Loss of Endogenously Cycling Adult Cardiomyocytes Worsens Myocardial Function

Abstract

Rationale: Endogenously cycling adult cardiomyocytes (CMs) increase after myocardial infarction (MI) but remain scare, and are generally thought not to contribute to myocardial function. However, this broadly held assumption has not been tested, mainly because of the lack of transgenic reporters that restrict Cre expression to adult CMs that reenter the cell cycle. Objective: We created and validated a new transgenic mouse, αMHC-MerDreMer-Ki67p-RoxedCre::Rox-Lox-tdTomato-eGFP (denoted αDKRC) that restricts Cre expression to cycling adult CMs and uniquely integrates spatial and temporal adult CM cycling events based on the DNA specificities of orthologous Dre- and Cre recombinases. We then created mice that expressed an inducible Diphtheria toxin (DTA), αDKRC::DTA mice, in adult cycling CMs and examined the effects of ablating these endogenously cycling CMs on myocardial function after Ischemic-Reperfusion (I/R) MI. Methods and Results: A tandem αDKRC transgene was designed, validated in cultured cells, and used to make transgenic mice. The αDKRC transgene integrated between MYH6 and MYH7 and did not disrupt expression of the surrounding genes. Compared to controls, αDKRC::RLTG mice treated with Tamoxifen expressed tdTomato+ in CMs with rare Bromodeoxyuridine (BrdU)+, eGFP+ CMs, consistent with reentry of the cell cycle. We then pre-treated αDKRC::RLTG mice with Tamoxifen to activate the reporter before sham or reperfusion (I/R) myocardial infarction (MI) surgeries. Compared to Sham surgery, the I/R MI group had increased single and paired eGFP+ CMs predominantly in the border zones (5.8 {plus minus} 0.5 vs. 3.3 {plus minus} 0.3 CMs per ten-micron section, N = 8-9 mice per group, n = 16-24 sections per mouse), indicative of cycled CMs. The single to paired eGFP+ CM ratio was ~9 to 1 (5.2 {plus minus} 0.4 single vs. 0.6 {plus minus} 0.2 paired CMs) in the I/R MI group after MI, suggesting that cycling CMs were more likely to undergo polyploidy than replication. The ablation of endogenously cycling adult CMs in αDKRC::DTA mice caused progressive worsening left ventricular chamber size and function after I/R MI, compared to controls. Conclusions: Although scarce, endogenously cycling adult CMs contribute to myocardial function after injury, suggesting that these cells may be physiologically relevant.

In Vivo Methods to Monitor Cardiomyocyte Proliferation

“…Importantly, the MADM mouse technology has limitations in addition to the efficiency of labeling recombination events that can potentially underestimate the quantifica- αMHC-MerDreMer-Ki67p-RoxedCre (αDKRC) mice. Advanced non-Cre DNA recombinases, such as Dre [77][78][79], VCre [80,81], SCre [80,81], and FLP [82,83] with DNA recognition sites other than LoxP, have unlocked the possibilities to engineer more sophisticated transgenic mice and interrogate temporal and lineage-specific cell biology [6,78,84]. Combining inducible non-Cre recombinases (MerDreMer) with modified Cre recombinases (RoxedCre) facilitates existing LoxP transgenics to answer previously unsolved questions in biology and disease.…”

“…In mice, the majority of cardiomyocytes are determined within the first week of postnatal life, followed by two waves of DNA synthesis leading to binucleation and increased ploidy, a measure of genome size [ 4 ]. A small fraction of cardiomyocytes re-enter the cell cycle in response to injury [ 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 ]; however, cytokinesis is incomplete after cardiomyocytes re-enter the cell cycle, leading to further polyploidization and limited generation of new cardiomyocytes [ 14 ]. Human cardiomyocyte turnover is ~0.04% in the first year of life and ~0.01% per year in adulthood based on nuclear bomb test-derived radioisotope decay estimates [ 4 , 5 , 15 , 16 ].…”

“…The incorporation of thymidine analogs and transgenic mice revealed spatial and regional distribution of cycled cardiomyocytes after injuries, such as myocardial infarction [ 6 , 7 , 10 , 34 ]. Generally, most cardiomyocyte cycling occurs in the border zones, defined as the regions of the heart nearest the infarcted area.…”

“…Cardiomyocyte binucleation may be a barrier to proliferation, limiting mammalian myocardial regeneration. Recently, we observed that ~90% of cardiomyocytes reentering the cell cycle did not produce neighboring cells, suggesting that cycling failed to proliferate and may have undergone endoreplication [ 6 ]. These observations were consistent with prior results in the field.…”

Induced Cardiomyocyte Proliferation: A Promising Approach to Cure Heart Failure

“…Furthermore, in vivo models [ 126 , 127 ] and hiPS-derived cardiomyocytes [ 128 ] have been generated to study cell cycle progression in the heart using FUCCI indicators. Interestingly, a recent study generated a Ki67-based lineage and traced mice to track the cell cycle activation in cardiomyocytes [ 129 ]. Finally, a recent preprint described the generation of an Aurora kinase B-based reporter to be used in identification of cardiomyocyte cell cycle activation in vitro and in vivo [ 36 ].…”