Adverse Impact of Diet-Induced Hypercholesterolemia on Cardiovascular Tissue Homeostasis in a Rabbit Model: time-Dependent Changes in Cardiac Parameters

In . J. Mol. Sci. 2013, 14, 19086-19108; doi:10.3390/ijms140919086 In e na ional Jou nal o Molecula Sciences ISSN 1422-0067 www.mdpi.com/jou nal/ijms A icle Ad e se Impac o Die -Induced Hype choles e olemia on Ca dio ascula Tissue Homeos asis in a Rabbi Model: Time-Dependen Changes in Ca diac Pa ame e s A ila Ke ész 1, Ma iann Bombicz 2, Daniel P iksz 2, Jozse Balla 3,4, Gyo gy Balla 3,5, Rudol Gesz elyi 2, Balazs Va ga 2, Da id D. Haines 2, A pad Tosaki 2 and Bela Juhasz 2,* 1 Depa men o Ca diology, Facul y o Medicine, Uni e si y o Deb ecen, Nagye dei k . 98, Deb ecen 4032, Hunga y; E-Mail: d .[email p o ec ed]m 2 Depa men o Pha macology, Facul y o Pha macy, Uni e si y o Deb ecen, Nagye dei k . 98, Deb ecen 4032, Hunga y; E-Mails: bom[email p o ec ed] (M.B.); [email p o ec ed] (D.P.); gesz elyi. udol @pha m.unideb.hu (R.G.); [email p o ec ed] (B.V.); [email p o ec ed] (D.D.H.); [email p o ec ed] (A.T.) 3 MTA-DE Vascula Biology, Th ombosis and Hemos asis Resea ch G oup, Hunga ian Academy o Sciences, Uni e si y o Deb ecen, Nagye dei k . 98, Deb ecen 4032, Hunga y; E-Mails: [email p o ec ed]b.hu (J.B.); [email protected] (G.B.) 4 Depa men o Neph ology, Medical and Heal h Science Cen e , Uni e si y o Deb ecen, Nagye dei k . 98, Deb ecen 4032, Hunga y 5 Depa men o Pedia ics, Uni e si y o Deb ecen, Nagye dei k . 98, Deb ecen 4032, Hunga y * Au ho o whom co espondence should be add essed; E-Mail: juha[email p o ec ed]; Tel./Fax: +36-52-255-586. Recei ed: 1 July 2013; in e ised o m: 31 July 2013 / Accep ed: 2 Augus 2013 / Published: 17 Sep embe 2013 Abs ac : The p esen s udy e alua es a hypo hesis ha die - ela ed hype choles e olemia inc eases oxida i e s ess- ela ed bu den o ca dio ascula issue, esul ing in p og essi ely inc eased mo ali y, along wi h de e io a ion o elec ophysiological and enzyma ic unc ion in abbi myoca dium. New Zealand whi e abbi s we e di ided in o ou g oups, de ined as ollows: GROUP I, choles e ol- ee abbi chow o 12 weeks; GROUP II, choles e ol- ee chow, 40 weeks; GROUP III, chow supplemen ed wi h 2% choles e ol, 12 weeks; GROUP IV, chow supplemen ed wi h 2% choles e ol, 40 weeks. A he 12 and 40 weeks ime poin s, animals in each o he a o emen ioned coho s we e subjec ed o echoca diog aphic measu emen s, ollowed by sac i ice. Signi ican de e io a ion in majo OPEN ACCESS In . J. Mol. Sci. 2013, 14 19087 ou come a iables measu ed in he p esen s udy we e obse ed only in animals main ained o 40 weeks on 2% choles e ol-supplemen ed chow, wi h much lesse ad e se e ec s no ed in animals ed high choles e ol die s o only 12 weeks. I was obse ed ha abbi s ecei ing high choles e ol die s o 40 weeks exhibi ed signi ican ly inc eased mo ali y, wo sened ejec ion ac ion and gene al de e io a ion o ca diac unc ions, along wi h inc eased a he oscle o ic plaque o ma ion and in a c size. Addi ionally, myoca dium o GROUP IV animals was obse ed o con ain lowe le els o heme oxygenase-1 (HO-1) and cy och ome c oxidase III (COX III) p o ein ela i e o he con ols. Keywo ds: hype choles e olemic abbi ; ca diac pa ame e s; echoca diog aphy; heme-oxygenase; cy och ome oxidase; VEGF 1. In oduc ion Ca dio ascula diseases emain he majo cause o mo bidi y and mo ali y in indus ialized Wes e n na ions. In hese diso de s, dea h mos o en occu s as a consequence o a he oscle osis, leading o s oke, ischemia, myoca dial in a c ion, hea ailu e and o he synd omes cha ac e ized by se e ely dys egula ed in lamma o y p ocesses and hei esul ing deg ada ion o issue unc ion [1,2]. A hallma k o hese synd omes is mic o ascula damage associa ed wi h a p og essi e decline in heal hy ca dio ascula issue homeos asis, which ypically is accompanied by diminished a e ial lexibili y and onse o age-associa ed pa hologies, especially ca dio ascula , neu ologic and kidney disease [3]. The associa ion o dyslipidemia and ischemic hea diseases a e well known. Ne e heless, a he ime o his w i ing, a de ailed desc ip ion o he e iology and molecula pa homechanisms o hype choles e olemia-induced a he oscle osis and i s complica ions emains o be de e mined [3]. Recen e idence sugges s ha mi ochond ia a e pa icula ly sensi i e o hype choles e olemia-associa ed oxida i e s ess; he e o e, he mo phological and unc ional analysis o mi ochond ia is expec ed o con ibu e o he p e en ion o and he apy o hype choles e olemia-induced diseases [4]. Hype choles e olemia is associa ed wi h ele a ed le els o malondialdehyde (MDA), a measu e o oxida i e s ess in abbi ao a [5–8]. Hype choles e olemia is also known o inc ease he p oduc ion o eac i e oxygen species (ROS) h ough a ious pa homechanisms. Mi ochond ial elec on- ans e complexes a e majo sou ces o ROS, and oxida i e damage o he elec on anspo complexes ha a e in close p oximi y o hese ROS sou ces, e.g., cy och ome c oxidase, a e expec ed o inhibi elec on anspo . Such inhibi ion inc eases elec on leakage, leading o u he ROS p oduc ion [9]. The esul ing in ensely oxida i e en i onmen may inac i a e cy och ome c oxidase, esul ing in issue damage—an example being doxo ubicin-induced ca diomyopa hy [10]. Physiologically ele an pa hological changes obse ed as a esul o long- e m hype choles e olemia may also occu as a esul o b ie hype choles e olemic episodes (days), wi h p o ound ad e se e ec s on endo helium-dependen unc ions o he mic oci cula ion, including dila ion o a e ioles, luid il a ion ac oss capilla ies and egula ion o leukocy e ec ui men in pos capilla y enules [11]. A u he consequence o hype choles e olemia-induced mic o ascula esponses may include enhanced In . J. Mol. Sci. 2013, 14 19088 ulne abili y o he mic o ascula u e o he ha m ul e ec s o ischemia and o he in lamma o y condi ions [11]. Mo eo e , inc eased sensi i i y o en icula ib illa ion exhibi ed by he hype choles e olemic hea is also obse ed and is associa ed wi h p olonged ac ion po en ial du a ion, longe QT (measu e o he ime be ween he s a o he Q wa e and he end o he T wa e in he hea 's elec ical cycle) in e als and inc eased epola iza ion dispe sion [12]. A e a he oscle o ic- ela ed e iologies, hea ailu e (HF) is he second majo con ibu o o he incidence o ca dio ascula dys unc ions [13]. HF is also a leading cause o dea h in middle- and high-income na ions [1]. Hea ailu e is no classed as a dis inc disease, bu gene ally conside ed o be a g oup o symp oms, which may be igge ed o exace ba ed by a majo a he oscle o ic condi ion, hype ension, al ula diseases, ca diomyopa hy and ad e se d ug e ec s [1]. Conges i e hea ailu e mani es s as le - o igh -sided o bi en icula , and he known majo isk ac o s include obesi y, hype ension, smoking and diabe es [14]. Cu en ly a ailable he apies may slow he p og ession o his pa hology, bu no cu e i [15]. Mo eo e , igh -sided hea ailu e is conside ed o be incu able, and he diso de is e ac o y o mos d ugs [15]. The o e all g im ou look o de ini i e ea men o ca dio ascula diso de s is ne e heless mi iga ed somewha by he esul s o ea men modali ies ex ac ed om adi ional and he bal medicine. P e ious s udies ha e demons a ed he signi ican ca dio ascula bene i s o a wide ange o plan ma e ials, including commonly used componen s o he human die , as well as na u al heal h p oduc s and nu aceu icals [16,17]. P e iously, he au ho s demons a ed ha sou che y seed ex ac , a na u al p oduc ha con ains a powe ul induce o heme oxygense-1, s ongly inhibi s ischemia- epe usion (IR) inju y [3,18,19]. S a egies ha augmen he cu en ly used pha maco he apies o e he ad an age o low- o-negligible oxici y, widesp ead a ailabili y and low p ice. Fo he a o emen ioned easons, cha ac e iza ion o he biological e ec s media ed by he bal p oduc s opens oppo uni ies in he apy o ca dio ascula diso de s. The p esen in es iga ion is expec ed o yield insigh in o mechanisms by which hype choles e olemia- ela ed oxida i e s ess deg ades he unc ion o c i ical mac omolecules and p omo es disease. Hype choles e olemia is a well-es ablished isk ac o o co ona y a e y disease and is ela ed o endo helial dys unc ion a bo h he mac o- and mic o- ascula le el. A signi ican inding o he p esen s udy is ha he ejec ion ac ion also was g ea ly diminished in animals main ained long- e m (40 weeks) on high dose choles e ol die (2%), wi h subsequen de elopmen o myoca dial in a c ion. The co e hypo hesis o he p esen in es iga ion is ha oxida i e s ess induced in a hype choles e olemic abbi model by sus ained choles e ol die s p omo es pa hological changes in ways ha may be e ealed by changes on he molecula le el. The in es iga o s examined he e ec s o hype choles e olemia on ca diac sys olic and dias olic pa ame e s using echoca diog aphy. Mechanis ic expe imen s we e conduc ed ha included measu emen o he ac i i y o cy op o ec i e enzymes, such as heme-oxygenase (HO-1), in luences on e ascula iza ion o ascula endo helial g ow h ac o (VEGF) p o ein, an iapop o ic in luences and he e ec s o die a y ea men s on mi ochond ial unc ion. Co olla y s udies also included assessmen o he ole o cy och ome c oxidase (COX) enzymes in egula ing ATP me abolism and hos ing adap i e, an i-in lamma o y p ocesses, including ROS sca enging. In . J. Mol. Sci. 2013, 14 19089 2. Resul s and Discussion Se um choles e ol le els in animals adminis e ed eed supplemen ed wi h 2% choles e ol (hype choles e olemic abbi s) and abbi s ecei ing no mal eed (con ols) we e e alua ed du ing he s udy pe iod. As shown in Figu e 1, o al se um choles e ol in hype choles e olemic animals p o ided wi h eed con aining 2% choles e ol was signi ican ly inc eased du ing he 40-week ime-cou se o he s udy ela i e o le els obse ed in con ol abbi s ecei ing no mal eed (p < 0.05). Figu e 1. Time-dependen se um choles e ol le el in no mal and hype choles e olemic abbi s. A e age o al se um choles e ol le els (mmol/L ± SEM) in wo g oups o abbi s (n = 4 pe g oup), each adminis e ed eed con aining 2% choles e ol, we e measu ed using he Ca dioCheck se um choles e ol analyze du ing a 12-week (sho pe iod) and 40-week (long pe iod) ime-cou se in hype choles e olemic abbi s (squa es) and non-hype choles e olemic con ol animals (diamonds). * p < 0.05 o compa ison o se um choles e ol in hype choles e olemic e sus non-hype choles e olemic abbi s. Al hough hepa ic unc ions we e no speci ically e alua ed in he p esen s udy, he known consequences o hype choles e olemia on he li e mus be conside ed in he con ex o he in luence o his o gan on ca diac heal h. Sus ained ele a ion in die a y choles e ol le els places a bu den on he li e ha con ibu es signi ican ly o ascula de e io a ion [20]. Dis up ion o no mal hepa ic ac i i y may ad e sely a ec ca diac heal h h ough nume ous pa hways. One example o he in e ac ion be ween hese wo o gan sys ems, which may eme ge as an a ea o de elopmen o no el he apies, is ha he dis up ion o no mal egula ion by he li e o pla ele -ac i a ing ac o ace yl-hyd olase may s ongly con ibu e o a he oscle osis, since his enzyme is low densi y lipop o ein (LDL)-associa ed [21]. I is he e o e likely ha o he e ec o s o ca dio ascula me abolism ha a e egula ed by he li e and a e high densi y lipop o ein (HDL)- and/o LDL-associa ed may con ibu e o ca diac pa hologies as a esul o hype lipidemia. Plaque co e age in ho acic a e ies is shown in Figu e 2. The ex en o plaque co e age in animals main ained on high choles e ol die s (HC) was signi ican ly g ea e han abbi s ed wi h no mal chow In . J. Mol. Sci. 2013, 14 19090 (con ol) (p < 0.05), in which negligible a he oscle o ic plaque accumula ion was no ed, as shown in he Figu e 2 pho og aphs o dissec ed a e ies. Mo eo e , as expec ed, animals ed high choles e ol die s o 40 weeks (HC long) exhibi ed signi ican ly highe plaque co e age han abbi s ecei ing he 2% choles e ol-supplemen ed eed o 12-week pe iods (HC sho ) (p < 0.05). Figu e 2. A e ial plaque co e age. (a) A he oscle o ic plaque co e age in Sudan III-s ained luminal sec ions o ho acic a e ies ha es ed om ou g oups o abbi s (n = 4 pe g oup), adminis e ed no mal eed (con ol) o eed supplemen ed wi h 2% choles e ol (HC, high choles e ol). Plaque co e age e alua ed ollowing a 12-week (sho ) o 40-week (long) ime-cou se is shown as he a e age pe cen age o plaque co e age in a e ies om each g oup o animals ± SEM; (b) The ex en o a he oscle osis in a e ial lumens is ep esen ed in his og am o m as pe cen age plaque co e age; and as pho og aphic images o ep esen a i e a e ial sec ions shown in he same ames as each his og am. * p < 0.05 o compa ison o pe cen age a he oscle o ic plaque co e age in a e ies o non-hype choles e olemic long (con ol long) abbi s e sus hype choles e olemic long (HC long) and hype choles e olemic sho (HC sho ) abbi s. # p < 0.05 o compa ison o pe cen age a he oscle o ic plaque co e age in a e ies o hype choles e olemic long (HC long) abbi s e sus hype choles e olemic sho (HC sho ) abbi s. (a) (b) The a e age ex en o in a c zones in con ol sho , con ol long, HC sho and HC long g oups is ep esen ed by his og ams in Figu e 3. These ou comes e eal ha in a c ed egions did no de elop in non-hype choles e olemic (con ol) animals, wi h only small in a c ed a eas o ming in hea s o abbi s ecei ing high choles e ol chow o 12-week pe iods (HC sho ). Con e sely, in a c ed egions we e no ed in hea s o abbi s ed wi h he 2% choles e ol-supplemen ed die o 40 weeks (HC long), which we e signi ican ly la ge han he in a c size o HC sho animals (p < 0.05). C on ol Sho Con ol Long HC Sho HC Long 0 20 40 60 80 100 * # * A he oscle osis co e age (%) C on ol Sho Con ol Long HC Sho HC Long 0 A he oscle o ic Plaque Co e age In . J. Mol. Sci. 2013, 14 19091 Figu e 3. To al in a c ed egion. (a) Hea s we e isola ed om ou g oups o abbi s (n = 4 pe g oup), adminis e ed no mal chow o 12 weeks (con ol sho ); no mal chow o 40 weeks (con ol long); high choles e ol (2%) chow du ing a 12-week ime-cou se (HC sho ); o high choles e ol chow du ing a 40-week ime-cou se (HC long). Isola ed hea s we e pe used in modi ied K ebs-Henselei bica bona e bu e using a Langendo appa a us. The o al in a c ed egion was de e mined by pe usion wi h iphenyl e azolium chlo ide (TTC) solu ion, ollowed by mic oscopic analysis o ans e se sec ions o each hea . A e age sizes o he in a c zone o hea s in each g oup ± SEM a e shown o each ea men g oup; (b) The magni ude o in a c zones measu e in hea s o animals subjec ed o selec ed ea men condi ions is displayed in his og am o m in he same ame spaces as pho og aphs o in a c ed hea sec ions. *p < 0.05 o compa ison o a e age % in a c size in HC long e sus HC sho g oup. # p < 0.05 o compa ison o a e age % in a c size in HC long e sus con ol long g oup. (a) (b) Hype choles e olemia deg ades homeos a ic p ocesses in ca dio ascula issue in ways ha engende epica dial and mic o ascula co ona y a e y disease. Ele a ed le els o se um choles e ol p omo e o ma ion o plaque deposi s in epica dial a e ies, which p og essi ely na ow essel lumen, dep i ing issues hey supply o oxygena ed blood. As myoca dial oxygen demand exceeds supply, in a c zones may de elop in egions o myoca dial issue supplied by occluded blood essels. In humans, he clinical p esen a ion o such p ocesses is e med “non-ST (connec s he QRS complex and he T wa e) ele a ion myoca dial in a c ion”. The pa hogenesis o his synd ome does no in ol e occlusion o he in a c - ela ed co ona y a e y; he e o e, no ST segmen ele a ion—a sign o o al co ona y occlusion—is ypically obse ed in elec oca diog aphy (ECG) e alua ions o a lic ed indi iduals. Fo non-ST ele a ion myoca dial in a c ion, he ex en o nec o ized issue is dependen on he size o he myoca dial egion supplied by he co ona y a e y dis al o he s eno ic lesion and he ime in e al o he insu icien oxygen supply. Ha ing se e ely s eno ic co ona y a e ies and s ained myoca dial nec o ic egions, he pa hophysiology o myoca dial in a c ion in HC long animals o he p esen s udy may be simila o non-ST segmen myoca dial in a c ion obse ed in humans. Fu he mo e, since he o al in a c ed egion o myoca dial mass inc eases, he sys olic unc ion o he C on ol Sho Con ol Long HC Sho HC Long 0 10 20 30 40 * # In a c Size (%) Con ol Sho Con ol Long HC Sho HC Long In a c Magni ude In . J. Mol. Sci. 2013, 14 19092 hea is a enua ed, causing a dec ease in ac ional sho ening and he ejec ion ac ion. Expe imen al liga ion o he co ona y a e y c ea es dis up ion o issue homeos asis ha esembles ST segmen ele a ion myoca dial in a c ion obse ed in humans when he co ona y a e y is o ally occluded by h ombus o ma ion a he ime o up u e o an a he oscle o ic plaque. This in a c ed egion is clea ly dis inguishable om no mal myoca dial issue and plain nec o ic a eas—and may, hus, be iden i ied wi h li le ambigui y, unlike in non-ST ele a ion cases. As shown in Figu es 4 and 5, he alues o ac ional sho ening (FS) and ejec ion ac ion (EF) o he le en icle we e signi ican ly educed in HC long animals ela i e o hei alues in non-hype choles e olemia con ol abbi s (p < 0.05) and o animals main ained o sho e ime pe iods on hype choles e olemia-inducing chow (HC sho ) (p < 0.05). These ou comes a e expec ed based on p e ious s udies, which e eal signi ican posi i e co ela ion be ween he occu ence o ca diac insu iciency and associa ed hea ailu e wi h possible le en icula hype ophy [22–24]. The a o emen ioned echoca diog aphic da a p o ides insigh in o he in luence o high choles e ol on he unde lying pa hogenesis o ca dio ascula disease and helps accoun o o he obse a ions desc ibed in he p esen epo , such as inc eased in a c magni ude in hype choles e olemia animals. No signi ican di e ences in G oups I–III we e obse ed. This expec a ion shows ha nei he ageing i sel no sho - e m hype choles e olemia a lic he sys olic and dias olic pa ame e s and no mal hea unc ion. Al hough, in animals ecei ing a long- e m choles e ol die , hese alues we e signi ican ly lowe (p < 0.05), which shows ha long- e m hype choles e olemia can de e io a e le en icle sys olic unc ion. As seen in Table 1, no signi ican di e ences in o he echoca diog aphic pa ame e s we e measu ed. I should be also no ed ha he le en icle (LV) masses o G oups II and IV we e highe han he LV masses o he sho - ea ed animals, al hough his only ep esen s ha he LV mass is co ela ed wi h inc easing body weigh , since hese animals we e i e mon hs olde a he momen o ex e mina ion. M-mode images demons a e c oss-sec ions o he hea swep by he ins umen a ion o e a de ined ime pe iod. This e alua ion is conduc ed by placing he cu so line on 2D images a he mid- en icula le el, wi h he la ges diame e in ei he he pa as e nal long o pa as e nal sho axis iew (on he M-mode pic u es a he uppe pa 2D iews wi h he cu so ; a he lowe pa , he acing a he cu so line could be seen). This echnique, which p o ides a e y high empo al esolu ion, allows he end sys olic [minimal diame e s, le en icula in e nal diame e a end-sys ole (LVIDs)] and end dias olic [maximal diame e , le en icula in e nal diame e a end-dias ole (LVIDd)] diame e s o be p ecisely measu ed. These pa ame e s a e used o calcula e ac ional sho ening [(LVIDd − LVIDs)/LVIDd] and ejec ion ac ion [(LVIDd − LVIDs)2/LVIDd2] (cubed o mula), which a e indica o s o le en icula emp ying capaci y. No mally, he hea ejec s mo e han 50% o i s end dias olic olume (ejec ion ac ion), and he diame e sho ens mo e han 25% (linea ejec ion ac ion o ac ional sho ening). When he myoca dial issue is damaged ( ypically by oxida i e s ess), i s con ac ile unc ion is a enua ed and emp ying capabili y dec eased. In . J. Mol. Sci. 2013, 14 19093 Figu e 4. F ac ional sho ening (FS) and ejec ion ac ion (EF) o le en icles. Echoca diog aphic measu emen s we e conduc ed on ou g oups o abbi s (n = 4 pe g oup), adminis e ed no mal chow o 12 weeks (con ol sho ); no mal chow o 40 weeks (con ol long); high choles e ol (2%) chow du ing a 12-week ime-cou se (HC sho ); o high choles e ol chow du ing a 40-week ime-cou se (HC long). Measu emen s we e made o ac ional sho ening (FS), de e mined as [le en icle end dias olic diame e (LVEDD) – le en icle end sys olic diame e (LVESD)]/LVEDD, and ejec ion ac ion (EF), calcula ed as (LVEDD 2 − LVESD 2 )/LVIDD 2 , as shown. * p < 0.05 o compa ison o a e age % FS o % EF wi h all o he g oups. Figu e 5. (a) M-mode image o con ol sho (CS) hea ; (b) M-mode image o con ol long (CL) hea ; (c) M-mode image o HC sho (HCS) hea ; (d) M-mode image o HC long (HCL) hea . (a) (b) In . J. Mol. Sci. 2013, 14 19094 Figu e 5. Con . (c) (d) Table 1. Values o M-mode and 2D echoca diog aphic a iables in New Zealand whi e abbi s. FS, ac ional sho ening o le en icle; EF, ejec ion ac ion, IVSd, in e en icula wall hickness in dias ole; IVSs, in e en icula wall hickness in sys ole; LVIDd, le en icula in e nal diame e a end-dias ole; LVIDs, le en icula in e nal diame e a end-sys ole; LVPWd, le en icula pos e io ( ee) wall hickness in dias ole; LVPWs, le en icula pos e io ( ee) wall hickness in sys ole; LV mass, mass o le en icle; (n = 4 in each g oup); * e e ence ange o heal hy New Zealand whi e abbi s [23]. S anda dized e e ence anges o HC sho and HC long alues in a hype choles e emic abbi model main ained unde he condi ions desc ibed in he p esen epo we e no ound in public domain li e a u e sou ces and acco dingly a e deno ed “na” below in Table 1. Con ol Sho Ca diac Pa ame e s Mean ± SD Minimum Maximum Re e ence ange * Coe icien o a ia ion (%) FS (%) 34.97 ± 5.085 25.77 44.44 17.63 o 40.16 14.54 EF (%) 60.08 ± 6.712 47.13 71.76 42.42 o 75.65 11.17 IVSd (mm) 2.81 ± 1.063 1.92 4.99 2.11 o 2.82 37.81 IVSs (mm) 4.308 ± 0.7011 3.23 5.63 2.98 o 5.92 16.27 LVIDd (mm) 17.07 ± 1.574 14.84 19.38 13.59 o 17.89 9.22 LVIDs (mm) 11.09 ± 1.173 8.95 12.93 8.51 o 13.63 10.57 LVPWd (mm) 3.102 ± 1.003 0.81 4.61 1.89 o 3.63 32.33 LVPWs (mm) 4.815 ± 1.173 2.93 6.42 2.50 o 5.43 24.35 LV mass (mg) 7532 ± 1707 3232 9911 na 22.67 Con ol Long Ca diac Pa ame e s Mean ± SD Minimum Maximum Re e ence ange * Coe icien o a ia ion (%) FS (%) 33.21 ± 4.035 28.07 40.61 17.63 o 40.16 12.15 EF (%) 57.63 ± 5.635 50.03 68.15 42.42 o 75.65 9.78 IVSd (mm) 2.474 ± 0.5083 1.86 3.89 2.11 o 2.82 20.55 IVSs (mm) 4.083 ± 0.7418 3.05 5.47 2.98 o 5.92 18.17 LVIDd (mm) 19.09 ± 3.852 12.83 25.95 13.59 o 17.89 20.18 In . J. Mol. Sci. 2013, 14 19101 likely ha in es iga ions o he ela ionship be ween s a in d ugs and p ocesses in which HO-1 is a pa icipan will yield no el s a egies o p e en ion and managemen o ca dio ascula disease. To al ac i i y o cy och ome c oxidase (COX) was e alua ed in he ca diac issue o es animals, wi h ou comes o hese expe imen s shown in Figu e 8. He e, he pa e ns o COX enzyme ac i i y pa alleled exp ession o COXIII p o ein wi h espec o he s a us o he animals om which ca diac issue was ha es ed o assay o COX p o ein using Wes e n blo (Figu e 7) o COX-media ed con e sion o educed e ocy och ome c o i s oxidized o m (Figu e 8). Le en icula issue aken om HC long (40 weeks) animals exhibi ed signi ican ly g ea e abili y o con e COX subs a e han hea issue om HC sho (12 weeks) abbi s, bu signi ican ly less han con ol animals ecei ing no mal chow (Figu e 8). Al hough da a p esen ed he e does no pe mi an e idence-based in e p e a ion o he mechanis ic basis o hese obse a ions, he e, oo, i is likely ha high le els o oxidized LDL esul ing om he hype choles e olemic condi ion o he animals has impai ed bo h unc ion and exp ession o c i ical mi ochond ial enzymes, including COX [29]. 3. Expe imen al Sec ion 3.1. Animals and Induc ion o Hype choles e olemia The expe imen s we e ca ied ou wi h adul male New Zealand abbi s wi h a body weigh ange o 2.0–2.5 kg. Animals ecei ed humane ca e in compliance wi h he “P inciples o Labo a o y Animal Ca e” o mula ed by he Na ional Socie y o Medical Resea ch, p epa ed by he Na ional Academy o Sciences (publica ion No. 86 23, e ised 1985). The abbi s we e p o ided wi h labo a o y oden chow (no mal) o chow en iched wi h 2.0% choles e ol (Godollo LTD, Budapes , Hunga y), daily o 40 weeks ad libi um. 3.2. Se um Choles e ol Measu emen Se um choles e ol le els in he enous blood o each animal we e measu ed using a Ca dioCheck se um choles e ol analyze (Poin O Ca e Diagnos ics, L d., A a mon, New Sou h Wales, Aus alia) a ime-poin s 0 (baseline), 4, 8, 12, 32 and 40 weeks ollowing ini ia ion o eeding. 3.3. Echoca diog aphy Echoca diog aphy was conduc ed unde ligh anes hesia (ke amine 15 mg/kg, xylazine 3 mg/kg, in amuscula (i.m.). The ches o each abbi was sha ed, and he animal was posi ioned in a do sal decubi us posi ion. Imaging o each hea was accomplished using a VEVO 770 High-Resolu ion In Vi o Mic o-Imaging Sys em (FUJIFILM VisualSonics, Inc., To on o, ON, Canada) wi h undamen al imaging modali y. Images we e s o ed on magne o-op ical disks o o -line analysis. Pa as e nal long axis iews we e ob ained and eco ded o ensu e ha he mi al and ao ic al es, as well as he apex we e isualized. The exac posi ion o he ansduce was adjus ed as necessa y o acqui e s anda d images. The pa as e nal sho axis iews we e eco ded a he mid-papilla y muscle le el. M-mode acings we e pe o med a he mid-papilla y muscle le el, ei he in pa as e nal long o sho axis iews. M-mode o isualiza ion and quan i ica ion o wall mo ion in ca dio ascula esea ch was used; single line acquisi ion allows o he e y high- empo al (1000 ps) esolu ion necessa y o In . J. Mol. Sci. 2013, 14 19102 analysis o LV unc ion. All measu emen s we e made by a single obse e , who was blind o he iden i y o he acings. All measu emen s we e a e aged o e h ee o i e consecu i e ca diac cycles. Echoca diog aphic measu emen s included sep al (IVSTD) and pos e io (PWTD) le en icula (LV) wall hickness in dias ole, LV ca i y size (end-dias olic (LVEDD) and end-sys olic (LVESD) dimensions and ao ic oo and le a ial an e opos e io diame e . F ac ional sho ening was compu ed as ollows: (LVEDD − LVESD)/LVEDD, and as global sys olic unc ion was balanced, he ejec ion ac ion (EF) was de i ed as EF = (LVEDD2 − LVESD2)/LVIDD2. Le en icula myoca dial mass was calcula ed using LVMass (T oy) = 1.05 × [(LVEDD + pos e io wall hickness a end dias ole (PWTD) + IVSTD) × 3 − (LVEDD) × 3]. 3.4. Rabbi Hea Isola ion Following he 12- and 40-week pe iod, du ing which he animals we e adminis e ed no mal o 2% choles e ol-en iched chow, hepa in (1000 IU/kg) and ke amine/xylazine (40/5 mg/kg) we e injec ed in a enously. Nex , ho aco omies we e pe o med on each animal; he hea s we e excised and placed in o ice-cold pe usion bu e . The ao as we e hen cannula ed, and hea s we e pe used acco ding o Langendo me hod o a 5-minu e washou pe iod a a cons an pe usion p essu e equi alen o 100 cm o wa e (10 kPa). The pe usion medium consis ed o a modi ied K ebs-Henselei bica bona e bu e : 118 mM NaCl, 4.7 mM KCl, 1.7 mM CaCl2, 25 mM NaHCO3, 0.36 mM KH2PO4, 1.2 mM MgSO4 and 10 mM glucose [32]. The le a ium was cannula ed, and he Langendo sys em was adap ed and swi ched o isola ed wo king hea s, as p e iously desc ibed in a a model [33], adap ed o abbi hea expe imen s [34]. The e ised p ocedu e used a le a ial illing p essu e o 17 cm (1.7 kPa) and ao ic a e load p essu e o 90 cm (9.0 kPa) o bu e . Ao ic low was measu ed by a calib a ed low me e (Gilmon Ins umen s, Ba ing on, IL, USA), and co ona y low a e was measu ed by a imed collec ion o he co ona y pe usa e ha d ipped om he hea . Following a 10-min ae obic pe usion o he hea , ca diac unc ion was eco ded and moni o ed h oughou he expe imen al pe iod by a compu e sys em ha moni o ed sil e elec odes and p essu e ansduce s connec ed di ec ly o he isola ed hea s (ADIns umen s, Powe Lab, Cas le Hill, Aus alia) in he no mal con ol and hype choles e olemic g oups. Be o e ischemia, and du ing epe usion, hea a e (HR), co ona y low (CF) and ao ic low (AF) a es we e egis e ed. Le en icula de eloped p essu e (LVDP) was also eco ded by he inse ion o a ca he e in o he le en icle ia he le a ium and mi al al e [32]. The hemodynamic pa ame e s we e egis e ed by a compu e acquisi ion sys em (ADIns umen s, Powe Lab, Cas le Hill, Aus alia). The isola ion o hea s, ischemia/ epe usion and he measu emen s o ca diac unc ion we e conduc ed ollowing 12 weeks o choles e ol-supplemen ed die , and compa isons we e made be ween he age-ma ched non-choles e olemic and hype choles e olemic g oups. Ca diac unc ion was egis e ed be o e he induc ion o no mo he mic global ischemia and du ing epe usion. 3.5. In a c Size De e mina ion 100 mL o 1% iphenyl e azolium chlo ide (TTC) solu ion in phospha e bu e (Na2HPO4 88 mM, NaH2PO4 1.8 mM) was adminis e ed ia he side a m o he ao ic cannula and, hen, s o ed a −70 °C o la e analysis. Hea s we e sliced ans e sely [35] in a plane pe pendicula o he apicobasal axis In . J. Mol. Sci. 2013, 14 19103 in o 2–3 mm hick sec ions, weigh ed, blo ed d y, placed in be ween mic oscope slides and scanned on a Hewle -Packa d Scanje 5p single pass la bed scanne (Hewle -Packa d, Palo Al o, CA, USA). Using NIH 1.61 image p ocessing so wa e (Hewle -Packa d, Palo Al o, CA, USA), each image was subjec ed o equi alen deg ees o backg ound sub ac ion, b igh ness and con as enhancemen o imp o ed cla i y and dis inc ness. The in a c a ea o each slice was aced, and he espec i e a eas we e calcula ed by pixel densi y analysis [36]. Wi h he use o he NIH Image 1.61 image p ocessing so wa e, each digi alized image was subjec ed o equi alen deg ees o backg ound sub ac ion and b igh ness/con as enhancemen o imp o ed cla i y. In a c size was exp essed as a pe cen age a io o he in a c zone o he isk zone (weigh o he le en icle). He e, iphenyl e azolium, (TTC) s aining, a di ec , pos -sac i ice app oach o imaging he in a c ed a eas, is used o a oid p oblems in clea ly demons a ing in a c ed zones, along wi h high cos in insic o ecen ly-de eloped non-in asi e imaging me hodologies used wi h li e animals [37,38]. P e iously, he au ho s ha e demons a ed he ele a ed occu ence o ca diac in a c ion in abbi s wi h die -induced hype choles e olemia e sus heal hy con ol animals. These s udies showed ha sys emic educ ion o oxida i e s ess by o al ea men wi h sou che y seed ex ac , an induce o heme oxygenase-1, esul ed in signi ican educ ion in he in a c size obse ed in hea s o hype choles e olemic abbi s ea ed wi h he ex ac e sus un ea ed con ols [3]. Occu ence o in a c ion obse ed in he p esen s udy may be accoun ed o by ele a ed a e ial plaque co e age obse ed in hype choles e olemic animals shown in Figu e 2. These esul s a e consis en wi h p e ious human s udies demons a ing ha p og essi e es ic ion o blood low o he hea and plaque-associa ed in lamma ion inc eases oxida i e s ess on ca diac issues and con ibu es o in a c ion [39,40]. 3.6. Analysis o A he oscle o ic Lesions Quan i ica ion o a y s eaks was pe o med wi h Sudan III s ain. Tho acic a e ies we e ha es ed, dissec ed ee o excess connec i e issue and a , insed wi h modi ied K ebs-Henselei bu e and ixed in 10% ( / ) bu e ed o malin. Ca o id a e ies we e hen opened longi udinally and exposed o 5 mg/mL Sudan III in 70% ( / ) isop opanol o 15 min in a wa e ba h a 37 °C, and he s ain was di e en ia ed wi h se e al inses o 70% isop opanol. The a e ies we e hen scanned, and he a he oscle o ic plaque was de e mined [32,36]. 3.7. Cy och ome c Oxidase (COX) Ac i i y The ac i i y o cy och ome c oxidase in abbi myoca dium was measu ed using a colo ime ic assay ki o oxida ion o cy och ome c by his enzyme (Sigma-Ald ich, S . Louis, MO, USA). B ie ly, mi ochond ia isola ed om eshly ha es ed hea muscle using he MITOISO1 ki (Sigma-Ald ich, S . Louis, MO, USA) we e ea ed wi h di hio h ei ol o educe cy och ome c, ollowed by COX-media ed eoxida ion o he molecule. COX ac i i y a oom empe a u e (~22 °C) in each sample was measu ed as a dec ease in abso bance o e ocy och ome c, a an abso bance wa eleng h o 550 nm (UV Helios Alpha S2 spec opho ome e , (UNICAM, Budapes , Hunga y) in i s con e sion om a educed o oxidized s a e. The COX ac i i y in any pa icula sample was epo ed as (−ΔA550/min). In . J. Mol. Sci. 2013, 14 19104 3.8. Wes e n Blo Analysis To al p o ein (100 μg) in he Clon ech Ex ac ion bu e was added o an equal olume o sodium dodecyl sul a e (SDS) bu e and boiled o 10 min be o e being sepa a ed on 12% SDS polyac ylamide gels in a unning bu e (25 mM T is, 192 mM glycine, 0.1% (w/ ) SDS, pH 8.3) a 120 V. The P ecision plus P o ein Kaleidoscope s anda ds (10 L) (Bio-Rad Labo a o ies, He cules, CA, USA) we e used as molecula -weigh s anda ds. The gel was ans e ed on o a ni ocellulose memb ane (Bio-Rad Labo a o ies, He cules, CA, USA) a 100 V and le o 1 h in a ans e bu e (25 mM T is base, 192 mM glycine, 20% ( / ) me hanol, pH 8.3). A e blocking he memb anes o 1 h in a T is-bu e ed saline (TBS-T) (50 mM T is, pH 7.5, 150 mM NaCl) con aining 0.1% ( / ) Tween-20 and 5% (w/ ) non a d y milk, blo s we e incuba ed o e nigh a 4 °C wi h he p ima y an ibody (HO-1, VEGF, COX III, COX IV, GAPDH). Memb anes we e washed 3 imes in TBS-T be o e being incuba ed o 1 h wi h ho se adish pe oxide (HRP)-conjuga ed seconda y an ibody dilu ed 1:2000 in TBS-T and 1% (w/ ) non a d y milk. De ec ion was made by au o adiog aphy o a iable leng hs o ime wi h medical X- ay ilm (Ag a-Ge ae N.V., Mo sel, Belgium). GAPDH (cy oplasm) and COX IV (mi ochond ial) we e used as he loading con ols (Sigma-Ald ich, S . Louis, MO, USA). Quan i a i e analysis o scanned Wes e n blo s o es ima e he le els o HO-1, VEGF, COX III, COX IV and GAPDH p o ein in each sample we e calcula ed using he Scion o Windows Densi ome y Image p og am, e sion Alpha 4.0.3.2 (Scion Co po a ion, Ma yland, MD, USA). Signal in ensi y o bands co esponding o each p o ein o in e es was es ima ed and epo ed in a bi a y uni s ± SEM. Fo hese expe imen s, he Scion Densi ome y Image p og am was selec ed as he mos cos -e ec i e o e alua ion o selec ed p o eins. The p oduc is public domain so wa e de i ed om an imaging p og am used ou inely by he Uni ed S a es Na ional Ins i u es o Heal h (NIH). I was selec ed o he p esen s udy based on i s di e se ange o applica ion and eliabili y. 3.9. S a is ics Resul s a e exp essed as he mean ± SEM (n = 4 in each g oup). One-way analysis o a iance was i s ca ied ou o es any di e ences be ween he mean alues o di e en g oups. I di e ences we e es ablished, he esul s be ween wo g oups we e compa ed by he Tukey es . Resul s we e conside ed o be signi ican i p < 0.05. 4. Conclusions The majo ou comes o he p esen s udy a e summa ized in Table 2. Expe imen s conduc ed by he au ho s es ablish clea co ela ion be ween he leng h o ime an animal’s ca dio ascula sys em is exposed o he hype choles e olemic s a e and dys egula ion o homeos a ic p ocesses c i ical o ca diac heal h. The indings epo ed he e u he p o ide a ame o e e ence o cha ac e iza ion o he mechanisms o ca diac disease associa ed wi h hype choles e olemia. In . J. Mol. Sci. 2013, 14 19105 Table 2. A summa y o he expe imen s conduc ed in his s udy and hei majo ou comes. In- ex igu e Desc ip ion o expe imen s Majo ou comes 1 Se um choles e ol e alua ed in abbi s ea ed o 12- and 40-week pe iods wi h 2% choles e ol-en iched eed. Signi ican ly highe choles e ol le els in animals ecei ing choles e ol-supplemen ed eed. 2 Plaque co e age e alua ed in ho acic a e ies. Signi ican ly ele a ed plaque co e age in blood essels o hype choles e olemia abbi s e sus heal hy con ols and sho - e m hype choles e olemia. 3 Ex en o in a c ed zones e alua ed in hea s o subjec animals. Non-hype choles e olemia: No in a c ion. 12-week high choles e ol: No/Minimal. 40-week high choles e ol: Ex ensi e. 4,5 Echoca diog aphic measu emen s made in hea s o subjec animals. Signi ican educ ion in le en icula ac ional sho ening (FS) and ejec ion ac ion (EF) in he hype choles e olemia long g oup. 6 Ao ic low measu emen s we e made in hea s o subjec animals. Signi ican educ ion in ao ic low o abbi s ea ed long- e m wi h high choles e ol die s. 7 Wes e n blo analysis o le en icula issue o media o s o ca dio ascula homeos asis. Con en o majo media o s in hea s o hype choles e olemia e sus con ol animals: COXIII, 12 week: signi ican ly dec eased in HC sho g oup e sus no mal COXIII, 40 week: signi ican ly ele a ed in HC long g oup e sus HC sho g oup. VEGF: no signi ican di e ences. HO-1, 12-week: HC sho g oup has signi ican ly lowe exp ession compa ed o no mal. HO-1, 40-week: signi ican ly lowe . HC long showed u he dec eased exp ession. 8 To al cy och ome c oxidase (COX) ac i i y was measu ed in le en icula issue o subjec animals. 12 week: signi ican ly dec eased in HC sho g oup e sus no mal. 40 week: signi ican ly ele a ed in HC long g oup e sus HC sho g oup. Acknowledgemen s This s udy was suppo ed by g an s om OTKA (78223, 104017, K-83478, K-75883), and in pa by he p ojec o New Hunga y De elopmen Plan, co- inanced by he Eu opean Union and he Eu opean Social Fund o TAMOP-4.2.2./B-10/1-2010-0024, and TAMOP-4.2.2.A-11/1/KONV-2012-0045. This esea ch was suppo ed by he Eu opean Union and he S a e o Hunga y, co- inanced by he Eu opean Social Fund in he amewo k o TAMOP 4.2.4. A/2-11-1-2012-0001 Na ional Excellence P og am. MTA-DE Vascula Biology, Th ombosis and Hemos asis Resea ch G oup is suppo ed by he Hunga ian Academy o Sciences (MTA-DE Vasc. Biol. 11003). The s udy was also inanced by he Uni e si y o Deb ecen. The au ho s a e since ely g a e ul o S ephanie C. Fox, J.D. o QueenBeeEdi o Bloom ield Connec icu , U.S.A. (sc jdqueenb[email p o ec ed]) o he ha d wo k in o ganizing, o ma ing, and edi ing his a icle. The au ho s con i m ha he e a e no con lic s o in e es wi h espec o any o he opic ma e ial p esen ed he ein. In . J. Mol. 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