DOI: 10.1161/CIRCULATIONAHA.115.016978
Inside Out Signaling: Moving the AT1 Receptor in To Get the Message Out
Running title: Zablocki et al.; Exosomal AT1R for intercellular communication
Daniela Zablocki, MS; Junichi Sadoshima, MD, PhD
Dept of Cell Biology and Molecular Medicine, Cardiovascular Research Institute, Medical Rutgers–New Jersey Med edic ed icaal School, Newark, NJ ic NJ
Address Add Ad dress for for Correspondence: Correspondence: d Junichi Sadoshima, MD, PhD Department of Cell Biology and Molecular Medicine, Cardiovascular Research Institute Rutgers–New Jersey Medical School 185 South Orange Avenue, Medical Science Building G-609 Newark, NJ 07103 Tel: 973-972-8619 Fax: 973-972-8919 E-mail: [email protected]
Journal Subject Code: Basic science research:[128] ACE/angiotensin receptors Key words:Editorial, angiotensin receptor, stretch
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DOI: 10.1161/CIRCULATIONAHA.115.016978
With cardiovascular disease remaining a major cause of morbidity and mortality in spite of recent therapeutic advances, the molecular basis of heart failure continues to be an active field of investigation. In particular, the renin-angiotensin system (RAS), which plays a major role in blood pressure control and cardiac hypertrophy, has been the subject of countless studies. The RAS comprises a complex set of proteolytic processing steps and interconnected signaling pathways, acting both systemically and locally, and involving multiple angiotensin (Ang) peptides and receptors. Of these, the most extensively studied are AngII, considered to be the major effector molecule of the RAS, and one of its receptors, the AngII type 1 receptor (AT1R). Two decades ago, AngII treatment and mechanical stretch were shown to have similar resulting effects upon cardiomyocyte signal transduction pathways and gene expression, re esu sult ltin lt ingg in in hypertrophic growth that was found to be primarily mediated by the AT1R1-4 . Later studies how wed tthat hatt me ha m chaanical stress can activate the AT ch AT1R 1 even in the absen nce of AngII5, with ligand showed mechanical absence bbinding bind i ding and mechanical meecha chanicall stress str tres esss causing es causin cau usin ingg distinct disttin inctt conformational con nform mat atio iona io nal changes na chaang ch nges es inn thee re rece receptor ceept ptor o 6. Th or Thee AT1R AT 1R is a G protein-coupled prootein-ccouupledd receptor recept re ptoor (GPCR) pt (G GPCR CR)) that thaat mediates med ediiatees the ed th he hypertrophic hype hy p rttro pe oph hic i effects effeccts off AngII AngIII by triggering rig igge geri ge ring ri ng downstream dow owns nstr ns trea tr eam ea m signaling sign si gnal gn alin al ingg through in thro th roug ro ugh gh coupled coup co uple ple ledd heterotrimeric hete he tero te rotr ro trim tr imer im eric er ic G pproteins. rottei ro eins ns. Bi ns Bind Binding ndin nd ingg of A in AngII ngII ng II also causes recruitment of ȕ-arrestin, which, in turn, leads to receptor desensitization and internalization. Ligand-independent or ȕ-arrestin biased activation of the receptor, on the other hand, is independent of G protein signaling and induces distinct conformational changes in the receptor and in ȕ-arrestin that trigger an alternative, cardioprotective downstream signaling pathway.7,8 What happens to the AT1R after internalization? In the current model, sorting of endosomal AT1R leads to one of three fates: interaction with other molecules to participate in intracellular signaling, resensitization and recycling of the receptor back to the plasma membrane
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DOI: 10.1161/CIRCULATIONAHA.115.016978
or lysosomal degradation.9 In this issue of Circulation, Pironti et al provide evidence of a fourth possibility, with potentially far-reaching consequences. Cardiac cells are capable of secreting multiple autocrine/paracrine factors, including AngII, thereby exerting effects beyond the individual cell. In addition, stress may promote release of exosomes, extracellular membrane vesicles capable of carrying a variety of cargoes, including proteins and nucleic acids, for transfer to other cells in the local environment or farther off via the systemic circulation. Exosomes, which are secreted by a variety of cell types and exist at high concentrations in the blood, originate from endosomal formation of intraluminal vesicles within multivesicular bodies, and are released from the cell by fusion of the multivesicular bodies with the cell membrane. In his study, using rigorous methods of purification and quantification, the authors show sho how w that that this exosome secretion increases significantly following either osmotic stretch or AngII treatment of HEK2 K293 K2 93T 93 T ce cells (w (with or without stable expressio io on of o AT1R) in vitro orr seven se HEK293T expression days of cardiac ppressure resssure overloa oadd inn wild oa willd type type (WT) (WT WT)) mice m ce in vvivo. mi ivo.. F urtthe herm rm more, ore ssome omee ooff the eexosomes om xoso xoso som mess we were re overload Furthermore, fo oun undd to con nta tain nA T1R, R, withh th the re ece c ptoor ddensity enssity in n th he cel ll me med diaa an nd se eru um in iincreasing crea eassingg ea found contain AT1R, receptor the cell media and serum ubs bsta tant ta ntia nt iall ia lly fo ll foll llow ll owin ingg st in stim imul im ulat lat atio ionn with io wiith oosmotic smot sm otic ot ic sstretch tret tr etch et ch oorr An AngI gIII in gI in vitro vit itro oorr pr it pres essu es sure re ooverload veerl verl rloa oadd in oa substantially following stimulation AngII pressure vivo. Transfer of these exosomes to WT HEK293T cells (which do not express AT1R) or AT1R knockout mice caused the recipients to become responsive to AngII treatment, demonstrating that the exosomal AT1Rs remain functional.10 Thus, it appears that not only is internalization not necessarily the end of the line for AT1Rs but it may actually be part of a process that allows cardiac cells to extend the reach of their signaling to more distant tissues (Figure 1). Packaging of AT1R into exosomes was found to be dependent upon the presence of ȕarrestin, and ȕ-arrestin2 in particular.10 This is not surprising given that ȕ-arrestin is known play an essential role in AT1R internalization by disrupting receptor/G protein interaction and
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recruiting components of the endocytic machinery.9 One question that has not yet been addressed, however, is precisely what role, if any, ȕ-arrestin2 plays in directing AT1R to exosomes rather than one of the alternate possible fates. Although global ȕ-arrestin2 knockout prevented AT1R packaging, exosomal release was unaffected.10 Nor does the increase in exosome release appear to be dependent upon the AT1R itself, since WT HEK293T cells also released an increased number of exosomes in response to osmotic stretch,10 although it might be helpful to confirm this observation in vivo by examining exosomal release in response to pressure overload in AT1R knockout mice. Both AngII and mechanical stress were used to stimulate exosome release, but these two stimuli have been previously shown to induce different ithh th it thee la latt tter tt er ȕ-arrestin conformations and trigger different downstream signaling pathways, w with latter being independent of ligand binding, at least in the short term.7 In the long term, however, some of the he effects eff ffec ects ec ts of stretch strretch and pressure overload ar st re al also mediated by AngI gII/ gI I AT1R signaling. are AngII/AT1R H Hypotonic yppotonic stress-induced stress ss-ind ss ndduced ed cc-fo c-fos foss up fo upregulation preeguulaation n inn ca cardiomyocytes ard diomy myoc my occyt ytess wa wass un unaf unaffected affectted bby y treatment treaatm tr tmen entt wi en with an nA AT1R T R an T1 antagonist, nta tago oni nist,111 bbut ut m mechanical eccha h ni niccall st stretch tretcch andd ppressure resssuuree ove overload verlloaad ha ve have avee be been eenn show shown own to ow o iinduce nduucee 12,13 ,13 ecr cret etio et ionn of A io ngII ng II.12, II W What, hatt, tthen, ha henn, iiss th he thee co comm common mmon mm on mechanism mec echa hani ha nissm by ni by which whi hich hi ch the the different dif iffe fere fe rent re nt types types yppes of of secretion AngII.
stimulus increase AT1R-containing exosome release, and how is the AT1R, rather than other receptors, specifically targeted for exocytosis? If AngII ligand binding is required, testing the effects of treatment with AT1R blockers or angiotensinogen knockout on AT1R packaging could be informative. Mechanical stretch induces internalization of the AT1R, which forms a more stable class B type interaction with ȕ-arrestin, but not of the ȕ1-adrenergic receptor, which forms a less stable class A type interaction,7 and this may perhaps account for the specific inclusion of AT1Rs. However, even under stimulated conditions, the AT1R density was less than 10 receptors/100 exosomes,10 suggesting that something else may be packaged into the exosomes
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DOI: 10.1161/CIRCULATIONAHA.115.016978
under these conditions as well. If so, what else are the exosomes carrying and why? As mentioned above, global knockout of ȕ-arrestin2 prevented transfer of AT1Rs to the exosomes during pressure overload in vivo. Similarly, cardiomyocyte-specific ȕ-arrestin2 knockout markedly reduced serum AT1R levels of tamoxifen-treated ȕarrestin2flox/flox/ĮMHCMerCreMer mice after seven days of pressure overload. Based on this observation, the authors concluded that cardiomyocytes are the major source of AT1Rcontaining exosomes during pressure overload in vivo.10 Cardiomyocytes have been shown to secrete a number of soluble factors that modulate the functions of surrounding cells, and it was recently demonstrated that regulation of Mediator complex subunit 13 (MED13) signaling in cardiomyocytes controls lipid utilization and oxygen consumption in white adipo ose ttissue issu is suee an su andd the adipose iver, presumably through circulating factors released by the heart.14 Nevertheless, exocytosis of liver, func cti tion onal on al rreceptors ecepto ec torrs would represent a significan to nt ex xtension of the mech ch hanistic network by which functional significant extension mechanistic ccardiomyocytes ard diomyocytess can caan exert exe xert rt control con ontr troll over ovverr other otherr cells cells and an nd tissues tisssue ti ssue uess att a distance. dista istaanc n e. However, How owev ever ev er, a er possibility po oss ssib i ility ex ib exis exists i tss th that hat th this con conclusion onnclu l sion on m may ay pprove rovve too bbee pr premature. rem matture ture r .N No o di direct ireect eevidence videncce is ppresented vi resennteed too confirm con onfi firm fi rm that tha hatt cardiomyocytes card ca rdio rd iomy io myoc ocyt oc ytes tes are are the the source sou ourc rcee of the rc the exosomes, exoso xo oso some mess, nor me nor is is there ther th eree any er an data data oon n th thee cardiac function of the cardiac-specific ȕ-arrestin2 knockout mice. It is, therefore, possible that the AT1R-containing exosomes are released from other cardiac cells as a secondary effect of changes in cardiac function following pressure overload. AT1R expression is relatively low in cardiomyocytes, whereas it is substantially higher in cardiac fibroblasts.15 Furthermore, cardiac fibroblasts have been shown to secrete cytokines and exosomes that induce hypertrophy in cardiomyocytes,16,17 raising the question of whether they could be involved in the observed AT1R-containg exosome release during pressure overload as well. The authors do not address a possible secondary effect of the cardiac-specific ȕ-arrestin2 knockout that makes interpretation
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of the data difficult, namely that, while cardiomyocyte levels of ȕ-arrestin2 were approximately 80% lower in the cardiac-specific ȕ-arrestin2 knockout mice than in WT mice, there was an approximately 4-fold increase in ȕ-arrestin2 levels in cardiac fibroblasts (see the supplementary data, Figure S3D).10 It is unclear how this might affect circulating AT1R levels. However, it may be that the exosomes are actually derived from cardiac fibroblasts rather than cardiomyocytes, and that it is the excess ȕ-arrestin2 in the fibroblasts of the knockout mice that is responsible for the decrease in circulating AT1R, possibly by allowing AT1R internalization but somehow preventing its exocytosis, rather than the decrease in cardiomyocyte ȕ-arrestin2. Further investigation of AT1R internalization and exosome tracking in the different cell types is required too address this issue. This study clearly represents a significant advance in understanding how the local cardiac S can can exert exer ex ert its er itts effects e fects systemically. Althoughh AngII ef AngII is secreted intoo the th circulation, the RAS espponsiveness of of target taarg rgett cells cel ells lss was wass until unt ntil il now now w thought thhough ght too be gh be dependent depe de pennden pe nden entt upon upoon their up the heir he ir intrinsic int ntri rinssic A ri T1R T1 R responsiveness AT1R deens nsit i ies. Itt no it nnow w ap ppear ars that ar at ccardiac ardia iacc ccells ia ellls ll ta ake ke pa artt in n ddetermining eter errmin nin ng no nnott ju just hhow ow muc uchh ooff tthe uc he densities. appears take part much effe ef fect fe ctor ct or molecule mol olec ecul ec ule le is released rel elea ease ea sedd but se butt also bu also how how ow capable cap apab able ab le its its targets tar arge gets ge ts are are of of responding resp re spon sp ondi on ding di ng to to it. it What What effector remains unclear at this point is the functional significance of this mechanism. Pironti et al used AT1R knockout mice to demonstrate that the AT1Rs in exosomes remain functional, causing increases in blood pressure and phosphorylation of extracellular signal-regulated kinase (ERK) in response to AngII treatment in vivo.10 However, they did not inject WT mice, so that it is unknown whether the introduction of exogenous AT1R-containing exosomes would have a significant additive effect over that of the endogenous receptors. Examination of receptor density in the tissues of WT and AT1R knockout mice revealed that the exosomes specifically target the heart and skeletal muscle but not the kidneys or lungs, and that, within the heart, they traffic to
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cardiomyocytes and the endothelial and smooth muscle cells of mesenteric vessels but not to cardiac fibroblasts.10 Exosomal markers reflect their parent cells, and it is thought that uptake depends upon binding of targeting peptides to receptors on recipient cells. Indeed, increasing targeting peptide stability was recently shown to significantly increase exosomal uptake.18 Furthermore, there is evidence that the endocytic pathway by which exosomes are internalized varies and may be cell type-specific.19,20 Thus, how the specific targeting of AT1R-exosomes is achieved and how they are taken up by the target cells remain to be elucidated. Likewise, the purpose of the receptor transfer has yet to be determined. The authors speculate that the transfer of AT1Rs may represent an attempt to offset receptor downregulation in the presence of ncreased AngII levels and that this may aggravate cardiac dysfunction and remod del elin ingg du in duri ring ri ng increased remodeling during pressure overload.10 If AT1R-containing exosome release is involved in the progression of heart failur ure, ur e, selectively sel elec ecti ec t veely inhibiting this process could represent rep eprresent a novel therapeutic theraape peutic modality. GPCR failure, kkinases kina i ases (GRKs) s)) ar are re rrequired e ui eq uire redd fo re forr GP GPCR CR iinternalization, ntterrnaalizaatiion n, and and pparoxetine, aro roxe xeti xe tine ti ne,, w ne which hicch spec sspecifically pec ecif ific if iccal ally y inhibits nhi h bits bi GRK GRK2, RK K2, 2 w was as re recently eceentlyy de demo demonstrated mo ons n trrat ated e tto o bbee ooff ggreater reeatter bbenefit enef en effit inn pr protecting rottecti ting ti ng thee hheart earrt aafter fterr myocardial m my yoc ocar oc ardi ar dial di al infarction inf nfar arct ar ctio ct ionn in mice io mic icee than than currently curre urrre rent ntly nt ly used use sedd ȕ se ȕ-blocker -blo blo lock cker ck er ttherapy. hera he rapy ra py.21 H However, owev ow ever er, AT er AT1R AT1R/ȕ1R//ȕ1R arrestin2-dependent ERK activation in response to mechanical stretch was shown to require GRK5 and GRK6 rather than GRK2.7 GRK5-specific small molecule inhibitors are currently under investigation22 and may prove to be of use in targeting pressure overload-induced AT1R exocytosis. On the other hand, knockout of either AT1R or ȕ-arrestin2 has been shown to decrease Akt phosphorylation and increase apoptosis in the mouse heart following mechanical stress, indicating that activation of the AT1R by mechanical stress triggers prosurvival signaling.7 This raises the possibility that the AT1R-exosome release, which is also triggered by mechanical stress in a ȕ-arrestin2-dependent manner, may also represent a protective
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mechanism. A recent study found that plasma exosomes collected from normal rats or humans were highly protective against cardiac ischemia/reperfusion injury in rats, supporting the idea that, at least under some conditions, exosomes may have a cardioprotective role.23 If so, it remains to be determined whether further upregulation of the process would be of therapeutic value for combating the progression of heart failure.
Acknowledgements: We sincerely thank Dr. Susmita Sahoo for her helpful suggestions.
Funding Sources: This work was supported in part by U.S. Public Health Service Grants HL102738, HL67724, HL91469, AG23039 and HL112330. This work was also supported by the Fondation Leducq Transatlantic Networks of Excellence.
Conflict of Interest Disclosures: None.
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Figure Legend: re L eg d: egend:
Figure 1. Schematic representation of the formation of AT1R-containing exosomes. Stimulation by AngII or mechanical stress activates the AT1R, triggering recruitment of ȕ-arrestin2. ȕarrestin2, in turn, recruits components of the endocytic machinery, leading to internalization of the AT1R. Endosomal AT1R is packaged into intraluminal vesicles within multivesicular bodies via a currently unknown mechanism, which are released as exosomes upon fusion of the multivesicular bodies with the cell membrane. Circulating AT1R-exosomes target the cardiomyocytes and mesenteric vessels in the heart, as well as skeletal muscle.
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Mechanical stress
AngII
Exosome release
AT1R 2 estin E-arr
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AT1R inhibitor E-arrestin2 t n2 ti 2
E-arrestin2 tin2 knockout out
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AP-2, A -2 AP 2, clathrin c at cl a hrin n
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Cell membrane
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Inside Out Signaling: Moving the AT1 Receptor in To Get the Message Out Daniela Zablocki and Junichi Sadoshima Circulation. published online May 20, 2015; Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2015 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7322. Online ISSN: 1524-4539
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Inside Out Signaling: Moving the AT1 Receptor in To Get the Message Out
Running title: Zablocki et al.; Exosomal AT1R for intercellular communication
Daniela Zablocki, MS; Junichi Sadoshima, MD, PhD
Dept of Cell Biology and Molecular Medicine, Cardiovascular Research Institute, Medical Rutgers–New Jersey Med edic ed icaal School, Newark, NJ ic NJ
Address Add Ad dress for for Correspondence: Correspondence: d Junichi Sadoshima, MD, PhD Department of Cell Biology and Molecular Medicine, Cardiovascular Research Institute Rutgers–New Jersey Medical School 185 South Orange Avenue, Medical Science Building G-609 Newark, NJ 07103 Tel: 973-972-8619 Fax: 973-972-8919 E-mail: [email protected]
Journal Subject Code: Basic science research:[128] ACE/angiotensin receptors Key words:Editorial, angiotensin receptor, stretch
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DOI: 10.1161/CIRCULATIONAHA.115.016978
With cardiovascular disease remaining a major cause of morbidity and mortality in spite of recent therapeutic advances, the molecular basis of heart failure continues to be an active field of investigation. In particular, the renin-angiotensin system (RAS), which plays a major role in blood pressure control and cardiac hypertrophy, has been the subject of countless studies. The RAS comprises a complex set of proteolytic processing steps and interconnected signaling pathways, acting both systemically and locally, and involving multiple angiotensin (Ang) peptides and receptors. Of these, the most extensively studied are AngII, considered to be the major effector molecule of the RAS, and one of its receptors, the AngII type 1 receptor (AT1R). Two decades ago, AngII treatment and mechanical stretch were shown to have similar resulting effects upon cardiomyocyte signal transduction pathways and gene expression, re esu sult ltin lt ingg in in hypertrophic growth that was found to be primarily mediated by the AT1R1-4 . Later studies how wed tthat hatt me ha m chaanical stress can activate the AT ch AT1R 1 even in the absen nce of AngII5, with ligand showed mechanical absence bbinding bind i ding and mechanical meecha chanicall stress str tres esss causing es causin cau usin ingg distinct disttin inctt conformational con nform mat atio iona io nal changes na chaang ch nges es inn thee re rece receptor ceept ptor o 6. Th or Thee AT1R AT 1R is a G protein-coupled prootein-ccouupledd receptor recept re ptoor (GPCR) pt (G GPCR CR)) that thaat mediates med ediiatees the ed th he hypertrophic hype hy p rttro pe oph hic i effects effeccts off AngII AngIII by triggering rig igge geri ge ring ri ng downstream dow owns nstr ns trea tr eam ea m signaling sign si gnal gn alin al ingg through in thro th roug ro ugh gh coupled coup co uple ple ledd heterotrimeric hete he tero te rotr ro trim tr imer im eric er ic G pproteins. rottei ro eins ns. Bi ns Bind Binding ndin nd ingg of A in AngII ngII ng II also causes recruitment of ȕ-arrestin, which, in turn, leads to receptor desensitization and internalization. Ligand-independent or ȕ-arrestin biased activation of the receptor, on the other hand, is independent of G protein signaling and induces distinct conformational changes in the receptor and in ȕ-arrestin that trigger an alternative, cardioprotective downstream signaling pathway.7,8 What happens to the AT1R after internalization? In the current model, sorting of endosomal AT1R leads to one of three fates: interaction with other molecules to participate in intracellular signaling, resensitization and recycling of the receptor back to the plasma membrane
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or lysosomal degradation.9 In this issue of Circulation, Pironti et al provide evidence of a fourth possibility, with potentially far-reaching consequences. Cardiac cells are capable of secreting multiple autocrine/paracrine factors, including AngII, thereby exerting effects beyond the individual cell. In addition, stress may promote release of exosomes, extracellular membrane vesicles capable of carrying a variety of cargoes, including proteins and nucleic acids, for transfer to other cells in the local environment or farther off via the systemic circulation. Exosomes, which are secreted by a variety of cell types and exist at high concentrations in the blood, originate from endosomal formation of intraluminal vesicles within multivesicular bodies, and are released from the cell by fusion of the multivesicular bodies with the cell membrane. In his study, using rigorous methods of purification and quantification, the authors show sho how w that that this exosome secretion increases significantly following either osmotic stretch or AngII treatment of HEK2 K293 K2 93T 93 T ce cells (w (with or without stable expressio io on of o AT1R) in vitro orr seven se HEK293T expression days of cardiac ppressure resssure overloa oadd inn wild oa willd type type (WT) (WT WT)) mice m ce in vvivo. mi ivo.. F urtthe herm rm more, ore ssome omee ooff the eexosomes om xoso xoso som mess we were re overload Furthermore, fo oun undd to con nta tain nA T1R, R, withh th the re ece c ptoor ddensity enssity in n th he cel ll me med diaa an nd se eru um in iincreasing crea eassingg ea found contain AT1R, receptor the cell media and serum ubs bsta tant ta ntia nt iall ia lly fo ll foll llow ll owin ingg st in stim imul im ulat lat atio ionn with io wiith oosmotic smot sm otic ot ic sstretch tret tr etch et ch oorr An AngI gIII in gI in vitro vit itro oorr pr it pres essu es sure re ooverload veerl verl rloa oadd in oa substantially following stimulation AngII pressure vivo. Transfer of these exosomes to WT HEK293T cells (which do not express AT1R) or AT1R knockout mice caused the recipients to become responsive to AngII treatment, demonstrating that the exosomal AT1Rs remain functional.10 Thus, it appears that not only is internalization not necessarily the end of the line for AT1Rs but it may actually be part of a process that allows cardiac cells to extend the reach of their signaling to more distant tissues (Figure 1). Packaging of AT1R into exosomes was found to be dependent upon the presence of ȕarrestin, and ȕ-arrestin2 in particular.10 This is not surprising given that ȕ-arrestin is known play an essential role in AT1R internalization by disrupting receptor/G protein interaction and
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recruiting components of the endocytic machinery.9 One question that has not yet been addressed, however, is precisely what role, if any, ȕ-arrestin2 plays in directing AT1R to exosomes rather than one of the alternate possible fates. Although global ȕ-arrestin2 knockout prevented AT1R packaging, exosomal release was unaffected.10 Nor does the increase in exosome release appear to be dependent upon the AT1R itself, since WT HEK293T cells also released an increased number of exosomes in response to osmotic stretch,10 although it might be helpful to confirm this observation in vivo by examining exosomal release in response to pressure overload in AT1R knockout mice. Both AngII and mechanical stress were used to stimulate exosome release, but these two stimuli have been previously shown to induce different ithh th it thee la latt tter tt er ȕ-arrestin conformations and trigger different downstream signaling pathways, w with latter being independent of ligand binding, at least in the short term.7 In the long term, however, some of the he effects eff ffec ects ec ts of stretch strretch and pressure overload ar st re al also mediated by AngI gII/ gI I AT1R signaling. are AngII/AT1R H Hypotonic yppotonic stress-induced stress ss-ind ss ndduced ed cc-fo c-fos foss up fo upregulation preeguulaation n inn ca cardiomyocytes ard diomy myoc my occyt ytess wa wass un unaf unaffected affectted bby y treatment treaatm tr tmen entt wi en with an nA AT1R T R an T1 antagonist, nta tago oni nist,111 bbut ut m mechanical eccha h ni niccall st stretch tretcch andd ppressure resssuuree ove overload verlloaad ha ve have avee be been eenn show shown own to ow o iinduce nduucee 12,13 ,13 ecr cret etio et ionn of A io ngII ng II.12, II W What, hatt, tthen, ha henn, iiss th he thee co comm common mmon mm on mechanism mec echa hani ha nissm by ni by which whi hich hi ch the the different dif iffe fere fe rent re nt types types yppes of of secretion AngII.
stimulus increase AT1R-containing exosome release, and how is the AT1R, rather than other receptors, specifically targeted for exocytosis? If AngII ligand binding is required, testing the effects of treatment with AT1R blockers or angiotensinogen knockout on AT1R packaging could be informative. Mechanical stretch induces internalization of the AT1R, which forms a more stable class B type interaction with ȕ-arrestin, but not of the ȕ1-adrenergic receptor, which forms a less stable class A type interaction,7 and this may perhaps account for the specific inclusion of AT1Rs. However, even under stimulated conditions, the AT1R density was less than 10 receptors/100 exosomes,10 suggesting that something else may be packaged into the exosomes
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under these conditions as well. If so, what else are the exosomes carrying and why? As mentioned above, global knockout of ȕ-arrestin2 prevented transfer of AT1Rs to the exosomes during pressure overload in vivo. Similarly, cardiomyocyte-specific ȕ-arrestin2 knockout markedly reduced serum AT1R levels of tamoxifen-treated ȕarrestin2flox/flox/ĮMHCMerCreMer mice after seven days of pressure overload. Based on this observation, the authors concluded that cardiomyocytes are the major source of AT1Rcontaining exosomes during pressure overload in vivo.10 Cardiomyocytes have been shown to secrete a number of soluble factors that modulate the functions of surrounding cells, and it was recently demonstrated that regulation of Mediator complex subunit 13 (MED13) signaling in cardiomyocytes controls lipid utilization and oxygen consumption in white adipo ose ttissue issu is suee an su andd the adipose iver, presumably through circulating factors released by the heart.14 Nevertheless, exocytosis of liver, func cti tion onal on al rreceptors ecepto ec torrs would represent a significan to nt ex xtension of the mech ch hanistic network by which functional significant extension mechanistic ccardiomyocytes ard diomyocytess can caan exert exe xert rt control con ontr troll over ovverr other otherr cells cells and an nd tissues tisssue ti ssue uess att a distance. dista istaanc n e. However, How owev ever ev er, a er possibility po oss ssib i ility ex ib exis exists i tss th that hat th this con conclusion onnclu l sion on m may ay pprove rovve too bbee pr premature. rem matture ture r .N No o di direct ireect eevidence videncce is ppresented vi resennteed too confirm con onfi firm fi rm that tha hatt cardiomyocytes card ca rdio rd iomy io myoc ocyt oc ytes tes are are the the source sou ourc rcee of the rc the exosomes, exoso xo oso some mess, nor me nor is is there ther th eree any er an data data oon n th thee cardiac function of the cardiac-specific ȕ-arrestin2 knockout mice. It is, therefore, possible that the AT1R-containing exosomes are released from other cardiac cells as a secondary effect of changes in cardiac function following pressure overload. AT1R expression is relatively low in cardiomyocytes, whereas it is substantially higher in cardiac fibroblasts.15 Furthermore, cardiac fibroblasts have been shown to secrete cytokines and exosomes that induce hypertrophy in cardiomyocytes,16,17 raising the question of whether they could be involved in the observed AT1R-containg exosome release during pressure overload as well. The authors do not address a possible secondary effect of the cardiac-specific ȕ-arrestin2 knockout that makes interpretation
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of the data difficult, namely that, while cardiomyocyte levels of ȕ-arrestin2 were approximately 80% lower in the cardiac-specific ȕ-arrestin2 knockout mice than in WT mice, there was an approximately 4-fold increase in ȕ-arrestin2 levels in cardiac fibroblasts (see the supplementary data, Figure S3D).10 It is unclear how this might affect circulating AT1R levels. However, it may be that the exosomes are actually derived from cardiac fibroblasts rather than cardiomyocytes, and that it is the excess ȕ-arrestin2 in the fibroblasts of the knockout mice that is responsible for the decrease in circulating AT1R, possibly by allowing AT1R internalization but somehow preventing its exocytosis, rather than the decrease in cardiomyocyte ȕ-arrestin2. Further investigation of AT1R internalization and exosome tracking in the different cell types is required too address this issue. This study clearly represents a significant advance in understanding how the local cardiac S can can exert exer ex ert its er itts effects e fects systemically. Althoughh AngII ef AngII is secreted intoo the th circulation, the RAS espponsiveness of of target taarg rgett cells cel ells lss was wass until unt ntil il now now w thought thhough ght too be gh be dependent depe de pennden pe nden entt upon upoon their up the heir he ir intrinsic int ntri rinssic A ri T1R T1 R responsiveness AT1R deens nsit i ies. Itt no it nnow w ap ppear ars that ar at ccardiac ardia iacc ccells ia ellls ll ta ake ke pa artt in n ddetermining eter errmin nin ng no nnott ju just hhow ow muc uchh ooff tthe uc he densities. appears take part much effe ef fect fe ctor ct or molecule mol olec ecul ec ule le is released rel elea ease ea sedd but se butt also bu also how how ow capable cap apab able ab le its its targets tar arge gets ge ts are are of of responding resp re spon sp ondi on ding di ng to to it. it What What effector remains unclear at this point is the functional significance of this mechanism. Pironti et al used AT1R knockout mice to demonstrate that the AT1Rs in exosomes remain functional, causing increases in blood pressure and phosphorylation of extracellular signal-regulated kinase (ERK) in response to AngII treatment in vivo.10 However, they did not inject WT mice, so that it is unknown whether the introduction of exogenous AT1R-containing exosomes would have a significant additive effect over that of the endogenous receptors. Examination of receptor density in the tissues of WT and AT1R knockout mice revealed that the exosomes specifically target the heart and skeletal muscle but not the kidneys or lungs, and that, within the heart, they traffic to
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cardiomyocytes and the endothelial and smooth muscle cells of mesenteric vessels but not to cardiac fibroblasts.10 Exosomal markers reflect their parent cells, and it is thought that uptake depends upon binding of targeting peptides to receptors on recipient cells. Indeed, increasing targeting peptide stability was recently shown to significantly increase exosomal uptake.18 Furthermore, there is evidence that the endocytic pathway by which exosomes are internalized varies and may be cell type-specific.19,20 Thus, how the specific targeting of AT1R-exosomes is achieved and how they are taken up by the target cells remain to be elucidated. Likewise, the purpose of the receptor transfer has yet to be determined. The authors speculate that the transfer of AT1Rs may represent an attempt to offset receptor downregulation in the presence of ncreased AngII levels and that this may aggravate cardiac dysfunction and remod del elin ingg du in duri ring ri ng increased remodeling during pressure overload.10 If AT1R-containing exosome release is involved in the progression of heart failur ure, ur e, selectively sel elec ecti ec t veely inhibiting this process could represent rep eprresent a novel therapeutic theraape peutic modality. GPCR failure, kkinases kina i ases (GRKs) s)) ar are re rrequired e ui eq uire redd fo re forr GP GPCR CR iinternalization, ntterrnaalizaatiion n, and and pparoxetine, aro roxe xeti xe tine ti ne,, w ne which hicch spec sspecifically pec ecif ific if iccal ally y inhibits nhi h bits bi GRK GRK2, RK K2, 2 w was as re recently eceentlyy de demo demonstrated mo ons n trrat ated e tto o bbee ooff ggreater reeatter bbenefit enef en effit inn pr protecting rottecti ting ti ng thee hheart earrt aafter fterr myocardial m my yoc ocar oc ardi ar dial di al infarction inf nfar arct ar ctio ct ionn in mice io mic icee than than currently curre urrre rent ntly nt ly used use sedd ȕ se ȕ-blocker -blo blo lock cker ck er ttherapy. hera he rapy ra py.21 H However, owev ow ever er, AT er AT1R AT1R/ȕ1R//ȕ1R arrestin2-dependent ERK activation in response to mechanical stretch was shown to require GRK5 and GRK6 rather than GRK2.7 GRK5-specific small molecule inhibitors are currently under investigation22 and may prove to be of use in targeting pressure overload-induced AT1R exocytosis. On the other hand, knockout of either AT1R or ȕ-arrestin2 has been shown to decrease Akt phosphorylation and increase apoptosis in the mouse heart following mechanical stress, indicating that activation of the AT1R by mechanical stress triggers prosurvival signaling.7 This raises the possibility that the AT1R-exosome release, which is also triggered by mechanical stress in a ȕ-arrestin2-dependent manner, may also represent a protective
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mechanism. A recent study found that plasma exosomes collected from normal rats or humans were highly protective against cardiac ischemia/reperfusion injury in rats, supporting the idea that, at least under some conditions, exosomes may have a cardioprotective role.23 If so, it remains to be determined whether further upregulation of the process would be of therapeutic value for combating the progression of heart failure.
Acknowledgements: We sincerely thank Dr. Susmita Sahoo for her helpful suggestions.
Funding Sources: This work was supported in part by U.S. Public Health Service Grants HL102738, HL67724, HL91469, AG23039 and HL112330. This work was also supported by the Fondation Leducq Transatlantic Networks of Excellence.
Conflict of Interest Disclosures: None.
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Figure Legend: re L eg d: egend:
Figure 1. Schematic representation of the formation of AT1R-containing exosomes. Stimulation by AngII or mechanical stress activates the AT1R, triggering recruitment of ȕ-arrestin2. ȕarrestin2, in turn, recruits components of the endocytic machinery, leading to internalization of the AT1R. Endosomal AT1R is packaged into intraluminal vesicles within multivesicular bodies via a currently unknown mechanism, which are released as exosomes upon fusion of the multivesicular bodies with the cell membrane. Circulating AT1R-exosomes target the cardiomyocytes and mesenteric vessels in the heart, as well as skeletal muscle.
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Mechanical stress
AngII
Exosome release
AT1R 2 estin E-arr
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AT1R inhibitor E-arrestin2 t n2 ti 2
E-arrestin2 tin2 knockout out
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AP-2, A -2 AP 2, clathrin c at cl a hrin n
? Multivesicular body
Cell membrane
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Inside Out Signaling: Moving the AT1 Receptor in To Get the Message Out Daniela Zablocki and Junichi Sadoshima Circulation. published online May 20, 2015; Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2015 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7322. Online ISSN: 1524-4539
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