Abstract 14678: Transcription Factor-Driven Conversion of Quiescent Cardiomyocytes to Pacemaker Cells
The heartbeat originates within the sinoatrial node (SAN), a small highly-specialized structure containing <10,000 genuine pacemaker cells. The ∼5 billion working cardiomyocytes downstream of the SAN remain quiescent when it fails, leading to circulatory collapse and fueling a $6B/year electronic pacemaker industry. To engineer faithful biological replicas of rare SAN cells as an alternative therapeutic strategy, we expressed TBX18, a gene critical for early SAN specification, in working cardiomyocytes in vitro, and in vivo in a model of bradycardia. Within days of transduction with TBX18, ventricular cardiomyocytes in culture (TBX18-NRVMs) developed spontaneous electrical firing physiologically indistinguishable from that of SAN cells. TBX18-NRVMs displayed a depolarized maximum diastolic potential (-47±10mV) relative to the resting membrane potential of GFP-NRVMs (-73±6 mV, p<0.05), with accompanying reduction in IK1. The expression level of the pacemaker-specific ion channel, HCN4, was increased in the TBX18-NRVMs compared to control. TBX18-NRCMs exhibited localized Ca2+ release events (LCRs) which preceded each whole-cell Ca2+ transient, replicating the LCRs observed in native SAN cells. Ryanodine suppressed the rate of spontaneous Ca2+ transients more strongly in Tbx18-NRVMs (47±6%) than in control (12±2%, p<0.05), indicative of greater dependence on calcium clock mechanisms in TBX18-NRVMs. Focal TBX18 gene transfer in the guinea-pig ventricle yielded ectopic pacemaker activity in vivo, correcting a bradycardic disease phenotype. Myocytes transduced in vivo (TBX18-VMs) acquired the cardinal tapering morphology, small cell volume with disorganized myofibrils and physiological automaticity of native SAN pacemaker cells, while controls remained rectangular and quiescent. We examined freshly-isolated TBX18-VMs 6-8 weeks after the in vivo gene transfer. Single cell RT-qPCR upon patch-clamp revealed that five out of six such cells maintained SAN-like automaticity even after TBX18 expression has waned, indicating faithful and long-term reprogramming. The creation of induced SAN-like pacemaker (iSAN) cells by Tbx18 gene transfer opens new prospects for bioengineered pacemakers.
- © 2012 by American Heart Association, Inc.