Phospho-mimic βIII-tubulin rescues microtubule and cardiac defects in Duchenne muscular dystrophy mice

Duchenne muscular dystrophy (DMD) cardiomyopathy is caused by mutations in the dystrophin gene and characterized by profound cytoskeletal disorganization, particularly pathological remodeling of the cardiomyocyte microtubule network. While the role of dystrophin in maintaining sarcolemmal integrity is well-established, its specific association with microtubule dynamics through post-translational modifications (PTMs) remains underinvestigated. Here, we identified βIII-tubulin Ser172 phosphorylation as a specific regulatory residue in cardiac DMD microtubule pathology. Using CRISPR-generated mdx mice carrying a phospho-mimic S172E mutation in βIII-tubulin, we demonstrate rescue of microtubule organization defects, including normalization of orthogonal microtubule misalignment, and restoration of physiological density and polymerization kinetics. Microtubule stabilization further improved secondary phenotypes, including connexin-43 (Cx43) phosphorylation and retention to intercalated discs, critical for electrical coupling and signal transduction. Furthermore, S172E expression protected against isoproterenol-induced arrhythmias and cardiac fibrosis in the mdx mouse model. Lentiviral expression of S172E-mutant βIVb-tubulin confirmed conservation of these effects across cardiac β-tubulin isoforms. Our findings reveal a regulatory link between microtubule dynamics and Cx43, establish β-tubulin Ser172 phosphorylation as a key regulator of microtubule dynamics in dystrophic hearts, and will help develop novel therapeutic strategies targeting microtubule stabilization in DMD cardiomyopathy.