QKI Deficiency Promotes FoxO1 Mediated Nitrosative Stress And Endoplasmic Reticulum Stress Contributing To Increased Vulnerability To Ischemic Injury In Diabetic Heart

BackgroundAs the improved standard of living today, diabetes is becoming more and more prevalent in the whole world. Among the varieties of causes of diabetic death, ischemia heart disease plays the most critical role. What’s worse, it shows more severe injury in the myocardial ischemia/reperfusion(I/R) stress. That is to say, hearts of diabetic individuals are susceptible to I/R injury. In addition, recent studies have revealed that, the disturbance of molecular metabolism in diabetes contributes to the decrease of diabetic cardioprotection. However, the mechanism underlying the impaired cardioprotection in diabetic hearts remains elusive.Quaking 5(QKI5), the member of STAR family, is known to link cell signaling to the cellular survival and QKI dysregulation may contribute to human diseases. Our previous study has shown that QKI5 can specifically reduce the activity of Fox O1 and protect heart from ischemia/reperfusion-induced injury, indicating the protection role of QKI5 in myocardiovascular metabolism. We hypothesized that the deficiency of QKI5 might contribute to the activation of Fox O1 and be related to the ischemic intolerance of diabetic hearts.AimsThe purpose of our current study was to investigate the role of QKI5 in diabetic myocardial ischemia/reperfusion injury and to identify new molecular mechanisms potentially contributive to the susceptibility to ischemic vulnerability of diabetic myocardium.MethodsMale leptin-deficient(ob/ob) mice and their wild-type(WT) C57BL/6 mice were subjected to myocardial I/R(30min/4h). Myocardial apoptosis was determined by TUNEL staining and caspase-3 activity. Myocardial infarct size was assessed by Evans blue-TTC double staining methods. Cardiac function was determined by echocardiography. Downregulation of Fox O1 and overexpression of QKI5 were used by selective Fox O1 si RNA and Adv-QKI5, respectively. Western blotting was used to evaluate the expression level of QKI5, Fox O1, nitrosative stress and ER stress. RNA-IP was used to identify the relationship between QKI5 and Fox O1 m RNAResults1. Compared with C57BL/6 mice, the general feature, IPGTT, ITT and echocardiographic measurement of ob/ob mice indicated that ob/ob mice can be used as diabetic mode. Diabetic ob/ob mice manifested profoundly greater cardiac injury than WT controls. MI/R-induced LDH release and infarct size were enlarged in ob/ob mice. 2. Total cardiac Fox O1 and nuclear Fox O1 expression was significantly higher in ob/ob mice than WT animals, while cytoplasmic Fox O1 in ob/ob mice hearts was significantly decreased as compared to WT animals. These results demonstrated thatcardiac FoxO1 is over-activated under conditions of diabetes. 3. There was significant nitrosative stress in ob/ob myocardium. Cardiomyocyte apoptosis was more severe in ob/ob comparing to WT myocardium when subjecting to MI/R treatment. It is noteworthy that although blockage of excess nitrosative stress from i NOS, treatment with 1400 W or M40401 effectively, rather than completely, reduced additional cardiomyocyte apoptosis in ob/ob animals. These results further suggest that there are other potential mechanisms involved in accelerating MI/R injury observed in diabetic heart. 4. Expression of CHOP, phosphorylation of PERK and e IF2α were all up-regulated in ob/ob myocardium comparing to WT one. It was showed that the ER stress related cleaved-caspase-12 was exacerbated in diabetic MI/R hearts, and inhibited by SAL treatment. But, treatment of SAL partially, rather than completely, reduced MI/R-induced caspase-3 activity in diabetic MI/R hearts. 5. To support that Fox O1 over-activation in diabetic hearts may engage both harmful stress to induced cardiomyocyte apoptosis, we knocked Fox O1 level in ob/ob mice hearts. Accordingly, the knockdown of Fox O1 effectively inhibited nitrosative stress and ER stress, then protected ob/ob hearts from I/R injury. 6. We first found that QKI5 was significantly down-regulated in diabetic mice. In order to investigate the protective role of QKI5, we upregulated the expression of QKI5 by intramyocardial injection of Ad-QKI5 in ob/ob heart availably inhibited the Fox O1 overexpression, resulting in decrease of nitrosative stress and ER stress, then improved the ischemic tolerance in diabetic hearts. 7. In order to detect the interaction between QKI5 and Fox O1, an RNA co-immunoprecipitation experiment and half-life of Fox O1 was performed. We found that overexpression of QKI5 inhibited the m RNA and protein levels of Fox O1. Half-life of Fox O1 m RNA was shortened in QKI5 overexpression. These data suggest that QKI5, as an RNA binding protein, destabilizes Fox O1 m RNA.Conclusions1. We first provide direct evidence linking QKI5 deficiency to Fox O1 overactivation inthe hearts of ob/ob mice. Moreover, as the selectively negative regulator of Fox O1, QKI5 plays a protective role in diabetic I/R injury. 2. The overexpression of QKI-5 repressed Fox O1 via decreasing its m RNA stability in cardiomyocytes. 3. Fox O1 aggravated I/R injury via induction of nitrosative stress and ER stress, highlighting the role of Fox O1 in I/R injury of diabetic myocardium.