The effect of antineoplastic drugs on commitment, differentiation and maturation of embryonic stem cells toward cardiomyocytes

Purpose: Embryonic stem cells (ES) represent a suitable model to study cardiotoxic effect of endogenous or exogenous substances. Indeed the cardiotoxic effect of antineoplastic therapy is a common hitch in treated patients and limits the efficacy of therapy. Recently it has been proposed that these effects, being cardiomyocytes terminally differentiated, may be due to an "extra-sensitive" population represented by resident progenitors of cardiomyocytes. This may also explain the relatively high and delayed incidence of cardiac complications in patients treated for childhood cancers. However, the sensitivity of stem cells to antineoplastic drugs has not been investigated. We aimed to study the effect of doxorubicin and imatinib on commitment, differentiation and maturation of stem cells toward cardiomyocytes. Methods: Mouse ES (mES, CGR8) cells were propagated in medium and differentiated into cardiomyocytes via formation of embryoid body (EB). We evaluated the effect of imatinib (1 and 0.1 M) and doxorubicin (Doxo, 0.1, 0.5 and 1 M)at different stage of the differentiation process. The effect of drugs on viability was studied by MTT (Thiazolyl Blue Tetrazolium Bromide) test. The effect of drugs on function were evaluated by measuring the percentage of beating EBs. To get information on the molecular mechanisms underlying or associated with the functional results, we used quantitative RT-PCRtoinvestigate the expression of transcription factor involved in cardiac differentiation (GATA-4, Nkx2.5, MEF2C). Results: Adding Doxo at the first day of differentiation processhindered EB formation. Thus, we added Doxo onto already formed EBs, by exposing EBs for 48 hours, starting from day 2 of EB formation.Also in this condition, Doxo showed a marked toxicity (MTT test) and prevented cell growth and the expression of cardiac specific markers. Surprisingly, when added to beating EBs (day 7-8), a 48-hour exposure to Doxo did not influence EB contractility. On the contrary, imatinib did not show any toxic effect on EB formation, cell growth and contractility. Conclusion: Antineoplastic drugs exert different effects on cardiac commitment and differentiation of mES. Moreover, the window for Doxo toxicity seems to be restricted to the early stages of cell commitment, namely EB formation. Thus, it seems important to verify the effects of Doxo on cell aggregation and proliferation as well as expression of early mesodermic transcription factors.