Cardiac angiogenic imbalance leads to peripartum cardiomyopathy

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1 oi:1.138/ntur114 Cri ngiogni imln ls to priprtum riomyopthy In S. Pttn 1,, Srosh Rn 3, Sji Shhul 4, Glnn C. Row 1, Cholsoon Jng 1, Lur Liu 1, Mihl R. Hkr 3, Juli S. Rh 3, John Mithll 4, Froz Mhmoo 4, Philip Hss 4, Citlin Frrll 1, Niol Koulisis 1, Eliyhu V. Khnkin 5, Suznn D. Burk 5,8, Igor Tuorh 6, Johnn Burshs 7, Fri l Mont 1, Dnis Hilikr-Klinr 7, S. Annth Krumnhi 5,8 & Zoltn Arny 1 Priprtum riomyopthy (PPCM) is n otn tl iss tht ts prgnnt womn who r nr livry, n it ours mor rquntly in womn with pr-lmpsi n/or multipl gsttion. Th tiology o PPCM, n why it is ssoit with pr-lmpsi, rmin unknown. Hr w show tht PPCM is ssoit with systmi ngiogni imln, ntut y pr-lmpsi. Mi tht lk ri PGC-1, powrul rgultor o ngiognsis, vlop prooun PPCM. Importntly, th PPCM is ntirly rsu y pro-ngiogni thrpis. In humns, th plnt in lt gsttion srts VEGF inhiitors lik solul FLT1 (sflt1), n this is ntut y multipl gsttion n pr-lmpsi. This nti-ngiogni nvironmnt is ompni y sulinil ri ysuntion, th xtnt o whih orrlts with irulting lvls o sflt1. Exognous sflt1 lon us istoli ysuntion in wil-typ mi, n prooun systoli ysuntion in mi lking ri PGC-1. Finlly, plsm smpls rom womn with PPCM ontin normlly high lvls o sflt1. Ths t init tht PPCM is minly vsulr iss, us y xss nti-ngiogni signlling in th priprtum prio. Th t lso xplin how lt prgnny poss thrt to ri homostsis, n why pr-lmpsi n multipl gsttion r importnt risk tors or th vlopmnt o PPCM. PPCM ts 1 in 3 to 1 in 3, prgnnis, with gogrphi hot spots o high inin, suh s Nigri n Hiti 1,. Th iss is hrtriz y systoli hrt ilur prsnting in th lst month o prgnny or th irst 5 months post-prtum. Although pproximtly hl o t womn rovr ri untion post-prtum, mny ptints progrss to hroni hrt ilur, ri trnsplnttion or th. Thus, PPCM n vstt othrwis hlthy young womn n thir innts. PPCM rmins iss o unknown tiology. Th onst lt in gsttion os not oini with inrs hmoynmi lo on th hrt, suggsting tht othr mhnisms r rsponsil. Rnt t hv suggst tht nti-ngiogni proltin rgmnts my hv n importnt rol in using th iss in som ptints 3. Risk tors or PPCM lso inlu pr-lmpsi n multipl gsttion, suggsting potntil mhnisti ovrlp with ths prosss 1,. PGC-1 is trnsriptionl otivtor tht rivs mitohonril iognsis n othr mtoli progrms in mny tissus, inluing th hrt 4,5.PGC-1 is highly xprss in th hrt, n mi lking PGC-1 glolly hv norml ri nrgti rsrvs n rspon poorly to strssul stimuli suh s trnsvrs orti ning 6,7. In ition to its rol in mitohonril homostsis, PGC-1 lso inus th xprssion n srtion o pro-ngiogni tors, suh s vsulr nothlil growth tor (VEGF), whih ls to ormtion o nw loo vssls 8,9. Although th ngiogni untion o PGC-1 hs n sri in skltl musl, its rol in ri tissu rmins unxplor. Cri-spii PGC-1 ltion ls to PPCM To stuy urthr th rol o PGC-1 in th hrt, w gnrt ri-spii PGC-1 knokout () mi (s Mthos). Whil stuying ths mi, w noti tht ml mi wr rtil, n livr norml littr sizs (not shown), ut invrily i tr on or two prgnnis (Fig. 1). Th hrts o ths mi wr lrg, ilt n iroti (Fig. 1 ), onsistnt with ilt riomyopthy. Twoimnsionl M-mo horiogrphy rvl ilt, poorly ontrtil hrts in mi tr thir son livry (Fig. 1). Lt vntriulr n-istoli imnsions (LVEDD) n lt vntriulr nsystoli imnsions (LVESD) wr mrkly nlrg, n rtionl shortning, irt msur o ri ontrtil untion, ws proounly prss (Fig. 1 i). Nulliprous mi, s wll s post-prtum ontrol mi, wr not t. Mls wr lso not t (Supplmntry Fig. 1). Thus, th sn o PGC-1 in riomyoyts ls to prooun PPCM in mi. PGC-1 rgults ngiognsis in ri tissu W hv rntly shown in skltl musl tht PGC-1 rgults ngiognsis y riving th xprssion o ngiogni tors lik VEGF 8,9. Anti-ngiogni thrpis, inluing ntiois tht nutrliz VEGF n smll-molul VEGF rptor inhiitors, r inrsingly ing us in onologil n ophthlmologil trtmnts, n riomyopthy n hrt ilur hv rntly n rogniz s importnt si ts 1,11, showing tht nti-ngiogni thrpy n hrmul to th hrt in humns. Impir VEGF signlling hs lso n link with ri ysuntion in mi 1,13. At th sm tim, lt prgnny is strong nti-ngiogni nvironmnt, prtly owing to th srtion y th plnt o nti-ngiogni tors, lik sflt1, tht in to n nutrliz solul mmrs o th VEGF mily 14. Ths osrvtions l us to postult tht PGC-1 rgults n ngiogni 1 Criovsulr Institut, Bth Isrl Donss Mil Cntr, Hrvr Mil Shool, 33 Brooklin Avnu, Boston, Msshustts 115, USA. Cntr or Vsulr Biology Rsrh, Bth Isrl Donss Mil Cntr, Hrvr Mil Shool, 33 Brooklin Avnu, Boston, Msshustts 115, USA. 3 Division o Mtrnl Ftl Miin/Dprtmnt o Osttris n Gynology, Bth Isrl Donss Mil Cntr, Hrvr Mil Shool, 33 Brooklin Avnu, Boston, Msshustts 115, USA. 4 Dprtmnt o Ansthsi n Critil Cr, Bth Isrl Donss Mil Cntr, Hrvr Mil Shool, 33 Brooklin Avnu, Boston, Msshustts 115, USA. 5 Division o Nphrology/Dprtmnt o Miin, Bth Isrl Donss Mil Cntr n Hrvr Mil Shool, 33 Brooklin Avnu, Boston, Msshustts 115, USA. 6 Dprtmnt o Criothori, Trnsplnttion n Vsulr Surgry, Mizinish Hohshul Hnnovr, Crl-Nurg Strss, D-365 Hnnovr, Grmny. 7 Dprtmnt o Criology n Angiology, MizinishHohshul Hnnovr, Crl-Nurg Strss, D-365 Hnnovr, Grmny. 8 Howr Hughs Mil Institut, 33 Brooklin Avnu, Boston, Msshustts 115, USA. Ths uthors ontriut qully to this work. 1 Mmilln Pulishrs Limit. All rights rsrv 17 MAY 1 VOL 485 NATURE 333

2 RESEARCH ARTICLE Survivl (%) 1 5 H&E Ml n ml Ml Fml Numr o prgnnis MT Hrt wight m Hrt wight : tiil lngth Figur 1 Mi lking ri PGC-1 vlop pri-prtum riomyopthy., Kpln Mir survivl urv in ml MHC-Cr: PGC- 1 lox/lox mi () vrsus ml mi or ontrol mi () o ithr gnr., Hmtoxylin n osin n Msson s trihrom stins o hrts rom post-prtum mi (), vrsus post-prtum mi (PP ).,, Hrt wight () n hrt wight:tiil lngth rtios () o mg mg mm 1 % h FS = LVEDD LVESD LVEDD LVESD LVEDD (PP CRE) FS LVESD g m PP CRE () i Bts pr min LVEDD Hrt rt nulliprous n mi, n n mi., Smpl M-mo horiogrms o mi n ontrol mi ontining th MHC Cr trnsgn lon (PP CRE). i, Ehoriogrphi msurs in mi o th init gnotyps, ithr nulliprous or post-prtum. n $ 5 or ll groups. P,.5. progrm in riomyoyts, n tht th sn o this progrm woul lv th hrts nlss to th nti-ngiogni stting o lt prgnny, thus ling th nimls to vlop PPCM. Consistnt with this i, ovrxprssion o PGC-1 in nontl rt vntriulr myoyts (NRVMs) strongly inu ngiogni gns involv in th tivtion n rruitmnt o nothlil lls (or xmpl, Vg) n murl lls (or xmpl, Pg), s wll s gns tht r involv in th mitohonril rspirtory hin 15 (or xmpl, Cys n Cox5) (Fig. ). Intrstingly, som ngiogni gns wr rprss (or xmpl, Bg), with pttrn o rprssion tht is similr to tht sn in skltl musl lls 8, initing tht PGC-1 rprogrms th ngiogni progrm in strotypil mnnr. Convrsly, PGC-1 short intrring RNA (sipgc-1) signiintly supprss th xprssion o Vg (Fig. ). Enothlil tivtion n migrtion is hllmrk o ngiognsis. As shown in Fig.,, ovrxprssion o PGC-1 in NRVMs l to mrk, os-pnnt, up to sixol inrs in th migrtion o th jnt humn umilil vin nothlil lls (HUVECs) in oultur systm. Aition o sflt1 ompltly nutrliz th inu nothlil migrtion, initing tht srt mmrs o th VEGF mily, proly VEGFA itsl, r ritil or th t. Convrsly, rprssion o PGC-1 in NRVMs y sirna signiintly rprss nothlil migrtion (Fig. ). Thus, PGC-1 ontrols n ngiogni progrm in riomyoyts. To tst whthr PGC-1 is rquir or this progrm in intt nimls, lvls o Vg n othr ngiogni tors wr msur in hrts rom PGC-1 mi. Th xprssion o Vg n Pg, n numr o othr ngiogni tors, ws rprss in ths hrts y s muh s 5% (Fig. 3). VEGFA protin ws rs y 3% in hrts (Fig. 3). Lvls o VEGFA r normlly tightly rgult, n vn glol hplo-insuiiny is lthl 16,17, unrsoring th signiin o 3% rop in protin lvls. Consistnt with ths inings, th pillry nsity o hrts, s msur y stining with th nothlil-spii mrkr CD31, ws rs y out 15% (Fig. 3, ). Thus, PGC-1 rgults oth vsulr nsity (ths t) n mitohonril untion 18 in th hrt, proviing n importnt rgultory link twn th livry o ul (through loo vssls) n its onsumption (y mitohonri).ths t suggst tht PPCM in ths mi might us y th omintion o hrt-spii vsulr t us y th sn o PGC-1, n th norml systmi ntingiogni nvironmnt o lt prgnny. In, th vsulr nsity in hrts rs y lmost 5% post-prtum (Fig. 3, ), whih is quivlnt to th rs in vsulr nsity sn in mi lking ri VEGF 19. Consistnt with this vsulr rrtion, prusion o post-prtum hrts ws proounly rs y nrly 5% ompr to wil-typ nimls, s trmin y mthoxyisoutylisonitril (MIBI) uptk n singl-photon mission omput tomogrphy n X-ry omput tomogrphy (SPE/) (Fig. 3, ). Pro-ngiogni thrpy rsus PPCM To tst irtly th i tht n ngiogni imln rivs PPCM in mi, rsu xprimnts wr rri out. In irst sris o xprimnts, ring mi wr ministr ily suutnous injtions o VEGF11 protin (1 mgkg 1 ), n isoorm o VEGFA, vrsus vhil ontrol (Supplmntry Fig. ). Th iy o VEGF11 injtions ws onirm y th prsn o VEGF11 in th srum, n roust tivtion o ri VEGFR phosphoryltion within 3 min o injtion (Supplmntry Fig., ). Th VEGF trtmnt l to improv survivl o th ring mls; thy wr l to surviv up to iv prgnnis (Supplmntry Fig. ). Howvr, pillry nsity ws only prtly rsu, ri ontrtility ws only mrginlly improv n th hrts rmin nlrg (Supplmntry Fig. g). Thus, VEGF ministrtion prtly rsu lthlity in multiprous nimls, ut it ws insuiint to ully rsu PPCM, suggsting tht othr pthwys r lso importnt. It hs rntly n shown tht STAT3 rgults mitohonril suproxi ismuts (MNSOD) n protts ginst rtiv oxygn spis (ROS) in th hrt 3. Asn o ri STAT3 n th onsqunt inrs in ROS l to inpproprit lvg o proltin to potnt nti-ngiogni 16-kD orm, n susqunt PPCM (Fig. 4). Importntly, th PPCM oul rsu y trtmnt with romoriptin, whih inhiits th srtion o proltin 334 NATURE VOL MAY 1 1 Mmilln Pulishrs Limit. All rights rsrv

3 RESEARCH 1 Cys NRVM 8 A-GFP 6 A-PGC1α 4 Rltiv mrna 6 4 A-GFP μl A-PGC1.1 μl A-PGC1 μl Vg 4 48 Rltiv mrna 3 Pg Bg Hours tr intion +sflt1 A-PGC1.5 μl +sflt1 HUVECs NRVM Migrt lls pr HPF Migrt Clls pr HFP A-GFP VEGF A-GFP + sflt Cox5 A-si Rltiv mrna 1..5 A- sipgc-1α 1. Vg.5 A-si A-PGC-1α Pg1 VEGF AsiPGC-1α A-PGC-1α + sflt1 Figur PGC-1 rgults n ngiogni progrm in riomyoyts.,, Rltiv xprssion o mitohonril gns (Cys n Cox5) n ngiogni gns (Vg, Pg n Bg) in NRVMs int with novirus xprssing PGC-1 (A-PGC-1) vrsus novirus xprssing grn luorsnt protin (A-GFP) () or sipgc-1 vrsus si ()., PGC-1 xprssion in NRVMs inus th migrtion o jnt nothlil lls, n th migrtion is lok y sflt1. Rprsnttiv imgs show phlloiin-stin nothlil lls tht hv migrt towrs th NRVMs. Th xprimntl prour using th Trnswll migrtion hmr is shown in th ottom pnl., Dt rom th prour in r quntii. HPF, high powr il., Knokown o PGC-1 inhiits th migrtion o nothlil lls. Error rs r 6 s..m. P,.5 vrsus ontrol. P,.5 vrsus lls not trt with sflt1. Rltiv mrna PP Pg1 CRE Vg Angpt1 Angpt Pg 1 Pg Fg 6 4 Cpillris pr HPF Rltiv VEGF CRE rom th pituitry gln. PGC-1 is known to inrs ROS svnging. In riomyoyts, ovrxprssion o PGC-1 strongly inu Mnso mrna n protin xprssion (Supplmntry Fig. 3, ). Convrsly, MNSOD ws rprss in PGC-1 hrts, n lvls o ROS wr inrs (Supplmntry Fig. 3, ). Thus, PGC-1 n STAT3 oth rgult MNSOD n ROS in riomyoyts, suggsting tht th sn o PGC-1 in th hrt my, lik th sn o STAT3, l to proltin-mit ntingiogni ts. Proltin h no irt ts on PGC-1 or VEGF xprssion in ri lls, n proltin lvls i not ir in hrt or srum twn wil-typ n nimls (Supplmntry Fig. 4). Ths osrvtions suggst tht PGC-1 rgults two sprt pro-ngiogni pthwys in th hrt VEGF pthwy n proltin pthwy n tht rrtion o oth pthwys in PGC-1 mi ls to PPCM (Fig. 4). To tst this i irtly, oth pthwys wr rsu simultnously: ring mi wr trt with ily suutnous injtions o VEGF protin n with romoriptin supplmnttion in th wtr (Fig. 4). This oul trtmnt rsult in omplt rsu o th PPCM in mls CD31 Rltiv MIBI uptk Figur 3 Mi lking ri PGC-1 hv ru mirovsulr nsity tht is worsn y prgnny.,, Rltiv mrna xprssion o svrl ngiogni tors () n VEGF protin lvls () in vrsus CRE ontrol hrts.,, Vsulr nsity in hrts rom nulliprous or post-prtum n mi. Rprsnttiv imgs stin or CD31 (lso known s PECAM) r shown () n quntii ().,, Ru ri MIBI uptk in vrsus nimls. Rprsnttiv SPE/ imgs r shown (), n quntii (). n $ 5 or ll groups. Error rs r 6 s..m. P,.5 vrsus ontrol. 1 Mmilln Pulishrs Limit. All rights rsrv 17 MAY 1 VOL 485 NATURE 335

4 RESEARCH ARTICLE Lt prgnny: PP CRE + Br/VEGF Bromoriptin Pituitry Proltin 3 kd 16 kd Miroirultion homostsis Plnt sflt1 ROS MNSOD VEGF STAT3 PGC-1α Criomyoyt Br m m Dily VEGF SC 1 μg kg 1 PP CRE Prntg m.1.1 FS LVEDD LVESD g h 1 m m m + Bromoriptin + Dily VEGF SC 1 μg kg 1 + Bromoriptin + VEGF + Br + Br/VEGF Figur 4 Comin trtmnt with VEGF n romoriptin rsus PPCM in PGC-1 mi., Shm o th propos rol o ri PGC- 1 in th rgultion o ri ngiognsis, n in ning ginst prgnny-inu nti-ngiogni tors., Exprimntl outlin. m, months; SC, suutnous., Smpl horiogrms rom, PP CRE mg 1 Hrt wight mg mm 1 5 Hrt wight tiil lngth n mi riving oth romoriptin n VEGF trtmnts (PP 1 Br/VEGF). h, Ehoriogrphi msurs ( ), hrt wight (g) n hrt wight:tiil lngth rtio (h), in PP mi o th init gnotyps. n $ 5 or ll groups. Error rs r 6 s..m. P,.5 vrsus PP CRE ontrol. P,.5 vrsus. (Fig. 4). Atr two prgnnis, hrt wights n ll horiogrphi inis o ri untion (rtionl shortning, LVEDD n LVESD) wr norml in mi (Fig. 4 h). Whn only romoriptin ws us, som o th lt vntriulr iltion ws prvnt, ut thr ws only miniml rsu o lt vntriulr untion, showing tht rsu o oth pthwys is nssry (Fig. 4 h). Togthr, ths t init tht PPCM n us y n ngiogni imln n vsulr ysuntion, t lst in ronts. Pr-lmpsi n ri ysuntion To tst th i tht nti-ngiogni signlling n us ri ysuntion in prgnnt womn, w stui womn with pr-lmpsi, in whom VEGF signlling is ompromis owing to high srum lvls o nti-ngiogni sflt1 1 (s Supplmntry Tl 1 or ptint hrtristis). Cri untion ws vlut non-invsivly y msuring th myoril prormn inx (MPI; lso known s th Ti inx) n othr inis o ri untion with ri horiogrphy. MPI msurs th rltiv urtion o isovolmi ontrtion n rlxtion (Fig. 5), n is snsitiv mrkr o istoli untion 4.Womn with pr-lmpsi h mrkly inrs srum lvls o sflt1 (Supplmntry Fig. 5, P 5.5), s prviously shown 1. Notly, womn with pr-lmpsi lso h mrkly norml MPI (Fig. 5 n Supplmntry Tl, P 5.1) n E/E9 (Fig. 5 n Supplmntry Tl, P 5.), nothr snsitiv msur o ri istoli ysuntion tht omprs rly istoli mitrl nnulus (E9)ntrnsmitrl(E) low vloitis 5. Morovr, th MPI orrlt with lvls o irulting sflt1 (Fig. 5, R 5.59, MPI = / 1, sflt1 (pg ml 1 ) ECG 1, 1, IV IVRT MPI MPI NL PE MPI E/E NL PE sflt1 Figur 5 Womn with pr-lmpsi hv prss ri untion tht orrlts with irulting sflt1 lvls, n sflt1 uss ri ysuntion in mi., Smpl tring o horiogrphi tissu Dopplr imging. IV, isovolmi ontrtion tim; IVRT, isovolmi rlxtion tim.,, Elvt MPI () n E/E9 () in womn with pr-lmpsi (PE) vrsus norml (NL) prgnnis. P 5.1 n P 5., rsptivly., Elvt MPI orrlts with sflt1 lvls. R P 5.3., Elvt MPI in prgnnt mi int with novirus xprssing sflt1. P 5.1, n $ 5 or ll groups. P,.5 vrsus ontrol. 336 NATURE VOL MAY 1 1 Mmilln Pulishrs Limit. All rights rsrv

5 RESEARCH P 5.3). Elvt loo prssur in th pr-lmpti womn (Supplmntry Tl 1) is unlikly to xplin th worsning MPI, us MPI is thought to rlt ri untion inpnntly o loo prssur, n prgnnt womn with similr mil lvtions o loo prssur ut without pr-lmpsi hv norml ri untion 6. Inst, ths t suggst tht lvt sflt1 uss th istoli ysuntion. To tst this i irtly, sflt1 ws livr systmilly to prgnnt mi y intrvnous injtion o noviruss xprssing sflt1, n MPI ws xmin using high-rsolution murin horiogrphy. sflt1 us signiint inrss in MPI in ths mi within 1 ys (Fig. 5). Ths t, tkn togthr with pulish osrvtions in ptints riving nti-ngiogni thrpis 1,11, strongly suggst tht lvt sflt1 uss ri ysuntion in womn with pr-lmpsi. Although th lt vntriulr ysuntion rovrs ollowing livry in mny ptints, son insult in som womn proly pripitts PPCM. sflt1 uss riomyopthy n is high in PPCM Th ov osrvtions strongly support th i tht PPCM n inu y xss nti-ngiogni signlling, inluing th high xprssion o sflt1 uring lt gsttion sn oth in womn 7 n mi (Supplmntry Fig. 5, P 5.9). To tst this i irtly, sflt1 ws livr systmilly to nulliprous mi, s ov. PGC- 1 mi tht riv sflt1 vlop prooun ri ilur within 3 wks; ths mi h inrs ri wight n mrk rss in rtionl shortning on horiogrphy (Fig. 6 ). This ws ompni y mrk rop in vsulr nsity (Supplmntry Fig. 6), lthough not in lrgr vssls (Supplmntry Fig. 7). Wil-typ mi lso show signiint, though lss xtnsiv, rss in vsulr nsity n ri untion tr xposur to 3 wks o sflt1. Thus, sflt1 lon is suiint, vn in th sn o prgnny, to us rmti riomyopthy in th stting o hrt tht is unl to withstn th nti-ngiogni insult. To invstigt urthr whthr lvt sflt1 lvls in humns oul ontriuting to PPCM, plsm rom womn with PPCM ws quir 4 6 wks post-prtum n sflt1 lvls wr msur. sflt1 lvls usully rturn to norml within 48 7 h tr livry 8. sflt1 lvls wr lvt in lrg sust o ths PPCM ptints (P 5.), rmining up to iv- or tnol highr thn th lvls in ontrol prtiipnts (Fig. 6). Post-prtum sflt1 lvls n rmin slightly highr in sujts with pr-lmpsi 9,3, ut th lvls oun hr r notly highr. Thus, th inings r onsistnt with th i tht sustntil prntg o PPCM sujts hv n xpos to pr-lmpsi, n tht srtion o sflt1 prsists inppropritly post-prtum. In, in our own institution, 33% o th lst 75 ss o PPCM wr ssoit with pr-lmpsi (Fig. 6, ), mrkly mor thn th popultion rt o 3 5% (r. 31). Th prsisting xtr-plntl sour o sflt1 in th post-prtum prio is not known, n my inlu plntl rmnnts, irulting mononulr lls 3 or sh synytil miroprtils 33. Disussion Our stuy shows tht ngiogni imln in th hrt uring th pri-prtum prio my l to PPCM in mi n in humns. Th t init tht PPCM is us y two-hit omintion o, irst, systmi nti-ngiogni signls uring lt prgnny n, son, host susptiility mrk y insuiint lol pro-ngiogni ns in th hrt. Th irst hit xplins why PPCM is iss o th lt gsttionl prio, whih is prisly whn irulting nti-ngiogni tors suh s sflt1 pk in prgnny 1,34. Othr pthwys, suh s proltin or xss ngiotnsin II signlling, my lso involv 3,35. Th irst hit is lso wors in pr-lmpsi, whih is hrtriz y mrkly lvt sflt1 lvls. Assoitions twn pr-lmpsi n PPCM hv n wll oumnt in mny popultions 1,,36 41 (Supplmntry Tl 3). Intrstingly, som stuis involving womn o Arin snt hv not oun n ssoition twn hyprtnsiv isorrs o prgnny n PPCM 4, suggsting tht thr is thni vriility in th pthognsis o PPCM. It is lso possil tht PPCM with n without ssoit pr-lmpsi hv irnt pthognss 43. Ovrll, our t suggst tht lvt sflt1 lvls in pr-lmpsi ontriut to t lst th PPCM tht is ssoit with pr-lmpsi. W urthr propos tht lvt sflt1 lvls in t prsnt hllng to th myorium in ll prgnnis, thus xplining why th pri-prtum prio puts womn t risk o vloping hrt ilur, vn in th sn o pr-lmpsi. Intrstingly, othr situtions o lvt sflt1 (twin prgnnis) n rurrnt xposurs to sflt1 (multipl prgnnis) r lso strong risk tors or PPCM vn in th sn o pr-lmpsi,43,44. Only minority o womn with pr-lmpsi vlops PPCM, onsistnt with th xistn o son hit. Anorml PGC-1 untion is suh n vnt in ronts, n it my lso son hit in th s o humns. A numr o prviously intii prosss my lso onstitut this son hit, inluing myoritis, immun tivtion, virl intion n/or utontiois 43. Intrstingly, PGC-1 xprssion is rprss y inlmmtory stts in th hrt n lswhr 45,46, suggsting tht mny o th ov prosss tht r implit in PPCM my prtly onvrg on PGC-1. Consistnt with this, w oun rprss PGC-1 xprssion in ri smpls rom womn with PPCM (Supplmntry Fig. 8). Anorml STAT3 untion n ROS proution 3 n gnti prispositions 47 my lso ontriuting tors. In onlusion, th t prsnt hr support th i tht PPCM is prtly two-hit vsulr iss u to imlns in ngiogni signlling, n tht nti-ngiogni stts suh s pr-lmpsi or multipl gsttion sustntilly worsn th svrity o th iss. Our t my xplin why prgnny triggrs PPCM, n lso th longstning pimiologil osrvtion tht pr-lmpsi is risk tor or vloping PPCM. Pro-ngiogni thrpis suh s xognous VEGF11, or rmovl o sflt1 itsl 48, my thror niil in PPCM. 1.8 m mg mm HW/TL.3 m..1 IgG sflt1 sflt1 1, sflt1 (pg ml) 1 1, 1 All livris Pr-lmpsi Figur 6 sflt1 is suiint to inu riomyopthy in mi, womn with PPCM hv lvt sflt1 lvls, n prlmpsi s risk tor or PPCM.,Hrt wight:tiil lngth rtios (), horiogrphi rtionl shortning () n LVESD ()in mi injt with novirus xprssing sflt1, vrsus ontrols., Elvt sflt1 lvls in postprtum womn with PPCM. P 5..,, Prvln o pr-lmpsi mong ll livris () n mong womn with PPCM ()t Hrvr thing hospitls in th prvious 9 yrs. FS LVESD PPCM PPCM PPCM + pr-lmpsi 1 Mmilln Pulishrs Limit. All rights rsrv 17 MAY 1 VOL 485 NATURE 337

6 RESEARCH ARTICLE METHODS SUMMARY All niml xprimnts wr prorm oring to prours pprov y th Institutionl Animl Cr n Us Committ. Humn solul VEGF11 ws git rom Sios. Bromoriptin trtmnts wr rri out s prviously sri 3. Humn stuis wr pprov y th institutionl rviw or o Bth Isrl Donss Mil Cntr. Inorm onsnt ws otin rom ll sujts. Angiogni tor ssys wr prorm with ommrilly vill ELISA ssys (R&D systms). Full Mthos n ny ssoit rrns r vill in th onlin vrsion o th ppr t Riv 6 Sptmr 11; pt 13 Mrh 1. Pulish onlin 9 My Prson, G. D. t l. Priprtum riomyopthy: Ntionl Hrt, Lung, n Bloo Institut n Oi o Rr Disss (Ntionl Instituts o Hlth) workshop rommntions n rviw. J. Am. M. Asso. 83, ().. Sliw, K., Ftt, J. & Elkym, U. Priprtum riomyopthy. Lnt 368, (6). 3. Hilikr-Klinr, D. t l. A thpsin D-lv 16 kd orm o proltin mits postprtum riomyopthy. Cll 18, (7). 4. Row, G. C., Jing, A. & Arny, Z. PGC-1 otivtors in ri vlopmnt n iss. Cir. Rs. 17, (1). 5. Hnshin, C. & Spiglmn, B. M. Proxisom prolirtor-tivt rptor gmm otivtor 1 otivtors, nrgy homostsis, n mtolism. Enor. Rv. 7, (6). 6. Arny, Z. t l. Trnsriptionl otivtor PGC-1 ontrols th nrgy stt n ontrtil untion o ri musl. Cll Mt. 1, (5). 7. Arny, Z. t l. Trnsvrs orti onstrition ls to lrt hrt ilur in mi lking PPAR- otivtor 1. Pro. Ntl A. Si. USA 13, (6). 8. Arny, Z. t l. HIF-inpnnt rgultion o VEGF n ngiognsis y th trnsriptionl otivtor PGC-1. Ntur 451, (8). 9. Chinsomoon, J. t l. Th trnsriptionl otivtor PGC-1 mits xrisinu ngiognsis in skltl musl. Pro. Ntl A. Si. USA 16, (9). 1. Kirk, R. Bvizum n hrt ilur. Ntur Rv. Clin. Onol. 8, 14 (11). 11. Uriz, I., Chng, S. & Moslhi, J. Rvrsil riomyopthy ssoit with sunitini n sorni. N. Engl. J. M. 365, (11). 1. My, D. t l. Trnsgni systm or onitionl inution n rsu o hroni myoril hirntion provis insights into gnomi progrms o hirntion. Pro. Ntl A. Si. USA 15, 8 87 (8). 13. Crmlit, P. t l. Impir myoril ngiognsis n ishmi riomyopthy in mi lking th vsulr nothlil growth tor isoorms VEGF164 n VEGF188. Ntur M. 5, (1999). 14. Bolh, Y., Sukhtm, V. P. & Krumnhi, S. A. Angiogni imln in th pthophysiology o prlmpsi: nwr insights. Smin. Nphrol. 4, (4). 15. Wu, Z. t l. Mhnisms ontrolling mitohonril iognsis n rspirtion through th thrmogni otivtor PGC-1. Cll 98, (1999). 16. Crmlit, P. t l. Anorml loo vssl vlopmnt n lthlity in mryos lking singl VEGF lll. Ntur 38, (1996). 17. Frrr, N. t l. Htrozygous mryoni lthlity inu y trgt intivtion o th VEGF gn. Ntur 38, (1996). 18. Lhmn, J. J. t l. Proxisom prolirtor-tivt rptor otivtor-1 promots ri mitohonril iognsis. J. Clin. Invst. 16, (). 19. Giorno, F. J. t l. A rimyoyt vsulr nothlil growth torprrin pthwy is rquir to mintin ri untion. Pro. Ntl A. Si. USA 98, (1).. St-Pirr, J. t l. Supprssion o rtiv oxygn spis n nurognrtion y th PGC-1 trnsriptionl otivtors. Cll 17, (6). 1. Lvin, R. J. t l. Cirulting ngiogni tors n th risk o prlmpsi. N. Engl. J. M. 35, (4).. Bruh, C. t l. Ti-inxinptintswith mil-to-mort ongstiv hrt ilur. Eur. Hrt J. 1, (). 3. Ti, C. t l. Nw inx o omin systolin istoli myorilprormn: simpl n rprouil msur o ri untion stuy in normls n ilt riomyopthy. J. Criol. 6, (1995). 4. Poulsn, S. H., Jnsn, S. E., Nilsn, J. C., Mollr, J. E. & Egstrup, K. Sril hngs n prognosti implitions o Dopplr-riv inx o omin lt vntriulr systoli n istoli myoril prormn in ut myoril inrtion. Am. J. Criol. 85, 19 5 (). 5. Ksnr, M. t l. Utility o Dopplr horiogrphy ntissu Dopplr imging in th stimtion o istoli untion in hrt ilur with norml jtion rtion: omprtiv Dopplr-onutn thtriztion stuy. Cirultion 116, (7). 6. Mlhiorr, K., Suthrln, G. R., Bltv, A., Lirti, M. & Thilgnthn, B. Mtrnl ri ysuntion n rmoling in womn with prlmpsi t trm. Hyprtnsion 57, (1). 7. Vnktsh, S. t l. Solul noglin ontriuts to th pthognsis o prlmpsi. Ntur M. 1, (6). 8. Mynr, S. E. t l. Exss plntl solul ms-lik tyrosin kins 1 (sflt1) my ontriut to nothlil ysuntion, hyprtnsion, n protinuri in prlmpsi. J. Clin. Invst. 111, (3). 9. Wol, M. t l. Prlmpsi n utur riovsulr iss: potntil rol o ltr ngiognsis n insulin rsistn. J. Clin. Enorinol. Mt. 89, (4). 3. Sxn, A. R. t l. Inrs snsitivity to ngiotnsin II is prsnt postprtum in womn with history o hyprtnsiv prgnny. Hyprtnsion 55, (1). 31. Rmn, C. W. & Srgnt, I. L. Ltst vns in unrstning prlmpsi. Sin 38, (5). 3. Rjkumr, A. t l. Extr-plntl xprssion o vsulr nothlil growth tor rptor-1, (Flt-1) n solul Flt-1 (sflt-1), y priphrl loo mononulr lls (PBMCs) in normotnsiv n prlmpti prgnnt womn. Plnt 6, (5). 33. Rjkumr, A. t l. Trnsriptionlly tiv synytil ggrgts in th mtrnl irultion my ontriut to irulting solul ms-lik tyrosin kins 1 in prlmpsi. Hyprtnsion 59, (1). 34. Noori, M., Donl, A. E., Anglkopoulou, A., Hingorni, A. D. & Willims, D. J. Prosptiv stuy o plntl ngiogni tors n mtrnl vsulr untion or n tr prlmpsi n gsttionl hyprtnsion. Cirultion 1, (11). 35. Hul, C. A. t l. Agonisti ngiotnsin II typ 1 rptor utontiois in postprtum womn with history o prlmpsi. Hyprtnsion 49, (7). 36. Cruz, M. O., Brillr, J. & Hir, J. U. Upt on priprtum riomyopthy. Ostt. Gynol. Clin. North Am. 37, (1). 37. Elkym, U. Clinil hrtristis o priprtum riomyopthy in th Unit Stts: ignosis, prognosis, n mngmnt. J. Am. Coll. Criol. 58, (11). 38. Dmkis, J. G. & Rhimtool, S. H. Priprtum riomyopthy. Cirultion 44, (1971). 39. Witlin, A. G., Mi, W. C. & Sii, B. M. Priprtum riomyopthy: n ominous ignosis. Am. J. Ostt. Gynol. 176, (1997). 4. Amos, A. M., Jr, W. A. & Russll, S. D. Improv outoms in priprtum riomyopthy with ontmporry. Am. Hrt J. 15, (6). 41. Goln, S. t l. Evlution o th linil rlvn o slin lt vntriulr jtion rtion s pritor o rovry or prsistn o svr ysuntion in womn in th Unit Stts with priprtum riomyopthy. J. Cr. Fil. 17, (11). 4. Ftt, J. D., Christi, L. G., Crrwy, R. D. & Murphy, J. G. Fiv-yr prosptiv stuy o th inin n prognosis o priprtum riomyopthy t singl institution. Myo Clin. Pro. 8, (5). 43. Ntusi, N. B. & Myosi, B. M. Atiology n risk tors o priprtum riomyopthy: systmti rviw. Int. J. Criol. 131, (9). 44. Bolh, Y. t l. Twin prgnny n th risk o prlmpsi: iggr plnt or rltiv ishmi? Am. J. Ostt. Gynol. 198, (8). 45. Shilling, J. t l. Toll-lik rptor-mit inlmmtory signling rprogrms ri nrgy mtolism y rprssing proxisom prolirtor-tivt rptor otivtor-1 signling. Cir. Hrt Fil. 4, (11). 46. Trn, M. t l. PGC-1 promots rovry tr ut kiny injury uring systmi inlmmtion in mi. J. Clin. Invst. 11, (11). 47. Horn, B.D., t l. Gnom-wi signiin n rplition o th hromosom 1p11. lous nr th PTHLH gn or priprtum riomyopthy. Cir. Criovs. Gnt. 4, Thhni, R. t l. Pilot stuy o xtrorporl rmovl o solul ms-lik tyrosin kins 1 in prlmpsi. Cirultion 14, (11). Supplmntry Inormtion is link to th onlin vrsion o th ppr t Aknowlgmnts S.R. is support y Hrvr Fulty DvlopmntnDivrsity Awr. S.S is support y John Hly Whit grnt. G.C.R. is support y Mrk Fllowship. M.R.H. is support y Hrvr Ctlyst, th Clinil n Trnsltionl Sin Cntr, n Hrvr Univrsity n ilit mi hlth r ntrs. S.A.K. is ninvstigtoro th HowrHughsMil Institut. Z.A. issupport y th NHLBI, th Smith Fmily Fountion, th Ellison Mil Fountion, th Mrh o Dims Fountion, n th Hrvr Stm Cll Institut. Author Contriutions I.S.P. prorm th mjority o th mous xprimntl work, with th ssistn o G.C.R, L.L., N.K. n C.F. Th linil MPI stuy ws prorm y S.R., S.S., J.S.R., M.R.H., J.M., F. M. n P.H. sflt1 msurmntswr prormys.r. MPI stuis wr prorm y E.V.K. n S.D.B. Th nothlil migrtion stuis wrprormyl.l. n C.J. Smpls romwomnwith PPCMwrproviy J.B., F..M., I.T. n D.H.-K. Ths uthors lso provi input on th mnusript. Th stuy ws oniv n suprvis y S.A.K. n Z.A. Th xprimntl prours wr sign y Z.A., who lso wrot th mnusript. All uthors r n pprov th inl mnusript. Author Inormtion Rprints n prmissions inormtion is vill t Th uthors lr ompting innil intrsts: tils ompny th ull-txt HTML vrsion o th ppr t Rrs r wlom to ommnt on th onlin vrsion o this rtil t Corrsponn n rqusts or mtrils shoul rss to Z.A. ([email protected]) or D.H.-K (hilikr.nis@mh-hnnovr.). 338 NATURE VOL MAY 1 1 Mmilln Pulishrs Limit. All rights rsrv

7 RESEARCH METHODS Animl stuis. All niml xprimnts wr prorm oring to prours pprov y th Institutionl Animl Cr n Us Committ. Mi ring lox llls o PGC-1 lnking xons 3 n 4, n mi ontining th -MHC::CRE trnsgn wr gits rom B. Spiglmn 49 n M. Shnir 5, rsptivly. Mi wr mintin on stnr ront how it with 1-h light n rk yls. For murin horiogrphy, th hst hir ws rmov with topil piltory gnt, n two-imnsionl imgs wr visuliz using Vivi FiV horiogrphy systm (GE Mil Systms) on mi tht wr not nsthtiz. Prstrnl short-xis projtions wr visuliz n M-mo rorings t th mi-vntriulr lvl wr ror. Hrt rt, LVEDD n LVESD wr msur in t lst thr ts rom t lst thr rorings n vrg, n lt vntriulr rtionl shortning ws thn lult (rtionl shortning 5 (LVEDD LVESD)/LVEDD). For th highrsolution MPI stuis, VisulSonis 1 horiogrphy mhin ws us on mi nsthtiz with isolurn, n th MPI ws lult using th mnuturr s sotwr progrm. SPE/ imging o mi ws prorm y th Longwoo Ar Smll Animl Imging Fility (SAIF). For th VEGF trtmnt stuis, humn VEGF11 (1 mgkg 1 ) ws injt suutnously ily, vrsus slin s th ontrol. For th romoriptin stuis, romoriptin ws to th rinking wtr. Mi wr r strting t th g o 8 wks whil riving ithr romoriptin in th rinking wtr or ily suutnous VEGF11, or oth. Clls n rgnts. All rgnts wr rom Sigm, unlss othrwis init. Humn solul VEGF11 ws git rom Sios. Stining o pillris ws prorm using nti-cd31 ntioy (BD Phrmingn) or isoltin B4 (Vtor L). Quntiition o pillris ws prorm omputtionlly, using Voloity sotwr (Improvision, PrkinElmr), on thr rnom ils hosn rom th sptum o trnsvrs stions rom th mi-hrt. Stining o rtriols ws prorm using nti-sma ntioy (Snt Cruz) n quntii similrly, using rnom low-powr ils. All quntiitions wr prorm linly. Isoltion n ultur o primry NRVMs ws prorm s sri. Clls wr int with novirus t multipliity o intion o 13 to 33,nmRNAxprssion ws msur 4 or 48 h ltr. Th novirus xprssing PGC-1 n sflt1 hv n sri 51,5. Proltin, VEGF n sflt1 ELISA ssys wr rom R&D Systms. Th thioritui i rtiv sustns (TBARS) ssy ws prorm on ri xtrts oring to th mnuturr s instrutions (Cymn). Gn xprssion stuis. Totl RNAs wr isolt rom mous tissu or ultur lls using th Trizol mtho (Invitrogn). Smpls or rl-tim polymrs hin rtion (PCR) nlyss wr rvrs trnsri (Invitrogn), n quntittiv rl-tim PCR rtions wr prorm on th omplmntry DNAs in th prsn o luorsnt y (SYBR grn) on BioR CFX 384 Touh rl-tim PCR ttion systm. DNA prouts o th xpt siz wr onirm or h primr pir. Enothlil migrtion ssy. NRVMs in 4-wll plts wr int with novirus xprssing GFP or PGC-1 or 34 h. ovin srum lumn (BSA) or sflt1 (1 ng ml 1 ) ws to th mi or 1 h. Thn, lls o HUVECs t wr put on th uppr omprtmnt o trnswlls (8.-mm por siz, Corning no. 34) pr-wrm with EBM mi ovrnight t 37 uc. HUVEC migrtion to th lowr omprtmnt o trnswlls ws msur tr 1 h. Migrt HUVECs wr ix with 4% prormlhy in PBS or min t 5 uc, lls rmin on th uppr omprtmnt wr rmov with otton sw. Clls wr lok with 5% BSA in PBS.% Twn (PBST) n stin with phlloin luorsin isothioynt in PBST or 4 h to visuliz ilmntous tin. Trnswll insrts wr wsh thr tims in PBST n mount onto slis with 49,6-imiino--phnylinol (DAPI) mounting mium. Humn stuis. Th institutionl rviw or o Bth Isrl Donss Mil Cntr in Boston pprov this stuy. Eligil womn wr nroll tr proviing writtn inorm onsnt rom Novmr 9 to My 1. Prgnnt womn t lst 18 yrs o g with singlton prgnny o t lst 4 wks n lss thn 41 wks, n ithr ignosis o pr-lmpsi or without ny hyprtnsiv isorr o prgnny wr ligil. Exlusion ritri inlu pr-xisting riovsulr iss, pulmonry iss n non-gsttionl its mllitus. Prtiipnts wr rruit tr mission to lour n livry, th nt-prtum loor or uring routin prntl visit. All linil t wr tkn rom mil rors. Th ignosis o pr-lmpsi ws s on th Ntionl High Bloo Prssur Eution Progrm Working Group inition, lso nors y th Amrin Congrss o Osttriins n Gynologists (ACOG). A mtrnl tl miin spilist onirm ll ignoss. Arhiv plsm smpls rom sujts with PPCM hv n prviously sri 3.Ptintsin oth stuis wr prominntly Cusin. Rtrosptiv nlyss o PPCM n pr-lmpsi in th Hrvr thing hospitls wr prorm using th Hrvr ShrHlth Rsrh Inormtion Ntwork (SHRINE) 53, -intii rpository o ggrgt ptint inormtion. Humn horiogrphy. Bsi trnsthori horiogrms wr prorm using Simns X-3 (Mountinviw) mhin, y two xprt horiogrphrs using P5-1 Trnsur. Imgs wr otin with th ptint lying in th lt ltrl uitus position n wr rport oring to th Amrin Soity o Ehoriogrphy guilins. Imgs wr stor in inloop ormt with thr ri yls o non-omprss t with ltroriogrm inormtion. Th horiogrphrs prorm omprhnsiv xmintion, whih inlu omplt two-imnsionl n olour low Dopplr ssssmnt o th lt vntril, right vntril n intr-ri vlvs. Spiilly: jtion rtion with visul quntittiv stimtion; trns-mitrl puls wv Dopplr (E n A wvs n lrtion tim); Dopplr tissu img (oth mil n ltrl mitrl nnulus wr intrrogt, n th inl vlu o pk vloity o E ws lult s th vrg o thr vloitis t h lotion); MPI, with th lultion prorm o-lin using Simns Syngo DICOM viwing sttion (Mountinviw). Th horiogrms wr -intii or lulting MPI. Ejtion rtion, MPI n E/E9 rtios wr lult. Eh img ws nlys lin y on o two horiogrphrs. Angiogni tor ssys. Womn onsnt to loo rw t th tim o th horiogrm. All smpls or th MPI stuy wr ollt in th nt-prtum or th livry, whrs smpls in th PPCM stuy wr ollt 4 6 wks post-prtum. Th smpls wr ntriug t 1,9g or 8 min n plsm ws ollt n stor t 8u C. Smpls wr rnomly orr n nlys y singl prson in lin shion. ELISA ssys or sflt1 wr prorm with ommrilly vill kits (R&D systms). All ssys wr prorm in uplit n vlus wr vrg. I.% irn ws osrv twn uplit vlus, th smpls wr r-nlys. Dt n sttistil nlysis. SAS 9. (SAS institut) ws us or t nlysis. All tsts wr two si, n P vlus o lss thn.5 wr onsir sttistilly signiint. Dt r prsnt s mn 6 stnr rror, or min n intrqurtil rngs, s init. Comprisons wr m using th two-til Stunt s t-tst or th non-prmtri Mnn Whitny tst, s init. 49. Hnshin, C. t l. Anorml gluos homostsis in skltl musl-spii PGC-1 knokout mi rvls skltl musl-pnrti ll rosstlk. J. Clin. Invst. 117, (7). 5. Agh, R. t l. Gn romintion in postmitoti lls. Trgt xprssion o Cr romins provoks ri-rstrit, sit-spii rrrngmnt in ult vntriulr musl in vivo. J. Clin. Invst. 1, (1997). 51. Puigsrvr, P. t l. A ol-inuil otivtor o nulr rptors link to ptiv thrmognsis. Cll 9, (1998). 5. Kuo, C. J. t l. Comprtiv vlution o th ntitumor tivity o ntingiogni protins livr y gn trnsr. Pro. Ntl A. Si. USA 98, (1). 53. Wr, G. M. t l. Th Shr Hlth Rsrh Inormtion Ntwork (SHRINE): prototyp rt qury tool or linil t rpositoris. J. Am. M. Inorm. Asso. 16, (9). 1 Mmilln Pulishrs Limit. All rights rsrv