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.

Keywords: Cell migration/adhesion; Genetic diseases; Muscle Biology; Therapeutics.

Figure 1. Ca²⁺-dependent annexin repair cap recruitment at the site of injury.

Myofibers were generated to express the Ca²⁺ indicator GCaMP5G (green), and time-lapse single-slice images were assessed at time points after membrane disruption. (A) GCaMP5G fluorescence was present at the site of injury, at 2 seconds (arrow), indicating the presence of Ca²⁺ immediately after damage at the site of injury (top panel). These data were validated with a non–protein-based Ca²⁺ indicator, Fluo-4 AM (green, bottom panel). (B) Time-lapse images of myofibers coelectroporated with GCaMP5G and annexin A6-tdTomato (A6, red). GCaMP5G fluorescence was present at the site of injury localized around the annexin A6–free zone (arrowhead) and at the annexin A6 cap (arrow). GCaMP5G colocalized (merge, yellow, arrow) with the annexin A6 repair cap. Scale bars: 5 μm. (C) Myofibers expressing fluorescently tagged annexins A1, A2, or A6 were injured at multiple Ca²⁺ concentrations. Annexin A1 and A6 repair cap size was reduced at 0.1 mM Ca²⁺ compared with 2 mM and 0.5 mM. Annexin A2 repair cap area was significantly reduced at 0.05 mM Ca²⁺ compared with 2 mM, 0.5 mM, and 0.1 mM Ca²⁺. (D) Cap kinetics were plotted as cap Feret diameter over a range of Ca²⁺ concentrations. Annexin A2 had a statistically significant leftward shift in K_m(1/2), followed by annexin A6 and then A1. Data are expressed as mean ± SEM. Differences were tested by 1-way ANOVA with Tukey’s multiple-comparisons test (C). *P < 0.05 (n = 5 myofibers per condition).

Figure 2. Annexin expression promoted release of blebs from the site if myofiber repair.

Myofibers were electroporated with the Ca²⁺ indicator GCaMP5G (green) with or without tdTomato-labeled annexin A1, annexin A2, or annexin A6. Ca²⁺ area and fluorescence were assessed after membrane damage. (A) High-magnification Z-projection images illustrate external blebs filled with the Ca²⁺ indicator emanating from the lesion when annexin A1, A2, or A6 was coexpressed and a corresponding reduction in Ca²⁺ indicator within the myofiber when compared with GCaMP5G alone (see panel B). (B) Membrane marked by FM 4-64 shows GCaMP5G-negative vesicles form in the absence of annexin overexpression. Scale bars: 5 μm. (C) Expression of annexin A6 or A2 resulted in an increased number of GCaMP5G-positive blebs. (D) Expression of annexin A6 resulted in the formation of the largest GCaMP5G-positive blebs. Data are expressed as mean ± SEM. Differences were tested by 1-way ANOVA with Tukey’s multiple-comparisons test. *P < 0.05 (n = 16 myofibers from n = 3 mice per condition).

Figure 3. Annexin expression reduced Ca²⁺ within the myofiber.

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).

Figure 4. Annexin A6 Ca²⁺-binding mutant reduced annexin repair cap recruitment and decreased myofiber membrane repair capacity.

(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).

Figure 5. Annexin A6 enhanced membrane repair capacity of healthy and dystrophic myofibers in vitro.

(A) Plasmid expression of annexin A6 in wild-type (WT) myofibers reduced FM 4-64 dye uptake, a marker of membrane damage, after laser-induced injury as compared with control myofibers. (B) Wild-type myofibers injured in the presence of extracellular recombinant annexin A6 (rANXA6) had significantly less FM 4-64 dye uptake compared with control myofibers. (C) Dystrophic (Dys) myofibers injured in the presence of rANXA6 had significantly less FM 4-64 dye uptake than control myofibers. Scale bars: 5 μm. Data are expressed as mean ± SEM. Differences were assessed by 2-tailed t test. *P < 0.05 (n = 10 myofibers from n = 3 mice per condition).

Figure 6. Ca²⁺ dependency of the protective effects of recombinant annexin A6.

(A and B) Wild-type myofibers were isolated and loaded with a fluorescence marker of membrane damage, FM 1-43 (green). Myofibers were pretreated with recombinant annexin A6 (rANXA6) and then damaged in 1 mM Ca²⁺ solution or 0 mM Ca²⁺ plus EGTA, a calcium chelator. FM 1-43 fluorescence uptake over time was significantly reduced at 1 mM Ca²⁺ compared with when EGTA was present. Scale bars: 5 μm. Data are expressed as mean ± SEM. Differences were tested by 2-way ANOVA with Bonferroni’s multiple-comparisons test. *P < 0.05 (n = 10 myofibers per condition; n = 3 mice per condition).

Figure 7. Local delivery using intramuscular injection of recombinant annexin A6 protected against muscle damage in vivo.

(A) Tibialis anterior muscles of wild-type mice were injected i.m. with recombinant human annexin A6 (rANXA6) or vehicle control and subsequently injured with cardiotoxin injection. (B) Gross imaging revealed decreased Evans blue dye (blue) uptake in rANXA6-pretreated muscle compared with the contralateral control muscle. (C) Immunofluorescence imaging revealed decreased dye uptake (red) in muscle pretreated with rANXA6. Surface plots of dye uptake depict reduced fluorescence in muscle pretreated with rANXA6. White dotted lines outline the muscle sections. (D) Tibialis anterior (TA) muscle pretreated with rANXA6 had a significant reduction, approximately 50%, of Evans blue dye fluorescence over muscle area compared with control muscle. Scale bars: 1 mm. Data are expressed as mean ± SEM. Differences were assessed by 2-tailed t test. *P < 0.05 (n = 3 mice per condition). EBD, Evans blue dye.

Figure 8. Systemic delivery using retro-orbital injection of recombinant annexin A6 protected against muscle damage in vivo.

(A) Wild-type mice were injected retro-orbitally (RO) with recombinant human annexin A6 (rANXA6) or control solution. Following this, muscles were damaged with cardiotoxin (CTX). (B and C) Immunofluorescence imaging revealed approximately 38% less dye uptake (red) in muscle pretreated with rANXA6. Dotted lines outline the tibialis anterior muscle sections (top panel). DAPI (blue) marks nuclei. Surface plots of dye uptake depict reduced fluorescence in muscle pretreated with rANXA6. (D) Whole-tissue spectroscopic analysis of injured gastrocnemius/soleus muscles revealed a 58% reduction in dye uptake with rANXA6 pretreatment compared with control muscle. Abs, absorbance at 620 nm. (E) Wild-type mice were injected intravenously with rANXA6 or control solution. Two hours later, tibialis anterior muscles were damaged with cardiotoxin. Muscles were harvested 7 days after injury. (F and G) Hematoxylin and eosin images were quantified and show a reduction in percentage myofiber damage (dotted lines), in rANXA6-treated mice compared with controls. Scale bars: 1 mm (B) and 500 μm (F). Data are expressed as mean ± SEM. Differences were assessed by 2-tailed t test. *P < 0.05. n = 3 mice, n = 6 legs per condition (B–D); n = 6 mice; n = 11 muscles per condition (F and G). EBD, Evans blue dye; TA, tibialis anterior.

Figure 9. Recombinant annexin A6 protected against muscle damage in a mouse model of muscular dystrophy in vivo.

(A and B) Sgcg-null mice, a model of limb girdle muscular dystrophy 2C, were injected intravenously with recombinant human annexin A6 (rANXA6) or BSA control solution 5 times over 48 hours. Prior to injections, serum creatine kinase (CK) was measured. Two hours after the fifth injection, mice were subjected to 60 minutes of downhill running. Thirty minutes after exercise, serum CK was measured. The fold change in CK after/before running was significantly reduced with rANXA6 administration compared with BSA-injected controls, consistent with a reduction in muscle injury from acute running. (C and D) Sgcg-null mice were injected intravenously every 3 days over 14 days with rANXA6 or control. On day 14, serum CK was evaluated. Serum CK in Sgcg-null mice treated with rANXA6 was lower than PBS control. (E) Sgcg-null mice were injected intravenously every 3 days for 14 days with rANXA6 or recombinant annexin A2. The serum CK fold change after/before treatment (day 14/day 0) was significantly reduced in Sgcg-null mice treated with recombinant annexin A6 compared with annexin A2. (F) Histological analysis of gastrocnemius/soleus muscles from Sgcg-null mice shown in part D injected with PBS or recombinant annexin A6. Low magnification is on the left and high magnification of boxed areas is on the right. Scale bars: 500 μm (left) and 50 μm (right). Data are expressed as mean ± SEM. Differences were assessed by 2-tailed t test (B and E). *P < 0.05 (n = 3 mice per condition, except part D, in which n = 2 WT controls, 1 Sgcg-null control, and 2 Sgcg-null treated mice).