Cardiac Troponin I-Interacting Kinase Affects Cardiomyocyte S-Phase Activity but Not Cardiomyocyte Proliferation

Abstract

Background: Identifying genetic variants that affect the level of cell cycle reentry and establishing the degree of cell cycle progression in those variants could help guide development of therapeutic interventions aimed at effecting cardiac regeneration. We observed that C57Bl6/NCR (B6N) mice have a marked increase in cardiomyocyte S-phase activity after permanent coronary artery ligation compared with infarcted DBA/2J (D2J) mice.

Methods: Cardiomyocyte cell cycle activity after infarction was monitored in D2J, (D2J×B6N)-F1, and (D2J×B6N)-F1×D2J backcross mice by means of bromodeoxyuridine or 5-ethynyl-2'-deoxyuridine incorporation using a nuclear-localized transgenic reporter to identify cardiomyocyte nuclei. Genome-wide quantitative trait locus analysis, fine scale genetic mapping, whole exome sequencing, and RNA sequencing analyses of the backcross mice were performed to identify the gene responsible for the elevated cardiomyocyte S-phase phenotype.

Results: (D2J×B6N)-F1 mice exhibited a 14-fold increase in cardiomyocyte S-phase activity in ventricular regions remote from infarct scar compared with D2J mice (0.798±0.09% versus 0.056±0.004%; P<0.001). Quantitative trait locus analysis of (D2J×B6N)-F1×D2J backcross mice revealed that the gene responsible for differential S-phase activity was located on the distal arm of chromosome 3 (logarithm of the odds score=6.38; P<0.001). Additional genetic and molecular analyses identified 3 potential candidates. Of these, Tnni3k (troponin I-interacting kinase) is expressed in B6N hearts but not in D2J hearts. Transgenic expression of TNNI3K in a D2J genetic background results in elevated cardiomyocyte S-phase activity after injury. Cardiomyocyte S-phase activity in both Tnni3k-expressing and Tnni3k-nonexpressing mice results in the formation of polyploid nuclei.

Conclusions: These data indicate that Tnni3k expression increases the level of cardiomyocyte S-phase activity after injury.

Keywords: cell cycle; myocytes, cardiac; regeneration.

Figure 1.

Pattern of cardiomyocyte DNA synthesis in D2J and B6N genetic backgrounds following infarction. (A) Example of an S-phase cardiomyocyte using the MHC-nLAC reporter. The first panel shows βGAL immune reactivity (red secondary antibody), the second panel shows BrdU immune reactivity (green secondary antibody), the third panel shows a merged image (yellow indicates co-localization of βGAL and BrdU immune reactivity, and the fourth panel shows the computer-generated ring to mark the anatomical position of the S-phase event. (B) Representative example of the pattern of cardiomyocyte S-phase activity in the heart from an infarcted D2J mouse. White rings indicate the anatomical positions of the S-phase cardiomyocyte nuclei; inset shows the same image with the brightness adjusted to facilitate visualization of the infarct anatomy at low magnification. (C) Representative example of the pattern of cardiomyocyte S-phase activity in the heart from an infarcted third-generation B6N backcross mouse. White rings indicate the anatomical positions of the S-phase cardiomyocyte nuclei; inset shows the same image with the brightness adjusted to facilitate visualization of the infarct anatomy at low magnification. (D) Computer-assisted parsing of an image of a heart into the ventricle remote from the infarct, the infarct border zone and the infarct (defined by the yellow, white and green traces, respectively). (E) Comparison of the cumulative cardiomyocyte S-phase labeling index in the remote ventricle (V), infarct border zone (BZ) and infarct (INF) myocardium after 14 days BrdU infusion post-injury. Yellow, white, and green dots indicate the V, BZ and INF labeling indices, respectively, per each one of 10 DJ2 mice and 17 (D2J × B6N)-F1 mice. Red symbols and vertical red lines indicate the mean labeling index and SEM, respectively, for each genotype. Kruskal – Wallis One Way Analysis on Ranks and Dunn’s method for Multiple Comparisons were used to compare labeling indices of each zone in D2J with each zone in (D2Jx B6N)-F1. p values for statistically significant differences are indicated.

Figure 2.

Mapping a locus which contributes to high levels of cardiomyocyte S-phase activity following infarction. The graph shows a genome-wide QTL linkage scan for cardiomyocyte S-phase activity using 28 animals from a [(D2J × B6N)-F1] × D2J backcross. Chromosomes 1 through X are indicated numerically on the x-axis. The y-axis shows the logarithm of the odds (LOD) score. Levels of significance (p < 0.05, 0.01, and 0.001) were determined by a permutation test; 1,000 permutations of the data. A single genomic region on chromosome 3 displays a highly significant linkage peak, with a LOD score of 6.38.

Figure 3.

Congenic mice heterozygous for the distal arm of chromosome 3 have high levels of cardiomyocyte S-phase activity post-injury. (A) SNP distribution map of the congenic mice; orange indicates homozygous D2J SNPs while purple indicates heterozygous D2J/B6N SNPs. (B) Representative example of a heart from a congenic mouse subjected to 14 days BrdU infusion immediately following permanent coronary artery ligation; a high level of cardiomyocyte S-phase activity is apparent in the remote myocardium (indicated by the white rings). Inset shows the same image with the brightness adjusted to facilitate visualization of the infarct anatomy at low magnification.

Figure 4.

Fine-scale mapping of a B6N allele which gives rise to high levels of cardiomyocyte S-phase activity following infarction. (A) SNP distribution on the distal arm of Chromosome 3 in [(D2J × B6N)-F1 × D2J] backcross mice with high rates of cardiomyocyte S-phase activity post-injury. Orange lines indicate chromosomal regions which are homozygous D2J, purple lines indicate chromosomal regions which are heterozygous D2J/B6N, and orange/purple dashes indicate chromosomal regions harboring a crossover event. (B) Proximal (in mouse #65761) and distal (in mouse #63328) boundary of the ROI as determined by WES analysis.

Figure 5.

Expression of human TNNI3K increases the level of cardiomyocyte S-phase activity in D2J mice following myocardial infarction. (A) Comparison of representative hearts from a control mouse and a TNNI3K^tg transgenic mouse which were subjected to 14 days BrdU infusion immediately following permanent coronary artery ligation (white rings indicate the position of S-phase cardiomyocyte nuclei). Insets show the same images with the brightness adjusted to facilitate visualization of the infarct anatomy at low magnification. (B) Cardiomyocyte S-phase labeling index in the remote myocardium after 14 days BrdU infusion in Control and TNNI3K^tg mice. Yellow markers indicate the labeling index for individual mice, while the wider horizontal red lines indicate the mean labeling index for a given genotype and the vertical red lines indicate the SEM; the asterisk indicates p <0.01 vs. Control mice by non-paired t-test. (C) Western blot demonstrating relative level of human TNNI3K protein expression in the TNNI3K^tg hearts.

Figure 6.

Cardiomyocyte S-phase activity culminates in the formation of polyploid nuclei in the presence or absence of TNNI3K. (A) Example of an S-phase positive nucleus used to quantitate DNA content. The top panel shows EdU incorporation (green signal), the second panel shows Hoechst 33342 staining of DNA (blue signal), the third panel shows βGAL immune reactivity (red secondary antibody) and the bottom panel shows a color combined image. (B) Quantitation of nuclear DNA content in D2J mice at 14 days post-injury (EdU(−), EdU negative; EdU(+), EdU positive; NON-CM, non-cardiomyocyte; M-CM, mononucleated cardiomyocyte; B-CM, binucleated cardiomyocyte). The box extends from the 25th to 75th percentile (endpoints of the interquartile range (IQR)) with a line drawn at the median (50th percentile). The upper whisker is the maximum value of the data that is within 1.5 times the IQR over the 75th percentile. The lower whisker is the minimum value of the data that is within 1.5 times the IQR under the 25th percentile.. Points beyond that are shown as individual dots. Data was compiled from 6 mice, and “n” indicates the total number of nuclei for each cell type. (C) Quantitation of nuclear DNA content in B6N mice at 14 days post-injury. Labels and graph specifics are the same as for Panel B. Data was compiled from 4 mice, and “n” indicates the total number of nuclei for each cell type.