Induced Pacemaker Cells Created by In Vivo Somatic Reprogramming: Phenotypic Comparison with Native Sinoatrial Node Cells

Abstract

Background: We report elsewhere in this meeting (N. Kapoor et al. abstract) that Tbx18, an embryonic T-box transcription factor, reprograms cultured cardiomyocytes to induced sinoatrial node (SAN) pacemaker cells. Here we sought to test the fidelity of reprogramming by expressing Tbx18 in adult ventricle and comparing the properties of Tbx18-transduced ventricular myocytes (Tbx-VMs) with those of GFP-transduced ventricular myocytes (as controls; control-VMs) and native SAN cells.Methods: Adenoviruses co-expressing Tbx18 and GFP, or GFP alone, were directly injected in the apex of the guinea pig heart (4×107cfu/heart). Five days later, the heart was harvested and cardiomyocytes were isolated from the site of virus injection.Results: Native SAN cells are smaller and leaner than nontransduced VMs: length-to-width (LtW) ratios equal 14.7±1.5 (n=28) and 7.6±0.7 (n=9), respectively. Freshly-isolated control-VMs maintained their native shape (LtW=7.4±0.9, n=4); in contrast, Tbx-VMs were leaner (LtW=16.0±1.0, n=12) than control-VMs and were often spindle-shaped, reproducing the morphological hallmark of SAN cells. Control-VMs had stable resting potentials at −76mV, with action potentials (APs) elicited only upon electrical stimulation. In contrast, spindle-shaped Tbx-VMs demonstrated diastolic depolarization (maximal diastolic potential=-59mV) and fired spontaneous APs at 26bpm.The whole-cell capacitance (a measure of cell size) of Tbx-VMs was smaller than that of control-VMs (40.8±3.6 vs. 119±16pF, respectively), but similar to that of native SAN cells (48.3±7.0pF). The electrophysiological and morphological features of Tbx-VMs generally resembled those of native SAN cells.Conclusion: Somatic gene transfer of Tbx18 in the ventricle in vivo yielded induced SAN (iSAN) cells which faithfully recapitulate the key phenotypic properties of genuine SAN cells. The in situ reprogramming was effective and swift (as little as five days), offering a novel approach to creating a biological pacemaker as an alternative to electronic devices.