Dif fe ren tial ex pres sion of HSP70 and ul tras truc tu re of heart and li ver tis suesof rats trea ted with adriamy cin: pro tec ti verole of L-car ni ti ne.

Mi rian Strauss and No raidys Po rras.

Sec ción de Bio lo gía Ce lu lar, Insti tu to de Me di ci na Tro pi cal, Uni ver si dad Cen tral de Ve ne zue la. Apar ta do Pos tal 47019, Ca ra cas 1041-A, Ve ne zue la.E-mail: mstrauss ve@yahoo.com

Key words: HSP70; ultrastructure; adriamycin; heart-liver; L-carnitine.

Ab stract. The anticancer drug adriamycin has been as so ci ated to tissular ox i da tive stress. In this re gard, the pro mo tion of anti-stress pro tein syn the sisby L-carnitine has been sug gested in rat adriamycin-in duced cardiomyopathyin the long-term. How ever, the citoprotective role of L-carnitine in car diacand hepatic tis sues af ter short-term adriamycin treat ment is un known. HSP70 in the supernatant of the ho mog e nized car diac and hepatic tis sues af tershort-term adriamycin treat ment was de ter mined by West ern blot anal y siswith and with out L-carnitine pro tec tion and com pared to the subcellularchar ac ter is tics of both tis sues by trans mis sion elec tron mi cro scopic anal y sis.Fe male Sprague-Dawley rats (n = 6), body weight 40-60g, were ran dom izedinto four groups: con trol, adriamycin, L-carnitine and L-carnitine-adriamycin.Sa line, adriamycin (15 mg/kg body weight) and L-carnitine (20 mg be foreadriamycin) were given in tra ve nously (0.1 mL). HSP70 ac cu mu la tion was dif -fer ent be tween the con trol and the adriamycin sam ples of both tis sues. HSP70 was higher in the liver than in the heart both with and with out L-carnitinepro tec tion. The nu clei of heart cells, in the adriamycin group showed al ter -ations in clud ing, form and ir reg u lar perinuclear cysternae with invaginationsof dif fer ent sizes that were not ob served in the L-carnitine–adriamycin sam -ples. Con sid er ing the dif fer en tial ex pres sion of HSP70 be tween liver andheart, our re sults may be im por tant for un der stand ing the role of these pro -teins in the adriamycin-in duced dis tinct lev els of or gan dam age and dys func -tion. We sug gest that L-carnitine ex og e nous ad min is tra tion might en hancethe re la tion ship be tween the cel lu lar en ergy state and the ac ti va tion of heatshock re sponse by an un known mech a nism. L-carnitine may en hance HSP70in a cel lu lar-type man ner.

Invest Clin 48(1): 33 - 43, 2007

Co rres pon ding author: Mi rian Strauss. Sec ción de Bio lo gía Ce lu lar, Insti tu to de Me di ci na Tro pi cal, Uni ver si dad Cen tral de Ve ne zue la. Apar ta do Pos tal 47019, Ca ra cas 1041-A, Ve ne zue la. Pho ne: +58-212-6053650, Fax:+58-212-2434685. E-mail: mstrauss ve@yahoo.com

Expre sión di fe ren cial de HSP70 y ul traes truc tu ra de te ji docar día co y he pá ti co de rata tra ta do con adria mi ci na: pa pel pro tec ti vo de la L-car ni ti na.Invest Clín 2007; 48(1): 33 - 43

Palabras clave: HSP70, ultraestructura, Adriamicina, corazón-hígado, L-carnitina.

Re su men. La adria mi ci na, dro ga an ti can ce ro sa, ha sido aso cia da con eles trés oxi da ti vo ti su lar. En este sen ti do, la pro mo ción de la sín te sis de pro teí -nas anti-es trés por L-car ni ti na ha sido su ge ri da en las car dio mio pa tías de lar -go pla zo in du ci das por adria mi ci na en ra tas. Sin em bar go, el pa pel ci to pro tec -ti vo de la L-car ni ti na en te ji do car dia co y he pá ti co, en la in to xi ca ción poradria mi ci na en el cor to pla zo, se des co no ce. La HSP70 pre sen te en los so bre -na dan tes de ho mo ge na tos de te ji do car dia co y he pá ti co lue go de tra ta mien tocon adria mi ci na a cor to pla zo y pro tec ción de L-car ni ti na, fue de ter mi na dame dian te téc ni cas de Wes tern blot y com pa ra da con las ca rac te rís ti cas sub ce -lu la res de am bos te ji dos. Ra tas hem bras Spra gue Daw ley (n = 6), de peso cor -po ral en tre 40-60g, fue ron dis tri bui das alea to ria men te en cua tro gru pos: con -trol, adria mi ci na, L-car ni ti na y L-car ni ti na-adria mi ci na; re ci bien do por vía in -tra ve no sa 0,1 mL de: so lu ción sa li na, 15 mg/kg de peso cor po ral, 20 mg pre -vio a la adria mi ci na y am bas, res pec ti va men te. Se de ter mi nó una acu mu la ción di fe ren cial de la HSP70 en tre las mues tras del gru po con trol y adria mi ci napara am bos te ji dos. La ma yor acu mu la ción de HSP70 fue en hí ga do, con y sinpro tec ción de L-car ni ti na. En el gru po adria mi ci na, las cé lu las car dia cas mos -tra ron nú cleos de for ma no re don dea da y cis ter na pe ri nu clear con in va gi na cio -nes de di fe ren te ta ma ño, ha llaz gos que no fue ron ob ser va dos en las mues trasdel gru po pro te gi do L-car ni ti na-adria mi ci na. To man do en cuen ta la ex pre sióndi fe ren cial de HSP70 en tre hí ga do y co ra zón, nues tros re sul ta dos pue den serim por tan tes para en ten der el pa pel de las pro teí nas de cho que tér mi co con tralos di fe ren tes ni ve les de ór ga no to xi ci dad in du ci da por adria mi ci na. Se su gie reque la ad mi nis tra ción exó ge na de L-car ni ti na po dría fa vo re cer la re la ción en treel es ta do ener gé ti co ce lu lar y la ac ti va ción de la res pues ta de cho que tér mi come dian te un me ca nis mo des co no ci do y de una ma ne ra te ji do de pen dien te.

Re ceived: 09-11-2005. Ac cepted: 04-05-2006.

IN TRO DUC TION

The widely used anticancer drugadriamycin (ADR) pro duces toxic ef fects innu mer ous or gans, the most se ri ous be ingheart dam age. Heart tox ic ity in hu man pa -tients is gen er ally ex pressed as cardio -myopathy fol low ing chronic ad min is tra tionwhereas the liver is rel a tively re sis tant to

dam age (1-3). ADR has been shown to be apo ten tial source of free rad i cals. In in vi trostud ies, quinone-con tain ing anticancer agents, in clud ing ADR, have been shown to formsemiquinone free rad i cal in ter me di ates inthe pres ence of cer tain flavin en zymes (4).ADR-in duced changes in mem brane func -tions have been shown to be ac com pa niedby lipid peroxidation in in vi tro as well as in

Investigación Clínica 48(1): 2007

34 Strauss and Porras

vivo stud ies (5, 6). This in creased per -oxidation of poly un sat u rated fatty ac ids isrec og nized as one of the pos si ble bio chem i -cal mech a nisms for the gen e sis of mem -brane in jury in the myocardium (7). Therole of ADR is as so ci ated to the flow of elec -trons from NADH to mo lec u lar ox y gen andthe pro duc tion of free rad i cals. In ter ac tionof ADR with cardiolipin, may con trib ute tothe de cline in cardiolipin-de pend ent en -zymes such as coenzyme A and cytochromeoxidase (7, 8). The car dio vas cu lar tox ic ityof ADR was at trib uted to a switch in the en -zy matic ac tiv ity of eNOS from a ni tric ox ide gen er at ing en zyme to a superoxide-gen er at -ing en zyme (i.e. NADHP oxidase ac tiv ity).In this sense, it have been dem on stratedthat heart tis sue re veals de pres sion in theac tiv i ties of anti-ox i da tive en zymes, in clud -ing catalase and dismutase superoxide (9).Hepatic lev els of glutatione (GSH) and GSH peroxidases I and II have been shown to berel a tively high and peroxidative dam age tohepatocytes was ob vi ated by GSH. In con -trast to the liver, it has been dem on stratedthat the heart has ex tremely low ac tiv i tiesof GSH peroxidase (10). In this re gard, it isbe lieved that con sti tu tive cel lu lar pro tec -tion against acute stress is pro vided by a va -ri ety of intracellular com po nents in clud ingthe antioxidative en zy matic sys tem,antioxidatives and per haps heat shock pro -teins (HSPs) (11). The HSPs con sti tute acel lu lar sys tem of en dog e nous pro tec tionwhich con trib utes for pro tein ho meo sta sisand is es sen tial for cel lu lar vi a bil ity (12).The over-ex pres sion of in di vid ual HSPs ei -ther in cul tured car diac cells in vi tro or inthe in tact heart has a pro tec tive ef fect. Inpar tic u lar, HSP70 con fers pro tec tion againstcar diac ischemic dam age and against a va ri -ety of ad verse en vi ron men tal con di tions in -clud ing cer tain anticancer drugs (13-17).The ex pres sion lev els of HSP70 in cells cor -re lated well with their sur vival fol low ingtreat ments with the tu mor ne cro sis fac tor,

staurosporine and ADR (18, 19). HSP70res cues cells from apoptosis down stream ofsome known anti-apoptotic pro teins such as caspase 3 (20, 21). How ever, HSP70 mRNAand pro tein are in duced in dif fer ent celltypes de pend ing on the se ver ity, the na tureof the stim u lus and the bio chem i cal char ac -ter is tics of the tis sue ex po sure to stress(22). An ex am ple of a cell-type spe cificstress re sponse may be rep re sented by thefind ings where rat liver sam ples showed thegreat est ac cu mu la tion of HSP72 af ter heatshock in com par i son to other tis sues in -clud ing the heart (23). On the other hand,L-carnitine (L-Car), a de riv a tive of nat u ralamino acid and a me tab o liz ing anti -oxidative agent, pro motes fatty acid ox i da -tion by translocating ac ti vated long-chainfatty acid into the ma trix of mi to chon dria(24). L-Car has a cardio pro tec tive roleagainst many toxic stresses in clud ing an ti -mony (25), and pro motes the ac cu mu la tion of HSP25 as a short-term re sult of L-Carpro tec tive strat egy, pre vi ous to ADR ad min -is tra tion which is as so ci ated in the longterm to al ter ations of much less se ver ity(26). Nev er the less, it is not known whetherthere is a re la tion ship be tween dif fer entheart and liver ADR subcellular toxic se ver -ity and dif fer ent ac cu mu la tion of HSP70with and with out the pro mo tion of HSP70by L-Car. Seek ing to iden tify mo lec u larclues re gard ing ADR-in duced cardio -toxicity, the aim of this study was to ana lyse HSP70 re sponse against mor pho log i cal fea -tures in heart and liver of short-termADR-treated rats. More over, given the po -ten tial pro tec tive role of L-Car againstheart pa thol o gies (26-28), we won der about its re la tion ship with HSP70 re sponse.

METH ODS

An i mals: Fe male Sprague-Dawley rats(40-60 g), ob tained from the InstitutoVenezolano de Investigaciones Científicas

Vol. 48(1): 33 - 43, 2007

HSP70 and ultrastrucuture of tis sues treated with adriamycin 35

(Ca ra cas, Ven e zuela), were used. The ratswere al lowed free ac cess to stan dard ro dent chow and wa ter ad li bi tum. The an i malswere main tained ac cord ing to the normsspec i fied in the “Guide to the Care and Use of Lab o ra tory An i mals” of the U.S. Na tional In sti tute of Health (NIH pub li ca tion No.85-23, re vised 1985).

MA TE RI ALS

Sigma Com pany (Mi ami, USA) was thesource of all bio chem i cal com pounds,monoclonal an ti bod ies and ADR. L-Car wasdo nated by Elmor Lab o ra to ries (Ca ra cas,Ven e zuela).

Ex per i men tal de signThe an i mals were ran dom ized in four

groups (n = 6): Con trol, ADR, L-Car andL-Car-ADR. The antitumoral drug was in -jected i.v. to an ac cu mu lated dose of 15mg/kg body wt, di vided into three subdoses of 5 mg/kg body wt (in 0.1 mL of ster ile wa -ter) ap plied at 3-day in ter vals. Con trol ratsre ceived sa line so lu tion (0.1 mL). L-Car was ad min is tered i.v. at a dose of 20 mg (0.1 mL)be fore each ADR subdose. 24 hours af terthe third ADR subdose heart and liver werere moved for sub se quent bio chem i cal andultrastructural study.

West ern blots anal y sisLeft ven tric u lar free wall and cen tral

hepatic lob ule sam ples were ho mog e nized(4°C; 1 mL ex trac tion buffer Tris-HCl20mM, EDTA 2mM, PMFS 1mM, pH 7.4) us -ing a Pot ter-Elevehjem tis sue grinder. TheHSP70 con tent in the supernatant of ho -mog e nized heart and hepatic tis sue was de -ter mined by West ern blot anal y sis (29).Pro tein sam ples were di luted with 4 xLaemmli buffer so lu tion. Equal amounts ofpro tein sam ples (5 µg per lane) were ap -plied to 1mm thick, 10% polyacrylamidegels and sep a rated by SDS-PAGE (30). The

equiv a lence of load ing con cen tra tions andthe ad e quacy of the sam ple prep a ra tionwere con firmed by vi su al iza tion of pro teinwith Coomassie blue stain. Pro teins weretrans ferred to nitro cellulose mem branewhich were washed in PBS with 0.3% low-fat milk to block non-spe cific bind ing sites and in cu bated at 4°C with mouse monoclonalIgG cross-re ac tive with HSP70 in a 1:5000di lu tion at room tem per a ture for 90 min.The immunodetection in cluded: block ing of pro tein bind ing sites, bind ing the pri maryan ti body (1:5000 HSP), wash ing un boundpri mary an ti body, bind ing the anti-IgG con -ju gate, wash ing to re move un bound con ju -gate and de tec tion by chemiluminescence.Pro tein immunoblots were scanned by 690Bio-Rad Den si tom e ter us ing the Multi-An a -lyst pro gram (Bio-Rad).

Tis sue prep a ra tion and ultrastructuralanal y sis: Sam ples (2mm) of free left ven -tric u lar wall and cen tral hepatic lob ulewere perfused with sa line so lu tion, fixed inKarnovsky (320 mosmol, pH 7.4, 2h, 4°C)and post-fixed in os mium acid (2% os miumtetroxide in Milloning buffer 0.12 M, 320mosmol, pH 7.4, 2h, 4°C). The sam pleswere de hy drated in graded in creas ing con -cen tra tions of ac e tone (50%, 70% + ura nyl, 80%, 95% 100%; 30 min each) and em bed -ded in poly mer iz ing ep oxy resin (60°C,48h). Af ter em bed ding, thin sec tions werecut (ultramicrotomy Reichert OmU3),stained with both sat u rated ura nyl ac e tate(45min, 60°C) and lead ci trate (3min,25°C), and then ex am ined with trans mis -sion elec tron mi cro scope (Hitachi H-300,75 Kv). The ultrastructural anal y sis wasonly of qual i ta tive type.

Sta tis ti cal anal y sisTo test the sig nif i cance of the data re -

sult ing from the op ti cal densitometry ofHSP70 rec og ni tion among the 4 ex per i men -tal groups, an anal y sis of vari ance by rangeor der ing (Kruskal-Wallis test) was used. Dif -

Investigación Clínica 48(1): 2007

36 Strauss and Porras

fer ences be tween groups were con sid eredsig nif i cant at p < 0.05.

RE SULTS

West ern blots anal y sisEx pres sion of HSP70 in heart and

liver: The anti-HSP70 monoclonal an ti bodyused in the pres ent in ves ti ga tion re actedonly with a sin gle band cor re spond ing tothe 70-kDa rat HSP70 pro tein in all groups(Figs. 1 and 2). Rep re sen ta tive de ter mi na -tions of sam ples ob tained from a pair of an i -mals in each group showed an ev i dent dif -fer en tial immunolabel rec og ni tion ofHSP70 be tween the sam ples from fourgroups (Con trol, ADR, L-Car, L-Car-ADR)and the two dif fer ent tis sues (car diac andhepatic). 24 hours af ter ADR toxic stress asshown in Fig. 1, a dif fer en tial pat tern ofHSP70 rec og ni tion was ev i dent. Par tic u -larly, the dif fer ence be tween Con trol heartand Con trol liver sam ples (lanes 1 and 2)was not as high as the dif fer ence be tweenADR ones, where the liver had ap par ently

twice as much HSP70 as the heart (lanes 3and 4). As shown in Fig. 2, L-Car heart sam -ples showed ap par ently lower ac cu mu la -tions of HSP70 than the cor re spond ing liver coun ter parts (lanes 1 and 2 re spec tively).In the heart L-Car-ADR group a de pressedex pres sion of HSP70 in con trast to the liver was ob served (lanes 3 and 4). Lower liverHSP70 rec og ni tion in L-Car-ADR than inADR groups, in con trast to heart (Fig. 1and 2, lane 3 and 4) was also ob served.Densitometric anal y sis in con di tions of lin -ear ity with re spect to pro tein con cen tra tion of the sam ple and time ex po sure of the pho -to graphic film cor rob o rated that the liverHSP70 amount was big ger than that of theheart in all the groups stud ied. Par tic u larly, Con trol, ADR, L-Car and L-Car-ADR liversam ples were 21%, 71%, 20% and 17% more than the amount de tected in the heart, re -spec tively.

Ultrastructural anal y sisCar diac ultrastructural anal y sis: Mi -

cro graphs of the free left ven tric u lar wall

Vol. 48(1): 33 - 43, 2007

HSP70 and ultrastrucuture of tis sues treated with adriamycin 37

Control Heart

Control Liver

ADR Heart

ADR Liver

kDa1 2 3 4

140

70

25

15

140

120

Control Heart

Control Liver

ADR Heart

ADR Liver

70

kDa

A B

Fig. 1. 1a: Gel stai ned with Coo mas sie Bri lliant Blue G-250 as a re cord of loa ding pro tein con trol (2,3 con trols and 4,5 ADR trea ted sam ples from heart and li ver, res pec ti vely). 1b: A re pre sen ta ti -ve Wes tern blot of hsp70 using two con ti guous la nes for each group analy zed. The two la nesiden ti fied by 1 and 2, whe reas the two la nes iden ti fied by 3 and 4 co rres pond to ADR sam plesfrom heart and li ver res pec ti vely. All la nes were loa ded with 5 µg of the par ti cu lar pro tein.

showed myo car dial cells with nu cleus ofnor mal ap pear ance in Con trol group(Fig. 3A) whereas ADR group (Fig. 3B) hadnu cleus al ter ation in clud ing form and ir reg -u lar perinuclear cysternae with invagin -ations of dif fer ent size. The L-Car group(Fig. 3C) re sem bled the Con trol, and theL-Car-ADR group (Fig. 3D) showed a con -tin u ous peri nuclear cysternae like Con troland L-Car, and few lipid drops in the cy to -plasm.

Hepatic ultrastructural anal y sis:Hepatic tis sue of the four groups (Fig. 4A,B,C,D re spec tively) showed hepatocyteswith nor mal ap pear ance.

DIS CUS SION

A dif fer en tial level of HSP70 ex pres -sion be tween liver and heart of short-termADR treated fe male rats was found in thepres ent study. HSP70 is be lieved to par tic i -pate in an ar ray of cel lu lar ac tiv i ties, in -clud ing cytoprotection (22). Ex po sure of

car diac myocytes to ox i da tive stress byH2O2 treat ment causes post-translationalmod i fi ca tion in two pro tein fam i lies in -volved in cytoprotection: the peroxiredoxins and two small heat shock pro tein fam ilymem bers: alfa Beta-crystallin and HSP25(31). In ADR cardio tox ic ity pro tected byL-Car, it has been sug gested that there is are la tion be tween HSP25 cel lu lar con tent inthe short term and cytoprotection ex -pressed in terms of a heart subcellular pa -thol ogy of less se ver ity, in the long term(26). A re dox mech a nism may be in volvedin the heat-shock sig nal path way in ox i da -tive stress, where in duc tion of HSP70 pro -tein has been cor re lated with a marked de -ple tion of intracellular bound thiols and ade crease in lipid peroxidation (32). Inastrocytes treated with acetyl-L-Car the in -duc tion of heme oxygenase-1 was re lated to the up-reg u la tion of HSP60 as well as highex pres sion of the re dox-sen si tive tran scrip -tion fac tor Nrf2 in the nu clear frac tion oftreated cells (33). Acetyl-L-Car ac tiv i ties in -

Investigación Clínica 48(1): 2007

38 Strauss and Porras

L-Car Liver

L-Car-ADR Heart

kDa

140

70

25

15

140

120

1 2 3 4 L-Car Heart

L-Car-ADR Liver

L-Car Heart

L-Car Liver

L-Car-ADR Heart

L-Car-ADR Liver

70

kDa

A B

Fig. 2. 2a: Gel stai ned with Coo mas sie Bri lliant Blue G-250 as a re cord of loa ding pro tein con trol (2,3 L-Car and 4,5 L-Car-ADR trea ted sam ples from heart and li ver, res pec ti vely). 2b: A re pre sen -ta ti ve Wes tern blot of hsp70 using two con ti guous la nes for each group analy zed. The two la -nes iden ti fied by 1 and 2 co rres pond to CAR sam ples from heart and li ver res pec ti vely, whe -reas the two la nes iden ti fied by 3 and 4 co rres pond to CAR-ADR sam ples from heart and li verres pec ti vely. All la nes were loa ded with 5 µg of the par ti cu lar pro tein.

clude acetylation of -NH2 and -OH func -tional groups in amino ac ids and N ter mi nal amino ac ids in pro teins; as well as, act ingas a mo lec u lar chaperone to larger mol e -cules (34). In ad dic tion, a stress-reg u latedpro tein, GRP58, a mem ber of thioredoxinsuperfamily, is a carnitine palmitoyl -transferase isoenzyme (35). On the other

hand, stress is ac com pa nied by changes inthe en ergy state. As a re sult, the pref er en -tial ox i da tion of car diac mi to chon drial DNA fol low ing acute in tox i ca tion with ADR mayac count for many of the bioenergetic def i -cits as so ci ated with the cardiotoxicity ob -served in vivo (36). Potencial mo lec u larmech a nisms of liver L-Car en dog e nous

Vol. 48(1): 33 - 43, 2007

HSP70 and ultrastrucuture of tis sues treated with adriamycin 39

Fig. 3. Mi cro graphs of the free left ven tri cu lar wall sho wed myo car dial cells with nu cleus of nor malap pea ran ce in Con trol group (Fig. 3A). ADR group (Fig. 3B) had nu cleus al te ra tion in clu dingform and irre gu lar pe ri nu clear cyster nae with in va gi na tions of dif fe rent size. The CAR group(Fig. 3C) re sem bled the Con trol, and the CAR-ADR group (Fig. 3D) sho wed a con ti nuous pe ri -nu clear cyster nae like Con trol and CAR, and few li pid drops in the cyto plasm. Bar = 0.7µm

biosynthesis might be re lated to thebioenergetic def i cits ob served in mi to chon -dria iso lated from heart, but no liver (37).A mod er ate de crease in intracellular ATPcor re lates with an at ten u a tion in HSF1 inthe heart and the res to ra tion of ATP leadsto greater ac ti va tion of HSF1 (38). In thisre gard, acetil-L-Car acts by stim u lat ing en -ergy me tab o lism. Futhermore, the re doxreg u la tion of mam ma lian HSF1 is es sen tialfor HSP gene ac ti va tion and pro tec tionfrom stress (39). It is pos si ble that HSP70plays a chaperone role in the pro cess of ri -

bo some biogenesis. The sta bi li za tion of ri -bo some as sem bly may have an im pact inthe pres er va tion of pro tein syn the sis dur ing stress and may be an im por tant com po nentof the gen eral mech a nism of cytoprotection me di ated by HSPs (40). How ever, fol low inga stress ful con di tion, HSP70 mRNA andpro tein are in duced in dif fer ent cell typesde pend ing on the se ver ity and the na ture of the stim u lus (22). In ad di tion, sex, causesdif fer ences in drug tox ic ity in rat, where fe -male Sprague-Dawley hearts have twice asmuch HSP70 as male hearts, due to

Investigación Clínica 48(1): 2007

40 Strauss and Porras

Fig. 4. He pa tic tis sue of the four groups (Fig.4 A,B,C,D res pec ti vely) sho wed he pa tocy tes with nor mal ap pea ran ce. Bar = 0.7µm.

upregulation by es tro gen (41). More over,cor re spon dence be tween the ex pres sionand/or ac cu mu la tion of dif fer ent HSPs hasbeen linked to the es tab lish ment of asynergic net work re lated to the main te -nance of pro tein sta bil ity (42). The provenstim u la tion ca pac ity of L-Car in re la tion toin creased HSP25 de tec tion in re sponse toADR (26), could in volve other mem bers ofthe HSP fam ily in clud ing HSP70, in bothheart and liver tis sue. In ad di tion in ADRcardiomyopathy a de pressed pro tein syn the -sis in clud ing HSPs has also been dem on -strated (43) in con trast to the hepatic re -sponse. The pres ence of HSPs may pre ventthe dam ag ing ef fects of stress by im ped ingthe pre cip i ta tion of de na tured pro teinscaused by the at tack (44). The dif fer ence in HSP ac cu mu la tion may al ter the ef fec tive -ness of the or gans to re spond to stress. Onthe other hand, it has been sug gested thatstress con trib utes to the pathogenesis ofproteinopathies through the stim u la tion ofpro tein ag gre ga tion (45). This may belinked to our find ings con cern ing the pres -ence of cardioproteinopathies ob served inthe heart in com par i son to their ap par entab sence in the liver of ADR treated rats(46). HSP70 con tent may con trib ute to thebio chem i cal dif fer ences which may de ter -mine tis sue sus cep ti bil ity. In con clu sion,the de ter mi na tion of the greater amount of HSP70 in liver than in heart ADR fe maletreated rats, and its cor re spon dence to thebig ger tisular pres er va tion in liver than inheart, may be im por tant for un der stand ingthe func tion of this pro tein in or gan dif fer -en tial dam age. How ever, since the bio chem -i cal ex per i ments were per formed with amonoclonal an ti body that rec og nized boththe con sti tu tive and in duc ible form ofHSP70, fur ther ex per i ments are re quiredus ing an ti bod ies for each form of HSP70with re spect to the cor re spon dence be -tween the HSP70 in duced form and thetoxic stress con di tion. We sug gest that

L-Car ex og e nous ad min is tra tion might en -hance the re la tion ship be tween cel lu lar en -ergy state and ac ti va tion of heat shock re -sponse by the pro mo tion of HSP70 in a cel -lu lar-type man ner.

ACKNOWLEDGEMENTS

This study was sup ported by Consejode Desarrollo Científico y Humanístico (PG09-00-5676-2004) of the Universidad Cen -tral de Ven e zuela and Laboratorios ELMORS.A. The au thors are grate ful to Prof. MarkGreg son for his help with Eng lish and toMarianela Ro dri guez for her help withultrastructural anal y sis.

REFERENCES

1. Doroshow JH, Locker GY, Myers CE. Ex -per i men tal an i mals model of ADRiamycincardiotoxicity. Can cer Treat Rep 1979;63:855-860.

2. Doroshow JH. Doxorubicin-in duced car -diac tox ic ity. N Eng J Med 1991;324:843-845.

3. Phillips FS, Gilladoga A, Marquardt H,Stern berg SS, Vidal PM. Some ob ser va -tions of the tox ic ity of ADRiamycin (NSC- 123127). Can cer Chemother Rep 1975;6:77-181.

4. Doroshow JH. Ef fect of Anthracycline An -ti bi ot ics on ox y gen rad i cal for ma tion in rat heart. Can cer Res 1983; 43:460-472.

5. Singal PK, Segstro RJ, Singh RP, KutrykMJ. Changes in lysosomal mor phol ogy and en zyme ac tiv i ties dur ing the de vel op mentof adriamycin-in duced cardiomyopathy.Can J Cardiol 1985; 1:139-147.

6. Singal PK, Pierce GN. Adriamycin stim u -lates low-af fin ity Ca2+ bind ing and lipidperoxidation but de presses myo car dialfunc tion. Am J Physiol 1986; 250:H419-25.

7. Singal PK, Deally CM, Wein berg LE.Subcellular Ef fects of Adriamycin in theheart: A con cise Re view. J Mol Cell Cardiol 1987; 19:817-828.

8. Iwamoto Y, Hansen IL, Por ter TH. In hi bi -tion of coenzyme Q10-en zymes, succin -

Vol. 48(1): 33 - 43, 2007

HSP70 and ultrastrucuture of tis sues treated with adriamycin 41

oxidase and NADH-oxidase by adriamycinand other qui nones hav ing antitumor ac -tiv ity. Biochem Biophys Res Commun1974; 58:633-638.

9. Doroshow JH, Locker GY, Myers CE. En -zy matic de fenses of the mouse heartagainst re ac tive ox y gen me tab o lites: al ter -ations pro duced by doxorubicin. J Clin In -vest 1980; 65:128-135.

10. Odom AL, Hatwig CA, Stan ley JS, Benson AM. Bio chem i cal de ter mi nants ofADRiamycin tox ic ity in mouse liver, heartand in tes tine. Biochem Pharmacol 1992;43:831-836.

11. Das DK, Maulik N, Engelman RM,Rousou JA, Deaton D, Flack JE. Sig nalTransduction path way lead ing to hsp 27and hsp70 gene ex pres sion dur ing myo car -dial ad ap ta tion to stress. An nals NY AcadSci 1999; 874:129-137.

12. Knowlton AA, Kapadia S, Torre-AmioneG, Durand JB, Bies R, Young J, Mann DL.Dif fer en tial ex pres sion of heat shock pro -teins in nor mal and fail ing hu man hearts.J Mol Cell Cardiol 1998; 30: 811-818.

13. Jayakumar J, Susuki K, Khan M,Smolenski RT, Farrell A, Latif N, RaiskyO, Abunasra H, Sammut IA, Murtuza B,Amrani M, Yacoub MH. Gene ther apy formyo car dial pro tec tion: transfection of do -nor hearts with heat shock pro tein 70gene pro tects car diac func tion againstischemia-reperfusion in jury. Cir cu la tion2000; 102:302-306.

14. Karlseder J, Wissing D, Holzer G, Orel L,Sliutz G, Auer H, Jaattela M, Si mon MM.Hsp70 overexpression me di ates the es cape of a doxorubicin-in duced G2 cell cy cle ar -rest. Biochem Biophys Res Commun 1996; 220:153-159.

15. Kawana K, Miyamoto Y, Tanonaka K,Han-no Y, Yoshida H, Takahashi M,Takeo S. Cytoprotective mech a nism ofheat shock pro tein 70 against hypoxia/ reoxygenation in jury. J Mol Cell Cardiol2000; 32:2229-2237.

16. Mar tin JL, Hickey E, Weber LA, Dillmann WH, Mestril R. In flu ence of phosphor -ylation and oligomerization on the pro tec -tive role of the small heat shock pro tein 27

in rat adult cardiomyocytes. Gene Expr1999; 7:349-355.

17. Okubo S, Wildner O, Shah MR, ChelliahJC, Hess ML, Kukreja RC. Gene trans ferof heat shock pro tein 70 re duces in farctsize in vivo af ter ischemia/reperfusion inthe rab bit heart. Cir cu la tion 2001; 103:877-881.

18. Mosser DD, Caron AW, Bourget L, Meriin AB, Sherman MY, Morimoto RI, MassieB. The chaperone func tion of hsp70 is re -quired for pro tec tion against stress-in -duced. Mol Cell Biol 2000; 19:7146-7159.

19. Nylandsted J, Gyrd-Hansen M, Daniel -ewicz A, Fehrenbacher N, Lademann U,Hoyer-Hansen M, Weber E, Multhoff G,Rohde M, Jaattela M. Heat shock pro tein70 pro motes cell sur vival by in hib it inglysosomal mem brane permeabilization. JExp Med. 2004; 200:425-435.

20. Jaattela M, Wissing D, Kokholm K,Kallunki T, Egeblad M. Hsp70 ex erts itsanti-apoptotic func tion down stream ofcaspase- 3-like pro teas es. EMBO J 1998;17: 6124-6134.

21. Leonardi R, Pannone G, Magro G, KudoY, Takata T, Lo Muzio L. Dif fer en tial ex -pres sion of heat shock pro tein 27 in nor -mal oral mu cosa, oral ep i the lial dysplasiaand scamous cell car ci noma. Oncol Rep2002; 9:261-266.

22. Rajdev S, Sharp FR. Stress pro tein as mo -lec u lar mark ers of neurotoxicity. ToxicolPathol 2000; 28:105-112.

23. Beck SC, Paidas CN, Tan H, Yang Jay, De Maio A. De pressed ex pres sion of the in -duc ible form of hsp70 (hsp70) in brain and heart af ter in vivo heat shock. Am J Physiol 1995; 269:R608-R613.

24. Bieber LL. Carnitine. Ann Rev Biochem1998; 57:261-283.

25. Alvarez M, Malécot CO, Gannier F,Lignon JM. An ti mony-in duced cardio -myopathy in guinea-pig and pro tec tion byL-carnitine. B J Pharmacol 2005; 144: 17- 27.

26. Strauss M, Anselmi G, Hermoso T, Tejero F. Carnitine pro motes heat shock pro teinsyn the sis in adriamycin-in duced cardio -myopathy in a neo na tal rat ex per i men tal

Investigación Clínica 48(1): 2007

42 Strauss and Porras

model. J Mol Cell Cardiol 1998; 30:2319-2325.

27. Pauly DF, Pepine CJ. The Role ofCarnitine in myo car dial dys func tion. Am JKid ney Dis 2003; 41:S35-43.

28. Sayed-Ahmed MM, Salman TM, GaballalHE, Abou El-Naga SA, Nicolai R, Calvani M.Propionil L-carnitine as pro tec tor againstadriamycin in duced cardiomyopathy 2001;43:513-520.

29. Towbin H, Staehelin T, Gordon J. Elec -tro pho retic trans fer of pro tein frompolyacrilamide gels to nitrocellulosesheets: pro ce dure and some ap pli ca tions.Proc Natl Acad Sci USA 1979; 76:4350- 4354.

30. Laemmli UK. Cleavege of struc tural pro -teins dur ing the as sem bly of the head ofbacteriophage T4. Na ture 1970; 227:680- 685.

31. Cullingford TE, Wait R, Clerk A, Sugden PH. Ef fects of ox i da tive stress on the car -diac myocyte proteome: mod i fi ca tions toperoxiredoxins and small heat shock pro -teins. J Mol Cell Cardiol 2006; 40:157- 162.

32. Calabrese V, Renis M, Calderone A,Russo A, Barcelllona ML, Rizza V. Stresspro tein and SH-groups in ox i dant-in ducedcell dam age al ter acute etanol ad min is tra -tion in rat. Free Radic Biol Med 1996;20:391-397.

33. Calabrese V, Ravagna A, Colombrita C,Scapagnini G, Guagliano E, Calvani M,Butterfield DA, Giuffrida Stella AM.Acetylcarnitine in duces heme oxygenase in rat astrocytes and pro tects against ox i da -tive stress: in volve ment of the tran scrip -tion fac tor Nrf2. J Neurosci Res. 2005;79:509-521.

34. Pettegrew JW, Le vine J, McClure RJ.Acetyl-L-Carnitine phys i cal-chem i cal, met -a bolic, and ther a peu tic prop er ties: rel e -vance for its mode of ac tion in Alz hei mer’s dis ease and ge ri at ric de pres sion. Mol Psy -chi a try 2000; 5:616-632.

35. Murthy MSR, Pande SV. A stress-reg u -lated pro tein, GRP58, a mem ber ofthioredoxin superfamily, is a carnitinepalmitoyl transferase isoenzyme. Biochem.J 1994; 304:31-34.

36. Palmeira CM, Serrano J, Kuehl DW,Wallace KB. Pref er en tial ox i da tion of car -diac mi to chon drial DNA fol low ing acute in -tox i ca tion with doxorubicin BiochimBiophys Acta 1997; 1321:101-106.

37. Wallace DC. Mi to chon drial ge net ics: a par -a digm for ag ing and de gen er a tive dis -eases? Sci ence 1992; 256:628-632.

38. Chang J, Knowlton AA, Xu F, Wasser JS.Activation of the heat shock re sponse: re -la tion ship to en ergy me tab o lites. A P-(31)P NMR study in rat hearts. Am J PhysiolHeart Circ Physiol 2001; 280:H426-433.

39. Ahn SG, Thiele DJ. Re dox reg u la tion ofmam ma lian heat shoack fac tor 1 is es sen -tial for Hsp gene ac ti va tion and pro tec tion from stress. Genes and De vel op ment 2003; 17:516-528.

40. Cornivelli L, Zeidan Q, De Maio A. HSP70 In ter acts with ri bo somal sub units ofthermotolerant cells. Shock 2003;20: 320-325.

41. Voss MR, Stallone JN, Li M, CornelussenRN, Knuefermann P, Knowlton AA. Gen -der dif fer ences in the ex pres sion of heatshock pro teins: the ef fect of es tro gen. AmJ Physiol Heart Circ Physiol 2003; 285: H687-92.

42. Coronato S, Di Girolano W, Salas M,Spinelli O, Laguens G. Biología de lasproteínas del choque térmico. Medicina(Bue nos Ai res) 1999; 59:477-486.

43. Löw-Friedrich I, von Bredow F, SchoeppeW. In vi tro stud ies of the cardiotoxicity ofchemo ther a peu tics. Che mo ther apy 1990;36:416-421.

44. De Maio A. Heat shock pro tein, ox y genrad i cals, and apoptosis: The con flict be -tween pro tec tion and de struc tion. CritCare Med 2000; 28:1679-1681.

45. Macario AJ, Coway de Macario E. Stressand Mo lec u lar Chaperones dis ease. Int JClin Lab Res 2000; 30:49-66.

46. Strauss M, Anselmi G. Is adriamycincardiomyopathy a proteinopathy subcell -ular dense con tent and de pressed ac cu mu -la tion of pro tected pro tein. In: Mi cros copyMeth ods in Pa thol ogy, Pro ceed ings of the15 th In ter na tional Con gress on Elec tronMi cros copy, Durban, South Af rica, 1-6 Sep -tem ber 2002.

Vol. 48(1): 33 - 43, 2007

HSP70 and ultrastrucuture of tis sues treated with adriamycin 43