Abstract 16331: Netrin-1 Abrogates Ischemia Reperfusion Induced Cardiac Mitochondrial Dysfunction via Attenuation Of NOX4 And NOS Uncoupling
Oxidative stress has been shown to mediate cardiac ischemia reperfusion (I/R) injury. We have previously characterized a novel pathway by which netrin-1 induces cardioprotection at least partially via a DCC/ERK1/2-dependent nitric oxide (NO) production. We have further shown that the protective effects of netrin-1 also occurs in vivo using a classic coronary artery ligation model of I/R. The present study aim to determine if netrin-1 also additionally regulates the oxidative stress pathways. We hypothesize that netrin-1 protects the heart partially via the attenuation of superoxide production derived from NADPH oxidase (NOX) and uncoupled nitric oxide synthase (NOS). To this end, hearts rapidly removed from 5-7 weeks old C57BL6 mice were perfused using a Langendorff system, and subjected to a 20 minute global ischemia, followed by a 60 minute reperfusion. Total superoxide production measured with electron spin resonance (ESR) shows a ~2.5 fold increase in I/R-injured hearts compared with controls (p<0.001), which was eliminated by netrin-1 perfusion to baseline. We then examined the expression level of NOX proteins as a possible source of superoxide, and found that NOX4 was increased by ~1.7 fold in I/R-injured hearts compared to controls, with netrin-1 reducing this overexpression to ~1.27 fold. Analysis of L-NAME sensitive superoxide production, an indicator of NOS (un)coupling activity, revealed that NOS was uncoupled by I/R, and recoupled by netrin-1 perfusion. We further found that siRNA-mediated NOX4 silencing was able to recouple NOS, reduce infarct size, and improve mitochondrial function. Finally, recoupling of NOS with sepiapterin was also effective in reducing infarct size and improving mitochondrial function in I/R-injured heart. In summary, our data demonstrate a novel pathway for netrin-1's cardioprotective effect, namely from a reduction of NOX4 expression, to the restoration of NOS function, to improving mitochondrial function and ultimately decrease in infarct size resulting from I/R injury. These data compliment our previous findings on netrin-1's pathway involving DCC, ERK1/2, eNOS activation and NO to give a more complete picture of the signaling events that occurs to mediate netrin-1 induced cardioprotection.
- © 2011 by American Heart Association, Inc.