{"http:\/\/dx.doi.org\/10.14288\/1.0389655":{"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool":[{"value":"Non UBC","type":"literal","lang":"en"},{"value":"Cellular and Physiological Sciences, Department of","type":"literal","lang":"en"}],"http:\/\/www.europeana.eu\/schemas\/edm\/dataProvider":[{"value":"DSpace","type":"literal","lang":"en"}],"https:\/\/open.library.ubc.ca\/terms#identifierCitation":[{"value":"Biomolecules 10 (3): 353 (2020)","type":"literal","lang":"en"}],"http:\/\/purl.org\/dc\/terms\/contributor":[{"value":"University of British Columbia. Life Sciences Institute","type":"literal","lang":"en"}],"http:\/\/purl.org\/dc\/terms\/creator":[{"value":"Freitas-Andrade, Moises","type":"literal","lang":"en"},{"value":"Bechberger, John","type":"literal","lang":"en"},{"value":"Wang, Jasmine","type":"literal","lang":"en"},{"value":"Yeung, Ken  K.C.","type":"literal","lang":"en"},{"value":"Whitehead, Shawn  N.","type":"literal","lang":"en"},{"value":"Shultz Hansen, Rie","type":"literal","lang":"en"},{"value":"Naus, Christian  C.","type":"literal","lang":"en"}],"http:\/\/purl.org\/dc\/terms\/issued":[{"value":"2020-03-27T18:21:38Z","type":"literal","lang":"en"},{"value":"2020-02-26","type":"literal","lang":"en"}],"http:\/\/purl.org\/dc\/terms\/description":[{"value":"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.","type":"literal","lang":"en"}],"http:\/\/www.europeana.eu\/schemas\/edm\/aggregatedCHO":[{"value":"https:\/\/circle.library.ubc.ca\/rest\/handle\/2429\/73822?expand=metadata","type":"literal","lang":"en"}],"http:\/\/www.w3.org\/2009\/08\/skos-reference\/skos.html#note":[{"value":"biomoleculesArticleDanegaptide Enhances Astrocyte Gap JunctionalCoupling and Reduces Ischemic Reperfusion BrainInjury in MiceMoises Freitas-Andrade 1, John Bechberger 1, Jasmine Wang 2, Ken K.C. Yeung 2,Shawn N. Whitehead 2, Rie Shultz Hansen 3 and Christian C. Naus 1,*1 Cellular & Physiological Sciences, Faculty of Medicine, Life Science Institute, University of British Columbia,Vancouver, BC V6T 1Z3, Canada; moisesfreitasandrade@gmail.com (M.F.-A.); jbechber@gmail.com (J.B.)2 Anatomy & Cell Biology, Western University, London, ON N6A 5C1, Canada; jwang464@uwo.ca (J.W.);kyeung@uwo.ca (K.K.C.Y.); Shawn.Whitehead@schulich.uwo.ca (S.N.W.)3 Zealand Pharma A\/S, 2860 Copenhagen, Denmark; Rha@zealandpharma.com* Correspondence: ccnaus@gmail.com; Tel.: +1-604-317-4214Received: 28 December 2019; Accepted: 21 February 2020; Published: 26 February 2020\u0001\u0002\u0003\u0001\u0004\u0005\u0006\u0007\b\u0001\u0001\u0002\u0003\u0004\u0005\u0006\u0007Abstract: Ischemic stroke is a complex and devastating event characterized by cell death resultingfrom 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 ofdanegaptide (ZP1609), an antiarrhythmic dipeptide that specifically enhances GJ conductance, in twodifferent rodent stroke models. In this study, danegaptide increased astrocytic Cx43 coupling withno significant effects on Cx43 hemichannel activity, in vitro. Using matrix-assisted laser desorptionionization imaging mass spectrometry (MALDI IMS) the presence of danegaptide within brain tissuesections were detected one hour after reperfusion indicating successful transport of the dipeptideacross the blood brain barrier. Furthermore, administration of danegaptide in a novel mouse brainischemia\/reperfusion model showed significant decrease in infarct volume. Taken together, this studyprovides evidence for the therapeutic potential of danegaptide in ischemia\/reperfusion stroke.Keywords: connexin43; stroke; gap junction; astrocytes; danegaptide1. IntroductionStroke is a debilitating event associated with either mortality or significant reduction in qualityof life. In ischemic stroke, occlusion of the middle cerebral artery (MCA) results in rapid loss ofneurons in the infarct core while the peri-infarct, also referred to as the penumbra, is a region ofinstability that could be recovered [1]. Within the peri-infarct, reactive astrocytes expressing highlevels of connexin43 (Cx43) in gap junctions (GJs) have been identified [2\u20135]. Several studies haveshown that reduction of Cx43 levels is associated with detrimental outcome in ischemic stroke [6\u20139].Collectively, these studies underscore Cx43 as a possible key player in brain ischemia and a potentialtarget for therapeutic strategies.In astrocytes, GJs are composed primarily of the channel protein Cx43 and, to a lesser extent,Cx30 and Cx26 [10\u201312]. Individual Cx43 proteins assemble into hexamers around a central hydrophilicpore to form transmembrane channels, termed connexons or hemichannels, which then couple withapposing connexons on neighboring cells forming a GJ intercellular channel. Individual GJs coalesceinto dense plaques that may contain thousands of channels. However, hemichannels can also functionas single cell membrane channels, particularly in pathological conditions [13]. GJ channels directlybridge the cytoplasm between coupled cells and permit the movement of ions and low molecularBiomolecules 2020, 10, 353; doi:10.3390\/biom10030353 www.mdpi.com\/journal\/biomoleculesBiomolecules 2020, 10, 353 2 of 12weight molecules (about 1\u20132 kDa) to neighboring cells or in the case of hemichannels, directly connectthe cell cytoplasm to the extracellular milieu [13,14]. In the brain, astrocytic cell networks play animportant role in homeostasis and repair after injury [15\u201317]. This coordinated interplay betweenastrocytes is achieved, in large part, through Cx43 channels.Permanent middle cerebral artery occlusion (pMCAO) in transgenic cre\/Cx43 floxed mice, whereCx43 is specifically deleted in astrocytes, resulted in significant increase in infarct volume, reductionin reactive astrocytes and an increase in apoptosis, 4 days after stroke insult [18]. Similarly, in Cx43heterozygous null (Cx43+\/\u2212) mice, the loss of a single Cx43 allele in these mice resulted in reducedastrocyte coupling that led to increased neuronal death [6]. Interestingly, an in vitro study showedthat Cx43 coupling between astrocytes delayed neuronal death in an oxygen\/glucose deprivationmodel [19]. The authors concluded that astrocytic gap junctional networks provide protection againsttissue damage during the acute phase of stroke [19]. This is consistent with the spatial buffering capacityassociated with astrocytes [20] and may indicate the ability of Cx43-coupled astrocyte-networks tobuffer or dissipate the toxic microenvironment affected by stroke.Several lines of evidence demonstrate that inflammatory factors and hypoxia promote Cx43hemichannel opening, resulting in both neuronal and astrocytic cell death [21\u201325]. We have recentlyshown that pharmacological inhibition of Cx43 hemichannels, without affecting Cx43 GJ coupling,is neuroprotective in stroke [26]. Similarly, Chen et al. (2019) showed neuroprotective effects byinhibiting Cx43 hemichannels in an ischemia\/reperfusion mouse model [27]. However, whetherincreasing astrocyte Cx43 coupling in brain ischemia is neuroprotective is unknown.Recent developments in Cx43 pharmacology have led to the synthesis of small molecules that canregulate Cx43 GJ coupling [28]. For example, the antiarrhythmic dipeptide ZP1609, referred to here asdanegaptide, has been used to promote Cx43 GJ coupling in cardiomyocytes in myocardial infarction,resulting in reduced infarct size [29\u201331]. More recently, danegaptide was reported to prevent Cx43 GJuncoupling and promote retinal vascular endothelial cell survival in diabetic retinopathy [32]. Whetherdanegaptide has a comparable beneficial effect in the ischemic brain is unknown.The aim of this study was to determine whether danegaptide affects Cx43 function in astrocytes andif danegaptide improves stroke outcome in mice. We show that danegaptide significantly increases Cx43GJ coupling in astrocytes while not affecting Cx43 hemichannel activity. Moreover, we demonstratethat danegaptide crosses the blood brain barrier (BBB) and administration after stroke\/reperfusionis neuroprotective.2. Materials and Methods2.1. Permanent Middle Cerebral Artery OcclusionAll procedures were approved by the Animal Care Committee of the University of British Columbia.Wildtype (WT) C57Bl\/6 mice from the Jackson laboratory (Bar Harbor, ME, USA) aged 3\u20134 monthswere used. Mice were maintained on a 12:12 h light:dark cycle, with food and water available adlibitum. Two different stroke models were performed: transient middle cerebral artery occlusion(tMACO) [33,34] and permanent middle cerebral artery occlusion (pMCAO) [18,35]. Briefly, the micewere anesthetized with sodium pentobarbital (65 mg\/kg i.p.) and given an opiate (Buprenorphine,0.1 mg\/kg) and a local analgesic (Bupivacaine, 0.1 mL at 0.25%) at the incision site prior to surgery.Body temperature was maintained at 37 \u25e6C during surgery and recovery. For the tMCAO procedure,we used the tandem ipsilateral common carotid artery (CCA) middle cerebral artery (MCA) occlusionstroke model [33,34]. With the mouse in supine position a midline incision was made and the CCAwas isolated and separated from the vagus nerve. The blood flow within the CCA was reduced witha 70-g pressure microclamp. The mouse was then rotated, and the head was held securely in placeusing a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). With the aid of a dissectingmicroscope (Helmut Hund GmbH, Wetzlar, Germany), a skin incision was made on the right side ofthe head from the anterior of the ear towards the corner of the eye horizontally and from the corner ofBiomolecules 2020, 10, 353 3 of 12the eye vertically 5 mm. The squamosal bone was exposed by gently pulling back the temporal muscle.Using a fine battery-powered drill (Dremel, London, ON, Canada), a small hole was made ~2 mm indiameter on the skull bone to remove dura and expose the MCA. A metal pin was then placed underthe MCA. The mouse was then placed in the supine position for temporary clamping of the CCA(Supplementary Figure S1). The CCA\/MCA was blocked for 1 h. However, at 50 min, saline (pH 7.4) or75 \u00b5g\/kg of danegaptide (Zealand Pharma, Copenhagen, Denmark) was injected intravenously into thetail vein [30]. The 75 \u00b5g\/kg dose was previously shown to be protective in pigs subjected to myocardialinfarcts [30]. After the 1 h of ischemia, the clamp on the CCA and wire under the MCA were removedto allow reperfusion (Supplementary Figure S2). Cerebral blood flow was measured according tomanufacturer\u2019s protocol using the moorFLPI-2 system (Moor Instruments Inc, Wilmington, DE, USA).Mice were subsequently treated with 300 \u00b5g\/kg of danegaptide by intraperitoneal (IP) injection at 1, 2,and 3 h after the initial injection. The larger 300 \u00b5g\/kg concentration of danegaptide administered viaIP is due to the fact that the absorption from this route is usually one-half to one-fourth as rapid asfrom the intravenous route [36]. Furthermore, the IP route was used in order to minimize animal stressand trauma due to repeated tail vein injection.For the pMCAO procedure, exposure of the MCA was similar to the method described above andpreviously reported [18,37]. Briefly, once the MCA was exposed, the blood vessel was then cauterizedabove and below the rhinal fissure using an electronic coagulator (Codman and Shurtleff Inc., Raynham,MA, USA). After visually confirming the absence of reperfusion through the MCA, the skin incisionwas closed with sutures. Mice were then given IP injection of either saline or danegaptide (1\u201310 mg\/kg)at 1, 2, 3 and 4 h post-pMCAO. Efforts were made to reduce the number of animals used and tominimize animal suffering.Following injections of compounds, mice were allowed to recover for 48 h post-stroke. Mice werethen anesthetized using a lethal dose of sodium pentobarbital (120\/kg) IP and transcardially perfusedwith phosphate-buffered saline (PBS). The brains were quickly removed and rapidly frozen at \u221280 \u25e6Ctill further processing for infarct visualization by immunohistochemistry [37].2.2. Quantification of Cerebral InfarctionA cryostat (HM 505E; Micron, Walldorf, Germany) was used to obtain 20 \u00b5m- and 10 \u00b5m-thicksections, collected at 200 \u00b5m intervals for infarct volume determination and immunohistochemistry.To prepare sections for measurement of infarct size, they were stained with 0.125% thionin (FisherScientific, Toronto, ON, Canada). Total infarct volumes were calculated using a stereological approachthrough the rostrocaudal extent of the infarct area, and corrected for edema as previously described [38].Images were analyzed by an observer blind to experimental conditions with ImageJ software (Bethesda,MD, USA, available at: http:\/\/rsbweb.nih.gov\/ij\/).2.3. Matrix-Assisted Laser Desorption\/Ionization Imaging Mass Spectrometry (MALDI-IMS)A CryoStar NX50 was used for all tissue sectioning. Tissue samples were mounted onto thetissue holder of the cryostat with water and coronal sections were obtained to 14 \u00b5m thicknesses at\u221225 \u25e6C. Each tissue section was approximately 5.5\u20139 mm by 7\u20139 mm. Once sliced, the sections werethaw mounted onto indium tin oxide (ITO) coated glass slides and placed in a desiccator for 10 min.A six-step wash first using, 70% ethanol, and 100% ethanol was employed. This was followed by amixture of 60% ethanol, 30% chloroform, and 10% acetic acid. Finally, another 100% ethanol, water,and 100% ethanol was used. Upon tissue acidification, the water wash was replaced with 0.2% TFA.The tissue was incubated in this solvent at room temperature for 2.5 min.2,5-dihydroxybenzoic acid (DHB) matrix was prepared to 20 mg\/mL in 20% EtOH, 15% 100 mMammonium citrate, and 1% phosphoric acid. DHB was applied onto sample sections using theautomated TM sprayer from HTX Technologies. Complete sample coverage was obtained using8 passes at a velocity of 1200 mm\/min, and a flow rate of 0.05 mL\/min. A final matrix density ofBiomolecules 2020, 10, 353 4 of 123.33 \u00d7 10\u22122 mg\/mm2 was achieved. A rehydration step was performed using a 5% acetic acid solution.The sample slide was placed in the rehydration chamber at 70 \u25e6C for 3.5 min.All MALDI-IMS sample analyses were performed on a Sciex TOF\/TOF 5800 MALDI massspectrometer (Toronto, ON, Canada). TOF\/TOF Series Explorer in the positive ion, reflectron mode andData Explorer were used for data acquisition and spectral processing, respectively. The Sciex TOF\/TOFImaging Acquisition Software (Toronto, ON, Canada) was used for region selection and image dataacquisition. A total sum of 600 shots\/spot and 30 shots\/spot were acquired for spectral and imagingdata acquisition, respectively, with a 1 kHz OptiBeam On-Axis Nd:YAG laser system (Toronto, ON,Canada). The open source MSiReader software (Toronto, ON, Canada) was used for imaging datamanipulations and analysis.2.4. Astrocyte Isolation and In Vitro HypoxiaWT astrocytes were isolated from early postnatal (P0-P1) cortices as previously described [39].Briefly, dissected cortices were triturated in DMEM (Sigma-Aldrich, Oakville, ON, Canada). The cellsuspension was passed through a 70 \u00b5m cell filter strainer and then seeded into flasks (2 cortices\/T75flask). Culture media (DMEM supplemented with 10% FBS, 10 units\/mL penicillin, and 10 \u00b5g\/mLstreptomycin) was replaced 3 days after plating and every second day thereafter. Primary astrocytesreached subconfluence at 7\u20138 days in vitro. Subconfluent cells were vigorously shaken to remove cellsloosely attached to the astrocyte monolayer (mainly oligodendrocytes and microglia). Astrocytes werethen harvested with trypsin-EDTA (Invitrogen Surrey, BC, Canada) and frozen in freezing medium(90% FBS, and 10% DMSO). Frozen astrocytes were thawed and plated on culture dishes. Cultures weremaintained for 5\u20137 days prior to experiments. All experiments were carried out on confluent astrocytesand performed independently at least three times. Astrocytes isolated from different breeding pairswere used for each set of experiments [39].2.5. In Vitro Scrape Loading Dye-Transfer AssayThe scrape-loading assay allows the introduction of a GJ-permeant fluorescent dye, such asLucifer Yellow CH (LY) into cells to monitor its propagation into GJ-coupled cells within minutes afterloading. Astrocytes were gently washed in PBS and then incubated at 37 \u25e6C for 20 min to increasingconcentrations of danegaptide (0.01, 0.1, 1.0 or 10.0 \u00b5g\/mL) in 1 mL of PBS. Dye entry was induced byscraping a confluent monolayer of astrocytes, incubated with 50 \u00b5L of the GJ permeable dye LY (0.5%)and GJ impermeable dye dextran-rhodamine (0.05%; 10 kDa) in PBS, with a scalpel blade and allowingthe dyes to incubate for 2 min. Excess dye was washed off with several rinses of PBS. The extent ofcoupling was determined by epifluorescence Zeiss Axioplan2 fluorescence microscope measuring thearea (in pixels) of LY diffusion subtracted by the area of the GJ impermeable reference dye, dextranrhodamine, with ImageJ software [40].2.6. Western Blot AnalysisProtein samples were isolated from separate cultures of astrocytes incubated with either 0.01, 0.1,1.0 or 10.0\u00b5g\/mL of danegaptide in 1 mL of PBS and resolved by sodium dodecyl sulfate-polyacrylamidegel electrophoresis as described previously [41]. Briefly, 40 \u00b5g of protein was separated on a 12%sodium dodecyl sulfate\u2013polyacrylamide gel and transferred to polyvinylidene difluoride membranes.Membranes were processed and incubated overnight at 4 \u25e6C with primary antibodies against Cx43(1:5000; catalog number: C6219; Sigma-Aldrich, Toronto, ON, Canada); mouse anti-\u03b3-tubulin (1:3000catalog number: T6557; Sigma-Aldrich, Canada), in Tris-buffered saline (TBS; Tris 50 mM, NaCl150 mM, pH 8.0) containing 0.05% Tween (TBST) in 1% non-fat dry milk. The membranes were washedand incubated with horseradish peroxidase-conjugated secondary antibody (Vector Laboratories Inc,Burlingame, CA, USA) 1\/5000 in TBST containing 5% non-fat dry milk. Immunoreactive proteins werevisualized by chemiluminescent solution (Super Signal West Pico, Pierce Biotechnology Inc, Surrey,BC, USA).Biomolecules 2020, 10, 353 5 of 122.7. Hemichannel AssayTime-lapse fluorescence imaging to measure hemichannel activity was performed as previouslydescribed [40]. Briefly, WT astrocytes in cell culture dishes were gently washed twice with Locke\u2019ssolution containing (in mM) 154 NaCl, 5.4 KCl, 2.3 CaCl2, and 5 HEPES, at pH 7.4 and incubatedin either 5 mM ethidium bromide (EtBr) or 5 mM EtBr and 1 \u00b5g\/mL danegaptide. Fluorescenceintensity was recorded every 30 s for 15 min with a Zeiss Axioplan2 fluorescence microscope. To inducehemichannel activity, control cells were incubated in Ca2+\/Mg2+-free solution and fluorescence intensitywas measured every 30 s for 15 min. Images of ethidium uptake were analyzed with the ImageJsoftware. For quantification, region of interest (ROI) was selected for at least 200 nuclei and ROI wasselected for background (no cells). Fluorescence for each ROI at each time point 0 to 10 min wasmeasured. Then, data was exported to Excel and background was subtracted for each ROI. Data wasthen exported to Graphpad software for statistical analysis.2.8. StatisticsAnimals for surgery and cell isolation were randomly selected and experimenter blinded to thegenotype and treatments. A one-way ANOVA (one factor) was used to compare multiple means.When appropriate, post-hoc comparisons were made (specified in figure legends). Unpaired t-testswere performed when comparing between two groups. All p-values \u22640.05 were considered statisticallysignificant. Statistics was performed using Prism 6 (Graph Pad, San Diego, CA, USA).3. Results3.1. Concentration-Dependent Increase in Cx43 Dye Coupling in Astrocytes Exposed to DanegaptideDanegaptide is a dipeptide chemically derived from the class of antiarrhythmic peptides suchas rotigaptide [28]. Similarly to rotigaptide, danegaptide has been reported to maintain Cx43 GJcoupling, probably by functional modifications of Cx43 that reduce GJ closing and prevent intercellularuncoupling [32,42]. To determine whether danegaptide exhibits a similar effect on reducing GJ closingin astrocytes, we incubated astrocytes with increasing concentrations (0.01, 0.1, 1.0 or 10.0 \u00b5g\/mL) ofdanegaptide in 1 mL PBS. A concentration-dependent increase in dye coupling was observed with asignificant (p = 0.0097) 1.8-fold increase at 1 \u00b5g of the drug (Figure 1A,B). However, at 10 \u00b5g, the levelof dye coupling was similar to control levels (Figure 1A,B).To determine whether danegaptide had an effect on Cx43 expression, astrocytes were similarlyincubated with increasing concentrations of the drug and subjected to Western blot. There were nomarked changes in Cx43 protein levels in astrocytes exposed to either 0.01, 0.1, 1.0 or 10.0 \u00b5g\/mL ofdanegaptide (Figure 1C). Collectively, these results are similar to what was previously reported for theantiarrhythmic peptide rotigaptide in cardiac myocytes and HeLa cells [31].3.2. Danegaptide Does Not Affect Hemichannel Activity in Astrocytes In VitroIn addition to the coupling function of Cx43, the occurrence of functional hemichannels(i.e., hemichannels that can be turned into the open state) composed of Cx43 was demonstratedin primary cultures of astrocytes in the absence of external Ca2+ [14,43]. To determine whether Cx43hemichannel activity is affected by danegaptide in astrocytes, dye uptake assay was performed onastrocytes in PBS. Decreasing extracellular divalent cations is a well-known stimulus for hemichannelopening; in line with this, we found that applying Ca2+- and Mg2+-free solution, on our positive controlastrocytes, triggered ethidium uptake in astrocytes (Figure 2). In contrast to the coupling effects ofdanegaptide, hemichannel activity was not affected when astrocytes were exposed to 1 \u00b5g of the drugin PBS (Figure 2).Biomolecules 2020, 10, 353 6 of 12Biomolecules 2020, 10, 353 6 of 13  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 \u00b5g\/mL danegaptide for 20 min followed by incubation with LY (green) and dextran rhodamine (red). Scale bar = 50 \u00b5m. (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 \u00b5g\/mL danegaptide for 20 min. One-way ANOVA followed by Dunnett\u2019s multiple comparisons test, **p = 0.0097; n = 4. Error bars represent mean \u00b1 SEM. (C) Representative immunoblot of Cx43 (upper blot) and \u03b3-tubulin (lower blot) from WT astrocytes exposed to either 0.01, 0.1, 1.0 or 10.0 \u00b5g\/mL danegaptide for 20 min, n = 3 independent experiments. 3.2. Danegaptide Does Not Affect Hemichannel Activity in Astrocytes In Vitro In addition to the coupling function of Cx43, the occurrence of functional hemichannels (i.e., hemichannels that can be turned into the open state) composed of Cx43 was demonstrated in primary cultures of astrocytes in the absence of external Ca2+ [14,43]. To determine whether Cx43 hemichannel activity is affected by danegaptide in astrocytes, dye uptake assay was performed on astrocytes in PBS. Decreasing extracellular divalent cations is a well-known stimulus for hemichannel opening; in line with this, we found that applying Ca2+- and Mg2+-free solution, on our positive control astrocytes, triggered ethidium uptake in astrocytes (Figure 2). In contrast to the coupling effects of danegaptide, hemichannel activity was not affected when astrocytes were exposed to 1 \u00b5g of the drug in PBS (Figure 2). Figure 1. (A) In vitro scrape-loading assay (red line shows scrape edge) showing WT astrocytes exposedto 0.01, 0.1, 1.0 or 10.0 \u00b5g\/mL danegaptide for 20 min followed by incubation with LY (green) anddextran rhodamine (red). Scale bar = 50 \u00b5m. (B) Quantification of total average diffusion area in LYpixels normalized to dextran rhodamine pixels in WT astrocytes subjected to either 0.01, 0.1, 1.0 or10.0 \u00b5g\/mL danegaptide for 20 min. One-way ANOVA followed by Dunnett\u2019s multiple comparisonstest, ** p = 0.0097; n = 4. Error bars represent mean \u00b1 SEM. (C) Representative immunoblot of Cx43(upper blot) and \u03b3-tubulin (lower blot) from WT astrocytes exposed to either 0.01, 0.1, 1.0 or 10.0 \u00b5g\/mLdanegaptide for 20 min, n = 3 independent experiments.Biomolecules 2020, 10, 353 7 of 13  Figure 2. Micrographs above show examples of EtBr uptake after 10 min in WT astrocytes exposed to either PBS, 1 \u00b5g\/mL danegaptide or Ca2+\/Mg2+-free solution (divalent free) to induce hemichannel opening after 10 min incubation. Scale bar = 100 \u00b5m Graph below shows representative time-lapse measurements of ethidium uptake in WT astrocytes exposed to 5 \u00b5M EtBr with either control (PBS), 1 \u00b5g\/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). 3.3. MALDI IMS Was Able to Detect the Presence of Danegaptide in Brain Tissue MALDI IMS analysis of intact tissue sections was used to determine whether danegaptide penetrated the brain parenchyma under ischemic conditions. A significant increase in signal intensity was observed for images of danegaptide-injected samples in comparison to those injected with saline (Figure 3A). These samples were extracted 1 h following IP injection of either saline or danegaptide solutions. Alternatively, mobility of danegaptide in ischemic conditions was also investigated using MALDI IMS. Samples were treated with tMCAO followed by reperfusion. Either saline or danegaptide solutions were injected 10 min prior to reperfusion for these samples. Based on the images obtained, signal of greater intensity could be seen permeating the entire tissue section for tissue samples injected with danegaptide in comparison to saline-injected samples (Figure 3A). This was further supported by the MS spectra of the tissue region (Figure 3B). The increase in signal intensity indicates successful transport of danegaptide even under ischemic conditions. This is consistent with a recent study showing that danegaptide crosses the BBB, under different stroke models, when IP injected [44]. Figure 2. Micrographs above show examples of EtBr uptake after 10 min in WT astrocytes exposed toeither PBS, 1 \u00b5g\/mL danegaptide or Ca2+\/Mg2+-free solution (divale t free) to induce hemichannelopening after 10 min incubation. Scale bar = 100 \u00b5m Graph below shows representative time-lapsemeasurements of ethidium uptake in WT astrocytes exposed to 5 \u00b5M EtBr with either control (PBS),1 \u00b5g\/mL danegaptide or Ca2+\/Mg2+-free solution (divalent free) to induce hemichannel opening. Linearregression performed in control and danegaptide reveals slopes are not significantly different (n = 3independent experiments).Biomolecules 2020, 10, 353 7 of 123.3. MALDI IMS Was Able to Detect the Presence of Danegaptide in Brain TissueMALDI IMS analysis of intact tissue sections was used to determine whether danegaptidepenetrated the brain parenchyma under ischemic conditions. A significant increase in signal intensitywas observed for images of danegaptide-injected samples in comparison to those injected with saline(Figure 3A). These samples were extracted 1 h following IP injection of either saline or danegaptidesolutions. Alternatively, mobility of danegaptide in ischemic conditions was also investigated usingMALDI IMS. Samples were treated with tMCAO followed by reperfusion. Either saline or danegaptidesolutions were injected 10 min prior to reperfusion for these samples. Based on the images obtained,signal of greater intensity could be seen permeating the entire tissue section for tissue samples injectedwith danegaptide in comparison to saline-injected samples (Figure 3A). This was further supported bythe MS spectra of the tissue region (Figure 3B). The increase in signal intensity indicates successfultransport of danegaptide even under ischemic conditions. This is consistent with a recent studyshowing that danegaptide crosses the BBB, under different stroke models, when IP injected [44].Biomolecules 2020, 10, 353 8 of 13  Figure 3. To assess danegaptide levels in the brain, 10 \u00b5m-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 \u00b5g\/kg of danegaptide was injected intravenously. After reperfusion, 300 \u00b5g\/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). 3.4. Danegaptide Reduced Infarct Volume in Mice Subjected to Ischemia\/Reperfusion Danegaptide is a small orally available therapeutic peptide that exhibits protective characteristics under ischemia\/reperfusion injury, in different tissue types [30,45]. To investigate whether danegaptide could be protective in stroke, mice were therapeutically treated with the drug under an ischemia\/reperfusion stroke model tMCAO. Brain ischemia was induced in mice by occluding the CCA and MCA. After 50 min of ischemia, saline or 75 \u00b5g\/kg [30] of danegaptide was administered via tail vein injection. After 60 min of ischemia, reperfusion was introduced and mice were treated with subsequent danegaptide at 1, 2 and 3 h after the initial injection. Mice were allowed to recover for 48 h and infarct volume was assessed. A significant (p = 0.0018) 42.2% reduction in infarct volume was measured in those mice treated with danegaptide, compared to controls (Figure 4). In a small cohort of animals, we tested whether danegaptide, at increasing concentrations (1, 6.5, or 10 mg\/kg), would have a similar protective effect in mice subjected to pMCAO. In contrast to the effects observed in tMCAO study, danegaptide did not provide significant protection in a pMCAO stroke model (Supplementary Figure 3). Figure 3. To assess danegaptide levels in the brain, 10 \u00b5m-thick coronal brain sections were thawmounted onto ITO glass slides and processed for imaging mass spectrometry in positive reflectronmode on the 5800 TOF\/TOF ABSciex instrument. Using mass spectrometry imaging-induced ionimages, the spatial distribution of danegaptide was observed using image comparison. (A) Sham salineinjected 1 h before tissue removal. (B) Sham peptide injected 1 h before tissue removal. (C) tMCAO 12 hsaline. (D) tMCAO 1 h peptide and (E) tMCAO 12 h peptide. (F) Spectral comparison throughout thebrain tissue sectioned from mice injected with the peptide, consistent with passage through the bloodbrain barrier. After 50 min of ischemia, saline or 75 \u00b5g\/kg of danegaptide was injected intravenously.After reperfusion, 300 \u00b5g\/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).3.4. Danegaptide Reduced Infarct Volume in Mice Subjected to Ischemia\/ReperfusionDanegaptide is a small orally available therapeutic peptide that exhibits protective characteristicsunder ischemia\/reperfusion injury, in different tissue types [30,45]. To investigate whetherdanegaptide could be protective in stroke, mice were ally treated wi h the drug under anis emia\/reperfusion stroke odel tMCAO.Brain ischemia was induced in mice by occluding th CCA and MCA. After 50 min of isc mia,saline or 75 \u00b5g\/kg [30] of danegaptide was a ministered via tail vein injection. After 60 min of ischemia,reperfusio was introduced and mice were treated with subs quent danegaptide at 1, 2 and 3 h after theinitial injection. Mice were allowed to recover for 48 h and infarct volume was assessed. A significantBiomolecules 2020, 10, 353 8 of 12(p = 0.0018) 42.2% reduction in infarct volume was measured in those mice treated with danegaptide,compared to controls (Figure 4).Biomolecules 2020, 10, x FOR PEER REVIEW 9 of 13  Figure 4. Quantification of infarct volume from WT (C57Bl\/6) mice 48 h after tMCAO treated with either saline or 75 \u00b5g\/kg danegaptide after 50 min of ischemia, intravenously. After reperfusion, 300 \u00b5g\/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. 4. Discussion Present findings indicate that danegaptide promotes astrocytic Cx43 coupling, while having no effect on Cx43 hemichannel activity under basal in vitro conditions. Danegaptide was also detected in the brain parenchyma, indicating BBB permeability of this peptide. Furthermore, pharmacological use of danegaptide in tMCAO was neuroprotective at 48 h after the stroke event (other time points were not examined in this study). Taken together, this study underscores danegaptide as a potential therapeutic against tMCAO and sets the stage for further study of this drug in the context of stroke. Danegaptide was developed as a dipeptide with similar characteristics to those of rotigaptide, that is, enhancing Cx43 GJ conductance in order to decrease ischemia-induced arrhythmias [46]. A recent study demonstrated that retinal vascular cell treated with danegaptide preserved Cx43 GJ intercellular communication, decreased cell death, and reduced cell monolayer permeability, in an in vitro model of diabetic retinopathy [32]. In our study, we show that under normal culture conditions, danegaptide promoted a concentration-dependent increase in astrocyte Cx43 GJ coupling. However, at higher concentrations (10 \u00b5g\/mL), the GJ coupling effect of danegaptide was diminished. Similarly, Kim et al. (2018) showed that rat retinal endothelial cells grown in high glucose resulted in Cx43 uncoupling; however, at 100 nM, danegaptide significantly increased Cx43 coupling [32]. Consistent with our results, at higher concentrations of danegaptide, the coupling effect was lost in rat retinal endothelial cells grown in high glucose [32]. We showed that these changes in coupling were not due to danegaptide-dependent effects on Cx43 expression. Similarly, others have shown that rotigaptide does not affect Cx43 protein levels in cardiac myocytes and HeLa cells expressing Cx43 [31]. While danegaptide increase Cx43 GJ communication between astrocytes, under normal culture conditions, surprisingly, danegaptide does not affect Cx43 hemichannel activity. By contrast, others have also shown that danegaptide reduces dye uptake in cultured C6 glioma cells [28]. The different effects of danegaptide on Cx43 GJ and hemichannels could be due to the sensitivity of the drug to the conformational changes of Cx43 between the GJ and hemichannel state. Whether danegaptide affects Cx43 hemichannels under pathological conditions is an important question to be addressed in future studies. A growing number of therapeutic targets against connexins are being developed [47]. However, delivery of pharmacological agents in the brain are a challenge due to the tight BBB. A previous study demonstrated that IP-injected danegaptide crosses the BBB and was clearly detected in ischemic Figure 4. Quantification of infarct volume from WT (C57Bl\/6) mice 48 h after tMCAO treated with eithersaline or 75 \u00b5g\/kg danegaptide after 50 min of ischemia, intravenously. After reperfusion, 300 \u00b5g\/kgof 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 anddanegaptide-treated mice. Pale blue region indicates infarct tissue. Scale bar = 2 mm.In a small cohort of animals, we tested whether danegaptide, at increasing concentrations (1, 6.5,or 10 mg\/kg), would have a similar protective effect in mice subjected to pMCAO. In contrast to theeffects observed in tMCAO study, danegaptide did not provide significant protection in a pMCAOstroke model (Suppleme tary Figure S3).4. DiscussionPresent findings indicate that danegaptide promotes astrocytic Cx43 coupling, while having noeffect on Cx43 hemichannel activity under basal in vitro conditions. Danegaptide was also detected inthe brain parenchyma, indicating BBB permeability of this peptide. Furthermore, pharmacologicaluse of danegaptide in tMCAO was neuroprotective at 48 h after the stroke event (other time pointswere not examined in this study). Taken together, this study underscores danegaptide as a potentialtherapeutic against tMCAO and sets the stage for further study of this drug in the context of stroke.Danegaptide was developed as a dipeptide with similar characteristics to those of rotigaptide,that is, enhancing Cx43 GJ conductance in order to decrease ischemia-induced arrhythmias [46].A recent study demonstrated that retinal vascular cell treated with danegaptide preserved Cx43 GJintercellular communication, decreased cell death, and reduced cell monolayer permeability, in anin vitro model of diabetic retinopathy [32]. In our study, we show that under normal culture conditions,danegaptide promoted a concentration-dependent increase in astrocyte Cx43 GJ coupling. However,at higher concentrations (10 \u00b5g\/mL), the GJ coupling effect of danegaptide was diminished. Similarly,Kim et al. (2018) showed that rat retinal endothelial cells grown in high glucose resulted in Cx43uncoupling; however, at 100 nM, danegaptide significantly increased Cx43 coupling [32]. Consistentwith our results, at higher concentrations of danegaptide, the coupling effect was lost in rat retinalendothelial cells grown in high glucose [32]. We showed that these changes in coupling were not dueto danegaptide-dependent effects on Cx43 expression. Similarly, others have shown that rotigaptidedoes not affect Cx43 protein levels in cardiac myocytes and HeLa cells expressing Cx43 [31].Biomolecules 2020, 10, 353 9 of 12While danegaptide increase Cx43 GJ communication between astrocytes, under normal cultureconditions, surprisingly, danegaptide does not affect Cx43 hemichannel activity. By contrast, othershave also shown that danegaptide reduces dye uptake in cultured C6 glioma cells [28]. The differenteffects of danegaptide on Cx43 GJ and hemichannels could be due to the sensitivity of the drug tothe conformational changes of Cx43 between the GJ and hemichannel state. Whether danegaptideaffects Cx43 hemichannels under pathological conditions is an important question to be addressed infuture studies.A growing number of therapeutic targets against connexins are being developed [47]. However,delivery of pharmacological agents in the brain are a challenge due to the tight BBB. A previousstudy demonstrated that IP-injected danegaptide crosses the BBB and was clearly detected in ischemicmouse brain tissue [44]. Similarly, using MALDI-IMS techniques, we observed danegaptide inbrain parenchyma in mice subjected to ischemia\/reperfusion. More importantly, we show here thatadministration of danegaptide during tMCAO is neuroprotective. In contrast, danegaptide did notsignificantly decrease infarct volume in mice subjected to pMCAO. These contrasting results mayreflect Cx43 behavior under different stroke models. For example, Mart\u00ednez et al. (2000) demonstratedthat rat cortical astrocytes subjected to 12 h hypoxia exhibited Cx43 dye coupling similar to controls.However, 15\u201330 min after reoxygenation, dye coupling was transiently reduced by approximately70%. The reduction in dye coupling occurred without changes in the levels of Cx43 [48]. Others haveshown similar effects of astrocytic Cx43 coupling in hypoxia\/reoxygenation experiments [25]. It ispossible that in the pMCAO model, Cx43 gap junctions remain coupled and, therefore, danegaptideprovides little effect. We recently reported that Cx43 coupling increases 2 h after pMCAO [26]. In thetMCAO model, reperfusion, which is similar to reoxygenation, results in increased Cx43 GJ uncoupling;in this situation, danegaptide would promote Cx43 GJ stabilization and coupling between astrocytes.The increased danegaptide-dependent coupling between astrocytes may buffer the detrimental effectsof reperfusion; namely, generation of both reactive oxygen species and proinflammatory factors. Both ofthese processes have been reported to reduce Cx43 intercellular communication [21,39].While the results of this study are promising and are consistent with other ischemia\/reperfusionexperiments in other organ systems [29,30,45,46], the detailed mechanism of action of danegaptide isnot fully elucidated [46] and requires further study5. ConclusionsThe findings presented here highlight danegaptide as a potential therapeutic peptide specificallyfor an ischemia\/reperfusion model of stroke. Manipulating astrocytic networks by pharmacologicallytargeting GJ channels may provide a novel strategy for therapeutic intervention. It is the goal of thisstudy to stimulate future research regarding this drug in the context of stroke.Supplementary Materials: The following are available online at http:\/\/www.mdpi.com\/2218-273X\/10\/3\/353\/s1,Figure S1: Diagram indicating method used to induce tMCAO. First, a 70-g clamp is applied to carotid (1). Second,a pin is inserted under MCA causing stroke (2). The pin (2) is tapered allowing for reducing blockage by reducingpressure via shifting the pin slowly away from MCA, Figure S2: Heat map of blood flow during tMCAO leftpanels. A reduction in blood flow is observed when clamp is applied to carotid. After pin is applied under MCA,indicated by black arrow (clearly seen in black and white image middle panels) blood flow is further reduced. Pinis slowly removed (10 min and 20 min) showing a slow return of blood flow. Once clamp is removed from carotid,a considerable increase in blood flow was observed at reperfusion. Blue colour represents low or no flow of blood.Right panel show coloured photos of surgical procedure, Figure S3: Photomicrographs of thionin-stained sections48 h after pMCAO in WT mice treated with either saline, 1 mg\/kg, 6.5 mg\/kg or 10 mg\/kg danegaptide, pale bluearea indicates the infarct delineated by black outline. Scale bar = 2 mm. Graph below indicates infarct volume 48 hafter pMCAO from WT mice treated either saline, 1 mg\/kg, 6.5 mg\/kg or 10 mg\/kg danegaptide (one-way ANOVAfollowed by Dunnett\u2019s multiple comparisons test; saline versus scrambled: saline vs. danegaptide (1 mg\/kg),p = 0.8639; saline vs. danegaptide (6.5 mg\/kg), p = 0.7579; saline vs. danegaptide (10.0 mg\/kg) p = 0.1528; saline:n = 6 mice; danegaptide (1 mg\/kg): n = 3 mice; danegaptide (6.5 mg\/kg): n = 3 mice; danegaptide (10.0 mg\/kg):n = 3). Error bars represent mean \u00b1 SEM.Author Contributions: Conceptualization, C.C.N., S.N.W. and R.S.H.; methodology, M.F.-A., J.B., J.W. andK.K.C.Y.; writing\u2014original draft preparation, M.F.-A. and C.C.N.; writing\u2014review and editing, C.C.N., S.N.W.,Biomolecules 2020, 10, 353 10 of 12J.B., J.W. and R.S.H.; supervision, C.C.N. and S.N.W.; project administration, C.C.N. and S.N.W.; fundingacquisition, C.C.N. and S.N.W. All authors have read and agreed to the published version of the manuscript.Funding: S.N.W.: NSERC and CFI. C.C.N. funded by Canadian Institutes of Health Research, Zealand Pharma,and the Canada Research Chairs program.Acknowledgments: We thank S. Wong and W.C. Sin for technical assistance.Conflicts of Interest: C.C.N. received a research grant from Zealand Pharma.References1. Del Zoppo, G.J.; Sharp, F.R.; Heiss, W.D.; Albers, G.W. Heterogeneity in the penumbra. J. Cereb. Blood FlowMetab. 2011, 31, 1836\u20131851. [CrossRef] [PubMed]2. 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