Fig 2: Argonaute 2-dependent suppression of TBX18 protein expression: miR-1-3p and miR-1b arrest TBX18 mRNA translation(A) Schematic representation of the RNA-induced silencing complex and Argonaute 2 (AGO2)-dependent TBX18 protein suppression. AGO2 binds to the cytosolic miRNA-processing machinery and complementary TBX18 mRNA at the 3' UTR. Acriflavine (ACF) inhibits AGO2 function in mammalian cells.(B) Representative western blot demonstrating sustained TBX18 protein expression up to 72 h post-transfection (single dose) by 2.5 µM ACF (top right). Quantification by densitometry analysis (bottom) (n = 12 in 48 h CMmTBX18 + ACF; n = 10 in 72 h CMmTBX18 + ACF; n = 6 in 48 h CMmEGFP + ACF; n = 6 in 72 h CMmEGFP; n = 3 in 48 h TBX18 adenovirus; and n = 2 in 72 h TBX18 adenovirus [biological replicates]).(C) Representative western blot demonstrating downregulated Cx43 protein expression up to 72 h post-transfection (top right). Quantification by densitometry analysis (bottom) (n = 12 in each experimental group and n = 2 in TBX18 adenovirus [biological replicates]).(D and E) (D) miRNome heatmap analysis of miRNA expression demonstrates a robust change 48 h after transfection with TBX18 chemically modified mRNA (middle left). (E) Volcano plot representing the most upregulated and downregulated miRs indicate that miR-1-3p and miR-1b are highly upregulated after TBX18 chemically modified RNA transfection. Prediction analysis demonstrating that miR1-3p and miR-1b are predicted to bind to TBX18 3' UTR (centered).(F) Representative TBX18 3' UTR luciferase assay results quantified by percentage of firefly/Renilla luciferase activity compared with no-insert control. Experimental conditions were as follows: group 1, empty vector; group 2, control untransfected; group 3, miR-1-3p/miR-1b mimc transfections; and group 4, a scramble sequence transfection (bottom right) (n = 4 in empty vector; n = 4 in control untransfected; n = 4 in miR-1-3p/miR-1b mimic treatment; and n = 4 in scramble sequence [biological replicates]).(G and H) Representative western blot demonstrating sustained TBX18 protein expression by co-transfecting NRVMs with TBX18 chemically modified RNA and antagomiRs to miR-1-3p and miR-1b (bottom left). Quantification by densitometry analysis (bottom) (n = 4 in TBX18, n = 2 in EGFP chemically modified mRNA, and n = 2 in TBX18 adenovirus [biological replicates]). Adenovirus expressing a TBX18 transgene was used as a positive control in (H). Representative western blot demonstrating HCN4 protein expression by co-transfecting NRVMS with TBX18 chemically modified RNA and antagomiRs to miR-1-3p and miR-1b (bottom middle). Quantification by densitometry analysis (bottom) (n = 6 in TBX18 and n = 6 in EGFP [biological replicates]).(I) Representative action potential traces from CMmEGFP- or CMmTBX18-treated NRVMs highlighting the gradual phase 4 depolarization in CMmTBX18.(J) Beating rate, action potential duration at 90% (APD90), and depolarization rate of the membrane potential are summarized.Data are represented as mean ± SEM. Statistical significance was determined using an independent t test.

Fig 3: TBX18 chemically modified mRNA biological pacemaker activity originates at the focal injection site and induces iSAN-like morphology in ventricular myocytes in vivo(A) Experimental overview indicating rats were transfected with 100 μg TBX18 chemically modified mRNA delivered with antagomiRs to miR-1-3p and miR-1b.(B) A representative fluorescent image with electrodes orientation for far-field electrogram.(C) Original ECG traces are shown before and after cutting high interventricular septum for induction of permanent AV block (AVB). Note, the time to first ventricular escape beat in the TBX18 chemically modified RNA-treated group is shorter than that in EGFP-treated group. Traces are representative (n = 3 for TBX18 and n = 4 in EGFP chemically modified mRNA [biological replicates]). Scale bars: pre-AVB, 5 s; post-AVB, 20 s; and inset, 80 s.(D) Average heart rate within 2 min before induction of AVB (n = 3). Average atrial beats within 2 min after induction of AVB (n = 3). Average ventricular beats within 2 min after AVB (n = 3).(E) Activation maps and action potentials derived from optical mapping are shown after AVB. Arrow indicating the earliest activation site is shown in red. The origin of all ventricular ectopic beats in the TBX18 chemically modified mRNA-treated group correlated with the injection site. Sinus nodal-like potentials are also found within the injection area in the TBX18 chemically modified mRNA-treated group. Scale bars: y, 0.25 ms normalized fluorescence units and x, 50 ms. Images are representative (n = 3 for each group [biological replicates]). Hearts were harvested 72 h post-transfection and processed for qRT-PCR and histology.(F) qRT-PCR demonstrates a 1,000-fold increase in TBX18 expression at the injection site 72 h post-transfection (n = 3 [biological replicates]).(G) Immunohistochemical micrographs demonstrate iSAN-like morphology of ventricular myocytes 72 h post-transfection with TBX18 chemically modified mRNA. Scale bar: 20 μm.Data are represented as mean ± SEM. Statistical significance was determined using an independent t test.