Abstract 16892: Tetrahydrobiopterin Increases Myocardial Tolerance to Ischemia/reperfusion Injury in Diabetes Mellitus and Hyperglycemia by Preventing eNOS Uncoupling
Tetrahydrobiopterin (BH4), an essential factor for endothelial nitric oxide synthase (eNOS), is significantly decreased in diabetes and hyperglycemia (HG). We hypothesized that modulation of cardiac BH4 in diabetes and HG alters myocardial tolerance to ischemia/reperfusion (I/R) injury by changing eNOS coupling. Type 2 diabetic db/db mice at the age of 12- 14 weeks showed severe obesity and significantly elevated plasma concentrations of glucose compared with C57BLKS/J control mice (420±11 vs. 200±17 mg/dl, n=8, P<0.05). Baseline cardiac BH4 concentrations measured by HPLC were significantly lower in db/db than C57BLKS/J mice (2.5±0.4 vs. 5.2±0.7 pmol/mg protein, n=5, P<0.05). Fractional shortening and mitral E/A ratio of left ventricle determined by echocardiography were significantly smaller in db/db than C57BLKS/J mice. In Langendorff-perfused hearts, left ventricular developed pressure and +dP/dt were significantly smaller in db/db than C57BLKS/J mice at baseline and during 2 h of reperfusion following 30 min of no-flow ischemia. Endothelial cells (ECs) were co-cultured with cardiomyocytes (CMs) and underwent 2 h of hypoxia followed by 2 h of reoxygenation. Addition of ECs to CMs at a ratio of 1:3 significantly increased NO production measured by ozone chemiluminesence and decreased lactate dehydrogenase activity in normoglycemic media compared to CMs alone. This EC-derived protection was abolished by prior incubation of ECs in hyperglycemic media. Interestingly, the addition of 100 μM sepiapterin (a BH4 precursor) and increased expression of GTP cyclohydrolase 1 in ECs by GTP cyclohydrolase 1 overexpressor shRNA increased endothelial BH4 levels and NO production and restored the protective effects of ECs in the presence of HG. The ratio of eNOS dimmer/monomer determined by non-denaturing, low-temperature SDS-PAGE was significantly decreased by HG but restored by both sepiapterin and GTP cyclohydrolase 1 overexpression in ECs. These results demonstrate that decreased cardiac BH4 and diabetic cardiomyopathy attenuate tolerance to I/R injury in diabetes, and that increased BH4 by pharmacological and genetic approaches prevents eNOS uncoupling and increases myocardial tolerance to I/R injury during HG.
- © 2012 by American Heart Association, Inc.