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 Therapeutics

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 3

Annexin expression reduced Ca2+ within the myofiber.

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Annexin expression reduced Ca2+ within the myofiber. Myofibers were elec...
Myofibers were electroporated with the Ca2+ indicator GCaMP5G (green) with or without tdTomato-labeled annexin A1, annexin A2, or annexin A6 (red imaging not shown in this image). Ca2+ area and fluorescence were assessed after membrane damage. (A) Time-lapse single-slice images reveal that coexpression of either annexin A1, A2, or A6 resulted in a significant reduction in GCaMP5G fluorescence (green) measured inside the myofiber at the site of injury over time. (B) Expression of either annexin A1, A2, or A6 resulted in a significant reduction in GCaMP5G fluorescence measured inside the myofiber at the site of injury over 240 seconds of imaging, with annexin A6 inducing the greatest reduction in GCaMP5G fluorescence. (C) Both annexin A2 and A6 contributed to the early reduction in GCaMP5G fluorescence, as seen by imaging during the first 20 seconds after injury. (D) Initial GCaMP5G mean fluorescence was not significantly different between groups. Scale bars: 5 μm. Data are expressed as mean ± SEM. Differences were tested by 2-way ANOVA with Bonferroni’s multiple-comparisons test (B and C) or 1-way ANOVA with Tukey’s multiple-comparisons test (D). * P < 0.05 (n = 9 myofibers from n = 3 mice per condition).