Abstract Body: Introduction: Somatic reprogramming of chamber cardiomyocytes by TBX18 has been shown to create induced pacemaker myocytes (iPMs). One of the defining features of the native sinoatrial node (SAN) is its collagen-rich extracellular matrix (ECM), which facilitates the node’s compact structure. However, the role of ECM organization in TBX18-iPMs has not been explored.
Hypothesis: We hypothesized that promoting an SAN-like ECM microenvironment enhances entrainment of TBX18-iPMs toward tissue-level rhythm generation.
Methods: Neonatal rat ventricular myocytes (NRVMs) were transduced with TBX18 or GFP (control) and cultured with/without ascorbic acid (AA) to promote collagen biosynthesis. In vivo validation was performed using a rat complete atrioventricular block (CAVB) model.
Results: AA treatment led to enhanced collagen fibrillar ECM in AA-treated iPMs (TBX18_AA) compared to untreated iPMs (TBX18_nt). Electron microscopy revealed that caveolae structures, ion channel-rich submicron membrane invaginations that are prominent in the native SAN myocytes, were more abundant in TBX18_AA than TBX18_nt monolayers (9.0±2.2 vs 5.5±1.9 caveolae/µm, P≤0.05, n≥12). The higher caveolae density was accompanied by upregulation of SAN-enriched ion channels (Hcn4, Cacna1d) and suppressed Gja1 expression which maintained SAN-like slow Ca²⁺ propagation in the iPMs compared to GFP_AA or GFP_nt (2.6±1.9 and 4.2±0.9 cm/s for TBX18_nt and TBX18_AA vs. 24.4±1.0 and 17.2±4.6 cm/s for GFP_nt and GFP_AA, n=3). ScRNA-seq identified upregulated G-protein coupled receptor-mediated cAMP signaling in TBX18_AA compared to TBX18_nt, which could enhance synchronization and entrainment of the iPMs. Multielectrode array recordings indicated TBX18_AA monolayers achieved syncytial, synchronous pacing (n=6/6 wells) while most TBX18_nt monolayers showed asynchronous activity (n=4/6 wells). Furthermore, the duration of continuous pacing without a pause was >30-fold longer in TBX18_AA than in TBX18_nt monolayers (18.0±16.4 vs 0.5±0.5 min, P≤0.05, n=6), demonstrating significantly enhanced pacemaker entrainment. Focal myocardial gene transfer of TBX18 in the left ventricular apex of CAVB rats with daily tail vein injection of AA enhanced de novo ventricular pacing compared to TBX18 animals injected with saline (HR≥200 bpm; 20.2±3.9 % vs 8.5±5.6 %, P≤0.05, n≥3).
Conclusions: A self-organized, SAN-like collagen ECM significantly improves entrainment of TBX18-iPMs in vitro and cardiac pacing in vivo.