Targeting Mechanically‐Activated NOS Signaling in Dystrophic Cardiomyopathy

Abstract

Patients deficient in dystrophin, a protein that links the cytoskeleton to the extracellular matrix via the dystrophin‐glycoprotein complex (DGC), exhibit muscular dystrophy, cardiomyopathy, and impaired muscle nitric oxide (NO) production. Our objective was to identify the mechanisms of how the DGC regulates cardiac muscle nitric oxide synthase (NOS) activity. Isolated adult wild‐type (WT) or dystrophin‐deficient mdx mouse cardiomyocytes were plated on deformable membranes, and live‐cell DAF‐FM dye fluorescence and biochemical approaches were used to investigate how the DGC regulates mechanically‐activated NO signaling. 1Hz cyclic stretch of WT cardiomyocytes significantly increased NO production and neuronal NOS (nNOS) S1412 phosphorylation. In contrast, stretch‐induced NO production and nNOS phosphorylation were impaired in mdx cardiomyocytes, suggesting that dystrophin regulates the mechanical activation of NOS. We found that inhibition of AMP‐activated protein kinase (AMPK) inhibited stretch‐induced NO production in WT cardiomyocytes, indicating that AMPK plays a critical role in mechanoactivation of NOS. Importantly, direct pharmacological activation of AMPK increased nNOS S1412 phosphorylation and was sufficient to enhance NO production in mdx cardiomyocytes. We conclude that the DGC regulates the mechanical activation of cardiac NOS, and that pharmacological AMPK activation may be a therapeutic strategy to restore NOS activity in dystrophin‐deficient cardiomyocytes. Supported by the Muscular Dystrophy Association, the University of Michigan CVRE Training Program, and NIH T32 GM‐08322.