Tbx18 Orchestrates Cytostructural Transdifferentiation of Cardiomyocytes to Pacemaker Cells by Recruiting the Epithelial–Mesenchymal Transition Program

Previously, we reported that heterologous expression
of an embryonic
transcription factor, Tbx18, reprograms ventricular cardiomyocytes
into induced pacemaker cells (Tbx18-iPMs), though the key pathways
are unknown. Here, we have used a tandem mass tag proteomic approach
to characterize the impact of Tbx18 on neonatal rat ventricular myocytes.
Tbx18 expression triggered vast proteome remodeling. Tbx18-iPMs exhibited
increased expression of known pacemaker ion channels, including Hcn4
and Cx45 as well as upregulation of the mechanosensitive ion channels
Piezo1, Trpp2 (PKD2), and TrpM7. Metabolic pathways were broadly downregulated,
as were ion channels associated with ventricular excitation–contraction
coupling. Tbx18-iPMs also exhibited extensive intracellular cytoskeletal
and extracellular matrix remodeling, including 96 differentially expressed
proteins associated with the epithelial-to-mesenchymal transition
(EMT). RNAseq extended coverage of low abundance transcription factors,
revealing upregulation of EMT-inducing Snai1, Snai2, Twist1, Twist2,
and Zeb2. Finally, network diffusion mapping of >200 transcriptional
regulators indicates EMT and heart development factors occupy adjacent
network neighborhoods downstream of Tbx18 but upstream of metabolic
control factors. In conclusion, transdifferentiation of cardiac myocytes
into pacemaker cells entails massive electrogenic, metabolic, and
cytostructural remodeling. Structural changes exhibit hallmarks of
the EMT. The results aid ongoing efforts to maximize the yield and
phenotypic stability of engineered biological pacemakers.