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.


Introduction
Despite the progress in cardiovascular disease treatment, the healthcare burden of ischemic heart failure (HF) is increasing worldwide [1]. Heart ischemia is originated by the critical obstruction of coronary arteries, leading to an imbalance between the consumption and supply of nutrients in the affected area in the heart. Ischemia causes cells lesions of different degrees depending on the extent of blood flow reduction and the length of the ischemic period, which influence the reduction in pH, ATP, and creatine phosphate, as well as increased levels of intracellular Na + and Ca 2+ concentrations, enlarged cell volume, and intracellular membranes disruption [2]. Treatment for myocardial ischemia involves the prompt and timely recovery of blood flow, which is known as myocardial reperfusion or revascularization and is necessary to save oxygen-deficient tissue (for a review, see [3,4]). Paradoxically, reperfusion causes additional injuries due to metabolites reaction with oxygen giving rise to reactive oxygen species (ROS) known as oxygen paradox [5]; stress of sarcoplasmic reticulum (SR), producing the accumulation of secondary metabolites and bad protein products; capillary non-reflow that leads to a worse local reperfusion [6][7][8]; and the mishandling of the intracellular Ca 2+ concentration ([Ca 2+ ] i ) [9]. The pathophysiological value of [Ca 2+ ] i homeostasis is well-recognized. Actually, one of the critical factors In heart, Ucn binds to CRFR2, which interacts with G-proteins activating different signaling pathways (PKC-MERK/ERK; cAMP-EPAC/PKA; PI3K/Akt) that acutely decrease cell death, improve Ca 2+ handling, enhance cell survival, and improve cardiac function. Ucn also activates transcription factors and stimulates miRNAs, release which regulates the expression of genes related to apoptosis, fibrosis, hypertrophy, and Ca 2+ homeostasis. The downregulation of these genes prevents the development of adverse cardiac remodeling, avoiding its progress toward heart failure.

Structure and Expression of Urocortin
Ucn is a mammalian peptide member of the corticotropin-releasing factor (CRF) family. Three isoforms of Ucn have been described so far, Ucn1, Ucn2, and Ucn3 [18][19][20][21], which differ in terms of their structure, expression, and affinity to CRF receptors. Ucn1 is a 40 amino acid (aa) peptide having 63% and 45% sequences identity with urotensin and Figure 1. Schematic model illustrating acute and long-term cardioprotection afforded by urocortin (Ucn) from I/R injuries. In heart, Ucn binds to CRFR2, which interacts with G-proteins activating different signaling pathways (PKC-MERK/ERK; cAMP-EPAC/PKA; PI3K/Akt) that acutely decrease cell death, improve Ca 2+ handling, enhance cell survival, and improve cardiac function. Ucn also activates transcription factors and stimulates miRNAs, release which regulates the expression of genes related to apoptosis, fibrosis, hypertrophy, and Ca 2+ homeostasis. The downregulation of these genes prevents the development of adverse cardiac remodeling, avoiding its progress toward heart failure.
Sequelae of acute myocardial infarction (AMI) are known to trigger the adverse cardiac remodeling, which is the basic mechanism underlying the progression to HF considered the end stage of various types of cardiovascular disease, including ischemic heart disease (IHD) [13]. Therefore, there is an urgent need for adjunct cardioprotection therapy to prevent the impact of I/R injuries on heart function [14,15].
In the last two decades, Urocortin (Ucn) isoforms, peptides related to stress, arose as potential therapeutic drugs to improve performances of heart in I/R and under HF [16,17]. This review aims to highlight Ucn's role in IHD and discusses the available clinical evidence of Ucn isoforms' therapeutic feature in HF.

Structure and Expression of Urocortin
Ucn is a mammalian peptide member of the corticotropin-releasing factor (CRF) family. Three isoforms of Ucn have been described so far, Ucn1, Ucn2, and Ucn3 [18][19][20][21], which differ in terms of their structure, expression, and affinity to CRF receptors. Ucn1 is a 40 amino acid (aa) peptide having 63% and 45% sequences identity with urotensin and CRF, respectively [18]. Ucn1 was first identified in the brain, where it is expressed mainly author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76].  cAMP, BNP, Nt-pro-BNP, adrenaline, ghrelin, noradrenaline, ADM, GH, LH, FSH, PRL, AVP, ET-1, PRA or aldosterone plasma levels unaltered  LVDV and LVSV, LVEF and mitral valve Doppler indices unaltered  Unaltered urine volume and urinary excretion of Ucn1, Na + , K + , CrCl, and cAMP Temporal ACTH and cortisol increase [62] Single-blind doseescalation design 8 males with stable congestive HF (LVEF ≥40%), NYHA class II-III. With medication Ucn2, 25-100 µg (0.5-2 µg/mL)  cAMP plasma levels increase  CO and HR increase  MAP and VR decrease  LVEF increase at high dose (100 µg)  Electrocardiogram alteration and arrhythmias not observed Temporal flushed during drug infusion [63] Single-blind doseescalation design 8 healthy unmedicated men Ucn2 ,25-100 µg (0.5-2 µg/mL)  cAMP and cGMP plasma levels increase  CO and HR increase dose dependent  DBP and MAP and VR decrease dose dependent  LVEF increase dose dependent  PRA, Ang II, norepinephrine, cGMP increase and epinephrine decrease at high dose  ACTH, cortisol, insulin, ghrelin, Nt pro-BNP, arginine vasopressin, ET-1, or ADM unaltered Urine Na + , K + , and CrCl decrease [73] Single-center, randomized, doubleblind, placebo-controlled trial 53 [76] Abbreviations: ACTH, adrenocorticotropic hormone; ADM, adrenomedullin; ANP, atrial natriuretic peptide; AngII, angiotensin II; AVP, arginine vasopressin BNP, brain natriuretic peptide; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanine monophosphate; CO, cardiac output; CI, cardiac index; CrCl, creatinine clearance; DBP, diastolic blood pressure; ET-1, endothelin-1; FSH, follicle-stimulating hormone; GH, growth hormone; HR, heart rate; LVEF, left ventricular ejection fraction; LH, luteinizing hormone; LVDV, left ventricular diastolic volume; LVSV, left ventricular systolic volume; MAP, mean arterial pressure; NYHA, New York Heart Association; Nt-pro-BNP, N-terminal pro-brain natriuretic peptide PRA, plasma renin activity; PRL, prolactin; SBP, systolic blood pressure; TSH, thyroid stimulating hormone; VR, vascular resistance. author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. Temporal ACTH and cortisol increase [62] Single-blind doseescalation design 8 males with stable congestive HF (LVEF ≥40%), NYHA class II-III. With medication Ucn2, 25-100 µg (0.5-2 µg/mL)  cAMP plasma levels increase  CO and HR increase  MAP and VR decrease  LVEF increase at high dose (100 µg)  Electrocardiogram alteration and arrhythmias not observed Temporal flushed during drug infusion [63] Single-blind doseescalation design 8 healthy unmedicated men Ucn2 ,25-100 µg (0.5-2 µg/mL)  cAMP and cGMP plasma levels increase  CO and HR increase dose dependent  DBP and MAP and VR decrease dose dependent  LVEF increase dose dependent  PRA, Ang II, norepinephrine, cGMP increase and epinephrine decrease at high dose  ACTH, cortisol, insulin, ghrelin, Nt pro-BNP, arginine vasopressin, ET-1, or ADM unaltered Urine Na + , K + , and CrCl decrease [73] Single-center, randomized, doubleblind, placebo-controlled trial 53 [76] Abbreviations: ACTH, adrenocorticotropic hormone; ADM, adrenomedullin; ANP, atrial natriuretic peptide; AngII, angiotensin II; AVP, arginine vasopressin BNP, brain natriuretic peptide; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanine monophosphate; CO, cardiac output; CI, cardiac index; CrCl, creatinine clearance; DBP, diastolic blood pressure; ET-1, endothelin-1; FSH, follicle-stimulating hormone; GH, growth hormone; HR, heart rate; LVEF, left ventricular ejection fraction; LH, luteinizing hormone; LVDV, left ventricular diastolic volume; LVSV, left ventricular systolic volume; MAP, mean arterial pressure; NYHA, New York Heart Association; Nt-pro-BNP, N-terminal pro-brain natriuretic peptide PRA, plasma renin activity; PRL, prolactin; SBP, systolic blood pressure; TSH, thyroid stimulating hormone; VR, vascular resistance. author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. Decrease in markers of cardiac remodeling [61] In a rat model of I/R, the i.v. infusion of Ucn2 (150 µg/Kg) right before heart reperfusion recovered significantly cardiac contractility and prevented fibrosis. Ucn2 administration recovered left ventricle ejection fraction and shortening, improved the amplitude of [Ca 2+ ] i transient, and modulated the expression of several proteins related to [Ca 2+ ] i homeostasis, such as TRPC5 and Orai1 channels [48]. Moreover, Ucn2 infusion in the same animal model prevented I/R dysregulation of miR-324-3p and miR-139-3p expression 1 week after I/R, which regulates the expression of different genes related to adverse remodeling. These studies confirmed that Ucn2 might provide long-lasting effects through post-transcriptional genes' modulation via miRNAs [55].
Interestingly, a recent study demonstrated that the Ucn3 gene transfected using adenoassociated viruse 8 (AAV8) in mice after 3 weeks of AMI improved Ca 2+ handling and left ventricle function, as compared with HF mice. In addition, mRNA expression of hypertrophy markers and stress as BNP, ANF, α-skeletal actin, and β-MHC was lower in mice transfected with Ucn3 gene than in HF mice, preventing adverse cardiac remodeling and apoptosis [61].

Therapeutic Values of Urocortin in Heart Failure
Preclinical studies proposed a positive therapeutic potential of Ucn's family in cardiovascular diseases since they increased cardiac output, left ventricular ejection fraction (LVEF) [62,63], and evoked vasodilation in human coronary arteries isolated from ischemic HF patients [64]. Therefore, the first investigations studied Ucn's value as a biochemical marker for the diagnosis and management of patients with HF [65][66][67] but also as therapeutic drug that might overcome HF symptoms [68], as summarized in Table 2. Clinical and translational studies about Ucn in AMI patients are still rare, although several reports investigated Ucn in HF patients, as reviewed recently [68]. One of the first clinical trials in HF patients with reduced ejection fraction (HFrEF) identified elevated levels of Ucn1, as compared to non-HF patients. The study demonstrated significant positive relationships between plasma Ucn1 levels and other circulating neurohormones known to be activated in this condition, such as ANP, NT-proANP, BNP, NT-proBNP, C-type natriuretic peptide, adrenomedullin, and endothelin 1 [67]. Plasma levels of Ucn1 in AMI patients are apparently higher than in control patients during the first 5 days after the heart attack. The high plasma level of Ucn1 on day 0 confirmed a significant and independent predictive value for mortality and adverse cardiac events. Of note, plasma Ucn1 levels are significantly elevated both in patients with non-ST elevation AMI (NSTEMI) and with ST elevation AMI (STEMI), comparing to control [69,70].
In the case of Ucn2, increased blood levels were observed in patients with systolic dysfunction but not in those with coronary artery disease without myocardial infarction [71]. A recent report used a new immunoassay for plasma NT-proUcn2 and observed modest but significant increased plasma concentrations in patients with HFrEF from different origins, including those with myocardial infarction, along with an inverse relationship to 2-year mortality in HF. This study concluded that perhaps the modest increase in NT-proUcn2 concentrations in HF may limit its utility as a diagnostic marker, but further investigations are required to confirm this statement [66].
On the other hand, independent reports analyzed the effect of the i.v. infusion of Ucn on hemodynamic parameters in healthy and HF patients. For instance, that i.v. administration of a bolus of 50 µg of Ucn1 to healthy volunteers, compared to placebo administration, resulted in increased plasma levels of corticotropin (ACTH), cortisol, ANP, and decreased in ghrelin level [72]. By contrast, Ucn1 infusion did not alter plasma levels of second messengers as cAMP, BNP, adrenaline, noradrenaline, endothelin, plasma renin activity, aldosterone, etc. [72]. Moreover, the administration of Ucn1 in patients with stable congestive heart failure (CHF) also increased levels of corticotropin and cortisol as compared to the placebo group, but without differences in ANP and ghrelin levels. Nevertheless, Ucn1 infusion did not change the heart rate, systolic and diastolic blood pressure, or the cardiac output [62,72]. No detectable differences in hemodynamic and renal effects were detected, either [62].  Abbreviations: ACTH, adrenocorticotropic hormone; ADM, adrenomedullin; ANP, atrial natriuretic peptide; AngII, angiotensin II; AVP, arginine vasopressin BNP, brain natriuretic peptide; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanine monophosphate; CO, cardiac output; CI, cardiac index; CrCl, creatinine clearance; DBP, diastolic blood pressure; ET-1, endothelin-1; FSH, follicle-stimulating hormone; GH, growth hormone; HR, heart rate; LVEF, left ventricular ejection fraction; LH, luteinizing hormone; LVDV, left ventricular diastolic volume; LVSV, left ventricular systolic volume; MAP, mean arterial pressure; NYHA, New York Heart Association; Nt-pro-BNP, N-terminal pro-brain natriuretic peptide PRA, plasma renin activity; PRL, prolactin; SBP, systolic blood pressure; TSH, thyroid stimulating hormone; VR, vascular resistance.
Unaltered urine volume and urinary excretion of Ucn1, Na + , K + , CrCl, and cAMP Temporal ACTH and cortisol increase [62] Single-blind dose-escalation design 8 males with stable congestive HF (LVEF ≥40%), NYHA class II-III. With medication Ucn2, 25-100 µg (0.5-2 µg/mL) min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. Abbreviations: ACTH, adrenocorticotropic hormone; ADM, adrenomedullin; ANP, atrial natriuretic peptide; AngII, angiotensin II; AVP, arginine vasopressin BNP, brain natriuretic peptide; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanine monophosphate; CO, cardiac output; CI, cardiac index; CrCl, creatinine clearance; DBP, diastolic blood pressure; ET-1, endothelin-1; FSH, follicle-stimulating hormone; GH, growth hormone; HR, heart rate; LVEF, left ventricular ejection frac-

Electrocardiogram alteration and arrhythmias not observed
Temporal flushed during drug infusion [63] Single-blind dose-escalation design 8 healthy unmedicated men Ucn2, 25-100 µg (0.5-2 µg/mL) β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. Abbreviations: ACTH, adrenocorticotropic hormone; ADM, adrenomedullin; ANP, atrial natriuretic peptide; AngII, angiotensin II; AVP, arginine vasopressin BNP, brain natriuretic peptide; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanine monophosphate; CO, cardiac output; CI, cardiac index; CrCl, creatinine clearance; DBP, diastolic blood pressure; ET-cAMP and cGMP plasma levels increase β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. Abbreviations: ACTH, adrenocorticotropic hormone; ADM, adrenomedullin; ANP, atrial natriuretic peptide; AngII, angiotensin II; AVP, arginine vasopressin BNP, brain natriuretic peptide; cAMP, cyclic adenosine monophosphate; cGMP, cyclic CO and HR increase dose dependent of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. Abbreviations: ACTH, adrenocorticotropic hormone; ADM, adrenomedullin; ANP, atrial natriuretic peptide; AngII, angiotensin II; AVP, arginine vasopressin BNP, brain natriuretic peptide; cAMP, cyclic adenosine monophosphate; cGMP, cyclic DBP and MAP and VR decrease dose dependent of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. Abbreviations: ACTH, adrenocorticotropic hormone; ADM, adrenomedullin; ANP, atrial natriuretic peptide; AngII, angio-LVEF increase dose dependent tients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. PRA, Ang II, norepinephrine, cGMP increase and epinephrine decrease at high dose author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. CO increase author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76]. author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76].  [74] DBP and VR decrease Erythema and hot feeling [76] Abbreviations: ACTH, adrenocorticotropic hormone; ADM, adrenomedullin; ANP, atrial natriuretic peptide; AngII, angiotensin II; AVP, arginine vasopressin BNP, brain natriuretic peptide; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanine monophosphate; CO, cardiac output; CI, cardiac index; CrCl, creatinine clearance; DBP, diastolic blood pressure; ET-1, endothelin-1; FSH, follicle-stimulating hormone; GH, growth hormone; HR, heart rate; LVEF, left ventricular ejection fraction; LH, luteinizing hormone; LVDV, left ventricular diastolic volume; LVSV, left ventricular systolic volume; MAP, mean arterial pressure; NYHA, New York Heart Association; Nt-pro-BNP, N-terminal pro-brain natriuretic peptide PRA, plasma renin activity; PRL, prolactin; SBP, systolic blood pressure; TSH, thyroid stimulating hormone; VR, vascular resistance.
Other studies evaluated the use of i.v. administration of Ucn2 to avoid Ucn1 effects on the central nervous system. For example, the i.v. administration of Ucn2 (25 and 100 µg) in healthy volunteers and in patients with congestive HF increased cardiac output, heart rate, and LVEF in a dose-dependent manner; meanwhile, it decreased systemic vascular resistances [63]. Similar findings on hemodynamic responses were observed in patients with congestive HF in NYHA II-III receiving the same doses of Ucn2 [73]. Later, a randomized controlled clinical trial using Ucn2 in acute decompensated HF, confirmed systemic vasodilation, and increased cardiac output without increasing the heart rate. Ucn2 administration was accompanied by a sustained fall in BNP levels and a transient decline in renal indices with a reduction in creatinine clearance, which was probably due to the pronounced falls in systemic arterial pressure [74]. Another clinical trial evaluated the effect of Ucn2 and Ucn3 in healthy volunteers and in patients with stable HF who did not respond to concomitant medical therapy. The study found that Ucn2 and Ucn3 (360 pg/min and 12 ng/min, respectively) caused vasodilatation and reduced peripheral vascular resistances; meanwhile, they increased the heart rate and cardiac output [75]. The author stated that Ucn3 exerted more marked hemodynamic effects than Ucn2 in HF patients but not in healthy subjects. However, the heart rate increase with the highest dose of Ucn3 was less pronounced in patients with HF, which was probably due to concomitant β-adrenoreceptor blocker therapy according to the authors' conclusion [75]. No episodes of arrhythmia or adverse effects, such as those previously witnessed in animal models, were observed in this study [75]. Interestingly, the i.v. infusion of Ucn3 (15 and 30 ng/kg/min) in HFrEF patients induced a significant increase in cardiac index following 60 min infusion and reduced systemic vascular resistances without significant effects on heart rate or systolic blood pressure [76].

Conclusions
To summarize, even if the effect of Ucn infusion in patients suffering AMI has not been addressed yet, data from clinical trials in HF patients provided evidence that Ucn isoforms efficiently decrease peripheral vascular resistances and the mean arterial pressure. The lowering of peripheral vascular resistances will be beneficial for AMI patients since it may promote a decrease in the afterload. At the same time, cardiac output increase by Ucn might be beneficial to compensate for cardiac myocyte loss during AMI, although this effect may also increase the oxygen consumption of the myocardium. Moreover, Ucn's ability to regulate the post-transcriptional and translational processes involved in cardiac remodeling is worth being deeply investigated. Actually, understanding the mechanisms and downstream targets of Ucn in preclinical studies holds promise to allow fine-tuning of the signaling pathway activated under I/R, which can be hopefully translated soon to clinical trials. Future detailed clinical trials using a large number of patients are eagerly needed to determine whether Ucn could be useful as a sensitive biomarker for the adverse cardiac remodeling post-AMI or as therapeutic drugs that will mitigate AMI injuries to avoid the evolution of adverse cardiac remodeling toward HF.