Zhizhen Lu

Metformin attenuates cardiac fibrosis by inhibiting the TGFβ1–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.

β-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 β‐adrenoceptors induced IL‐6 production in neonatal mouse cardiac fibroblasts (NMCFs) through a Gs/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 β‐adrenoceptor agonist isoprenaline in NMCFs.

Alteration of α1-adrenoceptor subtypes in aortas of 12-month-old spontaneously hypertensive rats

Alterations in α1-adrenoceptor subtypes in aortas from 12-month-old spontaneously hypertensive rats (SHR) were studied in functional studies and RNase protection assays. The norepinephrine-induced contraction, including maximum response and pD2 values, was not significantly different between the SHR and age-matched Kyoto Wistar (WKY) rats. The pA2 values of the α1D-adrenoceptor subtype-selective antagonist BMY7378 (8-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-8-azaspiro(4.5)decane-7,9-dionedihydrochloride) were increased from 8.10±0.12 in WKY rats to 8.45±0.13 in SHR (P 0.05). Preincubation of preparations in 50 μM chloroethylclonidine for 30 min irreversibly inhibited the norepinephrine-induced response more profoundly in aortas from SHR than in aortas from WKY rats. The results of RNase protection assays showed that mRNAs for α1A- and α1B-adrenoceptor subtypes were decreased and that mRNA for the α1D-adrenoceptor subtype was increased in aortas from SHR compared with WKY rats. The results suggested that the α1A-adrenoceptor subtype was decreased and the α1D-adrenoceptor subtype was increased in aortas of 12-month-old SHR.

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.

Metformin is a novel suppressor for transforming growth factor (TGF)-β1

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-β1 (TGF-β1), 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-β1 and found that metformin inhibits [125I]-TGF-β1 binding to its receptor. Furthermore, based on molecular docking and molecular dynamics simulations, metformin was predicted to interact with TGF-β1 at its receptor-binding domain. Single-molecule force spectroscopy revealed that metformin reduces the binding probability but not the binding force of TGF-β1 to its type II receptor. Consequently, metformin suppresses type II TGF-β1 receptor dimerization upon exposure to TGF-β1, which is essential for downstream signal transduction. Thus, our results indicate that metformin is a novel TGF-β suppressor with therapeutic potential for numerous diseases in which TGF-β1 hyperfunction is indicated.

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

Aims Rapid over-activation of β-adrenergic receptor (β-AR) upon stress leads to cardiac inflammation, a prevailing factor that underlies heart injury. However, mechanisms by which acute β-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 β-AR insult. Methods and results Male C57BL/6 mice were injected with a single dose of β-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 β-AR insult, which promoted early macrophage infiltration. Further investigation revealed that the rapid inflammasome-dependent activation of IL-18, but not IL-1β, was the critical up-stream regulator for elevated chemokine expression in the myocardium upon ISO induced β1-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 β-AR over-activation triggers cytokine cascades, macrophage infiltration and pathological cardiac remodelling. Blocking IL-18 at the early stage of β-AR insult can successfully prevent inflammatory responses and cardiac injuries.

Different roles of α1-adrenoceptor subtypes in mediating cardiomyocyte protein synthesis in neonatal rats

1. Three different α1‐adrenoceptor subtypes, designated α1A, α1B and α1D, have been cloned and identified pharmacologically in cardiomyocytes. In vitro studies have suggested that α1‐adrenoceptors play an important role in facilitating cardiac hypertrophy. However, it remains controversial as to which subtype of α1‐adrenoceptors is involved in this response. In the present study, we investigated the different role of each α1‐adrenoceptor subtype in mediating cardiomyocyte protein synthesis, which is a most important characteristic of cardiac hypertrophy in cultured neonatal rat cardiomyocytes.

Metformin attenuates angiotensin II‐induced TGFβ1 expression by targeting hepatocyte nuclear factor‐4‐α

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. TGFβ1 is a key cytokine mediating cardiac fibrosis. Here, we investigated the effects of metformin on TGFβ1 production induced by angiotensin II (AngII) and the underlying mechanisms.

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.

Mammalian Tolloid Alters Subcellular Localization, Internalization, and Signaling of α1a-Adrenergic Receptors

In the present study, we identified the CUB5 domain of mammalian Tolloid (mTLD) as a novel protein binding to α1A-adrenergic receptor (AR) using the yeast two-hybrid system. Whereas CUB5 did not couple to either α1B-AR or α1D-AR. It was determined that amino acids 322 to 359 of α1A-AR were the major binding region for CUB5. The direct interaction between α1A-AR cytoplasmic tail and CUB5 was discovered by glutathione S-transferase pull-down assay. We confirmed the interaction of mTLD with α1A-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 α1A-AR, it significantly altered the subcellular localization of the receptors. Moreover, mTLD reduced the level of cell surface α1A-ARs, delayed the initial rate of agonist-induced receptor internalization, and facilitated agonist-induced calcium transient. We have demonstrated that mTLD interacts with α1A-AR directly, alters the subcellular localization of receptor, and influences agonist-induced α1A-AR internalization and calcium signaling.