Frequency of mononuclear diploid cardiomyocytes underlies natural variation in heart regeneration

Abstract

Adult mammalian cardiomyocyte regeneration after injury is thought to be minimal. Mononuclear diploid cardiomyocytes (MNDCMs), a relatively small subpopulation in the adult heart, may account for the observed degree of regeneration, but this has not been tested. We surveyed 120 inbred mouse strains and found that the frequency of adult mononuclear cardiomyocytes was surprisingly variable (>7-fold). Cardiomyocyte proliferation and heart functional recovery after coronary artery ligation both correlated with pre-injury MNDCM content. Using genome-wide association, we identified Tnni3k as one gene that influences variation in this composition and demonstrated that Tnni3k knockout resulted in elevated MNDCM content and increased cardiomyocyte proliferation after injury. Reciprocally, overexpression of Tnni3k in zebrafish promoted cardiomyocyte polyploidization and compromised heart regeneration. Our results corroborate the relevance of MNDCMs in heart regeneration. Moreover, they imply that intrinsic heart regeneration is not limited nor uniform in all individuals, but rather is a variable trait influenced by multiple genes.

Figure 1

Variation in mononuclear diploid cardiomyocyte content predicts outcome after injury. (a) Distribution of 120 inbred mouse strains of the HMDP for frequency of mononuclear cardiomyocytes in the naive adult heart. See supplementary table 1 for full list of strains, including n-values. (b) Single-cell ventricular suspension stained with the vital dye LiveDead (green), for cardiac troponin T (cTnT) (red) and with DAPI (blue), identifying a small mononuclear cardiomyocyte (white arrowhead) next to a larger binucleated cardiomyocyte, with two dead cardiomyocytes also evident. Scale bar represents 50 μm. (c) DNA FISH using probes from the Hoxc (chr15, green; yellow arrowheads) and Sox2 (chr3, red; white arrowheads) loci; nuclei are stained with DAPI. Top, a binucleated cardiomyocyte with two diploid nuclei. Middle, a mononuclear cardiomyocyte with a tetraploid nucleus. Bottom, a mononuclear cardiomyocyte with a diploid nucleus. Scale bars in the cell view (left) represent 20 μm; scale bars in the nuclear view (right) represent 5 μm. (d) Quantification for four strains of the percentage of mononuclear (MN) cardiomyocytes that had diploid nuclei as assessed by FISH. n = 3 mice for all strains. One-way ANOVA P = 0.048; F = 4.14; P value on graph is Bonferroni correction. (e) Calculation of percentage of MNDCMs across four strains. One-way ANOVA P = 0.0027; P values on graph are Tukey HSD post hoc. (f) Ejection fraction measured by echocardiography on four strains at baseline, 3 d after myocardial infarction (MI) caused by coronary artery ligation and 28 d after ligation. See supplementary tables 2 and 3 for complete statistics. Strain A (n = 5 mice), SWR (n = 8 mice), C57Bl/6 (n = 4 mice) and SJL (n = 7 mice). (g) Percentage improvement 1 month after injury, calculated by (Ejection fraction at 28 d – Ejection fraction at 3 d)/(Ejection fraction at 3 d) for the four strains. One-way ANOVA P = 0.0004; F = 9.20; P values on graph are Bonferroni correction. (h) Quantification of the scar area 1 month after injury, represented as a percentage of the total left ventricle (LV) for the four strains. Strain A (n = 6), SWR (n = 8), C57Bl/6 (n = 4) and SJL (n = 7). One-way ANOVA P = 0.04; F = 3.29; P value on graph is Bonferroni corrected. All error bars are s.e.m.

Figure 2

Mononuclear diploid cardiomyocytes proliferate after injury. (a) Confocal image of an EdU⁺ (red) and Myl2⁺ (green) cardiomyocyte (white arrowheads). Left panel is the flattened z-stack. Right panel is the orthogonal view. Black arrowhead points to the same cell, indicating Myl2⁺ cytoplasm above and below the EdU⁺ nucleus. Scale bars represent 20 μm. (b) Quantification of percentage of EdU⁺ cardiomyocyte (CM) nuclei in the border zone of the left ventricle for the four strains. Strain A (n = 6 mice), SWR (n = 8 mice), C57Bl/6 (n = 4 mice) and SJL (n = 7 mice). One-way ANOVA P = 0.0002; F = 10.51; P values on graph are Bonferroni corrected. (c) A Myl2⁺EdU⁺ mononuclear cardiomyocyte (yellow arrowhead) identified in a ventricular cell preparation from a mouse exposed to EdU after coronary artery ligation. Scale bar represents 50 μm. (d) Flow cytometry plots (forward scatter, FSC-H, vs. DNA dye, DyeCycle Violet) of ventricular nuclei isolated from 1 strain A mouse exposed to EdU after coronary artery ligation. The different nuclear populations (cTnI⁺, cardiomyocyte; EdU⁻ or EdU⁺) and their ploidy (2N, 4N) and quantifications are indicated. Plots and values shown are one example of three independent experiments. (e) Average quantification of ploidy across nuclear populations from 3 independent strain A mice (as in d). All error bars represent s.e.m.

Figure 3

Tnni3k influences mononuclear diploid cardiomyocyte population size and cellular regenerative capacity after injury. (a) Manhattan plot of the genome-wide association analysis performed on the data for 120 HMDP strains shown in Figure 1a and supplementary table 1. Red line indicates the boundary for statistical significance (P = 4 × 10⁻⁶). (b) Chromosomal map of the 1.2-Mb locus on chromosome 3 corresponding to the most significant peak identified by genome-wide association analysis. Coordinates are for mouse genome build 37. (c) Immunoblot for Tnni3k and Gapdh on adult atrial tissue isolated from C57Bl/6 control and Myh6-Cre⁺ mice with varying copy numbers of the Tnni3k fl allele; WT, wild type. Both panels are from the same blot and exposed for the same length of time. (d) Frequency of mononuclear cardiomyocytes in Myh6-Cre⁺ mice with varying copy numbers of the Tnni3k fl allele. n = 4 mice for all genotypes. One-way ANOVA P = 0.003; F = 12.01; P values on graph are Bonferroni corrected. (e) Quantification of the number of diploid nuclei found within the mononuclear cardiomyocyte population, assessed by DNA FISH. n = 3 mice for both genotypes. Two-tailed Student t-test. (f) Calculation of MNDCM percent in the Myh6-Cre⁺, Tnni3k fl/+ genotype vs. the Tnni3k fl/fl genotype. Two-tailed Student t-test; t = 11.6. (g) Frequency of mononuclear cardiomyocytes in mice with varying copy numbers of Tnni3k null allele (global knockout). WT (n = 4 mice), heterozygote (het; n = 7 mice), knockout (KO; n = 4 mice). One-way ANOVA P = 2.43 × 10⁻⁵; F = 29.25; P values on graph are Bonferroni correction. (h) Quantification of EdU⁺ cardiomyocyte (CM) nuclei in the border zone of the left ventricle 1 month after coronary artery ligation in Tnni3k knockout (KO, n = 7) and WT (n = 6) littermates. Cardiomyocytes were identified as Myl2⁺ as in Figure 2a. Two-tailed Student t-test. (i) Distribution of cTnI⁺ nuclear ploidy as assessed by flow cytometry. KO (n = 4), control (CTRL; n = 2 heterozygotes and 1 WT). (j) Quantification of EdU⁺ cardiomyocytes that were mononuclear after infarction in both CTRL (n = 1 heterozygote and 1 WT) and KO (n = 2). Two-tailed Student t-test. (k) Calculation of the number of EdU⁺ cardiomyocytes completing cell division based on the analyses in i and j. Two-tailed Student t-test; t = 7.08. All error bars represent s.e.m.

Figure 4

Overexpression of Tnni3k induces polyploidization and impairs regeneration in zebrafish hearts. (a) Quantification of the percent of binucleated cardiomyocytes in single-cell suspensions isolated from adult control (Ctrl; n = 5) and mouse Tnni3k transgenic (n = 5) zebrafish hearts. (b) Total fluorescence calculated from DAPI-stained cardiomyocyte nuclei across single-cell suspensions isolated from control and mTnni3k transgenic adult zebrafish. Red points represent nuclei that had a total fluorescence intensity greater than 3 s.d. above the mean of control preparations and are therefore presumed to be polyploid. Values indicated in red above each sample are the percentage of nuclei presumed to be polyploid. Mean of all Tnni3k samples = 14.4 ± 2.8% compared to 0% from control samples (P < 0.001; two-tailed Student t-test). (c) Examples of zebrafish hearts sectioned and stained for acid fuchsin orange G (AFOG) 30 d post-ventricular resection (dpr) with scores “0” (no scar, fully regenerated), “1” (small residual scar on either the endocardial or epicardial surface), “2” (small transmural scar) or “3” (large transmural scar) indicated. Scale bars represent 200 μm. (d) Quantification of scar severity at 30 dpr across control (Ctrl, n = 18) and Tnni3k transgenic (n = 24) fish. Chi-squared test P = 0.042. (e) An example image of the injury border zone stained with Pcna (red) and Mef2 (green) at 7 dpr. White arrowheads indicate double-positive cardiomyocyte nuclei. Scale bar represents 100 μm. (f) Quantification of Pcna⁺ cardiomyocyte (CM) nuclei in the border zone of 7-dpr heart sections represented as a percent of the total number of Mef2⁺ cardiomyocyte nuclei. Ctrl (n = 11 fish) and Tnni3k transgenic (n = 10 fish). Two-tailed Student t-test. Error bars represent s.e.m.