Recombinant annexin A6 promotes membrane repair in a stem cell derived-cardiomyocyte model of dystrophic cardiomyopathy

Heart failure is a major source of mortality in Duchenne muscular dystrophy (DMD). DMD arises from mutations that ablate expression of the protein dystrophin, which render the plasma membrane unusually fragile and prone to disruption. In DMD patients, repeated mechanical stress leads to membrane damage and cardiomyocyte loss. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offer the opportunity to study specific mutations in the context of a human cell, but these models can be improved by adding physiologic stressors. We modeled the primary defect underlying DMD by applying equibiaxial mechanical strain to DMD iPSC-CMs. DMD iPSC-CMs demonstrated an increased susceptibility to equibiaxial strain after 2 hours at 10% strain relative to healthy control cells, measured as increased lactate dehydrogenase (LDH) release. After 24 hours, both DMD and healthy control iPSC-CMs showed evidence of injury with release of LDH and cardiac troponin T. We exposed iPSC-CMs to recombinant annexin A6, a protein resealing agent, and found reduced LDH and troponin release in DMD and control iPSC-CMs that had been subjected to 24 hour strain at 10%. We used aptamer protein profiling of media collected from DMD and control iPSC-CMs and compared these results to serum protein profiling from DMD patients. We found a strong correlation between the proteins in DMD patient serum and media from DMD iPSC-CMs subjected to mechanical stress. By developing an injury assay that specifically targets an underlying mechanism of injury seen in DMD-related cardiomyopathy, we demonstrated the potential therapeutic efficacy of the protein membrane resealer, recombinant annexin A6, for the treatment of DMD-related cardiomyopathy and general cardiac injury. ### Competing Interest Statement Grants/support: National Institutes of Health NS047726 (EM), AR052646 (EM), and F32HL154712 (DF), Parent Project Muscular Dystrophy, and Lakeside Discovery (EM, ARD). Conflicts: Northwestern University filed provisional patent applications #62/783,619 (ARD and EMM) and #63/309,925 (ARD, DEF, and EMM) on behalf of the authors and related to the content of this work. Relationship to industry: EMM consults for Amgen, AstraZeneca, Avidity, Cytokinetics, 4D Molecular Therapeutics, Jansson, Pfizer, PepGen, Stealth Biopharma, Tenaya and Invitae. EMM and ARD are the founders of Ikaika Therapeutics Inc. These relationships are unrelated to the content of this work. * ACE : Angiotensin converting enzyme DMD : Duchenne muscular dystrophy iPSC : Induced pluripotent stem cell iPSC-CMs : Induced pluripotent stem cell-derived cardiomyocytes LDH : Lactate dehydrogenase MG53 : Mitsugumin53