Circulation Journal REVIEW Circ J 2019; 83: 261 – 266 doi: 10.1253/circj.CJ-18-0428

Corticotropin-Releasing Hormone Family and Their Receptors in the Cardiovascular System

Mikito Takefuji, MD, PhD; Toyoaki Murohara, MD, PhD

The identification of corticotropin-releasing hormone (CRH) has led to the discovery of a growing family of ligands and receptors. CRH receptor 1 (CRHR1) and CRHR2 are mammalian G- coupled receptors (GPCRs) with high affinity for CRH and the CRH family of peptides. CRHR1 is predominantly expressed in the brain and plays a vital role in the hypothalamic-pituitary-adrenal (HPA) axis stress responses by secreting adrenal corticotropic hormone (ACTH). CRHR2 is predominantly expressed in the heart, and a CRHR2-specific ligand, urocortin 2 (UCN2), shows positive cardiac chronotropic and inotropic effects through 3´,5´-cyclic adenosine monophosphate (cAMP) signaling in response to CRHR2-mediated Gαs activation in mice and humans. Central administration of the CRH family of peptides increases mean arterial pressure through CRHR1 activation, whereas peripheral administration of the peptides decreases mean arterial pressure through CRHR2 activation. These observations have led to further investigations of CRHR2 as an important and unique GPCR in the physiological and pathological functioning of the cardiovascular (CV) system. Moreover, recent clinical trials demonstrate CRHR2 as a potentially therapeutic target in the treatment of heart failure. We present recent reviews of the role of CRHRs in basic CV physiology and in the pathophysiology of CV diseases.

Key Words: Corticotropin-releasing hormone receptor 2; G-protein coupled receptors; Heart failure; Urocortin

-protein coupled receptors (GPCRs) belong to the acid precursor.6 CRH plays a key role in regulating the largest and most diverse superfamily of cell surface basal and stress-induced pituitary-adrenal axis that G receptors. They react to extracellular stimuli and increases glucocorticoid and androgen secretion.7 CRH regulate cardiovascular (CV) function through cellular release in response to acute stress is essential for the survival G-protein-mediated signaling.1 GPCRs are a conserved of the organism, but CRH released as a result of exposure family of 7 transmembrane receptors that have been tar- to chronic stress may influence emotions and exert negative geted for drug therapy.2 GPCRs are involved in cardiac effects on the homeostasis of the organism’s physiological dysfunction and hypertension,3 and inhibitors of GPCRs functions.8 are widely used to treat patients with CV diseases such as Urocortin 1 (UCN1), -2, and -3 were identified as a 2nd heart failure and hypertension.4 Several studies have inves- mammalian CRH family of peptides. Vaughan et al identi- tigated various aspects of 2 GPCR families, β-adrenergic fied UCN1 in the Edinger-Westphal nucleus and lateral receptors and angiotensin II receptors, in CV diseases. superior olive regions of the rat brain as a mammalian There are approximately 800 GPCRs in humans, but the member of the mammalian CRH family of peptides.9 The role of most of the GPCRs in CV diseases remains unclear, peptide was named UCN1 because of its homology with suggesting that uncharacterized GPCRs have considerable fish urotensin and CRH. UCN2 (38 amino acids) and potential in the development of novel therapeutics for CV UCN3 (38 amino acids) were identified as members of diseases. This review focuses on corticotropin-releasing CRH-like peptides by searching databases hormone receptors (CRHRs) as potential therapeutic and cloning from human and mouse cDNA libraries.10,11 GPCRs in CV diseases, as well as their agonists and Simultaneously, human stresscopin (N-terminally 2-amino antagonists. acid extended UCN3) and stresscopin-related peptide (N-terminally 5-amino acid extended UCN2) were identified 12 Corticotropin-Releasing Hormone Family as members of the CRH family of peptides by Hsu et al. Although CRH mRNA is expressed widely throughout the CRH, also named corticotropin-releasing factor, is a peptide brain, UCN mRNA expression has restricted expression that was first characterized from the ovine hypothalamus.5 levels in the mammalian brain.13 In peripheral tissue, CRH is a 41-amino acid polypeptide (Figure) generated by UCN1 mRNA is broadly expressed in multiple organs, the cleavage of the C-terminus of pre-proCRH, a 196-amino including the pituitary, gastrointestinal tract, testis, heart,

Received April 11, 2018; revised manuscript received November 18, 2018; accepted December 5, 2018; J-STAGE Advance Publication released online December 22, 2018 Department of Cardiology, Nagoya University School of Medicine, Nagoya, Japan The Guest Editor for this article was Dr. Yoshihiko Saito. Mailing address: Mikito Takefuji, MD, PhD, Department of Cardiology, Nagoya University School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-8550, Japan. E-mail: [email protected]. ISSN-1346-9843 All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: [email protected]

Circulation Journal Vol.83, February 2019 262 TAKEFUJI M et al.

Figure. CRHR2-mediated cellular signaling in the cardiomyocyte. (A) Amino acid sequences of the CRH family of peptides. Identical residues are marked in red. (B) CRHR2 mediates cellular signaling through G-protein. ATP, adenosine triphosphate; cAMP, 3´,5´-cyclic adenosine monophosphate; CREB, cAMP response element binding protein; CRH, corticotropin-releasing hormone; CRHR, corticotropin-releasing hormone receptor; EPAC, exchange protein directly activated by cAMP; PKA, protein kinase A; PLN, phospholamban; RyR, ryanodine receptor; TnI, troponin I; UCN, urocortin.

and thymus,14 UCN2 mRNA is highly expressed in skin N termini (≈47%), which are responsible for their agonist and skeletal muscle,15 and UCN3 mRNA is expressed in selectivity.6,17 CRHR1 has a high affinity for CRH as well the skin and small intestine.11 The CRH family of peptides as UCN1, but not for UCN2 or UCN3 (Table).18,19 UCN1, regulates various physiological processes, including glucose -2, and -3 bind with considerably higher affinity for metabolism, CV regulation, immune function, and behavior. CRHR2 than for CRHR1. Therefore, UCN1 is considered The sites of mRNA expression of the CRH family of as an endogenous ligand for both CRHR1 and CRHR2, peptides have been well studied in various tissues; however, whereas UCN2 and UCN3 are generally considered as the organs or tissues from which the UCNs are released to endogenous ligands exclusive to CRHR2.18 mediate physiological and pathological processes remain CRHR1 mRNA is widely expressed in the mammalian largely unknown. brain with high levels of expression in the anterior pitu- itary;13 8 splice variants of CRHR1 mRNA have been CRHRs identified in humans. CRHR1α appears to have biological activity, but the physiological significance of the remaining CRH and the UCNs bind to CRHR1 and CRHR2, which CRHR1 variants is uncertain.20 CRHR2 is expressed as 3 belong to the secretin-like class B family of GPCRs. There splice variants: CRHR2α, CRHR2β, and CRHR2γ.21 are 18 class B GPCRs, which are characterized by a large mRNA of both CRHR2α and CRHR2γ is expressed in the extracellular N-terminal domain of 120–160 residues, which brain, whereas CRHR2β mRNA is widely expressed in is part of the binding site for agonists.16 Both CRHR1 and peripheral tissues, particularly the heart and skeletal CRHR2 are found in humans and other mammals, muscle.22–24 Pharmacological characterization of the CRHR2 and their receptors exhibit a 70% identity at the amino acid splicing variants revealed no major differences among level. Although the intracellular and transmembrane amino CRHR2α, -2β, and -2γ.18 The physiological and patho- acid sequences of CRHR1 and CRHR2 are highly homolo- logical roles of CRHR1 and CRHR2 may depend on their gous, a low degree of homology exists in their extracellular tissue distribution.

Circulation Journal Vol.83, February 2019 CRH Family 263

Table. CRHR1- and CRHR2-Mediated Cardiovascular Effects Primary location Ligand Cardiovascular effects CRHR1 Brain CRH Intravascular volume ↑ (human) UCN1 CRHR2 Brain UCN1 Cardiac contraction ↑ (mouse, human) Heart UCN2 Heart rate ↑ (sheep, human) Skeletal muscle UCN3 Cardiac remodeling ↑↓ (mouse) HUVEC (cell line) Vasodilation ↑ (mouse, rat, human) CRHR, corticotropin-releasing hormone receptor; HUVEC, human umbilical vein endothelial cell; UCN, urocortin.

Vascular Effects of CRHRs hearts isolated from Wistar rats showed that UCN1 induces Essentially, hyperactivity of the hypothalamic-pituitary- positive inotropic effects.38 adrenal (HPA) axis involving CRH has been associated In vivo, intravenous administration of UCN1 increased with elevated blood pressure and CV diseases.25,26 Central cardiac contractility analyzed by transthoracic echocardiog- administration of CRH increases mean arterial pressure in raphy in wild-type mice to approximately twice the baseline, the rat through the HPA axis,27 and treatment with anta- but had no effect on CRHR2−/− mice.31 Intravenous injec- larmin, a CRHR1 antagonist, decreases hypertension tion of UCN1 in healthy sheep increased heart rate, cardiac produced by intracerebroventricular injection of CRH in output, and cardiac contractility.39 Experiments involving the rat.28 Intracerebroventricular injection of α-helical administration of UCNs have revealed the inotropic effects CRH9-41, a non-selective CRHR antagonist, attenuates of CRHR2 in the heart, but in conventional CRHR2−/− stress-induced hypertension in rats.27 The mechanisms of mice and in cardiomyocyte-specific CRHR2 deficiency, HPA axis-induced hypertension probably involve several basal cardiac function remains unaffected.31,34 Thus, the processes, such as microvascular dysfunction and irre- physiological role of CRHR2 in basal cardiac function is versible reductions in nephron number by cortisol.29 still unclear. Although central administration of CRH increases Potential therapeutic effects of UCNs in CV diseases mean arterial pressure, peripheral administration of CRH have also been investigated. When mice were treated with decreases mean arterial pressure in rats.30 The hypotensive intraperitoneal injection of UCN2 prior to occlusion of the effect of intravenous injection of CRH in rats was left anterior descending coronary artery, the cardiac infarct 40 decreased by treatment with α-helical CRH9-41, but not size was reduced. Single intravenous bolus administration with antalarmin.28 Conventional CRHR2-deficient mice of UCN2 to a mouse with heart failure (muscle-specific (CRHR2−/−) show elevated basal mean arterial pressure LIM protein-deficient mice, a model of dilated cardiomy- and diastolic pressure compared with wild-type mice, and opathy) produced a significant enhancement of inotropic suppressed intravenous UCN1-induced blood pressure and lusitropic effects on left ventricular function and reduction.31 Intravenous administration of UCN2, which improved cardiac output.41 UCN1 treatment prevented binds with much higher affinity to CRHR2 than to CRHR1, further deterioration of cardiac dysfunction induced by reduced basal mean arterial pressure in rats, and treatment rapid left ventricular pacing in sheep. Infusion of UCNs with astressin2-B (a CRHR2 antagonist) blocked UCN2- into normal and failing hearts improved cardiac output. induced decreases in mean arterial pressure without influ- However, the cardiac effects of long-term activation of encing the basal mean arterial pressure.32 CRHRs have 2 CRHR2 remain unclear. Continuous UCN2 overexpression interesting effects on blood pressure: CRHR1 in the brain in the liver (>15-fold) using an adenovirus system increased elevates blood pressure and CRHR2 in peripheral tissues cardiac output in mice.42 Chronic treatment with UCN2 causes vasodilation. for 1 month in a mouse myocardial infarction model reduced infarct size and ameliorated cardiac remodeling Cardiac Effects of CRHRs with a decrease in mean blood pressure.43 Continuous RT-PCR analysis using human heart tissue shows that UCN2 infusion (a 2-fold increase in plasma UCN2) without CRHR2α is highly expressed in all 4 chambers of the heart, a significant effect on blood pressure resulted in cardiac whereas CRHR2β is weakly expressed only in the left dysfunction, whereas CRHR2 blockade suppressed pressure atrium, and the expression of CRHR1 in the human heart is overload-induced chronic cardiac dysfunction, suggesting unclear.33 A non-biased quantitative RT-PCR (qRT-PCR) that chronic UCN2 activation without vasorelaxation may analysis, which determined the copy numbers of 475 have cardiotoxic effects.34 The situation may be similar to GPCRs in adult murine cardiomyocytes, revealed that that seen with cardiac β-adrenergic receptors, which are CRHR2 was the 4th most abundantly expressed GPCR in also coupled to Gαs. Although adrenaline treatment is cardiomyocytes.34 In vitro, UCN2 and UCN3 significantly beneficial during the acute phase, chronic activation of increase myocyte contractility in a dose-dependent manner, β-adrenergic receptors are harmful and cardiotoxic in as characterized by increased fractional shortening and chronic heart failure. Beta-blockers have become a frontline peak systolic Ca2+ transients.35 In rabbit and mouse drug for the treatment of chronic heart failure. It is still ventricular myocytes, UCN2 mediates inotropic and lusi- unclear whether UCNs mediate protective effects of cardiac tropic effects via the cAMP- and Ca2+/calmodulin-CaMKII contractility through a direct effect on cardiac myocytes or signaling pathways, and also induces arrhythmogenic through reduction of blood pressure.44 effects.36,37 Ex vivo experiments using Langendorff-perfused

Circulation Journal Vol.83, February 2019 264 TAKEFUJI M et al.

CRHRs in Cellular Signaling failure; however, UCN assays have not been internationally GPCRs are coupled to heterotrimeric G-, resulting standardized.70 More accurate methods are required to in the activation of the corresponding cellular signaling examine circulating levels of endogenous UCNs in healthy pathways. The CRH family of peptides stably mediate subjects and patients with heart failure. cAMP signaling in HEK293 cells by expressing CRHR1 UCN1 infusion significantly elevates the circulation levels and CRHR2.45 CRHR-mediated cAMP signaling in of ACTH, cortisol, and ANP, but not BNP in healthy response to Gαs activation activates adenylyl cyclase and volunteers.71 Heart rate, cardiac output, blood pressure, protein kinase A (PKA), which in turn phosphorylate and ejection fraction are unchanged in healthy volunteers several proteins involved in excitation-contraction coupling, treated with UCN1. In 8 males with stable heart failure, including L-type Ca2+ channels, phospholamban, ryanodine brief intravenous UCN1 infusion increased corticotropin receptor (RyR), and troponin I in cardiomyocytes and cortisol without increasing ANP levels and hemody- (Figure).46,47 Gαs-mediated cellular signaling controls the namic effects.72 activity of well-known cardiac functions, stimulating Brief intravenous infusions of UCN2 in 8 healthy positive chronotropic, lusitropic, and inotropic effects.48 humans increased plasma UCN2 concentrations by 15- to Although transgenic overexpression of Gαs in mice resulted 60-fold and induced dose-related increases in cardiac in an enhanced efficacy of heart rate and cardiac contraction output, heart rate, and left ventricular ejection fraction.73 in response to catecholamines, the mice developed cardiac The administration of UCN2 in healthy humans decreased hypertrophy and dilation as they aged.49,50 The CRH diastolic blood pressure and mean arterial pressure, but peptide family is known to activate the PKA, CaMKII and not systolic blood pressure. UCN2 and UCN3 infusion AKT signaling pathways to temporarily increase cardiac caused arterial vasodilatation in 18 healthy volunteers, function,51 but the effects of chronic activation of these with the effects partly dependent on endothelial NO and pathways in the heart remains controversial.52–54 GPCR- cytochrome P450 metabolites of arachidonic acid.74 In 8 activated β-arrestin facilitates recycling and degradation of males with stable congestive heart failure, intravenous GPCRs, and also mediates G-protein-independent signaling UCN2 administration increased cardiac output substan- at GPCRs.55 The β-arrestin-dependent activation of the tially, secondary to decreased afterload through vasodila- ERK signaling confers cardioprotection in mice exposed tion.75 In patients with acute decompensated heart failure, to catecholamines.56 UCN stimulation may activate UCN2 infusion reduced blood pressure and increased car- β-arrestin-mediated signaling in cardiomyocytes. diac output.76 These findings suggest that brief intravenous Exchange protein directly activated by cAMP (EPAC) infusion of UCN2 may be a novel therapeutic approach to acts in parallel with, or independently of PKA to mediate treating acute heart failure, but further studies are necessary cAMP-induced effects in cellular contexts.57,58 EPAC causes to investigate the therapeutic effects of continuous infusion cardiac hypertrophy and remodeling in cardiomyocytes, of UCN2 against heart failure. but also induces the relaxation of vascular smooth muscle through Rap1 signaling and endothelial-dependent vascular Conclusions relaxation by activating endothelial nitric oxide synthase and production of nitric oxide (NO).59,60 Furthermore, CRH has been investigated as a primary hormone in stress UCN1 induces accumulation of cAMP through CRHR2 response, together with its receptor CRHR1. In addition in aortic smooth muscle cells.61 Removal of the endothelium to CRH, the role of UCN2 and CRHR2 in the CV system decreases the relaxant effect of UCN1 in rat coronary is garnering attention. Injection of UCNs revealed the arteries, indicating that endothelial-derived factors are inotropic effects of CRHR2 in the heart and on vascular involved in this process.62 UCN2 induces NO production tone, but the physiological role of endogenous UCNs and through cAMP- and Ca2+-mediated pathways in porcine CRHR2 and the identity of the organs and tissues secreting aortic endothelial cells.63 These findings suggest that UCNs, remain unclear. Furthermore, whether excessive or CRHR2 in the endothelium, as well as in aortic smooth insufficient activation of UCN2–CRHR2 is related to CV muscle, causes vasodilation, but further studies are neces- diseases needs to be determined. Further basic and clinical sary to understand the mechanisms of the CRHR2- investigations of UCNs and CRHRs can advance the mediated vascular relaxation. diagnosis of heart failure, as well as its treatment.

CRHR and Plasma UCNs in Humans Grants levels of CRHR1, CRHR2, CRH, UCN1, This work was supported by a Grant-in-Aid for Scientific Research UCN2, and UCN3 were examined by qPCR in 108 donors (C) from the Ministry of Education, Culture, Sports, Science, and for heart transplantation (control) and in 110 patients with Technology of Japan (M.T.). The authors declare that they have no heart failure.64 The qPCR analysis showed that cardiac competing interests. expression of CRHR1, CRH, and UCN3 was higher (P<0.001) and that of CRHR2 was lower (P=0.012) in the Conflict of Interest Statement patients than in the controls. The authors declare that they have no competing interests. Plasma UCN1 is elevated in heart failure, and UCN1 levels are related to clinical signs of heart failure and circu- References 65–67 lation levels of plasma natriuretic peptides (NPs). An 1. Katritch V, Cherezov V, Stevens RC. Structure-function of the increase in plasma UCN2 levels has been examined in G protein-coupled receptor superfamily. Annu Rev Pharm Toxicol patients with heart failure,34,51 and in patients with 2013; 53: 531 – 556. abdominal aortic aneurysm.68 Validated ELISA analysis 2. Hauser AS, Attwood MM, Rask-Andersen M, Schioth HB, Gloriam DE. Trends in GPCR drug discovery: New agents, showed that the plasma NT-proUCN2 concentration was targets and indications. Nat Rev Drug Discov 2017; 16: 829 – 842. 69 significantly increased with heart failure. These findings 3. Sato M. Roles of accessory proteins for heterotrimeric G-protein suggest that UCNs are elevated in patients with heart in the development of cardiovascular diseases. Circ J 2013; 77:

Circulation Journal Vol.83, February 2019 CRH Family 265

2455 – 2461. 24. Perrin M, Donaldson C, Chen R, Blount A, Berggren T, 4. Yancy CW, Jessup M, Bozkurt B, Butler J, Casey DE Jr, Colvin Bilezikjian L, et al. Identification of a second corticotropin- MM, et al. 2017 ACC/AHA/HFSA focused update of the 2013 releasing factor receptor gene and characterization of a cDNA ACCF/AHA guideline for the management of heart failure: A expressed in heart. Proc Natl Acad Sci USA 1995; 92: 2969 – 2973. report of the American College of Cardiology/American Heart 25. Rosmond R, Bjorntorp P. The hypothalamic-pituitary-adrenal Association Task Force on Clinical Practice Guidelines and the axis activity as a predictor of cardiovascular disease, type 2 Heart Failure Society of America. Circulation 2017; 136: e137 – diabetes and stroke. J Intern Med 2000; 247: 188 – 197. e161. 26. Brotman DJ, Golden SH, Wittstein IS. The cardiovascular toll 5. Vale W, Spiess J, Rivier C, Rivier J. Characterization of a of stress. Lancet 2007; 370: 1089 – 1100. 41-residue ovine hypothalamic peptide that stimulates secretion 27. Morimoto A, Nakamori T, Morimoto K, Tan N, Murakami N. of corticotropin and beta-endorphin. Science 1981; 213: 1394 – The central role of corticotrophin-releasing factor (CRF-41) in 1397. psychological stress in rats. J Physiol 1993; 460: 221 – 229. 6. Dautzenberg FM, Hauger RL. The CRF peptide family and 28. Briscoe RJ, Cabrera CL, Baird TJ, Rice KC, Woods JH. their receptors: Yet more partners discovered. Trends Pharmacol Antalarmin blockade of corticotropin releasing hormone-induced Sci 2002; 23: 71 – 77. hypertension in rats. Brain Res 2000; 881: 204 – 207. 7. Tsigos C, Chrousos GP. Hypothalamic-pituitary-adrenal axis, 29. Seckl JR, Meaney MJ. Glucocorticoid programming. Ann NY neuroendocrine factors and stress. J Psychsom Res 2002; 53: Acad Sci 2004; 1032: 63 – 84. 865 – 871. 30. Corder R, Turnill D, Ling N, Gaillard RC. Attenuation of 8. Rivier JE, Rivier CL. Corticotropin-releasing factor peptide corticotropin releasing factor-induced hypotension in anesthetized antagonists: Design, characterization and potential clinical rats with the CRF antagonist, alpha-helical CRF9-41: Comparison relevance. Front Endocrinol 2014; 35: 161 – 170. with effect on ACTH release. Peptides 1992; 13: 1 – 6. 9. Vaughan J, Donaldson C, Bittencourt J, Perrin MH, Lewis K, 31. Coste SC, Kesterson RA, Heldwein KA, Stevens SL, Heard AD, Sutton S, et al. Urocortin, a mammalian neuropeptide related to Hollis JH, et al. Abnormal adaptations to stress and impaired fish urotensin I and to corticotropin-releasing factor. Nature cardiovascular function in mice lacking corticotropin-releasing 1995; 378: 287 – 292. hormone receptor-2. Nature Genet 2000; 24: 403 – 409. 10. Reyes TM, Lewis K, Perrin MH, Kunitake KS, Vaughan J, 32. Chen CY, Doong ML, Rivier JE, Tache Y. Intravenous urocortin Arias CA, et al. Urocortin II: A member of the corticotropin- II decreases blood pressure through CRF(2) receptor in rats. releasing factor (CRF) neuropeptide family that is selectively Regul Pept 2003; 113: 125 – 130. bound by type 2 CRF receptors. Proc Natl Acad Sci USA 2001; 33. Kimura Y, Takahashi K, Totsune K, Muramatsu Y, Kaneko C, 98: 2843 – 2848. Darnel AD, et al. Expression of urocortin and corticotropin- 11. Lewis K, Li C, Perrin MH, Blount A, Kunitake K, Donaldson releasing factor receptor subtypes in the human heart. J Clin C, et al. Identification of urocortin II,I an additional member of Endocrinol Metab 2002; 87: 340 – 346. the corticotropin-releasing factor (CRF) family with high affinity 34. Tsuda T, Takefuji M, Wettschureck N, Kotani K, Morimoto R, for the CRF2 receptor. Proc Natl Acad Sci USA 2001; 98: Okumura T, et al. Corticotropin releasing hormone receptor 2 7570 – 7575. exacerbates chronic cardiac dysfunction. J Exp Med 2017; 214: 12. Hsu SY, Hsueh AJ. Human stresscopin and stresscopin-related 1877 – 1888. peptide are selective ligands for the type 2 corticotropin-releasing 35. Makarewich CA, Troupes CD, Schumacher SM, Gross P, Koch hormone receptor. Nat Med 2001; 7: 605 – 611. WJ, Crandall DL, et al. Comparative effects of urocortins and 13. Henckens MJ, Deussing JM, Chen A. Region-specific roles ofthe stresscopin on cardiac myocyte contractility. J Mol Cell Cardiol corticotropin-releasing factor-urocortin system in stress. Nat Rev 2015; 86: 179 – 186. Neurosci 2016; 17: 636 – 651. 36. Yang LZ, Kockskamper J, Khan S, Suarez J, Walther S, 14. Kageyama K, Bradbury MJ, Zhao L, Blount AL, Vale WW. Doleschal B, et al. cAMP- and Ca2+/calmodulin-dependent Urocortin messenger ribonucleic acid: Tissue distribution in protein kinases mediate inotropic, lusitropic and arrhythmogenic the rat and regulation in thymus by lipopolysaccharide and effects of urocortin 2 in mouse ventricular myocytes. Br J glucocorticoids. Endocrinology 1999; 140: 5651 – 5658. Pharmacol 2011; 162: 544 – 556. 15. Chen A, Blount A, Vaughan J, Brar B, Vale W. Urocortin II gene 37. Yang LZ, Kockskamper J, Heinzel FR, Hauber M, Walther S, is highly expressed in mouse skin and skeletal muscle tissues: Spiess J, et al. Urocortin II enhances contractility in rabbit Localization, basal expression in corticotropin-releasing factor ventricular myocytes via CRF(2) receptor-mediated stimulation receptor (CRFR) 1- and CRFR2-null mice, and regulation by of protein kinase A. Cardiovasc Res 2006; 69: 402 – 411. glucocorticoids. Endocrinology 2004; 145: 2445 – 2457. 38. Calderon-Sanchez E, Delgado C, Ruiz-Hurtado G, Dominguez- 16. de Graaf C, Song G, Cao C, Zhao Q, Wang MW, Wu B, et al. Rodriguez A, Cachofeiro V, Rodriguez-Moyano M, et al. Extending the structural view of class B GPCRs. Trends Biochem Urocortin induces positive inotropic effect in rat heart. Cardiovasc Sci 2017; 42: 946 – 960. Res 2009; 83: 717 – 725. 17. Inda C, Armando NG, Dos Santos Claro PA, Silberstein S. 39. Parkes DG, Vaughan J, Rivier J, Vale W, May CN. Cardiac Endocrinology and the brain: Corticotropin-releasing hormone inotropic actions of urocortin in conscious sheep. Am J Phisiol signaling. Endocr Connect 2017; 6: r99 – r120. 1997; 272: H2115 – H2122. 18. Hauger RL, Grigoriadis DE, Dallman MF, Plotsky PM, Vale 40. Li J, Qi D, Cheng H, Hu X, Miller EJ, Wu X, et al. Urocortin 2 WW, Dautzenberg FM. International Union of Pharmacology. autocrine/paracrine and pharmacologic effects to activate AMP- XXXVI: Current status of the nomenclature for receptors for activated protein kinase in the heart. Proc Natl Acad Sci USA corticotropin-releasing factor and their ligands. Pharmacol Rev 2013; 110: 16133 – 16138. 2003; 55: 21 – 26. 41. Bale TL, Hoshijima M, Gu Y, Dalton N, Anderson KR, Lee 19. Grace CR, Perrin MH, Cantle JP, Vale WW, Rivier JE, Riek R. KF, et al. The cardiovascular physiologic actions of urocortin II: Common and divergent structural features of a series of cortico- Acute effects in murine heart failure. Proc Natl Acad Sci USA tropin releasing factor-related peptides. J Am Chem Soc 2007; 2004; 101: 3697 – 3702. 129: 16102 – 16114. 42. Gao MH, Lai NC, Miyanohara A, Schilling JM, Suarez J, Tang 20. Hillhouse EW, Grammatopoulos DK. The molecular mechanisms T, et al. Intravenous adeno-associated virus serotype 8 encoding underlying the regulation of the biological activity of corticotropin- urocortin-2 provides sustained augmentation of left ventricular releasing hormone receptors: Implications for physiology and function in mice. Hum Gene Ther 2013; 24: 777 – 785. pathophysiology. Endocr Rev 2006; 27: 260 – 286. 43. Ellmers LJ, Scott NJ, Cameron VA, Richards AM, Rademaker 21. Bale TL, Vale WW. CRF and CRF receptors: Role in stress MT. Chronic urocortin 2 administration improves cardiac responsivity and other behaviors. Annu Rev Pharmacol Toxicol function and ameliorates cardiac remodeling after experimental 2004; 44: 525 – 557. myocardial infarction. J Cardiovasc Pharmacol 2015; 65: 269 – 22. Lovenberg TW, Chalmers DT, Liu C, De Souza EB. CRF2 alpha 275. and CRF2 beta receptor mRNAs are differentially distributed 44. Venkatasubramanian S, Newby DE, Lang NN. Urocortins in between the rat central nervous system and peripheral tissues. heart failure. Biochem Pharmacol 2010; 80: 289 – 296. Endocrinology 1995; 136: 4139 – 4142. 45. Dautzenberg FM, Py-Lang G, Higelin J, Fischer C, Wright MB, 23. Kostich WA, Chen A, Sperle K, Largent BL. Molecular identi- Huber G. Different binding modes of amphibian and human fication and analysis of a novel human corticotropin-releasing corticotropin-releasing factor type 1 and type 2 receptors: factor (CRF) receptor: The CRF2 gamma receptor. Mol Endocrinol Evidence for evolutionary differences. J Pharmacol Exp Ther 1998; 12: 1077 – 1085. 2001; 296: 113 – 120.

Circulation Journal Vol.83, February 2019 266 TAKEFUJI M et al.

46. Wettschureck N, Offermanns S. Mammalian G proteins and Pharmacol 2002; 135: 1467 – 1476. their cell type specific functions. Phisiol Rev 2005; 85: 1159 – 1204. 63. Grossini E, Molinari C, Mary DA, Uberti F, Ribichini F, 47. Salazar NC, Chen J, Rockman HA. Cardiac GPCRs: GPCR Caimmi PP, et al. Urocortin II induces nitric oxide production signaling in healthy and failing hearts. Biochim Biophys Acta through cAMP and Ca2+ related pathways in endothelial cells. 2007; 1768: 1006 – 1018. Cell Physiol Biochem 2009; 23: 87 – 96. 48. Rockman HA, Koch WJ, Lefkowitz RJ. Seven-transmembrane- 64. Pilbrow AP, Lewis KA, Perrin MH, Sweet WE, Moravec CS, spanning receptors and heart function. Nature 2002; 415: 206 – Tang WH, et al. Cardiac CRFR1 expression is elevated in human 212. heart failure and modulated by genetic variation and alternative 49. Iwase M, Bishop SP, Uechi M, Vatner DE, Shannon RP, Kudej splicing. Endocrinology 2016; 157: 4865 – 4874. RK, et al. Adverse effects of chronic endogenous sympathetic 65. Ng LL, Loke IW, O’Brien RJ, Squire IB, Davies JE. Plasma drive induced by cardiac GS alpha overexpression. Circ Res urocortin in human systolic heart failure. Clin Sci 2004; 106: 1996; 78: 517 – 524. 383 – 388. 50. Iwase M, Uechi M, Vatner DE, Asai K, Shannon RP, Kudej RK, 66. Wright SP, Doughty RN, Frampton CM, Gamble GD, Yandle et al. Cardiomyopathy induced by cardiac Gs alpha overexpres- TG, Richards AM. Plasma urocortin 1 in human heart failure. sion. Am J Physiol 1997; 272: H585 – H589. Circ Heart Fail 2009; 2: 465 – 471. 51. Adao R, Santos-Ribeiro D, Rademaker MT, Leite-Moreira AF, 67. Gruson D, Ahn SA, Ketelslegers JM, Rousseau MF. Circulating Bras-Silva C. Urocortin 2 in cardiovascular health and disease. levels of stress associated peptide Urocortin in heart failure Drug Discovery Today 2015; 20: 906 – 914. patients. Peptides 2010; 31: 354 – 356. 52. Shimizu I, Minamino T. Physiological and pathological cardiac 68. Emeto TI, Moxon JV, Biros E, Rush CM, Clancy P, Woodward L, hypertrophy. J Mol Cell Cardiol 2016; 97: 245 – 262. et al. Urocortin 2 is associated with abdominal aortic aneurysm 53. Anderson ME, Brown JH, Bers DM. CaMKII in myocardial and mediates anti-proliferative effects on vascular smooth muscle hypertrophy and heart failure. J Mol Cell Cardiol 2011; 51: cells via corticotrophin releasing factor receptor 2. Clin Sci 2014; 468 – 473. 126: 517 – 527. 54. Antos CL, Frey N, Marx SO, Reiken S, Gaburjakova M, 69. Liew OW, Yandle TG, Chong JP, Ng YX, Frampton CM, Ng Richardson JA, et al. Dilated cardiomyopathy and sudden death TP, et al. High-sensitivity sandwich ELISA for plasma NT- resulting from constitutive activation of protein kinase a. Circ proUcn2: Plasma concentrations and relationship to mortality in Res 2001; 89: 997 – 1004. heart failure. Clin Chem 2016; 62: 856 – 865. 55. Zhabyeyev P, Zhang H, Oudit GY. Is beta-arrestin 2 a magic 70. Rademaker MT, Richards AM. Urocortins: Actions in health bullet for heart failure treatment? Hypertension 2017; 70: 887 – and heart failure. Clin Chim Acta 2017; 474: 76 – 87. 889. 71. Davis ME, Pemberton CJ, Yandle TG, Lainchbury JG, 56. Patel PA, Tilley DG, Rockman HA. Beta-arrestin-mediated Rademaker MT, Nicholls MG, et al. Urocortin-1 infusion in signaling in the heart. Circ J 2008; 72: 1725 – 1729. normal humans. J Clin Endocrinol Metab 2004; 89: 1402 – 1409. 57. de Rooij J, Zwartkruis FJ, Verheijen MH, Cool RH, Nijman 72. Davis ME, Pemberton CJ, Yandle TG, Lainchbury JG, SM, Wittinghofer A, et al. Epac is a Rap1 guanine-nucleotide- Rademaker MT, Nicholls MG, et al. Effect of urocortin 1 infusion exchange factor directly activated by cyclic AMP. Nature 1998; in humans with stable congestive cardiac failure. Clin Sci 2005; 396: 474 – 477. 109: 381 – 388. 58. Kawasaki H, Springett GM, Mochizuki N, Toki S, Nakaya M, 73. Davis ME, Pemberton CJ, Yandle TG, Fisher SF, Lainchbury Matsuda M, et al. A family of cAMP-binding proteins that JG, Frampton CM, et al. Urocortin 2 infusion in healthy directly activate Rap1. Science 1998; 282: 2275 – 2279. humans: Hemodynamic, neurohormonal, and renal responses. J 59. Lezoualc’h F, Fazal L, Laudette M, Conte C. Cyclic AMP sensor Am Coll Cardiol 2007; 49: 461 – 471. EPAC proteins and their role in cardiovascular function and 74. Venkatasubramanian S, Griffiths ME, McLean SG, Miller MR, disease. Circ Res 2016; 118: 881 – 897. Luo R, Lang NN, et al. Vascular effects of urocortins 2 and 3 in 60. Roberts OL, Dart C. cAMP signalling in the vasculature: The healthy volunteers. J Am Heart Assoc 2013; 2: e004267. role of Epac (exchange protein directly activated by cAMP). 75. Davis ME, Pemberton CJ, Yandle TG, Fisher SF, Lainchbury Biochem Soc Trans 2014; 42: 89 – 97. JG, Frampton CM, et al. Urocortin 2 infusion in human heart 61. Kageyama K, Gaudriault GE, Suda T, Vale WW. Regulation of failure. Eur Heart J 2007; 28: 2589 – 2597. corticotropin-releasing factor receptor type 2β mRNA via cyclic 76. Chan WY, Frampton CM, Crozier IG, Troughton RW, AMP pathway in A7r5 aortic smooth muscle cells. Cell Signal Richards AM. Urocortin-2 infusion in acute decompensated 2003; 15: 17 – 25. heart failure: Findings from the UNICORN study (urocortin-2 62. Huang Y, Chan FL, Lau CW, Tsang SY, He GW, Chen ZY, et in the treatment of acute heart failure as an adjunct over conven- al. Urocortin-induced endothelium-dependent relaxation of rat tional therapy). JACC Heart Fail 2013; 1: 433 – 441. coronary artery: Role of nitric oxide and K+ channels. Br J

Circulation Journal Vol.83, February 2019