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 .
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011613 Ma ch 20, 2025 21 / 24
PLOS Gene icS Ca diac al es
Valida ion: Ch is ian Meye .
Visualiza ion: Ch is ian Meye .
W i ing – o iginal d a : Ch is ian Meye , Achim Paulula .
W i ing – e iew & edi ing: Ch is ian Meye , Achim Paulula .
Re e ences
1. Bazigou E, Lyons OTA, Smi h A, Venn GE, Cope C, B own NA, e al. Genes egula ing lymphangio-
genesis con ol enous al e o ma ion and main enance in mice. J Clin In es . 2011;121(8):2984–92.
h ps://doi.o g/10.1172/JCI58050 PMID: 21765212
2. Blomq is CG. Ca dio ascula adap a ion o weigh lessness. Med Sci Spo s Exe c. 1983;15(5):428–
3 1. h ps://doi.o g/10.1249/00005768-198315050-00015 PMID: 6645874
3. Meissne MH, Mone a G, Bu nand K, Glo iczki P, Loh JM, Lu ie F, e al. The hemodynamics and
diagnosis o enous disease. J Vasc Su g. 2007;46 Suppl S:4S–24S. h ps://doi.o g/10.1016/j.
j s.2007.09.043 PMID: 18068561
4. No dmeye S, Lee CB, Goube g i s L, Knosalla C, Be ge F, Falk V, e al. Ci cula o y e iciency in
pa ien s wi h se e e ao ic al e s enosis be o e and a e ao ic al e eplacemen . J Ca dio asc
Magn Reson. 2021;23(1):15. h ps://doi.o g/10.1186/s12968-020-00686-0 PMID: 33641670
5. Sacks MS, Da id Me yman W, Schmid DE. On he biomechanics o hea al e unc ion. J Biomech.
2009;42(12):1804–24. h ps://doi.o g/10.1016/j.jbiomech.2009.05.015 PMID: 19540499
6. Schmid -Nielsen K. Animal physiology. Adap a ion and en i onmen . 5 h edi ion. Camb idge Uni e si y
P ess; 2002
7. Schoen FJ. E ol ing concep s o ca diac al e dynamics: he con inuum o de elopmen , unc ional
s uc u e, pa hobiology, and issue enginee ing. Ci cula ion. 2008;118(18):1864–80. h ps://doi.
o g/10.1161/CIRCULATIONAHA.108.805911 PMID: 18955677
8. A ms ong EJ, Bischo J. Hea al e de elopmen : endo helial cell signaling and di e en ia ion. Ci c
Res. 2004;95(5):459–70. h ps://doi.o g/10.1161/01.RES.0000141146.95728.da PMID: 15345668
9. S ua AG, Williams A. Ma an’s synd ome and he hea . A ch Dis Child. 2007;92(4):351–6. h ps://doi.
o g/10.1136/adc.2006.097469 PMID: 17376944
10. Pal a S, Pai AM, Gill KS, Pai RG. New insigh s in o he p og ession o ao ic s enosis: implica ions o
seconda y p e en ion. Ci cula ion. 2000;101(21):2497–502. h ps://doi.o g/10.1161/01.ci .101.21.2497
PMID: 10831524
11. Rajamannan NM, Ge sh B, Bonow RO. Calci ic ao ic s enosis: om bench o he bedside--eme ging
clinical and cellula concep s. Hea . 2003;89(7):801–5. h ps://doi.o g/10.1136/hea .89.7.801 PMID:
12807865
12 . S ewa BF, Sisco ick D, Lind BK, Ga din JM, Go diene JS, Smi h VE, e al. Clinical ac o s
associa ed wi h calci ic ao ic al e disease. Ca dio ascula Heal h S udy. J Am Coll Ca diol.
1997;29(3):630–4. h ps://doi.o g/10.1016/s0735-1097(96)00563-3 PMID: 9060903
13 . S okes W. The diseases o he hea and he ao a. Philadelphia: Lindsay and Blakis on; 1855.
14 . Vas esaege MM, Delcou R. The na u al his o y o a he oscle osis. Ci cula ion. 1962;26:841–55.
h ps://doi.o g/10.1161/01.ci .26.5.841 PMID: 13996459
15 . Mancusi C, Bahlmann E, Basile C, Ge d s E. new e idence abou ao ic al e s enosis and ca dio as-
cula hemodynamics. High Blood P ess Ca dio asc P e . 2022;29(3):231–7. h ps://doi.o g/10.1007/
s40292-022-00520-x PMID: 35438477
16 . Popo ici-Baznosanu A. Bei äge zu Kenn nis des Ci cula ionssys ems de Insek en. Jena Z Med
Na u wiss. 1905;40:667–96.
1 7. Wasse hal LT. Func ional mo phology o he hea and o a new cephalic pulsa ile o gan in he
blow ly Callipho a icina (Dip e a: Callipho idae) and hei oles in hemolymph anspo and
acheal en ila ion. In J Insec Mo phol Emb yol. 1999;28(1–2):111–29. h ps://doi.o g/10.1016/
s0020-7322(99)00012-4
18 . We inge O. Das ci cula ionssys em de ipulidenla en mi besonde e be ücksich igung on ipula
selene. Z wiss Zool. 1927;129:453–82.
19 . Ande eck JW, King JG, Hillye JF. Con ac ion o he en al abdomen po en ia es ex aca diac e -
og ade hemolymph p opulsion in he mosqui o hemocoel. PLoS One. 2010;5(9):e12943. h ps://doi.
o g/10.1371/jou nal.pone.0012943 PMID: 20886066

PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011613 Ma ch 20, 2025 22 / 24
PLOS Gene icS Ca diac al es
20. Angioy A, Pie a P. Mechanism o bea e e sal in semi-in ac hea p epa a ions o he blow ly
Pho mia egina (Meigen). J Comp Physiol B. 1995;165(B):165–70.
21. Ge ould J. O de s o insec s wi h hea -bea e e sal. Biol Bull. 1933;64:424–31.
22. Sláma K. Physiology o hea bea e e sal in adul D osophila melanogas e (Dip e a: D osophilidae).
Eu J En omol. 2010;107:13–31.
23. Wasse hal LT. Pe iodic hea bea e e sals cause ca diogenic inspi a ion and expi a ion wi h coupled
spi acle leakage in es ing blow lies, Callipho a icina. J Exp Biol. 2014;217(P 9):1543–54. h ps://doi.
o g/10.1242/jeb.097238 PMID: 24436373
24. Zei ouni B, Séna o e S, Sé e ac D, Aknin C, Sémé i a M, Pe in L. Signalling pa hways in ol ed
in adul hea o ma ion e ealed by gene exp ession p o iling in D osophila. PLoS Gene .
2007;3(10):1907–21. h ps://doi.o g/10.1371/jou nal.pgen.0030174 PMID: 17937502
25. Lehmache C, Abeln B, Paulula A. The ul as uc u e o D osophila hea cells. A h opod S uc De .
2012;41(5):459–74. h ps://doi.o g/10.1016/j.asd.2012.02.002 PMID: 22426062
26. Lamme s K, Abeln B, Hüsken M, Lehmache C, Psa haki OE, Alco a E, e al. Fo ma ion and unc ion
o in aca diac al e cells in he D osophila hea . J Exp Biol. 2017;220(P 10):1852–63. h ps://doi.
o g/10.1242/jeb.156265 PMID: 28254880
2 7. Meye C, B ei sp eche L, Ba aille L, Vincen AJM, D echsle M, Meye H, e al. Fo ma ion
and unc ion o a highly specialised ype o o ganelle in ca diac al e cells. De elopmen .
2022;149(19):de 200701. h ps://doi.o g/10.1242/de .200701 PMID: 36189830
28. Meye C, D echsle M, Meye H, Paulula A. Di e en ia ion and unc ion o ca diac al es in he adul
D osopihila hea . J Exp Biol. 2023;226.
29. Choma MA, Su e MJ, Vakoc BJ, Bouma BE, Tea ney GJ. Hea wall elocime y and exogenous
con as -based ca diac low imaging in D osophila melanogas e using Dopple op ical cohe ence
omog aphy. J Biomed Op . 2010;15(5):056020. h ps://doi.o g/10.1117/1.3503418 PMID: 21054114
30. Choma M, Su e M, Vakoc B, Bouma B, Tea ney G. Physiological homology be ween D osophila
melanogas e and e eb a e ca dio ascula sys ems. Dis Mod Mech. 2011;4:411–20.
31. D echsle M, Schmid AC, Meye H, Paulula A. The conse ed ADAMTS-like p o ein lonely hea
media es ma ix o ma ion and ca diac issue in eg i y. PLoS Gene . 2013;9(7):e1003616. h ps://doi.
o g/10.1371/jou nal.pgen.1003616 PMID: 23874219
32. Glenn JD, King JG, Hillye JF. S uc u al mechanics o he mosqui o hea and i s unc ion in bidi ec-
ional hemolymph anspo . J Exp Biol. 2010;213(4):541–50. h ps://doi.o g/10.1242/jeb.035014 PMID:
20118304
33. Weismann CG, Blice-Baum A, Tong T, Li J, Huang BK, Jonas SM, e al. Mul i-modal and mul iscale
imaging app oaches e eal no el ca dio ascula pa hophysiology in D osophila melanogas e . Biol
Open. 2019;8(8):bio044339. h ps://doi.o g/10.1242/bio.044339 PMID: 31455664
34. Sidebo ham D, Le G ice IJ. Chap e 1 - physiology and pa hophysiology. In: Sidebo ham D, Mckee A,
Gillham M, Le y JH, edi o s. Ca dio ho acic c i ical ca e. Elese ie . 2007. p. 3–27.
35. O o CM. Val ula ao ic s enosis: disease se e i y and iming o in e en ion. J Am Coll Ca diol.
2006;47(11):2141–51. h ps://doi.o g/10.1016/j.jacc.2006.03.002 PMID: 16750677
36. Geng X, Cha B, Mahamud MR, S ini asan RS. In aluminal al es: de elopmen , unc ion and dis-
ease. Dis Model Mech. 2017;10(11):1273–87. h ps://doi.o g/10.1242/dmm.030825 PMID: 29125824
3 7. Saik ishnan N, Kuma G, Sawaya FJ, Le akis S, Yogana han AP. Accu a e assessmen o ao ic s eno-
sis: a e iew o diagnos ic modali ies and hemodynamics. Ci cula ion. 2014;129(2):244–53. h ps://doi.
o g/10.1161/CIRCULATIONAHA.113.002310 PMID: 24421359
38. Chin apalli RTV, Hillye JF. Hemolymph ci cula ion in insec ligh appendages: physiology o he wing
hea and ci cula o y low in he wings o he mosqui o Anopheles gambiae. J Exp Biol. 2016;219(P
24):3945–51. h ps://doi.o g/10.1242/jeb.148254 PMID: 27742896
39. League GP, Onuh OC, Hillye JF. Compa a i e s uc u al and unc ional analysis o he la al and
adul do sal essel and i s ole in hemolymph ci cula ion in he mosqui o Anopheles gambiae. J Exp
Biol. 2015;218(P 3):370–80. h ps://doi.o g/10.1242/jeb.114942 PMID: 25524976
40. Lee W-K, Socha JJ. Di ec isualiza ion o hemolymph low in he hea o a g asshoppe (Schis o-
ce ca ame icana). BMC Physiol. 2009;9:2. h ps://doi.o g/10.1186/1472-6793-9-2 PMID: 19272159
41. Babcock DT, B ock AR, Fish GS, Wang Y, Pe in L, K asnow MA, e al. Ci cula ing blood cells unc-
ion as a su eillance sys em o damaged issue in D osophila la ae. P oc Na l Acad Sci U S A.
2008;105(29):10017–22. h ps://doi.o g/10.1073/pnas.0709951105 PMID: 18632567
42. Hinde e S, Schenke-Layland K. Ca diac ib osis - A sho e iew o causes and he apeu ic s a e-
gies. Ad D ug Deli Re . 2019;146:77–82. h ps://doi.o g/10.1016/j.add .2019.05.011 PMID: 31158407
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011613 Ma ch 20, 2025 23 / 24
PLOS Gene icS Ca diac al es
43. Jensen MT, Fung K, Aung N, Sangh i MM, Chadala ada S, Pai a JM, e al. Changes in ca diac mo -
phology and unc ion in indi iduals wi h diabe es melli us: The UK biobank ca dio ascula magne ic
esonance subs udy. Ci c Ca dio asc Imaging. 2019;12(9):e009476. h ps://doi.o g/10.1161/CIRCIM-
AGING.119.009476 PMID: 31522551
44. Poggio P, Ca allo i L, Songia P, Di Minno A, Amb osino P, e al. Impac o al e mo phology on he
p e alence o co ona y a e y disease: A sys ema ic e iew and me a-analysis. J Am Hea Assoc.
2016;5.
45. G e e A, Bang C, Be g R, Egs up K, Rossebo A. Res ing hea a e and isk o ad e se ca dio ascu-
la ou comes in asymp oma ic ao ic s enosis: he SEAS s udy. In J Ca diol. 2015;180:122–8.
46. Fink M, Callol-Masso C, Chu A, Ruiz-Lozano P, Izpisua Belmon e JC, Giles W, e al. A new me hod
o de ec ion and quan i ica ion o hea bea pa ame e s in D osophila, zeb a ish, and emb yonic
mouse hea s. Bio echniques. 2009;46(2):101–13. h ps://doi.o g/10.2144/000113078 PMID: 19317655
4 7. Oco K, Ree es N, Wessells R, Fink M, Chen H. KCNQ po assium channel mu a ions cause ca diac
a hy hmias in D osophila ha mimic he e ec s o aging. P oc Na l Acad Sci U S A. 2007;104:3943–8.
48. Oco K, Fink M, Camma a o A, Be ns ein S, Bodme R. Semi-au oma ed op ical hea bea analysis
o small hea s. J Vis Exp imen s. 2009;31:pii: 1435.
49. Caldwell JC, Mille MM, Wing S, Soll DR, Ebe l DF. Dynamic analysis o la al locomo ion in D o-
sophila cho do onal o gan mu an s. P oc Na l Acad Sci U S A. 2003;100(26):16053–8. h ps://doi.
o g/10.1073/pnas.2535546100 PMID: 14673076
50. Nichols C, Becnel J, Pandey U. Me hods o assay D osophila beha io . J Vis Exp. 2012;61.
51. Pos Y, Paulula A. Muscle unc ion assessmen using a D osophila la ae c awling assay. Bio P o oc.
2018;8(14):e2933. h ps://doi.o g/10.21769/BioP o oc.2933 PMID: 34395754
52. Pass G. Accesso y pulsa ile o gans: e olu iona y inno a ions in insec s. Annu Re En omol.
2000;45:495–518. h ps://doi.o g/10.1146/annu e .en o.45.1.495 PMID: 10761587
53. Ro s ein B, Paulula A. On he mo phology o he D osophila hea . J Ca dio asc De Dis.
2016;3(2):15. h ps://doi.o g/10.3390/jcdd3020015 PMID: 29367564
54. B ooks DS, Vishal K, Kawakami J, Bouyain S, Geisb ech ER. Op imiza ion o w MT ck o moni o
D osophila la al locomo o ac i i y. J Insec Physiol. 2016;93–94:11–7. h ps://doi.o g/10.1016/j.jins-
phys.2016.07.007 PMID: 27430166
55. Husson SJ, Cos a WS, Schmi C, Go schalk A. Keeping ack o wo m acke s. Wo mBook; 2013.
1–17.
56. Bishop ic NH. E olu ion o he hea om bac e ia o man. Ann N Y Acad Sci. 2005;1047:13–29.
h ps://doi.o g/10.1196/annals.1341.002 PMID: 16093481
5 7. Polilo AA. Small is beau i ul: ea u es o he smalles insec s and limi s o minia u iza ion. Annu Re
En omol. 2015;60:103–21. h ps://doi.o g/10.1146/annu e -en o-010814-020924 PMID: 25341106
58. Campbell J, Be y JD, Liang Y. Ana omy and physiology o he small in es ine. ana omy and physiol-
ogy o he small in es ine. Shakel o d´s Su ge y o he Alimen a y T ac , 2 olume se . 8 h ed. 2019.
817–41.
59. Shin M, Lawson ND. Back and o h: His o y o and new insigh s on he e eb a e lympha ic al e.
De G ow h Di e . 2021;63(9):523–35. h ps://doi.o g/10.1111/dgd.12757 PMID: 34716915
60. McCoy J, Ba es M, Egge C, Sie o M, Cassidy S, Newman J, e al. Pa hophysiology o exe cise
in ole ance in ch onic diseases: he ole o diminished ca diac pe o mance in mi ochond ial and
hea ailu e pa ien s. Open Hea . 2017;4(2):e000632. h ps://doi.o g/10.1136/openh -2017-000632
PMID: 28878952
61. Baumga ne H, Hung J, Be mejo J, Chambe s JB, E angelis a A, G i in BP, e al. Echoca diog aphic
assessmen o al e s enosis: EAE/ASE ecommenda ions o clinical p ac ice. J Am Soc Echoca -
diog . 2009;22(1):1–23; quiz 101–2. h ps://doi.o g/10.1016/j.echo.2008.11.029 PMID: 19130998
62. Kamimu a D, Hans S, Suzuki T, Fox E, Hall M, e al. Delayed ime o peak eloci y is use ul o de ec -
ing se e e ao ic s enosis. J Am Hea Assoc. 2016;5.
63. Aminda i A, Sal ik L, Ki kkop u K, Yacoub M, Yalcin HC. Assessmen o calci ied ao ic al e lea -
le de o ma ions and blood low dynamics using luid-s uc u e in e ac ion modeling. In o m Med
Unlocked. 2017;9191–9. h ps://doi.o g/10.1016/j.imu.2017.09.001
64. Sláma K. Ex aca diac e sus ca diac haemocoelic pulsa ions in pupae o he mealwo m (Teneb io
moli o L.). J Insec Physiol. 2000;46(6):977–92. h ps://doi.o g/10.1016/s0022-1910(99)00208-5
PMID: 10802111
65. Sláma K. Mechanical aspec s o hea bea e e sal in pupae o Manduca sex a. J Insec Physiol.
2003;49(7):645–57. h ps://doi.o g/10.1016/s0022-1910(03)00065-9 PMID: 12837317
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011613 Ma ch 20, 2025 24 / 24
PLOS Gene icS Ca diac al es
66. Sláma K. A new look a he compa a i e physiology o insec and human hea s. J Insec Physiol.
2012;58(8):1072–81. h ps://doi.o g/10.1016/j.jinsphys.2012.04.014 PMID: 22609364
6 7. Cao Y, He S, Ding M, Gu W, Wang T, e al. Regula exe cise in D osophila p e en s age- ela ed ca -
diac dys unc ion caused by high a and hea -speci ic knockdown o skd. In J Mol Sci. 2023;24.
68. Nys o iak MA, Bha naga A. Ca dio ascula E ec s and Bene i s o Exe cise. F on Ca dio asc Med.
2018;5:135. h ps://doi.o g/10.3389/ c m.2018.00135 PMID: 30324108
69. Wen D-T, Zheng L, Yang F, Li H-Z, Hou W-Q. Endu ance exe cise p e en s high- a -die induced
hea and mobili y p ema u e aging and dsi 2 exp ession decline in aging D osophila. Onco a ge .
2017;9(7):7298–311. h ps://doi.o g/10.18632/onco a ge .23292 PMID: 29484111
70. Zheng L, Li QF, Ni L, Wang H, Ruan XC, Wu XS. Li e ime egula exe cise a ec s he inciden o di e -
en a hy hmias and imp o es o ganismal heal h in aging emale D osophila melanogas e . Bioge on-
ology. 2017;18(1):97–108. h ps://doi.o g/10.1007/s10522-016-9665-5 PMID: 27787741
71. Taghli-Lamallem O, Akasaka T, Hogg G, Nudel U, Ya e D, Chambe lain JS, e al. Dys ophin de i-
ciency in D osophila educes li espan and causes a dila ed ca diomyopa hy pheno ype. Aging Cell.
2008;7(2):237–49. h ps://doi.o g/10.1111/j.1474-9726.2008.00367.x PMID: 18221418
72. Zhu J, Fu Y, Ne le on M, Richman A, Han Z. High h oughpu in i o unc ional alida ion o candida e
congeni al hea disease genes in D osophila. eLi e. 2017;6. h ps://doi.o g/10.7554/eLi e.22617
73. Neely GG, Kuba K, Camma a o A, Isobe K, Amann S, Zhang L, e al. A global in i o D osophila
RNAi sc een iden i ies NOT3 as a conse ed egula o o hea unc ion. Cell. 2010;141(1):142–53.
h ps://doi.o g/10.1016/j.cell.2010.02.023 PMID: 20371351
74. Sláma K, Fa kaš R. Hea bea pa e ns du ing he pos emb yonic de elopmen o D osophila mela-
nogas e . J Insec Physiol. 2005;51(5):489–503. h ps://doi.o g/10.1016/j.jinsphys.2004.11.016 PMID:
15893996
75. Wasse hal LT. D osophila lies combine pe iodic hea bea e e sal wi h a ci cula ion in he an e io
body media ed by a newly disco e ed an e io pai o os ial al es and “ enous” channels. J Exp Biol.
2007;210(P 21):3707–19. h ps://doi.o g/10.1242/jeb.007864 PMID: 17951411
76. Hillye JF. Insec hea hy hmici y is modula ed by e olu iona ily conse ed neu opep ides and neu-
o ansmi e s. Cu Opin Insec Sci. 2018;29:41–8. h ps://doi.o g/10.1016/j.cois.2018.06.002 PMID:
30551824
7 7. Hillye JF. Insec immunology and hema opoiesis. De Comp Immunol. 2016;58:102–18. h ps://doi.
o g/10.1016/j.dci.2015.12.006 PMID: 26695127
78. Sellin J, Alb ech S, Kölsch V, Paulula A. Dynamics o hea di e en ia ion, isualized u ilizing hea
enhance elemen s o he D osophila melanogas e bHLH ansc ip ion ac o Hand. Gene Exp Pa -
e ns. 2006;6(4):360–75. h ps://doi.o g/10.1016/j.modgep.2005.09.012 PMID: 16455308
79. Mo in-Poula d I, Des alminil-Le ou neau M, Ba aillé L, F endo J-L, Leb e on G, Vanzo N, e al. Iden i-
ica ion o Bipo en ial Blood Cell/Neph ocy e P ogeni o s in D osophila: Ano he Rou e o Gene a ing
Blood P ogeni o s. F on Cell De Biol. 2022;10:834720. h ps://doi.o g/10.3389/ cell.2022.834720
PMID: 35237606
80. Meye C, Ba aillé L, D echsle M, Paulula A. Tailup exp ession in D osophila la al and adul ca diac
al e cells. Genesis. 2023;61(1–2):e23506. h ps://doi.o g/10.1002/d g.23506 PMID: 36546531
81. Wang S, Meye H, Ochoa-Espinosa A, Buchwald U, Onel S, Al enhein B, e al. GBF1 (Ga enzwe g)-
dependen sec e ion is equi ed o D osophila ubulogenesis. J Cell Sci. 2012;125(P 2):461–72.
h ps://doi.o g/10.1242/jcs.092551 PMID: 22302994
82. Lehmache C, Tögel M, Pass G, Paulula A. The D osophila wing hea s consis o syncy ial muscle
cells ha esemble adul soma ic muscles. A h opod S uc De . 2009;38(2):111–23. h ps://doi.
o g/10.1016/j.asd.2008.09.002 PMID: 18983940
83. Vogle G, Oco K. Visualizing he bea ing hea in D osophila. J Vis Exp. 2009;31e1425. h ps://doi.
o g/10.3791/1425
84. Oco K, Vogle G, Bodme R. Me hods o assess D osophila hea de elopmen , unc ion and aging.
Me hods. 2014;68(1):265–72. h ps://doi.o g/10.1016/j.yme h.2014.03.031 PMID: 24727147
85. Tine ez JY, Pe y N, Schindelin J, Hoopes GM, Reynolds GD, e al. T ackMa e: An open and ex ensi-
ble pla o m o single-pa icle acking. Me hods. 2017;115:80–90.