Non-canonical modulation of mitochondrial ROS and dynamics by Netrin-3 drives inflammasome activation in...

Heart failure is driven by pathological remodeling in which inflammation and mitochondrial dysfunction reinforce each other, yet the molecules that coordinate this interplay remain poorly defined. Netrin-3 is a guidance cue with no known function in the heart. We asked whether Netrin-3 acts in cardiomyocytes to affect mitochondrial integrity under inflammatory stress.

We used neonatal rat cardiomyocytes treated with nigericin to activate the NLRP3 inflammasome. Netrin-3 was overexpressed via adenovirus or knocked down by siRNA. Its subcellular distribution was tracked by fractionation and by immunofluorescence. Mitochondrial morphology, Drp1 Ser616 phosphorylation, Mfn2 protein levels, and ROS production were quantified. Inflammasome activation was evaluated through caspase-1 cleavage, ASC speck formation, and IL-1β release. In mice with transverse aortic constriction, we delivered AAV9-shNetrin-3 or a neutralizing antibody and assessed cardiac function, tissue histology, and molecular markers.

NLRP3 activation increased Netrin-3 expression and its mitochondrial fraction content, a pattern supported by immunofluorescence colocalization with TOM20. Netrin-3 overexpression was associated with greater mitochondrial fragmentation, higher Drp1 phosphorylation, lower Mfn2, and increased mitochondrial ROS. Netrin-3 knockdown had the opposite effects on these parameters. ROS scavenging with N-acetylcysteine or MitoTEMPO attenuated the Netrin-3-associated increases in caspase-1 cleavage and IL-1β secretion. In pressure-overloaded hearts, Netrin-3 was upregulated; its genetic or pharmacological inhibition correlated with improved ejection fraction and reduced hypertrophy, fibrosis, and macrophage infiltration.

These observations support a model in which Netrin-3 participates in coupling inflammasome signaling to mitochondrial alterations through a ROS-dependent route. Whether Netrin-3 inhibition has therapeutic value in heart failure warrants further investigation.