Abstract
Ischemic heart disease is the leading cause of mortality in diabetes mellitus and coronary endothelial dysfunction is implicated in the development and progression of cardiac ischemia. HuR (Human antigen R, also known as ELAVL1) is an RNA binding protein which binds to and stabilizes mRNAs that contain AU‐rich elements in 3′‐UTRs. HuR is ubiquitously expressed and regulates many cellular functions via altering mRNAs’ stability. In this study, we explore the role of HuR in coronary endothelial dysfunction in diabetic mice. Type 1 diabetes was induced in mice by high‐dose streptozotocin (STZ) injection and type 2 diabetes by low‐dose STZ injection with high‐fat diet. Mouse coronary endothelial cells (MCECs) were isolated using magnetic beads coated with CD31. The protein levels of HuR in the nuclear and cytosolic fraction were assessed by immunofluorescence staining and western blot. Ribonucleoprotein immunoprecipitation (RIP) was performed to determine the binding of our focus RNAs to HuR in ribonucleoprotein (RNP) complexes. Mitochondrial calcium concentration was measured by Rhod2‐AM staining. Our western blot data demonstrate that MCECs isolated from diabetic mice exhibited significantly lower concentration of HuR in the nuclear fraction, but not in cytoplasm, compared to control. We also confirmed that nuclear HuR expression was markedly reduced in diabetic MCECs using immunofluorescence staining. We predicted the target mRNAs of HuR and performed RIP for those mRNA. Among the mRNAs which we tested, hexokinase 2 (HK2) mRNA was detected in the RNP complex of HuR. Furthermore, we found that the binding of HuR to HK2 mRNA was significantly decreased in MCECs isolated from diabetic mice compared to control. Mitochondrial calcium concentration and the protein expression of voltage dependent anion channel (VDAC), which modulates calcium uptake into mitochondria, were significantly increased and HK2, an endogenous negative regulator of VDAC, was significantly decreased in diabetic MCECs in comparison to control. In addition, HK2 overexpression in diabetic MCECs attenuated mitochondrial calcium uptake toward control levels. In conclusion, our data suggest that decreased HuR level in the nucleus reduces the stability of HK2 mRNA, downregulates HK2 protein expression, and subsequently increases mitochondrial calcium uptake in diabetic MCECs. HuR might be a potential therapeutic target for ischemic heart disease in diabetic patients via restoring coronary endothelial function.Support or Funding InformationThis work was supported by National Heart, Lung, and Blood Institute Grant R01 HL‐115578 (A. Makino)
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