMs loaded with Rhod-2 resolved localized Ca2+ release events (LCRs) that preceded each whole-cell Ca2+ transient, mimicking LCRs observed in native SAN cells. LCRs were not detected in control NRCMs. Ryanodine (10 μM) suppressed the spontaneous beating rate by 47±6% in Tbx18-NRCMs but only by 12±2% in controls (p<0.05). A 2.3-fold increase in caffeine-releasable [Ca2+]i stores was observed in Tbx18-NRCMs which can be attributed to increased SERCA2a activity due to downregulation and phosphorylation of phospholamban (PLN), an inhibitor of SERCA. Tbx18 expression led to a relative increase in the protein level of phosphorylated PLN (Ser16) similar to the relative p-PLN increase in adult SAN. In addition, Tbx18-NRCMs shrank in size, exhibited sparse myofibrils, ceased to express connexin 43 and atrial natriuretic peptide and exhibited increased levels of cAMP and HCN4, reproducing key features of SAN cells. The data indicate that expression of a single transcription factor suffices to create induced pacemaker cells from working cardiomyocytes.
- © 2011 by American Heart Association, Inc.