Our data show for the first time that UII-induced VSMCs proliferation and CREB activation requires a complex signalling pathway that involves on the one hand SOCE mediated by STIM1, Orai1, and TRPC1, and on the other hand EGFR, ERK, and CaMK activation.

Abstract-Urocortin has been shown to produce vasodilatation in several arteries, but the precise mechanism of its action is still poorly understood. Here we demonstrate the role of store operated Ca 2ϩ entry (SOCE) regulated by Ca 2ϩ -independent phospholipase A 2 (iPLA 2 ) in phenylephrine hydrochloride (PE)-induced vasoconstriction, and we present the first evidence that urocortin induces relaxation by the modulation of SOCE and iPLA 2 in rat coronary artery. Urocortin produces an endothelium independent relaxation, and its effect is concentrationdependent (IC 50 Ϸ4.5 nmol/L). We show in coronary smooth muscle cells (SMCs) that urocortin inhibits iPLA 2 activation, a crucial step for SOC channel activation, and prevents Ca 2ϩ influx evoked by the emptying of the stores via a cAMP and protein kinase A (PKA)-dependent mechanism. Lysophophatidylcholine and lysophosphatidylinositol, products of iPLA 2 , exactly mimic the effect of the depletion of the stores in presence of urocortin. Furthermore, we report that long treatment with urocortin downregulates iPLA 2 mRNA and proteins expression in rat coronary smooth muscle cells. In summary, we propose a new mechanism of vasodilatation by urocortin which involves the regulation of iPLA 2 and SOCE via the stimulation of a cAMP/PKA-dependent signal transduction cascade in rat coronary artery. Key Words: urocortin Ⅲ iPLA 2 Ⅲ vasoconstriction Ⅲ store operated Ca 2ϩ entry Ⅲ cAMP-PKA C oronary artery smooth muscle cells (SMCs) regulate vascular tone influencing perfusion of the heart, peripheral resistance, and as a consequence heart function. Agonist induces a contraction of vascular SMCs by a rise in cytosolic free Ca 2ϩ concentration 1,2 because of a rapid Ca 2ϩ release by InsP 3 from intracellular stores and a transmembrane Ca 2ϩ influx through L-type voltage-dependent Ca 2ϩ channels or nonvoltage-gated channels such as store-operated Ca 2ϩ (SOC) channels. The relative contribution of each channel depends on the smooth muscle type. [2][3][4] The use of selective inhibitors of sarcoplasmic reticulum Ca 2ϩ -ATPase pump, as thapsigargin (TG), to activate SOC channels not only increases Ca 2ϩ influx but also enhances tone in a variety of SMCs. 3,5 Recently we showed Ca 2ϩ -independent phospholipase A 2 (iPLA 2 ) to be a crucial determinant of storeoperated Ca 2ϩ entry (SOCE). We demonstrated that the emptying of the stores activated iPLA 2 and its lysophospholipid products opened the SOC channels in aortic SMCs and nonexcitable cells. 6 -8 Thus iPLA 2 became a potential physiological target for regulation and fine-tuning of SOCE by other signaling cascades in SMCs.A few years ago a new 40-aa peptide, urocortin, 9,10 related to corticotropin-releasing factor (CRF) was described as a new player in cardiac control, 11,12 and was proposed to protect cardiac myocytes during ischemia/reperfusion by downregulating iPLA 2 expression. 13,14 Urocortin also emerged as a potent vasodilator peptide, and its mechanism of action appears to be complex, eg, vasodilatation has been reported...

Urocortin 1 and 2 (Ucn-1 and Ucn-2) have established protective actions against myocardial ischemia-reperfusion (I/R) injuries. However, little is known about their role in posttranscriptional regulation in the process of cardioprotection. Herein, we investigated whether microRNAs play a role in urocortin-induced cardioprotection. Administration of Ucn-1 and Ucn-2 at the beginning of reperfusion significantly restored cardiac function, as evidenced ex vivo in Langendorff-perfused rat hearts and in vivo in rat subjected to I/R. Experiments using microarray and qRT-PCR determined that the addition of Ucn-1 at reperfusion modulated the expression of several miRNAs with unknown role in cardiac protection. Ucn-1 enhanced the expression of miR-125a-3p, miR-324-3p; meanwhile it decreased miR-139-3p. Similarly, intravenous infusion of Ucn-2 in rat model of I/R mimicked the effect of Ucn-1 on miR-324-3p and miR-139-3p. The effect of Ucn-1 involves the activation of corticotropin-releasing factor receptor-2, Epac2 and ERK1/2. Moreover, the overexpression of miR-125a-3p, miR-324-3p and miR-139-3p promoted dysregulation of genes expression involved in cell death and apoptosis (BRCA1, BIM, STAT2), in cAMP and Ca2+ signaling (PDE4a, CASQ1), in cell stress (NFAT5, XBP1, MAP3K12) and in metabolism (CPT2, FoxO1, MTRF1, TAZ). Altogether, these data unveil a novel role of urocortin in myocardial protection, involving posttranscriptional regulation with miRNAs.

Calcium is an important second messenger required not only for the excitation-contraction coupling of the heart but also critical for the activation of cell signaling pathways involved in the adverse cardiac remodeling and consequently for the heart failure. Sustained neurohumoral activation, pressure-overload, or myocardial injury can cause pathologic hypertrophic growth of the heart followed by interstitial fibrosis. The consequent heart’s structural and molecular adaptation might elevate the risk of developing heart failure and malignant arrhythmia. Compelling evidences have demonstrated that Ca 2+ entry through TRP channels might play pivotal roles in cardiac function and pathology. TRP proteins are classified into six subfamilies: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPML (mucolipin), and TRPP (polycystin), which are activated by numerous physical and/or chemical stimuli. TRP channels participate to the handling of the intracellular Ca 2+ concentration in cardiac myocytes and are mediators of different cardiovascular alterations. This review provides an overview of the current knowledge of TRP proteins implication in the pathologic process of some frequent cardiac diseases associated with the adverse cardiac remodeling such as cardiac hypertrophy, fibrosis, and conduction alteration.

Aims: Urocortin-2 (Ucn-2) is a potent cardioprotector against Ischemia and Reperfusion (I/R) injuries. However, little is known about its role in the regulation of intracellular Ca2+ concentration ([Ca2+]i) under I/R. Here, we examined whether the addition of Ucn-2 in reperfusion promotes cardioprotection focusing on ([Ca2+]i handling.Methods and Results: Cardiac Wistar rat model of I/R was induced by transient ligation of the left coronary artery and experiments were conducted 1 week after surgery in tissue and adult cardiomyocytes isolated from risk and remote zones. We observed that I/R promoted significant alteration in cardiac contractility as well as an increase in hypertrophy and fibrosis in both zones. The study of confocal [Ca2+]i imaging in adult cardiomyocytes revealed that I/R decreased the amplitude of [Ca2+]i transient and cardiomyocytes contraction in risk and remote zones. Interestingly, intravenous infusion of Ucn-2 before heart’s reperfusion recovered significantly cardiac contractility and prevented fibrosis, but it didn’t affect cardiac hypertrophy. Moreover, Ucn-2 recovered the amplitude of [Ca2+]i transient and modulated the expression of several proteins related to [Ca2+]i homeostasis, such as TRPC5 and Orai1 channels. Using Neonatal Rat Ventricular Myocytes (NRVM) we demonstrated that Ucn-2 blunted I/R-induced Store Operated Ca2+ Entry (SOCE), decreased the expression of TRPC5 and Orai1 as well as their interaction in reperfusion.Conclusion: Our study provides the first evidences demonstrating that Ucn-2 addition at the onset of reperfusion attenuates I/R-induced adverse cardiac remodeling, involving the [Ca2+]i handling and inhibiting the expression and interaction between TRPC5 and Orai1.

Our results show that Ucn evokes potent positive inotropic and lusitropic effects mediated, at least in part, by an increase in I(CaL) and [Ca(2+)](i) transient amplitude. These effects may involve the activation of Epac, PKC, and MAPK signalling pathways.

AimsUrocortin-1 (Ucn-1) is an endogenous peptide that protects heart from ischemia and reperfusion (I/R) injuries. Ucn-1 is known to prevent cardiac cell death, but its role in the transcription of specific genes related to survival signaling pathway has not been fully defined. The aim of this study was to investigate the molecular signaling implicated in the improvement of cardiac myocytes survival induced by Ucn-1.Methods and ResultsUcn-1 administration before ischemia and at the onset of reperfusion, in rat hearts perfused in Langendorff system, fully recovered heart contractility and other hemodynamic parameters. Ucn-1 enhanced cell viability and decreased lactate dehydrogenase (LDH) release in adult cardiac myocytes subjected to simulated I/R. Annexin V-FITC/PI staining indicated that Ucn-1 promoted cell survival and decreased cell necrosis through Epac2 (exchange protein directly activated by cAMP) and ERK1/2 (extracellular signal–regulated kinases 1/2) activation. We determined that Ucn-1 shifted cell death from necrosis to apoptosis and activated caspases 9 and 3/7. Furthermore, mini-array, RT-qPCR and protein analyses of apoptotic genes showed that Ucn-1 upregulated the expression of CD40lg, Xiap and BAD in cells undergoing I/R, involving Epac2 and ERK1/2 activation.ConclusionsOur data indicate that Ucn-1 efficiently protected hearts from I/R damage by increasing the cell survival and stimulated apoptotic genes, CD40lg, Xiap and BAD, overexpression through the activation of Epac2 and ERK1/2.

Voltage-dependent Ca1.2 L-type Ca channels (LTCC) are the main route for calcium entry in vascular smooth muscle cells (VSMC). Several studies have also determined the relevant role of store-operated Ca channels (SOCC) in vascular tone regulation. Nevertheless, the role of Orai1- and TRPC1-dependent SOCC in vascular tone regulation and their possible interaction with Ca1.2 are still unknown. The current study sought to characterize the co-activation of SOCC and LTCC upon stimulation by agonists, and to determine the possible crosstalk between Orai1, TRPC1, and Ca1.2. Aorta rings and isolated VSMC obtained from wild type or smooth muscle-selective conditional Ca1.2 knock-out (Ca1.2) mice were used to study vascular contractility, intracellular Ca mobilization, and distribution of ion channels. We found that serotonin (5-HT) or store depletion with thapsigargin (TG) enhanced intracellular free Ca concentration ([Ca]) and stimulated aorta contraction. These responses were sensitive to LTCC and SOCC inhibitors. Also, 5-HT- and TG-induced responses were significantly attenuated in Ca1.2 mice. Furthermore, hyperpolarization induced with cromakalim or valinomycin significantly reduced both 5-HT and TG responses, whereas these responses were enhanced with LTCC agonist Bay-K-8644. Interestingly, in situ proximity ligation assay revealed that Ca1.2 interacts with Orai1 and TRPC1 in untreated VSMC. These interactions enhanced significantly after stimulation of cells with 5-HT and TG. Therefore, these data indicate for the first time a functional interaction between Orai1, TRPC1, and Ca1.2 channels in VSMC, confirming that upon agonist stimulation, vessel contraction involves Ca entry due to co-activation of Orai1- and TRPC1-dependent SOCC and LTCC.