Micro RNA array analysis in OLETF, a type 2 diabetic rats, and LETO, a non-diabetic control rats

AMP deaminase (AMPD) plays a crucial role in adenine nucleotide metabolism, and its upregulated activity contributes to diastolic dysfunction of the heart caused by type 2 diabetes mellitus (T2DM) via reduction of the adenine nucleotide pool. Here we examined the mechanism of AMPD upregulation by T2DM. The protein level of 90-kDa full-length AMPD3 was increased in whole myocardial lysates and in the sarcoplasmic reticulum (SR) fraction by 55% and 123%, respectively, in OLETF, a rat model of T2DM, compared to those in LETO, a non-diabetic control, though AMPD mRNA level was unchanged in OLETF. MicroRNA array analysis revealed downregulation (>50%) of 57 microRNAs in OLETF compared to those in LETO, among which miR-301b was predicted to interact with the 3’UTR of AMPD3 mRNA. The 90-kDa AMPD3 level was significantly increased by miR-301b inhibitor, but decreased by a miR-301b mimetic in H9c2 cells. A luciferase reporter assay indicated binding of miR-301b to the 3’UTR of AMPD3 mRNA. The results indicate that translational regulation by miR-301b mediates AMPD3 upregulation in the diabetic heart. Since ATP generated by SR-associated glycolytic enzymes contributes to Ca2+ uptake by SERCA2a, upregulated AMPD3 in the vicinity of the SR may be a therapeutic target for diabetic cardiomyopathy. Rats were anesthetized and ventilated as in in vivo hemodynamic experiments. Hearts of the rats were excised, rapidly frozen in liquid nitrogen, and stored at -80°C until further use. The expression of a set of 595 miRNAs in rat heart samples was examined using a Taqman® Rodent MiRNA Array v2.0 (Applied Biosystems, Foster City, CA).