Insulin regulation of heart function in aging fruit flies

Transcription

1 4 Nture Pulishing Group Insulin regultion of hert funtion in ging fruit flies Roert J Wessells, Erin Fitzgerld, Jmes R Cypser, Mr Ttr & Rolf Bodmer Insulin-IGF reeptor (InR) signling hs onserved role in regulting lifespn, ut little is known out the geneti ontrol of delining orgn funtion. Here, we desrie progressive hnges of hert funtion in ging fruit flies: from one to seven weeks of fly s ge, the resting hert rte dereses nd the rte of stress-indued hert filure inreses. These ge-relted hnges re minimized or sent in long-lived flies when systemi levels of insulin-like peptides re redued nd y muttions of the only reeptor, InR, or its sustrte, hio. Moreover, interfering with InR signling exlusively in the hert, y overexpressing the phosphtse dpten or the forkhed trnsription ftor dfoxo, prevents the deline in rdi performne with ge. Thus, insulin-igf signling influenes ge-dependent orgn physiology nd senesene diretly nd utonomously, in ddition to its systemi effet on lifespn. The ging fly hert is model for studying the genetis of ge-sensitive orgn-speifi pthology. Signl trnsdution y the InR pthwy hs een implited in lifespn regultion of multiple verterte nd inverterte niml models nd is therefore thought to e widely onserved mehnism in the ontrol of ging mong higher eukryotes. Beuse funtionl ging in inverterte models is rrely doumented 4, however, little is known out how this signling pthwy extends lifespn or how these longevity ftors ontrol the deline of orgns nd tissues. Do geneti mnipultions tht extend life retrd the deteriortion of orgn performne, nd if they do, to wht extent do the genes t through systemi physiology or through tion speifilly in the ffeted orgn? Conversely, do geneti mnipultions of longevityssoited genes exlusively in the orgn retrd its deteriortion? And in turn, how do suh orgn-speifi mnipultions ffet the overll lifespn nd mortlity rte of n orgnism? These questions re est ddressed in model orgnism in whih the mehnisms of senesene t the level of orgn funtion n e genetilly mnipulted nd seprted from systemi regultion of ging. Drosophil melnogster is well suited for this purpose, not only euse of its short lifespn nd geneti verstility, ut lso euse it ontins simple orgn with relevne to ging, the hert, whih is formed y highly onserved moleulr mehnisms nd undergoes, s in humns, physiologil hnges s it ges. We used two mesures to ssess ge-dependent physiologil hnges in rdi funtion tht our in D. melnogster: the hert rte, whih progressively dereses, nd the likelihood of stressindued hert filure, whih inreses two to four times over the dult lifespn 9,. These ge-relted hnges in hert funtion re virtully olished in flies when lifespn is extended either y redutions in systemi levels of insulin-igf like lignds or y muttions of InR or its insulin-reeptor sustrte hio. In ddition, impeding insulin-igf signling exlusively in the hert, y ugmenting the tivity of dpten or dfoxo, similrly hlts the norml ge-dependent deline in rdi performne, nd rdi-speifi tivtion of the InR pthwy uses the herts of young flies to perform in mnner typil of older flies. These dt provide first diret evidene tht insulin-igf signling impts ge-dependent orgn physiology nd senesene y ting utonomously in the orgn nd independently of systemi ontrol of lifespn. Furthermore, our oservtions show how insulin-like peptide signling, in ddition to its nonutonomous ontrol of lifespn, n serve s its own seondry hormone system in the tissue-utonomous ontrol of senesene. The geneti nlysis of rdi ging in the fly provides new pproh to eluidting the ontrol mehnisms of senesene nd orgn-speifi pthologies ssoited with it. RESULTS Age-dependent hnges in rdi performne of fruit flies Despite divergene in form nd funtion etween rthropod nd verterte herts, in insets, s in humns, rdi performne delines with ge 8. Thus, s is the se in hert development, the ell-signling nd hormone-regultory networks tht ontrol these prmeters during the ging proess will proly e onserved. To study the ontrol of orgn senesene in the geneti ontext of D. melnogster, we mesured hert rte nd response to rdi stress (Figs. ). The resting hert rte ws onstnt nd rhythmi t lte lrvl erly pupl stges (Fig.,), ws higher in young dults thn in lrve (Fig.,d) nd then grdully delined with dvning ge (P o.; Figs. d nd e,f nd dt not shown) 9,. Beuse the inrese in hert filure in the ging humn popultion ours preferentilly under duress, we imed to pproximte rdi stress in flies nd mesured the rdi response to ute stress in progressively older flies. For this purpose, we pplied externl eletril stimuli to pe fly s hert to high rte for predetermined mount of time (Fig. g,h). We lulted the perentge of herts The Burnhm Institute, Center for Neurosiene nd Aging, 9 Torrey Pines Rod, L Joll, Cliforni 9, USA. Deprtment of Eology nd Evolutionry Biology, Box G-W, Brown University, Providene, Rhode Islnd 9, USA. Correspondene should e ddressed to R.B. (rolf@urnhm.org). Pulished online Novemer 4; doi:.8/ng4 NATURE GENETICS VOLUME [ NUMBER [ DECEMBER 4

2 4 Nture Pulishing Group Figure D. melnogster hert rte hnges with ge. () A demonstrtion of edge-tring to delinete hert movements. The top tre represents the left edge of the hert (ompre red tringle to tht in ); the enter tre represents the right edge of the hert (ompre lue tringle to tht in ). The lower tre represents the sutrtive distne etween the two edges. () High-mgnifition imge of lrvl hert in distole. () High-mgnifition imge of lrvl hert in systole. (d) Plot of hert rtes from n outred wild-type (WT; yw Cnton S) strin y ge. Hert rte mesurements were mde t C nd 9 C for onseutive weeks. All flies were rered t the sme temperture ( C). Hert rte mesurement for oth genders t eh temperture is progressively lower with ge (ge min effet, F rtio ¼ 4, P o.). Temperture hs signifint effet on hert rte (temperture min effet, F rtio ¼ 4, P o.), lthough t dvned ges, flies tested t higher tempertures progressively lose their ility to et fster thn ohorts tested t lower temperture. n 4 for eh smple point. tht filed in response to this stress, s defined y rdi rrest or firilltion during or immeditely fter tretment (Supplementry Tles online). As expeted of progressive deteriortion in orgn funtion, the rte of ping-indued hert filure inresed s ontinuous funtion with ge (Fig. ) in oth sexes nd t two test tempertures (logisti regression: ge, w ¼ 48, P o.; temperture, w ¼., P ¼.8). The progression of filure risk signifintly deelerted t older ges mong femles (logisti regression: femles: ge, w ¼., P o.; (ge), w ¼.4, P ¼.; mles: ge, w ¼.4, P ¼.4; (ge), w ¼., P ¼.4), whih oinides with the well-hrterized deelertion of mortlity rte in fruit flies tht typilly ours etween nd weeks of ge 4,. The oinident leveling-off of these trjetories implies tht mortlity deelertion t dvned ges my result t lest in prt from redution in the rte of physiologil deteriortion. Redution in insulin-igf signling hlts rdi ging With this rdi model, we investigted rdi funtionl hnges in flies rrying muttions tht inrese life expetny. Gene muttions Hert rte (Hz) 4 tht redue insulin-igf signling improve lifespn in orgnisms rnging from the nemtode Cenorhditis elegns to mmmls. D. melnogster hs single insulin-like reeptor enoded y InR, nd signling through this reeptor regultes growth nd metolism, s well s reprodution nd lifespn 4,8. Speifi heterolleli genotypes of InR nd null muttions of the insulin-reeptor sustrte enoded y hio improve lifespn up to 8% nd 48%, respetively, y reduing mortlity rte ross ll dult ges 4,9,. We exmined ge-dependent rdi performne in flies rrying these muttions nd rrying lleles of InR tht influene growth ut not survivl (M.T. nd M. Plmer, unpulished oservtion). Among young flies, none of the InR lleles used signifint hnge in pingindued rdi filure or in hert rte (t week of ge; Figs. 4 d nd Supplementry Tles online). In old flies ( weeks of ge), however, the long-lived InR genotype, InR /E9, resulted in ping-indued hert filure rte s low s tht in young flies (Fig. 4d). Moreover, the tendeny towrd derese in hert rte with ge ws lso forestlled in these InR /E9 dults (Fig. ). d... WT mle () WT mle (9) WT femle () WT femle (9) Pup g d -week-old dult h e f -week-old dult s s Hz Figure D. melnogster hert ontrtions under norml nd stimulted onditions. () Wveform from pupl hert. () Power spetrum mesurement of pek frequeny of the hert wveform from. () Wveform from -week-old dult hert. (d) Power spetrum mesure of pek frequeny of the hert wveform from. (e) Wveform from -week-old dult hert. (f) Power spetrum mesure of pek frequeny of the hert wveform from e. (g) Wveform of hert undergoing externl eletril ping. (h) Wveform of hert fter ping hs esed. VOLUME [ NUMBER [ DECEMBER 4 NATURE GENETICS

3 4 Nture Pulishing Group WT mle () WT mle (9) WT femle () WT femle (9) 4 Figure Hert filure s funtion of ge fter externl eletril ping from outred wild-type offspring (WT; yw Cnton S). Experiments were done t C ndt9c for weeks. Test temperture lone hd no effet on filure rte (w ¼., P ¼.8). Ping-indued filure rte ws ge-dependent for oth genders t oth tempertures (w 4 4, P o.). There ws no signifint effet of gender on ge-dependent filure rtes during the first weeks of dulthood (logisti regression, sex-y-ge, w ¼.4, P ¼.49), ut femles hd somewht lower filure rte etween nd weeks of ge thn mles (logisti regression, sex-y-ge, (w ¼., P ¼.). n 4 9 for eh smple point. Next, we exmined flies homozygous with respet to null muttion in hio, the D. melnogster homolog of insulin-reeptor sustrte genes 4 (ref. ). In stoks where eh hio genotype (+/+, +/ nd /) segregtes mong silings in ommon geneti kground, hio / flies re smll nd live B% longer thn wildtype flies 9,.Likelong-livedInR /E9 flies, ging hio / flies hd low rtes of ping-indued hert filure nd rrest nd their hertet frequeny did not derese with ge (Fig. nd Supplementry Tles online). Heterozygotes, whih hve lifespn intermedite etween those of wild-type nd hio / flies, retined the wild-type pttern of stress-indued hert filure, nd their hert et deresed with ge (Fig. ). Mles nd femles of eh hio genotype hd similr ge-dependent ptterns in ll hert funtion ssys (Supplementry Tle online nd dt not shown). If redution in insulin signling n inrese longevity nd retrd rdi ging, then redued systemi level of insulin-like lignd should lso slow demogrphi ging nd rdi funtionl deline. Therefore, we lted the insulin-produing ells (IPCs), group of neurons oexpressing the insulin-like lignds Ilp, Ilp nd Ilp tht re loted in the hed nd projet to the trunk of the fly,. Altion of these IPCs delyed development nd yielded smll dults, s previously reported. Although men survivl ws not improved when the IPCs were lted (Fig. e), prmetri nlysis of the mortlity funtion showed tht the IPC-lted flies experiene high ge-independent risk of deth in ddition to low ge-dependent level of mortlity (Fig. f); their demogrphi ging is retrded ut msked t young ges, perhps euse the defiieny in insulin-lignd experiened during development is generlly deleterious. Among young IPC-lted dults the risk of rdi filure is somewht elevted (Fig. d). This filure rte inresed only slightly with ge, however, nd remined fr lower thn tht in ontrol herts (P o.; Fig. d), s would e expeted if insulin signling regultes rdi funtionl ging. Insulin-IGF signling nd rdi-utonomous ging Bsed on reent studies with invertertes s well s with mie, insulin-igf regultion of lifespn is thought to involve hnges of seondry hormone tivities nd of tissue physiology in response to hnges in insulin signling 4 9. From this view we expet redued insulin signling to influene rdi ging through systemi nd seondry systems. Therefore, we sked whether mnipulting the reeption of insulin-igf signls speifilly in the hert is suffiient to lter its funtionl ging hrteristis. To ugment insulin-igf signling in the hert, we overexpressed wild-type InR with rdi-speifi Gl4 driver (GMH; Fig., nd Supplementry Video online). This geneti mnipultion deresed the hert rte (Fig. ) nd inresed the ping-indued filure rte in young flies (Fig.,,e), ut used little progressive hnge with ge. Although ge still hd sttistilly signifint effet for oth mesures in these flies (see legend to Fig. ), the mgnitude of the gedependent hnge ws mrkedly redued (Figs. nd e; ompre with Figs. d nd ). In ontrst, when rdi-speifi insulin-igf signling ws ntgonized y expression of dpten, phosphtse tht inhiits the PI-kinse required for insulin-reeptor signl trnsdution,,the ge-dependent hnges in hert rte nd indued hert filure rte were onsiderly redued (Figs. nd ), similr to the redution seen in hio-null mutnts (Fig. ). Additionlly, rdi-speifi d... InR +/+ InR +/E9. InR 4/+ InR 4/E9 InR /+ InR /E9 InR /+ InR /E weeks weeks weeks weeks Figure 4 Redution in insulin reeptor signling improves hert performne t dvned ges. The effets of three different EMS-generted muttions nd wild-type llele of InR on ping-indued filure rte in young nd old flies were ompred. Eh muttion ws rossed to InR E9, nd the filure rtes of the resulting progeny were ompred with those of InR TM/+ s n internl ontrol. Dt re grouped in four pnels ( d), showing filure rtes of -week-old (lk r) nd -week-old (gry r) flies. The llele InR is long-lived when omplemented with InR E9 (reltive ge-speifi mortlity is redued y ftor of more thn ; M.T., unpulished oservtions). At week of ge, the filure rte of trns-heterozygotes (InR */E9 ) did not systemtilly differ from tht of the heterozygote or the wild-type. At weeks, ll lleles with norml lifespn (InR +, InR 4 nd InR ; ) hd similrly inresed rtes of hert filure (logisti regression: genotype min effet, w ¼., P 4.4; ge min effet, w ¼ 9., P o.; genotype-y-ge, w ¼., P 4.4). In ontrst, with ge, the long-lived InR /E9 (d) flies did not inrese their rte of rdi filure (logisti regression: genotype, w ¼, P ¼.; ge, w ¼., P ¼.8; genotype-y-ge, w ¼, P ¼.). Brs represent omined mle nd femle dt. NATURE GENETICS VOLUME [ NUMBER [ DECEMBER 4

4 4 Nture Pulishing Group +/+ +/ /.... Hert rte (Hz) hio / hio /+ InR /E9 4 weeks Survivorship.4. Control +/+ Control UAS-rpr /+ Control Ilp-Gl4 /+ Ilp-Gl4;UAS-rpr 4 Age (d) weeks d yw UAS-rpr Ilp-Gl4 e f.9 yw yw.8 Ilp-Gl4. UAS-rpr.8... * * In mortlity rte 4 hio / hio /+ InR /E9 Control Ilp-Gl4 4 Figure Redution in insulin-igf signling improves hert performne t dvned ges. () Plot of hert rte y ge for three long-lived genotypes over weeks. hio / mutnts hve miniml ut sttistilly signifint deline in hert rte with ge (F rtio ¼ 9., P ¼.). hio /+ mutnts show lrger deline in hert rte with ge (F rtio ¼, P o.), similr to the wild-type in Figure d. InR /E9 flies hve n intermedite deline in hert rte with ge (F rtio ¼ 48, P ¼.). n 4 for eh smple point. () Ping-indued filure rtes for hio / flies (n ¼ 8), ompred with hio /+ (n ¼ 4) flies nd oisogeni hio +/+ ontrols (n ¼ 4) t (lk) nd (gry) weeks of ge. hio +/+ nd hio +/, ut not hio /, flies hd inresed rdi filure rte with ge (logisti regression, genotype w ¼ 4, P o.; ge w ¼, P o.; genotype-y-ge, w ¼., P ¼.). Dt of two independent isogenized hio strins nd oth sexes re omined. () Plot of ping-indued filure rte y ge for three long-lived genotypes. In n independent repli test using the sme genotype fly lines s in Figures 4 nd,, filure rte ws mesured ross -week time ourse. hio /+ flies showed signifint ge-dependent inrese in filure rte (w 4, P o.). InR /E9 flies lso showed slight inrese in filure rte with ge (w ¼.9, P ¼.), wheres hio / flies showed no evidene of n ge-dependent hert filure rte (w ¼., P ¼.). As there ws no signifint gender effet for ny of the three genotypes, mle nd femle dt were omined. n 4 for - to -week smple points; n 4 for - nd -week smples. (d) Ping-indued filure rte of flies with ltion (y reper, rpr) of the IPCs expressing Ilp). At week nd weeks of ge, femles with ltion of IPCs (UAS-rpr;Ilp-Gl4) were ompred with ontrols with intt IPCs inluding prentl yw nd segregting UAS-rpr yw nd Ilp-Gl4 yw. The rte of rdi filure inreses with ge in oth groups, ut this hnge is signifintly less in IPC-lted flies (logisti regression: IPC ltion min effet w ¼., P ¼.; ge min effet w ¼ 8, P o.; IPC ltion-y-ge effet, w ¼., P ¼.). (e,f) Demogrphi ging of femles with lted IPCs (UAS-rpr;Ilp-Gl4) mesured s survivorship (in e reltive to eh ontrol genotypes) nd mortlity rte (in f reltive to ontrols with intt IPCs omined). Medin survivl did not differ sustntilly mong genotypes. The mortlity of the IPC-lted femles is est desried y Mkehm-Gompertz, where mortlity m x ¼ C + Aexp(Bx); the full model (C ¼.4, A ¼., B ¼.) is plotted s dshed line; the Gompertzonly omponent (Aexp(Bx)) is plotted s solid thin line, whih differs t eh prmeter from the Gompertz mortlity (hevy line) for the omined IPC-intt ontrols (P o.). Age (d) 4 8 expression of dfoxo, forkhed fmily trnsription ftor whose tivity is inhiited y insulin signling,,, ompletely olished the ge-ssoited inrese in filure rte (Fig. d). We otined similr results with expression of wild-type dfoxo or of dfoxo mutted t the three phosphoryltion sites trgeted y insulin signl trnsdution. Notly, we did not oserve n inrese in filure rte even t weeks of ge, when mny of the flies hve died, implying tht interferene with InR signling rogtes the deline of the hert s performne throughout life. DISCUSSION Tken together, these dt indite tht insulin signling in rdi tissue diretly influenes its funtionl ging, nd tht impeding this orgn-speifi response is suffiient to slow ge-relted hnges in rdi funtionl prmeters. We lso note tht none of the geneti mnipultions used hnge in hert rte t pupl stge (Supplementry Tle online nd dt not shown), suggesting tht the oserved phenomen re dult-speifi, lthough the possiility tht onsequenes of ltered development my mnifest themselves only in dults nnot e formlly exluded t this point. Even though hertspeifi mnipultion of insulin-igf signl trnsdution n slow rdi ging diretly nd utonomously, hnge in systemi insulin signling proly influenes orgn performne through dditionl venues s well. Indeed, long-lived InR or hio mutnts re defiient in dult synthesis of juvenile hormone (M.-P. Tu, C.-M. Yin & M.T., unpulished results), nd defiieny of this hormone is suffiient to inrese lifespn in numer of insets 9. Notly, we found tht the low filure rte of old hio mutnts in response to eletril ping ws resued (reverted to wild-type levels) y exposure to n exess of the insulin-dependent seond messenger, juvenile hormone, during dult stges only (R.J.W., M.T & R.B., unpulished oservtions). Therefore, insulin hs the potentil to influene rdi ging oth through utonomous effets in hert tissue nd through effets on seondry hormones. As expression of dpten or dfoxo in the hert retrds funtionl ging in n orgn-utonomous mnner, we my now egin to ddress 8 VOLUME [ NUMBER [ DECEMBER 4 NATURE GENETICS

5 . 4 Nture Pulishing Group Emryoni hert GMH(GFP) n outstnding prolem in the iology of ging: how would life expetny hnge if n intervention meliortes the progressive pthology of one speifi orgn or system? We found tht there ws no differene in lifespn etween flies with rdi-speifi overexpression of InR nd flies with rdi-speifi overexpression of dpten (Fig. 8). Thus, mnipulting rdi performne does not neessrily lter lifespn in flies, ut insted llows studies of orgnspeifi senesene unoupled from overll ging. Adult hert Hert rte (Hz).. UAS-dPTEN GMH UAS-InR GMH UAS-dFOXO( ) GMH 4 Figure Hert-speifi mnipultion of insulin-reeptor signling ffets ge-relted hnges in rdi physiology. () Emryoni expression of GMH-Gl4 shown with GFP. () Adult dominl expression pttern of GMH-Gl4 shown with GFP (see Supplementry Video online). () Hert rte of flies undergoing hert-speifi overexpression of genes in the insulin signling pthwy plotted ginst ge. Adult progeny of UAS-dPTEN GMH-Gl4 nd UAS-dFOXO GMH-Gl4 show onsistently high hert rte ross ll ges (men hert rtes ¼..8 Hz), whih delines slightly with ge for dfoxo (F rtio ¼ 8, P o.) ut not for dpten (F rtio ¼., P ¼.4). Progeny from UAS-InR GMH-Gl4 show onsistently low hert rte ross ll ges (men hert rte ¼. Hz), with no effet of ge (F rtio ¼.9, P ¼.4). In ll ses, mles nd femles do not differ nd their dt re omined. n 4 for eh smple point yw GMH weeks d UAS-InR UAS-InR yw GMH weeks UAS-dPTEN UAS-dPTEN UAS-dFOXO( ) UAS-dFOXO( ) yw GMH.9 yw GMH weeks weeks In ontrst to the ove finding with rdi-speifi mnipultion of InR signl trnsdution, impeding insulin signls in the hed ft ody, y expression of dfoxo, is ple of inresing lifespn, similr to Df in the entrl nervous system nd Df or Foxo in the intestine of C. elegns,9. Mnipulting insulin signling in the hed ft ody lso dereses neuronl insulin trnsription, implying tht insulin itself is seondry hormone ting diretly on peripherl tissues. Here we show tht ltion of the IPCs is suffiient to redue UAS-dPTEN GMH UAS-dFOXO GMH UAS-InR GMH 4 Figure Hert-speifi mnipultion of insulin-igf signling lters hert performne utonomously with ge. () Rte of hert filure fter externl ping of independent wild-type strins. s t week re similr (% to %) nd inresed t nd weeks ( % nd %, respetively; within eh genotype, t-test, P o.). n ¼ for - nd -week smples; n ¼ for -week smples; see Figure for similr filure rtes otined from progeny of wild-type ross yw Cnton S. The deline in rdi performne is eqully pronouned if rdi rrest is onsidered seprtely (P o.; Supplementry Fig. online). () in progeny from the ross UAS-InR GMH nd from the wild-type ontrol UAS-InR yw. Filure rte inreses with ge irrespetive of genotype (logisti regression: ge, w 4, P o.4; genotype-y-ge, w ¼.4, P ¼.; n 4 for eh smple). Note, however, the inresed filure rte t week of UAS-InR GMH ompred to UAS-InR yw. () in progeny from UAS-dPTEN GMH-Gl4 nd from the wild-type ontrol UAS-dPTEN yw. of UAS-dPTEN GMH-Gl4 progeny does not inrese with ge (w ¼.89, P ¼.), wheres the filure rte of the UAS-dPTEN yw progeny is ge-dependent (w ¼ 9., P ¼.; n 4 for eh smple; see lso Supplementry Fig. online). (d) of progeny from UAS-dFOXO GMH nd from the wild-type ontrol UAS-dFOXO yw. of UASdFOXO GMH did not signifintly inrese with ge (w ¼., P ¼.), wheres the filure rte of UAS-dFOXO yw is ge-dependent (w ¼ 8, P o.; ge-y-genotype effet, w ¼.8, P ¼.; see lso Supplementry Fig. online). (e) Independent repli experiment monitoring the filure rte weekly for weeks of flies with hert-speifi overexpression of InR, dpten or dfoxo. UAS-InR GMH-Gl4 hve high filure rte t ll ges (men filure rte ¼.8). In ontrst to hert-speifi InR overexpression, oth UAS-dPTEN GMH nd UAS-dFOXO GMH progeny showed low filure rte t ll ges (men filure rte ¼. nd., respetively), whih does vry with ge (w ¼., P ¼. nd w ¼.9, P ¼.9, respetively; n 4 for eh smple). e NATURE GENETICS VOLUME [ NUMBER [ DECEMBER 4 9

6 4 Nture Pulishing Group Survivorship UAS-InR GMH (F) UAS-dPTEN GMH (F) UAS-InR GMH (M) UAS-dPTEN GMH (M) 4 Age (d) Figure 8 Survivl urves of mle (M) nd femle (F) progeny from the ross of the hert-speifi driver GMH-Gl4 with UAS-InR or UAS-dPTEN. For eh sex survivl does not differ mong flies expressing InR or dpten (logrnk test strtified y sex, w ¼., P ¼.). demogrphi ging nd to slow the ge-dependent deline in rdi performne. Thus, insulin signling in the hed ft ody my nonutonomously influene ging y ffeting the systemi level of insulins produed y the IPCs, wheres irulting insulin proximlly medites the ge-dependent funtion of dult orgns nd physiology, here illustrted y the hert. The etiology of rdi funtionl ging in humns is diverse, with origins in irultion, in the nervous system nd in the musle tissue itself 8 ; this is proly the se for the fly s well. How the struture nd funtion of these tissues hnge with ging nd respond to insulin signls my help determine the fundmentl wys in whih postmitoti tissues senese. Although inverterte herts hve importnt physiologil differenes from verterte herts, nd thus my mnifest divergent ge-dependent physiologil end-points, suh endpoints, s is the se in hert development, re expeted to e ontrolled in similr fshion y homologous signling systems. We show here, for the first time to our knowledge, tht funtionl ging n e ttenuted in n orgn-utonomous fshion nd suggest tht inverterte geneti models offer new opportunities to disover venues for orgn-speifi tretments of ging-ssoited degenertion. METHODS Fly stoks. We otined w 8 flies from the Bloomington stok enter nd used them s wild-type ontrol. We otined the wild-type Cnton S flies from S. Oldhm (The Burnhm Institute). We used yw flies s the geneti kground for the Ilp-Gl4 line nd s nother wild-type ontrol. Both lines were dontion from E. Rulifson (University of Pennsylvni). We used two different UAS-dPTEN lines in these studies: UAS-dPTEN(III), whih ontins single insertion on the third hromosome, nd UAS-dPTEN(II), whih ontins two UAS-dPTEN insertions on the seond hromosome 4.Theresultsfor the two lines were indistinguishle. The results shown in Figures 8 nd Supplementry Tles online were generted using UAS-dPTEN(II). These lines were donted y B. Edgr (Fred Huthinson Cner Reserh Center). We isogenized hio (hio / ) flies in triplite lines, eh of whih ws krossed 8 genertions into n w kground. The EMS-generted InR lleles were donted y M. Frsh (Mount Sini Shool of Mediine), exept InR E9, whih ws donted y J. Jk (University of Connetiut).We used P element medited germline trnsformtion in yw kground to generte dfoxo strins with either UAS-dFOXO(X) (onstrut with PKB phosphoryltion muttions T44A, S9A nd S9A) or UAS-dFOXO(wt) (onstrut with wild-type DNA). GMH onstrution nd expression. The 9-p hert enhner frgment from the tinmn gene,8 (HE) ws loned into the P{GWB}vetor upstrem of the Gl4 sequenes (y T.V. Venktesh (formerly of University of Mihign; 8 urrently t Monsnto)). To drive expression speifilly in the mjor myordil ells of the dult hert 9, this driver ws ugmented with multiple opies of UAS-Gl4 element 4, thus llowing ontinued myordil expression one tivted. We rossed trnsgeni flies rrying HE-Gl4 to flies rrying UAS- GFP. We then rossed UAS-GFP;HE-Gl4 progeny to line rrying multiple insertions of UAS-Gl4. We llowed femles to reomine nd then rossed them to lner lines. We seleted progeny rrying UAS-GFP;HE-Gl4 nd multiple UAS-Gl4 insertions y virtue of their rdi-speifi fluoresene s oth lrve nd dults (see Fig.,). We nmed the resulting stok GMH. The myordil speifiity of GMH is further illustrted in Supplementry Video online. Hert rte mesurements. We ligned live video imges of pupl eting herts with rster lines in softwre from IonOptix to generte three simultneous tres. We nlyzed these tres y Fourier trnsformtion to generte mesurement of the pek frequeny of movement, whih is expressed s the hert rte. We lso used utoorreltion nlysis to provide mesurement of the degree of vriility in time etween ets. We seleted pupe t the erliest stge of puption when flies re still ut trnsprent ( h fter puprium formtion). We olleted dult flies s virgins, seprted them y gender nd then ged them on stndrd food t C to either or d. Although we pplied edge-tring to dult herts for demonstrtion purposes, it is frequently diffiult to lign ontrst edges on dults due to utiulr pigmenttion. Therefore, we routinely did dult hert ounts y visully ounting eh hert five times for s nd then verging the five ounts to generte the individul hert rte. We then verged the hert rtes for ll individuls to rete the vlues represented for prtiulr genotypes. Eletril ping. We pled flies etween two eletrodes touhing ondutive jelly spred over the eletrodes. We ped the hert with squre wve stimultor t 4 V nd Hz for s. Hert filure rte is defined s the perentge of flies tht enter either rdi rrest or firilltion during or immeditely fter ping. Filed reovery rte is defined s the numer of flies tht, fter ping-indued rrest or firilltion, return to norml, produtive hert et within min of esstion of ping. Totl reovery indites the proportion of flies with norml hertet min fter esstion of ping. Survivl. We ged mle nd virgin femle progeny of the rosses UAS- InR GMH nd UAS-dPTEN GMH seprtely in groups of flies eh on yest gluose food (% yest, % gluose) t C. We trnsferred flies to fresh food on lternte dys nd ounted the numers of ded flies fter trnsfer (Fig. 8). We initited three replite demogrphy ges for eh genotype (three ontrol (yw is used for wild-type, +): + +; UAS-rpr +; nd Ilp-Gl4 +; nd the ltion-experimentl: Ilp-Gl4 UAS-rpr) with newly elosed femles nd mles eh (Fig. e,f; for ge design see ref. 4). We provided fresh food nd ounted nd removed ded flies from ll ges every other dy. We lulted genotype life tles y the extint ohort method with replite ge dt omined. Note: Supplementry informtion is ville on the Nture Genetis wesite. ACKNOWLEDGMENTS We thnk B. Edgr, E. Hfen, E. Rulifson, B. Hssn, H. Bellen nd the Bloomington stok enter for sending flies nd K. Fitzgerld, A. Stroe nd M. Montero for tehnil ssistne with prt of the hert performne ssys. R.J.W. hs een supported y fellowship from the Amerin Hert Assoition. This work reeived support from grnts from Ntionl Institutes of Helth (Ntionl Institute on Aging) nd The Ellison Medil Foundtion to M.T. nd y Ntionl Institutes of Helth (Ntionl Hert, Lung nd Blood Institute nd Ntionl Institute on Aging) to R.B. COMPETING INTERESTS STATEMENT The uthors delre tht they hve no ompeting finnil interests. Reeived 4 August; epted Otoer 4 Pulished online t Gurente, L. & Kenyon, C. Geneti pthwys tht regulte geing in model orgnisms. Nture 48, (). 8 VOLUME [ NUMBER [ DECEMBER 4 NATURE GENETICS

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Wolkow, C.A., Kimur, K.D., Lee, M.S. & Ruvkun, G. Regultion of C. elegns lifespn y insulinlike signling in the nervous system. Siene 9, 4 ().. Gerish, B., Weitzel, C., Koer-Eizermnn, C., Rottiers, V. & Antei, A. A hormonl signling pthwy influening C. elegns metolism, reprodutive development, n life spn. Dev. Cell, 84 8 ().. Tu, M.-P., Yin, C.-M. & Ttr, M. Impired ovrin edysone synthesis of Drosophil melnogster insulin reeptor mutnts. Aging Cell, 8 (). 8. Bluher, M., Khn, B.B. & Khn, C.R. Extended longevity in mie lking the insulin reeptor in dipose tissue. Siene 99, 4 (). 9. Liin, N., Bermn, J.R. & Kenyon, C. Tissue-speifi tivities of C. elegns DAF- in the regultion of lifespn. Cell, 489 ().. Stmoli, V. et l. Negtive regultion of PKB/Akt-dependent ell survivl y the tumor suppressor PTEN. Cell 9, 9 9 (998).. Oldhm, S. et l. The Drosophil insulin/igf reeptor ontrols growth nd size y modulting PtdInsP() levels. Development 9, 4 49 ().. Puig, O., Mrr, M., Ruhf, M. & Tjin, R. Control of ell numer y Drosophil FOXO: downstrem nd feedk regultion of the insulin reeptor pthwy. Genes Dev., ().. Junger, M.A. et l. The Drosophil Forkhed trnsription ftor FOXO medites the redution in ell numer ssoited with redued insulin signling. J. Biol., (). 4. Go, X., Neufeld, T.P. & Pn, D. Drosophil PTEN regultes ell growth nd prolifertion through PIK-dependent nd independent pthwys. Dev. Biol., ().. Fernndez, R., Trini, D., Azpizu, N. Frsh, M. & Shlessinger, J. The Drosophil insulin reeptor homolog: gene essentil for emryoni development enodes two reeptor isoforms with different signling potentil. EMBO J., 84 (99).. Chen, C., Jk, J. & Groflo, R.S. The Drosophil insulin reeptor is required for norml growth. Endorinology, 84 8 (99).. Venktesh, T.V. et l. Crdi enhner tivity of the homeoox gene tinmn depends on CREB onsensus inding sites in Drosophil. Genesis, (). 8. Bodmer, R. The gene tinmn is required for speifition of the hert nd viserl musles in Drosophil. Development 8, 9 9 (99). 9. Molin, M.R. & Cripps, R.M. Osti, the inflow trts of the Drosophil hert, develop from genetilly distint suset of rdil ells. Meh. Dev. 9, 9 (). 4. Hssn, B.A. et l. tonl regultes neurite roriztion ut does not t s proneurl gene in the Drosophil rin. Neuron, 49 (). 4. Ttr, M., Priest, N. & Chien, S. Negligile Senesene during Reprodutive Dormny in Drosophil melnogster. Am. Nt. 8, 48 8 (). NATURE GENETICS VOLUME [ NUMBER [ DECEMBER 4 8