AAV9-mediated βIII-tubulin Ser172 phospho-mimic expression improves arrhythmic and inflammatory remodeling in dystrophic cardiomyopathy

<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>Duchenne muscular dystrophy (DMD) cardiomyopathy is characterized by progressive microtubule remodeling, connexin-43 (Cx43) dysregulation, and ventricular arrhythmias. We previously demonstrated phospho-mimic knock-in of βIII-tubulin S172E preserves microtubule organization and attenuates cardiac pathology in mdx mice. However, whether these protective effects can be reproduced using a clinically relevant gene-delivery strategy remains unknown.</jats:p> </jats:sec> <jats:sec> <jats:title>Methods and Results</jats:title> <jats:p>We generated a cardiomyocyte-specific adeno-associated virus serotype 9 (AAV9) vector expressing phospho-mimic βIII-tubulin (Tubb3-S172E) under the cardiac troponin T promoter and delivered it to 4-5-month-old wild-type and mdx mice. Cardiac Tubb3-S172E expression was confirmed by quantitative qPCR and immunoblotting. In mdx mice, AAV9-mediated Tubb3-S172E expression significantly reduced mononuclear inflammatory infiltration, restored Cx43 localization at intercalated discs, and attenuated isoproterenol-induced arrhythmia susceptibility. In contrast, cardiac fibrosis, Nav1.5 protein expression, and peak sodium current density were not significantly improved. Overexpression of wild-type βIII-tubulin in healthy hearts increased Cx43 lateralization and arrhythmia susceptibility, indicating that βIII-tubulin phosphorylation state rather than protein abundance determines its protective function.</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusions</jats:title> <jats:p>Cardiomyocyte-targeted delivery of phospho-mimic βIII-tubulin partially recapitulates the protective effects observed in the genetic S172E knock-in model. These findings identify βIII-tubulin Ser172 phosphorylation as a critical regulator of microtubule-dependent electrical remodeling and support therapeutic modulation of this pathway in Duchenne muscular dystrophy cardiomyopathy.</jats:p> </jats:sec> <jats:sec> <jats:title>Research Perspective</jats:title> <jats:list list-type="bullet"> <jats:list-item> <jats:p>Cardiomyocyte-targeted AAV9 delivery of phospho-mimic âIII-tubulin improves Cx43 organization, inflammatory remodeling, and arrhythmia susceptibility in dystrophic hearts, demonstrating that therapeutic modulation of βIII-tubulin Ser172 phosphorylation partially recapitulates the protective effects observed in the genetic S172E model.</jats:p> </jats:list-item> <jats:list-item> <jats:p>The dissociation between improved electrical remodeling and persistent Nav1.5 and fibrotic abnormalities suggests that βIII-tubulin Ser172 phosphorylation selectively regulates specific microtubule-dependent pathological pathways in dystrophic cardiomyopathy.</jats:p> </jats:list-item> <jats:list-item> <jats:p>Future studies should define the molecular mechanisms linking βIII-tubulin Ser172 phosphorylation to cardiomyocyte-immune cell communication and determine how this pathway coordinates electrical and inflammatory remodeling in dystrophic hearts.</jats:p> </jats:list-item> </jats:list> </jats:sec>