Neuronal nitric oxide synthase localizes to utrophin expressing intercalated discs and stabilizes their structural integrity

The neuronal nitric-oxide synthase (nNOS) splice variant nNOSμ is essential for skeletal muscle function. Its localization is dependent on dystrophin, which stabilizes the dystrophin glycoprotein complex (DGC) at the sarcolemma of skeletal muscle fibers. In Duchenne muscular dystrophy (DMD) dystrophin is absent and sarcolemmal nNOS is lost. This leads to functional ischemia due to a decrease in contraction-induced vasodilation. In cardiomyocytes, nNOSμ is believed to be the predominant NOS isoform. However, the association of nNOS with the DGC in the heart is unclear. Here, we report nNOS localization at the intercalated discs (IDs) of cardiomyocytes, where utrophin is highly expressed. In mdx, mdx:utr, nNOSμ knock-out (KO), and mdx:nNOSμ KO mice, we observed a gradual reduction of nNOS at IDs and disrupted ID morphology, compared to wild-type. In mdx:nNOSμ KO mice, but not in mdx or nNOSμ KO mice, we also observed an early development of cardiac fibrosis. These findings suggest that nNOS localization in the heart may not depend exclusively on the presence of dystrophin. Additionally, the β1 subunit of soluble guanylyl cyclase (sGC), responsible for the production of cGMP through nitric oxide (NO) signaling, was also detected at the IDs. Together, our results suggest a new role of nNOS at the IDs for the cGMP-dependent NO pathway and the maintenance of ID morphology.