SUMMARY Duchenne muscular dystrophy (DMD) is incurable. Lack of dystrophin is the culprit. While the ultimate solution to this devastating disease is to restore dystrophin, the scientific and medical communities actively seek to discover and fix key secondary factors. We study a corrective role of phosphorylated Connexin-43 (Cx43) as a potential secondary factor in DMD cardiomyopathy. Cx43 forms gap junction channels at the intercalated disc (ID) of the cardiomyocytes (CMs). The gap junction helps the heart to beat in unison. We found that dystrophic CMs exhibit pathological Cx43 remodeling – that is Cx43 lateralization away from the ID. Lateralized Cx43 proteins remain as hemichannels, allowing for an undesired passage of ions and metabolites to and from the extracellular space of the CM. Cx43 remodeling contributes to arrhythmic events, stress-induced lethality and long-term fibrotic cardiomyopathy in DMD. Our long-term goal is to improve cardiac function in DMD patients by identifying secondary targets for pharmaceutical agents to protect against heart failure. In this grant proposal, we postulate that the microtubule (MT) network is the connector between dystrophin loss and Cx43 remodeling and that phospho-Cx43 can fix the dystrophic MTs. Our central hypothesis is that Cx43 remodeling and pathology are linked to a dystrophic, disorganized microtubule that triggers X-ROS elevation and oxidation of CaMKII to affect the phospho-status of Cx43. Reciprocally, changes in the phospho-status of Cx43 affects the phospho-status of b-tubulin. As a result of this loop, Cx43/MT normalization corrects downstream effectors, with an ultimate impact on arrhythmias and long-term fibrosis. We also believe that, in addition of suppressing Cx43 pathological remodeling, phospho-Cx43 stabilizes critical members of the intercalated disc. We will first determine if Cx43 remodeling mediates pathology triggered by a dystrophic microtubule cytoskeleton (Aim1). We will investigate if a phospho-mimic form (serine 172) of b-tubulin phenocopies the beneficial effect of colchicine (an inhibitor of MT polymerization) mediated by phospho-Cx43 in dystrophic MTs. We will also test an alternative role for phospho-Cx43 as a scaffold stabilizer. We expect to demonstrate a regulatory interplay between MT and Cx43, in which one phospho-player rescues the other. We will then determine if and to what extent NOX2 and oxidized CaMKII promote Cx43 remodeling and pathology. We will also test if Cx43 is a direct target of CaMKII (Aim2). We expect that important aspects of the Cx43-mediated pathobiology of the hearts of dystrophic mice will be prevented when X-ROS and oxidized-CaMKII are targeted. Impactfully, the microtubules link the lack of dystrophin with Cx43 remodeling. Phospho-mimic mutations appear to correct the dystrophic microtubule hyperdynamics, suppress Cx43 remodeling, and to protect the intercalated disc. The study of phospho-Cx43 will lead to new treatments of DMD-cardiomyop...