Mitochondrial Cx43 Research Articles

Effect of sevoflurane postconditioning on mitochondrial connexin 43 during myocardial ischemia-reperfusion in rats and the role of mito-KATP channels: an in vitro experiment

Objective To investigate the effect of sevoflurane postconditioning on mitochondrial connexin 43 (Cx43) during myocardial ischemia-reperfusion (I/R) in isolated rat hearts and the role of mitochondrial ATP-sensitive potassium (mito-KATP) channels in it. Methods Forty-five adult male Sprague-Dawley rats, weighing 200-250 g, were used in the study.Their hearts were excised and retrogradely perfused with K-H solution in a Langendorff apparatus.The 45 isolated hearts were assigned into 5 groups (n=9 each) using a random number table: control group (group C), I/R group, sevoflurane postconditioning group (group Sev), and sevoflurane postconditioning plus 5-hydroxydecanoate (5-HD, a specific mito-KATP channel blocker) group (group Sev+ 5-HD) and I/R plus 5-HD group (group I/R+ 5-HD). The hearts were subjected to 20 min of global ischemia followed by 90 min of reperfusion to establish the model of myocardial I/R injury.From the beginning of reperfusion, the hearts were perfused with K-H solution saturated with 3% sevoflurane for 15 min in group Sev, with K-H solution saturated with 3% sevoflurane and containing 100 μmol/L 5-HD in group Sev+ 5-HD, and with K-H solution containing 100 μmol/L 5-HD in group 5-HD.The heart rate (HR), left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP) and the maximum rate of increase and decrease of ventricular pressure (±dp/dtmax) were recorded at the end of equilibration (T1) and 30 and 60 min of reperfusion (T2, 3). At 90 min of reperfusion, the myocardial infarct size was measured by TTC staining, and the expression of total Cx43 (tCx43) and phosphorylated Cx43 (p-Cx43) in mitochondria was determined by Western blot analysis.The percentage of myocardial infarct size and p-Cx43/tCx43 ratio were calculated. Results Compared with group C, the HR, LVDP and ±dp/dtmax were significantly decreased, and the LVEDP was increased at T2, 3, and the percentage of myocardial infarct size was increased in the other 4 groups, the expression of mitochondrial tCx43 and p-Cx43 was significantly down-regulated in I/R, Sev+ 5-HD and 5-HD groups (P 0.05). Compared with group I/R, the HR, LVDP and ±dp/dtmax were significantly increased, and the LVEDP was decreased at T2, 3, the percentage of myocardial infarct size was decreased, and the expression of mitochondrial tCx43 and p-Cx43 was up-regulated in group Sev (P 0.05). Compared with group Sev, the HR, LVDP and ±dp/dtmax were significantly decreased, and the LVEDP was increased at T2, 3, the percentage of myocardial infarct size was increased, and the expression of mitochondrial tCx43 and p-Cx43 was down-regulated in group Sev+ 5-HD (P 0.05). Conclusion The mechanism by which sevoflurane postconditioning attenuates myocardial I/R injury may be related to induction of mito-KATP channel opening and up-regulation of the expression of mitochondrial Cx43 in cardiomyocytes of rats. Key words: Anesthetics, inhalation; Ischemic postconditioning; Myocardial reperfusion injury; Mitochondria; Connexin 43; KATP channels

New protein-protein interactions of mitochondrial connexin 43 in mouse heart.

Connexin 43 (Cx43), the gap junction protein involved in cell‐to‐cell coupling in the heart, is also present in the subsarcolemmal fraction of cardiomyocyte mitochondria. It has been described to regulate mitochondrial potassium influx and respiration and to be important for ischaemic preconditioning protection, although the molecular effectors involved are not fully characterized. In this study, we looked for potential partners of mitochondrial Cx43 in an attempt to identify new molecular pathways for cardioprotection. Mass spectrometry analysis of native immunoprecipitated mitochondrial extracts showed that Cx43 interacts with several proteins related with mitochondrial function and metabolism. Among them, we selected for further analysis only those present in the subsarcolemmal mitochondrial fraction and known to be related with the respiratory chain. Apoptosis‐inducing factor (AIF) and the beta‐subunit of the electron‐transfer protein (ETFB), two proteins unrelated to date with Cx43, fulfilled these conditions, and their interaction with Cx43 was proven by direct and reverse co‐immunoprecipitation. Furthermore, a previously unknown molecular interaction between AIF and ETFB was established, and protein content and sub‐cellular localization appeared to be independent from the presence of Cx43. Our results identify new protein–protein interactions between AIF‐Cx43, ETFB‐Cx43 and AIF‐ETFB as possible players in the regulation of the mitochondrial redox state.

Interaction between connexin 43 and nitric oxide synthase in mice heart mitochondria.

Connexin 43 (Cx43), which is highly expressed in the heart and especially in cardiomyocytes, interferes with the expression of nitric oxide synthase (NOS) isoforms. Conversely, Cx43 gene expression is down-regulated by nitric oxide derived from the inducible NOS. Thus, a complex interplay between Cx43 and NOS expression appears to exist. As cardiac mitochondria are supposed to contain a NOS, we now investigated the expression of NOS isoforms and the nitric oxide production rate in isolated mitochondria of wild-type and Cx43-deficient (Cx43Cre-ER(T)/fl) mice hearts. Mitochondria were isolated from hearts using differential centrifugation and purified via Percoll gradient ultracentrifugation. Isolated mitochondria were stained with an antibody against the mitochondrial marker protein adenine-nucleotide-translocator (ANT) in combination with either a neuronal NOS (nNOS) or an inducible NOS (iNOS) antibody and analysed using confocal laser scanning microscopy. The nitric oxide formation was quantified in purified mitochondria using the oxyhaemoglobin assay. Co-localization of predominantly nNOS (nNOS: 93 ± 4.1%; iNOS: 24.6 ± 7.5%) with ANT was detected in isolated mitochondria of wild-type mice. In contrast, iNOS expression was increased in Cx43Cre-ER(T)/fl mitochondria (iNOS: 90.7 ± 3.2%; nNOS: 53.8 ± 17.5%). The mitochondrial nitric oxide formation was reduced in Cx43Cre-ER(T)/fl mitochondria (0.14 ± 0.02 nmol/min./mg protein) in comparison to wild-type mitochondria (0.24 ± 0.02 nmol/min./mg). These are the first data demonstrating, that a reduced mitochondrial Cx43 content is associated with a switch of the mitochondrial NOS isoform and the respective mitochondrial rate of nitric oxide formation.

Suppression of PKCε-mediated mitochondrial connexin 43 phosphorylation at serine 368 is involved in myocardial mitochondrial dysfunction in a rat model of dilated cardiomyopathy.

Mitochondrial connexin43(Cx43) is important in cardioprotection by ischemic preconditioning; however, whether mitochondrial Cx43 is involved in mitochondrial dysfunction in the pathogenesis of dilated cardiomyopathy (DCM) remains to be elucidated. The present study was performed to investigate the changes in expression and the phosphorylation state of mitochondrial Cx43 in a rat model of DCM, and to determine whether the altered phosphorylation state of mitochondrial Cx43 was involved in mitochondrial dysfunction. A rat model of DCM was generated by daily oral administration of furazolidone (FZD) for 30weeks. Reverse transcription polymerase chain reaction and western blot analysis revealed a decrease in the overall expression of Cx43, accompanied by reduced levels of serine 368‑phosphorylated‑Cx43 immunoreactivity in the myocardium and myocardial mitochondria. In addition, the mitochondrial membrane potential and the activities of cytochrome c oxidase, succinate dehydrogenase and protein kinase C(PKC)ε were all significantly reduced compared with those of the control group. Phorbol‑12‑myristate‑13‑acetate (PMA), a specific PKC activator, partially reversed the FZD‑induced mitochondrial Cx43 dephosphorylation at serine368 and mitochondrial dysfunction in the cardiomyocytes. However, pretreatment with 18β‑glycerrhetinic acid, a connexin channel inhibitor, eliminated the mitochondrial protective effect of PMA in the cardiomyocytes sparsely plated without cell to cell contact. These results suggested that dephosphorylation of mitochondrial Cx43 at serine368, due to the suppression of PKCε activity, may be a novel mechanism for mitochondrial dysfunction in the pathogenesis of DCM.

Doxorubicin-Mediated Cardiotoxicity: Role of Mitochondrial Connexin 43.

Doxorubicin is the highly effective anthracycline, but its clinical use is limited by cardiotoxicity and consequent dysfunction. It has been proposed that the etiology of this is related to mitochondrial dysfunction. Connexin 43 (Cx43), the principal protein building block of cardiac gap junctions and hemichannels, plays an important role in cardioprotection. Recent reports confirmed the presence of Cx43 in the mitochondria as well. In this study, the role of mitochondrial Cx43 was evaluated 3 or 6h after Doxorubicin administration to the rat heart cell line H9c2. Pharmacological inhibition of Hsp90 demonstrated that the mitochondrial Cx43 conferred cardioprotection by reducing cytosolic and mitochondrial reactive oxygen species production, mitochondrial calcium overload and mitochondrial membrane depolarization and cytochrome c release. In conclusion, our study demonstrates that Cx43 plays an important role in the protection of cardiac cells from Doxorubicin-induced toxicity.

S-nitrosation of mitochondrial connexin 43 regulates mitochondrial function.

S-nitrosation (SNO) of connexin 43 (Cx43)-formed channels modifies dye uptake in astrocytes and gap junctional communication in endothelial cells. Apart from forming channels at the plasma membrane of several cell types, Cx43 is also located at the inner membrane of myocardial subsarcolemmal mitochondria (SSM), but not in interfibrillar mitochondria (IFM). The absence or pharmacological blockade of mitochondrial Cx43 (mtCx43) reduces dye and potassium uptake. Lack of mtCx43 is associated with loss of endogenous cardioprotection by ischemic preconditioning (IPC), which is mediated by formation of reactive oxygen species (ROS). Whether or not mitochondrial Lucifer Yellow (LY), ion uptake, or ROS generation are affected by SNO of mtCx43 and whether or not cardioprotective interventions affect SNO of mtCx43 remains unknown. In SSM from rat hearts, application of NO donors (48 nmol to 1 mmol) increased LY uptake (0.5 mmol SNAP 38.4 ± 7.1 %, p < 0.05; 1 mmol GSNO 28.1 ± 7.4 %, p < 0.05) and the refilling rate of potassium (SNAP 227.9 ± 30.1 %, p < 0.05; GSNO 122.6 ± 28.1 %, p < 0.05). These effects were absent following blockade of Cx43 hemichannels by carbenoxolone as well as in IFM lacking Cx43. Unlike potassium, the sodium permeability was not affected by application of NO. Furthermore, mitochondrial ROS formation was increased following NO application compared to control SSM (0.5 mmol SNAP 22.9 ± 1.8 %, p < 0.05; 1 mmol GSNO 40.6 ± 7.1 %, p < 0.05), but decreased in NO treated IFM compared to control (0.5 mmol SNAP 14.4 ± 4 %, p < 0.05; 1 mmol GSNO 13.8 ± 4 %, p < 0.05). NO donor administration to isolated SSM increased SNO of mtCx43 by 109.2 ± 15.8 %. Nitrite application (48 nmol) to mice was also associated with elevated SNO of mtCx43 by 59.3 ± 18.2 % (p < 0.05). IPC by four cycles of 5 min of ischemia and 5 min of reperfusion increased SNO of mtCx43 by 41.6 ± 1.7 % (p < 0.05) when compared to control perfused rat hearts. These data suggest that SNO of mtCx43 increases mitochondrial permeability, especially for potassium and leads to increased ROS formation. The increased amount of SNO mtCx43 by IPC or nitrite administration may link NO and Cx43 in the signal transduction cascade of cardioprotective interventions.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-014-0433-x) contains supplementary material, which is available to authorized users.

The FGF-2-triggered protection of cardiac subsarcolemmal mitochondria from calcium overload is mitochondrial connexin 43-dependent

Fibroblast growth factor 2 (FGF-2) protects the heart from ischaemia- and reperfusion-induced cell death by a mechanism linked to protein kinase C (PKC)ε-mediated connexin 43 (Cx43) phosphorylation. Cx43 localizes predominantly to gap junctions, but has also been detected at subsarcolemmal (SSM), but not interfibrillar (IFM), mitochondria, where it is considered important for cardioprotection. We have now examined the effect of FGF-2 administration to the heart on resistance to calcium-induced permeability transition (mPTP) of isolated SSM vs. IFM suspensions, in relation to mitochondrial PKCε/Cx43 levels, phosphorylation, and the presence of peptide Gap27, a Cx43 channel blocker. FGF-2 perfusion increased resistance to calcium-induced mPTP in SSM and IFM suspensions by 2.9- and 1.7-fold, respectively, compared with their counterparts from vehicle-perfused hearts, assessed spectrophotometrically as cyclosporine A-inhibitable swelling. The salutary effect of FGF-2 was lost in SSM, but not in IFM, in the presence of Gap27. FGF-2 perfusion increased relative levels of PKCε, phospho(p) PKCε, and Tom-20 translocase in SSM and IFM, and of Cx43 in SSM. Phospho-serine (pS) 262- and pS368-Cx43 showed a 30- and 8-fold increase, respectively, in SSM from FGF-2-treated, compared with untreated, hearts. Stimulation of control SSM with phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased both calcium tolerance and mitochondrial Cx43 phosphorylation at S262 and S368. The PMA-induced phosphorylation of mitochondrial Cx43 was prevented by εV1-2, a PKCε-inhibiting peptide. SSM are more responsive than IFM to FGF-2-triggered protection from calcium-induced mPTP, by a mitochondrial Cx43 channel-mediated pathway, associated with mitochondrial Cx43 phosphorylation at PKCε target sites.

Connexin 43 impacts on mitochondrial potassium uptake

In cardiomyocytes, connexin 43 (Cx43) forms gap junctions and unopposed hemichannels at the plasma membrane, but the protein is also present at the inner membrane of subsarcolemmal mitochondria (SSM). Both inhibition and genetic ablation of Cx43 reduce ADP-stimulated complex 1 respiration. Since mitochondrial potassium influx impacts on oxygen consumption, we investigated whether or not inhibition or ablation of mitochondrial Cx43 alters mitochondrial potassium uptake. SSM were isolated from rat left ventricular myocardium and loaded with the potassium-sensitive dye PBFI (potassium-binding benzofuran isophthalate). Intramitochondrial potassium was replaced by tetraethylammonium. Mitochondria were incubated under control conditions or treated with 250 μM Gap19, a peptide that specifically inhibits Cx43-based hemichannels at plasma membranes. Subsequently, 140 mM KCl was added and the slope of the increase in PBFI fluorescence over time was calculated. The slope of the PBFI fluorescence of the control mitochondria was set to 100%. In the presence of Gap19, the mitochondrial potassium influx was reduced from 100 ± 11.6% in control mitochondria to 65.5 ± 10.7% (n = 6, p < 0.05). In addition to the pharmacological inhibition of Cx43, potassium influx was studied in mitochondria isolated from conditional Cx43 knockout mice. Here, the ablation of Cx43 was achieved by the injection of 4-hydroxytamoxifen (4-OHT; Cx43Cre-ER(T)/fl + 4-OHT). The mitochondria of the Cx43Cre-ER(T)/fl + 4-OHT mice contained 3 ± 1% Cx43 (n = 6) of that in control mitochondria (100 ± 11%, n = 8, p < 0.05). The ablation of Cx43 (n = 5) reduced the velocity of the potassium influx from 100 ± 11.2% in control mitochondria (n = 9) to 66.6 ± 5.5% (p < 0.05). Taken together, our data indicate that both pharmacological inhibition and genetic ablation of Cx43 reduce mitochondrial potassium influx.

Connexin 43 acts as a cytoprotective mediator of signal transduction by stimulating mitochondrial KATP channels in mouse cardiomyocytes

Potassium (K+) channels in the inner mitochondrial membrane influence cell function and survival. Increasing evidence indicates that multiple signaling pathways and pharmacological actions converge on mitochondrial ATP-sensitive K+ (mitoKATP) channels and PKC to confer cytoprotection against necrotic and apoptotic cell injury. However, the molecular structure of mitoKATP channels remains unresolved, and the mitochondrial phosphoprotein(s) that mediate cytoprotection by PKC remain to be determined. As mice deficient in the main sarcolemmal gap junction protein connexin 43 (Cx43) lack this cytoprotection, we set out to investigate a possible link among mitochondrial Cx43, mitoKATP channel function, and PKC activation. By patch-clamping the inner membrane of subsarcolemmal murine cardiac mitochondria, we found that genetic Cx43 deficiency, pharmacological connexin inhibition by carbenoxolone, and Cx43 blockade by the mimetic peptide 43GAP27 each substantially reduced diazoxide-mediated stimulation of mitoKATP channels. Suppression of mitochondrial Cx43 inhibited mitoKATP channel activation by PKC. MitoKATP channels of interfibrillar mitochondria, which do not contain any detectable Cx43, were insensitive to both PKC activation and diazoxide, further demonstrating the role of Cx43 in mitoKATP channel stimulation and the compartmentation of mitochondria in cell signaling. Our results define a role for mitochondrial Cx43 in protecting cardiac cells from death and provide a link between cytoprotective stimuli and mitoKATP channel opening, making Cx43 an attractive therapeutic target for protection against cell injury.

Downregulation of Mitochondrial Connexin 43 by High Glucose Triggers Mitochondrial Shape Change and Cytochrome c Release in Retinal Endothelial Cells

To determine connexin 43 (Cx43) localization in mitochondria and investigate the effects of high glucose (HG) on mitochondrial Cx43 (mtCx43) expression and whether altered mtCx43 channel activity is involved in promoting apoptosis in retinal endothelial cells. MtCx43 localization was determined using immunostaining, green fluorescent protein (GFP)-tagged Cx43 followed by confocal imaging, and Western blot analysis using protein isolated from mitochondria of rat retinal endothelial cells (RRECs). To assess HG effects on mtCx43 expression, RRECs were grown in normal (5 mM) or HG (30 mM) medium for 7 days, and mtCx43 protein level assessed by Western blot analysis. To determine if mtCx43 channel inhibition affected mitochondrial morphology, RRECs grown sparsely were left untreated or treated with β-glycerrhetinic acid (β-GA), an inhibitor of connexin channels, and imaged using confocal microscopy. Additionally, mitochondria isolated from RRECs were treated with β-GA, and cytochrome c release assessed by Western blot. Cx43 localization on the mitochondria of RRECs was confirmed with immunofluorescence staining using Cx43 antibody and GFP-tagged Cx43 imaged in live cells. Western blot analysis indicated that Cx43 was located primarily on the inner mitochondrial membrane, and mtCx43 protein level was significantly reduced in RRECs grown in HG condition. Treatment of RRECs with β-GA significantly decreased mtCx43 phosphorylation, induced mitochondrial fragmentation, and isolated mitochondria treated with β-GA showed increased cytochrome c release. HG-induced downregulation of mtCx43 protein resulting in decreased channel activity may promote mitochondrial morphology changes and cytochrome c release, suggesting a novel mechanism for hyperglycemia-induced apoptosis in diabetic retinopathy.

Mitochondrial connexin 43 impacts on respiratory complex I activity and mitochondrial oxygen consumption

Connexin 43 (Cx43) is present at the sarcolemma and the inner membrane of cardiomyocyte subsarcolemmal mitochondria (SSM). Lack or inhibition of mitochondrial Cx43 is associated with reduced mitochondrial potassium influx, which might affect mitochondrial respiration. Therefore, we analysed the importance of mitochondrial Cx43 for oxygen consumption. Acute inhibition of Cx43 in rat left ventricular (LV) SSM by 18α glycyrrhetinic acid (GA) or Cx43 mimetic peptides (Cx43-MP) reduced ADP-stimulated complex I respiration and ATP generation. Chronic reduction of Cx43 in conditional knockout mice (Cx43Cre-ER(T)/fl + 4-OHT, 5–10% of Cx43 protein compared with control Cx43fl/fl mitochondria) reduced ADP-stimulated complex I respiration of LV SSM to 47.8 ± 2.4 nmol O2/min.*mg protein (n = 8) from 61.9 ± 7.4 nmol O2/min.*mg protein in Cx43fl/fl mitochondria (n = 10, P < 0.05), while complex II respiration remained unchanged. The LV complex I activities (% of citrate synthase activity) of Cx43Cre-ER(T)/fl+4-OHT mice (16.1 ± 0.9%, n = 9) were lower than in Cx43fl/fl mice (19.8 ± 1.3%, n = 8, P < 0.05); complex II activities were similar between genotypes. Supporting the importance of Cx43 for respiration, in Cx43-overexpressing HL-1 cardiomyocytes complex I respiration was increased, whereas complex II respiration remained unaffected. Taken together, mitochondrial Cx43 is required for optimal complex I activity and respiration and thus mitochondrial ATP-production.

Role of connexin-43 in protective PI3K-Akt-GSK-3β signaling in cardiomyocytes

Sarcolemmal connexin-43 (Cx43) and mitochondrial Cx43 play distinct roles: formation of gap junctions and production of reactive oxygen species (ROS) for redox signaling. In this study, we examined the hypothesis that Cx43 contributes to activation of a major cytoprotective signal pathway, phosphoinositide 3-kinase (PI3K)-Akt-glycogen synthase kinase-3β (GSK-3β) signaling, in cardiomyocytes. A δ-opioid receptor agonist {[d-Ala(2),d-Leu(5)]enkephalin acetate (DADLE)}, endothelin-1 (ET-1), and insulin-like growth factor-1 (IGF-1) induced phosphorylation of Akt and GSK-3β in H9c2 cardiomyocytes. Reduction of Cx43 protein to 20% of the normal level by Cx43 small interfering RNA abolished phosphorylation of Akt and GSK-3β induced by DADLE or ET-1 but not that induced by IGF-1. DADLE and IGF-1 protected H9c2 cells from necrosis after treatment with H(2)O(2) or antimycin A. The protection by DADLE or ET-1, but not that by IGF-1, was lost by reduction of Cx43 protein expression. In contrast to Akt and GSK-3β, PKC-ε, ERK and p38 mitogen-activated protein kinase were phosphorylated by ET-1 in Cx43-knocked-down cells. Like diazoxide, an activator of the mitochondrial ATP-sensitive K(+) channel, DADLE and ET-1 induced significant ROS production in mitochondria, although such an effect was not observed for IGF-1. Cx43 knockdown did not attenuate the mitochondrial ROS production by DADLE or ET-1. Cx43 was coimmunoprecipitated with the β-subunit of G protein (Gβ), and knockdown of Gβ mimicked the effect of Cx43 knockdown on ET-1-induced phosphorylation of Akt and GSK-3β. These results suggest that Cx43 contributes to activation of class I(B) PI3K in PI3K-Akt-GSK-3β signaling possibly as a cofactor of Gβ in cardiomyocytes.

Fibroblast growth factor‐2 exerts protective effects on cardiac mitochondria

Fibroblast growth factor‐2 (FGF‐2) is a potent cardioprotective agent, by a mechanism linked to connexin43 (Cx43) phosphorylation at protein kinase C (PKC) target serines such as S262. Here we have examined the effects of FGF‐2 on mitochondrial resistance to calcium‐ induced opening of the permeability transition pore (swelling), in relation to cardiac mitochondrial Cx43 levels and phosphorylation.Subsarcolemmal mitochondria isolated from FGF‐2‐treated hearts: (a) showed 1.5–2 fold increase in total Cx43, PKCε, TOM20 translocase, and a over 30‐fold increase in phospho‐S262‐Cx43, compared to control mitochondria obtained from untreated hearts; (b) were more resistant to calcium‐induced swelling and cytochrome C release; (c) were resistant to calcium‐induced mitochondrial Cx43 degradation. Calpeptin, a potent inhibitor of the calcium activated protease calpain, prevented Cx43 degradation in control mitochondria. Stimulation of control mitochondria with the phorbol‐ester PMA, or FGF‐2, increased both mitochondrial Cx43 phosphorylation at S262 and resistance to calcium‐induced swelling / cytochrome c release.Our data imply that FGF‐2 cardioprotection is mediated by increasing mitochondrial resistance to calcium damage, via Cx43 translocation to mitochondria and mitochondrial Cx43 phosphorylation.This work is supported by Heart and Stroke Foundation of Canada and St. Boniface Hospital Research Foundation.

Localization of connexin 43 in mitochondria of ovarian cancer cells

Connexin 43 (Cx43) is a gap junctional protein typically known for its channel forming role in intercellular communication between many types of cells. It has been reported that Cx43 proteins are localized in mitochondrial membranes from cardiovascular cells, Sca‐1+ stem cells, and rat brain tissues. In those various cell types Cx43 seemingly has a cytoprotective role during physiological stress. The present studies were undertaken to determine whether Cx43 protein is present in mitochondria and could possibly play a similar role in human ovarian cancer cells (OVCAR‐3 and SK‐OV‐3 cell lines). Western blot analysis demonstrated that the relative amount of Cx43 in mitochondrial pellets was enriched over that in other fractions. This was validated by comparison with patterns of established mitochondrial proteins such as voltage‐dependent anion channel and cytochrome c oxidase subunit IV. Immunogold transmission electron microscopy confirmed localization of Cx43 in the mitochondria. Additional findings that the level of mitochondrial Cx43 channel protein was specifically reduced with induction of apoptosis by etoposide are compatible with the possibility that it has a role in mitochondrial homeostasis in these ovarian cancer cells. This project was funded by the TTUHSC Department of Cell Biology and Biochemistry and the South Plains Oncology Consortium.

GSK3β Transfers Cytoprotective Signaling through Connexin 43 onto Mitochondrial ATP-Sensitive K+ Channels

Background: Ischemic preconditioning involves activation of signaling pathways, including activation of protein kinase C (PKC) and inhibition of glycogen synthase kinase 3beta (GSK3β). Opening of mitochondrial ATP-sensitive K+ (mitoKATP) channels has been proposed to play a critical role in ischemic preconditioning. Recently, we demonstrated that connexin 43 (Cx43) found in the inner mitochondrial membrane, is essential for cytoprotective signal transduction onto mitoKATP channels.Objectives: To dissect signal transduction targeted at mitoKATP channels and to possibly identify a functional link between mitoKATP channel activation, mitochondrial Cx43 and GSK3β activity.Methods: We used direct single-channel patch-clamp recordings of cardiac mitoplasts from C57/Bl6J mice and homozygous GSK3β-S9A mice with cardiac-specific expression of a constitutively active form of GSK3β, and performed Western blots and co-immunoprecipitation of isolated mitochondria.Results: MitoKATP channel activity was stimulated by the GSK3β small molecule inhibitor SB216763, which increased the open probability compared to control. The Cx43 inhibitor carbenoxolone and the Cx43 mimetic peptide 43GAP27 significantly reduced mitoKATP channel activation by SB216763, supporting the notion that GSK3 transfers cytoprotective signaling via Cx43 onto mitoKATP channels. In GSK3β-S9A mitoplasts mitoKATP currents could be recorded with similar single-channel properties as in wildtype mitochondria. Single-channel activation of mitoKATP by the PKC activator PMA was significantly attenuated in GSK3β-S9A mitoplasts. Western blot analysis revealed reduced phosphorylation of mitochondrial Cx43 at the PKC phosphorylation site Ser368 upon PMA application in mitochondria from GSK3β-S9A mice versus wildtype. Furthermore Immunoprecipitation of Cx43 from isolated mitochondria revealed a signal for GSK3β, while immunoprecipitation of GSK3β also showed a signal for Cx43, indicating an association of these proteins.Conclusions: PKC transmits a signal onto mitoKATP channels through GSK3β. This GSK3β signal transduction on mitoKATP channels is transferred via Cx43 within a multiprotein signaling module.

Heat shock protein 90 mediates anti-apoptotic effect of diazoxide by preventing the cleavage of Bid in hypothermic preservation rat hearts

Successful organ preservation is the premise for clinical organ transplantation. The present study investigated whether heat shock protein 90 (Hsp90) is important in the anti-apoptotic effect of diazoxide in hypothermic preservation rat hearts. Isolated rat hearts were preserved in Celsior solution, with or without diazoxide, for 3 to 9 hours, followed by 60 minutes of reperfusion. Cell apoptosis was assessed by terminal deoxynucleotide transferase-mediated deoxy uridine triphosphate nick-end labeling. The left ventricular developed pressure (LVDP) was recorded. Expression of Hsp90 protein and cleavage of Bid were detected by Western blot and polymerase chain reaction. After hypothermic preservation for 3 to 9 hours, the LVDP recovery rate significantly decreased and cardiomyocyte apoptosis index increased in a time-dependent manner. When compared with the 9-hour preservation group, Celsior solution supplemented with diazoxide significantly enhanced the LVDP recovery rate and decreased the apoptosis index. The cleavage of Bid increased after 9 hours of hypothermic preservation, which was inhibited by Celsior solution supplemented with diazoxide. Hypothermic preservation of rat hearts for 9 hours decreased the expression of Hsp90, whereas diazoxide supplementation significantly increased the expression of Hsp90. The Hsp90 inhibitor 17-allylamino-17-demethoxy-geldanamycin inhibited the diazoxide-induced decrease in cleavage of Bid, improvement of cardiac function, and decrease of apoptosis. Hsp90 inhibitor had no effect on the diazoxide-induced increase of total Cx43 protein expression in hearts preserved 9 hours, but inhibited the diazoxide-induced increase of mitochondrial Cx43 protein level. Hsp90 might mediate diazoxide-induced cardioprotection against apoptosis in hypothermic preservation heart by preventing the cleavage of Bid.

Cholesterol diet leads to attenuation of ischemic preconditioning-induced cardiac protection: the role of connexin 43

Cardioprotection by ischemic preconditioning (IP) was abolished in connexin 43 (Cx43)-deficient mice due to loss of Cx43 located in mitochondria rather than at the sarcolemma. IP is lost in hyperlipidemic rat hearts as well. Since changes in mitochondrial Cx43 in hyperlipidemia have not yet been analyzed, we determined total and mitochondrial Cx43 levels in male Wistar rats fed a laboratory chow enriched with 2% cholesterol or normal chow for 12 wk. Hearts were isolated and perfused according to Langendorff. After a 10-min perfusion, myocardial tissue cholesterol, superoxide, and nitrotyrosine contents were measured and Cx43 content in whole heart homogenate and a mitochondrial fraction determined. In the cholesterol-fed group, tissue cholesterol and superoxide formation was increased (P < 0.05), while total Cx43 content remained unchanged. Mitochondrial total and dephosphorylated Cx43 content decreased. Hearts were subjected to an IP protocol (3 × 5 min ischemia-reperfusion) or time-matched aerobic perfusion followed by 30-min global ischemia and 5-min reperfusion. IP reduced infarct size in normal but not in cholesterol-fed rats. At 5-min reperfusion following 30-min global ischemia, the total and dephosphorylated mitochondrial Cx43 content was increased, which was abolished by IP in both normal and high-cholesterol diet. In conclusion, loss of cardioprotection by IP in hyperlipidemia is associated with a redistribution of both sarcolemmal and mitochondrial Cx43.

Glycogen synthase kinase 3β transfers cytoprotective signaling through connexin 43 onto mitochondrial ATP-sensitive K + channels

Despite compelling evidence supporting key roles for glycogen synthase kinase 3β (GSK3β), mitochondrial adenosine triphosphate-sensitive K(+) (mitoK(ATP)) channels, and mitochondrial connexin 43 (Cx43) in cytoprotection, it is not clear how these signaling modules are linked mechanistically. By patch-clamping the inner membrane of murine cardiac mitochondria, we found that inhibition of GSK3β activated mitoK(ATP). PKC activation and protein phosphatase 2a inhibition increased the open probability of mitoK(ATP) channels through GSK3β, and this GSK3β signal was mediated via mitochondrial Cx43. Moreover, (i) PKC-induced phosphorylation of mitochondrial Cx43 was reduced in GSK3β-S9A mice; (ii) Cx43 and GSK3β proteins associated in mitochondria; and (iii) SB216763-mediated reduction of infarct size was abolished in Cx43 KO mice in vivo, consistent with the notion that GSK3β inhibition results in mitoK(ATP) opening via mitochondrial Cx43. We therefore directly targeted mitochondrial Cx43 by the Cx43 C-terminal binding peptide RRNYRRNY for cardioprotection, circumventing further upstream pathways. RRNYRRNY activated mitoK(ATP) channels via Cx43. We directly recorded mitochondrial Cx43 channels that were activated by RRNYRRNY and blocked by the Cx43 mimetic peptide (43)GAP27. RRNYRRNY rendered isolated cardiomyocytes in vitro and the heart in vivo resistant to ischemia/reperfusion injury, indicating that mitochondrial Cx43- and/or mitoK(ATP)-mediated reduction of infarct size was not undermined by RRNYRRNY-related opening of sarcolemmal Cx43 channels. Our results demonstrate that GSK3β transfers cytoprotective signaling through mitochondrial Cx43 onto mitoK(ATP) channels and that Cx43 functions as a channel in mitochondria, being an attractive target for drug treatment against cardiomyocyte injury.

Calcium-induced permeability transition in rat brain mitochondria is promoted by carbenoxolone through targeting connexin43

Carbenoxolone (Cbx), a substance from medicinal licorice, is used for antiinflammatory treatments. We investigated the mechanism of action of Cbx on Ca(2+)-induced permeability transition pore (PTP) opening in synaptic and nonsynaptic rat brain mitochondria (RBM), as well as in rat liver mitochondria (RLM), in an attempt to identify the molecular target of Cbx in mitochondria. Exposure to threshold Ca(2+) load induced PTP opening, as seen by sudden Ca(2+) efflux from the mitochondrial matrix and membrane potential collapse. In synaptic RBM, Cbx (1 μM) facilitated the Ca(2+)-induced, cyclosporine A-sensitive PTP opening, while in nonsynaptic mitochondria the Cbx threshold concentration was higher. A well-known molecular target of Cbx is the connexin (Cx) family, gap junction proteins. Moreover, Cx43 was previously found in heart mitochondria and attributed to the preconditioning mechanism of protection. Thus, we hypothesized that Cx43 might be a target for Cbx in brain mitochondria. For the first time, we detected Cx43 by Western blot in RBM, but Cx43 was absent in RLM. Interestingly, two anti-Cx43 antibodies, directed against amino acids 252 to 270 of rat Cx43, abolished the Cbx-induced enhancement of PTP opening in total RBM and in synaptic mitochondria, but not in RLM. In total RBM and in synaptic mitochondria, PTP caused dephosphorylation of Cx43 at serine 368. The phosphorylation level of serine 368 was decreased at threshold calcium concentration and additionally in the combined presence of Cbx in synaptic mitochondria. In conclusion, active mitochondrial Cx43 appears to counteract the Ca(2+)-induced PTP opening and thus might inhibit the PTP-ensuing mitochondrial demise and cell death. Consequently, we suggest that activity of Cx43 in brain mitochondria represents a novel molecular target for protection.

Mitochondria-specific transgenic overexpression of connexin-43 simulates preconditioning-induced cytoprotection of stem cells.

We previously reported that preconditioning of stem cells with insulin-like growth factor-1 (IGF-1) translocated connexin-43 (Cx-43) into mitochondria, causing cytoprotection. We posit that these preconditioning effects could be simulated by mitochondria-specific overexpression of Cx-43. During IGF-1-induced preconditioning of C57black/6 mouse bone marrow stem cell antigen-1(+) (Sca-1(+)) cells, Cx-43 was mainly translocated onto the mitochondrial inner membrane, which was abrogated by an extracellular signal-regulated kinases 1 and 2 (ERK1/2) blocker PD98059. To investigate the role of mitochondrial Cx-43, we successfully designed a vector coding for full-length mouse Cx-43 with a mitochondria-targeting sequence (mito-Cx-43) and cloned into a shuttle vector (pShuttle-IRES-hrGFP-1) for mitochondria-specific overexpression of Cx-43 (mito-Cx-43). Sca-1(+) cells with mito-Cx-43 reduced cytosolic accumulation of cytochrome c, lowered caspase-3 activity, and improved survival during index oxygen-glucose deprivation as determined by terminal deoxynucleotidyl transferase dUTP nick-end labelling and lactate dehydrogenase assays. Computational analysis revealed a B-cell lymphoma-2 (Bcl-2) homology domain-3 (BH3) motif in Cx-43 with a conserved pattern of amino acids consistent with the Bcl-2 family that regulated cytochrome c release. Moreover, computational secondary structure prediction indicated an extended α-helix in this region, a known condition for BH3-driven protein-protein interactions. Cx-43 translocation into mitochondria during preconditioning was ERK1/2-dependent. Expression of mito-Cx-43 simulated the cytoprotective effects of preconditioning in stem cells. Structural features of Cx-43 were shared with the Bcl-2 family as determined by computational analysis.