• Decreased junctophilin-2 levels are associated with cardiac t-tubule derangements in mdx mice, the mouse model of Duchenne muscular dystrophy (DMD).

• Reduced junctophilin-2 protein levels correlate with increases in total microtubule content in mdx hearts.

• Colchicine-mediated microtubule depolymerization increases junctophilin-2 protein levels and improves localization patterns which, in turn, are associated with t-tubule reorganization and reduced calcium sparks.

• This study identifies microtubule-mediated misregulation of junctophilin-2 as a novel molecular mechanism in Duchenne cardiomyopathy.