Cardioprotective Utility of Urocortin in Myocardial ...

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sists of fish urotensin I, frog sauvagine and urocortins I, II and III. Urocortin I (Ucn) is a 40-amino acid peptide that was first isolated from rat midbrain and shares a ...
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REVIEW ARTICLE

Cardioprotective Utility of Urocortin in Myocardial Ischemia-Reperfusion Injury: Where do We Stand? Craig Basman1#, Pratik Agrawal2#, Richard Knight2, Louis Saravolatz2, Chad McRee3, Carol Chen-Scarabelli4, Jagat Narula5^ and Tiziano Scarabelli*3,4^ 1

Lenox Hill Hospital, North Shore LIJ Health System, New York, NY, USA; 2St John Hospital and Medical Center, Wayne State University Medical School, Detroit, MI, US; 3University of Alabama at Birmingham (UAB) Medical Center, Birmingham, Alabama, USA; 4Birmingham Veterans Affairs Medical Center, University of Alabama at Birmingham, Birmingham, Alabama, USA; 5Mount Sinai Medical Center, Icahn School of Medicine at Mount Sinai, New York, NY, USA

ARTICLE HISTORY Received: May 22, 2015 Revised: November 24, 2015 Accepted: August 03, 2016 DOI: 10.2174/18744672106661702231014 22

Abstract: There has been a constant pursuit for development of newer therapies which can contribute to the relatively nascent field of cardioprotection in the setting of myocardial ischemia-reperfusion injury. One novel cardioprotective agent among others, that has shown promising results in the limited number of research studies undertaken till now, is Urocortin. Urocortins are peptides belonging to the Corticotropin-Releasing Hormone family. Acting through a variety of downstream mechanisms, urocortin has been shown to alter cellular metabolism and modulate the mechanism of cell death occurring as a result of ischemia-reperfusion injury. New evidence continues to accumulate in support of urocortin’s beneficial role in cytoprotection. We present here an updated review largely focused on the various mechanisms through which urocortin alters cellular metabolism, and discuss the clinical potential of urocortin’s cardioprotective ability in myocardial ischemia-reperfusion injury.

Keywords: Urocortin, ischemia-reperfusion, cytoprotection, corticotropins, cell death. INTRODUCTION The Urocortins are part of the Corticotropin Releasing Hormone (CRH) family of peptides. CRH was the first member of this peptide family to be identified in 1981. It was purified from ovine hypothalamus and was found to mediate the autonomic, endocrine and behavioral responses to stress by inducing release of adrenocorticotropic hormone (ACTH) from the pituitary [1]. The CRH family also consists of fish urotensin I, frog sauvagine and urocortins I, II and III. Urocortin I (Ucn) is a 40-amino acid peptide that was first isolated from rat midbrain and shares a 45% sequence identity with CRH [2]. The structures of human and mouse Ucn genes were discovered in 1998, and it was subsequently also cloned in sheep and Syrian hamsters [3-5]. Urocortin II (stresscopin-related peptide) and urocortin III (stresscopin) were discovered more recently. CRH and the *Address correspondence to this author at the University of Alabama at Birmingham (UAB) Medical Center, Birmingham, Division of Cardiovascular Disease, Alabama, USA; Tel: +1 205 679 1801; Fax: +1 205 936 9890; E-mail: [email protected] #Dr. Basman and Dr. Agrawal equally contributed as first authors to the writing of this manuscript. ^Dr. Narula and Dr. Scarabelli equally contributed as senior authors to the editing of this manuscript.

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three urocortins show similarities in their molecular and biochemical aspects, but have distinct as well as overlapping functions [2, 6, 7]. CRH and the urocortins interact with two classes of CRH receptors, CRH receptor-1 (CRH-R1) and CRH receptor-2 (CRH-R2), both of which can be expressed as different isoforms. Both these receptors possess a seven transmembrane helical domain and elicit several downstream signaling responses [8-10]. CRH-R1 is predominantly expressed in the brain and pituitary, while CRH-R2 isoforms are found in the brain and peripheral organs, which includes high expression in the heart [11] (Fig. 1). In a study by Kimura et al, RTPCR detected expression of the CRH-R2 isoform throughout the entire human heart, while CRH-R2 was only weakly detected in the heart (expressed in some left atria/ventricles and one right ventricle) [12]. In contrast, the rat heart had higher levels of the beta isoform of CRH-R2, which was located in all four chambers [12]. These findings suggest that the cardiac activity of endogenously produced Ucn is mediated by activation of CRH-R2 receptors in the cardiovascular system. Like CRH-R2, Ucn expression has been shown in several peripheral organs, especially in the heart.

© 2017 Bentham Science Publishers

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Fig. (1). Schematic diagram depicting interaction of CRH and urocortins with CRH receptors type-1 and -2, both of which are seventransmembrane helix receptors. (Ucn = Urocortin; CRH = Corticotropin Releasing Hormon; MEK 1/2 = Mitogen Activated Protein Kinase Kinase; PI3K = Phosphatidyl Inositol 3-OH Kinase; PKC = Protein Kinase C; Katp = ATP sensitive potassium channel; iPLA2 = calcium independent phospholipase A2).

While CRH binds predominantly to CRH-R1 isoforms, Ucn binds to both CRH-R1 and CRH-R2, though with higher affinity to CRH-R2 [13]. Urocortins II and III are effectively exclusive ligands for CRH-R2, however this paper will focus solely on Urocortin I (Ucn). The role of Ucn in cardioprotection is becoming increasingly evident, as several studies have demonstrated a Ucn-mediated cardioprotective role against myocyte death elicited by ischemia-reperfusion (I/R) injury and this review paper will focus on the mechanism and function of Ucn in myocardial infarction. GENOMIC LOCATION The human urocortin genes are located on chromosome 2p23-2p21, and the mouse urocortin gene is located on chromosome 5. Each consists of 2 exons: Exon-1 codes for the 5`-untranslated region, while the entire coding region is confined to exon-2 [3]. The rat urocortin gene is located on chromosome 6q12, and its coding region is also confined to exon-2 [14]. These genomic structures are both similar to the CRH gene whose coding region is found on its second exon [15], and supports the notion of CRH and urocortin emergence by gene duplication. Human, mouse and rat urocortin genes are highly homologous (~90% between human and mouse nucleotide identity in the coding regions of mature peptides). The mouse and rat mature peptides have a 97% identical coding nucleotide sequence [3]. Most of the studies on Ucn involve the human, mouse or rat.

Regulation of Urocortin Gene Expression In vitro, simulated ischemia yielded an increase in Ucn mRNA and peptide levels [16]. Several consensus transcription factor-binding sites have been identified in each of the Ucn promoters including a cyclic AMP response element, GATA-binding sites, and a C/EBP-binding site and BRn-2binding sites. I/R injury has been shown to enhance expression of the transcription factors C/EBP beta and gamma, resulting in increased urocortin transcription [16]. In accordance to this, the mature urocortin peptide was rapidly released and able to exert its downstream effects after binding to the cardiac CRH-R2 receptor [16]. ACUTE MECHANISMS OF CARDIOPROTECTION THROUGH KINASE ACTIVATION Ucn, which binds to CRH-R2, yields a downstream reaction that activates major cellular kinases; including mitogenactivated protein kinase (MAPK), phosphatidylinositol 3-OH kinase (Pl3K) and protein kinase C (PKC) [17]. These kinases modify the activity of several mitochondrial channels that are key for the induction of apoptosis [18-20] (Fig. 2). Mitogen-Activated Protein Kinase (MAPK) Ucn activation of MAPK might result in cardioprotection by several different mechanisms. Two members of the MAP Kinase cascade, extracellular signal-regulated kinases 1 and 2 (ERK 1/2 or p42/44) are activated after phosphorylation by

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Fig. (2). Multiple mechanisms of action through which urocortin ultimately leads to cardioprotection, subsequent to activation of CRH receptor type-2 in the heart. (CRH = Corticotropin Releasing Hormon; MEK 1/2 = Mitogen Activated Protein Kinase Kinase; PI3K = Phosphatidyl Inositol 3-OH Kinase; PKC = Protein Kinase C; MAPK = Mitogen Activated Protein Kinase; CT-1 = Cardiotropin-1; Hsp90 = Heat Shock Protein 90; Akt = Protein Kinase B; MPTP = Mitochondrial Permeability Transition Pore; Katp = ATP sensitive potassium channel; iPLA2 = calcium independent phospholipase A2; LPC = Lysophosphotidylcholine).

Mitogen-activated protein kinase kinase (MEK 1/2) [19]. Activation of the ERK 1/2 cascades results in several possible cardioprotective mechanisms such as: 1) Phosphorylation of the pro-apoptotic protein BAD, 2) Inhibition of the conformational change in BAX required for its translocation into the mitochondria, and 3) Decreased expression of the proapoptotic protein BIM [19]. In one study which exhibited Ucn’s cardioprotective effects as a result of the ERK 1/2 cascade, Ucn infusion was shown to prevent cell death when administered to myocytes prior to and during I/R. Subsequently, a MEK 1/2 inhibitor (PD98059) was applied to cultured myocytes prior to Ucn infusion. The cells were then subjected to I/R which resulted in substantially less cell survival than when Ucn was infused without the MEK 1/2 inhibition [21]. This study showed that the ERK 1/2 pathways have a major role in Ucn cardioprotection. Recently, our group showed that in mice heart, Src tyrosine kinase is involved in the urocortin-induced activation of ERK 1/2. Short-term treatment with Ucn caused rapid phosphorylation of Src, which serves as an upstream modulator of ERK 1/2 activation. In one experiment which was designed to investigate Ucn’s activation of ERK 1/2, a Src kinase inhibitor (PP2) was administered after a period of hypoxia/reoxygenation. This resulted in reduction of urocortin-induced phosphorylation of ERK 1/2, which was associated with drastically reduced cardioprotective effects on myocytes [22]. After Src was identified as an upstream modulator of the MAP Kinase cascade, our group found that Ucn-mediated cardioprotection was activated through a Src tyrosine kinase-

STAT3 pathway. STAT3 has been associated with cardioprotective effects of several agents, including resveratrol [23, 24]. Src kinase is a known inducer of STAT3 transcription. We found that in HL-1 cardiac cells, Ucn induced nuclear translocation of tyrosine 705 phosphorylated STAT3, which was inhibited when myocytes were pretreated with PP2 [25]. This shows that the Src tyrosine kinase-STAT3 pathway is activated by Ucn, and likely also contributes to Ucnmediated cardioprotection. Another theory for Ucn’s cardioprotective effects through the MAP Kinase cascade is by production of heat shock protein (Hsp90) and cardiotropin-1 (CT-1). Hsp90 is a cardioprotective agent, which is also detected in cardiac cells exposed to Ucn; and a MEK 1/2 inhibitor (PD98059) prevents the Ucn-mediated increase in Hsp90 expression. Thus, MEK 1/2 activation likely leads to cardioprotection by also increasing expression of Hsp90 [26]. Like Ucn’s relationship to Hsp90, a different study showed that Ucn enhances mRNA expression of another cardioprotective agent, cardiotropin-1 (CT-1). Ucn produces CT-1 through activation of the p42/44 (ERK 1/2) pathway [27]. This data shows that Ucn produces two cardioprotective agents, Hsp90 and CT-1, from downstream activation via the MAP Kinase cascade. Phosphatidyl-Inositol 3-OH Kinase (PI3K) Ucn binding to CRH-R2 also results in the activation of the PI3K/Akt pathway. Protein kinase B (Akt) is known to inactivate the peptide Bcl-2-associated death domain (BAD), which neutralizes the anti-apoptotic effect of Bcl2 and BclXl [18]. When Ucn binds to its receptor, it leads to PI3K

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recruitment to the membrane which then activates the protein kinase Akt. Akt further down regulates pro-apoptotic genes and enhances cell survival. When a PI3K inhibitor is applied to human myocytes exposed to Ucn, the cardioprotective activity normally provided by Ucn is lost [28]. This suggests that the PI3K/Akt signaling pathway is also involved in the cardioprotective effects of Ucn. Protein Kinase C (PKC) The PKC family has significant involvement in I/R injury. One isoform, PKC, has been identified as an isoenzyme involved in cardioprotection; while another isoform, PKC, has been linked to deleterious effects on the heart [20]. PKC activation induces a pathway that results in the inactivation of the pro-apoptotic protein BAD and an increase in expression of the anti-apoptotic Bcl2 [29, 30]. Alternatively, the PKC isoform has been found to increase myocyte apoptosis and necrosis [20]. In mouse studies, Ucn caused PKC relocation from the cytosol to the mitochondria and provided cardioprotection. Furthermore, when PKC was inhibited by an inhibitor peptide, Ucn-mediated cardioprotection was lost [31]. This data showed that the cardioprotective effect of Ucn is also reliant on PKC. Diabetes is an independent risk factor for death in patients having cardiac surgery under cardiopulmonary bypass [32]. Our recent study compared right atrial biopsies of diabetic (DM) and non-diabetic patients before and after cardiopulmonary bypass. Post-cardioplegic Ucn levels were 50% lower in DM patients than in non-DM patients. In accordance, apoptosis was more prevalent in the DM heart. In non-DM patients, cardioplegia increased PKC mRNA and protein with no over-expression of PKC. Conversely, in DM patients the reverse was found: there was PKC overexpression after cardioplegia and no increase inexpression of PKC [33]. This inability to increase Ucn expression in the DM heart in response to the I/R injury inherent in cardiopulmonary bypass may well contribute to this paradoxical over-expression of PKC instead of PKC, and may be an important pathological mechanism why DM patients have worse postoperative outcomes after cardiac surgery. CHRONIC MECHANISMS In addition to the ‘acute’ protein-kinase mediated effects, Ucn also significantly alters the expression of several genes, notably the ATP sensitive potassium channel (Katp), calcium-independent phospholipase A2 (iPLA2) and PKC (described above). These mechanisms contribute to Ucn’s cardioprotection through a more “chronic” modulatory effect. Though these genes are functionally different from each other, they all form part of a signaling network which is involved in regulation of the mitochondrial apoptotic pathway. ATP Sensitive Potassium Channel (Katp) Katp channels are known to be cardioprotective [34]. Normally, the Katp channels remain closed, but when ATP declines, for example during I/R, Katp channels open to replenish the cell’s ATP [35]. The Katp channels are transmembrane proteins formed of two subunits, a component of the sulfonylurea receptor (SUR) family (which contains the ATP/ADP binding site) and one of two channel pore-

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forming proteins, Kir6.1 and Kir6.2. The Kir6.1-containing channel is expressed in the mitochondria, and Ucn treatment enhances the expression of Kir6.1, but does not influence Kir6.2 expression [37]. In both isolated cardiac cells and the intact heart the cardioprotective effects of Ucn were blocked when both generalized and mitochondrial-specific Katp channel blockers were administered [37]. This data suggests that Ucn-mediated cardioprotection involves the Kir6.1 subunit of Katp channels, which act to keep the channel open during ischemia-reperfusion. Calcium Independent Phospholipase-A2 (iPLA2) iPLA2 catalyzes the formation of arachidonic acid into a substrate for cyclooxygenases to produce prostaglandins and other inflammatory mediators, and Ucn is known to reduce its expression. Lysophosphatidylcholine (LPC), a potent cardiotoxic agent, is produced during the process of this reaction. During I/R, levels of iPLA2 are increased which leads to accumulation of LPC [38]. In rabbit hearts, catalytically competent iPLA2 was increasingly expressed following I/R when compared to its activity in control hearts; [39] and cardiac damage as measured by infarct size was significantly increased when LPC was applied during ischemia. However, Ucn pre-treatment reversed the increase in infarct size, and so did bromoenol lactone (an irreversible iPLA2 inhibitor) [40]. This suggests that Ucn’s ability to downregulate iPLA2 might provide cardioprotection by decreasing LPC accumulation. There is other evidence that suggests that iPLA2 antagonizes Katp channel activity [41]. Hence, Ucn-induced downregulation of iPLA2 likely has more than one mechanism for providing cardioprotection. UCN EFFECTS ON APOPTOSIS AND NECROSIS DURING ISCHEMIA/ REPERFUSION Ucn’s beneficial role on myocytes exposed to I/R likely results from its role in modulating both apoptosis and necrosis. Following a period of ischemia, the ADP/ATP ratio largely determines the fate of the cell. Cells remain viable below a ratio of 0.11, with apoptosis occurring between a ratio of 0.11-1, and necrosis occurring when the ratio is above 1 [42]. Necrosis is even more hazardous than apoptosis because it triggers an inflammatory reaction which releases proteolytic enzymes that scavenge necrotic cells and may extend their lysis to surrounding normal tissues. This leads to larger infarcted areas in the heart and ultimately a more profound fibrotic scar. This phenomenon is referred to as ‘infarct expansion’ and is extremely common [43]. Infarct expansion increases mortality rates and incidence of nonfatal complications such as heart failure and aneurysm formation [44]. Unlike necrosis, apoptosis does not contribute to infarct expansion. Our group has shown that administration of Ucn is associated with a reduction in the overall number of myocytes dying by both necrosis and apoptosis. Furthermore, apoptotic death is promoted over a necrotic fate, which likely plays a role in Ucn’s cardioprotective effects [45]. CPK is released from necrotic myocytes, and is used as a marker of cardiac damage in ischemic patients. In our study, Ucn infusion during I/R was shown to decrease CPK levels [46]. These findings indicate that cardioprotection by Ucn likely

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involves its inherent ability to decrease overall myocyte death and to transform myocytes, that would potentially become necrotic, into a less hazardous fate.

by endogenous Ucn [49]. In the light of these findings, perhaps inclusion of exogenous Ucn in cardioplegic solutions could reduce the extent of myocyte apoptosis in patients undergoing similar procedures.

Cardioprotection via Evasion of Mitochondrial Damage

Our group also showed, using an animal model, that Ucn is released into the blood by viable cardiac myocytes when exposed to ischemia. After short periods of ischemia and before cell death had occurred, Ucn levels rose. In contrast, when ischemia was continued long enough to result in cell death, Ucn levels fell. There was an inverse relationship with Ucn and the extent of myocardial damage [50]. These findings suggest that Ucn serum levels may help in the diagnosis of cardiac chest pain arising due to brief periods of myocardial ischemia, as an alternative to troponin and CPK which are released only from dying myocytes. Perhaps further research may demonstrate that Ucn expression is a more sensitive marker to detect sub-lethal ischemia than conventional biomarkers.

It may be hypothesized that the mitochondria are a favored target of Ucn, and that Ucn-induced cardioprotection is mainly attributed to its ability to reduce mitochondrial damage [17]. The mitochondrial permeability transition pore (MPTP) is a nonspecific channel of the inner mitochondrial membrane which opens as a result of calcium overload. During I/R, experiments have shown that calcium accumulates and MPTP channels open, furthering the extent of cardiac injury. When an inhibitor of MPTP such as cyclosporine-A or sanglifehrin A is administered, further I/R injury is prevented [47]. A study with Langendorff-perfused rat hearts showed that pretreatment with Ucn inhibited opening of MPTP during reperfusion, and decreased the amount of oxidative stress [48]. Hence, the amount of cell death following pretreatment with Ucn was significantly less when subjected to I/R, than in cells without pre-treatment with Ucn [48]. This suggests that prevention of MPTP opening is yet another a cardioprotective mechanism of Ucn. Scope of Ucn-Mediated Cardioprotection after IschemiaReperfusion Injury With the growing number of patients receiving cardiopulmonary bypass surgery and percutaneous transluminal coronary angioplasty (PTCA), it is important that we continue to develop therapies to combat I/R injury. In one study, addition of exogenous urocortin ex vivo during hypoxia and reoxygenation reduced cardiac damage in isolated rat heart [21]. Infusion of urocortin was shown to reduce actual infarct size in the rat heart, both ex vivo and in vivo [21]. Additionally, while beta blockers and calcium channel antagonists provide cardioprotection at the cost of depressing myocardial contractility, Ucn notably protects against I/R injury without significantly affecting basal myocardial contractility [46]. After I/R injury, developed pressure (defined as the difference between LV systolic and diastolic pressure) drops as diastolic pressure rises. When Ucn is infused, the postischemic rise of diastolic pressure in the LV is reduced [46]. Partial to complete recovery of diastolic pressure is seen irrespective of whether Ucn is infused before the ischemic event, during ischemia and just before reperfusion, or during reperfusion alone [46]. The mechanism of these Ucnmediated hemodynamic benefits during I/R is still unclear, but the results are encouraging. The data that Ucn infusion during reperfusion alone improved outcomes is especially promising. This could be clinically useful during iatrogenic reperfusion, where the culprit artery is reopened via a pharmacological or interventional approach. On-pump cardiac surgery with cardioplegic arrest inevitably exposes the heart to I/R. Our group found that Ucn expression, when measured following mitochondria-initiated myocyte apoptosis induced by cardioplegic arrest, was proportional to the duration of cardiac arrest [49]. Viable myocytes expressed greater cytosolic amounts of Ucn, which was not detected in dying myocytes. This study demonstrates that in on-pump cardioplegic arrest, I/R injury might be limited

In one of the few human studies, Phrommintikul et al followed 66 patients with acute myocardial infarction (AMI). They found plasma Ucn levels to be elevated for 5 days from the onset of AMI. Furthermore, a higher level within 1 day of presentation was associated with augmented mortality rates. When combined with NT-proBNP, Ucn improved prognostic performance in patients with acute myocardial infarction [51]. UROCORTIN AND CARDIAC HYPERTROPHY: NOT ALL SUNSHINE Although Ucn has shown promising results with regard to cardioprotection during ischemia- reperfusion in model systems, there is some concern that it might cause cardiac hypertrophy if given over prolonged periods. In neonatal and adult cardiac myocytes, Ucn was identified to have hypertrophic effects mediated by the same pathway activated by CT1 [52]. In hypertensive rats with hypertrophic hearts, Ucn expression was found to be elevated compared to control subjects [53]. These results are inconclusive as to whether Ucn induces hypertrophy, or if the overexpression is from increased myocardial oxygen demand. In addition, hypertrophy is known to be a gradual process; and hence it would be unlikely that Ucn therapy for a short duration would cause cardiac hypertrophy. Furthermore, since hypertrophy seems to be mediated through CT-1 and not through the p42/44 pathway, future therapies involving selective activation of the p42/44 pathway could prevent this potential side effect. CONCLUSION Ucn plays an important role in altering cellular metabolism and regulating apoptosis and necrosis in cardiac myocytes exposed to ischemia-reperfusion. After Ucn binds to the CRH-R2 receptor, it has ‘acute’ protein-kinase mediated effects, and also ‘chronic’ modulatory effects that provide cardioprotection during I/R through various downstream mechanisms. Our research suggests that Ucn plays an important role in regulating cardiac necrosis and ‘infarct expansion’, without depressing myocardial contractility. With today’s population, on-pump cardiac bypass and PTCA are a prevalent cause of iatrogenic ischemia and reperfusion, and it is important that we minimize ‘infarct expansion’. Further

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studies are needed in man to define the precise diagnostic and therapeutic role of Ucn in the prevention of cardiac damage during ischemia-reperfusion.

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DISCLOSURE The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. No writing assistance was utilized in the production of this manuscript. CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest.

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