Asparagine Synthetase Marks a Distinct Dependency Threshold for Cardiomyocyte Dedifferentiation.

Abstract

Adult mammalian cardiomyocytes have limited proliferative capacity, but in specifically induced contexts they traverse through cell-cycle reentry, offering the potential for heart regeneration. Endogenous cardiomyocyte proliferation is preceded by cardiomyocyte dedifferentiation (CMDD), wherein adult cardiomyocytes revert to a less matured state that is distinct from the classical myocardial fetal stress gene response associated with heart failure. However, very little is known about CMDD as a defined cardiomyocyte cell state in transition. Here, we leveraged 2 models of in vitro cultured adult mouse cardiomyocytes and in vivo adeno-associated virus serotype 9 cardiomyocyte-targeted delivery of reprogramming factors (<i>Oct4</i>, <i>Sox2</i>, <i>Klf4</i>, and <i>Myc</i>) in adult mice to study CMDD. We profiled their transcriptomes using RNA sequencing, in combination with multiple published data sets, with the aim of identifying a common denominator for tracking CMDD. RNA sequencing and integrated analysis identified <i>Asparagine Synthetase</i> (<i>Asns</i>) as a unique molecular marker gene well correlated with CMDD, required for increased asparagine and also for distinct fluxes in other amino acids. Although <i>Asns</i> overexpression in <i>Oct4</i>, <i>Sox2</i>, <i>Klf4</i>, and <i>Myc</i> cardiomyocytes augmented hallmarks of CMDD, <i>Asns</i> deficiency led to defective regeneration in the neonatal mouse myocardial infarction model, increased cell death of cultured adult cardiomyocytes, and reduced cell cycle in <i>Oct4</i>, <i>Sox2</i>, <i>Klf4</i>, and <i>Myc</i> cardiomyocytes, at least in part through disrupting the mammalian target of rapamycin complex 1 pathway. We discovered a novel gene <i>Asns</i> as both a molecular marker and an essential mediator, marking a distinct threshold that appears in common for at least 4 models of CMDD, and revealing an <i>Asns</i>/mammalian target of rapamycin complex 1 axis dependency for dedifferentiating cardiomyocytes. Further study will be needed to extrapolate and assess its relevance to other cell state transitions as well as in heart regeneration.