Danegaptide Enhances Astrocyte Gap Junctional Coupling and Reduces Ischemic Reperfusion Brain Injury in Mice

Abstract

Ischemic stroke is a complex and devastating event characterized by cell death resulting from a transient or permanent arterial occlusion. Astrocytic connexin43 (Cx43) gap junction (GJ) proteins have been reported to impact neuronal survival in ischemic conditions. Consequently, Cx43 could be a potential target for therapeutic approaches to stroke. We examined the effect of danegaptide (ZP1609), an antiarrhythmic dipeptide that specifically enhances GJ conductance, in two different rodent stroke models. In this study, danegaptide increased astrocytic Cx43 coupling with no significant effects on Cx43 hemichannel activity, in vitro. Using matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) the presence of danegaptide within brain tissue sections were detected one hour after reperfusion indicating successful transport of the dipeptide across the blood brain barrier. Furthermore, administration of danegaptide in a novel mouse brain ischemia/reperfusion model showed significant decrease in infarct volume. Taken together, this study provides evidence for the therapeutic potential of danegaptide in ischemia/reperfusion stroke.

Keywords: astrocytes; connexin43; danegaptide; gap junction; stroke.

Figure 1

(A) In vitro scrape-loading assay (red line shows scrape edge) showing WT astrocytes exposed to 0.01, 0.1, 1.0 or 10.0 µg/mL danegaptide for 20 min followed by incubation with LY (green) and dextran rhodamine (red). Scale bar = 50 μm. (B) Quantification of total average diffusion area in LY pixels normalized to dextran rhodamine pixels in WT astrocytes subjected to either 0.01, 0.1, 1.0 or 10.0 µg/mL danegaptide for 20 min. One-way ANOVA followed by Dunnett’s multiple comparisons test, ** p = 0.0097; n = 4. Error bars represent mean ± SEM. (C) Representative immunoblot of Cx43 (upper blot) and γ-tubulin (lower blot) from WT astrocytes exposed to either 0.01, 0.1, 1.0 or 10.0 µg/mL danegaptide for 20 min, n = 3 independent experiments.

Figure 2

Micrographs above show examples of EtBr uptake after 10 min in WT astrocytes exposed to either PBS, 1 µg/mL danegaptide or Ca²⁺/Mg²⁺-free solution (divalent free) to induce hemichannel opening after 10 min incubation. Scale bar = 100 µm Graph below shows representative time-lapse measurements of ethidium uptake in WT astrocytes exposed to 5 μM EtBr with either control (PBS), 1 µg/mL danegaptide or Ca²⁺/Mg²⁺-free solution (divalent free) to induce hemichannel opening. Linear regression performed in control and danegaptide reveals slopes are not significantly different (n = 3 independent experiments).

Figure 3

To assess danegaptide levels in the brain, 10 μm-thick coronal brain sections were thaw mounted onto ITO glass slides and processed for imaging mass spectrometry in positive reflectron mode on the 5800 TOF/TOF ABSciex instrument. Using mass spectrometry imaging-induced ion images, the spatial distribution of danegaptide was observed using image comparison. (A) Sham saline injected 1 h before tissue removal. (B) Sham peptide injected 1 h before tissue removal. (C) tMCAO 12 h saline. (D) tMCAO 1 h peptide and (E) tMCAO 12 h peptide. (F) Spectral comparison throughout the brain tissue sectioned from mice injected with the peptide, consistent with passage through the blood brain barrier. After 50 min of ischemia, saline or 75 µg/kg of danegaptide was injected intravenously. After reperfusion, 300 µg/kg of danegaptide was delivered by IP at 1, 2, and 3 h after the initial injection. Pseudo colour scale: blue low levels and yellow high level of signal. (n = 5).

Figure 4

Quantification of infarct volume from WT (C57Bl/6) mice 48 h after tMCAO treated with either saline or 75 µg/kg danegaptide after 50 min of ischemia, intravenously. After reperfusion, 300 µg/kg of danegaptide was delivered by IP at 1, 2, and 3 h after the initial injection. (unpaired t-test; saline: n = 10 mice; danegaptide: n = 10 mice). Photomicrographs of thionin-stained sections from saline and danegaptide-treated mice. Pale blue region indicates infarct tissue. Scale bar = 2 mm.