Fig. 3: Cardiomyocyte Replenishment after ISO-induced CM loss.

a Representative confocal microscopy images of BrdU (left) and EdU (middle) incorporation in the apical endocardium 28 days after ISO in wt C57BL/6J mice implanted with subcutaneous mini-pumps to systemically release BrdU or EdU for 28 days. Right, representative confocal microscopy of BrdU incorporation from epicardium to endocardium of the LV Apex 28 days after ISO in wt mice implanted with subcutaneous mini-pumps to systemically release BrdU for 28 days. It is evident the intensity gradient of BrdU incorporation from the epicardium to the endocardium. Scale bar = 50 μm. b Number of newly generated BrdU^pos or EdU^pos CMs at 7 and 28 days in saline or ISO-treated wt mice implanted with subcutaneous mini-pumps to systemically release EDU or BrdU for 7 or 28 days, respectively. n = 7 per group, *p < 0.05 vs. Saline, #p < 0.05 vs. Epi, §p < 0.05 vs. Myo, and †p < 0.05 vs. EdU (one-way ANOVA analysis with Tukey’s multiple comparison test). c Representative reconstruction of three overlapping confocal microscopy images (left) and a high magnification field (×3 zoom from a ×63 microscopy objective, right) showing newly formed cardiomyocytes (arrowheads) whose nuclei are unambiguously identified by PCM-1 (white fluorescence) while being BrdU-positive (green) 28 days after ISO injury in the apical sub-endocardium. DAPI (Blue) depicts cell nuclei. d Cardiac nuclei are identified by FACS analysis based on DAPI labelling (left panel). Fluorescent gating allows the separation of cardiomyocyte nuclei (PCM-1-positive) and non-cardiomyocyte (PCM-1-negative) nuclei in cardiac nuclei preparations isolated from digested heart tissue (left-mid panel). Representative plots show the number of BrdU-labelled cardiomyocyte nuclei (gated on PCM-1-positive nuclei) 28 after saline (right-mid panel) or Isoproterenol (ISO, right panel) injection. It should be noted that the number of BrdU^pos cardiomyocyte nuclei should be corrected when extrapolating it to number of cardiomyocytes considering that by the vast majority of adult cardiomyocytes in the adult murine hearts are binucleated. Scale bars = 50 µm. All data are mean ± SD