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E2F6 attenuates the hypoxia mimetic cobalt chloride induced apoptosis of cardiomyocytes.
Introduction: The E2F pathway governs cell growth, differentiation, and death in all cell types. To interrogate the role of this pathway in postnatal myocardium we generated a transgenic mouse model (Tg) with cardiac specific expression of the repressor E2F6 which deregulates the E2F response (1). The Tg mice developed a E2F6 dose dependent dilated cardiomyopathy in the absence of any hypertrophy or cell death (1,2). Here we examined whether E2F6 serves as an anti-apoptotic agent to attenuate cell death of postnatal cardiomyocytes to drugs such as doxorubicin (dox) and the hypoxia mimetic agent cobalt chloride (CoCl2).
Methods/Results: Microarray analysis of wild type (Wt) E2F6- transgenic (Tg) mice revealed an increase in genes which regulate the DNA damage response which was confirmed by RT-qPCR: Chek1 kinase (3.6 fold, p-value: 0.0005), Rad51 (2 fold, p-value: 0.01), and Blm2 (4 fold, p-value: 0.006). Western blot analysis of Chek1 confirmed a threefold up-regulation at the protein level (p-value: 0.022). Neonatal cardiomyocytes (NCM) were isolated from Wt and Tg mouse myocardium at post-natal day 1 and treated with dox or CoCl2. At 500nM dox induced caspase 3 cleavage and p53 acetylation in both Wt and Tg NCM to a similar extent. E2F6 expression was down-regulated following 3 hours of dox exposure and completely abolished after 24hr. In HeLa cells, dox also targeted the endogenous degradation of E2F6 (50% reduction, p-value: 0.04; with 500nM dox; 90% reduction, p-value: 0.008 with 1uM), while the apoptotic E2F1 was up-regulated (1.4 fold p:0.04; 500nM dox and 2 fold,p-value:0.004 by 1uM). Treatment of NCM with the CoCl2 revealed that E2F6 attenuated the induction of caspase 3 cleavage by 50 fold (p-value:0.02) as well as the diminishing the bax: bcl2 ratio. CoCl2 treatment of Wt NCM resulted in a two fold increase in the number of TUNEL positive cells while E2F6 Tg NCM were unaffected. Unlike dox, CoCl2 treatment did not inhibit E2F6 levels in NCM further demonstrating that E2F6 is anti-apoptotic.
Conclusion: The data demonstrate that E2F6 protects against the drug induced apoptosis of cardiomyocytes. Further, dox appears to confer cell death via post-transcriptional mechanisms targeting the degradation of E2F6 and upregulation of the proapoptotic E2F1.
1. Westendorp B, et al. (2012). FASEB J. 26: 2569-2579.
2. Major JL et al. (2015). J Mol Cell Cardiol. 84: 179-190.