Despite an established role of mitochondrial dysfunction in cardiac ischemia/reperfusion (I/R) injury, the upstream activators have remained incompletely defined. We have recently identified an innovative role of exogenously applied netrin-1 in cardioprotection, which is mediated by increased nitric oxide (NO) bioavailability. Here, we tested the hypothesis that this "pharmacological" treatment of netrin-1 preserves mitochondrial function via novel mechanisms that are NO dependent. Freshly isolated C57BL6 mouse hearts were perfused using a Langendorff system, and subjected to a 20min global ischemia/60min reperfusion, in the presence or absence of netrin-1. I/R induced marked increases in infarct size, total superoxide and hydrogen peroxide production, activity and protein abundance of NADPH oxidase (NOX) isoform 4 (NOX4), as well as impaired mitochondrial integrity and function, all of which were attenuated by netrin-1. This protective effect of netrin-1 is attributed to cGMP, a downstream effector of NO. The protein levels of NOX1 and NOX2 were however unaffected, and infarct size from NOX1 and NOX2 knockouts was not different from wild type animals. Scavenging of NO with PTIO reversed inhibitory effects of netrin-1 on NOX4, while NO donor attenuated NOX4 protein abundance. In vivo NOX4 RNAi, or sepiapterin perfusion, resulted in recoupling of NOS, decreased infarct size, and blockade of dysfunctional mitochondrial swelling and mitochondrial superoxide production. These data demonstrate that netrin-1 induces cardioprotection through inhibition of NOX4 activity, which leads to recoupling of NOS, augmented NO bioavailability, reduction in oxidative stress, and ultimately preservation of mitochondrial function. The NO-dependent NOX4 inhibition connects with our previously established pathway of DCC/ERK1/2/eNOS/NO/DCC feed-forward mechanism, to maintain NOS in the coupling state to attenuate oxidative stress to preserve mitochondrial function. These findings may promote development of novel therapeutics for cardiac I/R injury. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease".
Keywords: Ischemia/reperfusion (I/R) injury; Mitochondrial dysfunction; NADPH oxidase isoform 4 (NOX4); NO synthase (NOS); NOS uncoupling; Netrin-1.
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