Author(s): Alexis R. Demonbreun, Northwestern University; Dominic E. Fullenkamp, Northwestern University; Jodi L. Curtin, Northwestern University; Nina L. Rieser, Northwestern University; Lauren A. Vaught, Northwestern University; Elizabeth M. McNally, Northwestern University //

ABSTRACT: Background and Methods:    Many monogenic disorders, including the muscular dystrophies, display phenotypic variability despite the same disease-causing mutation. To identify genetic modifiers of muscular dystrophy as future therapeutic targets, we used quantitative trait locus mapping and whole genome sequencing in a mouse model of muscular dystrophy. This approach uncovered a modifier locus on chromosome 11 associated with increased skeletal and cardiac muscle membrane damage.  Whole genome and RNA sequencing identified Anxa6, encoding annexin A6, as a modifier gene.  Annexin A6 (ANXA6) belongs to the annexin family of calcium-dependent phospholipid binding proteins that facilitate membrane repair, accumulating at sites of damage.  A synonymous variant in exon 11 creates a cryptic splice donor resulting in a truncated annexin A6 protein, referred to as ANXA6N32.  This polymorphism is present in both the C57 and DBA/2J background strains, but not in the 129 strain.  In vivo, the presence of ANXA6N32 was associated with reduced membrane-associated annexin A6 in damaged myofibers and increased dye uptake.  To define the molecular machinery that directs muscle repair, we developed a method to visualize membrane resealing and repair in real-time in live myofibers.  This method uses electroporation to express fluorescently tagged proteins combined with high-resolution confocal microscopy.  In this system, muscle fibers are wounded with a confocal laser and then imaged to observe the resealing process in real-time.  More recently, we developed a cell-based assay in which patient-derived induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are plated on flexible silicone membranes and subjected to equibiaxial strain as physiologic mechanical stressor to better model in vivo membrane insult.     Results:  Within seconds of laser-wounding, endogenous annexin A6 formed a repair cap at the site of muscle membrane injury.  Cell-intrinsic overexpression of annexin A6 enhanced membrane repair after laser injury evidenced by decreased dye uptake after insult.  Moreover, treatment with exogenously-added recombinant annexin A6 protein enhanced membrane repair capacity.  In contrast, overexpression of ANXA6N32 disrupted formation of the annexin A6 cap at the membrane lesion, decreasing repair capacity.  In vivo administration of recombinant annexin A6 protein reduced cardiotoxin-induced muscle damage, visualized by reduced dye uptake and improved histopathology compared to control treated mice.  Furthermore, recombinant annexin A6 was sufficient to enhance muscle membrane repair after laser-injury in dystrophic mice harboring the deleterious A6 allele.  In addition to improved skeletal muscle repair, recombinant annexin A6 was sufficient to reduce the release of troponin T and lactate dehydrogenase (LDH) from flex-injured iPSC-CMs, clinically-relevant biomarkers of injury, indicating enhanced repair in human cardiac cells.    Conclusions:  These data demonstrate that annexin A6 promotes membrane resealing and identifies annexin A6 as a potential therapeutic target to enhance membrane repair capacity to treat both skeletal and cardiac muscle in muscular dystrophy.

Source of Funding: This work was supported by National Institutes of Health NS047726 (EMM), AR052646 (EMM) and F32 HL154712 (DEF).  Additional funding was through Lakeside Discovery (ARD and EMM).