Inhibition of adenylyl cyclase 8 prevents the upregulation of Orai1 channel, which improves cardiac function after myocardial infarction.

The upregulation of Orai1 and subsequent store-operated Ca2+ entry (SOCE) has been associated with adverse cardiac remodeling and heart failure (HF). However, the mechanism underlying Orai1 upregulation and its role in myocardial infarction remains unclear. Our study investigated the role of Orai1 in activating adenylyl cyclase 8 (AC8) and cyclic AMP (cAMP) response element-binding protein (CREB), as well as its contribution to cardiac dysfunction induced by ischemia and reperfusion (I/R). We found that I/R evoked an increase in the expression of Orai1 and AC8 in rats' hearts, resulting in a substantial rise in diastolic Ca2+ concentration ([Ca2+]i), and reduced ventricular contractions. The expression of Orai1 and AC8 was also increased in ventricular biopsies of post-ischemic HF patients. Mechanistically, we demonstrate that I/R activation of Orai1 stimulated AC8, which produced cAMP and phosphorylated CREB. Subsequently, p-CREB activated the ORAI1 promoter, resulting in Orai1 upregulation and SOCE exacerbation. Intramyocardial administration of AAV9 carrying AC8 short hairpin RNA decreased the expression of AC8, Orai1 and CREB, which restored diastolic [Ca2+]i and improved cardiac contraction. Therefore, our data suggests that the axis composed by Orai1/AC8/CREB plays a critical role in I/R-induced cardiac dysfunction, representing a potential new therapeutic target to limit the progression of the disease toward HF.

Cardiac protection induced by urocortin-2 enables the regulation of apoptosis and fibrosis after ischemia and reperfusion involving miR-29a modulation.

Urocortin-2 (Ucn-2) has demonstrated cardioprotective actions against myocardial ischemia-reperfusion (I/R) injuries. Herein, we explored the protective role of Ucn-2 through microRNAs (miRNAs) post-transcriptional regulation of apoptotic and pro-fibrotic genes. We determined that the intravenous administration of Ucn-2 before heart reperfusion in a Wistar rat model of I/R recovered cardiac contractility and decreased fibrosis, lactate dehydrogenase release, and apoptosis. The infusion of Ucn-2 also inhibited the upregulation of 6 miRNAs in revascularized heart. The in silico analysis indicated that miR-29a and miR-451_1∗ are predicted to target many apoptotic and fibrotic genes. Accordingly, the transfection of neonatal rat ventricular myocytes with mimics overexpressing miR-29a, but not miR-451_1∗, prevented I/R-induced expression of pro- and anti-apoptotic genes such as Apaf-1, Hmox-1, and Cycs, as well as pro-fibrotic genes Col-I and Col-III. We also confirmed that Hmox-1, target of miR-29a, is highly expressed at the mRNA and protein levels in adult rat heart under I/R, whereas, Ucn-2 abolished I/R-induced mRNA and protein upregulation of HMOX-1. Interestingly, a significant upregulation of Hmox-1 was observed in the ventricle of ischemic patients with heart failure, correlating negatively with the left ventricle ejection fraction. Altogether, these data indicate that Ucn-2, through miR-29a regulation, provides long-lasting cardioprotection, involving the post-transcriptional regulation of apoptotic and fibrotic genes.

Urocortin Role in Ischemia Cardioprotection and the Adverse Cardiac Remodeling.

Despite the considerable progress in strategies of myocardial protection, ischemic heart diseases (IHD) and consequent heart failure (HF) remain the main cause of mortality worldwide. Several procedures are used routinely to guarantee the prompt and successful reestablishment of blood flow to preserve the myocardial viability of infarcted hearts from ischemia injuries. However, ischemic heart reperfusion/revascularization triggers additional damages that occur when oxygen-rich blood re-enters the vulnerable myocardial tissue, which is a phenomenon known as ischemia and reperfusion (I/R) syndrome. Complications of I/R injuries provoke the adverse cardiac remodeling, involving inflammation, mishandling of Ca2+ homeostasis, apoptotic genes activation, cardiac myocytes loss, etc., which often progress toward HF. Therefore, there is an urgent need to develop new cardioprotective therapies for IHD and HF. Compelling evidence from animal studies and pilot clinical trials in HF patients suggest that urocortin (Ucn) isoforms, which are peptides associated with stress and belonging to the corticotropin releasing factor family, have promising potential to improve cardiovascular functions by targeting many signaling pathways at different molecular levels. This review highlights the current knowledge on the role of urocortin isoforms in cardioprotection, focusing on its acute and long-term effects.

Role of Orai1 and L-type CaV1.2 channels in Endothelin-1 mediated coronary contraction under ischemia and reperfusion.

Ischemia and Reperfusion (I/R) injuries are associated with coronary artery hypercontracture. They are mainly originated by an exacerbated response to agonists released by endothelium such as Endothelin (ET-1), involving the alteration in intracellular calcium handling. Recent evidences have highlighted the implication of Store-Operated Calcium Channels (SOCC) in intracellular calcium homeostasis in coronary artery. However, little is known about the role of SOCC in the regulation of coronary vascular tone under I/R. The aim of this study was to evaluate the role of SOCC and l-type Ca2+ channels (LTCC) in coronary artery vasoconstriction originated by ET-1 in I/R. We used Left Anterior Descendent coronary artery (LAD) rings, isolated from Wistar rats, to study the contractility and intracellular Ca2+ concentration ([Ca2+]i) under a simulated I/R protocol. We observed that responses to high-KCL induced depolarization and caffeine-induced Ca2+ release are attenuated in coronary artery under I/R. Furthermore, ET-1 addition in ischemia promotes transient and small rise of [Ca2+]i and coronary vascular tone. Meanwhile, these effects are significantly potentiated during reperfusion. The resulting ET-1-induced vasoconstrictions and [Ca2+]i increase were abolished by; GSK-7975A and gadolinium, inhibitors of SOCC; and nifedipine a widely used inhibitor of LTCC. Interestingly, using in situ Proximity Ligation Assay (PLA) in isolated coronary smooth muscle cells we found significant colocalization of LTCC CaV1.2 isoform with Orai1, the pore forming subunit of SOCC, and TRPC1 under I/R. Our data suggest that hypercontraction of coronary artery induced by ET-1 after I/R involves the co-activation of LTCC and SOCC, which colocalize significantly in the sarcolemma of coronary smooth muscle cells.

TRP Channels: Current Perspectives in the Adverse Cardiac Remodeling.

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 Ca2+ 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 Ca2+ 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.

Urocortin-2 Prevents Dysregulation of Ca2+ Homeostasis and Improves Early Cardiac Remodeling After Ischemia and Reperfusion.

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.

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.

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.

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.

Efecto cardioprotector de la ranolazina en el proceso de isquemia-reperfusión en cardiomiocitos de rata adultos / Cardioprotective effect of ranolazine in the process of ischemia-reperfusion in adult rat cardiomyocytes

Introducción y objetivos: La ranolazina se emplea como tratamiento complementario de la angina en pacientes sintomáticos insuficientemente controlados con los tratamientos antianginosos de primera línea. La ranolazina inhibe los canales de sodio operados por voltaje, lo cual indica su posible intervención en el proceso de reperfusión al prevenir la sobrecarga de sodio y calcio que se produce durante la isquemia. En este estudio, se ha caracterizado el efecto de la ranolazina en la homeostasis del calcio en miocitos cardiacos adultos de ratas a las que se aplicó un protocolo de isquemia y reperfusión simuladas. Métodos: Se evaluaron los efectos de la ranolazina en los cambios de la concentración de calcio intracelular en diferentes momentos empleando electroestimulación de campo. El estudio del calcio intracelular se llevó a cabo mediante microfluorimetría utilizando el indicador fluorescente Fura-2 y por microscopia confocal utilizando el indicador Fluo-3. Resultados: Se observó que los cardiomiocitos a los que se aplicaba la isquemia-reperfusión mostraban un aumento de la concentración de calcio diastólica y una disminución de la amplitud de los transitorios de calcio intracelular. La aplicación de la ranolazina durante la isquemia mejoró significativamente la regulación del calcio evitando la sobrecarga de calcio intracelular, reduciendo la concentración de calcio diastólica, aumentando la carga de calcio en el retículo sarcoplásmico y preservando la amplitud del transitorio de calcio intracelular, lo cual se reflejaba en una recuperación satisfactoria en el proceso de acoplamiento de excitación-contracción durante la reperfusión. Sin embargo, estos efectos de la ranolazina no se produjeron cuando el fármaco se aplicó solo durante la reperfusión o cuando se aplicó tanto en la isquemia como en la reperfusión. Conclusiones: La ranolazina muestra unos efectos favorables en los cardiomiocitos expuestos a isquemia-reperfusión, pero solo cuando se aplica durante la isquemia. Este efecto se alcanza mejorando la regulación del calcio durante la isquemia (AU)

Introduction and objectives: Ranolazine is used as a complementary treatment for angina in symptomatic patients who are inadequately controlled with first-line antianginal therapies. Ranolazine inhibits sodium voltage-dependent channels, suggesting their possible involvement in the reperfusion process by preventing the sodium and calcium overload that occurs during ischemia. In this study, we characterized the effect of ranolazine on calcium homeostasis in isolated adult cardiac myocytes from rats subjected to a simulated ischemia and reperfusion protocol. Methods: The effects of ranolazine on changes in intracellular calcium concentration were evaluated at different times using field electrostimulation. The study of intracellular calcium was performed using microfluorimetry with the fluorescent indicator, Fura-2, and by confocal microscopy with the indicator, Fluo-3. Results: We found that cardiomyocytes subjected to ischemia-reperfusion showed an increase in the diastolic calcium concentration and a decrease in the amplitude of intracellular calcium transients. The application of ranolazine during ischemia significantly improved intracellular calcium handling, preventing intracellular calcium overload, decreasing the diastolic calcium concentration, increasing the sarcoplasmic reticulum calcium load, and preserving the amplitude of the intracellular calcium transient, which was reflected by successful recovery in the process of excitation-contraction coupling during reperfusion. However, these effects of ranolazine did not occur when it was applied during reperfusion or when applied in both ischemia and reperfusion. Conclusions: Ranolazine shows beneficial effects in cardiomyocytes exposed to ischemia/reperfusion but only when applied during ischemia. This effect is achieved through its improvement of calcium handling during ischemia (AU)

Cardioprotective Effect of Ranolazine in the Process of Ischemia-reperfusion in Adult Rat Cardiomyocytes.

INTRODUCTION AND OBJECTIVES: Ranolazine is used as a complementary treatment for angina in symptomatic patients who are inadequately controlled with first-line antianginal therapies. Ranolazine inhibits sodium voltage-dependent channels, suggesting their possible involvement in the reperfusion process by preventing the sodium and calcium overload that occurs during ischemia. In this study, we characterized the effect of ranolazine on calcium homeostasis in isolated adult cardiac myocytes from rats subjected to a simulated ischemia and reperfusion protocol. METHODS: The effects of ranolazine on changes in intracellular calcium concentration were evaluated at different times using field electrostimulation. The study of intracellular calcium was performed using microfluorimetry with the fluorescent indicator, Fura-2, and by confocal microscopy with the indicator, Fluo-3. RESULTS: We found that cardiomyocytes subjected to ischemia-reperfusion showed an increase in the diastolic calcium concentration and a decrease in the amplitude of intracellular calcium transients. The application of ranolazine during ischemia significantly improved intracellular calcium handling, preventing intracellular calcium overload, decreasing the diastolic calcium concentration, increasing the sarcoplasmic reticulum calcium load, and preserving the amplitude of the intracellular calcium transient, which was reflected by successful recovery in the process of excitation-contraction coupling during reperfusion. However, these effects of ranolazine did not occur when it was applied during reperfusion or when applied in both ischemia and reperfusion. CONCLUSIONS: Ranolazine shows beneficial effects in cardiomyocytes exposed to ischemia/reperfusion but only when applied during ischemia. This effect is achieved through its improvement of calcium handling during ischemia.

Low-dose combination of Rho kinase and L-type Ca(2+) 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.

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.

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 Ca(2+) entry (SOCE) signaling pathway in UII-induced coronary artery vasoconstriction. METHODS AND RESULTS: We used a combination of isometric tension measurement, Ca(2+) imaging, pharmacology, and molecular approaches to study UII-mediated rat coronary artery vasoconstriction and intracellular Ca(2+) mobilization in coronary smooth muscle cells. We found that UII promoted dose-dependent vasoconstriction and elicited Ca(2+) and Mn(2+) 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 Ca(2+)-independent phospholipase A(2)ß 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 A(2) activation. Furthermore, lysophosphatidylcholine, an independent phospholipase A(2) product, activated Orai1 but not STIM1-dependent contraction and SOCE. CONCLUSIONS: Here, we demonstrated that different critical players of SOCE signaling pathway are required for UII-induced vasoconstriction of rat coronary artery.

Cardioprotective action of urocortin in postconditioning involves recovery of intracellular calcium handling.

Ischemia/reperfusion (I/R) damage in the heart occurs mainly during the first minutes of reperfusion. Urocortin (Ucn) is a member of the corticotrophin-releasing factor that has been identified as a potent endogenous cardioprotector peptide when used in pre- and postconditioning protocols. However, the underlying mechanisms are not completely elucidated. Here, we focused on intracellular calcium ([Ca(2+)](i)) handling by Ucn when applied in early reperfusion. We used Langendorff-perfused rat hearts to determine hemodynamic parameters, and confocal microscopy to study global [Ca(2+)](i) transients evoked by electrical stimulation in isolated cardiomyocytes loaded with fluorescence Ca(2+) dye fluo-3AM. We found that the acute application of Ucn at the onset of reperfusion, in isolated hearts submitted to ischemia, fully recovered the hearts contractility and relaxation. In isolated cardiac myocytes, following ischemia we observed that the diastolic [Ca(2+)](i) was increased, the systolic [Ca(2+)](i) transients amplitude were depressed and sarcoplasmic reticulum (SR) Ca(2+) load was reduced. These effects were correlated to a decrease in the Na(+)/Ca(2+) exchanger (NCX) activity. Importantly, Ucn applied at reperfusion produced a complete recovery in diastolic [Ca(2+)](i) and global [Ca(2+)](i) transient amplitude, which were due to NCX activity improvement. In conclusion, we demonstrated that [Ca(2+)](i) handling play an essential role in postconditioning action of Ucn.

Mechanisms underlying the activation of L-type calcium channels by urocortin in rat ventricular myocytes.

AIMS: The aim of this study was to elucidate the signalling pathways implicated in the modulation of cardiac L-type Ca(2+) channels by urocortin (Ucn) in ventricular myocytes. METHODS AND RESULTS: Adult rat ventricular myocytes were stimulated in vitro with Ucn for 20-40 min. L-type calcium currents (I(CaL)) were measured with the patch-clamp technique, whereas quantification of activation of extracellular signal-regulated kinases 1/2 (ERK1/2) was assessed by sandwich-ELISA. Ucn induced a significant increase in I(CaL) density that was not prevented by the protein kinase A (PKA) inhibitor KT-5720 or the non-selective antagonist of guanine nucleotide exchange factor brefeldin A. The Ucn effect was antagonized by astressin, a corticotropin-releasing factor receptor-2 (CRF-R2) antagonist, and significantly reduced by protein kinase C (PKC) and ERK1/2 inhibitors. The cyclic AMP (cAMP) analogue 8-pCPT-2'OMe-cAMP, which selectively activates the exchange protein activated by cAMP (Epac), was ineffective in modifying I(CaL). Analysis of phospho-ERK1/2 showed that Ucn induced a significant activation of the ERK1/2 pathway in ventricular myocytes and this effect was prevented by pre-incubation with PKC inhibitors. CONCLUSION: The present study provides evidence of new mechanisms involved in the modulation of L-type Ca(2+) channels by Ucn in adult ventricular myocytes. We propose that the marked increase in I(CaL) density induced by Ucn is mediated through CRF-R2 and involves PKC-dependent activation of the ERK1/2 pathway, whereas PKA and Epac signalling are not implicated.

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.

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.

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 approximately = 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.