Valve cells are crucial for efficient cardiac performance in Drosophila

PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011613 Ma ch 20, 2025 1 / 24 OPEN ACCESS Ci a ion: Meye C, Paulula A (2025) Val e cells a e c ucial o e icien ca diac pe o mance in D osophila. PLoS Gene 21(3): e1011613. h ps://doi.o g/10.1371/jou nal.pgen.1011613 Edi o : John Ewe , Uni e sidad de Valpa aiso, CHILE Recei ed: Decembe 17, 2024 Accep ed: Feb ua y 7, 2025 Published: Ma ch 20, 2025 Pee Re iew His o y: PLOS ecognizes he bene i s o anspa ency in he pee e iew p ocess; he e o e, we enable he publica ion o all o he con en o pee e iew and au ho esponses alongside inal, published a icles. The edi o ial his o y o his a icle is a ailable he e: h ps://doi.o g/10.1371/jou nal. pgen.1011613 Copy igh : © 2025 Meye , Paulula . This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Da a a ailabili y s a emen : All ele an da a a e wi hin he pape and i s Suppo ing In o ma ion iles. RESEARCH ARTICLE Val e cells a e c ucial o e icien ca diac pe o mance in D osophila Ch is ian Meye 1, Achim Paulula 1,2* 1 Depa men o Biology/Chemis y, Zoology & De elopmen al Biology, Osnab ück Uni e si y, Osnab ück, Ge many, 2 Cen e o Cellula Nanoanaly ics (CellNanOs), Osnab ück Uni e si y, Osnab ück, Ge many * [email p o ec ed] Abs ac Blood low in me azoans is egula ed by he ac i i y o he hea . The open ci cula o y sys em o insec s consis s o ela i ely ew s uc u al elemen s ha de e mine ca diac pe o mance ia hei coo dina ed in e play. One o hese elemen s is he in aca diac al e be ween he ao a and he en icle. In D osophila, i is buil by only wo cells, whose unique his ology ep esen s an e olu iona y no el y. While he de elopmen and di e en ia ion o hese highly specialised cells ha e been elucida ed p e iously, hei physiological impac on hea pe o mance is s ill unsol ed. The p esen s udy in es iga ed he physiological conse- quences o ca diac al e mal o ma ion in D osophila. We show ha ca diac pe o mance is educed i al es a e mal o med o damaged. Less blood is anspo ed h ough he hea p ope , esul ing in a dec eased o e all anspo capaci y. A educed luminal opening was iden i ied as a main eason o he dec eased hea pe o mance in he absence o unc- ional al es. In aca diac hemolymph low was isualised a he al e egion by mic opa i- cle injec ion and e ealed cha ac e is ic simila i ies o al e blood low in e eb a es. Based on ou da a, we p opose a model on how he D osophila in aca diac al es suppo p ope hemolymph low and dis ibu ion, he eby op imising gene al hea pe o mance. Au ho summa y Blood low in me azoans is egula ed by he hea ’s ac i i y. Insec s possess an open ci cula o y sys em wi h a ew s uc u al componen s ha collec i ely in luence ca diac pe o mance. A key elemen is he in aca diac al e be ween he ao a and he en icle, which in D osophila consis s o only wo specialised cells, ep esen ing an e olu iona y inno a ion. While he de elopmen o hese cells has been s udied, hei physiological impac on hea unc ion is less unde s ood. This s udy in es iga es he consequences o ca diac al e mal o ma ion in D osophila. Ou indings indica e ca diac pe o mance declines when he al es a e mal o med o damaged, leading o dec eased blood ans- po and lowe o e all capaci y. A educed luminal opening con ibu es signi ican ly o diminished hea pe o mance wi hou unc ional al es. Mo eo e , he isualisa ion o in aca diac luid low using mic opa icle injec ion e eals simila i ies o blood low in e eb a es. Based on hese insigh s, we p opose a model de ailing how in aca diac al es op imise luid low and enhance insec hea pe o mance. PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011613 Ma ch 20, 2025 2 / 24 PLOS Gene icS Ca diac al es In oduc ion Ca diac al es con ol blood low di ec ionali y, he eby enhancing pumping agains highe p essu es and g a i y, hus boos ing ca diac pe o mance [1–7]. The mo phology and unc- ion o ca diac al es di e signi ican ly among animals due o adap ion o en i onmen al condi ions. Fo example, mammalian mul icellula al es, sepa a ing he hea chambe s, con- sis o connec i e issue wi h collagen and elas in as main molecula cons i uen s. In con as , enous al es cons i u e cellula laps lined wi h a hin ma ix [8]. In e eb a es, and p esumably in all animals ha bou ing a closed ci cula o y sys em, he di ec ionali y o blood low is egula ed by in aca diac al es, which a e cons uc ed as lap- like s uc u es ha open and close he luminal space o he essel. Howe e , ce ain disease symp oms a e g ounded in he mal o ma ion o ca diac al es. Fo example, pa ien s su e ing om Ma an synd ome exhibi myoca dial in a c ion caused by he ailu e o p ope al e unc ion, esul ing in he na owing o he hea lumen (s enosis) and consequen ly comp o- mising hea unc ion [9]. Fu he mo e, ao ic a he oscle osis leads o igidi ica ion o he connec i e issue and o ma ion o calcium deposi s on he al es in all mammals, bi ds, ep- iles and ish and may inally also lead o s enosis [10–14]. As a consequence, se e e ca diac al e mal o ma ions d ama ically impac ca diac ou pu and o e all heal h in gene al [15]. In e eb a e hea s, like in Dip e a, exhibi a he simple al es. They a e o med by simple s uc u es like muscle pilla s, la ge spongy cells o cellula pads [16–18]. D osophila, like all insec s, possess an open ci cula o y sys em. The mo emen o he hemolymph, he insec blood, is d i en bo h by he ac i i y o ca diac muscles and by con ac ion o he body wall muscles. Addi ionally, hea bea e e sal, changing he low di ec ion o hemolymph unde ce ain physiological condi ions, has also been desc ibed in many insec s [19–23]. As a conse- quence, he in aca diac al es si ua ed a he ansi ion o he wide-luminal en icle and he na ow-luminal ao a, mus unc ion as wo-way ga es, which open and close o s eaming in bo h di ec ions. In he p esen s udy, we in es iga ed he ole o D osophila in aca diac al es in hemolymph dis ibu ion, hea pe o mance and he ex en o which hey con ibu e o he con ol o low p ope ies wi hin he hea . Only in ecen yea s, ligh has been shed on he complex biogenesis and unc ion o hese in aca diac al es and hei unique ul as uc u e [24–28]. Ou p e ious wo k in oduced a ious ools o s udying he D osophila al e cells. We iden i ied wo al e-speci ic epo e lines [27,28] and se e al c i ical genes, which, upon down- egula ion o exp ession as cons i u i ely ac i e a ian s, induce di e en ypes o mal- o ma ions in al e cells, wi hou a ec ing neighbou ing ca diac cells [27]. We now imp o ed and ex ended p e iously es ablished me hods o analyse hemolymph low in li ing and in ac animals. The combina ion o di e en imaging me hods allowed us o analyse he physiologi- cal consequences o al e ca diac mal o ma ion in D osophila. Based on ou expe imen al da a, we pos ula e a model in which he in aca diac al es e ol ed in D osophila o ensu e luminal closu e upon hea bea ing accompanied by a high de o ma ion capabili y needed o ully open he hea lumen. These ea u es allow e icien hemolymph dis ibu ion in he animal. A educ ion o he luminal opening, caused by al e damage in mu an s, educes he amoun o ci cula ing hemolymph, educes o e all i ness and nega i ely impac hemolymph dynamics in he ly hea . We, he e o e p esen ou cu en s udy as an essen ial i s s ep o u u e wo k wi h a ocus on analysing he physiological signi icance o he insec hea o he animal as a whole. Resul s P e iously we demons a ed ha al e cells u ilise exis ing endosomal pa hways o gene - a e la ge in acellula memb anous ca i ies, he al osomes (Fig 1C and 1D; [27]). Gene ic Funding: This wo k was suppo ed by g an s om he Deu sche Fo schungsgemeinscha o A.P. (PA 517/13-1 & PA 517/13-2, SFB 1557). We also acknowledge he suppo o he Open Access Publishing Fund o he Osnab ück Uni e si y. The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip . Compe ing in e es s: The au ho s ha e decla ed ha no compe ing in e es s exis . PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011613 Ma ch 20, 2025 3 / 24 PLOS Gene icS Ca diac al es manipula ion o he Rab5-dependen ea ly endocy osis, he Rab4/Rab11-dependen endo- somal ecycling pa hways o componen s o he cy oskele on, like Spec in, a ec p ope al osome biogenesis and lead o di e en al e pheno ypes (Fig 1; [27]). Pheno ypes a e cha ac e ized by an o e all educ ion o al e cell size, a educ ion in he numbe o Fig 1. Mal o ma ions o ca diac al es in D osophila. (A) Open ci cula o y sys em in he D osophila la a. Hemolymph en e s he hea cham- be ia pai s o os ia cells, and is pumped an e io ly h ough he ao a. I hen ci cula es in he open body ca i y and een e s he hea again. The in e sec ion o ao a and hea chambe is sepa a ed by a pai o in aca diac al es. The di ec ion o he hemolymph low is depic ed by ed a ows. (B) Du ing dias ole hemolymph (depic ed in g ey) en e s he hea chambe ia os ia cells. Val e cells cons i u e a oundish shape and seal he hea lumen. Upon sys ole, he hea chambe con ac s, al e cells possess an elonga ed shape opening he hea lumen and hemolymph is pumped an e io ly (see S1 Video). (C) Schema ic illus a ion o ca diac al es du ing dias ole and sys ole. Upon dias ole, al e cells p ess hei cell bodies in o each o he and seal he hea lumen. Upon sys ole al es become elonga ed, he eby opening he hea lumen. (D, D’) In con ol animals, al es a e cha ac e ised by a la ge oundish cell body and 2-4 la ge al osomes. (E, E’) Upon knockdown o ab11 al osomes a e almos absen om al es. (F, F’) Ec opic exp ession o a cons i u i ely ac i e o m o Rab4 leads o an inc eased numbe o smalle al osomes. (G, G’) Knockdown o alpha- spec in leads o a collapse o al e cells and al osomes. (H, H’) Inhibi ion o ab5-media ed endocy osis esul s in signi ican ly smalle al es cells and he absence o al osomes. h ps://doi.o g/10.1371/jou nal.pgen.1011613.g001 PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011613 Ma ch 20, 2025 4 / 24 PLOS Gene icS Ca diac al es cha ac e is ic al osomes pe al e cell, as well as an o e all educ ion o he size o he al osomes. Fo ins ance, knockdown o ab11 leads o a educ ion o al e size by 50% and he o al numbe o al osomes pe cell is educed om 3-4 al osomes o 1 o he comple e absence o his s uc u e occu s. These mo phological changes come along wi h changes in he al es ope a ing mode, as damaged al es show educed de o ma ion capabili ies upon hea bea ing [27]. Toge he wi h ou al e-speci ic Gal4 d i e lines, his now gi es us he unique oppo u- ni y o manipula e al e cells wi hou a ec ing o he ca diac cells. Thus, o he i s ime, we can expe imen ally analyse he in luence o al e cells on hemolymph ci cula ion and hea pe o mance in insec s. Damaged al es lead o a educ ion in ca diac pe o mance To in es iga e ca diac ou pu and hemolymph pumping e iciency, we pe o med dye- angiog aphy. Dye angiog aphy has been p e iously es ablished o isualise and measu e hemolymph s eaming in D osophila and Anopheles (Fig 2A) [29–33]. The me hod elies on he injec ion o a ace dye in o he abdominal body ca i y o li ing specimens. The dye is sucked in o he hea chambe and pumped an e io ly upon hy hmic hea bea ing. We used he accumula ion o he dye in he head egion o he animal as ead-ou o ca diac e iciency (Fig 2) [30,31]. As p e iously epo ed, changing he exp ession le els o Rab4, Rab5, Rab11 o alpha- Spec in, speci ically in al e cells, led o mal o med al es o di e en deg ees. We measu ed he ca diac pe o mance in con ol mu an animals, and ound signi ican ly less dye accu- mula ing in he head egion in any o he al e mu an s compa ed o he con ol (Fig 2D-J). Accumula ion o he ace was educed by 40% o 50% in ab11 knockdown, alpha spec in knockdown and Rab4 o e exp essing animals (Fig 2J). Concu en o p e ious indings, knock-down o ab5, which leads o he mos se e e damage in al e cell mo phology (Fig 1H), also caused he mos d ama ic decline in pumping e iciency by 80% o 90% (Fig 2J and 2I). The e o e, ou esul s clea ly demons a e ha educed ca diac ou pu and hemolymph pumping e iciency is due o damaged ca diac al es. Why is his he case? Luminal closu e and hemolymph eloci ies A e analysing he impac o al e ana omy on he global mo ion o hemolymph in he body, we nex add essed he mo ion o hemolymph wi hin he hea ube i sel . The ime- es ic ed luminal closu e o he hea lumen, ensu ing he con olled hemolymph illing o he hea chambe , cons i u es he mos undamen al unc ion o ac i e ca diac al es in he D osophila hea [26–28] (Fig 1A-C). In D osophila, hemolymph is anspo ed by he hy hmic anspo o hemolymph om he hea chambe in o he an e io ao a and a e wa ds in o he open body ca i y [30]. As a esul o he hy hmic open/close cycles o he al e, hemolymph is no anspo ed in a con inuous cu en , bu a he in indi idual uni s (Fig 3A-C). In o de o es ima e he low pa e n wi hin he hea , we p ecisely selec ed di e en egions o in e es (ROIs) wi hin he ao a, and moni o ed he passing o single ace uni s o e ime. This allowed us o es ima e luminal closing capabili ies o he al e, he eloci y o he anspo ed hemolymph, as well as he hemolymph “package” size [30]. Measu ing image in ensi ies in single ROIs esul ed in pe iodic oscillog ams, e lec ing he mo ion o he ace and he pe iodic open/close cycles o he al e. In con ol animals, in ensi y alues pe iodically al e na e be ween peaks o high and low in ensi y, e lec ing he abili y o al es o close he luminal space be ween hea bea s. Al hough he bulk mo ion o hemolymph was d ama ically a ec ed, we ound ha hemolymph anspo wi hin he hea PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011613 Ma ch 20, 2025 5 / 24 PLOS Gene icS Ca diac al es appea ed no mal in mos al e mu an lines, showing nea ly iden ical e iciency (Fig 3D-H). Ne e heless, based on pixel in ensi ies he amoun o ace dye anspo ed by each con- ac ion cycle was educed in hese al e mu an s, explaining he impac on he bulk mo ion measu ed be o e (Fig 3J). Oscillog ams om ab11 knockdown animals showed simila peaks o high and low in ensi ies, indica ing p ope ime- es ic ed luminal closu e o he al es bu in addi ion passages o low in ensi ies a e p esen , ep esen ing ca diac a es s (Fig 3F). Mos d ama ically, oscillog ams ob ained om ab5 mu an animals, which display he mos se e e mal o med al es, showed no pe iodic change o in ensi y a all (Fig 3J). Thus, hese animals comple ely ail o sus ain an e icien hemolymph anspo wi hin he hea . O e ime, he ao a was only e y slowly illed by he dye, mos likely d i en by di usion (Fig 3I). As a esul , we conclude ha less hemolymph is anspo ed pe hea bea i al es a e mal o med, leading o a educed hemolymph dis ibu ion (Fig 2J). S eaming eloci y inside he hea In humans i has been shown ha ca diac and ascula al es con ol ao ic blood eloci y. In heal hy people, ao ic blood eloci ies a y unde di e en ci cums ances. Fo ins ance, Fig 2. Ca diac pe o mance and hemolymph dis ibu ion a e signi ican ly educed upon al e mal o ma ion. (A) Scheme illus a ing he me hodology o dye angiog aphy analysis. Black ink is injec ed in he pos e io end o ea ly whi e p epupae. T ace dye accumula es o e ime in he an e io egion due o he con inuous pumping ac i i y o he hea (see S2 Video). (B, C) Ligh mic oscopic illus a ion o a con ol animal 5 sec and 30 sec a e injec ion, espec- i ely. Injec ed dye accumula es in he an e io egion (as e isk). (D-I) Digi al sub ac ion and colou -coded pixel in ensi y o he ace dye in con ols (D, E) and al e mu an animals (F-I). (J) S a is ical analysis o he dye angiog aphy. A e 30 seconds signi ican ly less dye was anspo ed o he an e io end in al e mu an s, compa ed o con ol lines. N= 10 animals pe geno ype. Two- ailed S uden ’s - es *P < 0.05, ***P < 0.001. h ps://doi.o g/10.1371/jou nal.pgen.1011613.g002 PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011613 Ma ch 20, 2025 6 / 24 PLOS Gene icS Ca diac al es Fig 3. Val e closing capabili y and hemolymph low analysis. (A) Scheme illus a ing expe imen al se up o al e closing e iciency and hemolymph low analysis. A egion o in e es (ROI 1) is se an e io ly o ca diac al es. Pixel in ensi y in ROI 1 inc eases i hemolymph packages pass and dec eases, i al es close a e each hea bea , espec i ely. Hemolymph eloci y was calcula ed using wo egions o in e es (ROI1 & ROI 2) in he ao a. The espec i e ime PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011613 Ma ch 20, 2025 7 / 24 PLOS Gene icS Ca diac al es a es peak eloci y is abou 1 m/sec, which can inc eases o mo e han 3 m/sec du ing exe cise [34]. In addi ion, ao ic eloci y measu emen s ha e been used as eadou o he classi ica ion o he se e i y o s enosis pheno ypes [35]. This led us o assume ha mal o - ma ions in he D osophila al e cells may also lead o abe an ao ic hemolymph eloci ies. Thus, we measu ed ao ic hemolymph eloci ies based on he oscillog ams ob ained om he dye angiog aphy expe imen s (Fig 3A and [30]. We ound ha ca diac al e mal o - ma ion does no impac ao ic hemolymph eloci ies (Fig 3K). Ao ic eloci ies in heal hy animals was es ima ed as ~5,2 ± 1,5 mm/sec, while upon al e damage ao ic eloci ies anged om ~4 o 6 ± 2,5 mm/sec (Fig 3K). These esul s a e compa able o ao ic eloci ies p e iously epo ed [30]. The luminal dis ance in he open s a e is signi ican ly educed in al e mu an s P e iously we demons a ed ha he de o mabili y o he al e cells is c i ical o hei mode o ac ion. We specula ed ha in e e ing wi h he ou genes egula ing al osome o ma- ion, ab4, ab11, alpha-spec in o ab5, may lead o a educed de o mabili y o hese cells upon hea bea ing [27]. A educed de o ma ion capabili y o al es is o en associa ed wi h a highly educed luminal opening upon sys ole and hus opening du a ion ime [36,37]. Fo his eason, we in es iga ed he closing cha ac e is ics and mo ili y o he heal hy and mal o med al es in semi-in ac hea p epa a ions in mo e de ail (Fig 4A-F). We ound ha he luminal dis ance in he peak open s a e, when he hemolymph is pumped an e io ly, is signi ican ly educed in all al e mu an s analysed (Fig 4Q). In e es ingly, al e cells o ab5 knockdown animals we e unable o close he hea lumen in semi-in ac p epa a ions p op- e ly. The e o e, al e opening ime could no be es ima ed. Howe e , measu ing he ime om he ini ial luminal opening un il he end o a luminal closing, which includes he peak opening phase, we ound no di e ences be ween con ol g oups (Fig 4G-K and 4R) and al e mu an s (Fig 4L-P and 4R). Ou esul s show ha he du a ion o he o e all opening ime emains una ec ed, bu he maximal luminal opening du ing peak opening phase is con- side ably educed. This indica es ha mal o med al e cells impac he a io o he di e en opening and closing phases o he al e du ing a comple e hea cycle, bu do no in luence hea hy hm. P esence o in ac al e cells is c i ical o con olling physical s eaming pa ame e s inside he hea ube In o de o analyse he dynamics o hemolymph s eaming in li ing insec s, se e al me hods ha e been es ablished. These include pa icle injec ion in o in ac D osophila and Anopheles specimens [26,38,39], mic obubble injec ion in o Schis oce ca ame icana [40] o acking o GFP-labelled hemocy es in D osophila [41]. Howe e , con inuous da kening o he cu icle o one hemolymph ace packages eaching peak pixel in ensi y in he wo ROIs was calcula ed o he dis ance o he wo ROIs. (B) B igh ield illumina ion showing injec ed whi e p e-pupa and hemolymph low in he ao a. (C) Digi al sub ac ion and colo -coded pixel in ensi y, same animal as in (B). (D-I) Val e closing capabili y. Upon hea bea hemolymph packages pass ROI1 (loca ed an e io ly o ca diac al es), eaches peak in ensi y and alls down o a baseline again, indica ing hemolymph anspo and p ope al e luminal closu e in con ol animals and in ab11 knockdown, spec in knockdown and Rab4 o e exp ession lines (D-H, Scheme in D highligh s cha ac e is ic imepoin s o he pumped dye package passing he ROI, as e isks ep esen espec i e poin s in he oscillog am. (I) In ab5 knockdown animals no ac i e hemolymph anspo was de ec ed. (J) Analysis o hemolymph low in he ao a. Mean pixel in ensi y o he dye package is signi ican ly educed upon al e mal o ma ion, indica ing less hemolymph is pumped (Scheme illus a es dye package in he ao a and i s a ea analyzed). Fo ab5 knockdown animals no ace package was o med and could be analyzed. N= 5 animals pe geno ype. (K) Hemo- lymph ao a eloci y is una ec ed i al es a e mal o med. Fo ab5 knockdown animals, hemolymph eloci y could no be de e mined. N= 10 animals pe geno ype. (J) Two- ailed S uden ’s - es , (K) ab11 KD Mann-Whi ney es *P < 0.05, **P < 0.01, ***P < 0.001. h ps://doi.o g/10.1371/jou nal.pgen.1011613.g003 PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011613 Ma ch 20, 2025 8 / 24 PLOS Gene icS Ca diac al es Fig 4. Val e luminal dis ance and opening ime upon al e mal o ma ion. (A-F, Q) Val e luminal opening is signi ican ly educed, i al es a e mal- o med, bu he ime o al es in he open s a e is simila o con ol animals (R). (G-K) Rep esen a i e ames om highspeed ideos o dissec ed 3 d ins a la a o con ol and Rab4 o e exp ession animals (L-P), showing one opening cycle o al es. Val es depic ed in yellow, hea lumen depic ed in ed. N= 10 animals pe geno ype. Two- ailed S uden ’s - es *P < 0.05, **P < 0.01, ***P < 0.001. h ps://doi.o g/10.1371/jou nal.pgen.1011613.g004 PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011613 Ma ch 20, 2025 9 / 24 PLOS Gene icS Ca diac al es Fig 5. Pa icle s eaming a he al e. Selec ed ames o a se ies highligh ing al es posi ion upon hea bea ing (A-B). Val es elonga e and sho en upon hea bea ing leading o a dis inc na owed lumen in he hea ube (Box in B). T acks o injec ed pa icles in he hea ube passing con ol (C) and mu an al es (D) (As e isk indica es posi ion o one al e). Pa icles lowing in he ou e lumen ge pa ially de lec ed by al es physical p esence, while pa icles in he cen e o he lumen a e una ec ed. All pa icles each highes eloci y when passing he al e cells (C-D). (E-F) Scheme showing speed eloci ies o all acks analyzed. Red box ep esen s he a ea occupied by al e cells upon hea bea ing. (G) Pa icle speed a he posi ion o he al es is highly inc eased upon al e damage. Mann-Whi ney es ***P < 0.001. (H) Model illus a ing hemolymph low in wild ype and al e mu an s. Wild ypic al es a e highly de o mable and open he hea lumen, allowing unobs uc ed low. In con as , in al e mu an s hea lumen is na ow and low is obs uc ed. Highe s eaming eloci ies a e p esen upon al e damage. h ps://doi.o g/10.1371/jou nal.pgen.1011613.g005 PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011613 Ma ch 20, 2025 16 / 24 PLOS Gene icS Ca diac al es A de imen al e ec o a educed hemolymph dis ibu ion on animal i ness appea s likely. Physical ac i i y is highly bene icial o ca dio ascula heal h and may p e en age- ela ed ca - diac dys unc ion in lies and men [67–70]. In con as , ca diac diseases o en lead o educed ca diac pe o mance and highe exe cise in ole ance [60]. In e es ingly, hemolymph dis i- bu ion in D osophila is p ima ily d i en by he ac i i y o he hea , bu also by he o e all con ac ion ac i i y o he body wall muscles [52,53]. Animals lacking a ully unc ional hea indeed show educed i ness o limi ed li espan, e.g., seen in pe ica din o lonely hea mu an s [31,71,72]. Mo eo e , many genes a ec ing hea di e en ia ion led o simila pheno ypes i he lies a e ea ed by s ess, e.g., a highe empe a u e [73]. Ou p esen s udy ocused on he con ibu ion o he al e cells o hea pe o mance and i ness. We ound ha a educed hea pe o mance caused by damaged al es nega i ely impac s endu ance o la ae. In addi ion, he animals may y o compensa e loss o ci cula ion e iciency by inc easing body mo e- men , i.e., bending and u ning. We conclude ha upon e olu ion in aca diac al es e ol ed o boos ca diac pe o mance, i.e., o enhance hemolymph dis ibu ion in insec hea s. In D osophila in aca diac al es de eloped by modula ion o al eady exis ing molecula pa hways, gene a ing a highly special- ised cell wi h unique cellula o ganelles. We showed ha his s uc u e is ideally sui ed o ul il i s main asks: 1) E ec i ely seal he hea lumen upon dias ole, enabling p ope sucking in o hemolymph in o he hea chambe . 2) E ec i ely de o m upon sys ole, enabling unobs uc ed hemolymph low and dis ibu ion by ully opening he hea lumen. This idea is u he suppo ed by s udies poin ing o ca diac al es as hemodynamic al es [30]. The esis ance o he al e dec eases a a ce ain p essu e and inally leads o he opening o he hea lumen. The p esence o he al osomal compa men enables al e cells o de o m p ope ly upon hemodynamic p essu e. In con as , he absence o al osomes will inc ease al e p essu e esis ance and educe hei de o ma ion capabili y, esul ing in a na ow lumen. As a esul , he hea canno pump e icien ly agains his highe p essu e and educed luminal opening. The e m “ al e” s ands o a dedica ed s uc u e ha enables unidi ec ional luid low by a mechanism ha is eminiscen laps. Flaps close a lumen as he luid lows in he opposi e di ec ion agains he lap. This is no he case o he in aca diac al e cells in D osophila. We belie e ha he p ima y unc ion o hese al es is no o ep esen a “one-way- al e”, bu indeed a hemodynamic al e as p e iously sugges ed [30]. This hypo hesis is now suppo ed by a ious obse a ions p esen ed in his and p e ious s udies: 1) The absence o dye in he ao a a he end o sys ole, indica es no hemolymph p essu e an e io o and agains he al es. The absence o a backwa d o ien a ed p essu e agains he al e [30]. 2) When la ae a e dissec ed o obse e he hea bea , he body in e io and he in e play o muscles, body wall mo emen , ca diac ac i i y and ci cula ion a e los . Unde hese ci - cums ances we obse ed a pe iodically occu ing e og ade low. Howe e , his e og ade low does no lead o a luminal hea closu e by he al es [26]. 3) The la al ca diac al e pe sis s in o adul hood. Two addi ional al es, each consis ing o wo indi idual al e cells, a e o med du ing me amo phosis and all al es ha e a simila mo phology as he la al in aca diac al e [25,28]. Howe e , in adul D osophila, al es pe mi a pe iodically occu ing e og ade low [22,74,75]. PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011613 Ma ch 20, 2025 17 / 24 PLOS Gene icS Ca diac al es 4) Bidi ec ional hemolymph low is epo ed o a a ie y o insec species and is based on addi ional con ac ile egions wi hin he hea in combina ion wi h an e io ly and pos- e io ly loca ed excu en openings [22,74–76]. I has been specula ed ha bidi ec ional hemolymph low migh be bene icial o cleaning he luminal compa men o he hea om cell deb is and o he ma e ial emains [77]. Al hough a numbe o ques ions emain o be add essed, he p esen s udy highligh s o he i s ime he necessi y and physiological impo ance o ca diac al es in an in e eb a e model o ganism. He eby, we p esen a aluable se o ools and me hods o u u e s udies on hemodynamics in insec s and on he ole o al es cells in ci cula ion. Ma e ials and me hods Fly s ocks and gene ics The oll-Gal4 and handC-GFP lines used in his s udy we e made by ou labo a o y [78]. The ollowing lines we e ob ained om he Blooming on D osophila S ock Cen- e (BDSC) a Indiana Uni e si y: 76E11-Gal4 (RRID:BDSC_39933), UAS-Rab4pQ67L (RRID:BDSC_9770) and UAScd4 d::GFP (RRID:BDSC_35836). The ollowing RNAi lines we e ob ained om he VIENNA D osophila Resou ce Cen e ( ) o Blooming on D osoph- ila S ock Cen e (BL): ab5 103945, ab11 108382 and spec in 42053. As a con ol s ains espec i e al e speci ic d i e s oll-Gal4 and 76E11-Gal4 we e used. The exp ession pa e ns o he Gal4-d i e lines used he ein we e p e iously desc ibed: oll enhance [26] and 76E11 [79] and [80]). Fly husband y was ca ied ou as desc ibed p e iously [81]. An ibodies and eagen s An ibodies we e used o de ec Spec in (1:20; De elopmen al S udies Hyb idoma Bank, USA), Monoclonal abbi an i-GFP (1:2000) was om Abcam (Ab6556) and monoclonal mouse an i- GFP (3E6, 1:500) was om In i ogen (A-11120; The mo Fishe Scien i ic). Seconda y an ibod- ies used we e an i-mouse Cy2, an i-mouse Cy3 and an i- abbi Cy2 (1:200; Diano a, Ge many). Immunos aining o al e cells o hi d ins a la ae Thi d ins a wande ing la ae we e dissec ed ollowing p e iously published p o ocols [25,27,82]. B ie ly, animals we e pinned down on hei do sal side on a Sylga d pla e, co e ed wi h PBS, opened en ally and Visce a we e ca e ully emo ed. Specimens we e ixed in 4% me hanol- ee pa a o maldehyde. A e washing s eps, samples we e pe meabilised wi h 1% T i on in PBS, ollowed by h ee addi ional washing s eps. A e wa ds, nonspeci ic epi opes we e blocked by incuba ion in sa u a ion bu e . P ima y an ibodies we e dilu ed in PBS bu - e and incuba ed o e nigh a 4 °C unde cons an shaking be o e he solu ion was emo ed and eplaced by BBT bu e o ho ough washing. Seconda y an ibodies, wi h coupled luo o- pho es, we e dilu ed in PBS and incuba ed a oom empe a u e in he da k o 2 h. Unbound an ibodies we e emo ed by h ee washing s eps in PBS. Finally, samples we e embedded in Fluo omoun -GTM (The mo Fishe Scien i ic) wi h DAPI o Ro iMoun Fluo Ca e wi h DAPI (Ro h) and imaged wi h a Zeiss LSM 800 lase scanning mic oscope. Images we e anal- ysed using Fiji ImageJ so wa e. Dye angiog aphy and hemolymph accumula ion The basic p o ocol was modi ied a e [30] and [31]. B ie ly, selec ed whi e p e-pupae we e glued on a mic oscope slide using double sided Sco ch ape. Injec ions we e pe o med using small cus om made glass capilla ies equipped on a mic o-manipula o connec ed o an PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011613 Ma ch 20, 2025 18 / 24 PLOS Gene icS Ca diac al es Eppendo Fem oJe mic o-injec ion sys em. Injec ion o black ink (Pelican) in o he pos e- io body pa was done by a single injec ion o 0,1 sec a 2 psi. Accumula ion o he dye was eco ded using a Leica WILD MZ8 s e eomic oscope equipped wi h a Basle acA2000-165uc came a and ansmi ed ligh illumina ion. Videos we e cap u es o 30 seconds a a ame a e o ~100 ames/s and con e ed o 8-bi g ayscale ideos in ImageJ. Backg ound was subs ac ed by deduc ing pixel in ensi ies om he i s ideo ame om all ollowing ames. Accumula ion o dye was analyzed in an an e io 15x15 pixel ROI using ImageJ and he “Plo Z-axis p o ile”- ool. 10 animals pe geno ype we e analyzed and he mean dye accumula ion was es ima ed. Da a we e analyzed using an unpai ed wo- ailed S uden ’s - es . Val e closing capabili y Val e closing capabili y was de e mined om dye angiog aphy ideos (see sec ion abo e). In b ie a 15 x 15 pixel ROI was se an e io o he al e cells and pixel in ensi y was mea- su ed o i e seconds wi h ImageJ using he Plo z-axis- ool. Backg ound in ensi y is mos ly in luenced by he indi idual da kening o he animal’s cu icle and was hus calcula ed o each animal p io o dye injec ion, espec i ely. Each ime he dye bulk passes he ROI and s eams an e io ly, his leads o a ime-dependen peak inc ease in pixel in ensi y, which a e wa ds goes down o a baseline, i al es close p ope ly. Hemolymph bulk analysis Fo hemolymph bulk analysis ideos om dye angiog aphy we e aken and p ocessed wi h ImageJ. Mean hemolymph bulk pixel In ensi y was measu ed om single dye packages wi hin he ao a, using ImageJ Wand-T acing-Tool o ou line he en i e a ea o he dye package. Fi e animals pe geno ype we e e alua ed and o each animal i e dye bulk packages we e ana- lyzed, espec i ely. Da a we e analyzed using an unpai ed wo- ailed S uden ’s - es . Hemolymph ao a eloci y Hemolymph eloci y in 3 d la al ao a was de e mined om dye angiog aphy analysis and as p e iously desc ibed [30]. In b ie , wo ROI wi h a size o 15 x15 Pixel we e se in he pos- e io and an e io pa o he ao a, espec i ely. Veloci y o hemolymph was calcula ed by measu ing he dis ance be ween he ROI di ided by he ime i akes o one hemolymph bulk o each peak pixel in ensi y in bo h ROI. Fo each geno ype 10 la ae and 3 ca diac cycles we e analyzed and mean eloci ies pe animal we e calcula ed. Da a we e analyzed using an unpai ed wo- ailed S uden ’s - es and Mann-Whi ney es . Val e luminal dis ances and opening ime Fo analysis highspeed ideos o semi-in ac hea p epa a ions we e used. Luminal dis ances o al es we e calcula ed manually by measu ing he minimum luminal dis ance be ween opposing al e cells in he ully open s a e in a 90° angle and om he cy oplasmic bo de o al e cells. Fo each geno ype 8 animals and 5 hea bea s we e analysed, o ab5 knockdown lines 5 animals we e analysed. Val e opening ime was measu ed by calcula ing he ime be ween wo closed al e s a es, espec i ely. Fo each animal mean luminal opening ime was calcula ed om 10 single hea bea s and 10 animals pe geno ype we e analyzed. Da a we e analyzed using an unpai ed wo- ailed S uden ’s - es . Hemolymph eloci y a al es Fo analysis o s eaming eloci ies a al e le el, we injec ed ed luo escen polys y ene pa i- cles wi h a diame e o 1,2 µm (2% w/ in a i icial hemolymph; mic oPa icles GmbH, Be lin, PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011613 Ma ch 20, 2025 19 / 24 PLOS Gene icS Ca diac al es Ge many) wi h he same se up used o dye injec ion expe imen s desc ibed abo e. Pa icle low was eco ded using a Zeiss LSM 5 Pascal con ocal mic oscope equipped wi h a Basle acA800-510uc came a and an ebq 100 ligh lamp wi h adequa e il e se s. Highspeed ideos a 200 ps we e cap u ed o 10 seconds. Pa icles we e manually acked and eloci ies analyzed wi h ImageJ and he Manual T acking wi h T ackma e-Plugin. Posi ion o al es upon hea bea ing was es ima ed om handC-GFP epo e signal and pa icle s eaming eloci ies o all acks wi hin he al e egion we e es ima ed. Da a we e analyzed using nonpa ame ic Mann-Whi ney- es . Ca diac myo ibe o ganiza ion Wande ing 3 d ins a la ae we e collec ed and p epa ed in PBS con aining 8% MgCl2, ixed o 1 h in 4% Fo maldehyde a oom empe a u e, pe meabilized o 1 h in 1% T i on PBS p io o incuba ion o 1 h a RT in 1:100 Phalloidin TRITC solu ion. Images we e cap u ed wi h a Zeis LSM800 con ocal mic oscope. Myo ibe densi y and o ien a ion we e analyzed in a single 100 µm x 50 µm ROI in abdominal segmen i e (A5) be ween pai s o unc ional os ia cells. Myo ibe densi y was es ima ed by h eshold-based measu emen s o he hea a ea in ela ion o myo ibe co e age, espec i ely. Myo ibe o ien a ion was measu ed in ImageJ using Di ec ionali y- ool and Fou ie -componen s me hod wi h de aul se ings. Hea A ea was calcula ed om o hogonal sec ions acqui ed om con o- cal s acks using ImageJ o hogonal iew - unc ion. Sec ions om he abdominal segmen A5 we e aken and analyzed wi h ImageJ polygon selec ion- ool o ou line hea a ea. N = 10 animals pe geno ype we e in es iga ed and da a we e analyzed using an unpai ed wo- ailed S uden ’s - es . Analysis o ca diac pa ame e using SOHA-me hod Semi-in ac hea p epa a ions we e done as p e iously desc ibed [26,46]. In b ie , hi d ins a wande ing-la ae we e dissec ed in a i icial hemolymph om he en al side and isce a was emo ed [83]. Specimens we e allowed o eco e o a leas 5 min. Videos we e cap u ed wi h Basle piA640-210gm high-speed came a a 200 ames s-1, moun ed on an Olympus BX41TF s e eomic oscope. Reco dings we e done using pylon-Viewe so wa e (Basle AG) a 23 °C o ensu e cons an condi ions o all specimens. Videos we e u he p ocessed wi h ImageJ and hea pa ame e s we e examined ia semi-au oma ic op ical hea bea analysis (SOHA; [46,84]); N = 10 animals pe geno ype we e in es iga ed, o ab5 knockdown lines i e animals, and da a we e analyzed using an unpai ed wo- ailed S uden ’s - es . La al c awling assay La al c awling analysis was done as p e iously desc ibed [51]. In b ie , wande ing hi d ins a la ae we e collec ed om ubes and placed indi idually on a 92 x 16 mm polys y ene, humid- i ied pe i dish. Posi ioning o la ae causes sho pa alysis and immobiliza ion o la ae, he e o e imaging was s a ed a e he i s ini ial head mo emen s we e no iced. C awl- ing la ae we e imaged o 60 seconds using a Nikon D5200 digi al came a wi h a 40 mm 1:2,8 mm Nikon AF-S Mic o NIKKOR objec i e equipped o Kaise RS1 copys and wi h scale and p o ided wi h ou 3000 K opal lamps. La ae ha eached he end o he pe i dish and s a ed o climb up he wall du ing eco ding, we e disca ded. Videos we e u he p ocessed and analyzed using ImageJ and he T ackma e-plugin [85]. Fo la al shape analysis ImageJ’s Shape Desc ip o s and h eshold-based measu emen s we e pe o med. The majo and mino axis o a i ed ellipse desc ibing he la als shape was calcula ed o each ame. Roundess PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011613 Ma ch 20, 2025 20 / 24 PLOS Gene icS Ca diac al es o la al shape was hen calcula ed as he in e se o he aspec a io: (4*a ea/(π*majo axis²). Values app oaching 0,0 indica e an inc easingly elonga ed shape [54,55]. Fo each geno ype 15 la ae we e analysed and da a we e analyzed using an unpai ed wo- ailed S uden ’s - es . Suppo ing in o ma ion S1 Fig. Ca diac al e mal o ma ion impac s ca diac pa ame e . Knockdown o ab11 esul s in educed dias olic and sys olic diame e s and an inc eased a hy hmia index. Ec opic exp ession o Rab4 inc eases he numbe o al osomes and educes hei size, bu ca diac pa ame e we e una ec ed. Knockdown o spec in educed al e cells de o ma ion capabil- i y and luminal dis ance, as well as ac ional sho ening. Down egula ion o ab5 nega i ely a ec ed cell and al osomal size, as well as numbe o al osomes. In addi ion, hea a e, dia- s olic and sys olic diame e and ac ional sho ening we e signi ican ly educed. Ca diac mal- o ma ion leads o educed ca diac pumping e iciency and hemolymph dis ibu ion. N = 10 animals pe geno ype we e in es iga ed, o ab5 knockdown lines i e animals, and da a we e analyzed using an unpai ed wo- ailed S uden ’s - es *P < 0.05, **P < 0.01, ***P < 0.001. (TIFF) S1 Da a. In he Excel able p o ided, all p ima y da a measu ed and analyzed o he esul s p esen ed he e a e lis ed acco ding o he co esponding igu e able. (XLSX) S1 Video. 3 d Ins a D osophila la al hea hy hmically bea ing. In low ac s (os ia cells) upon sys ole due o ca diac chambe con ac ion. In aca diac al es open du ing sys ole due o an inc eased o wa d-o ien a ed hemolymph p essu e. (MP4) S2 Video. Scheme illus a ing he me hodology o dye angiog aphy analysis. Black ink is injec ed in he pos e io end o ea ly whi e p epupae. T ace dye accumula es o e ime in he an e io egion due o he con inuous pumping ac i i y o he hea . (MP4) Acknowledgemen s We hank Ma ina Biede mann, Ke s in E zold and Mech hild K abusch o excellen echni- cal assis ance and he Blooming on D osophila S ock Cen e and Vienna D osophila Resou ce Cen e o p o iding s ocks essen ial o his wo k. Fu he mo e, we hank ou s uden Maike Spielmeye o suppo ing us wi h expe imen al p epa a ion and imaging. We also hank PD D . Heiko Ha en and D . Maik D echsle o a ious expe imen al suppo and c i ical ead- ing o he manusc ip . Au ho con ibu ions Concep ualiza ion: Ch is ian Meye , Achim Paulula . Fo mal analysis: Ch is ian Meye , Achim Paulula . Funding acquisi ion: Achim Paulula . In es iga ion: Ch is ian Meye . Me hodology: Ch is ian Meye . P ojec adminis a ion: Achim Paulula . Supe ision: Achim Paulula . 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