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

Abstract

Background: Identifying genetic variants which impact 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 following permanent coronary artery ligation as compared to infarcted DBA/2J (D2J) mice. Methods: Cardiomyocyte cell cycle activity post-infarction was monitored in D2J, (D2J x B6N)-F1 and [(D2J x B6N)-F1 x D2J] backcross mice via bromodeoxyuridine or 5-ethynyl-2' -deoxyuridine incorporation, using a nuclear-localized transgenic reporter to identify cardiomyocyte nuclei. Genome-wide quantitative trait locus (QTL) analysis, fine scale genetic mapping, whole exome sequencing and RNA-seq analyses of the backcross mice were performed to identify the gene responsible for the elevated cardiomyocyte S-phase phenotype. Results: (D2J x B6N)-F1 mice exhibited a 14-fold increase in cardiomyocyte S-phase activity in ventricular regions remote from infarct scar as compared to D2J mice (0.798 ± 0.09% vs. 0.056 ± 0.004%; p < 0.001). QTL analysis of [(D2J x B6N)-F1 x D2J] backcross mice revealed that the gene responsible for differential S-phase activity was located on the distal arm of Chromosome 3 (LOD score = 6.38; p < 0.001). Additional genetic and molecular analyses identified 3 potential candidates. Of these, troponin I-interacting kinase ( Tnni3k ) 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 post-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 following injury.

Hypoxia to Stimulate Cardiomyocyte Proliferation After MI? A Word of Caution

“…11 They noted a similar increase in HW/ BW, but only a nonsignificant trend toward increase in cardiomyocyte size. Noteworthy is the reported 10% EdU labeling rate of cardiomyocytes, magnitudes higher 9 It is difficult to explain and points to methodological problems.…”

“…The Field group reported a BrdU/EdU labeling index in mice (over 14 days) of 0.6% and calculated that such rate would replace 50% of the heart´s cardiomyocyte over 569 days. 9 Obviously, 0.1% renewal in 28 days would need years to account for a doubling of cardiomyocyte numbers. One could argue that Nakada et al employed low doses of oral BrdU that underestimated the true DNA synthesis rate.…”

“…Noteworthy is the reported 10% EdU labeling rate of cardiomyocytes, magnitudes higher than the Johnson et al and Nakada et al reports and other articles in the field. 9 It is difficult to explain and points to methodological problems.…”

A Tedious Journey: Cardiomyocyte Proliferation Requires More Than S-Phase Entry and Loss of Polyploidization

“…Alternatively, TNNI3K might be involved in the regulation of metabolic processes and oxidative stress. Reuter et al 8 speculated that increased levels of oxidative stress render TNNI3K-expressing cardiomyocytes more permissive to cell-cycle reentry. However, it has been reported that enhanced DNA damage attributable to increased reactive oxygen species production prevents DNA replication, resulting in cell-cycle arrest.…”

“…In this issue of Circulation , Reuter et al 8 and Yu et al 9 addressed these questions by studying cardiac troponin I–interacting kinase (TNNI3K)–dependent S-phase activity and E2 promoter binding factor (E2F) 7 and 8–dependent polyploidization of cardiomyocytes, respectively (Figure). Reuter et al observed pronounced differences in cardiomyocyte S-phase activity after permanent coronary artery ligation in mice of different genetic backgrounds.…”