Abstract Body (Do not enter title and authors here): Duchenne Muscular Dystrophy (DMD) is a severe X-linked neuromuscular disorder predominantly affecting males, causing progressive muscle degeneration and premature death due to heart and skeletal muscle dysfunction. It is a rare genetic disease caused by mutations in the dmd gene, leading to the absence of dystrophin, a crucial protein for muscle integrity. Currently, there is no permanent cure for DMD, but steroid-based treatments alleviate symptoms like inflammation and muscle weakness. However, DMD-related heart complications, particularly dystrophic cardiomyopathy, are the primary cause of mortality by age 20. Connexin-43 (Cx43) is a gap junction protein responsible for electrical signaling in the heart. In the healthy condition, Cx43 is located at the intercalated disc and forms an intercellular channel with adjacent cardiomyocytes; In the DMD condition, Cx43 hemichannel accumulates at the peripheral site of cardiomyocytes and further exacerbates cardiac dysfunction, which is called Cx43 pathological lateralization. Our previous research indicated that phospho-mimic Cx43 at the serine triplet sites S325, S328, and S330 in mdx hearts significantly reduced Cx43 lateralization and improved cardiac function. Intriguingly, the phospho-mimic Cx43 also reduced the pathological hyperdensity and arrangement of microtubules in mdx cardiomyocytes. Colchicine treatment, known to affect microtubule polymerization, also improved cardiac function and Cx43 localization in mdx mice. These findings suggest a regulatory link between microtubules and Cx43 that is potentially beneficial for DMD treatment. To delve deeper, we generated a mouse model with a beta-tubulin mutation at the serine 172 site, which was replaced by glutamic acid. The S172 site of beta-tubulin is important for participating in GTP binding, which further regulates the polymerization of microtubules similar to colchicine’s effects. Our preliminary results showed reduced arrhythmias and Cx43 lateralization in mdx hearts with the S172E tubulin mutation, as well as an upregulated level of phospho-Cx43. Further investigation will explore how this mutation affects protein regulation and electrophysiology, and elucidate its mechanism in microtubule polymerization. These studies investigate the regulatory link between microtubules and Cx43, potentially suggesting a novel therapeutic target for DMD-associated cardiomyopathy.