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  • br Materials and methods br

    2019-07-08


    Materials and methods
    Results
    Discussion In this study, we demonstrate that SIRT1-mediated deacetylation is required for IPO to activate the Akt signaling pathway to confer cardioprotection. Reduced SIRT1 and increased Akt acetylation are responsible for the loss of IPO-mediated cardioprotection in diabetes. Up-regulation of SIRT1 restores IPO-mediated protective effects in diabetic hearts via deacetylation-dependent activation of Akt. To be the best of our knowledge, this is the first study demonstrating that the reduction of SIRT1 blunts the cardioprotective effects of IPO in diabetes by impairing the Akt signaling pathway (Fig. 12). SIRT1 is a nicotinamide Chloramphenicol dinucleotide (NAD+)-dependent protein deacetylase regulating many important physiologic processes including cell survival, metabolism and energy balance [29]. Previous studies from our lab and others have demonstrated that up-regulation of SIRT1 protects the heart against I/R injury in non-diabetic and diabetic animals, while cardiac specific SIRT1 knockout exacerbates myocardial injury caused by I/R [17,30]. Nadtochiy et al. have found that ischemic preconditioning (IPC) enhances SIRT1-mediated lysine deacetylation and the protective effects of IPC is blunted by SIRT1 knockdown (SIRT1+/−) or specific SIRT1 inhibitor splitomicin [31,32]. Nevertheless, Adam et al. have reported that IPC-mediated protection against MI/R is not prevented by SIRT1 inhibitor SIII [33]. The inconsistencies between these studies may be due to variations in the animal models and/or inhibitor specific. In addition, Potenza et al. have demonstrated that inhibition of SIRT1 abrogates the protective effects of adiponectin preconditioning against MI/R injury [34], suggesting that SIRT1 plays an important role in the cardioprotective effects of pharmacological preconditioning with adiponectin. Since the onset of ischemia is an acute and unpredictable event, the clinical use of ischemic pre-conditioning is limited and current attention is focused largely on IPO to alleviate MI/R injury [35]. However, the role of SIRT1 in IPO-mediated cardioprotection, especially in blunted cardioprotection of IPO in diabetes is still largely unknown. Our study demonstrated for the first time that up-regulation of SIRT1 in diabetic hearts restored IPO-mediated protective effects. Interestingly, even without diabetes, SIRT1 deficiency alone is sufficient to blunt the protective effects of IPO. Both gain- and loss-of-function experiments indicate that the reduction of SIRT1 is responsible for the blunted protective effects of IPO in diabetic hearts. Numerous studies have identified a number of signaling pathways are involved in the myocardial protection of IPO [[36], [37], [38]]. Among these pathways, the activation of Akt by phosphorylation is considered as an initial step that induces phosphorylation of downstream kinases such as GSK-3β to inhibit the opening of mitochondrial permeability transition pore and reduce the necrotic effects of reperfusion [6,39]. Loss of IPO-mediated cardioprotection has been previously described in both type 1 and type 2 diabetic animal models [10,11,40]. The complete or partial failure of IPO to reduce MI/R injury in diabetes is attributed to disrupted signaling pathways. Xue et al. showed that phosphorylated Akt was decreased in STZ-induced type 1 diabetic rats compared with that in control rats at baseline [41]. Bouhidel et al. reported that phosphorylated Akt was increased in type 2 diabetic ob/ob mice at baseline [10]. In our study, the expression of basal phosphorylated Akt was not significantly changed in HFD-STZ-induced diabetic sham hearts compared with non-diabetic sham hearts. These inconsistent findings may be due to different diabetic animal models, which may represent different situations in diabetic subjects. Our study observed that there was also no significant change in the expression of total Akt between diabetic and non-diabetic sham hearts, it is then speculated that post-translational modifications may hinder IPO-induced Akt phosphorylation in diabetes.