Abstract 16412: Altered Nitroso/Redox Balance in Dystrophic Cardiomyopathy is Associated With NOS1 Uncoupling
Introduction: Dystrophic cardiomyopathy is the cardiac manifestation of Duchenne muscular dystrophy. Hearts from the mdx mouse, the mouse model of this disease, exhibit increased oxidative stress due to the up-regulation of NADPH oxidase.
Hypothesis: We tested the hypothesis that this oxidative stress disrupts the bioavailability of tetrahydrobiopterin (H4B) leading to defective nitric oxide (NO) production associated with NOS1 uncoupling.
Methods: We used mdx mice with established cardiomyopathy. Real-time PCR was used to evaluate NADPH oxidase -NOX2- and western blots were performed to assess protein expression or phospholamban (PLB) phosphorylation. NO production was assessed by either nitrate + nitrite measurement in heart homogenate or DAF-2 epifluorescence in myocytes. The degree of S-nitrosylation in myocytes was measured using immunofluorescence.
Results: We found a five-fold increased expression of NOX2 as well as increased superoxide production compared to wild type (WT; p<0.05 vs. WT) in mdx hearts. PLB phospho-Serine 16 was decreased (p<0.05 vs. WT) and PLB phospho-Threonine 17 was augmented (p<0.01 vs. WT) in mdx hearts and these changes were restored to normal by specifically inhibiting NOX2 with 20 microM VAS2870. In addition, NOS1 expression was increased eight-fold in mdx hearts compared to WT (p<0.05 vs. WT). Furthermore, cardiac NO production was reduced in mdx (p<0.05 vs. WT) and also was restored by VAS2870 in cardiomyocytes. There was less S-nitrosylation in mdx myocytes, which consistently showed decreased levels of S-nitroscysteines compared to WT. This paradoxical effect could be due to NOS1 uncoupling. To test this possibility, we treated isolated cardiomyocytes with 300 microM H4B and observed that NO production in mdx recovered to WT levels and S-nitrosylation levels were restored toward normal. H4B treatment also increased PLB phospho-Serine 16 but did not affect PLB phospho-Threonine 17.
Conclusions: These results suggest that NOX2-derived oxidative stress results in H4B oxidation, causing NOS1 uncoupling. Diminished NOS1 activity has functional implications for mdx cardiomyocyte performance.
Author Disclosures: R.A. Dulce: None. V. Mayo: None. Y. Cao: None. A.V. Treuer: None. D.R. Gonzalez: None. J.M. Hare: Employment; Modest; Vestion. Ownership Interest; Modest; Biscayne. Ownership Interest; Significant; Vestion Inc.
- © 2014 by American Heart Association, Inc.