Abstract 21200: Netrin-1 Attenuates Superoxide Production in Ischemia/Reperfusion-Injured Heart: Roles of NADPH Oxidase 4, Uncoupled eNOS, and Mitochondrion
Introduction: Netrin-1 is an axon-guiding protein. Others and we have shown that netrin-1 stimulates angiogenesis in adult endothelial cells. In addition, we have recently demonstrated that netrin-1 functions as an extremely potent cardioprotective agent.
Hypothesis: Netrin-1 inhibits superoxide production in ischemia/reperfusion (I/R)-injured hearts via attenuation of NAD(P)H oxidase 4 (NOX4), uncoupled eNOS and mitochondrion.
Methods and Results: Mouse hearts isolated from C57BL6 mice were perfused using a Langendorff system, and subjected to 20 min ischemia and 1 hr reperfusion. L-NAME-sensitive superoxide production (reflective of eNOS uncoupling activity), and superoxide production from membrane fractions and isolated mitochondria, was determined by electron spin resonance. Pre-perfusion with netrin-1 markedly reduced infarct size of I/R-injured hearts (42.5±3.6% to 21.8±4.9%), via upregulation of nitric oxide production. This phenomenon is now further confirmed using an in vivo model of myocardial ischemic injury (30 min coronary occlusion followed by 24 hr reperfusion). Total superoxide production in I/R-injured hearts was substantially increased by 2.5-fold, which was completely attenuated to baseline by netrin−1. Intriguingly, netrin-1 treatment abolished eNOS uncoupling in I/R-injured hearts. Pre-perfusion with DCC-antibody, or MEK1/2 inhibitor U0126, completely reversed netrin-1's protective effects on eNOS uncoupling. The robust upregulation of NAD(P)H oxidase 4 (NOX4) by I/R, and the I/R-provoked increase in membrane superoxide production, were both attenuated by netrin−1. NOX1 or NOX2 expression however, was unaffected. We have previous shown that NOX lies upstream of uncoupled eNOS (Proc Natl Acad Sci U S A, 2005). These data seem to suggest that netrin-1 may protect eNOS from being uncoupled via attenuation of NOX4. Furthermore, mitochondrial production of superoxide was also diminished by netrin−1.
Conclusions: Our data demonstrate that netrin-1 inhibits superoxide production in I/R-injured hearts via attenuation of NOX4, uncoupled eNOS, and mitochondrion. This response is critical in preserving nitric oxide bioavailability.
- © 2010 by American Heart Association, Inc.