Emerging pathways of communication between the heart and non-cardiac organs

Eugenio Hardy-Randoand Carlos Fernandez-Patron

1 Biotechnology Laboratory,Study Center for Research and Biological Evaluations,Institute of Pharmacy and Foods,University of Havana,Havana PO Box 430,Cuba;

2 Department of Biochemistry,Cardiovascular Research Centre,Mazankowski Alberta Heart Institute,Facultyof Medicine and Dentistry,University of Alberta,Edmonton,AB T6G 2H7,Canada.

AbstractThe breakthrough discovery of cardiac natriuretic peptidesprovided the fi rstdirectdemonstration of the connection between theheart and the kidneys for the maintenance of sodiuMand volumehomeostasis in health and disease.Yet,little isstill known about how theheart and other organs cross-talk.Here,we review three physiological mechanisms of communication linking the heart to other organs through:i)cardiac natriuretic peptides,ii)the MicroRNA-208a/mediator complex subunit-13 axis and iii)the matrix metalloproteinase-2(MMP-2)/C-C motif chemokine ligand-7/cardiac secreted phospholipase A2(sPLA2)axis-a pathway which likely applies to the many cytokines,which are cleaved and regulated by MMP-2.Wealso suggest experimental strategies to answer still open questionson the latter pathway.In short,we review evidenceshow ing how the cardiac secretome in fluences themetabolic and in flammatory status of non-cardiac organs as well as the heart.

Keywords:heart,liver,metabolism,in flammation,natriuretic peptides,MicroRNA,matrix metalloproteinase

Introduction

Diseases with strong metabolic and in flammatory components(ischeMic heart disease,arthritis,neurodegeneration and cancer)are leading causes of morbidity,extremely high costs associated with health care,and mortality world wide[1].Given the incomplete understanding of these diseases,new studies to decipher organ-speci fi c cross-talk between in flammation and metabolic pathways are constantly needed.

This paper aims to:(i)introduce historical background for the groundbreaking notion that the heart exertsan endocrine function[2-5],(ii)review brie fly two other recent mechanisms-the cardiac-speci fic micro-RNA (MiR)-208a/mediator coMp lex subunit-13(MED13)axis[6]and the matrix metalloproteinase 2(MMP-2)/C-C motif chemokine ligand 7(CCL7)/cardiac secretory phospholipase A2(sPLA2)axis-by which the heart modulates lipid metabolisMin noncardiac organs(e.g.,the liver)[7-9]and(iii)suggest experimental approaches to elucidate the mechanisMthat regulates the cardiac-speci fi c origin of sPLA2 in MMP-2 de fi ciency as well as delineate the biocheMical pathway by which the dyad of MMP-2 and monocytechemotactic protein 3(MCP-3)/CCL7 modulates cholesterol homeostasis in the heart and other organs.Outputs of this latter line of research have the potential to open up new avenues for modulating cholesterol metabolism[e.g.,at the levels of MMP-2 activity or signaling mediators positioned downstreaMof C-C chemokine receptor type 2(CCR2)]in in flammatory and metabolic disorders.

The heart has an endocrine function consisting of the release of natriuretic peptides

Rows 1 to 8 in Table 1 show selected discoveries related to the ability of the heart atrial muscle cells of mammals to synthesize,store within speci fi c atrial granules,and release cardiac natriuretic peptides(cNPs).The mechanisMof action of cNPs is summarized in Box 1.These fi ndings by the research teaMof Dr.de Bold were the fi rst direct demonstration that the hearthasan endocrine function(summarized in Table1,rows 1 to 8).In Table 2,we summarize selected observationsdescribing how the cNPsysteMin fluences hypertrophic grow th,fi brosis,and cardiac remodeling/dysfunction.For a summary of the in fluences of cNPs on adipose tissue biology and metabolism,please see Table 3.

A cardiac-speci fi c MiR-208a/MED13 axis further connects the heart with other organs

Recentstudies[6,50-51]have identi fi ed cardiac-speci fi c MiR-208a as a negative regulator of the subunit 13 of mediator complex MED13(summarized in Table1,row 10).MED13 regulates the transcription of many nuclear receptor genes involved in fatty acid oxidation as well as in fluencing the activity of as-yet-unidenti fi ed secreted/circulating factors,which connect the activity of cardiac MED13 with the metabolisMand energy homeostasisprograMof non-cardiac organs,such as the liver and adipose tissue(row 10 of Table 1).

MMP-2 de fi ciency is associated with elevated secreted/circulating cardiac PLA2 activity

In 2015,a possibly new endocrine systeMwas postulated,by which theheart in fluencescardio-hepatic lipid metabolism,hepatic sensitivity to dietary cholesterol,systemic in flammatory status,severity of fever and energy expenditure[7-9].By investigating the pathophysiological consequencesof MMP-2 de fi ciency in MMP-2 null(Mmp2-/-)mice,it was found that MMP-2 governs the secretion of a highly pro-in flammatory cardiac-speci fi c phospholipase A2 activity(named‘cardiac’sPLA2).This fi nding provides a plausible and novel mechanism that could explain,at least partially,why human MMP-2 de fi ciency results in pediatric in flammatory arthritis with relentless bone loss,in flammation,cardiac developmental defects and other metabolic abnormalities such as hirsutisMand dwar fi sm[7-9].Two years after the identi fi cation of the MMP-2/cardiac sPLA2 axis[7-9],thereare key questions which warrant further investigation including:What is the molecular identity of the MMP-2-regulated sPLA2?What deterMines the cardiac origin of this sPLA2 in MMP-2 de fi ciency?We address these questions in the sections below.

Cardiac sPLA2 may belong to the faMily of classical secreted phospholipasesbut itsmolecular composition is unknown

Up to now unsuccessful,previousattempts to identify cardiac sPLA2 have used targeted time-resolved immuno fluorescence assays(TRIFA)[8]or RT-PCR with reagents targeting the 31 different PLA2s(including classical and atypical,cytosolic and secreted enzymes)[7-8]as well as conventional mass spectrometry,which is not inherently quantitative(the authors’unpublished data).Activity inhibition studies have suggested that cardiac sPLA2 may be a mixture ofindoxam-resistant(e.g.,PLA 2G1B,PLA 2G2D,PLA2G2F,PLA2G10)and indoxam-sensitive(e.g.,PLA2G2E,PLA2G5)sPLA2s or a new member of the sPLA2 family[8].

To date,identifying the isoforms responsible for cardiac sPLA2 activity hasbeen challenging calling for unbiased,highly sensitive and quantitative identi fi cation strategies such as a proteoMics approach coupled with stable isotope-labelling with aMino acids in vivo(mouse SILAC),a technique that has revolutionized the fi eld of quantitative proteomics making it feasible to quantitate protein expression in mouse organs in two states[52-53].Applying such a strategy(Box 2)has the added advantages of enabling the identi fi cation and quanti fi cation of all PLA2s deregulated(up-or downregulated)in MMP-2-de fi ciency along with any other proteoMics abnormalities.These resultant proteomic signature of MMP-2 de fi ciency could serve as biomarker of disease activity or as new therapeutic target in patients.

MMP-2,CCL7 and organ-hoMing immune cells govern cardiac sPLA2 release in an organ-speci fi c fashion

Recent studies indicate that CCL7(a small pro-Box 1 On the biology of cardiac natriuretic peptides

andchloride(≥30-fold),theurinevolume(～10-fold),the acommonprecursor,CardionatrinIV;involvingbothANPandBNPtocontrolphysiologicalfunctionssuchas andventriclethat Discoveryofcardiacnatriureticpeptidesandtheheart-speci fi cmiR-208a/MED13axis Author’smainresultsandconclusions Theatrialextract(i)decreasesbloodpressureandslightlyincreaseshematocrits;(ii)rapidlyincreasesthe concentrationandurinaryexcretionofsodium potassiumexcretion(two-fold);(iii)containsapotentnatriureticandchloriureticfactor,whichstrongly inhibitstherenaltubularNaCIreabsorption.Natriuresisanddiuresisisinducedbyatrialextractsfromallmammalianspecies,frogatrialandventricular extracts,henventricleextracts(onlydiuresis),andnotbyhenatrialextractsorrattissueextractsotherthan theatrialextract;(ii)Natriureticactivityisrestrictedtoheart.CardionatrinI,whichalsohaseffectonvascularsmoothmuscletone,hasamolecularmassof5.1kDaby urea-SDS-PAGEand49aminoacidresiduesoneofwhichiscysteine.(i)CardionatrinIV,consistingof126aminoacids,hasamolecularmassof19kDabyurea-SDS-PAGE,and beginsimmediatelyafterthesignalpeptidesequenceofprocardionatrinatresidue25.Itdoesnotcontain residues151and152,whicharearginines;(ii)CardionatrinIIIbeginsatresidue73andCardionatrinIbegins atresidue123;(iii)CardionatrinsI,III,manycleavagefragmentsthereofandnumerousversionsofthe carboxylterminalportionofCardionatrinIareproductsderivedfrom(iv)CardionatrinsI-IVpeptidesarederivedfrompreprocardionatrin,acommonprecursorof152aminoacids(intherat);(v)ThebiologicallyactivesequencesoftheatrialnatriureticfactoriscontainedintheCOOHterminalportionofthemolecule.BNPcontains26aminoacidresidues,twoCysresidues,sevenaminoacidsubstitutionsandoneadditionof(Arg)comparedtoα-ANP;(ii)BNPpossessesdiuretic-natriuretic(e.g.,increaseinurineoutput,Na+,K+,Cl-excretion)andhypotensive(decreaseinmeanbloodpressure)responsessimilartothatofANP;(iii)Theremaybeadualmechanism waterintakeandsaltappetite.BNPisspeci fi callylocalizedinonlysomeofthesecretorygranulesofthehumanatrium containANP,asshownwithdifferentpatients(withaorticregurgitation,mitralregurgitationorautoptic);(ii)TheatriumisthemajorproductionsiteofBNP;(iii)Together,ANPandBNPallowsthehumanheartto regulatebloodpressureandbody fl uid.(i)PosttranslationalprocessingofproBNPisrequiredforsecretionandbioactivity-thisprocessisimpaired inpatientswithheartfailureleadingtobiologicallyinactiveBNP;(ii)proBNP-derivedfragments(e.g.,the intactandglycosylatedformsofprecursorproBNP,NH2-terminal-truncatedBNPform3-32)circulatein humanplasmainadditiontobioactiveBNP1-32;(iii)Inplasmaofpatientswithheartfailure,asigni fi cant portionofimmunoreactiveB-typerelatedpeptidesiscomprisedofintactorglycosylatedformsofproBNPthissuggeststhatplasmaproteasescleavethecirculatingproBNPtoproducebiologicallyactiveBNP;(iv)In experimentalmodelsandinpatientswithchronicheartfailure,aresistancetoANPandBNPisobserved;possiblemechanismsofresistancetobiologicaleffectsofANPandBNPmayoperateat:a)thepre-receptor level(e.g.,existenceofinactivenatriureticpeptidesinplasma,increaseininactivationanddegradationof activenatriureticpeptides,decreasedrenal fi ltration),b)receptorlevel(e.g.,downregulationofNPR-Aand NPR-Bintargettissues,alteredANP/BNPreceptorbindingordesensitization,orc)post-receptorlevel(e.g.,alteredintracellularsignaling).(i)BothHUVECandH9c2musclecellsexpressCNP(150and200bp);whichcanbecon fi rmedinneonatal ratprimarycardiomyocytes;(ii)CNPcanbeimmunodetectedinbothH9c2cells(byradioimmunologic assay)andcardiomyocytesofpighearts;(iii)CNPisconstitutivelyexpressedincardiomyocytes.ANP;Whataretheacuterenaleffectsoftheextractofratatrialmyocardium?Moleculesinvestigated;Researchquestion;Authors(deBoldAJ etal.,1981)[2]ANP;Canextractsfromothersourcesinduceanatriureticanddiureticresponse?(deBoldAJandSalernoTA,1983)[3]ANP;Whatarethemoleculesresponsiblefortheseactivities?(deBoldAJandFlynnTG,1983)[4]ANP;WhatisthecommonprecursorofCardionatrinIandotheratrialpeptides?etal.,1985)[5]Table1(FlynnTG BNP;Identi fi cationinporcinebrainofanovelnatriureticpeptide(SudohT etal.,1988)[10]BNP;WhatistheintracellularlocalizationofBNPinhumancardiacmyocytes?(NakamuraS etal.,1991)[11]ANPandBNP;Natriureticpeptidescirculateinblood(Clerico etal.,2011andcitationstherein)[12]CNP;ArecardiomyocytesabletoproduceCNP?(DelRyS etal.,2011)[13]

cascade),d)from releaseofBNP(c)regulateenergy(i)Theproduction/releaseofcNPsisstimulatedby:a)AngII,ET1,α-adrenergicagonists,cytokinessuchas IL-1,IL-6andTNF-α,andlipopolysaccharide(allofwhichsignalthroughoutNF-kBactivatedbyMAPK),in fl ux),f)thyroidhormones(throughthyroidhormoneregulatoryelement),g)corticosteroids(through triglycerideandcholesterollevels);(ii)miR-208aisa b)argininevasopressin(throughCa2+in fl uxandPKC),c)GFs(signalingthroughMAPK mice;(iii)MED13 dioxideproduction)in prostaglandins(throughPLC,IP3,PKC,andMLCK),e)chromograninB(throughNF-kBandIP3/Ca2+ventricularcardiomyocytesisstimulatedbyin fl ammation,ventricularhypertrophy,and fi brosis;(iii)Evenin isolatedandculturedventricularcells,myocardialischemiacaninducethesynthesis/secretionofBNPandits h)estrogens;(ii)Theproductionand relatedpeptides;(iv)BothANPandBNPtranscriptionmaybeactivatedbythehypoxia-induciblefactor-1a Author’smainresultsandconclusions improved(i)Pharmacologicinhibitionofthecardiac-speci fi cmiR-208aconfersresistancetodiet-inducedobesity(e.g.,responsiveelement),and smallervisceralWATandsubscapularBAT,normalglucoseresponse,lowerfastinginsulinlevels)with(whichisinducedunderlowoxygenconditions).consumption,carbon negativetranscriptionalregulatorofMED13intheheart.AmongthefunctionsofMED13are:(a)toinhibit bene fi cialmetaboliceffects(e.g.,reducedserum expressionofmetabolicgenesregulatedbyNRs(e.g.,Gpd2,Thrsp,Cidea,Elovl6,Eno1,PPARγTkt),(b)to controlwhole-bodymetabolichomeostasis(e.g.,αMHC-Med13TGmice,withincreasedcardiacexpression ofMED13,showenhancedmetabolicrate,diminishedserumtriglycerideandcholesterollevels,resistanceto diet-inducedobesityincludinglessfatmassversusWTlittermates,reducedvisceralWATandsubscapular BATmassaswellaslessadipocytesizeandlesslipidaccumulation,improvedglucose(tolerance)response,whole-body insulinsensitivity,and levels,and oxygen dodecylsulfate-polyacrylamidegelelectrophoresis;BNP:brainnatriureticpeptide;Table1Discoveryofcardiacnatriureticpeptidesandtheheart-speci fi cmiR-208a/MED13axis(continued)glucocorticoid loweredplasmalipid expenditure(e.g.,increased de fi ciencyintheheartincreasessusceptibilitytometabolicsyndromeanddiet-inducedobesityinmice,as shownwithMed13cardiacknockoutmiceversusMed13fl/fl littermatesonHFdiet.(iv)Circulatingfactors mayrelayMED13activityfromthehearttootherorgansbutthesefactorsremainelusive.Moleculesinvestigated;Researchquestion;Authors ANPandBNP;Biologicalfactorsandpathophysiologicalmechanismsthatstimulatetheproduction/releaseofnatriureticpeptides(Clerico etal.,2011andcitationstherein)[12]Cardiacspeci fi cmiR-208aandMED13;HowdoescardiacMED13in fl uencewholebody metabolism?(GrueterCE etal.,2012)[6]Abbreviations:miR-208a:microRNA-208a;MED13:mediatorcomplexsubunit13;ANP:atrialnatriureticpeptide;SDS-PAGE:sodium proBNP:ventricularcirculatinginactiveprecursorofBNP;CNP:C-typenatriureticpeptide;HUVEC:humanumbilicalveinendothelialcell;NRs:nuclearreceptors;TG:transgenic;Gpd2:glycerol-3-phosphatedehydrogenase2;Thrsp:thyroidhormoneresponsive;Cidea:celldeath-inducingDFFA-likeeffectorA;Elovl6:ELOVLfattyacidelongase6;Eno1:Enolase1;PPARγ:peroxisomeproliferatoractivatedreceptorgamma;Tkt:Transketolase;WT:wild-type;WAT:whiteadiposetissue;BAT:brownadiposetissue;HF:highfat;AngII:angiotensinII;ET1:endothelin-1;NF-kB:nuclearfactorkappa-light-chain-enhancerofactivatedBcells;MAPK:p38mitogen-activated proteinkinase;PKC:proteinkinaseC;GFs:growthfactors;MLCK:myosinlightchainkinase;PLC:phospholipaseC.

As illustrated with ANP in the scheme,ANP and BNP are synthesized as preprohormones,stored as prohormones in secretory granules,processed into mature forms,and continuously secreted froMthe heart[14];CNP is also constitutively synthesized within the heart[13].Picomolar concentrationsare found in theplasmaof healthy subjects:ANP(3.2-19.5 pmol/l),BNP(1.4-14.5 pmol/L),CNP(1-6 pmol/L)[15-18].The main release mechanisMis exocytosis of vesicles budding froMimmature atrial granules[19-20].Release is stimulated by mechanical stretch of atrial muscle,change in hemodynamic load,sympathetic stimulationand avariety of agonists and pathophysiological mechanisms(Table 1,row 9).Natriuretic peptide receptor(NPR)-A,NPR-B and NPR-C mediate effects of cardiac natriuretic peptides.Binding to NPR-A and NPR-B,which are guanylyl cyclase-coupled receptors,catalyzes the conversion of guanosine-5'-triphosphate(GTP)to cyclic guanosine monophosphate(cGMP)[21].Elevated levelsof cGMPelicit variousbiological actions through different effectors(e.g.,cGMP-gated ion channels,cGMP-dependentprotein kinases,cGMP-regulated cyclic nucleotide phosphodiesterases)[22].Cardiac natriuretic peptides impact natriuresis,diuresis,improve glomerular fi ltration rate,suppress the reninangiotensin-aldosterone system,inhibit plasma renin activity,induce systemic vasodilation,and arterial hypotension.Target systems include heart,arteries,kidney,brain,liver,gut[23-39]as well as adipose tissue(thusalso enhancing lipolysis),skeletal muscle(where they increase oxidative capacity and mitochondrial biogenesis),and pancreas(thus improving insulin secretion).Thus,cardiac natriuretic peptides link the functions of the heart and other non-cardiac organs[40-43].LBD:ligand binding domain;KHD:kinasehomology domain;HR:hinge region;GCD:guanylyl cyclase domain;PKG:protein kinase G;PDE:phosphodiesterase;CNG:cyclic nucleotidegated ion channels;cAMP:cyclic AMP;IP3:inositol trisphosphate;ERK:extracellular-signal-regulated kinase;IL-6:interleukin 6.

Table 2 Mechanisms linking the cardiac natriuretic systeMand heart issues and involving MMPs/cytokines

Effectsofthecardiacnatriureticpeptidesysteminthecardio-adiposeaxis theneteffectofnatriureticpeptidesonadiposeandmuscletissues;(xi)TheinhibitoryeffectinducedbyANP-inducedlipolysisiscounteractedby isup-regulated(mRNAandprotein);(xvi)Natriureticpeptidesprotecthumanskeletalmuscleagainstpalmitate-inducedlipotoxicityandinsulin Selectedobservations(i)NPR-Cisthesecondlargestexpressedreceptorinadiposetissue;(ii)NPR-Cexpressioninadiposetissueisstronglyandselectivelydownregulatedbyfasting;(iii)Higherclearance and/ordiminishedactivityofcNPsinthesubcutaneousadiposetissueofobesehypertensivepatients;(iv)DecreaseeffectivenessofANPinobesepremenopausalwomen;(v)Increased effects(cGMP,natriuresis,diuresis,bloodpressure)ofANPonobesehypertensivepatientsafteravery-low-caloriediet;(vi)Inobesehypertensivesubjects,avariantofthepromoterof the NPR-C geneislinkedto:a)higherbloodpressure,b)lowerANPlevels,c)augmentedabdominalcircumference,andd)theriskofdevelopingabdominalobesityinmale;(vii)ANP inhibitstheproliferationofhumanvisceraldifferentiatedpre-adipocytesandmatureadipocytes;(viii)Natriureticpeptidesarestronglipolytichormones,apropertythatisenhancedby dietandweightloss;(ix)Natriureticpeptidesregulatethesupplyofnon-esteri fi edfattyacidstotheworkingmuscles(bothcardiacandskeletal);(x)Animprovedmetabolisminthe ANP-stimulatedsecretionofadiponectin;(xii)ThesecretionofcytokinessuchasIL-6andTNF-αandseveralchemokinesaswellasadipokines(leptin,retinol-bindingprotein-4)heartandmuscleswouldresultfrom involvedinin fl ammationandinsulinresistanceisinhibitedbyANP;(xiii)Thebiologicalactivity(localandsystemic)ofnatriureticpeptidesisincreasedbydownregulationofNPR-C duetofastingandcalorierestriction;(xiv)Natriureticpeptidescouldbehaveasanti-obesitypeptidesinthepresenceofuncouplingactivityinvolvingnon-esteri fi edfattyacidsand mitochondrialburningforATPsynthesis;(xv)AngIImayhamperthepositiveeffectsofnatriureticpeptides;(xvi)Inobesesubjectswithandwithouthypertensionorheartfailure,circulatinglevelsofnatriureticpeptidesandNH2-terminal-proANPandNH2-terminal-proBNParereduced;(xvii)ReducedNH2terminal-proBNPlevelscorrelatewithhepatic steatosisandhigherhematocritvaluesinelderlypatients.(i)ThereisaninversecorrelationbetweenBMI(e.g.,≥30kg/m2)andplasmaBNPandNH2-terminal-proBNPvalues,bothinhealthysubjectsandinpatientswithheartfailure;(ii)The increasedexpressionofNPR-CintheadiposetissuemayleadtotheperipheralclearanceofBNP;nevertheless,thisincreaseinNPR-Cshouldhavenoin fl uenceinmodulatingtheNH2-terminal-proBNPlevelsinobesesubjects.(iii)SomealterationsofperipheraldegradationofANPandBNPinindividualshavingthecorinI555(P568)allele;(iv)Theproductionand secretionofBNPmayberegulatedbythegonadalfunctionincludingtheestrogens/androgenscirculatingratio;(v)Theactivityofcardiacendocrinefunctionisdepressedbythe presenceofoneormorecirculatingortissuebioactivesubstances(e.g.,leptin);(vi)Anef fi cacious(standard)pharmacologicaltreatmentofobeseindividualsorpatientswith hypertensionand/ortype-2diabetesmellitustendstodecreasesigni fi cantlytheproduction/secretionofBNPandNT-proBNP;(vii)TheinversecorrelationbetweenBMIandBNP valuesinpatientswithheartfailure(e.g.,withhemodynamicimpairment,activationofcounteractingneurohormone,increasedcirculatinglevelsofcytokines)maybepartially explainedbytheeffectofthemalnutrition-in fl ammationcomplexsyndrome.(i)Natriureticpeptidespromotesfatmobilizationandutilizationaswellasregulateenergymetabolismandexpenditure;(ii)Inobesity,metabolicsyndromeandtype2diabetesthereisa stateofnatriureticpeptidesystemde fi ciency(natriuretichandicap);(iii)Contributingfactorstothenatriuretichandicaparetheelevatedlevelsofglucose,insulin,fattyacids,proin fl ammatorycytokines;(iv)Thenatriuretichandicapoccursdespiteahigherleft-ventricularmassandend-diastolicpressureinpeoplewithobesitycomparedtoleanindividuals;(v)HumanandmouseadiposetissueexpressNPR-C;highfatfeedingup-regulatesNPR-CandreducesNPRAmRNA-ascon fi rmedinstudieswithmice;(vi)Obesediabeticdb/dbmice show:a)whitefat,brownfatandskeletalmusclescontainingstronglyup-regulatedNPR-Cproteinlevels,b)down-regulatedNPRAproteinlevels,andc)astrongdecreaseofplasma BNPlevels;(vii)Micewithdiet-inducedobesityshowreducedNPRAexpression(mRNAandprotein);(viii)Insulinup-regulatesNPR-Cexpressioninadipocytes;(ix)ANPinfusionin healthyvolunteersincreasesadiponectinlevels;(x)Natriureticpeptidesinducetheexpression/secretionofadiponectininculturedhumanadipocytes;(xi)InhealthyJapanesemen,serumNH2terminal-proBNPisinverselyassociatedwithmetabolicriskandpositivelywithadiponectin;(xii)Systemicinsulinsensitivityisenhancedbyadiponectinandreducedby IL-6andTNF-αsecretionfromadiposetissue;(xiii)Humansvolunteersshowabloodglucose-loweringeffectofinfusedBNPtoanoralglucosechallenge;(xiv)Inhumans,whole-body insulinsensitivitycorrelates(positively)withNPR-Aexpressioninadiposetissueandskeletalmuscle;(xv)Inadipocytesofobeseindividualswithandwithouttype2diabetes,NPR-A isdown-regulatedandNPR-C robustlinkbetweennatriureticpeptidesignalingandtype2diabetesisdemonstratedbystudieswithtransgenicmiceofthenatriureticpeptidesystem.resistance;(xvii)A(i)Adipokines(e.g.,leptin)candifferentlyregulatetheproduction/secretionofANPandBNPbycardiomyocytesthroughoutmultiplemetabolicpathways;thesepathwaysareactivated orinhibiteddependingondifferentpathophysiologicalconditions;(ii)Acrosstalkexitsbetweenthecardiacendocrinefunctionandadiposetissue(e.g.,ANPandBNPregulatefat tissuefunctionandgrowthaswellasexertingpotentlipolyticeffectsinhumanfatcells,ANPincreasesproductionofadiponectin);(iii)ANPgeneexpressionisincreasedinadosedependentmannerbyfemalesexsteroids;(iv)EstradiolandprogesteronearerequiredfornormalANPgeneexpressionbyratcardiomyocytes;(v)Inpostmenopausalwomen,a stimulatoryactionontheproduction/secretionofANPandBNPisinducedbyhormonereplacementtherapywithfemalesteroidhormones;(vi)Thereisdebateontheeffectsof androgensontheproduction/secretionofANPandBNP(e.g.,inatrialculturedmyocytesofnewbornrats,thesynthesisandsecretionofbothventricularandatrialANPisstimulatedby testosterone;whereasincastratedmalerats,theconcentrationofANPinplasmaandANPatrialstoresisincreased;testosteronereplacementreducesANPlevelsinplasma,butnotin ANPatrialstores;blockingtheandrogenreceptorand,toalesserextent,suppressingandrogeninmenwithprostatecancer,increasesNH2-terminal-proBNPlevelsinplasma;other studies[e.g.,theDallasHeartStudy]inhumanssuggestthatestrogensmaystimulatewhereasandrogensinhibittheproduction/secretionofANPandBNP).in natriureticpeptidesystem Question Table3 Theroleofthecardiac adiposetissuebiologyand(Sarzani etal.,metabolism 2008andcitationstherein)[47]Pathophysiologicallinks betweenobesity(visceralfat distribution)andcardiac endocrinefunction(low plasmaBNPlevels)(Clerico etal.,2012andcitations therein)[48]Effectsofnatriureticpeptide inobesity,metabolic system syndromeandtype2 diabetes(Moro,2016and citationstherein)[49]Thelinkbetweenendocrine function,adiposetissue,and sexsteroidhormones(Clerico etal.,2011andcitations therein)[12]Abbreviations:ATP:adenosine5’-triphosphate;BMI:bodymassindex.

Box 2 On the molecular constituents of cardiac sPLA2

Since the fi rst identi fi cation ofits activity[7],the molecular identity of the MMP-2-regulated cardiac speci fi c secreted PLA2 has remained elusive[7-8].However,application of an unbiased quantitative proteomicsapproach could speed up the identi fi cation and enable quantitation of cardiac sPLA2 in biological samples.Proposed pillars of a quantitative proteoMics approach to identify cardiac sPLA2 froMMmp2-/-mice include:(i)Use stable isotope labeling by aMino acids(SILAC)[52-55]labeling integrated into the isolation strategy.(ii)BLAST-screen the identi fi ed cardiac sPLA2 against all known mouse and human PLA2s.(iii)Pursue a targeted"expression cloning strategy"where the cDNA of the identi fi ed enzyme is fi rst expressed in,e.g.,HEK-293,cellsand next CCL7-induction of PLA2 activity is measured-the hypothesized pathway by which CCL7 elicits sPLA2 release[8]is depicted in Box 3.

Box 3 On the mechanisMthat regulates the cardiac-speci fi c origin of sPLA2

Postulated heart/liver axis.The red arrows trace the MMP-2-regulated CCL7-mediated pathway leading to the release of pro-in flammatory cytokines as well as secretion of cardiac sPLA2 and downstreaMlipid mediators(Lyso-phosphatidylcholine[PC],arachidonic acid[C20:4],prostaglandin E2[PGE2]).Released into circulation,these mediators reach other organs where they in fluence the in flammatory status and lipid metabolism.Why sPLA2 issecreted froMthe heart and not the liver in MMP-2 de fi ciency?We postulate that the CD45+immune cells which reside in the liver of Mmp2-/-mice express the CC-chemokine receptors(CCR)to which CCL7 binds[56-57].These speci fi c immune cells scavenge and consequently reduce the levels of free CCL7 below the threshold necessary for induction of sPLA2 by hepatocytes.This would not occur in the heart of Mmp2-/-Mice because of lack of signi fi cant immune cell homing[8](as depicted in the enclosed Figure to the right,bottom).Published and unpublished observations by the authors support this notion including that:(i)CCL7 induces the transcription of classical sPLA2 isoenzymesaswell assPLA2 activity in Hepa-1c1c7 cells.(ii)CCR-expressing CD45+immune cells are found homing to the liver(but not theheart)of Mmp2-/-Mice,compared to Mmp2+/+Mice[8].in flammatory cytokine which is normally cleaved and inactivated by MMP-2)serves as stimulus for cardiacspeci fi c release of sPLA2 activity[8].This notion is supported by(a)ex vivo assays data[7],show ing that CCL7 stimulates sPLA2 release froMcardiac,but not hepatic tissue and(b)the normalization of the cardiohepatic lipid metabolic phenotype of MMP-2-de fi cient mice injected with neutralizing CCL7 antibodies,but not with isotype-matched non-immune IgG[8].However,since CCL7 receptors are expressed on immune cells,cardiomyocytes and hepatocytes[56-57],it is paradoxical that the liver of Mmp2-/-Mice does not exhibit elevated sPLA2 activity,whereas the heart of Mmp2-/-mice does,compared to w ild-type mice.In Box 3 we propose a mechanisMthat may clarify what makes cardiac sPLA2"cardiac"in origin.

Box 4 On the MMP-2 regulated CCL7-mediated modulation of lipid homeostasis

MMP-2 cleavage of CCL7 could in fluence lipid homeostasis through at least two pathways:Postulated pathway 1:Modulation of prostaglandin production by the CCL7/cardiac sPLA2 axis[7-8].(i)Cardiac sPLA2 activity,whose release isstimulated by CCL7,reachesnon-cardiac organs,such as the liver,through the circulation;(ii)Cardiac sPLA2 hydrolysesmembranephospholipidsin the liver releasing the fatty acid esteri fi ed at carbon-2(typically,arachidonic acid,C20:4)and leaving behind a lysophospholipid(e.g.,lyso-PC);(iii)Arachidonic acid is next converted into prostaglandin E2 by cyclooxygenases(as depicted in Box 3).

Postulated pathway 2:Modulation of cholesterol metabolisMby the CCL7/tumor necrosis factor(TNF)-α/adenosine monophosphate-activated protein kinase(AMPK)axis.As illustrated,CCL7 can in fluence cholesterol homeostasis in targetorgans,including the heart and liver.Several key signaling events are:(i)CCL7 binds and activates CCR2[56].(ii)A classical G-proteincoupled receptor-like calcium-dependent signaling cascade is elicited and pro-in flammatory genes,including TNF-α,areactivated.(iii)Transcriptional induction of protein phosphatase 2C(PP2C)expression is stimulated;(iv)PPC2 reduces phosphorylation of active(phosphorylated)AMPK[58];however,reduced AMPK phosphorylation can also be provoked by an interaction between AMPK and phosphoinositide3-kinaseenhancer A-aubiquitously expressed GTPaseand proto-oncogene-as recently reported[59].(v)Metabolic enzymesincluding 3-hydroxy-3-methylglutaryl-coenzyme A reductase(HMGCR)are phosphorylated e.g.,HMGCR in Ser 871)and inactivated.(vi)Mevalonate-the precursor ofisoprenoidsand sterols issynthesized.Thispathway can be probed at various levelssuch asby antagonizing theactionsof MCP-3/CCL7 and TNF-αwith neutralizing monoclonal antibodies(mAb),by RNA interference to knockdown PP2C expression(PP2C siRNA)and by activating AMPK(A-769662).

In fluence of the heart-centric MMP-2/CCL7/sPLA2 axis on lipid metabolism

A still-open question is whether MMP-2-mediated proteolysis of cytokines,such as CCL7,perturbs lipid metabolisMvia CCL7-receptor signaling pathways?To answer this question,Box 4 describes two pathways by which theheart in fluenceshepatic lipid metabolisMand in flammation.

Future studies w ill provide precision to the fi rst pathway described in Box 4,including the molecular identity(aMino acid sequence)of the enzyme isoforms responsible for cardiac sPLA2 activity(Box 2)and deciphering the mechanisMthat regulates the cardiac-speci fi c origin of sPLA2 in MMP-2 de fi ciency(Box 3).

Conclusions

Some three decades ago,Dr.de Bold and colleagues identi fi ed endogenous peptide-hormones(ANP)which they found to stimulatea rapid and massivediuresisand natriuresis when injected in rats.Since this pioneering discovery,which demonstrated directly the endocrine function of the heart,there have been several new discoveries.These include but arenot limited to:(i)the identi fi cation of other endogenous peptides(e.g.,BNP,CNP)with natriuretic and vasodilator activity,(ii)the roleof CNPsashormones thatcan target variousorgans(e.g.,the liver,brain,pancreas and intestine-not just the kidney)to in fluencemetabolism,(iii)the roleof the cardiac-speci fi c MiR-208a/MED13 axis to control whole body metabolism,(iv)a MMP-2/CCL7/sPLA2-mediated role played by the heart in in flammation and metabolism.These latter fi ndings are potentially relevant for:(a)Conditions where MMP-2 activity is reduced by inactivating mutations(or polymorphisms)of MMP2 gene or medicinal drugs with MMPinhibitory actions(although little is known about the prevalence of disorders caused by reduced MMP-2 activity)and(b)Disorders in which the expression of MMPs is deregulated-such as ischeMic heart disease,arthritis,cancer,type 2 diabetes,obesity,hypercholesteroleMia.Together,these discoveries could be vital for the diagnosis and for the design of new medicines for treating in flammatory and metabolic disorders.

Acknow ledgments

CFP is supported by a Natural Sciences and Engineering Council of Canada Discovery Grant and by funding froMthe University of A lberta Hospital Foundation for Medical Research.EHR was partially supported by a Visiting Scientist Fund froMUniversity of A lberta International.