Abstract Wed067: The microtubule network regulates NOX2-mediated oxidative stress and Cx43 remodelling in Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is a debilitating genetic disorder primarily affecting skeletal and cardiac muscles, driven by mutations in the dystrophin gene. While significant strides have been made in understanding DMD pathology, therapeutic options remain limited as dystrophin is one of the largest genes. Thus, it is challenging to encapsulate it in any gene vector. Emerging evidence implicates microtubule (MT) network dysfunction as a critical mediator of pathological changes in dystrophic hearts, including Cx43 hemichannel lateralization, increased oxidative stress, and inflammation. Cx43 composes integral membrane hemichannels that are important for forming gap junctions at the intercalated discs of cardiomyocytes to ensure synchronous contraction and proper functioning of the heart. Our research has revealed that dystrophic cardiomyocytes display pathological remodelling of Cx43 hemichannels away from the intercalated discs due to hypo phosphorylation of Cx43 C-terminal serine residues S325/S328/S330. Mdx mice engineered to substitute the Cx43 serine triplet with phospho-mimicking glutamic acids (mdx:Cx43S3E) exhibited resistance to Cx43 remodelling, reduced ROS (Reactive Oxygen Species) generation by NOX2 (X-ROS) and reduction in microtubule hyper density. The microtubule network in DMD hearts is disorganized and functionally impaired thereby improperly trafficking Cx43 hemichannels to lateral sites. This results in dysregulated transport of ions and metabolites likely contributing to the emergence of arrhythmias and heart failure associated with DMD. We have identified the microtubule network as a functional link between the loss of dystrophin and the remodelling of Cx43 in DMD hearts. We discovered that the correction of the hyper-dense and disorganized dystrophic microtubules via Colchicine (inhibitor of MT polymerisation) treatment resulted in decreased ROS in mdx hearts. At the molecular level, we have found that hypo phosphorylation at single serine residue at the 172nd position of β-TubulinIII of the MT in DMD hearts. By utilising a phospho-mimic β-tubulinIII wherein the serine residue at the 172nd position is replaced by a glutamic acid residue, we could enhance MT structural organisation and Cx43 remodelling. Our study highlights how microtubules underlie dysfunction in DMD and paves the way for a potential therapeutic target for DMD-associated cardiomyopathy.