Extended Data Fig. 2: Systematic assessment of miR-199a-3p expression after AAV6-mediated transduction.

a, Schematic representation of pig heart sectioning for histological and molecular studies. After arrest in diastole, the heart was excised and the pericardial sac removed. AAV injection sites, which were marked with coloured epicardial sutures during surgery, were further traced with a green waterproof paint. Four 1-cm thick transversal slices were cut starting from the base to the apex (1–4). Each slice was subsequently divided into 2–8 regions, each one labelled with a capital letter, and then into additional sub-regions (letters plus numbers) for targeted molecular and histological analyses. Sectors H, T and C corresponded to the infarct border zone, where the vectors were administered, whereas sector L was considered representative of the remote zone. b, Injection and infarct border segments for each slice were divided into smaller fragments (dashed lines) to accurately assess the levels of expression of the transgene at 12 days after transduction. The syringe indicates the injection sites. c, For each slice and segment, the graphs show real-time PCR quantifications of the mature miR-199a-3p expressed as fold change over endogenous levels (AAV6-control). One representative animal is shown out of four analysed in the same systematic manner with comparable results. d, In situ hybridization of pig heart sections for the detection of miR-199a expression at the single-cell level. Each of the sectors indicated in b was tested by in situ hybridization using LNA probes detecting miR-199a-3p or U6 snRNA, or a probe with the same nucleotide composition as the one against miR-199a-3p but with a scrambled sequence (scramble). Expression of miR-199a-3p was robust in cardiomyocytes and specific for the injected areas throughout the LV. One representative animal is shown out of four analysed in the same systematic manner with comparable results. Scale bar, 100 µm.