Eva Calderón-Sánchez

Role of Ca2+-Independent Phospholipase A2 and Store-Operated Pathway in Urocortin-Induced Vasodilatation of Rat Coronary Artery

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 Ca2+ entry (SOCE) regulated by Ca2+-independent phospholipase A2 (iPLA2) in phenylephrine hydrochloride (PE)-induced vasoconstriction, and we present the first evidence that urocortin induces relaxation by the modulation of SOCE and iPLA2 in rat coronary artery. Urocortin produces an endothelium independent relaxation, and its effect is concentration-dependent (IC50≈4.5 nmol/L). We show in coronary smooth muscle cells (SMCs) that urocortin inhibits iPLA2 activation, a crucial step for SOC channel activation, and prevents Ca2+ influx evoked by the emptying of the stores via a cAMP and protein kinase A (PKA)-dependent mechanism. Lysophophatidylcholine and lysophosphatidylinositol, products of iPLA2, exactly mimic the effect of the depletion of the stores in presence of urocortin. Furthermore, we report that long treatment with urocortin downregulates iPLA2 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 iPLA2 and SOCE via the stimulation of a cAMP/PKA-dependent signal transduction cascade in rat coronary artery.

Urotensin-II promotes vascular smooth muscle cell proliferation through store-operated calcium entry and EGFR transactivation.

AIMS Urotensin-II (UII) is a vasoactive peptide that promotes vascular smooth muscle cells (VSMCs) proliferation and is involved in the pathogenesis of atherosclerosis, restenosis, and vascular remodelling. This study aimed to determine the role of calcium (Ca(2+))-dependent signalling and alternative signalling pathways in UII-evoked VSMCs proliferation focusing on store-operated Ca(2+) entry (SOCE) and epithelium growth factor receptor (EGFR) transactivation. METHODS AND RESULTS We used primary cultures of VSMCs isolated from Wistar rat aorta to investigate the effects of UII on intracellular Ca(2+) mobilization, and proliferation determined by the 5-bromo-2-deoxyuridine (BrdU) assay. We found that UII enhanced intracellular Ca(2+) concentration ([Ca(2+)]i) which was significantly reduced by classical SOCE inhibitors and by knockdown of essential components of the SOCE such as stromal interaction molecule 1 (STIM1), Orai1, or TRPC1. Moreover, UII activated a Gd(3+)-sensitive current with similar features of the Ca(2+) release-activated Ca(2+) current (ICRAC). Additionally, UII stimulated VSMCs proliferation and Ca(2+)/cAMP response element-binding protein (CREB) activation through the SOCE pathway that involved STIM1, Orai1, and TRPC1. Co-immunoprecipitation experiments showed that UII promoted the association between Orai1 and STIM1, and between Orai1 and TRPC1. Moreover, we determined that EGFR transactivation, extracellular signal-regulated kinase (ERK) and Ca(2+)/calmodulin-dependent kinase (CaMK) signalling pathways were involved in both UII-mediated Ca(2+) influx, CREB activation and VSMCs proliferation. CONCLUSION 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.

Urocortin induces positive inotropic effect in rat heart.

AIMS The aim of this study is to evaluate the positive inotropic effect of urocortin (Ucn) and to characterize its signalling pathways. METHODS AND RESULTS Contractility was measured in ex vivo Langendorff-perfused hearts isolated from Wistar rats. Isolated ventricular cardiomyocytes were used to analyse intracellular calcium ([Ca(2+)](i)) transients evoked by electrical stimulation and L-type Ca(2+) current by confocal microscopy and whole-cell patch-clamping, respectively. The application of Ucn to perfused hearts induced progressive, sustained, and potent inotropic and lusitropic effects that were dose-dependent with an EC(50) of approximately 8 nM. Ucn effects were independent of protein kinase A (PKA) activation but were significantly reduced by protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) inhibitors and by brefeldin A, an antagonist of guanine nucleotide exchange factor, suggested to be an inhibitor of exchange protein activated by cAMP (Epac). These whole-organ effects were correlated with the inotropic effects observed in isolated cells: Ucn increased I(CaL) density, [Ca(2+)](i) transients, cell shortening and Ca(2+) content of sarcoplasmic reticulum. CONCLUSION 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.

Urotensin-II Signaling Mechanism in Rat Coronary Artery: Role of STIM1 and Orai1-Dependent Store Operated Calcium Influx in Vasoconstriction

Objective—Human urotensin-II (UII) is considered the most potentendogenous vasoconstrictor discovered to date, although the precise mechanism activated downstream of its receptor UTS2R in blood vessels remains elusive. The aim of this study was to determine the role of the store operated Ca2+ entry (SOCE) signaling pathway in UII-induced coronary artery vasoconstriction. Methods and Results—We used a combination of isometric tension measurement, Ca2+ imaging, pharmacology, and molecular approaches to study UII-mediated rat coronary artery vasoconstriction and intracellular Ca2+ mobilization in coronary smooth muscle cells. We found that UII promoted dose-dependent vasoconstriction and elicited Ca2+ and Mn2+ influx, which were sensitive to classical SOCE inhibitors. In addition, knockdown of either STIM1 or Orai1 essentially inhibited UII-mediated SOCE and prevented UII but not high-KCL evoked contraction in transfected coronary artery. Moreover, we found that Ca2+-independent phospholipase A2&bgr; was involved in UII effects and that is colocalized with STIM1 in different submembrane compartments. Importantly, STIM1 but not Orai1 downregulation inhibits significantly independent phospholipase A2 activation. Furthermore, lysophosphatidylcholine, an independent phospholipase A2 product, activated Orai1 but not STIM1-dependent contraction and SOCE. Conclusion—Here, we demonstrated that different critical players of SOCE signaling pathway are required for UII-induced vasoconstriction of rat coronary artery.

Hypoxia inhibits vasoconstriction induced by metabotropic Ca2+ channel-induced Ca2+ release in mammalian coronary arteries

AIMS We have previously described in rat basilar arterial myocytes that in the absence of extracellular Ca(2+) influx, activation of L-type Ca(2+) channels stimulates a metabotropic cascade leading to Ca(2+) release from the sarcoplasmic reticulum (SR) and contraction [a calcium channel-induced Ca(2+) release (CCICR) mechanism]. On the other hand, it is known that hypoxia reduces Ca(2+) channel activity in coronary myocytes. In the present study, we have investigated whether CCICR is present in coronary arterial myocytes and whether arterial ring contraction induced by CCICR can be inhibited by hypoxia. METHODS AND RESULTS Isometric force, arterial diameter, cytosolic [Ca(2+)] and electrical activity were recorded on mammalian (porcine, rat, and human) coronary artery preparations (dispersed myocytes, arterial rings, and intact arterial segments). In the absence of extracellular Ca(2+), Ca(2+) channel activation increased cytosolic [Ca(2+)] in isolated myocytes and contracted arterial rings. This contraction was suppressed by antagonists of L-type Ca(2+) channels and by inhibiting Ca(2+) release from the SR. Hypoxia induced dilatation of coronary arterial rings pre-contracted by activation of Ca(2+) channels in the absence of extracellular Ca(2+). This effect was present although K(ATP) channels and Rho kinase were blocked by glibenclamide and Y27632, respectively. CONCLUSION We show that Ca(2+) channel activation can induce metabotropic coronary arterial ring contraction in the absence of extracellular Ca(2+) and that this CCICR mechanism is inhibited by hypoxia. Thus, besides reduction of Ca(2+) entry through Ca(2+) channels, hypoxia seems to induce coronary vasorelaxation by inhibition of metabotropic CCICR.

Urocortin-1 Mediated Cardioprotection Involves XIAP and CD40-Ligand Recovery: Role of EPAC2 and ERK1/2

Aims Urocortin-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 Results Ucn-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. Conclusions Our 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.

Immunophilins and Cardiovascular Complications

Immunophilins belong to a highly conserved family of proteins with cis-trans peptidyl-prolyl isomerase activity, generally classified by their ability to selectively bind specific immunosuppressive drugs, thereby regulating their activity. Immunophilins include Cyclophilins (CyPs), which are specific targets of the immunosuppressant drug cyclosporin A (CsA); FKBPs (FK506-binding proteins), that are sensitive to both FK506 (tacrolimus) and rapamycin (sirolimus); and FCBPs which are sensitive to CsA and FK506. Immunophilins are expressed in multiple human tissues, including brain, heart, kidney, liver and lung and regulate functions as diverse as intracellular calcium signaling, protein transport, protein folding and gene transcription. In particular, immunophilins play key functional roles in the cardiovascular system, where they can associate with proteins such as ryanodine and IP3 receptors (RyR and IP3R), calcineurin, and mitochondrial permeability transition pore (MPTP) and Heat-shock proteins-caveolin-cholesterol complex and regulate their function. The biological importance of immunophilins is further revealed by the pathophysiology, as they have been implicated in several cardiovascular diseases, including vascular stenosis, atherosclerosis, heart failure and arrhythmias. This review summarizes some of the most recent studies on immunophilins and focuses on their roles in the mechanisms underlying the cardiovascular disease.

Orai1 and TRPC1 Proteins Co-localize with CaV1.2 Channels to Form a Signal Complex in Vascular Smooth Muscle Cells

Voltage-dependent CaV1.2 L-type Ca2+ 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 Ca2+ channels (SOCC) in vascular tone regulation. Nevertheless, the role of Orai1- and TRPC1-dependent SOCC in vascular tone regulation and their possible interaction with CaV1.2 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 CaV1.2. Aorta rings and isolated VSMC obtained from wild type or smooth muscle-selective conditional CaV1.2 knock-out (CaV1.2KO) mice were used to study vascular contractility, intracellular Ca2+ mobilization, and distribution of ion channels. We found that serotonin (5-HT) or store depletion with thapsigargin (TG) enhanced intracellular free Ca2+ concentration ([Ca2+]i) and stimulated aorta contraction. These responses were sensitive to LTCC and SOCC inhibitors. Also, 5-HT- and TG-induced responses were significantly attenuated in CaV1.2KO 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 CaV1.2 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 CaV1.2 channels in VSMC, confirming that upon agonist stimulation, vessel contraction involves Ca2+ entry due to co-activation of Orai1- and TRPC1-dependent SOCC and LTCC.

Low-dose combination of Rho kinase and L-type Ca2+ channel antagonists for selective inhibition of depolarization-induced sustained arterial contraction

L-type Ca(2+) channels (LTCCs) are involved in the maintenance of tonic arterial contractions and regulate the RhoA/Rho-associated kinase (ROCK) sensitization cascade. We have tested effects of individual and combined low concentrations of LTCCs and ROCK inhibitors to produce arterial relaxation without the adverse side effects of LTCCs antagonists. We have also studied whether this pharmacological strategy alters Ca(2+)-dependent electrical properties of isolated arterial and cardiac myocytes as well as cardiac contractility. Rat basilar, human carotid and coronary arterial rings were mounted on a small-vessel myograph to measure isometric tension and cardiac contractility was measured in Langendorff-perfused rat heart. Simultaneous cytosolic Ca(2+) concentration and arterial diameter were measured in intact pressurized arteries loaded with Fura-2. Patch-clamp techniques were used to measure electrical properties in isolated cardiac and arterial myocytes. Low concentrations of LTCCs and ROCK inhibitors reduced the tonic component of moderate depolarization-evoked contraction, leaving the phasic component practically unaltered. This selective vasorelaxant effect was more marked when the LTCCs and ROCK inhibitors were applied together. In the concentration range used (nM), Ca(2+) currents in arterial myocytes, cardiac action potentials and heart contractility were unaffected by this pharmacological approach. In conclusion, low doses of LTCCs and ROCK inhibitors could be used to selectively relax precontracted arteries in pathologic conditions such as hypertension, and cerebral or coronary spasms with minor side effects on physiological contractile properties of vascular and cardiac myocytes.

miR-125a, miR-139 and miR-324 contribute to Urocortin protection against myocardial ischemia-reperfusion injury

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.