Relaxin' the stiffened heart and arteries: the therapeutic potential for relaxin in the treatment of cardiovascular disease

Associate editor: P. Molenaar

‘Relaxin’ the stiffened heart and arteries: The therapeutic potentialfor relaxin in the treatment of cardiovascular disease

Chrishan S. Samuel a,b, Xiao-Jun Du c, Ross A.D. Bathgate a,b, Roger J. Summers d,!

a Howard Florey Institute, University of Melbourne, Victoria 3010, Australiab Department of Biochemistry and Molecular Biology, University of Melbourne, Victoria 3010, Australia

c Baker Heart Research Institute, 75 Commercial Road, Melbourne, Victoria 3004, Australiad Department of Pharmacology, Monash University, PO Box 13E, Clayton, Victoria 3800, Australia

Abstract

Although originally characterised as a reproductive hormone, relaxin has emerged as a multi-functional endocrine and paracrine factor thatplays a number of important roles in several organs, including the normal and diseased cardiovascular system. The recent discovery of the H3/relaxin-3 gene, and the elusive receptors for relaxin (Relaxin family peptide receptor; RXFP1) and relaxin-3 (RXFP3/RXFP4) have led to the re-classification of a distinct relaxin peptide/receptor family. Additionally, the identification of relaxin and RXFP1 mRNA and/or relaxin bindingsites in the heart and blood vessels has confirmed that the cardiovascular system is a target for relaxin peptides. While evidence for the productionof relaxins within the cardiovascular system is limited, several studies have established that the relaxin genes are upregulated in the diseasedhuman and rodent heart where they likely act as cardioprotective agents. The ability of relaxin to protect the heart is most likely mediated via itsantifibrotic, anti-hypertrophic, anti-inflammatory and vasodilatory actions, but it may also directly stimulate myocardial regeneration and repair.This review describes relaxin and its primary receptor (RXFP1) in relation to the roles and effects of relaxin in the normal and pathologicalcardiovascular system. It is becoming increasingly clear that relaxin has a number of diverse physiological and pathological roles in thecardiovascular system that may have important therapeutic and clinical implications.© 2006 Elsevier Inc. All rights reserved.

Keywords: H1 relaxin; H2 relaxin; H3 relaxin; LGR7; RXFP1

Abbreviations: !-SMA, !-smooth muscle actin; Ang II, angiotensin II; ANP, atrial natriuretic peptide; AT, angiotensin receptor; BFGF, basic fibroblast growthfactor; BP, binding protein; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; CHF, congestive heart failure; ECM, extracellularmatrix; Erk, extracellular signal-regulated kinase; ET-1, endothelin-1; FSH, follicle-stimulating hormone; FSHR, follicle-stimulating hormone receptor; GPCR, G-protein coupled receptor; GR, glucocorticoid receptor; GREAT, G-protein coupled receptor affecting testis descent; H1, H2, H3 relaxin, human gene-1, -2, -3 relaxin;IBMX, isobutylmethylxanthine; IGF-I, insulin-like growth factor-I; INSL3–6, insulin-like peptide 3–6; IL-1", interleukin-1"; KO, knockout; LDLa, low densitylipoprotein class A module; LGR, leucine-rich repeat containing G-protein coupled receptor; LH, luteinizing hormone; LHR, luteinizing hormone receptor; LRR,leucine-rich repeats; LV, left ventricle; MAPK, mitogen-activated protein kinase; MI, myocardial infarct; MMP, matrix metalloproteinase; NO, nitric oxide; NOS,nitric oxide synthase; NPR-A, natriuretic peptide receptor-A; NRCM, neonatal rat cardiac myocytes; PCR, polymerase chain reaction; PDE, phosphodiesterases; PI3-K, phosphoinositide-3 kinase; PKA, protein kinase-A; PKC#, protein kinase-C$; PTX, pertussis toxin; RT, reverse transcription; RXFP1–4, relaxin family peptidereceptor 1–4; SALPR, somatostatin and angiotensin-like peptide receptor; SEAP, secreted alkaline phosphatase; SHR, spontaneously hypertensive rat; TGF-",transforming growth factor-"; THP-1, human monocytic cell line; TSH, thyroid-stimulating hormone; TSHR, thyroid-stimulating hormone receptor; VEGF, vascularendothelial growth factor; VSMC, vascular smooth muscle cell.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5302. Relaxin family peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 531

Pharmacology & Therapeutics 112 (2006) 529–552www.elsevier.com/locate/pharmthera

! Corresponding author. Tel.: +61 3 9905 1440; fax: +61 3 9905 8192.E-mail address: [email protected] (R.J. Summers).

0163-7258/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.pharmthera.2005.05.012

mailto:[email protected]

http://dx.doi.org/10.1016/j.pharmthera.2005.05.012

3. Relaxin family peptide receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5313.1. Discovery of relaxin family peptide receptors . . . . . . . . . . . . . . . . . . . 5313.2. The RXFP1 receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5323.3. Features of relaxin receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533

3.3.1. General features of leucine rich repeat containing receptors . . . . . . . 5333.3.2. Functional domains of the RXFP1 receptor . . . . . . . . . . . . . . . 534

3.4. Signalling pathways activated by relaxin . . . . . . . . . . . . . . . . . . . . . 5343.4.1. Cyclic adenosine monophosphate generation in response

to RXFP1 activation . . . . . . . . . . . . . . . . . . . . . . . . . . . 5343.4.2. Mitogen-activated protein kinase activation in response

to RXFP1 stimulation. . . . . . . . . . . . . . . . . . . . . . . . . . . 5353.4.3. Interaction between relaxin and nitric oxide signalling . . . . . . . . . . 5363.4.4. Relaxin and glucocorticoid signalling. . . . . . . . . . . . . . . . . . . 537

3.5. Regulation of relaxin-family peptide receptors. . . . . . . . . . . . . . . . . . . 5374. Relaxin as a hormone relevant to the cardiovascular system . . . . . . . . . . . . . . . 537

4.1. The heart as a target organ for relaxin . . . . . . . . . . . . . . . . . . . . . . . 5374.2. Production of relaxin peptides in the cardiovascular system . . . . . . . . . . . . 5384.3. Alterations of relaxin and its primary receptor, RXFP1, in heart disease . . . . . 539

5. The effects of relaxin in the cardiovascular system . . . . . . . . . . . . . . . . . . . . 5395.1. Chronotropic and inotropic actions on the heart . . . . . . . . . . . . . . . . . . 5395.2. Hemodynamic regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5395.3. Regulation of myocardial hypertrophy . . . . . . . . . . . . . . . . . . . . . . . 5415.4. Myocardial protection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5425.5. Post-infarct healing and myocardial regeneration . . . . . . . . . . . . . . . . . 5435.6. Antifibrotic actions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543

6. The therapeutic potential of relaxin in heart disease and future researchdirections and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 546

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547

1. Introduction

The peptide hormone relaxin was one of the first peptides tobe discovered following studies by Frederick Hisaw in 1926 onthe modifications of the pelvic girdle during pregnancy (Hisaw,1926). He noted that the injection of serum from pregnantguinea pigs or rabbits into virgin guinea pigs shortly after estruspromoted a noticeable relaxation of the pubic ligament. Furtherstudies by Hisaw resulted in the isolation of a crude extract thatcontained the factor responsible for the relaxation effect and thishormone was named relaxin (Fevold et al., 1930). It was notuntil the 1970s that the structure of the relaxin hormone wasfinally determined and shown to be a 2-chain peptide withfeatures in common with insulin (Fig. 1). Subsequent cloning ofrelaxin genes in the 1980s has highlighted this similarity toinsulin and the common B, C and A chain prohormonestructure.

The past 20 years has seen rapid growth in ourunderstanding of the relaxin peptide, its evolution, structureand function (see Bathgate et al., 2006b for review). It hasbeen established that relaxin is essential for numerous aspectsof pregnancy including the modifications of the pelvic girdlein many mammalian species as anticipated from the originalstudies of Hisaw. However it has also become clear that inaddition to its roles in pregnancy relaxin also affects non-pregnant animals. A potential role for relaxin in cardiovascularfunction was suggested as early as 1967 by Frederick Hisaw

when he noted in studies using castrated monkeys that theadministration of relaxin caused marked morphologicalchanges in endometrial endothelial cells consistent withhypertrophy and hyperplasia (Hisaw et al., 1967). It hassince been clearly demonstrated that relaxin can affect bloodvessel structure and function (Conrad & Novak, 2004).Furthermore, it is now firmly established that these effectstogether with actions on the kidney, heart and the braincontribute to the cardiovascular changes seen during

Fig. 1. Representation of the structure of H2 relaxin based on the crystalstructure (Eigenbrot et al., 1991). The conserved residues incorporating therelaxin receptor binding motif (Arg-X-X-X-Arg-X-X-Ile/Val) are shown as wellas the separate A and B chains.

530 C.S. Samuel et al. / Pharmacology & Therapeutics 112 (2006) 529–552

pregnancy in most species (Conrad, 2004). Moreover, there isnow evidence that relaxin is produced by the heart and actslocally through its receptor as a cardioprotective agent(Dschietzig et al., 2001b) and as a regulator of extracellularmatrix (ECM) turnover (Samuel, 2005). Indeed relaxin hasbeen used in numerous animal models to successfully treatcardiac fibrosis and has enormous potential as an agent to treatcardiac fibrosis in humans. The roles of relaxin in thecardiovascular system and its potential use as a drug to treatcardiovascular disease will be outlined in detail over thecoming pages.

2. Relaxin family peptides

It is now well established that relaxin is a member of a familyof peptide hormones that diverged from insulin early in vertebrateevolution to form the relaxin peptide family (Hsu, 2003;Wilkinson et al., 2005). This peptide family is encoded by 7genes in humans, the relaxin genes RLN1, RLN2 and RLN3 andthe insulin-like peptide genes INSL3, INSL4, INSL5 and INSL6.Although the peptides display relatively low primary amino acidsequence homology (Fig. 2), phylogenetic analysis indicates thatthey evolved from a RLN3 ancestral gene (Hsu, 2003; Wilkinsonet al., 2005). Furthermore, most of the peptides of this familyinteract with related G-protein coupled receptors (GPCR) (out-lined below). Importantly, the peptides that make up this familyhave distinct expression profiles and biological functions. Thusrelaxin-3 is expressed predominantly in the brain and has putativeactions as a neurotransmitter (Burazin et al., 2002; Bathgate et al.,2003; Tanaka et al., 2005). INSL3 is expressed in the testis andovary and is essential for testicular descent in the male (Nef &Parada, 1999; Zimmermann et al., 1999). Additionally, INSL3has roles in oocyte maturation and male germ cell survival(Kawamura et al., 2004). INSL4-6 are predominantly expressedin the placenta, gut and testis, respectively, but their physiologicalroles are currently unknown.

Unlike humans and higher primates most species have only 2relaxin genes, corresponding to RLN2 and RLN3. The productof the human RLN2 gene, human gene-2 relaxin (H2 relaxin) isthe functional ortholog of the gene product from non-primatespecies that is termed “species” relaxin (e.g. rat relaxin, mouserelaxin). The function of the product of the human RLN1 gene,human gene-1 relaxin (H1 relaxin) is unknown, and indeed anative H1 relaxin peptide has yet to be isolated. Hence

throughout this review “relaxin” will refer to H2 relaxin andnon-primate “species” relaxin.

3. Relaxin family peptide receptors

3.1. Discovery of relaxin family peptide receptors

The receptors for relaxin, relaxin-3, INSL3 and INSL5 wereidentified recently (Table 1). Relaxin and INSL3 receptors are asubgroup (type C) of the family of leucine-rich repeatcontaining G-protein coupled receptors (LGR) that include thereceptors for follicle-stimulating hormone (FSH), luteinizinghormone (LH), and thyroid-stimulating hormone (TSH). Byusing inferences from similar phenotypic expression followingmutation and inactivation of INSL3 (Nef & Parada, 1999;Zimmermann et al., 1999) and a transgenic insertional mutationin mouse chromosome 5, an orphan LGR designated either G-protein coupled receptor affecting testis descent (GREAT)(Overbeek et al., 2001) or LGR8 (Hsu et al., 2002; Kumagai etal., 2002) was identified as the INSL3 receptor. The discoverythat the orphan receptor LGR7 was the relaxin receptor waslargely attributable to the pursuit of a hunch raised by thecombination of the similarity of the structure of LGR7 to LGR8and the similarity of the structure of relaxin to INSL3 (Hsu etal., 2002). LGR7 and LGR8, which are 757 (Hsu et al., 2002)and 737 (Overbeek et al., 2001) amino acids in length,respectively, share about 60% amino acid sequence identityand contain 10 leucine-rich repeats (LRR) in their large N-terminal extracellular domain. Two orphan GPCR designatedGPCR135 and GPCR142 were recently proposed as putativereceptors for relaxin-3 (Liu et al., 2003a, 2003b). Both receptorsbelong to the type I family of GPCRs. Unlike LGR7 and LGR8,GPCR135 and GPCR142 have short N-terminal extracellulardomains, and they contain only 469 and 374 amino acidresidues, respectively. CHO-K1 cells transfected with orphanreceptor GPCR135, which is also known as somatostatin andangiotensin-like peptide receptor (SALPR; Matsumoto et al.,2000), were used to identify relaxin-3 as a ligand in porcinebrain extracts (Liu et al., 2003b). GPCR142, the remainingorphan GPCR receptor that shares the closest amino acidsequence identity to GPCR135 (43%), was also shown to be areceptor for relaxin-3 (Liu et al., 2003b). More recentexperiments have demonstrated that GPCR142 is likely thereceptor for INSL5 (Liu et al., 2005). Thus most of the receptors

Fig. 2. Alignment of A and B chain sequences of all human relaxin-like peptides. Conserved amino acid residues are boxed in black and conservative amino acidsubstitutions are boxed and shaded. Gaps have been introduced to aid the alignment where necessary. The chains are numbered according to the H2 relaxin sequence.Reproduced from Bathgate et al. (2006a).

531C.S. Samuel et al. / Pharmacology & Therapeutics 112 (2006) 529–552

and cognate ligands for the relaxin family peptides have beenidentified. No specific receptors have yet been identified forINSL4 and INSL6. It has recently been recommended by theIUPHAR-NC (Bathgate et al., 2006a) that LGR7, LGR8,GPCR135 and GPCR142 now be known as relaxin familypeptide receptors 1–4 (RXFP1–4), respectively (Table 1). Onlythe RXFP1 receptor for which relaxin is the cognate ligand iscurrently of interest in terms of effects on the cardiovascularsystem. The closely related RXFP2 receptor will be mentionedoccasionally because of its role in the discovery of RXFP1receptors.

3.2. The RXFP1 receptor

Due to their 2-chain structure, the relaxin and INSL3peptides were originally thought to belong to the insulin ligandfamily and several of the relaxin paralogs have been namedINSL3–6 based on their order of discovery. Based on thehypothesized coevolution of peptide ligands and their receptors,it was originally believed that the receptor for relaxin wasrelated to the known insulin receptors and thus likely to be atyrosine kinase.

Recent advances in genome sequencing has facilitated theidentification of novel genes based on their sequence related-ness to known genes in the hormonal signalling pathway (Hsu etal., 2000). Searches for paralogs of the known gonadotropin andthyrotropin receptors led to the identification of a group ofGPCR called LGRs. LGRs resemble glycoprotein hormonereceptors (Hsu et al., 1998, 2000) that are mosaic proteins thatcontain an extracellular domain with multiple LRR that areimportant in ligand binding, and a typical GPCR 7-transmem-brane domain. Studies of LGRs from different species suggestthat 3 LGR subtypes (A, B and C) evolved during the earlyevolution of metazoans and that each subtype of LGR shares asimilar LRR domain and a unique hinge region between theLRR and the transmembrane region (Fig. 3) (Hsu, 2003). TheType A LGRs include the follicle-stimulating hormone receptor(FSHR), the luteinizing hormone receptor (LHR) and thethyroid-stimulating hormone receptor (TSHR), important forsignalling of the heterodimeric glycoprotein hormones FSH,LH, and TSH, respectively. In mammals, the Type B LGRscomprise 3 orphan GPCRs, LGR4–6. By contrast, Type CLGRs have only 2 members, RXFP1 and RXFP2 (Table 1).Because Type A LGR and the coevolved genes encodingglycoprotein hormone subunits could be traced to bothnematodes and insects, it was concluded that the 3 LGRsubtypes evolved before the emergence of vertebrates and

nematodes (Hsu et al., 2000). Therefore, the Type C LGRsignalling pathway represents one of the earliest forms of GPCRsignalling. The Type C LGR ectodomain consists of a lowdensity lipoprotein class A (LDLa) module, followed by analternatively spliced flanking region, and LRR. Cysteine richregions form “caps” at each end of the LRR, and these “caps”have been demonstrated to be an integral part of the LRRstructure in many proteins (Kobe & Kajava, 2001). Theectodomain is connected to the 7-transmembrane spanningregions followed by the C-terminal tail (Fig. 3). Based ondifferential splicing in the ectodomain, many different isoformsof the receptors can be generated (Muda et al., 2005; Scott et al.,2005b). In mouse, rat and pig, an exon 4-deleted RXFP1 splicevariant has been discovered (Scott et al., 2005b) where removalof exon 4 causes a frame-shift resulting in a premature stopcodon and production of a truncated protein (RXFP1-truncate)that encodes primarily the LDLa module without the LRR ortransmembrane region. When cells were co-transfected withplasmids containing RXFP1 and the secreted RXFP1-truncate,relaxin induced RXFP1 signalling was significantly reducedsuggesting that this protein acts as a functional antagonist invitro and may be an endogenous regulator of RXFP1 function.

A comparison of phenotypes of mice deficient in INSL3 (Nef& Parada, 1999; Zimmermann et al., 1999) and mice lacking a550-kb region of chromosome 3 which contained the RXFP2gene (Overbeek et al., 2001) led to the hypothesis that relaxin

Fig. 3. Schematic representation showing the putative structure of the RXFP1receptor. The major structural features include 7-transmembrane spanningdomains, hinge-like region, extracellular LRR, and LDLa module. The modelwas constructed based on the structures of bovine rhodopsin (Okada &Palczewski, 2001) for the transmembrane domains, FSH receptor (Fan &Hendrickson, 2005) for the LRR and complement-like repeat CR8 from the low-density lipoprotein receptor-related protein (Huang et al., 1999) for the LDLamodule. The LDLa module structure contains a Ca2+ molecule which isnecessary for the structure (Hopkins et al., 2005).

Table 1Receptors for relaxin family peptides

OfficialIUPHARnomenclature

Previousnames

Aminoacids

Ligand Humangenesymbol

Humangene ID

RXFP1 LGR7, RX1 757 H2 relaxin LGR7 59,350RXFP2 LGR8, RX2 754 INSL3 LGR8 122,042RXFP3 GPCR135, SALPR 469 H3 relaxin RLN3R1 51,289RXFP4 GPCR142, GPR100 374 INSL5 RLN3R2 339,403


family peptides were cognate ligands for Type C LGRs (Hsu etal., 2002). Functional studies established that porcine relaxin(Hsu et al., 2002) and H2 relaxin (Sudo et al., 2003) activateboth RXFP1 and RXFP2 to increase cyclic adenosinemonophosphate (cAMP) with similar potency and efficacy.RXFP1 and RXFP2 have more than 700 residues, share about60% amino acid sequence identity and contain 10 LRR in theirextracellular domain. RXFP1 transcripts are found in repro-ductive tissues, as well as brain, kidney, heart, and lung, whereactions of relaxin have been reported. The rat and mouseorthologs of RXFP1 have recently been cloned (Scott et al.,2004), and are able to bind H2 relaxin with high affinity andwhen activated increase cAMP accumulation. Both receptorshave a higher affinity for rat relaxin than the human receptor andalso bind human gene-3 relaxin (H3 relaxin) with high affinity(Scott et al., 2005a). Examination of the closely related relaxin-3 and INSL3 has demonstrated that these peptides canselectively activate RXFP1 and RXFP2, respectively (Kumagaiet al., 2002; Sudo et al., 2003; Bathgate et al., 2006c), althoughrelaxin-3 also potently activates RXFP3/4. The ectodomains ofboth RXFP1 and RXFP2 are important for ligand binding, as inthe case of Type A LGR (Osuga et al., 1997). When a solubleligand-binding region of RXFP1 (RXFP1-BP) was adminis-tered subcutaneously to antagonize endogenous circulatingrelaxin during the last 4 days of pregnancy, delivery wasdelayed by 27 hr and nipple development was retarded (Hsu etal., 2002). It is likely that RXFP1-BP blocks the action ofrelaxin in late pregnancy by acting as a relaxin binding protein(BP) and sequestering circulating relaxin. The phenotype isconsistent with earlier work on relaxin (Zhao et al., 1999) andRXFP1 (Krajnc-Franken et al., 2004) null mice and with studiesthat show delivery of pups is prolonged in rats treated with amonoclonal antibody against relaxin (Lao-Guico & Sherwood,1988). The phenotype is less pronounced in relaxin knockout(KO) mice where some were observed to have difficulties ingiving birth (Zhao et al., 1999) perhaps reflecting partialcompensation for the lack of relaxin during development andmaturation.

Relaxin is therefore the cognate ligand for RXFP1 and,although relaxin peptides from some species will activateRXFP2 at high (supra-physiological) concentrations, ratrelaxin does not activate human, rat or mouse RXFP2,indicating that in rodents, relaxin is not an RXFP2 ligand(Scott et al., 2005a). Studies crossing INSL3 overexpressingmice, RXFP2 and RXFP1 KO mice establish that RXFP2 isthe only receptor for INSL3 and there is no interaction (at leastin the gubernaculum) between the INSL3/RXFP2 and relaxin/RXFP1 signalling systems in vivo (Bogatcheva et al., 2003;Kamat et al., 2004).

Thus, in spite of their structural similarity, relaxin and insulinfamily peptides act through independent signalling pathways:the relaxin group activates GPCRs whereas the insulin groupactivates tyrosine kinases. Phylogenetic analysis of LGR andcoevolved relaxin family peptides from different metazoanssuggests that, whereas the number of relaxin receptors remainedconstant during vertebrate evolution, the ancestral gene forrelaxin duplicated multiple times in a vertebrate branch-specific

manner (Hsu, 2003). Therefore, relaxin family peptides fromdifferent branches of vertebrates may have adapted to distinctphysiological roles via a limited number of receptor genes.

3.3. Features of relaxin receptors

3.3.1. General features ofleucine rich repeat containing receptors

There are 3 LGR subgroups that all have multiple LRR buthave differences in their ectodomain features including theunique “hinge” region linking the LRR to the transmembranedomains (Fig. 3). Type A ectodomains contain 9 LRR andinclude the receptors for FSH, LH and TSH. Type Bectodomains contain 17 LRR and are currently orphanreceptors. Type C ectodomains contain 10 LRR and an N-terminal LDLa module and are the RXFP1 (LGR7) and RXFP2(LGR8) receptors (Hsu, 2003). RXFP1 and RXFP2 receptorsare highly conserved across species, with more than 90%sequence similarity observed between rodent and primatereceptors. Both receptors have N-linked glycosylation sitesand activate adenylate cyclase to cause cAMP accumulation andstimulation of protein kinase-A (PKA) pathways (Hsu, 2003).The LRR region of these receptors is made up of individualrepeats consisting of a "-strand and an !-helix connected by aturn. The crystal structure of the ectodomain of the related FSHreceptor has been solved (Fan & Hendrickson, 2005). The LRRof the FSH receptor form an elongated tube like structure, whichis slightly curved and FSH binds in a hand clasp fashion to the"-strands which make up the inner concave surface of the LRR.There is a differential distribution of electrostatic charge acrossthe surfaces of the LRR region that is important for ligandrecognition (Vassart et al., 2004). The crystallized FSH/FSHreceptor complex demonstrates that the ligand receptor interfacecontains an exceptionally high-buried charge density. There aremany direct interactions between acidic residues in the LRR andbasic residues in FSH.

It has been proposed that LGR have an inherently noisytransmembrane domain (i.e. they display constitutive activity)that is inhibited by the ectodomain that acts as a tethered inverseagonist to actively ‘switch off’ receptor activation. Once thehormone is bound to the LGR the ectodomain switches from atethered inverse agonist to a full agonist with the LDLa domaininteracting with the transmembrane region of the receptor(Vassart et al., 2004).

It has been suggested that the interaction of glycoproteinhormone receptors with their cognate ligands does not requirethe 7-TM region since there is high affinity binding to thesoluble ectodomain (Vassart et al., 2004; Fan & Hendrickson,2005). However, mutations of the exoloops of the 7-TM regionindicate that exoloop 2 and the hinge region have an importantrole in ligand recognition, but their importance lies in bindingand positioning of the ligand for interaction with the LRR ratherthan directly causing activation of the receptor (Vassart et al.,2004). The role of the 7-TM region is also indicated by naturallyoccurring polymorphisms of the FSH receptor involving the 7-TM region that show increased sensitivity to hCG andconstitutive activity (Vassart et al., 2004). Currently there are


no known polymorphisms of RXFP1–4 receptors with similarcharacteristics.

Like other glycoprotein hormone receptors, the ectodomainsof RXFP1 and RXFP2 are also a primary site of ligand binding.Ectodomain-only LGR7 (7BP, RXFP1-BP) and RXFP2 (8BP,RXFP2-BP) CD8 membrane anchored receptors demonstratehigh affinity binding of relaxin and INSL3 (Halls et al., 2005c).The soluble form of RXFP1-BP is released by the action ofthrombin on a thrombin cleavage site located at the RXFP1-CD8 junction and will also bind relaxin and blocks its actions atthe RXFP1 receptor presumably by acting as a relaxin BP (Hsuet al., 2002). Indeed, RXFP1-BP acted in the same manner asanti-relaxin antibodies (Hsu et al., 2002) and the effects werenot observed if RXFP1-BP was boiled prior to addition to thesystem (Hsu et al., 2002). However, also like the glycoproteinhormone receptors, the 7-TM region has a role in RXFP1 andRXFP2 binding (Halls et al., 2005c; see below). The RXFP1receptors therefore have a complex extracellular domaincontaining an LRR region that forms the primary binding siteand a LDLa module at the N-terminus that is important forsignal transduction. The e2 region of the TM region of thereceptor contains a low-affinity secondary binding site.

3.3.2. Functional domains of the RXFP1 receptorThe RXFP1 receptor is a LRR-containing GPCR with a

complex ectodomain (Fig. 3). A number of regions have beenidentified that are critical for ligand binding and signaltransduction. These are the LDLa module, the LRR andexoloop 2 of the transmembrane (TM) domain (Fig. 3). Studiescarried out using the wild type receptors, RXFP1 and RXFP2chimeras and membrane anchored ectodomains show that H2relaxin activates both RXFP1 and RXFP2 whereas H3 relaxinactivates only RXFP1 but not RXFP2 (Sudo et al., 2003). ForRXFP1 the primary binding site is located in the LRR regionsince the RXFP1-BP membrane anchored ectodomain displayshigh affinity binding for [33P] H2 relaxin (Halls et al., 2005c;Yan et al., 2005). Loss of the LDLa module does not affectbinding but signalling is abolished (Bathgate & Hsueh,unpublished data). Furthermore, optimal binding and signaltransduction is only observed in receptors having an intact TMdomain. The chimeric receptor LGR7/8 (RXFP1/2) containingthe ectodomain of RXFP1 receptor and the TM domain ofRXFP2 displays decreased cAMP production in response to H3relaxin and a reduced ability of H3 relaxin to compete for [33P]H2 relaxin binding (Sudo et al., 2003). Replacement of exoloop2 in the chimera with that of the RXFP1 receptor restored bothH3 relaxin binding and cAMP production (Sudo et al., 2003).This suggests that the TM2 loop has a role to play in bindingand signal transduction. This is supported by recent studies(Halls et al., 2005c) demonstrating in the RXFP1/RXFP2receptor chimera that there is reduced affinity and efficacy ofH2 relaxin and increased affinity and efficacy of INSL3. In theRXFP2 (LGR8) receptor the evidence for the presence of 2binding sites is much stronger since [33P] H2 relaxin identifies 2binding sites with differing affinities (Halls et al., 2005c). Thecorresponding RXFP2-BP (LGR8-BP) displays only a singlehigh affinity site (Halls et al., 2005c).

Thus in the RXFP1 receptor the LRR forms the primarybinding site for the cognate ligand relaxin. For RXFP1 it hasbeen suggested that the contact motif is RXXXRXX(I/V) (Sudoet al., 2003). It has recently been demonstrated that this motifinteracts with specific residues on the inner "-sheets of the LRRof RXFP1 (Bullesbach & Schwabe, 2005). Arg B-13 interactswith Glu277 and Asp279 of human RXFP1 whereas Arg B-16interacts with Glu233 and Asp231. Hydrophobic interactionsbetween Ile B20 and various amino acids on the inner "-sheetsof the LRR likely stabilize the binding interaction. It is likelythat interaction of this motif with the LRR would then allow theligand to make contact with exoloop 2 of the TM domain thatcauses the conformation of the ectodomain to change allowingthe LDLa module to direct G-protein coupling and subsequentlyadenylate cyclase activation.

Examination of the amino-acid sequence of the RXFP1receptor reveals N-glycosylation sites in the ectodomain and thesize of RXFP1, and RXFP1-BP on Western blots is consistentwith the ectodomain being highly glycosylated (Hsu et al.,2002; Kumagai et al., 2002; Yan et al., 2005). The C-terminalregion of RXFP1 has been examined for Ser, Thr and Tyrresidues that could represent sites for protein kinase phosphor-ylation. The RXFP1 receptor has 2 conserved motifs in the C-terminal tail that fulfil these criteria, an RQRKSMDSK motifimmediately distal to helix 8, and a SQSTRLNSYS motif at theC-terminus. These are similar to predicted and/or consensussequences for protein kinase-C (PKC) isozymes, includingPKC#. In particular, the C-terminal motif has the required Arg atthe −3 position and a hydrophobic residue at +1 (Nishikawa etal., 1997).

The suggestions regarding phosphorylation of the RXFP1receptor are qualified by the fact that there are known kinasesubstrates that lack the typical consensus sequences andhypotheses about receptor phosphorylation will have to betested by experimental approaches including use of inhibitors,direct measurements of phosphorylation in the presence orabsence of dominant-negative kinase mutants, and study ofreceptor mutants lacking the putative target residues.

3.4. Signalling pathways activated by relaxin

3.4.1. Cyclic adenosine monophosphategeneration in response to RXFP1 activation

Early studies of the biochemical effects of relaxin demon-strated increases in cAMP in target tissues (Braddon, 1978;Cheah & Sherwood, 1980; Sanborn et al., 1980). Increasedintracellular cAMP was observed in mouse pubic symphysis(Braddon, 1978), rat uterus (Cheah & Sherwood, 1980; Judsonet al., 1980; Sanborn et al., 1980) and in cultures of rat (Hsu etal., 1985) and rhesus monkey myometrium (Kramer et al.,1990), human endometrium (Chen et al., 1988; Fei et al., 1990),rat anterior pituitary cell (Cronin et al., 1987) and humanmonocytic cell line (THP-1) monocytes (Parsell et al., 1996).The increases in cAMP are important components in relaxin-induced myometrial inhibition in the rat (Dodge et al., 1999)and decidualization of human endometrial stromal cells (Huanget al., 1987; Tabanelli et al., 1992). Although relaxin stimulation


is clearly linked to increased cAMP in many tissues it is notclear exactly how relaxin activates adenylate cyclase. Thereseems to be at least some G-protein component of this increasein cAMP, consistent with the relaxin receptor RXFP1 being aGPCR (Hsu et al., 2002). cAMP elevation in THP-1 and humanendometrial stromal cells can be blocked by inhibitors oftyrosine kinase or mitogen-activated protein kinase (MAPK)(Bartsch et al., 2001) and relaxin appears to activate MAPK inthese cells (Zhang et al., 2002) although the activation issporadic and can be inhibited by blocking PKA suggesting thatit is a downstream event that follows elevation of intracellularcAMP (Ivell et al., personal communication). One suggestionfrom experiments in human endothelial stromal cells is thatrelaxin stimulation leads to the inhibition of a cell-specificphosphodiesterase (PDE) to induce sustained increases incAMP (Bartsch et al., 2004). In THP-1 cells, the findings areequivocal with some studies indicating that the increase incAMP is associated with PDE inhibition (Ivell et al., 2005) andothers showing that blockade of PDE by general inhibitorisobutylmethylxanthine (IBMX) or the PDE8 inhibitor dipyr-idamole fails to alter the cAMP response to relaxin (Nguyen etal., 2003). In HEK293 cells expressing RXFP1 and a secretedalkaline phosphatase (SEAP) reporter gene containing a cAMPresponse element, relaxin caused a marked response commen-surate with an increase in intracellular cAMP (Halls et al.,2005a, 2006). In THP-1 cells or HEK293T cells expressingRXFP1 receptors cAMP responses to relaxin during the first5 min of stimulation were not sensitive to phosphoinositide-3kinase (PI3-K) inhibition whereas responses to longer periodsof stimulation were inhibited by wortmannin or LY294002 (Fig.4) (Nguyen et al., 2003; Halls et al., 2005c, 2006). In HEK293cells it was of interest that the PI3-K inhibitor sensitivecomponent of the response was abolished by pretreatment ofcells with pertussis toxin (PTX) suggesting that PI3-Kactivation in HEK293 cells expressing RXFP1 may be via "%subunits derived from Gi (Fig. 4) (Halls et al., 2005c, 2006).PTX sensitivity was not a feature of the responses in THP-1cells, suggesting that if activation of PI3-K is by "% subunits

then they may be derived from G-proteins other than Gi in THP-1 cells (Nguyen et al., 2003). There is now (Nguyen &Dessauer, 2005) evidence that PKC# is the next step in PI3-Kmediated increases in cAMP production since inhibition of PKCwith chelerythrine reduced and activation with sphingomyeli-nase increased cAMP response to relaxin. Relaxin stimulatedtranslocation of PKC# to the cell membrane in a number of celllines, an effect that was inhibited by PKC# antisenseoligonucleotide treatment that reduced PKC# mRNA andprotein levels. The antisense oligonucleotide also reduced therelaxin-induced increase in cAMP levels and the remainingresponse was insensitive to blockade by PI3-K inhibitors(Nguyen & Dessauer, 2005). The cAMP response to activationof RXFP1 receptors is clearly complex and can depend to someextent on the cell type and conditions of study (Halls et al.,2006; Fig. 5).

The positive inotropic and chronotropic effects of relaxin arealso equivocal. Rabbit sinoatrial node cells respond to relaxinand the effects are consistent with increases in intracellularcAMP and activation of cAMP dependent protein kinase (Hanet al., 1994). Relaxin also stimulates the secretion of atrialnatriuretic peptide (ANP) from isolated perfused rat hearts (Tothet al., 1996). These effects were mediated through PKC,although treatment with a cAMP-dependent protein kinaseinhibitor also blocked ANP secretion as well as the chronotropicresponse, suggesting an involvement of cAMP. Measurement ofcAMP levels in atria which are likely to reflect the inotropicresponse demonstrate only weak coupling to cAMP (Kompa etal., 2002). It is feasible that the chronotropic response of the fewpacemaker cells in the SA node is mediated by cAMP whereasthe increased force of contraction of atrial myocytes utilisesanother mechanism.

3.4.2. Mitogen-activated protein kinaseactivation in response to RXFP1 stimulation

The complexity of the relaxin response is emphasised by theaction of relaxin in other cell types where it is not associatedwith increases in cAMP but may still involve some component

Fig. 4. Time-course of cAMP accumulation in HEK293T cells stably expressing the RXFP1 receptor. On the left cAMP accumulation in response to H2 relaxinstimulation (30 nM) was measured over various times up to 40 min in the presence and absence of the PI3K inhibitors LY294002 (10 &M, 30 min pre-incubation) orwortmannin (0.1 &M, 30 min pre-incubation). cAMP levels are expressed as a percentage of the forskolin (0.1 mM) response after 30 min incubation. Symbolsrepresent means, and vertical bars S.E. mean for 5 separate experiments performed in duplicate. !p<.05 versus H2 relaxin alone at RXFP1 (unpaired t-test). On theright the effect of PTX pretreatment (100 ng/mL, 16 hr) on cAMP accumulation in response to relaxin (30 nM) (modified from Halls et al., 2005b).


of G-protein coupled signalling. In rat ventricular fibroblastsrelaxin causes only a weak transient increase in cAMP (Samuelet al., 2004a) and studies in human lower uterine segmentfibroblasts showed no increase in cAMP but do show MAPKactivation (Palejwala et al., 1998, 2001). Thus, in fibroblasts,cAMP does not appear to be the predominant signallingmechanism but may be linked to MAPK signalling which exertsthe biological response.

Relaxin also activates extracellular signal-regulated kinase(Erk)1/2 in a variety of cells known to express RXFP1 receptors(Zhang et al., 2002). In human endothelial stromal cells, THP-1cells and in primary cultures of human coronary artery andpulmonary artery smooth muscle cells addition of H2 relaxinproduced rapid (<5 min) phosphorylation of Erk1/2 with nochange in levels of total Erk and the effect was blocked byinhibition of MEK (Zhang et al., 2002) (Fig. 5). No evidencewas obtained for activation of Akt or JNK in these cells byrelaxin. The transcription factor CREB was also activated byrelaxin in endothelial stromal cells but whether this results fromthe Erk1/2 cascade or cAMP/PKA pathway is not clear atpresent (Zhang et al., 2002). Erk1/2 activation by relaxin hasalso been demonstrated in HeLa cells and human umbilical veinendothelial cells although the much longer time course of theeffect (45–90 min) suggests that this may not be the result of a

direct interaction with relaxin receptors (Dschietzig et al.,2003).

3.4.3. Interaction between relaxin and nitric oxide signallingThere is also considerable evidence that relaxin can act on

cells both acutely and chronically by increasing the expressionand or activity of nitric oxide synthase (NOS) (Nistri & Bani,2003). Two mechanisms have been suggested, activation ofNOSIII by a GPCR mechanisms and induction of NOSIImediated by cAMP (Fig. 5). Activation of NOSIII by relaxinhas been suggested to involve stimulation of RXFP1,interaction of G-protein "% subunits with PI3-K followed byactivation of Akt and subsequently NOSIII by phosphorylationat Ser1179. Increased expression of NOSII would likely involvethe increase in cAMP levels, activation of PKA, phosphoryla-tion and inactivation of I'B thus promoting increased NOSIIexpression via NF-'B (Nistri & Bani, 2003). Recent studiesalso suggest that renal vasodilation in response to relaxininvolves endothelin receptors. This mechanism involvesupregulation of matrix metalloproteinase (MMP) 2 activity byrelaxin, followed by increased processing of big ET to yieldbioactive ET1–32 leading via ETB receptors to activation ofNOSIII and increased synthesis of nitric oxide (NO) (Fig. 6)(Conrad & Novak, 2004).

Fig. 5. Potential signalling pathways utilised by the RXFP1 receptor in a variety of cell types. The RXFP1 receptor can couple to adenylate cyclase (AC) via Gs andalso couples to PTX sensitive G-proteins. G-protein "% subunits then activate PI3-K that in turn activates PKC# (Halls et al., 2006) which can translocate to the cellmembrane (Nguyen & Dessauer, 2005) to stimulate adenylate cyclase (AC V/VI) and enhance cAMP production. The effect of "% subunits can be blocked by co-expression of "-adrenergic receptor kinase c-terminus ("-ARK-ct) which acts as a "% scavenger or by the inhibitors of PI3-K, wortmannin and LY294002. In somecells PI3-K may also switch on the protein kinase B (PKB/Akt) pathway to increase activation of NOSIII. cAMP will activate PKAwhich can phosphorylate manysignalling proteins but also enhance gene transcription of NOSII. Transactivation of tyrosine kinase receptors such as the epidermal growth factor receptor (EGFR) byAG1478 can also activate PI3-K that will switch on the P42/P44 (Erk1/2) MAPK pathway. There is also evidence that relaxin can directly activate nuclear GR. Notethat it is highly likely that the relative importance of the various mechanisms indicated will vary with the cell type in which the receptor is expressed (modified fromBathgate et al., 2006a).


3.4.4. Relaxin and glucocorticoid signallingRelaxin has also been reported to interact with the

glucocorticoid receptor (GR). Treatment with relaxin reducesthe production of inflammatory cytokines by human macro-phages in response to endotoxin (Dschietzig et al., 2004) andthe effect is blocked by the GR antagonist RU486. Relaxinenters intact cells and is concentrated in the nucleus where itinduces GR activation, nuclear translocation and DNA binding(Fig. 5). The mechanism of entry in unknown but probably doesnot involve internalisation of relaxin bound to RXFP1 since thereceptor does not appear to internalise (Bathgate, unpublishedobservations). Relaxin may therefore internalise by endocytosisor it may be a cell-penetrating peptide (Langel, 2002). Onentering the cell relaxin appears to act as a GR agonist andcompetes with GR agonists for binding to the GR. Sincerelaxins modified to have no action at the RXFP1 receptor retainactivity at GR, the effects appear to be mediated independentlyof an action at RXFP1.

3.5. Regulation of relaxin-family peptide receptors

Very few studies have been conducted to examine theregulation of RXFP receptors. Most information is available forRXFP1 and has mostly been obtained in studies not specificallydesigned to examine receptor regulation. Thus, in a study ofrelaxin receptor signalling in a rat model of cardiac failure, itwas shown that RXFP1 mRNA was decreased and thiscorresponded with a reduced response to relaxin in animals infailure (Kompa et al., 2002). In male rats, treatment with 17-"-oestradiol decreases the number of [33P] H2 relaxin bindingsites in atria and cerebral cortex whereas the same treatment infemale rats had no effect in these tissues but increased thenumber of binding sites in uterine myometrium (Osheroff et al.,1992; Tan et al., 1999). More recently studies in humanendometrium have shown marked increases in [33P] H2 relaxin

binding and RXFP1 mRNA expression in the secretory phase ofthe menstrual cycle although the agent responsible for thechanges was not identified (Bond et al., 2004).

4. Relaxin as a hormonerelevant to the cardiovascular system

4.1. The heart as a target organ for relaxin

Since the discovery of high-affinity binding sites for H2relaxin in the cardiac atria of male and female rats (Osheroff etal., 1992; Osheroff & Ho, 1993; Tan et al., 1999) as early as1 day after birth, many subsequent studies have demonstratedthat the heart and vessels are target organs for relaxin. Relaxinreceptor density in the atria (Bmax, 5.9 fmol/mg protein) ishigher than that in the uterine myometrium (Bmax, 2.9 fmol/mgprotein) (Tan et al., 1999) and these receptors were primarilylocalized to atrial cardiomyocytes (Osheroff, 1995). The affinityof H2 relaxin binding to the atria (pKD 8.92) was similar to thatin the cerebral cortex (pKD 8.79) and uterus (pKD 8.79) (Tan etal., 1999) suggesting that the same receptor is involved in all 3tissues. In addition, the administration of estrogen to female ratshad no effect on relaxin receptor density in the atria, butincreased relaxin receptor numbers in the uterus (Tan et al.,1999) suggesting that any relaxin-induced effects on the heartare not mediated by estrogen in females. The binding studiesalso suggest that relaxin receptors are primarily found in theatria rather than the ventricles (Osheroff et al., 1992; Osheroff &Ho, 1993; Tan et al., 1999). However, more recent studies haveestablished that RXFP1 receptor mRNA expression occurs inthe atria and/or ventricles of rats (Hsu et al., 2002; Kompa et al.,2002; Samuel et al., 2004a), mice (Bathgate et al., 2002) andhumans (Hsu et al., 2002). More specifically, RXFP1 receptortranscripts were identified in atrial and ventricular myocytes andfibroblasts, but not in vascular smooth muscle cells (VSMC)

Fig. 6. Interaction between relaxin and endothelin. High circulating relaxin levels in pregnancy interact with the RXFP1 receptor to cause increased expression ofMMP2 that acts on big ET-1 to produce ET1–32 to act on the ETB receptor. Activation of the ETB receptor switches on guanylate cyclase to generate increasedproduction of NO to cause vasodilatation (Conrad & Novak, 2004). The mechanism involved in the relaxin-mediated increase in expression of MMP2 may involveactivation of PKC# that is known to occur following RXFP1 stimulation.


(Samuel et al., 2004a). While the physiological significance ofRXFP1 gene transcripts in the ventricles has yet to be fullyelucidated, it has been demonstrated that the high density ofrelaxin receptors in the atria are involved in the inotropic andchronotropic responses to relaxin (Kompa et al., 2002; seeSection 5.1).

4.2. Production of relaxinpeptides in the cardiovascular system

A full understanding of the role of relaxin in thecardiovascular system has been hindered by lack of evidencefor a locally synthesised ligand, particularly in the normal/unchallenged heart. Although recent studies identified relaxingene transcripts in the mammalian heart (Fig. 7), these are onlydetected by reverse transcription (RT)-polymerase chainreaction (PCR), suggesting that expression levels are extremelylow. In human atria and ventricles both H1 and H2 relaxin, butnot H3 relaxin, are constitutively expressed, and are signifi-cantly elevated in patients with congestive heart failure (CHF)(Dschietzig et al., 2001b) (Fig. 7). H1 and H2 relaxin mRNAwas also identified in mammary arteries and saphenous veins

and were thought to be produced by myocytes and interstitialcells from the hearts of CHF patients (Dschietzig et al., 2001b).Confirmation of relaxin synthesis in the human heart wasprovided by Western blot analysis of heart tissue showing an18-kDa prorelaxin band (Dschietzig et al., 2001b), whereas themature 6-kDa relaxin was undetectable. Relaxin (Bathgate etal., 2002; Du et al., 2003) and relaxin-3 (Bathgate et al., 2002)mRNA expression have also been detected in the mouse atriaand ventricles by RT-PCR. In the rat however, only relaxin-3gene transcripts have been identified in the atria and ventricles,specifically in myocytes, fibroblasts and VSMC (Kompa et al.,2002; Samuel et al., 2004a) (Fig. 7). While an earlier study alsodemonstrated immunoreactive relaxin levels in rat atrialcardiomyocytes (Taylor & Clark, 1994), it was not clear if theantibody used was able to distinguish between relaxin andrelaxin-3 or between the mature and pro-forms of relaxin.Additionally, an alternatively spliced 101 base-pair (relaxin)PCR product has been identified in blood vessels of the rat(Gunnersen et al., 1995). These findings demonstrate that theheart is a potential source of relaxin, and also a target organ forrelaxin with a high density of relaxin receptors, particularly inthe atria.

Fig. 7. Relaxin expression in the rat and human heart. RT-PCR showing rat relaxin and relaxin-3 mRNA expression in RNA from rat neonatal cardiac cells. In A,ethidium bromide-stained PCR products of rat relaxin (396 bp) and relaxin-3 (498 bp). Samples consist of a DNA standard (lane 1) and duplicate PCR products fromrat atrial myocytes (lanes 2 and 3), atrial fibroblasts (lanes 4 and 5), ventricular myocytes (lanes 6 and 7), ventricular fibroblasts (lanes 8 and 9), and human VSMC(lanes 10 and 11). cDNA from a late pregnant rat ovary and rat brain were used as positive controls (lane 12) for rat relaxin and relaxin-3, respectively, whereas waterreplaced cDNA in negative control reactions (lane 13). Glyceraldehyde-3-phosphate dehydrogenase products were used as controls for quality and equal loading of ratrelaxin, and relaxin-3 (reproduced from Samuel et al., 2004a with permission; Copyright 2004, The Endocrine Society). In B, H1 and H2 mRNA are constitutivelyexpressed and significantly elevated in right atria and left ventricles from patients with CHF. At left RT-PCR; center Southern blot of PCR products from left ventricles.Lanes 1–5, controls; 6–10, dilated cardiomyopathy (DCM); 11–13, CHF secondary to ischemic heart disease (IHD); right RT-PCR from right atria. Lanes: 1–8,controls; 9–12, dilated cardiomyopathy. Expected sizes of bands are: H1 and H2, 473 bp; splicing form of H1 and H2, 574 bp; GAPDH, 900 bp. For left ventricles andright atria, quantitation (as obtained by autoradiography of Southern blots) is indicated for the different bands as fold activity compared with the mean of controls; therare bands of the larger splicing variant are not considered (reproduced with permission from Dschietzig et al., 2001b).


4.3. Alterations of relaxin and itsprimary receptor, RXFP1, in heart disease

A number of recent studies suggest that while the expressionof relaxin is low or undetectable in the normal heart, relaxinplays a prominent role in the diseased heart. Relaxin may act asa novel compensatory hormone in humans, since the circulatingand myocardial expression of H1 and H2 relaxin is significantlyelevated in patients with CHF (Armbruster et al., 2001;Dschietzig et al., 2001b) due to ischemic or dilated cardiomy-opathy (Fig. 7). The increase in plasma relaxin concentrationsand myocardial expression of H1 and H2 relaxin in patientswith CHF showed a positive correlation with the severity ofdisease (Dschietzig et al., 2001b), while vasodilatory therapywith hemodynamic improvements caused a decline in circulat-ing relaxin. However, other studies have either failed todemonstrate elevated circulating relaxin in patients with CHF(Kupari et al., 2005) or a link between circulating relaxin levelsand prognosis (Fisher et al., 2003), questioning whether relaxincould consistently be used to predict prognosis in patients withCHF. In separate studies, there was an inverse correlationbetween tissue levels of relaxin and the powerful vasoconstric-tor, endothelin-1 (ET-1) (Dschietzig et al., 2001b, 2003),confirming the vasodilatory actions of relaxin. A potentialfinding of interest emanating from these studies was thedetection of relaxin in the circulation of male patients.

Consistent with findings in humans, we have demonstratedthat the mouse relaxin gene is significantly upregulated in amodel of pressure overload-induced myocardial hypertrophy,caused by constriction of the thoracic aorta (Samuel & Du,unpublished data). Interestingly, following the discovery of H3relaxin (and its rodent equivalent, relaxin-3) (Bathgate et al.,2002), we have been unable to detect relaxin gene transcripts inthe rat heart (Kompa et al., 2002; Samuel et al., 2004a), incontrast to earlier studies that identified low levels of relaxinmRNA in the rat myocardium (Gunnersen et al., 1995;Dschietzig et al., 2001b). This may have been caused by thedifferent primers being used to detect rat relaxin mRNA in eachstudy, having different efficiencies in amplifying the cDNAproducts. On the other hand, relaxin-3 gene expression wasconsistently identified in atrial and ventricular myocytes(Kompa et al., 2002; Samuel et al., 2004a), fibroblasts, andVSMC by RT-PCR (Samuel et al., 2004a), suggesting thatrelaxin-3 is the predominant form of relaxin in the ratcardiovascular system. In accord with studies in the humanand mouse, relaxin-3 was upregulated primarily in the atria in arat model of CHF induced by myocardial infarction (MI)(Kompa et al., 2002). In addition increased left ventricular enddiastolic pressure (5–25 mm Hg) in rats also increased relaxinmRNA levels in both the atrium and ventricle (Dschietzig et al.,2001b). Thus, the overall indications are that the relaxins aremarkedly upregulated in the diseased heart under pathologicalconditions although the relaxin concerned may vary withspecies.

In contrast to the several studies that examined alteredrelaxin expression in the normal and diseased heart, little isknown about RXFP1 expression in the human and rodent heart,

under pathological conditions. RXFP1 mRNA levels weredecreased in both the atria and left ventricles (LV) in the rat MImodel (Kompa et al., 2002). However, it is yet to be establishedwhether the high circulating or tissue levels of relaxin in heartfailure are associated with downregulation or internalisation ofRXFP1 receptors.

5. The effects of relaxin in the cardiovascular system

5.1. Chronotropic and inotropic actions on the heart

The heart is clearly a target organ for relaxin in rodents. Asdescribed above, the atria of male and female rats possess highaffinity binding sites for relaxin (Osheroff et al., 1992; Osheroff& Ho, 1993; Tan et al., 1999) and RXFP1 mRNA has beendetected in the rat (Hsu et al., 2000; Kompa et al., 2002), mouse(Krajnc-Franken et al., 2004), and human (Hsu et al., 2002)heart. Numerous in vitro and in vivo studies demonstrate thatrelaxin has potent, direct and concentration-dependent chron-otropic and inotropic effects on the rat heart. The positivechronotropic effects of relaxin have been reported in perfusedintact hearts (Thomas & Vandlen, 1993; Bani Sacchi et al.,1995; Coulson et al., 1996; Toth et al., 1996) and isolated rightatria (Kakouris et al., 1992; Ward et al., 1992; Wade et al., 1994;Tan et al., 1998; Mathieu et al., 2001) and inotropic effects inleft atria (Kakouris et al., 1992; Ward et al., 1992; Wade et al.,1994; Tan et al., 1998; Mathieu et al., 2001). The chronotropiceffects are accompanied by the secretion of ANP in isolatedperfused rat hearts (Toth et al., 1996). In rat atrial myocytesrelaxin inhibits outward potassium currents, increases actionpotential duration, and enhances Ca2+ entry (Piedras-Renteria etal., 1997a, 1997b). In rabbit sinoatrial node cells, relaxinincreased the rate of action potentials and L-type Ca2+ current(Han et al., 1994) by a PKA dependent mechanism. Howeverrelaxin has only a minor effect on cAMP levels in rat atria(Kompa et al., 2002) perhaps related to the known ability of theRXFP1 receptor to activate both Gs and Gi (Fig. 4). Theseactions of relaxin on the heart are largely confined to rodentsand the hormone has no positive inotropic effect in the atria ofsheep (Bathgate et al., 2001) or humans (Summers & Castro,unpublished observations). In accord with these findingsradiolabelled relaxin does not bind specifically to sheep orhuman atria (Tan & Summers, unpublished observations).

5.2. Hemodynamic regulation

In many, but not all species, the circulating levels of relaxinincrease during the late stages of pregnancy (Sherwood et al.,1984; Sherwood, 2004) where it is generally agreed that relaxinplays a pivotal role in the compensatory regulation ofcirculation and blood volume via actions on the kidney,vasculature and central nervous system. It has been speculatedthat there is an association between inadequate relaxin levels oractions and pregnancy-related hypertension and preeclampsia(Kristiansson & Wang, 2001; Davison et al., 2004).

The renal vasodilatory properties of relaxin have beenthoroughly studied (Danielson et al., 1999; Conrad & Novak,


2004). Whilst in vitro studies show that relaxin dilates small andlarger arteries in reproductive as well as non-reproductiveorgans including kidney, heart, liver and gut (Bani Sacchi et al.,1995; Di Bello et al., 1995; Novak et al., 2002), chronicadministration of relaxin to rodents causes vasodilatation ofsmall renal arteries resulting in a significant reduction ineffective renal vascular resistance and increased glomerularfiltration rate and effective renal plasma flow (Fig. 8)(Danielson et al., 1999; Novak et al., 2002; Danielson &Conrad, 2003). Relaxin also attenuates the vasoconstriction inresponse to ET-1, angiotensin II (Ang II) and catecholamines(Massicotte et al., 1989; Danielson et al., 1999; Skott & Carter,2002; Dschietzig et al., 2003). However, it has yet to be

determined whether relaxin indirectly or directly regulates bothsodium and water retention by the kidney during pregnancy. Inhealthy men, hemodynamic disturbances produced by volumeoverload or the tilt test significantly increased the renal secretionof relaxin suggesting that it has a role in the maintenance ofcirculatory homeostasis (Heringlake et al., 2004). Exercise perse did not alter the circulating levels of relaxin (Kruger et al.,2004).

Although earlier studies failed to show hemodynamic effectsof relaxin in pregnant or non-pregnant animals (Ahokas et al.,1989; Ward et al., 1991), more recent studies suggest thatrelaxin induces vasodilatation through activation of NOS (BaniSacchi et al., 1995; Bani et al., 1998a; Failli et al., 2002). In

Fig. 8. Changes in glomerular filtration rate (A), effective renal plasma flow (B), and plasma osmolality (C) at times up to 24 hr during infusion of recombinant H2relaxin (4 &g/hr) using osmotic minipumps. Each bar represents mean±SE (n=6) rats each for the vehicle (20 mM sodium acetate, pH 5.0) and recombinant H2 relaxintreatments. !p<.05 vs. vehicle (reproduced with permission from Danielson and Conrad, 2003).


cultured VSMC, relaxin (1 nM–1 &M) concentration-depen-dently increased expression and activity of NOSII thus causingincreases in cyclic guanosine monophosphate (cGMP), reducedintracellular Ca2+ levels, and VSMC relaxation (Bani et al.,1998a). However relaxin has little effect on NOSIII expression(Failli et al., 2002) although vasodilation of coronary arteriesoccurs following acute exposure to relaxin (1–10 nM) inperfused rat (Bani et al., 1998b) or guinea-pig hearts (Di Bello etal., 1995; Masini et al., 1997). This action is blocked by NOSinhibitors, suggesting an NO-dependent mechanism.

Several studies suggest that the action of relaxin may involveupregulation and activation of ETB receptors (Conrad et al.,1999; Danielson et al., 2000; Dschietzig et al., 2003). Invascular endothelial cells or VSMC it was suggested that relaxinupregulates expression of ETB receptors (Dschietzig et al.,2003), although others failed to see a change in ETB receptorexpression in cultured vascular endothelial cells (Fig. 6)(Conrad, 2004). However, an obligatory role for the ETB

receptors in mediating vasodilation of small renal arteries inresponse to relaxin was indicated by the absence of this effect inETB receptor deficient rats (Jeyabalan et al., 2003). Interestinglythe vasodilatory effect of relaxin was blocked by MMPinhibitors (Jeyabalan et al., 2003). It was proposed (Conrad &Novak, 2004) that in vascular endothelial cells, relaxinupregulates MMP activity that is responsible for cleavage ofbig ET-1 to ET1–32. Activation of ETB subsequently leads toenhanced NOS activity with an increase in NO generation(Jeyabalan et al., 2003; Conrad & Novak, 2004). Thus, theupregulation of MMP, an action of relaxin previously regardedas important for matrix remodeling, also plays a pivotal role inmediating the vasodilatory effect of the peptide (Fig. 6).

There is also evidence to demonstrate that chronic relaxinalters arterial mechanical properties. Global arterial compliancewas determined in conscious rats by simultaneous measurementof flow and by the diastolic decay of aortic pulsatile pressure.Ten-day treatment with relaxin significantly reduced peripheralvascular resistance and increased arterial compliance andcardiac output in vivo, while increasing the passive complianceof small renal arteries in vitro (Conrad et al., 2004). Theseeffects occurred in the absence of a significant change in meanarterial pressure.

In rat models of hypertensive disease, such as spontane-ously hypertensive rats (SHR) and hypoxia-induced pulmo-nary hypertension (Tozzi et al., 2005), relaxin treatmentsignificantly reduces vascular resistance. In SHRs, thevasodilatory effect is seen in blood vessels in vitro (Dschietziget al., 2005; Failli et al., 2005) and is associated with reducedblood pressure (St Louis & Massicotte, 1985) or increasedcardiac output and global arterial compliance in vivo (Debrahet al., 2005). Pre-treatment of SHR mesenteric arteries withrelaxin also significantly blunted the vasoconstriction inresponse to arginine vasopressin or norepinephrine (Massicotteet al., 1989). In the pulmonary hypertensive model, treatmentwith relaxin (0.05 and 0.24 mg/kg/day for 10 days) reducedright ventricular pressure and, at the higher dose, loweredcollagen content in pulmonary arterial walls (Tozzi et al.,2005).

In addition to these direct vasodilatory and vascularremodeling actions, blood volume is regulated by relaxin viaa centrally mediated mechanism to regulate water intake(Sinnayah et al., 1999; Hornsby et al., 2001; Sunn et al.,2002) as well as by regulation of renal function (Dschietzig &Stangl, 2003; Conrad, 2004). These effects are particularlyimportant for the transition of the circulation from the non-pregnant to the pregnant condition during early pregnancy andpotentially important in the maintenance of homeostasis duringlate pregnancy. In the rat, intracerebroventricular injection ofrelaxin stimulates water intake (Sinnayah et al., 1999; Hornsbyet al., 2001). This action is blocked by the angiotensin receptor(AT1) antagonist losartan, suggesting that relaxin activates thecentral Ang II/AT1 system (Sinnayah et al., 1999). This action isalso associated with upregulation of Fos in the subfornicalorgan suggesting that this area of the brain is a site of action ofrelaxin (Sunn et al., 2002). Centrally administered relaxin alsoexerts hemodynamic actions with increases in blood pressureand heart rate (Yang et al., 1995; Parry et al., 1998). This bloodpressure response to relaxin involves the central release ofoxytocin and vasopressin (Parry et al., 1998; Sunn et al., 2002).

Thus, relaxin regulates circulatory homeostasis throughseveral mechanisms: firstly, it reduces vascular tone potentiallyby enhancing NO-dependent relaxation thereby antagonizingthe effect of several vasoconstrictors; secondly, it controls thebalance of blood volume through its central actions on waterintake and actions on the kidney; thirdly, relaxin is able toremodel the vasculature and this action is particularly importantin diseases such as hypertension.

5.3. Regulation of myocardial hypertrophy

In heart disease, there is evidence for an upregulation of therelaxin system in the myocardium (Dschietzig et al., 2001a;Kompa et al., 2002; Fisher et al., 2003; Zhang et al., 2005)(authors' unpublished data). A significant correlation betweenthe levels of both tissue and circulating relaxin (Dschietzig etal., 2001b) or relaxin-3 levels (Zhang et al., 2005) and theseverity of cardiac dysfunction has been documented bothclinically and experimentally. In some patients with cardiomy-opathy and CHF, the circulating level of H2 relaxin is increased5–8 folds and shows a positive correlation with the severity ofheart failure (Dschietzig et al., 2001b) although the physiolog-ical significance of the increase remains unclear. In relaxin KOmice, there was no significant change in cardiac weight betweenaged KO and wildtype mice of the same gender (Du et al.,2003), suggesting that an absence of endogenous relaxin per sedoes not cause hypertrophy in the mouse. However, in micewith constriction of the transverse aorta for 7 weeks, there was a70% increase in the left ventricular weight, and a 3-fold increasein relaxin expression in the hypertrophied LV (Samuel & Du,unpublished data). In SHRs there is a significant elevation ofrelaxin mRNA and protein expression in the heart (Dschietzig etal., 2005). A strong negative correlation was observed in cardiactissues (from 12-month-old SHR) between relaxin protein andorgan weight normalized by control values as the hypertrophyindex (Dschietzig et al., 2005). In a recent study, treatment of 9-


to 10-month-old SHR with relaxin (0.5 mg/kg/day for 14 days)(Lekgabe et al., 2005) failed to alter LV weight, myocyte sizeand expression of hypertrophy-related genes such as ANP.However, the mild hypertrophy in SHR at this age might limitthe chance of any inhibitory effect. However, there is as yet nodirect evidence for an action of relaxin in regulating cardiachypertrophy, nor does relaxin treatment change myocardialhypertrophy.

Based on the known actions of relaxin on the heart, it has thepotential to influence hypertrophic growth. Relaxin stimulatessynthesis and release of ANP in the heart (Toth et al., 1996;Dschietzig et al., 2005). In the perfused rat heart, an increase ofmechanical load is associated with upregulation of relaxin(Dschietzig et al., 2001b). Stimulation of natriuretic peptidereceptor-A (NPR-A) through ANP activates guanylyl cyclasecausing cGMP accumulation and activation of PKG (Takimotoet al., 2005). Recent studies have shown that disruption of pro-ANP (Franco et al., 2004), pro-ANP convertase corin (Chan etal., 2005) or NPR-A (Kishimoto et al., 2001) all lead to ahypertrophic phenotype. Conversely, activation of PKGstrongly inhibits hypertrophic signalling in mice with pres-sure-overload (Takimoto et al., 2005). There is also strongevidence that relaxin inhibits the activation, proliferation andfunction of cardiac fibroblasts (see Section 5.6). Interestingly, invitro studies have provided evidence that activated fibroblastscontribute to the development of hypertrophy by secreting anumber of factors such as growth factors and cytokines that areknown to be pro-hypertrophic, including Ang II, transforminggrowth factor-" (TGF-"), interleukin (IL)-6 (Harada et al.,1997; Ancey et al., 2002; Fredj et al., 2005a, 2005b). Studies onfibroblasts also consistently show that relaxin antagonises theactions of ET-1, insulin-like growth factor-I (IGF-1) and Ang II(Samuel et al., 2004a) all of which are known to be importantmediators of hypertrophic signalling (Yamazaki et al., 1998).

Based on these considerations, the effect of relaxin onhypertrophy has been examined using cultured neonatal ratcardiac myocytes (NRCM). Relaxin (16.7 nM) had no effect onhypertrophy in response to phenylephrine (50 &M). However,NRCM hypertrophy induced by fibroblast-conditioned mediumwas significantly inhibited by relaxin (Moore and Du,unpublished observations). In this study, relaxin also suppressedthe expression of !-smooth muscle actin (!-SMA) and Aktphosphorylation in cultured fibroblasts. Thus, further studies arerequired to extend these in vitro observations into in vivomodels, including relaxin KO mice.

5.4. Myocardial protection

There is evidence that relaxin possesses cardiac protectiveactions in myocardial ischemia and anaphylaxis. In in vivomodels of ischemia/reperfusion (Bani et al., 1998b; Perna et al.,2005), it has been shown that acute administration of relaxin torats (0.3–0.4 &g/kg i.v.) or pigs (1.25–5 &g/kg i.v.) significantlyreduced myocardial injury, as determined by reduced leakage ofmyocyte-specific enzymes or proteins, accumulation of perox-idative products, intracellular Ca2+ overload, and morpholog-ical abnormalities (Bani et al., 1998b; Perna et al., 2005).

Ischemic myocardium also showed less accumulation of blood-derived inflammatory cells in the presence of relaxin (Bani etal., 1998b; Perna et al., 2005). In the rat study, there appeared tobe a reduced severity of arrhythmias and better survivalfollowing the ischemia/reperfusion procedure (Bani et al.,1998b). Relaxin treatment caused a reduction of infarct size in 2studies as well as a reduction in myocyte and endothelial cellapoptosis in pigs (Bani et al., 1998b; Perna et al., 2005).Interestingly, the anti-apoptotic action of relaxin was also seenin reproductive tissues (Zhao et al., 2001; Samuel et al., 2003a).In a model utilising isoproterenol-induced cardiac injury, it wasreported that treatment with relaxin-3 (0.2 and 2 &g/kg/day) for10 days reduced release of intra-myocyte enzymes, loweredplasma level of ET, cardiac content of malondialdehyde(MDA), regional inflammatory cell infiltration and fibrosis(Zhang et al., 2005). These effects of relaxin, seen in rodent orpig models in vivo, resulted in a better preservation of cardiacfunction (Perna et al., 2005; Zhang et al., 2005).

The mechanisms involved in the protective effects of relaxinare likely to include preservation of coronary vascular integrity,antioxidant actions, and the prevention of inflammatoryneutrophil accumulation and subsequent release of pro-inflammatory cytokines. In an earlier study in perfusedguinea-pig hearts, administration of relaxin (20 ng/mL) to theperfusate provided cardiac protection (Masini et al., 1997).Thus, direct protection and antagonism of blood-derivedfactors, such as inflammatory cells, are involved in cardiacprotection produced by relaxin. There is also evidence foractions of relaxin on platelet aggregation (Bani et al., 1995),neurotrophil activation and adhesion to vascular endothelialcells (Nistri et al., 2003; Masini et al., 2004), together withactivation of NOS and increased NO production by coronaryvessels (Bani Sacchi et al., 1995; Conrad, 2004). Relaxin alsostabilises mast cells (Masini et al., 1994; Masini et al., 2002),and reduces release of factors such as histamine, leukotrienes,prostanoids, cytokines, chemokines as well as proteases(Krishnaswamy et al., 2005). These mast cell-derived mediatorscontribute to ischemia/reperfusion injury in cardiac or skeletalmuscles (Gilles et al., 2003; Bortolotto et al., 2004; Abonia etal., 2005).

In summary, these studies provide evidence that relaxinprotects against ischemia/reperfusion injury. The mechanismsresponsible include antagonism of inflammation, cell death,oxidative stress and calcium overload. However, the molecularmechanisms that underpin these effects are largely undefined.Our recent findings in cultured cardiomyocytes show thatapoptosis due to H2O2-mediated oxidative stress was markedlyprevented by pre-treatment with nanomolar concentrations ofrelaxin. This protective action was associated with elevatedratio of Bcl2/Bax and Akt, a molecule down-stream of PI3-Kknown to possess anti-apoptotic actions (authors' unpublisheddata). Further studies are essential to investigate the role of theelevated levels of relaxin in cardiomyopathy (Dschietzig et al.,2001b), SHR (Dschietzig et al., 2005), catecholamine-mediated cardiomyopathy (Zhang et al., 2005) as well aspressure-overload induced hypertrophy (authors' unpublisheddata).


5.5. Post-infarct healing and myocardial regeneration

Current concepts underlying the cell therapy of heart diseasehave been derived using myocardial infarct models that areassociated with fibrosis in the infarcted area. Injury to thehuman heart is followed by healing which predominately occursby scar formation, whereas in other species such as zebrafish(Raya et al., 2004) or MRL mice (Leferovich et al., 2001),regeneration is predominant and fibrosis is suppressed. In MRLmice, complete healing in the ear hole wound model isassociated with higher levels of regional MMP (Gourevitch etal., 2003). There is also evidence that cell therapy reduces theamount of scar formation to some extent (Kudo et al., 2003).Several studies show a low survival rate (<10%) of implantedstem cells after introduction into the normal or infarctedmyocardium (Muller-Ehmsen et al., 2002). In addition, theprocess of scar formation is associated with the end of the time-window allowing for stem cell homing and proliferation (Lu etal., 2004). An additional issue is stem cell integration since it isgenerally agreed that scar tissue forms a barrier to the properintegration of implanted cells. Scar formation, a commonconsequence of myocardial infarction, thus may be an importantfactor in preventing reparative growth of the myocardium. Thusmodulation of the fibrotic response of host tissue in conjunctionwith cell implantation has the potential to produce significantbenefits in the field of cell therapy, and is we believe an areaworthy of further study.

The well-documented antifibrotic action of relaxin suggeststhat by inhibiting the process of scar formation it might facilitatestem cell therapy. Relaxin has been shown to decrease fibrosisin several models. In a transgenic mouse displaying cardiac-specific over-expression of "2-adrenoceptors there are patho-logical changes that resemble those observed in ischaemiccardiomyopathy (Gao et al., 2003) and isoproterenol-inducedcardiomyopathy in the rat (Zhang et al., 2005). Collagen over-expression in "2-adrenoceptor transgenic mice is reduced byrelaxin administration as is cardiac fibrosis in isoproterenol-induced cardiomyopathy in the rat (Samuel et al., 2004a; Zhanget al., 2005). Other properties of relaxin may also contribute tothe tissue regeneration effect. A recent study reported thataddition of relaxin to skeletal myoblasts and adult cardiomyo-cytes in co-culture improved cell–cell integration (Formigli etal., 2005). A pro-angiogenic action of relaxin has been observedin non-cardiac tissues such as the endometrium or certain typesof cancers, where relaxin promotes vascular endothelial growthfactor (VEGF) expression causing either increased menstrualbleeding or tumor growth (Unemori et al., 1999; Palejwala etal., 2002; Silvertown et al., 2003). In ischaemic wound sites,relaxin can stimulate vasogenesis by the upregulation of VEGFand basic fibroblast growth factor (BFGF), and facilitate woundhealing (Unemori et al., 2000). However in a study (Ryan et al.,2005) that monitored the expression of VEGF receptor 2 at askin wound site using luciferase as a reporter gene there was nodemonstrable effect of relaxin. A recent study has shown thatfollowing laceration injury of skeletal muscle, relaxin givenregionally inhibited fibrosis and promoted muscle regeneration(Negishi et al., 2005). However, it remains to be determined

whether the pro-healing actions of relaxin are dependent on thetype of injury (e.g. ischemic or non-ischemic). A furtherpossibility is that relaxin is able to enhance the expression andsignalling of IGF-1 (Huang et al., 1992; Ohleth et al., 1997) thatis well known to promote muscle regeneration (Musaro et al.,2004; Saetrum Opgaard & Wang, 2005). Future studies arewarranted to test the potential role of relaxin treatment inpromoting cardiac and skeletal muscle regeneration (Fig. 9).

5.6. Antifibrotic actions

The most consistently reported biological effect of relaxin isits ability to stimulate the breakdown of collagen, thepredominant protein in most organs of the body (Samuel,2005). The collagens are the major structural elements oftissues, and provide tensile properties and a scaffold for cellattachment and migration. After injury the heart undergoesremodelling, utilising processes such as myocyte hypertrophy,necrosis and proliferation and differentiation of ECM-produc-ing fibroblasts, leading to increased ECM (and collagen)production and ultimately, interstitial fibrosis (cardiac scarring).Fibrosis is stimulated by a number of growth factors, cytokinesand endogenous regulators, and is associated with stiffening ofthe arteries and increased ventricular chamber stiffness, leadingto altered myocardial structure and cardiac dysfunction. There isincreasing evidence that relaxin provides a rapid and potentmeans of inhibiting the onset of cardiac fibrosis and decreasingestablished myocardial scarring (Samuel et al., 2004a; Lekgabeet al., 2005). Furthermore, recent studies in the relaxin gene-KO(relaxin-KO) mouse suggest that relaxin is an importantendogenous regulator of collagen turnover in the developingand ageing heart (Du et al., 2003) and in other organs (Samuel etal., 2003b, 2004b, 2005b).

Studies of the aging of relaxin-KO mice provide strongevidence that relaxin is an important, naturally occurringantifibrotic agent. Male relaxin-KO mice develop an age-relatedprogression of cardiac fibrosis, characterised by increases intype I collagen mRNA expression (from 6 months of age),collagen content and collagen concentration (from 9 months ofage) in the ventricles (Du et al., 2003). These mice also displayatrial hypertrophy, left ventricular diastolic dysfunction andincreased ventricular chamber stiffness, although their blood

Fig. 9. Post-infarct healing of the heart is primarily by scar formation. Thisprocess occurs rapidly and inhibits potential myocardial regeneration. Thefibrotic environment inhibits stem cell homing or migration, proliferation anddifferentiation, and coupling with native cardiomyocytes. Formation of maturescar tissue therefore makes cell therapy difficult. We hypothesise that the use ofrelaxin to inhibit or delay scar formation will promote myocardial regeneration.


pressure, cardiac contractile function and response to "-adrenoceptor stimulation remained unaltered (Du et al., 2003).Interestingly, these cardiac phenotypes were not observed infemale relaxin-KO mice at ages of up to 2 years, which mayresult from (i) a male gender-restricted phenotype as reported inother gene-KO models (Du, 2004); (ii) the presence ofandrogens in males that enhance the progression of cardiacfibrosis (Gao et al., 2003); or (iii) the presence of femalehormones or sex steroids that compensate for the absence ofrelaxin in female relaxin-KO mice. Fibrosis also occurs in thekidneys of male relaxin-KO mice (Samuel et al., 2004b), whilstin other organs such as the lung (Samuel et al., 2003b) and skin(Samuel et al., 2003a), fibrosis occurs in both males and

females, with the onset and severity of disease being delayed infemales. These findings suggest that endogenous relaxinprotects the ageing heart and other organs from the developmentof fibrosis, and that its effects are differentially influenced invarious organs by gender.

While the cardiac phenotypes of RXFP1 receptor-KO micehave yet to be established, these animals display manysimilarities to the relaxin-KO mouse with respect to collagenregulation. Both relaxin-KO (Zhao et al., 1999; Zhao et al.,2000) and RXFP1 receptor-KO (Kamat et al., 2004; Krajnc-Franken et al., 2004) mice display impaired nipple andmammary gland development (lactation) and to a lesser extentprolonged pregnancies and impaired ability to deliver their

Fig. 10. Modulation of collagen synthesis, degradation and deposition by recombinant H2 relaxin. Biosynthetically labelled interstitial collagens (A) from untreatedcardiac fibroblasts (2!105/cm2) and cells treated with either recombinant H2 relaxin (100 ng/mL) alone, TGF-" (1 ng/mL) alone or TGF-" (1 ng/mL) and recombinantH2 relaxin (100 ng/mL), or with Ang II (5!10−7 M) alone or Ang II (5!10−7 M) and recombinant H2 relaxin (100 ng/mL), were measured from the media samplesafter 72 hr of culture. Shown are representative figures of triplicate samples from 3 separate experiments. MMP-2 and -9 expression and activity were determined bygelatin zymography (B) of media from untreated cultures and cells treated with either TGF-" (2 ng/mL) or TGF-" (2 ng/mL) and recombinant H2 relaxin (100 ng/mL)over 72 hr. Shown is a representative zymograph of duplicate samples from each group, from 4 sets of samples/group. Also shown are the mean±SE ‘relative ODMMP-2’ of the total MMP-2 (derived from the latent and active forms of MMP-2), as determined by densitometry scanning. Collagen content of cell layers (C) fromuntreated fibroblasts and cells treated with recombinant H2 relaxin (100 ng/mL) alone, TGF-" (2 ng/mL) alone or TGF-" (2 ng/mL) and recombinant H2 relaxin(100 ng/mL) or from untreated atrial fibroblasts and cells treated with Ang II (10−7 M) alone or Ang II (10−7 M) and recombinant H2 relaxin (100 ng/mL), after 72 hrof culture were also measured. Results are presented as the mean±SE ‘relative collagen content’ from 3 to 4 separate experiments. !p<.05 and !!p<.01 comparedwith values from untreated cells. †p<.05 compared with values from TGF-" or Ang II treated cells. Additionally, recombinant H2 relaxin (100 ng/mL)-treatment oflow-density cells (density 5/mm2; these cells are more active and rapidly differentiate into myofibroblasts) over 7 days caused an inhibition of collagen deposition.Results are presented as the mean±SE ‘relative collagen content’ from 3 separate experiments (6 assays per group from each experiment). †p<.05 compared withvalues from untreated cells (reproduced with permission from Samuel et al., 2004a; Copyright 2004, The Endocrine Society).


pups, associated with increased collagen in the nipples and inseveral female reproductive organs. Male relaxin-KO (Samuelet al., 2003a) and RXFP1 receptor-KO mice (Krajnc-Franken etal., 2004) also display azoospermia and a reduction in fertility,associated with increased collagen in several male reproductiveorgans, while both KO display an age-related progression ofpulmonary fibrosis (Samuel et al., 2003b; Kamat et al., 2004).These findings suggest that many of the antifibrotic or collagen-related actions of relaxin are mediated via the RXFP1 receptor,which will have important implications for future researchtargeting prevention and reversal of fibrosis, a hallmark of thediseased heart.

Relaxin not only causes collagen remodelling in severalreproductive organs in preparation for parturition (Sherwood,2004; Bathgate et al., 2006b), but also acts on non-reproductivecells and tissues to inhibit fibrosis at several levels (reviewed in(Bathgate et al., 2003; Samuel, 2005; Bathgate et al., 2006b).The administration of H2 relaxin alone (100 ng/mL) to neonatalrat cardiac fibroblasts had no effect on basal cell function orcollagen expression (Samuel et al., 2004a). However, H2relaxin treatment significantly inhibited TGF-", Ang II orinsulin growth factor I-stimulated cardiac fibroblast prolifera-tion, differentiation, interstitial collagen synthesis and deposi-tion (Fig. 10). This effect most likely involved MMP-2-inducedcollagen degradation since there was an increase in MMP-2expression when H2 relaxin was applied to cells that werestimulated with the pro-fibrotic factors, Ang II or TGF-" (Fig.10) (Samuel et al., 2004a). Additionally, the decrease in atrialand ventricular collagen may have been associated with arelaxin-induced decrease in the expression of the tissueinhibitors of matrix metalloproteinases (TIMP), which usuallyact to inhibit the activation of the MMP. While the effects ofrelaxin on TIMP expression from these cardiac cells is yet to beestablished, relaxin has been shown to inhibit TIMP expressionwhen administered to other primary fibroblast cultures(Unemori & Amento, 1990). These findings (on cardiacfibroblasts) are consistent with studies on stimulated (but notnormal) fibroblast cultures from other organs such as the skin

(Unemori & Amento, 1990), lung (Unemori et al., 1996), liver(Williams et al., 2001; Bennett et al., 2003) and kidney(Masterson et al., 2004) in which H2 relaxin administrationinhibited newly formed collagen synthesis and secretion, whileincreasing MMP-induced collagen degradation, causing de-creased collagen deposition.

Relaxin has also inhibited or reversed fibrosis in every invivo animal model of disease investigated to date (Lekgabe etal., 2005; Samuel, 2005; Samuel et al., 2005a; Bathgate et al.,2006b), although the effectiveness and potency varies with theorgan and disease phase studied. Relaxin was shown to decreaseleft ventricular fibrosis in 3 models of fibrotic cardiomyopathy,associated with cardiac dysfunction and heart failure; caused byrelaxin-deficiency (Samuel et al., 2004a), transgenic cardiacoverexpression of "2-adrenoceptors (Samuel et al., 2004a) orhypertension (Lekgabe et al., 2005) (Fig. 11). More recently, H3relaxin has also been shown to have antifibrotic actions, anddecreased cardiac fibrosis associated with an isoprotenerol-induced model of myocardial ischemic injury (Zhang et al.,2005). These effects are likely mediated by RXFP1 as H3relaxin increased MMP expression in RXFP1 expressing ratcardiac fibroblasts (Bathgate et al., 2006c). Consistent with itseffects in other models of fibrosis (Samuel, 2005; Samuel et al.,2005a; Bathgate et al., 2006b), relaxin rapidly, selectively andalmost completely reversed cardiac fibrosis, when given to SHRduring the early onset of disease (Lekgabe et al., 2005), but wasless effective in relaxin-KO mice or "2-adrenoceptor over-expressing mice in decreasing established scarring of the LV(Samuel et al., 2004a). An important observation in this studywas that relaxin targeted the diseased myocardium (LV) and didnot influence non-affected regions of the heart or other organs(Lekgabe et al., 2005), consistent with in vitro studies (Samuelet al., 2004a) showing that relaxin does not affect basal collagenexpression. These findings demonstrate that relaxin is a potent,rapidly acting and safe antifibrotic agent, that has importanttherapeutic potential for the treatment of cardiac disordersassociated with fibrosis. Further work is required to determinethe optimal dosage, timing and route of administration of

Fig. 11. The effects of recombinant H2 relaxin treatment on collagen accumulation in 3 models of cardiac fibrosis in vivo. Collagen content/dry weight ventriculartissue was determined in 12-month-old relaxin wild-type (WT) mice (n=8), relaxin-KO mice treated with vehicle alone (n=4) and relaxin-KO mice treated with500 &g/kg/day recombinant H2 relaxin (n=4) for 14 days (A); from 5-month-old WT mice (n=8), "2-adrenoceptor transgenic (TG) mice treated with vehicle alone(n=8) or with recombinant H2 relaxin (n=8) for 14 days (B); and from 9-month-old (normotensive) Wistar Kyoto (WKY) rats (n=9) and SHR treated with vehiclealone (n=8) or with recombinant H2 relaxin (n=8) for 14 days (C). !p<.05 compared with values from WT mice/WKY rats. #p<.05 compared with values fromrelaxin-KO mice/"2-adrenoceptor TG mice/SHR treated with vehicle alone (reproduced with permission from Samuel et al., 2004a, Copyright 2004, The EndocrineSociety (A, B); Lekgabe et al., 2005 Copyright 2005, The American Heart Association (C)).


relaxin to the diseased heart in different stages of fibrosis. It willalso be important to know the regulation and activation ofRXFP1 receptor upon ligand binding in diseased states and thesignalling mechanisms by which relaxin exerts its antifibroticactions in the cardiovascular system under pathologicalconditions before relaxin can successfully be used clinically.

6. The therapeutic potential of relaxin in heartdisease and future research directions and perspectives

The antifibrotic action of relaxin has been well documentedby studies in vitro and in vivo on non-cardiovascular organs andmore recently on the heart (see Section 5.6). This action ischaracterized by the high potency of relaxin, a rapid action andan action confined to abnormal production of collagen. Thiscomplex pattern of activity of relaxin may reflect effects onseveral stages of collagen turnover (Fig. 12). Several recentstudies have shown that administration of relaxin for a shortperiod either prevented or reversed myocardial fibrosis inseveral models of cardiac fibrosis with different aetiologies. Itwould be of considerable interest to examine if these effects ofrelaxin could be reproduced in large animal models of cardiacfibrosis and more important in preliminary clinical trials. The

outcomes from these studies would clearly facilitate thetransition of relaxin from experimental curiosity to novelantifibrotic therapy.

The cardiac protective action in ischemia/reperfusion injuryhas been documented in rodents and in pigs (summarised inTable 2). In these studies (Section 5.4), the cardiac protectiveeffect was observed in several assays showing that relaxin wascapable of preventing cell injury, inflammation, oxidative stress,apoptosis, calcium overload and ventricular dysfunction. Theseexperimental findings clearly encourage the design of clinicaltrials testing the efficacy of relaxin in patients with ischemicheart attack.

Formation of scar tissue is closely associated with healing inthe mammalian heart following myocardial infarction. In therecent years, it has been shown that under certain circumstances,the infarcted heart has a potential to regenerate, to some degree,functional cardiomyocytes (for review, see Laflamme & Murry,2005; Leri et al., 2005). However there are several majorchallenges to such tissue regeneration, including the progressiveloss of stem cells and inadequate electrical and functionalsynchronization of the newly developed native myocytes. Animportant factor that reduces the impact of tissue regeneration isfibrosis (Olson, 2004; Laflamme & Murry, 2005). Therefore,the known actions of relaxin to inhibit fibrosis and apoptosis, tofacilitate angiogenesis, to activate the PI3-K pathway (known tobe important in myocardial regeneration), and to regulateinflammation, may make a substantial contribution to myocar-dial regeneration (Fig. 9).

Although the upregulation of relaxin in heart disease isobserved in both clinical and experimental studies (see Section4.3), the implications of these changes are not clear. It also hasto be established whether relaxin levels are altered in otherdisease conditions, such as hypertension and atherosclerosis.No studies have yet been carried out that examine whetherchanges in relaxin levels are associated with changes in relaxinreceptor numbers in the diseased heart and vessels. Consideringthe established beneficial actions of relaxin in the cardiovascu-lar system, the elevated circulating and organ levels of thepeptide hormone could be significant or they might merelyreflect an overall neurohormonal responses to disease. Deter-mination of answers to these key questions will be vital for ourunderstanding of the roles of relaxin in cardiovascular disease.The increasing availability of genetically manipulated mousemodels will facilitate research in this area.

Fig. 12. A summary of the antifibrotic actions of relaxin. The extent of fibrosis isdetermined by the balance between matrix synthesis and matrix degradation. It iswell documented that relaxin stimulates matrix metalloproteinase-inducedmatrix degradation, while inhibiting expression of the tissue inhibitors of MMP(TIMP) and matrix synthesis to alter this balance and reverse the fibroticprocess. It should be noted that the inhibitory actions of relaxin only occur intissues/cells associated with an abnormal accumulation of matrix proteins/collagen, but not in organs with normal collagen content.

Table 2Cardiovascular protection afforded by relaxin in various disease models

Models Inflammation Tissue injury Calciumoverload

Mast cellactivity

Coronaryblood flow

Cardiacfibrosis

Oxidativestress

I/R in vivo (pigs) (Perna et al., 2005) ↓ ↓ IS, apoptosis ↓ ↓ ↓I/R in vivo (rat heart) (Bani et al., 1998b) ↓ ↓ IS ↓Allergic response (Masini et al., 2002) ↓ ↓Cardiac toxicity of isoproterenol (Zhang et al., 2005) ↓ ↓ enzyme marker ↓ ↓I/R in vitro (guinea-pig heart) (Masini et al., 1997) ↓ ↓ ↓ ↓ via NO ↓SHR (Lekgabe et al., 2005) ↓Transgenic cardiomyopathy (Samuel et al., 2004a) ↓

I/R: ischemia/reperfusion; IS: infarct size; SHR: spontaneously hypertensive rats; NO nitric oxide.


Currently, there is limited knowledge of the signallingmechanisms utilised by relaxin in the cardiovascular system.Although studies in in vitro cell systems have demonstratedthat RXFP1 signalling is complex and involves numerouspathways it is yet to be established if the same mechanisms areutilised by cells in the cardiovascular system. It will also beimportant to build on studies that show interactions of relaxinwith other hormones, such as ET-1, Ang II, and estrogen.Progress in this area would significantly advance ourunderstanding of the physiological and potential therapeuticroles of relaxin.

Although there are an increasing number of reports on thecardiac actions of relaxin, the vascular effects of relaxin,particularly in diseased states, is largely unexplored. It is clearfrom experimental studies that relaxin exerts a significantinfluence on vascular function via NOS, and by interacting withother peptides such as ET-1 and Ang II. Considering theinhibitory effects of relaxin on the inflammatory response aswell as on matrix remodelling, it is likely that relaxin caninfluence the vasculature under conditions of hypertension oratherosclerosis, diseases that are associated with structuralchanges in the vessel wall, endothelial dysfunction and regionalinflammation. Further studies are warranted to explore thesepossibilities.

Relaxin may also have a role in treating the increasedcardiovascular risk associated with ageing in post-menopausalwomen. A decrease in the effects of estrogens has long beenregarded as the responsible factor for the rise in cardiovascularevents that occur following menopause. However, recentclinical trials have failed to document a cardiovascularprotective action of hormone replacement therapy (Grady etal., 2002; Manson et al., 2003). One possible interpretation ofthese findings is that the decline in the levels of other hormones,including relaxin, after menopause might contribute to theincrease in cardiovascular risk. Further research is required toaddress such a possibility.

The effects summarised in this review suggest that relaxinis a cardioprotective agent that is upregulated in response tocardiovascular disease and able to induce a number of effects,to inhibit inflammation and cardiac fibrosis and promote tissuemaintenance and regeneration. Elucidation of the signallingmechanisms utilised by relaxin to induce its various effectsand extension of the studies on animal models to humandisease will be crucial to further establishing its clinicalpotential.

Acknowledgments

This work was supported in part by a National Health andMedical Research Council (NH&MRC) Block Grant to theHoward Florey Institute (983001), NH&MRC project grants toRADB and RJS (300012), an Australian Research Council(ARC) Linkage Grant (LP0211545) (RJS) and a NationalHeart Foundation Grant (G04M1524) to XJD and CSS. XJDis an NH&MRC Fellow (225108). The authors would like tothank Prof. Geoffrey Tregear for his advice and encourage-

ment and Dr. Bronwyn Evans who analysed RXFP1–4receptors for putative protein kinase phosphorylation sites.

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Relaxin' the stiffened heart and arteries: the therapeutic potential for relaxin in the treatment of cardiovascular disease

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