Metformin attenuates angiotensin II-induced TGFß1 expression by targeting hepatocyte nuclear factor-4-α.

BACKGROUND AND PURPOSE: Metformin, a small molecule, antihyperglycaemic agent, is a well-known activator of AMP-activated protein kinase (AMPK) and protects against cardiac fibrosis. However, the underlying mechanisms remain elusive. TGFß1 is a key cytokine mediating cardiac fibrosis. Here, we investigated the effects of metformin on TGFß1 production induced by angiotensin II (AngII) and the underlying mechanisms. EXPERIMENTAL APPROACH: Wild-type and AMPKα2-/- C57BL/6 mice were injected s.c. with metformin or saline and infused with AngII (3 mg·kg-1 ·day-1 ) for 7 days. Adult mouse cardiac fibroblasts (CFs) were isolated for in vitro experiments. KEY RESULTS: In CFs, metformin inhibited AngII-induced TGFß1 expression via AMPK activation. Analysis using bioinformatics predicted a potential hepatocyte nuclear factor 4α (HNF4α)-binding site in the promoter region of the Tgfb1 gene. Overexpressing HNF4α increased TGFß1 expression in CFs. HNF4α siRNA attenuated AngII-induced TGFß1 production and cardiac fibrosis in vitro and in vivo. Metformin inhibited the AngII-induced increases in HNF4α protein expression and binding to the Tgfb1 promoter in CFs. In vivo, metformin blocked the AngII-induced increase in cardiac HNF4α protein levels in wild-type mice but not in AMPKα2-/- mice. Consequently, metformin inhibited AngII-induced TGFß1 production and cardiac fibrosis in wild-type mice but not in AMPKα2-/- mice. CONCLUSIONS AND IMPLICATIONS: HNF4α mediates AngII-induced TGFß1 transcription and cardiac fibrosis. Metformin inhibits AngII-induced HNF4α expression via AMPK activation, thus decreasing TGFß1 transcription and cardiac fibrosis. These findings reveal a novel antifibrotic mechanism of action of metformin and identify HNF4α as a new potential therapeutic target for cardiac fibrosis. LINKED ARTICLES: This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc.

IL-18 cleavage triggers cardiac inflammation and fibrosis upon ß-adrenergic insult.

Aims: Rapid over-activation of ß-adrenergic receptor (ß-AR) upon stress leads to cardiac inflammation, a prevailing factor that underlies heart injury. However, mechanisms by which acute ß-AR stimulation induce cardiac inflammation still remain unknown. Here, we set out to identify the crucial role of inflammasome/interleukin (IL)-18 in initiating and maintaining cardiac inflammatory cascades upon ß-AR insult. Methods and results: Male C57BL/6 mice were injected with a single dose of ß-AR agonist, isoproterenol (ISO, 5 mg/kg body weight) or saline subcutaneously. Cytokine array profiling demonstrated that chemokines dominated the initial cytokines upregulation specifically within the heart upon ß-AR insult, which promoted early macrophage infiltration. Further investigation revealed that the rapid inflammasome-dependent activation of IL-18, but not IL-1ß, was the critical up-stream regulator for elevated chemokine expression in the myocardium upon ISO induced ß1-AR-ROS signalling. Indeed, a positive correlation was observed between the serum levels of norepinephrine and IL-18 in patients with chest pain. Genetic deletion of IL-18 or the up-stream inflammasome component NLRP3 significantly attenuated ISO-induced chemokine expression and macrophage infiltration. In addition, IL-18 neutralizing antibodies selectively abated ISO-induced chemokines, proinflammatory cytokines and adhesion molecules but not growth factors. Moreover, blocking IL-18 early after ISO treatment effectively attenuated cardiac inflammation and fibrosis. Conclusion: Inflammasome-dependent activation of IL-18 within the myocardium upon acute ß-AR over-activation triggers cytokine cascades, macrophage infiltration and pathological cardiac remodelling. Blocking IL-18 at the early stage of ß-AR insult can successfully prevent inflammatory responses and cardiac injuries.

Metformin is a novel suppressor for transforming growth factor (TGF)-ß1.

Metformin is a widely used first-line antidiabetic drug that has been shown to protect against a variety of specific diseases in addition to diabetes, including cardiovascular disorders, polycystic ovary syndrome, and cancer. However, the precise mechanisms underlying the diverse therapeutic effects of metformin remain elusive. Here, we report that transforming growth factor-ß1 (TGF-ß1), which is involved in the pathogenesis of numerous diseases, is a novel target of metformin. Using a surface plasmon resonance-based assay, we identified the direct binding of metformin to TGF-ß1 and found that metformin inhibits [(125)I]-TGF-ß1 binding to its receptor. Furthermore, based on molecular docking and molecular dynamics simulations, metformin was predicted to interact with TGF-ß1 at its receptor-binding domain. Single-molecule force spectroscopy revealed that metformin reduces the binding probability but not the binding force of TGF-ß1 to its type II receptor. Consequently, metformin suppresses type II TGF-ß1 receptor dimerization upon exposure to TGF-ß1, which is essential for downstream signal transduction. Thus, our results indicate that metformin is a novel TGF-ß suppressor with therapeutic potential for numerous diseases in which TGF-ß1 hyperfunction is indicated.

Speckle Tracking Based Strain Analysis Is Sensitive for Early Detection of Pathological Cardiac Hypertrophy.

Cardiac hypertrophy is a key pathological process of many cardiac diseases. However, early detection of cardiac hypertrophy is difficult by the currently used non-invasive method and new approaches are in urgent need for efficient diagnosis of cardiac malfunction. Here we report that speckle tracking-based strain analysis is more sensitive than conventional echocardiography for early detection of pathological cardiac hypertrophy in the isoproterenol (ISO) mouse model. Pathological hypertrophy was induced by a single subcutaneous injection of ISO. Physiological cardiac hypertrophy was established by daily treadmill exercise for six weeks. Strain analysis, including radial strain (RS), radial strain rate (RSR) and longitudinal strain (LS), showed marked decrease as early as 3 days after ISO injection. Moreover, unlike the regional changes in cardiac infarction, strain analysis revealed global cardiac dysfunction that affects the entire heart in ISO-induced hypertrophy. In contrast, conventional echocardiography, only detected altered E/E', an index reflecting cardiac diastolic function, at 7 days after ISO injection. No change was detected on fractional shortening (FS), E/A and E'/A' at 3 days or 7 days after ISO injection. Interestingly, strain analysis revealed cardiac dysfunction only in ISO-induced pathological hypertrophy but not the physiological hypertrophy induced by exercise. Taken together, our study indicates that strain analysis offers a more sensitive approach for early detection of cardiac dysfunction than conventional echocardiography. Moreover, multiple strain readouts distinguish pathological cardiac hypertrophy from physiological hypertrophy.

Activation of α1B-adrenoceptors contributes to intermittent hypobaric hypoxia-improved postischemic myocardial performance via inhibiting MMP-2 activation.

Inhibition of matrix metalloproteinases-2 (MMP-2) activation renders cardioprotection from ischemia/reperfusion (I/R) injury; however, the signaling pathways involved have not been fully understood. Intermittent hypobaric hypoxia (IHH) has been shown to enhance myocardial tolerance to I/R injury via triggering intrinsic adaptive responses. Here we investigated whether IHH protects the heart against I/R injury via the regulation of MMP-2 and how the MMP-2 is regulated. IHH (Po2 = 84 mmHg, 4-h/day, 4 wk) improved postischemic myocardial contractile performance, lactate dehydrogenase (LDH) release, and infarct size in isolated perfused rat hearts. Moreover, IHH reversed I/R-induced MMP-2 activation and release, disorders in the levels of MMP-2 regulators, peroxynitrite (ONOO(-)) and tissue inhibitor of metalloproteinase-4 (TIMP-4), and loss of the MMP-2 targets α-actinin and troponin I. This protection was mimicked, but not augmented, by a MMP inhibitor doxycycline and lost by the α1-adrenoceptor (AR) antagonist prazosin. Furthermore, IHH increased myocardial α1A-AR and α1B-AR density but not α1D-AR after I/R. Concomitantly, IHH further enhanced the translocation of PKC epsilon (PKCε) and decreased the release of mitochondrial cytochrome c due to I/R via the activation of α1B-AR but not α1A-AR or α1D-AR. IHH-conferred cardioprotection in the postischemic contractile function, LDH release, MMP-2 activation, and nitrotyrosine as well as TIMP-4 contents were mimicked but not additive by α1-AR stimulation with phenylephrine and were abolished by an α1B-AR antagonist chloroethylclonidine and a PKCε inhibitor PKCε V1-2. These findings demonstrate that IHH exerts cardioprotection through attenuating excess ONOO(-) biosynthesis and TIMP-4 loss and sequential MMP-2 activation via the activation of α1B-AR/PKCε pathway.

Echocardiographic assessment of ß-adrenoceptor stimulation-induced heart failure with reduced heart rate in mice.

1. Chronic injection with the ß-adrenoceptor (ß-AR) agonist isoproterenol (ISO) has been commonly used as an animal model of ß-AR-induced cardiac remodelling and heart failure. This ISO-treated model usually exhibits significantly decreased conscious heart rate (HR). However, the HR in treatment groups is usually adjusted to the same levels by anaesthesia to assess cardiac geometry and function. In the present study, we report a method of echocardiographic assessment that represents the true cardiac geometry and function under conditions of ISO withdrawal. 2. Briefly, C57BL/6 mice were treated with 5 mg/kg per day ISO for 12 weeks. Cardiac geometry and function were assessed by high-resolution echocardiography in vehicle (saline) - and ISO-treated mice that were either conscious or anaesthetized using different concentrations of isoflurane. 3. The cardiac ß-AR response was decreased in ISO-treated mice, as evidenced by markedly decreased conscious HR. Vehicle- and ISO-treated mice did not differ in terms of cardiac geometry or function when HR was adjusted to the same level (400 b.p.m.) in both treatment groups, but cardiac geometry and function did differ when a low (1%) rather than high (1.5% or 2%) isoflurane concentration was used to adjust HR. Furthermore, 3 day ISO withdrawal eliminated the difference in conscious HR between the two groups. In addition, the groups differed in cardiac geometry and function regardless of the isoflurane concentration used. 4. In conclusion, using isoflurane to decrease the HR of treated groups to the same level may mask left ventricular dysfunction in ISO-treated mice. Withdrawal of ISO eliminated the difference in basal HR between the ISO-treated and control groups on echocardiography, allowing a more accurate assessment of cardiac pathological and functional changes.

ß-Adrenergic receptors stimulate interleukin-6 production through Epac-dependent activation of PKCδ/p38 MAPK signalling in neonatal mouse cardiac fibroblasts.

BACKGROUND AND PURPOSE IL-6 plays crucial roles in cardiac hypertrophy, cardiac fibrosis and heart failure. Activation of ß-adrenoceptors induced IL-6 production in neonatal mouse cardiac fibroblasts (NMCFs) through a G(s) /adenylate cyclase/cAMP/p38 MAPK pathway but independent of PKA. However, how cAMP activates p38 MAPK is still not defined. In this study, we have assessed the role of the exchange protein directly activated by cAMP (Epac) and PKCδ in p38 MAPK activation and IL-6 production by stimulated by the ß-adrenoceptor agonist isoprenaline in NMCFs. EXPERIMENTAL APPROACH The IL-6 concentration in cell culture supernatants was measured by ELISA. The levels of phosphorylated and total p38 MAPK and PKCδ were determined by Western blot analysis. The translocation of PKCδ was determined by immunoblotting the soluble and particulate fractions. Expression of Epac1 or PKCδ was knocked down by the corresponding, adenovirus-mediated, small hairpin RNA (shRNA). RESULTS In NMCFs, activation of ß-adrenoceptors enhanced PKCδ phosphorylation and translocation. Furthermore, knock-down of the PKCδ isoform using an adenovirus-mediated shRNA markedly down-regulated IL-6 induction by NMCFs stimulated with isoprenaline. Moreover, knock-down of Epac1 confirmed that Epac1 was upstream of PKCδ in IL-6 production. Additionally, both Epac1 and PKCδ mediated the p38 MAPK activation induced by isoprenaline. CONCLUSIONS AND IMPLICATIONS ß-Adrenoceptor agonists activate a cAMP/Epac/PKCδ/p38 MAPK pathway to produce IL-6 in NMCFs. This study identifies Epac as the link between cAMP and p38 MAPK signalling pathways and demonstrates that PKCδ can function as a novel downstream effector of this ß-adrenoceptor/cAMP/Epac pathway.

Transactivated EGFR mediates α1-AR-induced STAT3 activation and cardiac hypertrophy.

α(1)-Adrenergic receptor (α(1)-AR) is a crucial mediator of cardiac hypertrophy. Although numerous intracellular pathways have been implicated in α(1)-AR-induced hypertrophy, its precise mechanism remains elusive. We aimed to determine whether α(1)-AR induces cardiac hypertrophy through a novel signaling pathway-α(1)-AR/epidermal growth factor receptor (EGFR)/signal transducer and activator of transcription 3 (STAT3). The activation of STAT3 by α(1)-AR was first demonstrated by tyrosine phosphorylation, nuclear translocation, DNA binding, and transcriptional activity in neonatal Sprague-Dawley rat cardiomyocytes. Activated STAT3 showed an essential role in α(1)-AR-induced cardiomyocyte hypertrophic growth, as assessed by treatment with STAT3 inhibitory peptide and lentivirus-STAT3 small interfering RNA. The results were further confirmed by in vivo experiments involving intraperitoneal injection of the STAT3 inhibitor WP1066 significantly inhibiting phenylephrine-infusion-induced heart hypertrophy in male C57BL/6 mice. Furthermore, the α(1)-AR-activated STAT3 was associated with transactivation of EGFR because inhibition of EGFR with the selective inhibitor AG1478 prevented α(1)-AR-induced STAT3 tyrosine phosphorylation and its transcriptional activity, as well as cardiac hypertrophy. In summary, these results suggest that α(1)-AR induces the activation of STAT3, mainly through transactivation of EGFR, which plays an important role in α(1)-AR-induced cardiac hypertrophy.

Intercellular transportation of quantum dots mediated by membrane nanotubes.

In this work, we reported that the quantum dot (QD) nanoparticles could be actively transported in the membrane nanotubes between cardiac myocytes. Single particle imaging and tracking of QDs revealed that most QDs moved in a bidirectional mode along the membrane nanotubes with a mean velocity of 1.23 mum/s. The results suggested that QDs moving in the nanotubes were coordinately motivated by molecular motors. It provides new information for the study of intercellular transportation of nanoparticles.

Developmental changes in the geometry, function and responsiveness of the mouse heart to beta-adrenergic stimulation as determined by high-resolution echocardiography.

1. The mouse is the preferred species for gene targeting as a tool for research into the heart and heart development. The developmental features of the geometry and function of the heart in young mice are not well defined and cardiac functional responses following stimulation of beta-adrenoceptors have not been investigated. 2. Using the VisualSonic (Toronto, ON, Canada) high-resolution ultrasound system, we investigated male C57BL/6 mice at 0.5-18 weeks of age. Echocardiography was performed at baseline and repeated after administration of the beta-adrenoceptor agonist isoproterenol (4 microg/kg). 3. The geometry of the left ventricle became mature 2 weeks after birth. A significant decline in left ventricular contractile function occurred at 2-3 weeks of age. 4. Inotropic and chronotropic responses to isoproterenol were significantly weaker in mice at a weaning age < 2 weeks compared with adult mice (heart rate increment 3 +/- 3% vs 32 +/- 4%, respectively; fractional shortening increment 19 +/- 5% vs 78 +/- 8%, respectively; P < 0.001). 5. In conclusion, significant changes occur in mice from birth until weaning with respect to the topography, function and beta-adrenoceptor responsiveness of the heart. The results of the present study provide a reference point for future studies in genotyping cardiac function during the postnatal phase in genetically engineered mice.

Metformin attenuates cardiac fibrosis by inhibiting the TGFbeta1-Smad3 signalling pathway.

AIMS: The mechanism of the cardioprotective action of metformin is incompletely understood. We determined the role of metformin in cardiac fibrosis and investigated the mechanism. METHODS AND RESULTS: Ten-week-old male mice (C57BL/6) were subjected to left ventricular pressure overload by transverse aortic constriction. Mice received metformin (200 mg/kg/day) or normal saline for 6 weeks. Metformin inhibited cardiac fibrosis (fibrosis area/total heart area: 0.6 +/- 0.3 vs. 3.6 +/- 0.9%, P < 0.01) induced by pressure overload and improved cardiac diastolic function (left ventricular end-diastolic pressure: 5.2 +/- 0.9 vs. 11.0 +/- 1.6 mmHg, P < 0.05). Metformin inhibited the pressure overload-induced transforming growth factor (TGF)-beta(1) production in mouse hearts and the TGF-beta(1)-induced collagen synthesis in cultured adult mouse cardiac fibroblasts (CFs). Metformin suppressed the phosphorylation of Smad3 in response to TGF-beta(1) in CFs. Metformin also inhibited the nuclear translocation and transcriptional activity of Smad3 in CFs. CONCLUSION: Metformin inhibited cardiac fibrosis induced by pressure overload in vivo and inhibited collagen synthesis in CFs probably via inhibition of the TGF-beta(1)-Smad3 signalling pathway. These findings provide a new mechanism for the cardioprotective effects of metformin.

14-3-3 inhibits insulin-like growth factor-I-induced proliferation of cardiac fibroblasts via a phosphatidylinositol 3-kinase-dependent pathway.

1. Insulin-like growth factor (IGF)-I plays an important role in the pathogenesis of heart disease and has been shown to strongly induce the proliferation of cardiac fibroblasts (CFs). It remains unknown whether 14-3-3 proteins, which are associated the regulation of signal transduction, affect IGF-I-induced CF proliferation. 2. In the present study, we investigated the effects of 14-3-3 proteins on CF proliferation in response to IGF-I. Proliferation of CFs was determined by cell counting and a bromodeoxyuridine incorporation assay. Phosphorylation of signalling molecules was evaluated by western blottling. Activity of nuclear factor of activated T cells (NFAT) was examined using a dual luciferase reporter gene assay and immunofluorescence. 3. It was found that adenovirus-mediated transfection of YFP-R18 peptide (AdR18), a known inhibitor of 14-3-3, significantly enhanced IGF-I-induced CF proliferation. This potentiation arose from an increase in phosphorylation of phosphatidylinositol 3-kinase (PI3-K) and AKT (protein kinase B), inactivation of glycogen synthesis kinase (GSK) 3beta and increased NFAT activity. 4. Collectively, the results of the present study suggest that 14-3-3 proteins inhibit IGF-I-induced CF proliferation via a PI3-K-dependent NFAT signalling pathway. This finding may contribute to our understanding of the function of 14-3-3 proteins in the heart.

Chlorination diversifies Cimicifuga racemosa triterpene glycosides.

Extracts from the roots and rhizomes of black cohosh (Cimicifuga racemosa) are widely used as dietary supplements to alleviate menopausal symptoms. State-of-the-art quality control measures involve phytochemical fingerprinting of the triterpene glycosides for species identification and chemical standardization by HPLC. In the course of developing materials and methods for standardization procedures, the major C. racemosa triterpene glycoside (1) was isolated and initially thought to be cimicifugoside (2). Detailed HR-LC-MS and 1D and 2D NMR analysis of 1 and 2 unambiguously revealed that 1 is the chlorine-containing derivative of 2, namely, 25-chlorodeoxycimigenol-3-O-beta-d-xyloside. Accordingly, HPLC profiles of black cohosh preparations require revision of the assignments of the chlorinated (1) and nonchlorinated (2) pair. Besides explaining the substantial shift in polarity (DeltatR[RP-18] ca. 20 min), 25-deoxychlorination opens a new pathway of structural diversification in triterpene glycoside chemistry. As chemical conversion of 2 into 1 could be demonstrated, deoxychlorination may be interpreted as artifact formation. Simultaneously, however, it is a potentially significant pathway for the gastric in vivo conversion ("nature's prodrug") of the relatively polar triterpene glycosides into significantly less polar chlorinated derivatives with altered pharmacological properties.

alpha1-adrenergic receptors activate AMP-activated protein kinase in rat hearts.

To test the hypothesis that AMP-activated protein kinase (AMPK) is possibly the downstream signaling molecule of certain subtypes of adrenergic receptor (AR) in the heart, we evaluated AMPK activation mediated by ARs in H9C2 cells, a rat cardiac source cell line, and rat hearts. The AMPK-alpha subunit and the phosphorylation level of Thr(172)-AMPK-alpha subunit were subjected to Western blot analysis. Osmotic minipumps filled with norepinephrine (NE), phenylephrine (PE) or vehicle [0.01% (W/V) vitamin C solution] were implanted into male Sprague-Dawley rats subcutaneously. The pumps delivered NE or PE continuously at the rate of 0.2 mg/kg per hour. After 7-day infusion, the activity of AMPK was examined following immunoprecipitation with anti-AMPK-alpha antibody. At the cellular level, we found that NE elevated AMPK phosphorylation level in a dose- and time-dependent manner, with the maximal effect at 10 micromol/L NE after 10-minute treatment. This effect was insensitive to propranolol, a specific beta-AR antagonist, but abolished by prazosin, an alpha(1)-AR antagonist, suggesting that alpha(1)-AR but not beta-AR mediated the phosphorylation of AMPK. Moreover, the results from rat models of 7-day-infusion of AR agonists demonstrated that the activity of AMPK was significantly higher in NE (7.4-fold) and PE (6.0-fold) infusion groups than that in the vehicle group (P<0.05, n=6). On the other hand, no obvious cardiac hypertrophy and tissue fibrosis changes were observed in PE-infused rats. Taken together, our results demonstrate that alpha(1)-AR stimulation enhances the activity of AMPK, indicating an important role of alpha(1)-AR stimulation in the regulation of AMPK in the heart. Understanding the activation of AMPK mediated by alpha(1)-AR might have clinical implications in the therapy of heart failure.

Heterogeneous transportation of alpha1B-adrenoceptor in living cells.

The heterogeneous motion of alpha(1B)-adrenoceptor (alpha(1B)-AR) was visualized in living cells with BODIPY-labeled antagonist of AR by single molecule fluorescence microscopy at high spatial resolution. The moving trajectory was reconstructed by precise localization (better than 20 nm) with a least-square fit of a two-dimensional Gaussian point spread function to each single spot. Trajectory analysis revealed two apparent groups of movements: directed motion and hindered motion. The directed motion had speeds higher than 0.1 mum/s. The histogram of diffusion coefficients of the hindered motion showed distinction between the cell membrane and the cytoplasm: the diffusion coefficient was lower near the cell membrane than in the internal cytoplasm, suggesting that alpha(1B)-AR was located or trapped in different networks, which was consistent with the natural distribution of cytoskeleton in living cells. These results suggested that the heterogeneity in the motion of alpha(1B)-AR in living cell might be associated with different localizations of cell skeleton proteins in the cell, which could provide molecular insight of AR regulation in living cells.

Mammalian tolloid alters subcellular localization, internalization, and signaling of alpha(1a)-adrenergic receptors.

In the present study, we identified the CUB5 domain of mammalian Tolloid (mTLD) as a novel protein binding to alpha(1A)-adrenergic receptor (AR) using the yeast two-hybrid system. Whereas CUB5 did not couple to either alpha(1B)-AR or alpha(1D)-AR. It was determined that amino acids 322 to 359 of alpha(1A)-AR were the major binding region for CUB5. The direct interaction between alpha(1A)-AR cytoplasmic tail and CUB5 was discovered by glutathione S-transferase pull-down assay. We confirmed the interaction of mTLD with alpha(1A)-AR in human embryonic kidney (HEK) 293 cells by immunoprecipitation, immunofluorescence, and fluorescence resonance energy transfer. Although mTLD did not affect the density and affinity of receptors in crudely prepared membranes from HEK293 cells stably expressing alpha(1A)-AR, it significantly altered the subcellular localization of the receptors. Moreover, mTLD reduced the level of cell surface alpha(1A)-ARs, delayed the initial rate of agonist-induced receptor internalization, and facilitated agonist-induced calcium transient. We have demonstrated that mTLD interacts with alpha(1A)-AR directly, alters the subcellular localization of receptor, and influences agonist-induced alpha(1A)-AR internalization and calcium signaling.

Noncanonical cAMP pathway and p38 MAPK mediate beta2-adrenergic receptor-induced IL-6 production in neonatal mouse cardiac fibroblasts.

We previously reported that cardiac fibroblasts, but not cardiomyocytes, are served as the predominant source of IL-6 after isoproterenol stimulation in mouse myocardium. The present study investigated the molecular mechanism of isoproterenol-mediated secretion of IL-6 in mouse cardiac fibroblasts. Treatment of cells with isoproterenol-induced a time-dependent accumulation of IL-6, which was mediated by beta(2)-adrenergic receptor (AR), the preponderant beta-AR subtype in cardiac fibroblasts. Isoproterenol-induced secretion of IL-6 was mainly mediated by Gs-AC-cAMP signaling cascade and could be negatively regulated by Gi and PI3K. Surprisingly, the effect of cAMP was independent of protein kinase A and the exchange protein directly activated by cAMP (Epac)-Rap1 pathway and suggests the existence of a novel cAMP-dependent mechanism. p38 MAPK inhibitor SB203580, but not extracellular regulated protein kinase inhibitor, abrogated isoproterenol-induced IL-6 release in cardiac fibroblasts and mouse myocardium. Interestingly, p38 MAPK could also be positively regulated by Gs-AC-cAMP but negatively regulated by Gi-PI3K pathway. Finally, multiple transcription factors (AP-1, C/EBP, NF-kappaB and CREB) regulating the IL-6 gene are activated in response to isoproterenol stimulation, which may provide essential linkage between upstream cAMP-p38 MAPK signaling cascade and downstream IL-6 gene transcription. The present results suggest that beta(2)-AR mediates IL-6 production through a noncanonical cAMP responsible pathway and p38 MAPK.

AICAR stimulates IL-6 production via p38 MAPK in cardiac fibroblasts in adult mice: a possible role for AMPK.

Though known as a sensor of energy balance, AMP-activated protein kinase (AMPK) was recently shown to limit damage and apoptotic activity and contribute to the late preconditioning in heart. Interleukin-6 was also reported to involve in anti-apoptosis and cardio-protection in myocardium. Interestingly, both AMPK activity and IL-6 level were increased in response to ischemia, hypertrophy and oxidative stress. To determine whether AMPK activation will promote IL-6 production, cardiac fibroblasts (CFs) from mice were incubated with AMPK activator, 5-aminoimidazole-4-carboxamide-1-4-ribofuranoside (AICAR). The results demonstrated that AICAR time and dose-dependently stimulated IL-6 production by ELISA and immunofluorescence. Pretreatment with p38 mitogen-activated protein kinase (MAPK) inhibitor blocked AICAR-induced IL-6 production; furthermore, AICAR-activated p38 MAPK phosphorylation by Western blot. To confirm that the increase in IL-6 production is ascribed to AMPK activation, we used another known AMPK activator, metformin. It also dose-dependently potentiated IL-6 production in CFs, and this potentiation could be reversed by p38 MAPK inhibitor. In conclusion, AMPK activation promoted IL-6 production in CFs via p38 MAPK-dependent pathway.

Different roles of alpha1-adrenoceptor subtypes in mediating cardiomyocyte protein synthesis in neonatal rats.

1. Three different alpha1-adrenoceptor subtypes, designated alpha1A, alpha1B and alpha1D, have been cloned and identified pharmacologically in cardiomyocytes. In vitro studies have suggested that alpha1-adrenoceptors play an important role in facilitating cardiac hypertrophy. However, it remains controversial as to which subtype of alpha1-adrenoceptors is involved in this response. In the present study, we investigated the different role of each alpha1-adrenoceptor subtype in mediating cardiomyocyte protein synthesis, which is a most important characteristic of cardiac hypertrophy in cultured neonatal rat cardiomyocytes. 2. Cardiomyocyte hypertrophy was monitored by the following characteristic phenotypic changes: (i) an increase in protein synthesis; (ii) an increase in total protein content; and (iii) an increase in cardiomyocyte size. 3. The role of each alpha1-adrenoceptor subtype in mediating cardiomyocyte protein synthesis was investigated by the effect of specific alpha1-adrenoceptor subtype-selective antagonists on noradrenaline-induced [3H]-leucine incorporation. In addition, pKB values for alpha1-adrenoceptor subtype-selective antagonists were calculated and compared with the corresponding pKi values to further identify their effects. 4. Activation of alpha1-adrenoceptors by phenylephrine or noradrenaline in the presence of propranolol significantly increased [3H]-leucine incorporation, protein content and cell size. 5. Pre-incubating cardiomyocytes with 5-methyl-urapidil, RS 17053 or WB 4101 significantly inhibited noradrenaline-induced [3H]-leucine incorporation. However, there was no effect when cardiomyocytes were pre-incubated with BMY 7378. The correlation coefficients between pKB values for alpha1-adrenoceptor subtype-selective antagonists and pKi values obtained from cloned alpha1A-, alpha1B- or alpha1D-adrenoceptors were 0.92 (P <0.01), 0.66 (P >0.05) and 0.24 (P >0.05), respectively. 6. Our results suggest that the alpha1-adrenoceptor is dominantly responsible for adrenergic hypertrophy of cultured cardiomyocytes in neonatal rats. The efficiency in mediating cardiomyocyte protein synthesis is alpha1A > alpha1B >> alpha1D.

[Effects of beta-adrenoceptor activation on metabolism in neonatal rat cardiomyocytes].

The aim of the present study was to investigate the effects of beta-adrenergic receptor (beta-AR) activation on metabolism in cultured neonatal rat cardiomyocytes. The protein synthesis and total protein content of cardiomyocytes were determined by [(3)H]-leucine incorporation and BCA protein content assay. Cardiomyocyte glucose uptake was measured by [(3)H]-2-deoxy-D-glucose uptake analysis. Adenosine monophosphate activated protein kinase (AMPK) phosphorylation was detected by Western blot. The results showed that sustained stimulation with isoproterenol (ISO), a beta-adrenoceptor agonist, had no effect on [(3)H]-leucine incorporation and total protein content in cardiomyocytes. With beta-AR activation by ISO or NE (pretreated with a selective blocker of the alpha(1)-adrenoceptor prazosin) for 48 h, both the glucose uptake and AMPK phosphorylation increased significantly compared with unstimulated cardiomyocytes. These results suggest that although sustained beta-AR activation has no effect on cardiomyocyte protein metabolism, glucose uptake and AMPK activity are increased significantly. The role of these beta-AR activation-induced changes in cardiac hypertrophy remains to be further investigated.