Ablation of endogenously cycling adult cardiomyocytes worsens myocardial function after injury

Abstract

RationaleEndogenously 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. ObjectiveWe created and validated a new transgenic mouse, MHC-MerDreMer-Ki67p-RoxedCre::Rox-Lox-tdTomato-eGFP (denoted DKRC) 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), DKRC::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 ResultsA tandem DKRC transgene was designed, validated in cultured cells, and used to make transgenic mice. The DKRC transgene integrated between MYH6 and MYH7 and did not disrupt expression of the surrounding genes. Compared to controls, DKRC::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 DKRC::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 {+/-} 0.5 vs. 3.3 {+/-} 0.3 CMs per ten-micron section, N = 8 mice, n = 16 sections per mouse), indicative of cycled CMs. The single to paired eGFP+ CM ratio was [~]9 to 1 (5.2 {+/-} 0.4 single vs. 0.6 {+/-} 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 DKRC::DTA mice caused progressive worsening left ventricular chamber size and function after I/R MI, compared to controls. ConclusionsAlthough scarce, endogenously cycling adult CMs contribute to myocardial function after injury, suggesting that these cells may be physiologically relevant.