A broadly applicable method for quantifying cardiomyocyte cell division identifies proliferative events following myocardial infarction

Figures

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  Figure 1. Single-cell ventricular preparations allow for collection of multiple types of data (A) Experimental workflow from thymidine analog labeling to Langendorff digestion and to staining and imaging. (B–F) A single preparation can be used for multiple analyses, including (B) assessment of CM ploidy independent of thymidine analog status, (C) assessment of CM division, (D) quantification of total CM number, (E) total cycling CMs, and (F) measurement of CM dimensions. Figure created with BioRender.

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  Figure 2. Non-CM nuclei are not frequently included in ploidy analysis (A-A’) Representative images of CM single-cell suspensions labeled for N-Cad (magenta), PCM1 (red), and DAPI (white). In A’, yellow arrowheads point to non-CM nuclei, which overlay on top of a CM. White arrowheads point to PCM1+ CM nuclei. Scale bar: 100 μm. (B) Table showing quantifications done by three observers that indicate the frequency in which a nucleus was called “CM,” but that proved not to be positive for PCM1. SD = 0.17%. (C) A single cell from a suspension immunofluorescently labeled with DAPI (white), CX43 (magenta), NKX2-5 (red), and cTnT (green). Scale bar: 100 μm.

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  Figure 3. Inclusion of under-digested and/or broken CMs in ploidy analysis is rare (A-A’) Representative images of CM single-cell suspensions labeled for CX43 (magenta), cTnT (green), and DAPI (white). An intercalated disc is visible as a break in sarcomere continuity (A, white boxed inset), and proves to stain positive for CX43 (A’). Scale bar: 100 μm. (B) Table showing quantifications assessed by three observers that indicate the frequency with which CMs were included in the analysis, but later were identified as having CX43 staining within the CM boundaries (potentially indicating two cells still joined together) (column 3; SD = 0.67%) or, to not have CX43 staining at both ends (a potentially broken CM) (column 4; SD = 0.29%). (C–F’) Representative images of CM single cells from suspensions immunofluorescently labeled with DAPI (white), N-Cad (magenta), and Myl2 (green). (C–C’) A trinucleated cell with continuous sarcomere staining (inset), which contrasts the visible intercalated disc in (A) and (D). (D–D’) Two asymmetrically aligned binucleated cells with N-Cad in the middle (yellow arrowheads), along with a contiguous tetranucleated CM with no N-Cad staining in the middle (white…

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  Figure 4. Dynamic cell cycle and cellular hypertrophy across 2 weeks post-MI (A) Schematic depicting time line of MI and thymidine analog labeling windows. Ventricular suspensions collected 17 days post-injury via Langendorff perfusion. Schematic created using BioRender. (B) Representative images of EdU (cyan), BrdU (magenta), Myl2 (green), and DAPI (white) staining depicting an EdU-only+ binucleated CM (left), a BrdU-only+ mononuclear CM (center), and a double-positive mononuclear CM (right). Yellow arrowheads, non-CM nuclei. White arrowheads, CMs. Scale bar: 50 μm. (C) Total EdU+ and BrdU+ CMs identified in single-cell suspension of ventricular tissue post-MI between two inbred strains (C57BL/6J and A/J). N = 5 animals for each strain. Data represented in log2 scale. p values calculated by two-way ANOVA with Šidák’s post hoc test. Average across 5 biological replicates ± SEM. (D) Quantifications of EdU+ and BrdU+ CMs broken down by total DNA content considering both nucleation and nuclear ploidy (i.e., 2N [1 × 2N], 4N [1 × 4N and 2 × 2N], 8N [1 × 8N, 2 × 4N, trinucleated = 8N; and tetranucleated = 8N], or 16N). p values calculated by a two-way multivariate ANOVA (MANOVA) with least…

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  Figure 5. Dual-thymidine analog labeling following MI unveils cell division solely in the border zone (A) Frequency of Edu-BrdU double-positive CMs represented as a percentage of the CMs that had undergone DNA synthesis in the first week (i.e., were EdU+). Data are further broken down into their final DNA content (2N, 4N, 8N, and 16N, respectively), indicated by gradations of grayscale. N = 5 biological replicates for each strain. (B) Four-chamber schematic with the infarct (solid gray), border zone (blue outline), and remote zone (black outline) designations indicated. Schematic created using BioRender. (C) Representative images of EdU (cyan), BrdU (magenta), PCM1 (red), and DAPI (white) staining of intact myocardial tissue isolated 17 days post-MI. The top row is a double-labeled CM (indicated by white arrowhead) found in the remote zone (RZ) of a C57BL/6J heart. The center row is a double-labeled CM found in the infarct/border zone (I/BZ) of a C57BL/6J heart. The bottom row is a double-labeled CM found in the infarct/border zone (I/BZ) of an A/J heart. Scale bar: 20 μm.