Depletion Of Rbl2 Exacerbates Cardiomyocyte Apoptosis And Ischemia-reperfusion Injury By Augmenting E2f1-mediated Bnip3 Expression

Background: Reperfusion therapy, an effective treatment for myocardial infarction, triggers ischemia-reperfusion (I/R) injury and eventually may result in heart failure. Retinoblastoma-like 2 (Rbl2), a major retinoblastoma family member expressed in the heart, maintains the postmitotic state of adult cardiac myocytes. However, the role of Rbl2 in myocardial I/R injury remains unclear. We hypothesize that Rbl2 deficiency exacerbates myocardial injury following I/R. Methods and results: Wild type C57BL/6 (8–10-week, male) mice were subjected to 30 min of ischemia followed reperfusion. I/R induced phosphorylation of Rbl2 at Ser952, which has been associated with Rbl2 protein inactivation. To determine the role of Rbl2 in vivo, Rbl2-deficient mice and wild-type littermates were subjected to I/R and infarct size was evaluated by Evans blue/TTC staining. Rbl2 deficiency significantly increased infarct size at 24 h post I/R when compared with wild-type littermate controls. Echocardiography and Masson’s trichrome staining revealed that Rbl2 deficiency exacerbated I/R-induced cardiac dysfunction and fibrosis. Moreover, ablation of Rbl2 exacerbated I/R-induced cardiomyocyte apoptosis, as evidenced by the increased TUNEL positive signal. Consistently, knockdown of Rbl2 augmented H 2 O 2 -induced cleavage of PARP and caspase 3 in neonatal rat cardiomyocytes , suggesting that depletion of Rbl2 exacerbated oxidative stress-induced cardiomyocyte apoptosis. Mechanistically, both I/R and H 2 O 2 induced expression of the pro-apoptotic protein BNIP3, which was augmented by depletion of Rbl2. Since the BNIP3 promoter contains an E2F-binding site, we further examined the levels of the transcriptional activator E2F1 and the transcriptional repressor E2F4. Western blotting revealed that disruption of Rbl2 reduced E2F4 but increased E2F1 levels in mouse heart both at baseline and following I/R. Conclusion: Our findings suggest that Rbl2 deficiency exacerbates cardiomyocyte apoptosis and ischemia-reperfusion injury by augmenting E2F1-mediated BNIP3 expression.