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Patients with coronary artery disease show high serum levels of interleukin (IL)-27, a novel member of the IL-6 family. However, the function of IL-27 in hearts suffering ischemia/reperfusion (IR) injury is unclear. Here, we showed increased expression of mRNA for the IL-27 subunits, EBI3 and p28, in rat hearts after 40 min of coronary ligation and release for 7 days. This increase was associated with a peak in the release of the cardiac enzyme, creatine kinase-MB, on day 2 post-release. Moreover, levels of IL-27 receptor subunit gp130 mRNA, but not those of subunit WSX-1 mRNA, decreased in post-ischemic hearts. These results suggest that increased IL-27 production may compensate for receptor downregulation during myocardial recovery. Lactate dehydrogenase release and crystal violet staining revealed that IL-27 or IL-6 significantly attenuated severe hypoxia (SH, 2 % O2)-induced cell damage in H9c2 cardiomyoblasts and primary rat neonatal cardiomyocytes. Incubating cardiomyocytes with IL-27 or IL-6 resulted in time-dependent activation of signal transducers and activators of transcription 3 (STAT3). Interestingly, IL-27-induced STAT3 activation was attenuated by pre-treatment with a gp130-neutralizing antibody. Blocking gp130 also reduced the cytoprotective effects of IL-27 or IL-6. Moreover, IL-27-mediated protection against SH was blocked by stattic, a small-molecule inhibitor of STAT3. IL-27 markedly improved post-ischemic recovery and reduced tissue damage in isolated perfused hearts when administered 5 min before reperfusion. These results indicate that IL-27 protects the myocardium against IR injury and facilitates the recovery of damaged cardiomyocytes via the gp130/STAT3 pathway.

Purpose of Review: Excessive production of fibrosis is a feature of hypertension-induced renal injury. Activation of RhoA/Rho-kinase (ROCK) axis has been shown in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. We assessed whether selective endothelin receptor blockers can attenuate renal fibrosis by inhibiting RhoA/ROCK axis in DOCA-salt rats., Methods: At 4 weeks after the start of DOCA-salt treatment and uninephrectomization, male Wistar rats were randomized into three groups for 4 weeks: vehicle, ABT-627 (endothelin-A receptor inhibitor) and A192621 (endothelin-B receptor inhibitor)., Results: DOCA-salt was characterized by increased blood pressure, decreased renal function, increased proteinuria, increased glomerulosclerosis and tubulointerstitial fibrosis with myofibroblast accumulation, increased renal endothelin-1 levels and RhoA activity along with increased expression of connective tissue growth factor at both mRNA and protein levels as compared with uninephrectomized control male Wistar rats. Treatment with a selective mineralocorticoid receptor antagonist, eplerenone, ameliorated proteinuria. Impaired renal function and histological changes were overcome by treatment with ABT-627, but not with A192621. The beneficial effects of bosentan, a nonspecific endothelin receptor blocker, on proteinuria, RhoA activity, and connective tissue growth factor levels were similar to ABT-627. Furthermore, in an isolated perfuse kidney, a RhoA inhibitor, C3 exoenzyme, and two ROCK inhibitors, fasudil and Y-27632, significantly attenuated connective tissue growth factor levels., Conclusions: These results indicate that DOCA-salt elevates renal endothelin-1 levels and RhoA activity via activation of mineralocorticoid receptor, resulting in renal fibrosis and proteinuria. Endothelin-A receptor blockade can attenuate DOCA-salt-induced renal fibrosis probably through the inhibition of RhoA/ROCK activity and connective tissue growth factor expression.

HYS-32 [4-(3,4-dimethoxyphenyl)-3-(naphthalen-2-yl)-2(5H)-furanone] is a new analogue of the anti-tumor compound combretastatin A-4 containing a cis-stilbene moiety. In this study, we investigated its effects on Cx43 gap junction intercellular communication (GJIC) and the signaling pathway involved in rat primary astrocytes. Western blot analyses showed that HYS-32 dose- and time-dependently upregulated Cx43 expression. A confocal microscopic study and scrape-loading/dye transfer analyses demonstrated that HYS-32 (5μM) induced microtubule coiling, accumulation of Cx43 in gap junction plaques, and increased GJIC in astrocytes. The HYS-32-induced microtubule coiling and Cx43 accumulation in gap junction plaques was reversed when HYS-32 was removed. Treatment of astrocytes with cycloheximide resulted in time-dependent degradation of by co-treatment with HYS-32 by increasing the half-life of Cx43. Co-treatment with HYS-32 also prevented the LPS-induced downregulation of Cx43 and inhibition of GJIC in astrocytes. HYS-32 induced activation of PKC, ERK, and JNK, and co-treatment with the PKC inhibitor Go6976 or the ERK inhibitor PD98059, but not the JNK inhibitor SP600125, prevented the HYS-32-induced increase in Cx43 expression and GJIC. Go6976 suppressed the HYS-32-induced PKC phosphorylation and increase in phospho-ERK levels, while PD98059 did not prevent the HYS-32-induced increase in phospho-PKC levels, suggesting that PKC is an upstream effector of ERK. In conclusion, our results show that HYS-32 increases the half-life of Cx43 and enhances Cx43 expression and GJIC in astrocytes via a PKC-ERK signaling cascade. These novel biological effects of HYS-32 on astrocyte gap junctions support its potential for therapeutic use as a protective agent for the central nervous system., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We have demonstrated that activation of ATP-sensitive potassium (K(ATP)) channels can attenuate sympathetic hyperinnervation. Sildenafil, a phosphodiesterase-5 inhibitor, has been shown to provide a preconditioning-like cardioprotective effect via opening of K(ATP) channels. The aim of this study was to investigate whether chronic administration of sildenafil attenuates cardiac sympathetic hyperinnervation after myocardial infarction through activation of K(ATP) channels and to compare it with the nitric oxide donor isosorbide dinitrate. Male Wistar infarcted rats induced by ligation of the anterior descending artery were randomized to either vehicle, nicorandil, sildenafil, isosorbide dinitrate, glibenclamide, or a combination of nicorandil and glibenclamide, or sildenafil and glibenclamide. Myocardial norepinephrine levels revealed a significant elevation in vehicle-treated rats compared with sham-operated rats, consistent with sympathetic hyperinnervation after infarction assessed by immunohistochemical analysis for tyrosine hydroxylase, growth associated factor 43 and neurofilament and by protein expression and mRNA of nerve growth factor. Sympathetic hyperinnervation was reduced after administering either nicorandil or sildenafil. Arrhythmic scores during programmed stimulation in the sildenafil-treated rats were significantly lower than those treated with the vehicle. Furthermore, the beneficial effects of sildenafil-induced were reversed by the addition of either glibenclamide or 5-hydroxydecanoate, implicating mitochondrial K(ATP) channels as the relevant target. Isosorbide dinitrate failed to confer similar antiarrhythmia. 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one, a soluble guanylyl cyclase inhibitor, did not influence the effect of sildenafil on the nerve growth factor. These data indicate that sildenafil after infarction attenuated sympathetic hyperinnervation and arrhythmias by activation of mitochondrial K(ATP) channels through a guanylyl cyclase-cGMP-independent pathway., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Hypoxic injury to cardiomyocytes is a stress that causes cardiac pathology through cardiac-restricted gene expression. SRF (serum-response factor) and myocardin are important for cardiomyocyte growth and differentiation in response to myocardial injuries. Previous studies have indicated that AngII (angiotensin II) stimulates both myocardin expression and cardiomyocyte hypertrophy. In the present study, we evaluated the expression of myocardin and AngII after hypoxia in regulating gene transcription in neonatal cardiomyocytes. Cultured rat neonatal cardiomyocytes were subjected to hypoxia, and the expression of myocardin and AngII were evaluated. Different signal transduction pathway inhibitors were used to identify the pathway(s) responsible for myocardin expression. An EMSA (electrophoretic mobility-shift assay) was used to identify myocardin/SRF binding, and a luciferase assay was used to identify transcriptional activity of myocardin/SRF in neonatal cardiomyocytes. Both myocardin and AngII expression increased after hypoxia, with AngII appearing at an earlier time point than myocardin. Myocardin expression was stimulated by AngII and ERK (extracellular-signal-regulated kinase) phosphorylation, but was suppressed by an ARB (AngII type 1 receptor blocker), an ERK pathway inhibitor and myocardin siRNA (small interfering RNA). AngII increased both myocardin expression and transcription in neonatal cardiomyocytes. Binding of myocardin/SRF was identified using an EMSA, and a luciferase assay indicated the transcription of myocardin/SRF in neonatal cardiomyocytes. Increased BNP (B-type natriuretic peptide), MHC (myosin heavy chain) and [(3)H]proline incorporation into cardiomyocytes was identified after hypoxia with the presence of myocardin in hypertrophic cardiomyocytes. In conclusion, hypoxia in cardiomyocytes increased myocardin expression, which is mediated by the induction of AngII and the ERK pathway, to cause cardiomyocyte hypertrophy. Myocardial hypertrophy was identified as an increase in transcriptional activities, elevated hypertrophic and cardiomyocyte phenotype markers, and morphological hypertrophic changes in cardiomyocytes.

Caveolin-3, the major caveolin isoform in cardiomyocytes, plays an important role in the rapid signaling pathways initiated by stimulation of the membrane-associated molecules. To examine the role of caveolin-3 in regulating estrogen receptor alpha in cardiomyocytes, we investigate whether the membrane estrogen receptor alpha associates with caveolin-3 and whether this association is linked to the 17beta-estradiol-mediated signals. In control cardiomyocytes, following discontinuous sucrose gradient centrifugation, caveolin-3 was found predominantly in the lipid raft buoyant fractions, whereas it was distributed to both the buoyant and non-lipid raft heavy fractions following metabolic inhibition treatment. Confocal microscopy showed that estrogen receptor alpha co-localized with caveolin-3 on the plasma membrane of neonatal and adult rat cardiomyocytes. This membrane labeling of estrogen receptor alpha was not seen following treatment with the cholesterol-depleting agent methyl-beta-cyclodextrin (5mM), whereas metabolic inhibition had little effect on the membrane distribution of estrogen receptor alpha. Metabolic inhibition induced tyrosine phosphorylation of caveolin-3 and decreased its association with estrogen receptor alpha, both effects being mediated via a Src activation mechanism, since they were inhibited by the selective tyrosine kinase inhibitor PP2. Metabolic inhibition also induced tyrosine phosphorylation of connexin43 and increased its association with c-Src, both effects being prevented by 17beta-estradiol (200 nM). The effect of 17beta-estradiol on metabolic inhibition-induced tyrosine phosphorylation of connexin43 was inhibited by the specific estrogen receptor antagonist ICI182780. These data identify cardiac caveolin-3 as juxtamembrane scaffolding for estrogen receptor alpha docking at caveolae, which provide a unique compartment for conveying 17beta-estradiol-elicited, rapid signaling to regulate connexin43 phosphorylation during ischemia.

18Beta-glycyrrhetinic acid (18beta-GA) regulates serine/threonine dephosphorylation of connexin43 (Cx43). Phospho-specific antibodies were used here to determine the effect of 18beta-GA on serine 368-phosphorylated Cx43 (pSer368Cx43) in cultured rat neonatal cardiomyocytes by immunofluorescence microscopy and immunoblot analyses. 18beta-GA caused a time-dependent increase in pSer368Cx43 levels and induced gap junction disassembly, shown by a change in pSer368Cx43 immunostaining from large aggregates to dispersed punctates at cell-cell contact areas. 18beta-GA also induced a time-dependent increase in the levels of serine 729-phosphorylated PKCepsilon, the active form of PKCepsilon. The 18beta-GA-induced increase in pSer368Cx43 levels and changes in pSer368Cx43 staining pattern were abolished by the PKC inhibitor, chelerythrine. Furthermore, 18beta-GA increased the co-immunoprecipitation of Cx43 with PKCepsilon. However, the 18beta-GA-induced increase in pSer368Cx43 levels and increased association of Cx43 with PKCepsilon were inhibited by co-treatment with the protein phosphatase type 1 and type 2A inhibitor, calyculin A. We conclude that 18beta-GA induces Ser368 phosphorylation of Cx43 via PKCepsilon.

The mechanism by which 18beta-glycyrrhetinic acid regulates gap junction intercellular communication (GJIC) remains poorly understood. In this study, treatment of cultured rat neonatal cardiomyocytes with 18beta-glycyrrhetinic acid resulted in dose-dependent inhibition of GJIC as assessed by fluorescent dye transfer analysis. 18beta-Glycyrrhetinic acid induced time-dependent serine/threonine dephosphorylation and redistribution of connexin43 (Cx43) in cardiomyocytes and the induced Cx43 dephosphorylation was prevented by the protein phosphatase inhibitor, calyculin A. However, functional analyses showed that the inhibitory effect of 18beta-glycyrrhetinic acid on dye spreading among cardiomyocytes was not blocked by calyculin A, but was blocked by the Src-selective tyrosine kinase inhibitor, PP2. 18beta-Glycyrrhetinic acid also induced an increase in the levels of phosphorylated Src, and this effect was prevented by PP2. Immunoprecipitation using anti-Cx43 and anti-p-Src antibodies showed that 18beta-glycyrrhetinic acid increased the association between p-Src and Cx43 and induced tyrosine phosphorylation of Cx43. We conclude that the inhibitory effect of 18beta-glycyrrhetinic acid on GJIC in cardiomyocytes involves Src-mediated tyrosine phosphorylation of Cx43.

The effects of 17beta-estradiol (E2) on gap junction intercellular communication (GJIC) were assessed by Lucifer yellow dye coupling in cultured neonatal rat cardiomyocytes after metabolic inhibition (MI) using potassium cyanide and sodium iodoacetate. MI significantly reduced dye coupling of cardiomyocytes to 8.5% +/- 0.6% of control levels, and pretreatment with E2, but not its inactive isomer 17alpha-estradiol, dose-dependently (EC(50) = 0.41 microM) increased the dye coupling up to 76% +/- 15% of control levels. The effect of E2 on MI-induced dye uncoupling was abolished by tamoxifen, a potent estrogen receptor (ER) antagonist. The ligand, E2-BSA-FITC, labeled the cardiomyocyte surface, whereas BSA-FITC did not, suggesting the presence of membrane-associated E2 receptors. Double immunofluorescence microscopy showed that MI-induced the accumulation of non-phosphorylated Cx43 at the gap junction and that this was prevented by E2 pretreatment. Labeling of Lucifer yellow-microinjected cardiomyocytes with antibodies specific for Ser368-phosphorylated Cx43 (Ser368Cx43) or non-phosphorylated Cx43 confirmed that E2 reduced the MI-induced inhibition of dye coupling and accumulation of non-phosphorylated Cx43 concomitant with the reappearance of Ser368Cx43 at the gap junction. MI caused a decrease in Ser368Cx43 protein levels, and pretreatment with E2 significantly increased the levels of Ser368Cx43. Inhibition of protein kinase C (PKC) with chelerythrine blocked the E2-induced increase of Ser368Cx43 levels in MI-treated cardiomyocytes. These results suggest that E2 attenuates the inhibitory effect of MI on GJIC in cardiomyocytes by affecting the phosphorylation of Cx43, possibly mediated by activation of PKC via a membrane-associated signaling mechanism.

Gap junctions are intercellular communicating channels responsible for the synchronized activity of cardiomyocytes. Recent studies have shown that the membrane-associated guanylate kinase protein, zonula occludens-1 (ZO-1) can bind to catenins in epithelial cells and act as an adapter for the transport of the connexin isotype, Cx43 during gap junction formation. The significance of catenins in the development of gap junctions and whether complexes between catenins and ZO-1 are formed in cardiomyocytes are not clear. In this study, immunofluorescence and confocal microscopy showed sequential redistribution of alpha-catenin, beta-catenin, ZO-1, and Cx43 to the plasma membrane when rat cardiomyocytes were cultured in low Ca(2+) (<5 microM) medium, then shifted to 1.8 mM Ca(2+) medium (Ca(2+) switch). Diffuse cytoplasmic staining of alpha-catenin, beta-catenin, ZO-1, and Cx43 was seen in the cytoplasm when cardiomyocytes were cultured in low Ca(2+) medium. Staining of alpha-catenin, beta-catenin, and ZO-1 was detected at the plasma membrane of cell-cell contact sites 10 min after Ca(2+) switch, whereas Cx43 staining was first detected, colocalized with ZO-1 at the plasma membrane, 30 min after Ca(2+) switch. Distinct junctional and extensive cytoplasmic staining of alpha-catenin, beta-catenin, ZO-1, and Cx43 was seen 2 h after Ca(2+) switch. Immunoprecipitation of Triton X-100 cardiomyocyte extracts using anti-beta-catenin antibodies showed that beta-catenin was associated with alpha-catenin, ZO-1, and Cx43 at 2 h after Ca(2+) switch. Intracellular application of antisera against alpha-catenin, beta-catenin, or ZO-1 by electroporation of cardiomyocytes cultured in low Ca(2+) medium inhibited the redistribution of Cx43 to the plasma membrane following Ca(2+) switch. These results suggest the formation of a catenin-ZO-1-Cx43 complex in rat cardiomyocytes and that binding of catenins to ZO-1 is required for Cx43 transport to the plasma membrane during the assembly of gap junctions., (Copyright 2002 Wiley-Liss, Inc.)

Costameres, vinculin-containing structures found in skeletal and cardiac muscle, are thought to anchor the Z-discs of the peripheral myofibrils to the sarcolemma. Several lines of evidence indicate that two different sets of costameres, integrin- and N-cadherin-based, are present in cardiac muscles. In this study, immunoblot analysis was used to study the expression of N-cadherin, alpha-catenin, beta-catenin, vinculin, talin, and laminin in rat cardiac muscles at embryonic days 15 and 19, the day of birth (postnatal day 0), postnatal weeks 1, 2, 3, and 4, and in the adult. Double immunofluorescence microscopy was performed to study the spatial and temporal distribution of these two sets of costameres in rat cardiomyocytes. Costameric staining for N-cadherin, codistributed with beta-catenin, was strong from embryonic day 15 up to postnatal week 2, gradually decreased after postnatal week 3, and was undetectable at postnatal week 4 and in the adult. Confocal microscopy showed that N-cadherin colocalized with alpha-actinin at cortical myofibrils. Double-labeling of beta-catenin and talin indicated the coexistence of N-cadherin/catenin- and integrin/talin-based costameres in rat cardiac muscle. Although beta-catenin and vinculin were co-localized at the costamere of cardiomyocytes from embryonic day 15 to postnatal week 3, staining for beta-catenin or talin was mutually exclusive at all stages examined. These results demonstrate the simultaneous, but mutually exclusive, existence of N-cadherin/catenin- and integrin/talin-based costameres in rat cardiomyocytes between late embryonic stages and postnatal week 3, while only integrin/talin-based costameres were found in adult rats. The N-cadherin/catenin-based costameres in rat cardiac muscles may play a role in myofibrillogenesis similar to that of their counterparts in cultured cardiomyocytes., (Copyright 2002 Wiley‐Liss, Inc.)