Durante, M.

Frank, S.

Helm, A.

Ritter, S.

GSI Repository

Cellular radiation response of mouse embryonic stem cell derivedcardiomyocytes ∗S. Frank1,2, A. Helm1, M. Durante1,2, and S. Ritter† ,11GSI, Darmstadt, Germany; 2Technische Universita¨t Darmstadt, GermanyTo examine putative adverse effects of ionizing radia-tion on electrophysiological properties of cardiomyocytes,we recently performed a series of experiments with mouseembryonic stem cell derived cardiomyocytes (mESC-CM).For these studies mESC-CM were generated via embryoidbody (EB) formation. Yet, this system proved to be unsuit-able due to a low number of cardiomyocytes formed andpronounced inter- and intra-experimental variations [1]. Toovercome these limitations pure mESC-CM (Cor.At car-diomyocytes, Axiogenesis) have been used in subsequentelectrophysiological studies [2]. As a complement we alsoexamined the cellular radiation response of Cor.At cells.For the experiments cells were seeded in chamber slides.Two days after seeding cells were irradiated with 0.5, 1or 2 Gy X-rays (250 kV, 16 mA, 1.5 Gy/min). Radiationeffects were examined at 1, 2, 3 and 4 days after expo-sure. Apoptosis, micronucleus formation and binucleationwere analyzed by means of a DAPI nuclear staining; whilethe expression of connexin 43 (Cx 43) and troponin T wasmeasured by immunocytochemistry (Figure 2 B, C). Ourexperiments show a dose- and time-dependent increase inthe number of cells with micronuclei and in the number ofapoptotic cells. Exemplarily, in figure 1 the apoptotic re-sponse of control and irradiated cultures (2 Gy X-rays) isplotted. In the control the spontaneous frequency of apop-totic cells was ≤8%. At 1 day after exposure to 2 Gy X-rays the number of apoptotic cells was as high as in thecontrol samples, but it increased to 17% and 33% at day 2and 4 after exposure. Interestingly, the number of binucle-ated cells, indicating maturation, rose in all samples withtime and was not affected in the dose range examined (datanot shown). Additionally, we analyzed the expression ofCx 43 (Figure 2 C), a main compound of cardiac gap junc-tions, in comparison to the expression of troponin T (Figure2 B), a structural cardiac protein. An up-regulation of Cx43 expression after an exposure to carbon ions has beenobserved for rabbit cardiomyocytes resulting in improvedconductivity [3]. The analysis performed so far revealedpronounced sample variations, i.e. no consistent pictureemerged. Altogether, our first experiments show that dosesup to 2 Gy X-rays do not affect the maturation of Cor.Atcardiomyocytes. However, a dose-dependent increase inthe number of apoptotic cells and cells carrying micronu-clei was detected. Due to the high sample variations, nofirm conclusions on the impact of X-rays on the expression∗The research leading to these results has received funding fromthe Euratom Seventh Framework Programm under grant agreement n◦295823 (PROCARDIO)† s.ritter@gsi.deof Cx 43 and subsequently cellular communication can bedrawn. In further studies refined technique will be usedto study the expression of specific cardiac genes and firstexperiments with high LET radiation will be performed.Figure 1: Percentage of apoptotic Cor.At cardiomyocytesin unirradiated or X-ray irradiated cultures. Cells were ex-posed at day 0.Figure 2: Immunofluorescence staining of Cor.At cells forconnexin 43 (A) and troponin T (B), nuclei in (C) arestained with DAPI; merged images are shown in (D). Bin-ucleated cells are indicated by an arrowhead in (C, D).References[1] Materna et al., GSI Scientific Report 2012, 441[2] Helm et al., GSI Scientific Report 2013 (this issue)[3] Amino et al., Cardiovasc Res, 2006, 72:412-421BIOPHYSICS-20 GSI SCIENTIFIC REPORT 2013222 doi:10.15120/GR-2014-1-BIOPHYSICS-20

Cellular radiation response of mouse embryonic stem cell derived cardiomyocytes

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Cellular radiation response of mouse embryonic stem cell derived cardiomyocytes

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