Mouse embryonic stem cells irradiated with γ-rays differentiate into cardiomyocytes but with altered contractile properties

Abstract

Embryonic stem cells (ESCs) for their derivation from the inner cell mass of a blastocyst represent a valuable in vitro model to investigate the effects of ionizing radiation on early embryonic cellular response. Following irradiation, both human and mouse ESCs (mESCs) maintain their pluripotent status and the capacity to differentiate into embryoid bodies and to form teratomas. Although informative of the maintenance of a pluripotent status, these studies never investigated the capability of irradiated ESCs to form specific differentiated phenotypes. Here, for the first time, 5Gy-irradiated mESCs were differentiated into cardiomyocytes, thus allowing the analysis of the long-term effects of ionizing radiations on the differentiation potential of a pluripotent stem cell population. On treated mESCs, 96h after irradiation, a genome-wide expression analysis was first performed in order to determine whether the treatment influenced gene expression of the surviving mESCs. Microarrays analysis showed that only 186 genes were differentially expressed in treated mESCs compared to control cells; a quarter of these genes were involved in cellular differentiation, with three main gene networks emerging, including cardiogenesis. Based on these results, we differentiated irradiated mESCs into cardiomyocytes. On day 5, 8 and 12 of differentiation, treated cells showed a significant alteration (qRT-PCR) of the expression of marker genes (Gata-4, Nkx-2.5, Tnnc1 and Alpk3) when compared to control cells. At day 15 of differentiation, although the organization of sarcomeric α-actinin and troponin T proteins appeared similar in cardiomyocytes differentiated from either mock or treated cells, the video evaluation of the kinematics and dynamics of the beating cardiac syncytium evidenced altered contractile properties of cardiomyocytes derived from irradiated mESCs. This alteration correlated with significant reduction of Connexin 43 foci. Our results indicate that mESCs populations that survive an ionizing irradiation treatment are capable to differentiate into cardiomyocytes, but they have altered contractile properties.

Keywords: Cardiomyocyte; Embryonic stem cells; Ionizing radiation; Transcriptome.