Abstract Body (Do not enter title and authors here): Introduction
The sinoatrial node (SAN) is a compact structure with rich extracellular matrix (ECM) with a high content of fibrillar collagen. We have previously demonstrated reprogramming of chamber cardiomyocytes to induced pacemaker cells (iPMs) by TBX18 gene transfer. We sought to develop an in vitro tissue model of the SAN by enhancing ECM architecture of iPMs.
Methods
Monolayers of neonatal rat ventricular myocytes (NRVMs) were transduced with an adenoviral vector expressing either GFP or TBX18. The monolayers were cultured with/without L-ascorbic acid (AA, 375 uM), a co-factor for lysyl hydroxylase which is essential for collagen biosynthesis.

Results
Immunostaining indicated that collagen 1 (Col1) expression was confined to intracellular domain with little evidence for fibrillar collagen in both GFP and TBX18-NRVMs. AA treatment led to significant fibrillogenesis of Col1 ECM in both TBX18 and GFP-NRVMs with longer and more prominent collagen fibers in TBX18-NRVMs by d5. Within one week of gene transfer and AA treatment, TBX18-NRVMs spontaneously formed 3D tissue-like structures. Western blot confirmed that Col1 protein was more abundant in TBX18+AA than in GFP+AA by 101.4% (p<0.05, n=3), but was comparable between AA-treated and not treated TBX18-NRVMs, indicating SAN-like tissue formation was facilitated by collagen fibrillar organization rather than production. Transmission electron microscopy and immunostaining against alpha sarcomeric actinin indicated that AA-treated TBX18-NRVMs developed a multilayer, 3D tissue structure that was thicker compared to untreated TBX18-NRVMs or treated GFP-NRVMs. Caveolae are submicron sarcolemmal invaginations known to be densely populated in the native SAN ultrastructure. Caveolae density was significantly higher in AA-treated TBX18-NRVMs (9.0 ± 2.2 caveolae/μm) than nontreated TBX18 or GFP controls (5.5±1.9 or 3.8±1.0, p<0.001, n=11/group). Protein expression of Hcn4 ion channels remained comparable between AA treated and untreated TBX18-NRVMs (p<0.05, n=3). Automaticity of AA-treated, 3D tissue-like TBX18-NRVMs (38.4± 9.1 bpm, n=12) was faster than that of untreated TBX18 or GFP control (19.6±31.0 or 12.8±35.2 bpm, n=12, p<0.001), indicating functionally superior model of the SAN.

Conclusion
Facilitating fibrillar ECM organization enables self-organized, 3D tissue-like pacemaker structure in TBX18-reprogrammed iPMs. Our data provide a new platform for building engineered SAN models.