Mechanical stress-evoked but angiotensin II-independent activation of angiotensin II type 1 receptor induces cardiac hypertrophy through calcineurin pathway

Mechanical stress can induce cardiac hypertrophy through angiotensin II (AngII) type 1 (AT₁) receptor independently of AngII, however, the intracellular mechanisms remain largely indeterminate. Since calcineurin, a Ca²⁺-dependent phosphatase, plays a critical role in pressure overload-induced cardiac hypertrophy, we therefore, asked whether calcineurin is involved in the AT₁ receptor-mediated but AngII-independent cardiac hypertrophy. Mechanical stretch failed to elicit hypertrophic responses in COS7 cells co-transfected with plasmid of AT₁ receptor and siRNA of calcineurin. Mechanical stresses for 2 weeks in vivo and for 24 h in vitro significantly induced upregulation of calcineurin expression and hypertrophic responses, such as the increases in cardiomyocytes size and specific gene expressions, in cardiomyocytes of angiotensinogen gene knockout (ATG^−/−) mice, both of which were significantly suppressed by a specific calcineurin inhibitor FK506, suggesting a critical role of calcineurin in mechanical stress-induced cardiac hypertrophy in the ATG^−/− mice. Furthermore, an AT₁ receptor blocker Losartan not only attenuated cardiac hypertrophy but also abrogated upregulation of cardiac calcineurin expression induced by mechanical stresses in the AngII-lacking mice, indicating that calcineurin expression is regulated by AT₁ receptor without the involvement of AngII after mechanical stress. These findings collectively suggest that mechanical stress-evoked but AngII-independent activation of AT₁ receptor induces cardiac hypertrophy through calcineurin pathway.     

Involvement of calcium-sensing receptor in ischemia/reperfusion-induced apoptosis in rat cardiomyocytes

The calcium-sensing receptor (CaR) is a seven-transmembrane G-protein coupled receptor, which activates intracellular effectors, for example, it causes inositol phosphate (IP) accumulation to increase the release of intracellular calcium. Although intracellular calcium overload has been implicated in the cardiac ischemia/reperfusion (I/R)-induced apoptosis, the role of CaR in the induction of apoptosis has not been fully understood. This study tested the hypothesis that CaR is involved in I/R cardiomyocyte apoptosis by increasing [Ca2+]i. The isolated rat hearts were subjected to 40-min ischemia followed by 2 h of reperfusion, meanwhile GdCl3 was added to reperfusion solution. The expression of CaR increased at the exposure to GdCl3 during I/R. By laser confocal microscopy, it was observed that the intracellular calcium was significantly increased and exhibited a Deltapsim, as monitored by 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolcarbocyanine iodide (JC-1) during reperfusion with GdCl3. Furthermore, the number of apoptotic cells was significantly increased as shown by TUNEL assay. Typical apoptotic cells were observed with transmission electron microscopy in I/R with GdCl3 but not in the control group. The expression of cytosolic cytochrome c and activated caspase-9 and caspase-3 was significantly increased whereas the expression of mitochondrial cytochrome c significantly decreased in I/R with GdCl3 in comparison to the control. In conclusion, these results suggest that CaR is involved in the induction of cardiomyocyte apoptosis during ischemia/reperfusion through activation of cytochrome c-caspase-3 signaling pathway.     

Discovery of evocalcet,a next-generation calcium-sensing receptor agonist for the treatment of hyperparathyroidism

The calcium-sensing receptor (CaSR) plays an important role in sensing extracellular calcium ions and regulating parathyroid hormone secretion by parathyroid gland cells, and the receptor is a suitable target for the treatment of hyperparathyroidism. Cinacalcet hydrochloride is a representative CaSR agonist which widely used for the hyperparathyroidism. However, it has several issues to clinical use, such as nausea/vomiting and strong inhibition of CYP2D6. We tried to improve these issues of cinacalcet for a new pharmaceutical agent as a preferable CaSR agonist. Optimization from cinacalcet resulted in the identification of pyrrolidine compounds and successfully led to the discovery of evocalcet as an oral allosteric CaSR agonist. Evocalcet, which exhibited highly favorable profiles such as CaSR agonistic activity and good DMPK profiles, will provide a novel therapeutic option for secondary hyperparathyroidism.     

Inhibition of farnesylpyrophosphate synthase prevents angiotensin II-induced hypertrophic responses in rat neonatal cardiomyocytes: Involvement of the RhoA/Rho kinase pathway

The RhoA/Rho-kinase (ROCK) pathway is involved in angiotensin (Ang) II-induced cardiac hypertrophy. However, it is still unclear whether inhibition of farnesylpyrophosphate (FPP) synthase can attenuate Ang II-induced hypertrophic responses, and whether it involves the RhoA/ROCK pathway. The anti-hypertrophic effects of inhibition of FPP synthase with alendronate in Ang II-cultured neonatal cardiomyocytes were partially reversed by geranylgeranyol (GGOH) and were mimicked by GGTI-286, a geranylgeranyl transferase-I inhibitor, C3 exoenzyme, an inhibitor of Rho, or Y-27632, an inhibitor of ROCK. Pull-down assay showed alendronate reduced-active RhoA by Ang II was also partially antagonized by GGOH. This study revealed that the inhibition of FPP synthase by alendronate reduces RhoA activation by diminishing geranylgeranylation which prevents Ang II-induced hypertrophic responses in neonatal cardiomyocytes.

Structured summary

MINT-7260047: Rhotekin-RBD (uniprotkb:Q9BST9) physically interacts (MI:0915) with Rhoa (uniprotkb:P61589) by pull down (MI:0096)     

Identification and functionality of proteomes secreted by rat cardiac stem cells and neonatal cardiomyocytes

In the heart, the proteomes secreted by both cardiac stem cells (CSCs) and cardiac myocytes could act synergistically, but the identification and functionality of the proteins comprising the individual secretomes have not yet been described. In this study, we have identified proteins present in the media obtained from cultured rat CSCs and from cultured neonatal rat ventricular myocytes (NRVMs) and compared them with proteins identified in the media alone. Briefly, 83 unique proteins were identified after analysis by RPLC and MS. In total 49 and 23% were NRVM‐specific or CSC‐specific proteins, respectively, and 63% of total 83 proteins were integral plasma membrane and/or known secreted proteins. Fifteen proteins met our criteria for paracrine/autocrine factors: (i) robust protein identification, (ii) cell specific and (iii) known to be secreted. Most of these proteins have not been previously linked to stem cells. NRVM‐specific proteins atrial natriuretic factor (ANP) and connective tissue growth factor, and CSC‐specific protein interleukin‐1 receptor‐like 1 (ST2) were found to affect rat CSC proliferation. These findings suggest that relative concentration of each protein may be crucial for cellular intertalk and for the final outcome of cardiac cell therapy.     

Autoantibody against Cardiac β1-Adrenoceptor Induces Apoptosis in Cultured Neonatal Rat Cardiomyocytes

To clarify whether apoptosis is involved in the injury processes induced by autoantibodyagainst cardiac β_1-adrenoceptor,we investigated the biological and apoptotic effects of antibodies on culturedneonatal rat cardiomyocytes.Wistar rats were immunized with peptides corresponding to the second extra-cellular loop of the β_1-adrenoceptor to induce the production of anti-β_1-adrenoceptor antibodies in the sera.Immunoglobulin(Ig)G in the sera was detected using synthetic antigen enzyme-linked immunosorbentassay and purified using the diethylaminoethyl cellulose ion exchange technique.Apoptosis of cardiomyo-cytes was evaluated using agarose gel electrophoresis and flow cytometry.Our results showed that thepositive serum IgG greatly increased the beating rates of cardiomyocytes and showed an"agonist-like"activity.Furthermore,positive serum IgG induced cardiomyocyte apoptosis after treatment with β_1-adrenoceptor overstimulation for 48h.The effects of monoclonal antibody against β_-adrenoceptor werealso found to be similar to those of positive serum IgG.It was suggested that the autoantibody could inducecardiomyocyte apoptosis by excessive stimulation of β_1-adrenoceptor.     

Angiotensin II receptor binding in the locus ceruleus of spontaneously hypertensive rats and Wistar-Kyoto rats: A quantitative autoradiographic study with references to hypertension and cardiac/testicular hypertrophy

The locus ceruleus (LC) contains a high density of angiotensin II (All) receptors. The role of All receptors at the LC in genetic hypertension and organ function is unclear. Spontaneously hypertensive (SHR) rats and Wistar-Kyoto (WKY) rats were studied, and blood pressure of animals was measured using the tail-cuff method. Animals were decapitated and the heart weight (HW) and testicular weight (TW) of animals measured. All receptor binding was carried out by incubating the LC tissue sections with 200 pM [¹²⁵I]-All receptor ligand, and measured using quantitative autoradiography. Results showed that the HW/BW ratio was significantly higher in SHR rats than WKY rats. However, the TW/BW ratio was higher in SHR rats than WKY rats only at two hypertensive stages, whereas All receptor binding capacity in the LC was also statistically higher in SHR rats than WKY rats. Results indicated that cardiac and testicular hypertrophies were related to higher All receptor binding in the LC of SHR rats, when compared with WKY rats. Interestingly, the literature shows that there is an LC-testes axis. In conclusion, this study indicated that All receptors in the LC are associated with genetic hypertension, and testicular weight could be a reasonable index for essential hypertension.     

Activation of AP-1 through reactive oxygen species by angiotensin II in rat cardiomyocytes

Cardiovascular pathogenesis induced by angiotensin II (Ang-II) is a complex process often connected to oxidative stress. In the present study we show that, 4 h after addition, Ang-II induces a four- to fivefold increase in AP-1 activity in cultured neonatal rat cardiomyocytes and that the intracellular level of reactive oxygen species (ROS) correlates with the extent of AP-1 binding activity. Ang-II stimulated ROS generation in rat cardiomyocytes in a dose- and time-dependent manner. These effects of Ang-II were suppressed by the Ang-II receptor type I (AT₁) inhibitor CV-11974 as well as by the antioxidants diphenylene iodonium (DPI) and N-acetyl-l-cysteine (NAC), but not by AT₂ antagonist PD 122319. Furthermore, Ang-II induced a two- to threefold increase in protein synthesis and cell size during 12–24 h, which could be inhibited by CV-11974 as well as by DPI and NAC. Because the rat cardiomyocytes strongly expressed gp91^phox, this suggests that ROS generated in a gp91-containing NADPH oxidase are involved in signal transduction leading to AP-1 activation. Together, these findings indicate that Ang-II elicits the activation of the redox-sensitive AP-1 via ROS through AT₁, resulting in effects on cardiomyocyte function such as hypertrophy.     

VEGF increases the proliferative capacity and eNOS/NO levels of endothelial progenitor cells through the calcineurin/NFAT signalling pathway

We have investigated whether VEGF (vascular endothelial growth factor) regulates the proliferative capacity and eNOS (endothelial nitric oxide synthase)/NO (nitric oxide) pathway of EPCs (endothelial progenitor cells) by activating CaN (calcineurin)/NFAT (nuclear factor of activated T-cells) signalling. EPCs were obtained from cultured mononuclear cells isolated from the peripheral blood of healthy adults. Treatment with VEGF (50 ng/ml) potently promoted CaN enzymatic activity, activation of NFAT2, cell proliferation, eNOS protein expression and NO production. Pretreatment with cyclosporin A (10 μg/ml), a pharmacological inhibitor of CaN or 11R-VIVIT, a special inhibitor of NFAT, completely abrogated the aforementioned effects of VEGF treatment and increased apoptosis. The results indicate that VEGF treatment promotes the proliferative capacity of human EPCs by activating CaN/NFAT signalling leading to increased eNOS protein expression and NO production.     

Effects of urocortin II on neonatal rat cardiac myocytes and non-myocytes

Urocortin (Ucn) II and III, homologous peptides of Ucn that are specific ligands for corticotropin-releasing hormone (CRH) type 2 receptor (CRH-R2), have recently been identified. The present study was designed to elucidate the effects of Ucn II, which is predominantly expressed in rodent heart, on neonatal rat cardiac myocytes (MCs) and cardiac non-myocytes (NMCs). Ucn II increased the incorporation of [³H]-leucine into MCs, as well as the accumulation of cAMP and the secretion of atrial natriuretic peptide. However, no significant changes were demonstrated in NMCs or an MC/NMC co-culture system. The effects of Ucn II were attenuated by astressin2-B, a specific antagonist of CRH-R2, and/or H89, an inhibitor of protein kinase A (PKA). These results indicate that Ucn II may be another endogenous cardiovascular substance that acts via CRH-R2 and the cAMP-dependent PKA pathway.     

苹果多酚通过下调钙敏感受体改善大鼠低氧性肺动脉高压的作用

目的：探究苹果多酚通过下调钙敏感受体（CaSR）表达改善大鼠低氧性肺动脉高压的作用。方法：36只雄性SD大鼠随机分为对照组、低氧诱导组和苹果多酚干预组（n=12）。低氧诱导组和苹果多酚干预组大鼠每天在低氧舱（氧浓度为10%）中间断低氧暴露8 h,对照组在正常环境下饲养,为期四周。建模同时,苹果多酚干预组每日用苹果多酚按200 mg/kg的剂量灌胃,对照组、低氧诱导组用等量生理盐水灌胃,每日1次,共持续四周。建模完成后,检测各组大鼠的肺动脉平均压（mPAP）、肺血管阻力（PVR）、右心室肥厚指数（RVHI）、肺动脉中膜厚度（MT）、WA%、WT%及肺动脉组织中CaSR表达量。结果：低氧暴露组大鼠mPAP、PVR、RVHI、MT、WA%、WT%及肺动脉组织中CaSR表达量均较对照组明显升高（P<0.01）;苹果多酚干预组可改善上述效应。结论：苹果多酚能有效预防大鼠低氧性肺动脉高压,其机制与下调CaSR的表达有关。     

Reciprocal regulation of miR-23a and lysophosphatidic acid receptor signaling in cardiomyocyte hypertrophy

Earlier, our study demonstrated that lysophosphatidic acid (LPA) receptor mediated cardiomyocyte hypertrophy. However, the subtype-specific functions for LPA1 and LPA3 receptors in LPA-induced hypertrophy have not been distinguished. Growing evidence indicates that microRNAs (miRNAs) are involved in the pathogenesis of cardiac hypertrophy by down-regulating target molecules. The present work therefore aimed at elucidating the functions mediated by different subtypes of LPA receptors and investigating the modulatory role of miRNAs during LPA induced hypertrophy. Experiments were done with cultured neonatal rat cardiomyocytes (NRCMs) exposed to LPA and we showed that knockdown of LPA1 by small interfering RNA (siRNA) enhanced LPA-induced cardiomyocyte hypertrophy, whereas LPA3 silencing repressed hypertrophy. miR-23a, a pro-hypertrophic miRNA, was up-regulated by LPA in cardiomyocytes and its down-regulation reduced LPA-induced cardiomyocyte hypertrophy. Importantly, luciferase reporter assay confirmed LPA1 to be a target of miR-23a, indicating that miR-23a is involved in mediating the LPA-induced cardiomyocyte hypertrophy by targeting LPA1. In addition, knockdown of LPA3, but not LPA1, eliminated miR-23a elevation induced by LPA. And PI3K inhibitor, LY294002, effectively prevented LPA-induced miR-23a expression in cardiomyocytes, suggesting that LPA might induce miR-23a elevation by activating LPA3 and PI3K/AKT pathway. These findings identified opposite subtype-specific functions for LPA1 and LPA3 in mediating cardiomyocyte hypertrophy and indicated LPA1 to be a target of miR-23a, which discloses a link between miR-23a and the LPA receptor signaling in cardiomyocyte hypertrophy.     

Angiotensin II stimulates DNA synthesis of rat pancreatic stellate cells by activating ERK through EGF receptor transactivation

Although angiotensin II (Ang II) is known to participate in pancreatic fibrosis, little is known as to the mechanism by which Ang II promotes pancreatic fibrosis. To elucidate the mechanism, we examined the action of Ang II on the proliferation of rat pancreatic stellate cells (PSCs) that play central roles in pancreatic fibrosis. Immunocytochemistry and Western blotting demonstrated that both Ang II type 1 and type 2 receptors were expressed in PSCs. [3H]Thymidine incorporation assay revealed that Ang II enhanced DNA synthesis in PSCs, which was blocked by Ang II type 1 receptor antagonist losartan. Western blotting using anti-phospho-epidermal growth factor (EGF) receptor and anti-phospho-extracellular signal regulated kinase (ERK) antibodies showed that Ang II-activated EGF receptor and ERK. Both EGF receptor kinase inhibitor AG1478 and MEK1 inhibitor PD98059 attenuated ERK activation and DNA synthesis enhanced by Ang II. These results indicate that Ang II stimulates PSC proliferation through EGF receptor transactivation-ERK activation pathway.     

In vivo imaging of oxidative stress in the kidney of diabetic mice and its normalization by angiotensin II type 1 receptor blocker

This study was undertaken to evaluate oxidative stress in the kidney of diabetic mice by electron spin resonance (ESR) imaging technique. Oxidative stress in the kidney was evaluated as organ-specific reducing activity with the signal decay rates of carbamoyl-PROXYL probe using ESR imaging. The signal decay rates were significantly faster in corresponding image pixels of the kidneys of streptozotocin-induced diabetic mice than in those of controls. This technique further demonstrated that administration of angiotensin II type 1 receptor blocker (ARB), olmesartan (5 mg/kg), completely restored the signal decay rates in the diabetic kidneys to control values. In conclusion, this study provided for the first time the in vivo evidence for increased oxidative stress in the kidneys of diabetic mice and its normalization by ARB as evaluated by ESR imaging. This technique would be useful as a means of further elucidating the role of oxidative stress in diabetic nephropathy.     

Phosphoinositide hydrolysis increase by angiotensin-(1--7) in neonatal rat brain

Angiotensin (Ang)-(1–7) is an endogenous peptide hormone of the renin–angiotensin system which exerts diverse biological actions, some of them counterregulate Ang II effects. In the present study potential effect of Ang-(1–7) on phosphoinositide (PI) turnover was evaluated in neonatal rat brain. Cerebral cortex prisms of seven-day-old rats were preloaded with [³H]myoinositol, incubated with additions during 30 min and later [³H]inositol-phosphates (IPs) accumulation quantified. It was observed that PI hydrolysis enhanced 30% to 60% in the presence of 0.01 nM to 100 nM Ang-(1–7). Neither 10 nM [D-Ala⁷]Ang-(1–7), an Ang-(1–7) specific antagonist, nor 10 nM losartan, an angiotensin II type 1 (AT₁) receptor antagonist, blocked the effect of 0.1 nM Ang-(1–7) on PI metabolism. The effect of 0.1 nM Ang-(1–7) on PI hydrolysis was not reduced but it was even significantly increased in the simultaneous presence of [D-Ala⁷]Ang-(1–7) or losartan. PI turnover enhancement achieved with 0.1 nM Ang-(1–7) decreased roughly 30% in the presence of 10 nM PD 123319, an angiotensin II type 2 (AT₂) receptor antagonist. The antagonists alone also enhanced PI turnover. Present findings showing an increase in PI turnover by Ang-(1–7) represent a novel action for this peptide and suggest that it exerts a function in this signaling system in neonatal rat brain, an effect involving, at least partially, angiotensin AT₂ receptors.     

Telmisartan prevents hepatic fibrosis and enzyme-altered lesions in liver cirrhosis rat induced by a choline-deficient L-amino acid-defined diet

Rennin-angiotensin system is involved in liver fibrogenesis through activating hepatic stellate cells (HSCs). Telmisartan (Tel) is an angiotensin II type 1 receptor antagonist, could function as a selective peroxisome proliferator-activated receptor γ activator. Here we studied the effect of Tel on liver fibrosis, pre-neoplastic lesions in vivo and primary HSCs in vitro. In vivo study, we used the choline-deficient l-amino acid-defined (CDAA)-diet induced rat NASH model. The rats were fed the CDAA diet for 8 weeks to induce liver fibrosis and pre-neoplastic lesions, and then co-administrated with Tel for another 10 weeks. Tel prevented liver fibrogenesis and pre-neoplastic lesions by down-regulating TGFβ1 and TIMP-1, 2 and increasing MMP-13 expression. Tel inhibited HSCs activation and proliferation. These results suggested that Tel could be a promising drug for NASH related liver fibrosis.