Recombinant annexin A6 promotes membrane repair and protects against muscle injury
Alexis R. Demonbreun, … , David Y. Barefield, Elizabeth M. McNally
Alexis R. Demonbreun, … , David Y. Barefield, Elizabeth M. McNally
Published September 23, 2019
Citation Information: J Clin Invest. 2019;129(11):4657-4670. https://doi.org/10.1172/JCI128840.
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Research Article Muscle biology

Recombinant annexin A6 promotes membrane repair and protects against muscle injury

Abstract

Membrane repair is essential to cell survival. In skeletal muscle, injury often associates with plasma membrane disruption. Additionally, muscular dystrophy is linked to mutations in genes that produce fragile membranes or reduce membrane repair. Methods to enhance repair and reduce susceptibility to injury could benefit muscle in both acute and chronic injury settings. Annexins are a family of membrane-associated Ca2+-binding proteins implicated in repair, and annexin A6 was previously identified as a genetic modifier of muscle injury and disease. Annexin A6 forms the repair cap over the site of membrane disruption. To elucidate how annexins facilitate repair, we visualized annexin cap formation during injury. We found that annexin cap size positively correlated with increasing Ca2+ concentrations. We also found that annexin overexpression promoted external blebs enriched in Ca2+ and correlated with a reduction of intracellular Ca2+ at the injury site. Annexin A6 overexpression reduced membrane injury, consistent with enhanced repair. Treatment with recombinant annexin A6 protected against acute muscle injury in vitro and in vivo. Moreover, administration of recombinant annexin A6 in a model of muscular dystrophy reduced serum creatinine kinase, a biomarker of disease. These data identify annexins as mediators of membrane-associated Ca2+ release during membrane repair and annexin A6 as a therapeutic target to enhance membrane repair capacity.

Authors

Alexis R. Demonbreun, Katherine S. Fallon, Claire C. Oosterbaan, Elena Bogdanovic, James L. Warner, Jordan J. Sell, Patrick G. Page, Mattia Quattrocelli, David Y. Barefield, Elizabeth M. McNally

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Figure 4

Annexin A6 Ca2+-binding mutant reduced annexin repair cap recruitment and decreased myofiber membrane repair capacity.

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Annexin A6 Ca2+-binding mutant reduced annexin repair cap recruitment an...
(A) Myofibers were coelectroporated with wild-type-tdTomato (red labels) and either wild-type-GFP or mutant-GFP (green labels) annexin constructs, and cap size was assessed after membrane damage; only the red channel is shown to demonstrate the effect on wild-type annexin. (B) Coexpression of mutant annexin A6E233A was sufficient to reduce wild-type annexin A6 cap assembly. Cap kinetics were plotted as cap Feret diameter over a range of Ca2+ concentrations, from 0–2 mM. (C) Coexpression of annexin A6E233A was sufficient to significantly reduce the cap area of coexpressed annexin A1, A2, and A6. *P < 0.05 for WT + WT vs. WT + mutant. (D) Myofibers were electroporated with annexin A6–GFP or mutant A6E233A–GFP. Annexin A6E233A cap area (small arrow) was significantly smaller compared with annexin A6 (large arrowhead), correlating with increased FM 4-64 fluorescence area (large arrowhead). Scale bars: 5 μm. Data are expressed as mean ± SEM. Differences were assessed by 2-tailed t test (A, C, and D). *P < 0.05 (n = 4–18 myofibers from n = 3 mice per condition).