Epac is a guanine nucleotide exchange protein that is directly activated by cAMP, but whose cardiac cellular functions remain unclear. It is important to understand cardiac Epac signaling, because it is activated in parallel to classical cAMP-dependent signaling via protein kinase A. In addition to activating contraction, Ca2+ is a key cardiac transcription regulator (excitation-transcription coupling). It is unknown how myocyte Ca2+ signals are decoded in cardiac myocytes to control nuclear transcription. We examine Epac actions on cytosolic ([Ca2+]i) and intranuclear ([Ca2+]n) Ca2+ homeostasis, focusing on whether Epac alters [Ca2+]n and activates a prohypertrophic program in cardiomyocytes. Adult rat cardiomyocytes, loaded with fluo-3 were viewed by confocal microscopy during electrical field stimulation at 1 Hz. Acute Epac activation by 8-pCPT increased Ca2+ sparks and diastolic [Ca2+]i, but decreased systolic [Ca2+]i. The effects on diastolic [Ca2+]i and Ca2+ spark frequency were dependent on phospholipase C (PLC), inositol 1,3,5 triphosphate receptor (IP3R) and CaMKII activation. Interestingly, Epac preferentially increased [Ca2+]n during both diastole and systole, correlating with the perinuclear expression pattern of Epac. Moreover, Epac activation induced histone deacetylase 5 (HDAC5) nuclear export, with consequent activation of the prohypertrophic transcription factor MEF2. These data provide the first evidence that the cAMP-binding protein Epac modulates cardiac nuclear Ca2+ signaling by increasing [Ca2+]n through PLC, IP3R and CaMKII activation, and initiates a prohypertrophic program via HDAC5 nuclear export and subsequent activation of the transcription factor MEF2.

Cardiac, skeletal, and smooth muscle cells shared the common feature of contraction in response to different stimuli. Agonist-induced muscle's contraction is triggered by a cytosolic free Ca2+ concentration increase due to a rapid Ca2+ release from intracellular stores and a transmembrane Ca2+ influx, mainly through L-type Ca2+ channels. Compelling evidences have demonstrated that Ca2+ might also enter through other cationic channels such as Store-Operated Ca2+ Channels (SOCCs), involved in several physiological functions and pathological conditions. The opening of SOCCs is regulated by the filling state of the intracellular Ca2+ store, the sarcoplasmic reticulum, which communicates to the plasma membrane channels through the Stromal Interaction Molecule 1/2 (STIM1/2) protein. In muscle cells, SOCCs can be mainly non-selective cation channels formed by Orai1 and other members of the Transient Receptor Potential-Canonical (TRPC) channels family, as well as highly selective Ca2+ Release-Activated Ca2+ (CRAC) channels, formed exclusively by subunits of Orai proteins likely organized in macromolecular complexes. This review summarizes the current knowledge of the complex role of Store Operated Calcium Entry (SOCE) pathways and related proteins in the function of cardiac, skeletal, and vascular smooth muscle cells.

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.

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.

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