Abstract 10007: AMP Deaminase in Mitochondria-Associated ER Membranes Contributes to Reduction of the Threshold for Mitochondrial Permeability Transition in Type 2 Diabetic Hearts

Introduction: Despite the growing clinical burden of diabetic cardiomyopathy, there is no specific treatment for the disorder. We previously demonstrated that increased activity of AMP deaminase (AMPD) contributes to diastolic dysfunction in diabetic myocardium through ATP depletion and augmentation of xanthine oxidase-mediated ROS production. Additionally, the threshold for mitochondrial permeability transition (mPT), a major mechanism of cell necrosis, was found to be reduced by diabetes. Hypothesis: Increased localization of AMPD in mitochondria-associated ER membranes (MAMs) lowers the threshold for mPT, via accelerated Ca 2+ transport from the ER, by interaction with IP3 receptor (IP3R), VDAC and/or mitochondrial Ca 2+ uniporter (MCU). Methods and Results: The 90-kDa full-length and 70-kDa N-terminus-truncated form of AMPD3, a main isoform in rat hearts, were detected in the myocardium. The 90-kDa full-length AMPD3 was exclusively observed in outer mitochondrial membranes (OMMs), and both 90-kDa AMPD3 and 70-kDa AMPD3 were observed in MAMs. The 90-kDa AMPD3 levels in OMMs and MAMs were significantly higher in OLETF, type 2 diabetic rats, than in LETO, non-diabetic control rats, while 70-kDa AMPD3 levels were comparable in OLETF and LETO. The area of the MAM quantified by using electron micrographs was 57% larger in OLETF than in LETO (p<0.05). Immunoprecipitation experiments showed that 90-kDa AMPD3 was associated with VDAC, whereas 70-kDa AMPD3 did not interact with IP3R, VDAC or MCU. Ca 2+ retention capacity (CRC), an index of the threshold for mPT, analyzed in MAM-containing crude mitochondria was 21% lower in OLETF than in LETO (p<0.05). Although an inhibitor of AMPD (cpd3) had no effect on CRC, knockdown of 90-kDa AMPD3 significantly ameliorated H 2 O 2 -induced loss of mitochondrial membrane potential that was determined by TMRE in cultured isolated cardiomyocytes. Conclusions: The results suggest that 90-kDa AMPD3 in MAMs plays a role in regulation of the mPT threshold in a deaminase activity-independent manner and that increased AMPD3 in MAMs contributes to mitochondrial respiratory dysfunction in diabetic myocardium.