Fig. 1: Evaluating cardiomyocyte nuclei ploidy in postmortem human heart.

a,b,d, Representative flow cytometry analysis of cardiac nuclei with Alexa Fluor 488 conjugate cTroponin T with careful doublet exclusion for 1278 (a), 5657 (b), and 1465 (d). c, DNA content histograms of heart cell nuclei with varied ages. The first peak indicates diploid (2n) nuclei, the second peak indicates tetraploid (4n) nuclei and the third and fourth peaks indicate multiploid (>4n) nuclei. d,e, Evaluation of cardiomyocyte nuclei ploidy by flow cytometry (d) and Amnis imaging flow cytometry (e), showing diploid (2N), tetraploid (4N) and multiploid (multiple N) cardiomyocyte nuclei proportion in case 1465. f, Representative photomicrographs of isolated cardiomyocyte nuclei (n = 4 independent experiments; images from 500 cells per experiment were examined from n = 20 cases), confirming DNA content of a single tetraploid and multiploid cardiomyocyte nuclei. Scale bar, 20 µm. g, Flow cytometry sorted cardiomyocyte nuclei karyotyping confirming cardiomyocyte chromosomes numbers, 46 (2n), 92 (4n) and 138 (>4n) in cardiomyocyte nuclei isolated from case 1465 (n = 6; 15–20 nuclei counted each time). Scale bar, 10 µm. h, Representative ddPCR analysis from flow cytometry sorted nuclei (n = 4, from 12 cases). cTroponin-T-positive 2n, 4n and greater than 4n are highly enriched for PLN and MYH7 (cardiac markers), whereas cTroponin-T-negative 2n nuclei express PDGFB or CD31 (markers for fibroblast and endothelial cells). The bottom clusters on the plot represent the negative droplets and the upper clusters represent the droplets that are positive for the respective reference assay. The plus sign indicates cells sorted from cTroponin-T-positive staining population; the dash indicates cells sorted from cTroponin-T-negative population; the blank box indicates that cells were sorted based solely on ploidy status.