Effects of Voluntary Physical Activity and Endurance Training on Cardiac Mitochondrial Function of Rats Sub-Chronically Treated with Doxorubicin

E ec s o Volun a y Physical Ac i i y and Endu ance T aining on Ca diac Mi ochond ial Func ion o Ra s Sub-Ch onically T ea ed wi h Doxo ubicin Disse a ion submi ed o he Facul y o Spo s, Uni e si y o Po o o ob ain he 2nd cycle in Physical Ac i i y o Elde ly, unde he dec ee-law no. 74/2006 o 24 Ma ch Supe iso s. P o esso Dou o An ónio Ascensão P o esso Dou o José Magalhães Diogo Nuno Ma iani Felix Po o, June 2013 Ma iani, D. (2013). E ec s o Volun a y Physical Ac i i y and Endu ance T aining on Ca diac Mi ochond ial Func ion o Ra s Sub-Ch onically T ea ed wi h Doxo ubicin. Po o: D. Ma iani. Mas e hesis p esen ed o he Facul y o Spo , Uni e si y o Po o. KEY-WORDS: EXERCISE; HEART; BIOENERGETICS; MITOCHONDRIAL FUNCTION; DOXORUBICIN. FUNDING The p esen wo k was suppo ed by a esea ch g an om he FCT (PTDC/DTP/DES/1071/2012 – FCOMP-01-0124-FEDER-028617) and om IJUP (PP_IJUP2011 253) o An ónio Ascensão; om Resea ch Cen e In Physical Ac i i y, Heal h And Leisu e (CIAFEL) I&D UNIT (PEST- OE/SAU/UI0617/2011). DEDICATÓRIA Ao papá, à mamã, ao Gonçalo e à Ely. AGRADECIMENTOS Ago a que inalizo o mes ado, gos a ia de exp essa os meus p o undos e since os ag adecimen os: Aos P o esso es José Magalhães e An ónio Ascensão, pela disponibilidade semp e demons ada na o ien ação des e es udo, pelos sábios ensinamen os que me ansmi i am, pelo incen i o, po es a em semp e p esen es, pela paciência, amabilidade e pelos momen os de ca inho e amizade semp e demons ados. Ao Cen o de In es igação em A i idade Física, Saúde e Laze (CIAFEL) po odo o apoio e colabo ação p es ados na ealização des e es udo. A odo o g upo de abalho em especial à Inês, à Es ela e à Mané, que me acompanha am em odos os momen os do p o ocolo expe imen al des e es udo, pela disponibilidade, pelo incen i o e pela amizade. Inês, ob igada po odas as explicações, pela paciência e po es a es semp e disponí el! E a odas as pessoas e amigos que di e a ou indi e amen e con ibuí am pa a a conc e ização des e abalho. ix Table o Con en s! 1.!In oduc ion .................................................................................................................... 1! 2. S a e o a ........................................................................................................................ 3! 2.1- Age e ec s on ca diac unc ion ................................................................................. 3! 2.1.1 Aging e ec s on ca diac mi ochond ia unc ion ....................................................... 8! 2.2. Exe cise and ca diop o ec ion ................................................................................. 16! 2.2.1 Exe cise and ca diac mi ochond ial adap a ions ................................................... 18! 2.2.1.1. Mo phological and biochemical adap a ions ...................................................... 18! 2.2.1.2. Mi ochond ial biogenesis ................................................................................... 20! 2.2.1.3. Oxida i e s ess and an ioxidan capaci y ......................................................... 21! 2.2.1.4. Cell dea h pa hways .......................................................................................... 24! 2.3. Doxo ubicin: he apeu ic agen s. ca dio oxici y .................................................... 25! 2.3.1 Ca diac mi ochond ial oxici y induced by DOX ..................................................... 27! 2.3.1.1 Mo phological e idences .................................................................................... 28! 2.3.1.2 Inc eased oxida i e s ess .................................................................................. 29! 2.3.1.3 Inc eased suscep ibili y o apop osis .................................................................. 30! 2.4. Exe cise as a he apeu ic and p e en i e s a egy agains DOX-induced ca dio oxici y. .................................................................................................................. 31! 2.4.1 Acu e exe cise ....................................................................................................... 31! 2.4.2 Ch onic exe cise .................................................................................................... 32! 3. Aim ................................................................................................................................. 37! 4. Ma e ials and me hods ................................................................................................... 39! 4.1 Reagen s .................................................................................................................. 39! 4.2 Animals ..................................................................................................................... 39! 4.3 Exe cise p o ocols .................................................................................................... 40! 4.3.1 Endu ance aining p o ocol ................................................................................... 40! x ii Abb e ia ions and Symbols VO2max Maximal Oxygen Up ake A.M. Be o e Midday AIF Apop osis Inducing Fac o AMPK Adenosine Monophospha e-Ac i a ed P o ein Kinase ANT Adenosine Nucleo ide T anslocase ATP Adenosine T iphospha e Ca2+ Calcium Ion CAT Ca alase CHF Conges i e Hea Failu e CS Ci a e Syn hase Cyc D Cyclophilin D DNA Deoxy ibonucleic Acid DOX Doxo ubicin DTNB Be a Dys ob e in E Exe cise ER Endoplasmic Re iculum FW F ee Wheel G Glu ama e GPX Glu a hione Pe oxidase GSH Glu a hione h Hou s H2O2 Hyd ogen Pe oxide HSP Hea Shock P o eins IFM In e myo ib illa Mi ochond ia IR Ischemia Repe usion KCl Po assium Chlo ide Kg Kilog am KH2PO4 Monopo assium Phospha e m Me o M Mala e x iii MDA Malondialdehyde MFN Mi o usin mg Millig am min Minu e ml Millili e mM Millimola MnSOD Manganese Supe oxide Dismu ase MPTP Mi ocohnd ial Pe meabili y T ansi ion Po e m DNA Mi ochond ial Deoxy ibonucleic Acid MΩ Megaohm NaCl Sodium Chlo ide NADH Reduced Nico inamide Adenine Dinucleo ide NADPH Reduced Nico inamide Adenine Dinucleo ide Phospha e nmol Nanomol NO Ni ic Oxide NS Non-Signi ican O2 Oxygen O2- Supe oxide Radical ºC Deg ee Celsius OH. Hyd oxyl Radical PGC P oli e a o -Ac i a ed Recep o Gamma RCR Respi a o y Con ol Ra io RNA Ribonucleic Acid ROS Reac i e Oxygen Species SAL Saline SEM S anda d E o O The Mean -SH Sul hyd yl g oups SOD Supe oxide Dismu ase SSM Subsa colemmal Mi ochond ia T T ea men Time TM T eadmill TNF Tumo Nec osis Fac o xix TPP+ Te aphenylphosphonium VDAC Vol age Dependen Anion Channel (A-VO2)di A e io enous oxygen di e ence Δᴪ T ansmemb ane Elec ical Po en ial 1 1. In oduc ion Doxo ubicin (DOX, o ad iamycin) is a highly e ec i e an ibio ic used o ea se e al ypes o malignancies. Un o una ely, he clinical use o DOX is limi ed by he occu ence o a dose- ela ed ca diac oxici y ha esul s in li e- h ea ening ca diomyopa hy. DOX-induced ca diomyocy e dys unc ion is associa ed wi h inc eased le els o oxida i e damage in ol ing mi ochond ial bioene ge ics collapse in he p ocess (Wallace 2007). Ac ually, sub-ch onic DOX ea ed a s e eal de ec s on hea mi ochond ial unc ion, which a e accompanied by comp omised mi ochond ial elec on anspo chain ac i i y and inc eased oxida i e s ess and damage (Be hiaume, Oli ei a e al. 2005, San os, Mo eno e al. 2002). Among he s a egies ad ised o coun e ac he ca diac side e ec s associa ed wi h DOX ea men , physical exe cise has been s udied and ecommended as a non-pha macological ool agains myoca dial inju y (Ascensao, Fe ei a e al. 2007, Ascensao, Lumini-Oli ei a e al. 2011, Ascensao, Oli ei a e al. 2012). P e ious wo k has sugges ed ha he ad an age o bo h acu e (Ascensao, Lumini-Oli ei a e al. 2010, Wonde s, Hydock e al. 2008) and ch onic exe cise models (Ascensao, Fe ei a e al. 2006, Ascensao, Magalhaes e al. 2005, Ascensao, Magalhaes e al. 2005, Chicco, Hydock e al. 2006, Chicco, Schneide e al. 2005, 2006) on he p econdi ioning o DOX- ea ed a s include he p o ec ion o ca diac issue and mi ochond ia agains induced impai men s. Mo eo e , ecen s udies in es iga ing he e ec s o exe cise pe o med du ing and ollowing models o la e-onse ca dio oxici y caused by DOX p o ide e idence o exe cise-induced ca diop o ec ion in bo h adul and ju enile a models (Haywa d, Lien e al. 2012, Hydock, Lien e al. 2012). Howe e , he cellula and molecula mechanisms unde lying his p o ec i e pheno ype induced by exe cise a e s ill elusi e. In pa icula , whe he pe u ba ions in hea mi ochond ial oxida i e phospho yla ion capaci y and p o-oxidan edox modi ica ions associa ed wi h cumula i e DOX adminis a ion a e modula ed by long- e m physical exe cise pe o med du ing and a e ea men s is ye unknown. 2 We he e o e aimed o analyze he e ec s o wo ypes o long- e m exe cise wi h dis inc cha ac e is ics, pe o med be o e and du ing he o e all DOX ea men , on ca diac mi ochond ial bioene ge ics. Hea mi ochond ial espi a o y pa ame e s associa ed wi h oxygen consump ion, ansmemb ane elec ical po en ial and osmo ic swelling du ing mi ochond ial pe meabili y ansi ion po e (MPTP) induc ion as well as ma ke s o oxida i e s ess (sul hyd yl g oups (-SH) and malondialdehyde (MDA) con en s) we e de e mined. 3 2. S a e o a 2.1- Age e ec s on ca diac unc ion Aging can be cha ac e ized as a ime dependen decline o maximal unc ionali y ha a ec s issues and o gans o he whole body (Figuei edo, Mo a e al. 2008). The a e age human li e span has ma kedly inc eased in mode n socie y, a ac la gely a ibu ed o ad ances in medical and he apeu ic sciences ha ha e success ully educed he se e i y o se e al diseased condi ions (Chaudha y, El-Sikh y e al. 2011). Howe e , elde ly indi iduals con inue o su e he g ea es bu den om ca dio ascula disease, including co ona y hea disease ha emains he leading cause o dea h in indus ialized coun ies (Wei 2004). Tha can be unde s ood as he esul o se e al mo phophysiological, s uc u al and unc ional al e a ions, which we will u he add ess in de ail (Table 1). The aging-induced inc ease on ascula s i ness, sep um and myoca dium hickness and ib osis, is associa ed, among o he s, wi h: i) dec eased myocy e numbe ; ii) inc easing ca diomyocy e size wi h al e a ions in calcium (Ca2+) homeos asis and iii) inc eased collagen ibe s deposi ion, leading o ca diac dias olic dys unc ion, inc eased a e load, and loss o a e ial and hea compliance (S ai and Laka a 2012). Impo an ly, hese physiological- ela ed impai men s along wi h unchanged ca i y size and inc eased le en icle hype ophy a e he majo cha ac e is ic o aging hea (Chaudha y, El-Sikh y e al. 2011). In ac , Dai e al (2009) epo ed ha aging le en icle mass index inc eased by a ound 75% compa ed o a young adul g oup, indica ing he inc ease p e alence o le en icula pa hological hype ophy wi h age. I was also epo ed educ ion in dias olic unc ion, as well as wo sening o myoca dial pe o mance index (Dai, San ana e al. 2009). Fu he mo e, he conduc ion sys em also unde go some s uc u al al e a ions leading o hea dys unc ion, including a accumula ion a ound sinoa ial node, which c ea es a pa ial o comple e sepa a ion o he node om a ial issue (S ai and Laka a 2012), a ma ked dec eased in peacemake s numbe cells 4 (Wei 2004) and an inc eased calci ica ion in conduc ion sys em leading ul ima ely o a io en icula conduc ion lock. Slow and p olonged sys ole and dias ole a e o he aging- ela ed hea ea u es usually ela ed o an abe an Ca2+ handling (S ai and Laka a 2012). The dec eased dias olic unc ion, which alls linea ly wi h aging a a a e o abou 6–7% pe decade (Ga es, Tanaka e al. 2003), is associa ed o a dec ease in dias olic illing epo ed a es and unde s ess condi ions, as some daily asks ha p omo e an acu e inc ease o physical demands. In addi ion, he degene a ion o ca diac al ula appa a us is commonly epo ed, being al ula annula dila ion ound in he majo i y o olde pe sons and is also associa ed wi h concomi an co ona y a e y calci ica ion (Wei 2004). Table 1. E ec s o aging on ca dio ascula sys em Mo phophysiological changes S uc u al Changes Func ional Changes Re e ence Hea and Vascula sys em ↓ Ca diomyocy es numbe ↑ Ca diomyocy es size ↑ Collagen deposi ion ↑ Fibe s deposi ion ↓ Ca diomyocy es unc ion ↑ Sep um and Myoca dium s i ness ↓ Hea compliance ↑ Vascula hickness ↓ Vascula compliance ↑ Sys olic p essu e ↓ Dias olic unc ion ↓ max hea a e ↓ max ca diac ou pu ↑ A e load P olonged sys ole and dias ole Ho a and Uchida (2010), Reynolds (2004), S ai and Laka a (2012), T. Shioi (2012), Taylo , Cable e al. (2004) Conduc ion sys em ↑ Fib osis and a accumula ion (SA node) ↓ Peacemake s cells numbe ↑ Conduc ion sys em calci ica ion ↓ Ven icula compliance ↑ Hea hy hm dis u bances ↑ Risk a io en icula conduc ion lock ↑ Risk a ial a hy hmias, ↑ Risk a ial ib illa ion ↑ Risk achyca dia ↑ Ven icula a hy hmias Ho a and Uchida (2010), S ai and Laka a (2012) Val ula sys em ↑ Val ula annula dila a ion Co ona y a e y calci ica ion Reynolds (2004) (↑) – inc ease; (↓) – dec ease ↓ VO2 max 5 O e all, hese aging ela ed hea mo phophysiological, s uc u al and unc ional al e a ions esul in inc eased sys olic p essu e, one majo isk ac o s o de elopmen o a he oscle osis, hype ension and s oke, and a e ial ib illa ion (No h and Sinclai 2012). As a consequence o all hose age-associa ed al e a ions, ca diac unc ion declines maximal unc ionali y, being ha , unde basal condi ions his decline may no be de ec ed (Wessells and Bodme 2007). The mos used s anda d index o ca dio espi a o y i ness is he maximal oxygen consump ion (VO2max), which declines app oxima ely 10% pe decade s a ing a 20 o 30 yea s old (Souza 2012). VO2max can be es ima ed by he Fick’s equa ion: Equa ion 1: VO2max=Q×(A-VO2)di Q is ca diac ou pu , and is he p oduc o max hea a e and s oke olume, (A- VO2)di is he a e io enous di e ence o oxygen. As i is obse ed in igu e 1, based on Fick´s equa ion, ae obic exe cise capaci y depends on ca diac ou pu and a e io enous di e ence (Seeley, S ephens e al. 2005). Reduc ions in peak hea a e, pe iphe al oxygen u iliza ion and s oke olume appea o media e he age-associa ed decline in VO2max. Ac ually, impai men s in ca diac illing and inc eased a e load leads o a dec ease in hea a e, in a s ess si ua ion (Taylo , Cable e al. 2004) being he majo esponsible o much o he age-associa ed dec ease in maximal ca diac ou pu . Reduc ions in muscle oxygen deli e y and he e o e (A-VO2)di a e mainly due o educed and maldis ibu ion o ca diac ou pu . Also, a decline in skele al muscle oxida i e capaci y wi h aging is epo ed, due in pa o mi ochond ial dys unc ion, which appea s o play a pa icula ly impo an ole in old age, whe e skele al muscle VO2max is obse ed o decline by app oxima ely 50% e en unde condi ions o simila oxygen deli e y as young adul muscle (Be ik and Hepple 2008). 12 Besides nucleus, mi ochond ia a e he only o ganelles in animal cells ha possess hei own DNA, he m DNA, as well as ela ed ansc ip ional and ansla ional syn hesis machine y. m DNA is localized in ma ix wi h physical p oximi y o he mi ochond ial espi a o y chain (B a ic and T i uno ic 2010). Howe e , mi ochond ia canno be syn hesized de no o, ins ead hey eplica e in he cy osolic compa men h ough a p ocess o di ision ( o e s see Chaudha y, El-Sikh y e al. 2011). Impo an ly, he p o eins ha a e encoded by m DNA a e i al o no mal mi ochond ial unc ion and m DNA does no ha e he p o ein p o ec ion as nuclea DNA and has less e ec i e epai mechanisms (Desle , Ma cke e al. 2011). The e o e, mu a ions in m DNA h ough oxida i e s ess a ec he exp ession and in eg i y o oxida i e phospho yla ion complexes and can cause mi ochond ial dys unc ion and inc eased ROS p oduc ion (Wallace 2010). So, i is no able he in e ela ionship be ween oxida i e phospho yla ion complexes, ROS le els, m DNA mu a ions and ul ima ely cell dea h. Fu he mo e, mu a ions in m DNA and he esul an decline in mi ochond ial ac i i y obse ed in aged issues a e esponsible o he inc eased gene a ion o ROS, which in u n, will u he nega i ely impac mi ochond ia causing u he m DNA damage. This “ icious cycle” concep pos ula ed ha accumula ion o m DNA mu a ions is exponen ial and associa ed wi h massi e inc ease in ROS p oduc ion (Lenaz 1998). Being mi ochond ia s a egic o ganelles essen ial o cell unc ion and homeos asis, p o iding ene gy o he cell (Chaudha y, El-Sikh y e al. 2011), hey mus unde go some dynamic mechanisms. Mi ochond ial u no e and dea h can occu ia se e al p ocesses ha a e sugges ed o be in e ela ed namely apop osis, nec osis, au ophagy and ela ed dynamics o o ganelles. In his dynamic ne wo k biogenesis, usion and ission a e closely associa ed mechanisms (Chen and Knowl on 2011). In ac , apop osis is media ed by wo pa hways: he ex insic and he in insic pa hways, and bo h ha e been desc ibed in ca diac myocy es (Whelan, Kaplinskiy e al. 2010). The ex insic apop o ic pa hway can be igge ed by Fas ligand o umo nec osis ac o (TNF)-α, which a e exp essed in ca diac 13 myocy es and ha e been implica ed in ca dio ascula pa hology (Whelan, Kaplinskiy e al. 2010). In insic pa hways in ol e he pa icipa ion o endoplasmic e iculum (ER) and/o mi ochond ia (K oeme , Galluzzi e al. 2007). Impai men in mi ochond ial in eg i y, dynamics o me abolic ac i i y may esul in a ange o dele e ious e ec s o he cell, such as educed ATP p oduc ion, ele a ed cy osolic Ca2+, inc eased ROS elease, elease o p oapop o ic ac o s as cy oc ome c o caspases ac i a ion and Bax ansloca ion, igge ing cell dea h (Chen and Knowl on 2011). In mammalian cells, apop osis is egula ed by a a ie y o ac o s ha a e essen ially ei he p o-li e o p o-dea h (Goldspink, Bu nis on e al. 2003). Quan i a ion o he exp ession o genes in ol ed in he apop o ic pa hway migh ep esen a good index o he p obabili y o a cell o unde go apop osis. As he numbe o Bax-exp essing cells d ama ically inc eases in le en icula hype ophy and le en icula dys unc ion (Condo elli, Mo isco e al. 1999, G een and Reed 1998), i is sugges ed ha mi ochond ion is he p ima y o ganelle media ing he in insic apop o ic pa hway in hese condi ions (Chiong, Wang e al. 2011) Also, i excessi e Ca2+ en e s o mi ochond ia and enhanced oxida i e s ess condi ions a e p esen , a phenomenon known as pe meabili y ansi ion may occu (Ascensao, Lumini-Oli ei a e al. 2011). The mi ochond ial pe meabili y ansi ion is cha ac e ized by he loss o he impe meabili y o he mi ochond ial memb anes and i is sugges ed ha his condi ion is media ed by he o ma ion and opening o p o ein complex-like po es in he inne mi ochond ial memb ane, he mi ochond ial pe meabili y ansi ion po e (MPTP) ( o e s see Ascensao, Lumini-Oli ei a e al. 2011). Inc eased p o-oxidan gene a ion causing oxida i e s ess is one condi ion ha augmen ed he suscep ibili y o he opening o hese po es and he elease o p o-apop o ic p o eins wi hin mi ochond ia as cy och ome c, SMAC/DIABLO and he apop osis inducing ac o (AIF), which will ac i a e he caspase- ela ed apop o ic pa hways. I is sugges ed ha he elease o hese p o eins is dependen on he o ma ion and opening o MPTP ha c oss he inne and he ou e memb anes leading o he loss o 14 mi ochond ial memb ane po en ial (Δᴪ), inc eased mi ochond ial osmo ic swelling and up u e o he ou e mi ochond ial memb ane, which leads o dea h (Ascensao, Lumini-Oli ei a e al. 2011). I is belie ed ha he s uc u e and egula ion o his mul i-p o ein complex comp ises he ou e memb ane ol age- dependen anion channel (VDAC) as well as he inne memb ane adenine nucleo ide ansloca o (ANT) and cyclophilin D (Cyc D). Myocy e loss has been shown o occu in he aged a hea and o p ecede he occu ence o en icula dys unc ion (An e sa, Hile e al. 1986), being apop o ic ca diomyocy e dea h p esen unde di e en condi ions in humans (Hauns e e and Izumo 1998, Na ula, Haide e al. 1996, Oli e i, Abbi e al. 1997). Among he excess o biological phenomena a ec ed by aging, he mal unc ion and dec ease o biogenesis o mi ochond ial biogenesis seems o exe some o he mos po en e ec s on he o ganism (Lopez-Lluch, I us a e al. 2008). I biogenesis is a ec ed, i is easonable o expec ha mi ochond ial u no e mus be slowe and he accumula ion o modi ied lipids, p o eins and DNA mus also inc ease, u he agg a a ing he condi ions esul ing on de icien ac i i y o aged mi ochond ia (Lopez-Lluch, I us a e al. 2008). The p ecise eason o he dec ease in he a e o mi ochond ial biogenesis du ing aging is cu en ly unknown. Howe e , i seems ha bo h, ex a- and in a-cellula egula o y ac o s o mi ochond ial biogenesis a e implica ed. Speci ically, pe oxisome p oli e a o -ac i a ed ecep o gamma coac i a o (PGC1-α) has been shown o ac as a common in acellula media o du ing mi ochond ial biogenesis induced by ho monal ac o s (Wei zel, Iwen e al. 2003), and adenosine monophospha e-ac i a ed p o ein kinase (AMPK) an in acellula egula o o mi ochond ial biogenesis, which ac i i y appea s o be one o he main ac o s associa ed wi h de icien mi ochond ial biogenesis (Reznick, Zong e al. 2007). PGC amily membe s ha e gained pa icula in e es because o hei abili y o d i e i ually all mechanisms o mi ochond ial biogenesis in he hea , including mi ochond ial numbe , mi ochond ial espi a ion, exp ession o oxida i e phospho yla ion and a y acids oxida ion genes, and ROS le els (Lehman, Ba ge e al. 2000). Dec eased PGC-1α exp ession has been linked o he de elopmen o hea ailu e in mouse models ( o e s see Moslehi, DePinho e 15 al. 2012), and decline in mi ochond ial biogenesis and mi ochond ial p o ein quali y con ol in ca diac muscle was ound in aging (Kol ai, Ha e al. 2012) In addi ion, i s well known ha mi ochond ia a e dynamic o ganelles ha cons an ly unde go ission and usion and i has been ound o be ib an o ganelles ha con inuously di ide and use wi hin he cell and ha e unc ions ex ending beyond ene gy p oduc ion, including cell signaling (Liesa, Palacin e al. 2009). Dis up ion o ission and/o usion can also lead o cellula dys unc ion and o apop osis. This dynamic mechanism is egula ed by p o eins con olling ission, such as hFis1 and D p1, and usion, such as mi o usin 1 and 2 (MFN1 and MFN2) and OPA1. The co ec unc ion o hese p o eins seems o be c i ical o no mal mi ochond ial ac i i y, and hei de egula ion is associa ed wi h se e al pa hologic condi ions (Lopez-Lluch, I us a e al. 2008). Indeed, he impai men s on ission- ela ed p o ein hFis1 has been associa ed wi h he p ocess o senescence in mammalian cell cul u es (Lee, Jeong e al. 2007). Mo eo e , deple ion o hFis1 by RNA in e e ence (RNAi) induces d ama ic changes in mi ochond ial s uc u e, including he enla gemen and la ening o he o ganelle. Fu he mo e, elimina ion o any o he mi ochond ial usion p o eins as MFN1, MFN2 o OPA1, induces mi ochond ial agmen a ion, as expec ed, being ha down- egula ion o Opa1 exp ession in cells by RNAi esul s in spon aneous apop osis ( o e s see Chen and Knowl on 2011). O e all, de ec s in he mi ochond ial ission/ usion machine y and so loss o he symme y be ween usion and ission (Hoppins, Edlich e al. 2011) may con ibu e o he decline in mi ochond ial unc ion du ing aging. Howe e , se e al undamen al ques ions emain o be answe ed (Bossy-We zel, Ba soum e al. 2003). In he nex sec ion, he oles o physical exe cise as a s a egy o imp o e ca diac unc ion in adul and old subjec s as well as he mi ochond ial-media ed mechanisms associa ed wi h exe cise-induced ca diop o ec ion will be add essed. 16 2.2. Exe cise and ca diop o ec ion Ca diac damage is a majo con ibu o o mo bidi y and mo ali y in indus ialized coun ies; so i becomes impo an o de elop s a egies ha esul in ca diop o ec i e pheno ype. In his ega d, se e al app oaches ha e been in es iga ed and physical ac i i y has been shown o be an impo an coun e measu e o p o ec agains myoca dial inju ies (Bowles, Fa a e al. 1992, Ha is and S a nes 2001, Powe s, Demi el e al. 1998, Powe s, Quind y e al. 2004). In ac , ca dio espi a o y i ness is in e sely ela ed o ca dio ascula and all-cause mo ali y and i has c ucial ole p e en ing hea inju y (Kokkinos, Mye s e al. 2010). Some epo s had pos ula ed exe cise- induced bene i s based on dec eases o some isk ac o s o de elop ca diac and myoca dium impai men s such as bodymass index, body weigh , wais ci cum e ence, abdominal and isce al a and consequen ly insulin esis ance, iglyce ide le els, blood p essu e and, in gene al, me abolic synd ome- ela ed pa ame e s ( o e s see Golbidi and Lahe 2012). The e o e, ch onic ae obic exe cise is able, no only, o imp o e ca dio ascula unc ion in young heal hy subjec s, bu also, and mos impo an ly, in olde people and hose wi h ca dio ascula isk ac o s (Hamb ech , Fiehn e al. 1998). The s udy o he mechanisms esponsible o exe cise-induced ca diop o ec ion has been ongoing o o e decades and mo phological and biochemical/molecula al e a ions ha e been conside ed as pu a i e mechanisms o exe cise-induced ca diop o ec ion. Those include mo phological adap a ions o hea and co ona y a e ies, induc ion o myoca dial HSPs, inc eased myoca dial cyclooxygenase-2 ac i i y, ele a ed ER s ess p o eins, ni ic oxide p oduc ion, imp o ed unc ion o sa colemmal and/o mi ochond ial adenosine iphospha e (ATP)-sensi i e po assium channels and inc eased myoca dial an ioxidan capaci y ( o e s see Ka azis 2009). Fo ins ance, exe cise induces ascula emodeling and so, mo phological al e a ions in co ona y a e ies h ough angiogenesis and a e iogenesis (Leung, Yung e al. 2008). He e, ni ic oxide (NO) assumes impo an oles due i s an i-in lamma o y, asodila o and pla ele inhibi o y e ec s (Landmesse and 17 D exle 2005). Also, NO p o ec s agains inschemia- ep e usion (IR) inju y in such a way ha he hea esponds o ischemia using ni ic oxide species in a ha monized manne and hese mechanisms could be based on inhibi ion o Ca2+ in lux in o myocy es, an agonism o β-ad enegic s imula ion, educ ion in ca diac oxygen consump ion and abili y o inc ease he exp ession o HSP70 ( o e s see Golbidi and Lahe 2011). HSPs p o ec cell agains oxida i e inju y and apop osis (Polla, Kan engwa e al. 1996) and u he mo e, enhance eco e y om acu e myoca dial cellula inju y p o ec ing hea o subsequen inju y ( o e s see Powe s, Locke e al. 2001) by p omo ing es o a ion o dys unc ional enzymes and p e en ing agg ega ion o se e ely dena u ed p o eins. The majo i y o e idences indica e ha membe s o he 70-kDa amily a e he cy op o ec i e p o eins mos esponsible o cell p o ec ion ( o e s see Powe s, Locke e al. 2001). The exp ession o HSP70 in ca diomyocy es is associa ed wi h inc eased cell su i al and p o ec ion agains ischemic damage and i s now well es ablished ha acu e and ch onic exe cise a e able o induce inc eases in he exp ession o HSP70 (K egel and Moseley 1996, Powe s, Demi el e al. 1998), al hough in a empe a u e-dependen “ ashion”. The imp o ed unc ion o sa colemmal ATP sensi i e po assium channels (Powe s, Quind y e al. 2008) and ele a ed ER s ess p o eins a e o he impo an exe cise-induced ca diop o ec ion- ela ed al e a ions and bo h ha e special ele ance du ing a ca diac insul (Golbidi and Lahe 2012). The ER s ess p o eins help cellula homeos asis by main aining in acellula Ca2+ egula ion and p o ein olding du ing IR inju y (Logue, Gus a sson e al. 2005). Conside ing he impo ance o mi ochond ial machine y in he main enance o ca diac unc ion, mi ochond ial-media ed mechanisms ha e also been associa ed wi h exe cise induced ca diop o ec ion phenomenon. This opic will be u he discussed in he nex sec ions. 18 2.2.1 Exe cise and ca diac mi ochond ial adap a ions As p e iously men ioned, mi ochond ial adap a ions may play a c i ical ole in exe cise-induced p o ec ion agains ca dio ascula impai men s. The mechanisms behind his phenomenon emain unclea , howe e i may be ela ed o mo phological and biochemical adap a ions including biogenesis, an ioxidan p oduc ion o esis ance o cell dea h pa hways (Ascensao, Fe ei a e al. 2007, Ka azis, Al a ez e al. 2009, Ka azis, McClung e al. 2008). Those will be b ie ly add essed in he ollowing sec ion. 2.2.1.1. Mo phological and biochemical adap a ions Hea is a highly oxida i e o gan, wi h a low a e o cell g ow h and slow u no e o p o eins; so, i would be expec ed ha hea migh ha e a limi ed abili y o adap o acu e and/o ch onic condi ions. Al hough he e a e sca cely add essed ou comes abou exe cise-induced ca diac mi ochond ial mo phological adap a ions in heal hy hea s, he same canno be s a ed ega ding hea s unde dele e ious condi ions. In ac , unde dele e ious condi ions as DOX ea men , diabe es, o aging, mo phological impai men s on hea mi ochond ia ha e been epo ed (Ascensao, Oli ei a e al. 2012, Sea ls, Smi no a e al. 2004). Despi e his ac , in a s udy whe e ained animals we e submi ed o an endu ance swimming aining p og am, while he non- ained we e no engaged in any exe cise p og am Ascensao e al. (2006) epo ed ha endu ance swimming aining pe se caused no able changes in myoca dial s uc u e seen as an appa en inc eased glycogen con en , in e cala ed discs showing a no o ious scalloped appea ance and e iden signs o mi ochond ia biogenesis wi h ele a ed numbe o enc oached mi ochond ia pe ibe a ea, p obably esul ing in an inc eased olume/densi y o mi ochond ia. Also, mi ochond ia di ision, mild and ocal loss o c is ae densi y and o ganiza ion as minimal deg ada ion by-p oduc s, we e also p esen in non- ea ed ained hea s. Ano he s udy de eloped by Sea ls e al. (2004) 19 wi h ype I diabe es a s ound in ype I diabe es a s ha 9 weeks o mode a e exe cise is able o e e se some o he pheno ype o diabe ic ca diomyopa hy. Speci ically, he mi ochond ial quali y, cy oplasmic a ea, and collagen c oss- sec ional a ea e u ned owa d non-diabe ic alues wi h exe cise. Mi ochond ial biochemical adap a ions o exe cise we e also epo ed and se e al app oaches ha e been ying o p o e he exe cise-induced modi ica ions o some pa ame e s in ol ed in mi ochond ial bioene ge ics. Judge e al. (2005) epo ed ha wheel unning had no e ec on mi ochond ial p o ein yield, a es o oxygen consump ion (s a es 4 and 3) o RCR. This was no en i ely su p ising gi en ha o he s udies ha e shown ha , unlike skele al muscle, oxida i e capaci y o ca diac muscle is no inc eased in esponse o eadmill aining, an exe cise p o ocol ha is ypically much mo e in ense han olun a y wheel unning ( o e s see Judge, Jang e al. 2005). Also, S a nes e al. (2007) epo ed ha endu ance aining has no impac on mi ochond ial oxida i e phospho yla ion, being ha all oxida i e phospho yla ion pa ame e s we e simila in endu ance ained and seden a y a s. Because he hea is highly oxida i e, i is no expec ed o be as esponsi e o exe cise-induced inc eases in oxida i e capaci y as skele al muscle. Al hough, se e al epo s sugges ed ha exe cise pe se is able o imp o e mi ochond ial p o ein yield, a es o oxygen consump ion as well as ca diac unc ion ( o e s see Ascensao, Fe ei a e al. 2007, Ascensao, Magalhaes e al. 2006, Chicco, Schneide e al. 2006, Powe s, Lennon e al. 2002). Mo eo e , Ka asis e al. (2009) epo ed ha he abundance o se e al p o eins in ol ed in bioene ge ics was al e ed ollowing endu ance exe cise in bo h SS and IMF mi ochond ia. Mos speci ically, he p o ein le els o se e al p o eins in ol ed in β-oxida ion o a y acids we e inc eased ollowing epea ed bou s o endu ance exe cise. I is o especial conce n as du ing he de elopmen o hea disease o gene ic de ec s in mi ochond ial a y acid β-oxida ion he myoca dial ene gy sou ce swi ches om a y acid β-oxida ion o glycolysis. Also, he e a e some con o e sial epo s abou endu ance exe cise bene i s agains Ca2+-induced mi ochond ial dys unc ion. In ac , while S a nes e al. (2007) ound ha endu ance exe cise aining had no in luence when mi ochond ia was challenged wi h iden ical Ca2+ 20 concen a ions, being ha mi ochond ia om ained and seden a y animals displayed simila declines in ATP p oduc ion, F ench e al. (2008) ound ha exe cise-induced p o ec ion agains IR inju y, in pa , by a enua ing IR-induced oxida i e modi ica ion o impo an Ca2+-handling p o eins and p e en ing subsequen calpain ac i a ion. In his s udy, MnSOD may had an impo an ole as, he esul s suppo ha exe cise-induced inc eases in myoca dial MnSOD ac i i y a enua e he oxida ion and deg ada ion o Ca2+-handling p o eins, p e en ing calpain ac i a ion du ing IR. I has special conce n because calpain ac i a ion wi hin he myoca dium has been di ec ly linked wi h IR-induced nec o ic and apop o ic ca diac myocy e dea h (F ench, Hamil on e al. 2008). Fu he mo e, se e al epo s sugges ha exe cise pe se is able o enhance ca diac mi ochond ia Ca2+ up ake capaci y wi hou MPTP induc ion (Ascensao, Lumini-Oli ei a e al. 2011, Ka azis, McClung e al. 2008, Ma cil, Bou duas e al. 2006). The e o e, exe cise may induce bene icial mo phological and biochemical adap a ions in mi ochond ia, which can be ansla ed in a ca diac pheno ype mo e unc ional and p o ec ed agains dele e ious s imuli. 2.2.1.2. Mi ochond ial biogenesis Due o he high-ene gy demand o he hea , a decline in mi ochond ial unc ion can esul in a de e io a ion o ca diac pe o mance (Li, Muhl eld e al. 2011) and can lead o a wide a ie y o pa hophysiological condi ions. Acco ding o some epo s, he mal unc ion o mi ochond ia and he dec ease o mi ochond ial biogenesis, oge he wi h inc eased oxida i e damage, seem o exe some o he mos dele e ious e ec s on he o ganism (Gua en e 2008, Lopez-Lluch, I us a e al. 2008). Fo una ely, al hough con o e sial, hea seems o ha e he abili y o al e some gene exp ession p o ile and pheno ype o p oduce new and mo e unc ional mi ochond ia (Lee and Wei 2007). Regula exe cise o inc eased ene gy demand a e impo an s imuli ha lead o inc eased mi ochond ia biogenesis ( o e s see Lopez-Lluch, I us a e al. 2008). In ac , inc eases in cy osolic Ca2+ le els induced by exe cise s imula es 21 calmodulin kinase which hen p omo es PGC1-α exp ession (Wu, Kana ous e al. 2002) ha has been shown o be a majo egula o o mi ochond ial biogenesis (Rodge s, Le in e al. 2005). Also, Geng e al. (2010) showed ha in skele al muscle-speci ic PGC-1α knockou mice, exe cise-induced mi ochond ial biogenesis and angiogenesis we e signi ican ly a enua ed. Howe e , exe cise- induced mi ochond ial biogenesis on ca diac muscle is s ill no ully illusi e. In ac , al hough a signi ican amoun o expe imen al da a on mechanisms in ol ed in exe cise-induced mi ochond ial biogenesis has been ob ained in skele al muscle, less in o ma ion is a ailable abou he hea . In his ega d, Li e al. (2011) epo ed no changes in mi ochond ial biogenesis on le en icle a e 3 mon hs o endu ance exe cise. Ac ually, in con as o skele al muscle in which adap i e changes in olume ac ion o mi ochond ia can eadily occu , his is no so equen in ca diac muscle (Hood, Balaban e al. 1994). So, he exe cise-induced bene i s on hea mi ochond ia biogenesis a e s ill con o e sial. 2.2.1.3. Oxida i e s ess and an ioxidan capaci y Wi h exe cise pe o mance, inc eased O2 consump ion c ea es a o able condi ions o inc eased gene a ion o ROS, an ine i able consequence ha may inc ease oxida i e s ess a he o ganelle, cell, and issue (Ji, Leeuwenbu gh e al. 1998). Howe e , i hese s imuli a e epea ed o e ime, i may ha e a s ong modula ing e ec on a ious de ense sys ems in ca diac cells (Powe s, Lennon e al. 2002). Also, in some dele e ious condi ions ha lead o ROS p oduc ion, as IR-induced myoca dial inju y, DOX adminis a ion o aging, physical exe cise as been epo ed o induce bene ic coun e measu es. In ac , IR-induced myoca dial inju y is mani es ed due o he complex in e ac ion o nume ous ac o s bu ROS gene a ed by mi ochond ia du ing IR inju y a e belie ed o play key ole in his p ocess ( o e s see Honda, Ko ge e al. 2005). Du ing IR, mi ochond ial ROS gene a ion can lead o inc eased gene al oxida i e s ess and consequen ly Ca2+ o e load, which could be o 28 Un o una ely, mi ochond ia ha e been iden i ied as p ima y DOX a ge o ganelles, and hei in ol emen is e idenced by he esul s o many s udies epo ing unc ional and mo phological al e a ions, as will be b ie ly desc ibed. Indeed, i has been sugges ed ha ca diomyocy e dys unc ion induced by DOX ea men is ela ed o inc eased le els o ROS-induced damage and apop o ic cellula dea h, in ol ing mi ochond ia in he p ocess (Jung and Reszka 2001) culmina ing in he dis up ion o majo unc ions (Jung and Reszka 2001). The nex sec ions will b ie ly desc ibe some DOX-induced mi ochond ial dys unc ions such as mo phological e idences, inc eased oxida i e s ess and suscep ibili y o apop osis. 2.3.1.1 Mo phological e idences As a consequence o acu e o ch onic DOX ea men , ul as uc u al al e a ions in a ca diomyocy es ha e been epo ed (Ascensao, Fe ei a e al. 2007, Ascensao, Oli ei a e al. 2012, Yilmaz, A essahin e al. 2006, Zhou, S a ko e al. 2001). These include nuclea swelling associa ed wi h dis up ion o nuclea memb ane s uc u e, a ma ked in e s i ial and cellula edema, pe inuclea acuola ion, diso ganiza ion and degene a ion o he myoca dium, loss o myo ib ils, dis ension o he sa coplasmic e iculum, sligh enla gemen o he T- ubules and myo ib illa damage and loss ( o e s see Ca alho, San os e al. 2009, Zhou, S a ko e al. 2001). Also, in mice ca diomyocy es, DOX-induced acu e al e a ions in mi ochond ia we e obse ed, such as acuoliza ion, myelin deposi ion, dis up ion o memb ane and o ganelle degene a ion wi h c is ae degene a ion, in ami ochond ial acuoles as well as myelin igu es (Ascensao, Fe ei a e al. 2007, Ascensao, Oli ei a e al. 2012, Sa dao, Oli ei a e al. 2009). These ul as uc u al al e a ions a e dose-dependen being ha highe DOX concen a ions p omo e mo e p o ound cellula al e a ions (Ca alho, San os e 29 al. 2009) and hose ul as uc u al inju ies a e no epai ed a e cessa ion o DOX ea men becoming e en mo e ex ensi e. 2.3.1.2 Inc eased oxida i e s ess One o he mos known DOX-induced side e ec s is he inc eased oxida i e s ess and i seems o be an e en ha occu s bo h acu ely and ch onically (Ascensao, Oli ei a e al. 2012, Wallace 2003). I has been sugges ed ha DOX accumula ion in mi ochond ia leads o a edox cycling by complex I o he mi ochond ial espi a o y chain, whe e single elec ons a e ans e ed o DOX (Ascensao, Oli ei a e al. 2012). As p e iously men ioned, he possible bu con o e sial exis ence o a speci ic NADH dehyd ogenase able o s a his edox cycling may ha e an impo an ole. DOX en e s mi ochond ia and eac s wi h mi ochond ial complex I o o m semiquinone adical in e media es, which is a sho -li ed me aboli e and can eac wi h O2 p oducing ROS ( o e s see Ca alho, San os e al. 2009). Then, ROS can eac wi h mi ochond ial biomolecules in he icini y, which include lipids, p o eins and nuclei acids. Fu he mo e, inc eased ca diac oxida i e s ess associa ed wi h DOX oxici y leads o he deple ion o educing equi alen s, impai men in oxida i e phospho yla ion wi h consequen decline in ATP, and in e e ence wi h cellula Ca2+ homeos asis (Wallace 2003). Also, DOX is known o eac wi h mi ochond ial m DNA o ming adduc s ha in e e e wi h p o eins exp ession, lipid oxida ion and no mal mi ochond ial unc ion, which in u n u he inc eases ROS p oduc ion (Sa dao, Pe ei a e al. 2008). This “ icious cycle” pos ula ed ha accumula ion o m DNA mu a ions is exponen ial and associa ed wi h massi e inc ease in ROS p oduc ion (Lenaz 1998). As p e iously desc ibed, hea mi ochond ia a e impo an a ge o DOX, accumula ing he d ug a ela i ely high concen a ions. So, i is no su p ising ha hese o ganelles a e especially suscep ible o DOX-induced oxida i e damage, and a he same ime, hey a e also impo an sou ces o DOX-induced ROS (Ascensao, Oli ei a e al. 2012). On o he hand, s udies show an up egula ion o an ioxidan de enses, 30 sugges ing an adap i e esponse o cells o oxida i e unbalance p omo ed by DOX (Yilmaz, A essahin e al. 2006). In e es ingly, he an ioxidan capaci y seems o inc ease signi ican ly ollowing DOX ea men in young bu no in old Fische sugges ing an inc ease in DOX-induced oxida i e damage wi h age (P i sos and Ma 2000). Despi e his ac , is no able ha , al hough he hea has ela i ely low an ioxidan capaci y, i shows an up egula ion as an adap i e esponse o his oxida i e unbalance. Mo eo e , as a esponse o his cellula s ess, HSP 60 and HSP 70 inc eased in hea s and mi ochond ia om DOX ea ed animals and cells (Ascensao, Magalhaes e al. 2005, Ka azis, Smude e al. 2010). 2.3.1.3 Inc eased suscep ibili y o apop osis Ano he impo an DOX-induced al e a ion on cell is he inc eased suscep ibili y o igge apop osis. DOX oxici y leads o impai men s on cellula Ca2+ homeos asis, which leads o inc eased suscep ibili y o he MPTP opening (Oli ei a and Wallace 2006) ha is cha ac e ized by he loss o he impe meabili y ha cha ac e izes he inne mi ochond ial memb ane, as i was p e iously desc ibed. This complex p ocess is media ed by he o ma ion and opening o p o ein complex-like po es, he MPTP (Ascensao, Lumini-Oli ei a e al. 2011, Lumini-Oli ei a, Magalhaes e al. 2011). As al eady men ioned, one o he consequences o he MPTP induc ion, besides he dis u bance o cell and mi ochond ial Ca2+ homeos asis, is he elease o cy och ome c and some o he s p o-apop o ic p o eins, wi h consequen ini ia ion o apop o ic cascades. Also, he inc eased oxida i e s ess induced by DOX, is able o in e e e wi h mi ochond ial unc ionali y, which leads o apop osis. Indeed, DOX-induced ca diomyocy es apop osis has been sugges ed o occu bo h acu ely and ch onically (Ca alho, San os e al. 2009). As p e iously men ioned, i s impo an o de elop a s a egy ha esul s in a ca diop o ec i e pheno ype agains DOX-induced impai men s. In his ega d, physical exe cise in i s a ious o ms has been shown o be an e ec i e 31 in e en ion ha can coun e ac he acu e and ch onic dele e ious insul s o he myoca dium, which includes DOX ea men . The ollowing sec ions will b ie ly discuss his issue. 2.4. Exe cise as a he apeu ic and p e en i e s a egy agains DOX- induced ca dio oxici y. Exe cise has been conside ed he mos e ec i e s a egy o p omo e a heal hy li es yle and i s bene i s agains DOX- ela ed impai men s a e e iden (Ascensao, Lumini-Oli ei a e al. 2011, Ascensao, Magalhaes e al. 2005, Ascensao, Oli ei a e al. 2012, Chicco, Schneide e al. 2005, Em e and Bowles 2008, Haywa d, Lien e al. 2012, Ka azis, Smude e al. 2010, Powe s, Lennon e al. 2002, Wonde s, Hydock e al. 2008). Al hough he mos s udied o m o exe cise in DOX ea ed animals is he ch onic exe cise, acu e exe cise also seems o p o ide bene icial e ec s. Mi ochond ial adap a ions may play c i ical ole in exe cise-induced p o ec ion agains DOX-induced ca diac impai men s (Ascensao, Fe ei a e al. 2007, Ascensao, Oli ei a e al. 2012). The e ec s o exe cise on DOX- ea ed mi ochond ia a e highligh ed in able 2. 2.4.1 Acu e exe cise Acu e exe cise b oadly e e s o a single bou o exe cise pe o med only once and i was al eady p o ed o be e ec i e agains DOX-induced impai men s. In ac , Wonde s e al. (2008) epo ed ha an acu e bou o eadmill unning pe o med 24 hou s p io o DOX injec ion a enua ed he hemodynamic impai men obse ed a e acu e DOX adminis a ion and educed le en icula lipid pe oxida ion. In addi ion, Ascensao e al. (2011) showed ha an acu e bou o eadmill exe cise p o ec s agains ca diac mi ochond ial dys unc ion, p ese ing mi ochond ial phospho yla ion capaci y and a enua ing DOX-induced dec eased ole ance o MPTP induc ion. In he same s udy, i was 32 obse ed ha acu e exe cise p e en ed he dec eased ca diac mi ochond ial unc ion de ec ed as impai ed s a e 3, phospho yla i e lag-phase and maximal ansmemb ane po en ial. Also, acu e exe cise p e en ed he inhibi o y e ec s o DOX ea men on he ac i i y o ca diac mi ochond ial espi a o y chain complexes I and V, and on inc eased caspase-3 and -9 ac i i ies. Fu he mo e, i has also been desc ibed ha acu e exe cise may con ibu e o diminished ee adical p oduc ion ( o e s see Ascensao, Oli ei a e al. 2012). Howe e , u he esea ch is needed o cla i y he exac mechanisms by which an acu e exe cise induces a p o ec i e pheno ype in DOX- ea ed ca diac mi ochond ia. 2.4.2 Ch onic exe cise Unlike acu e exe cise, ch onic exe cise has been ha dly s udied and he esul s consis en ly demons a e ha is able o an agonize DOX-induced ca diac impai men s (Ascensao, Magalhaes e al. 2005, Chicco, Schneide e al. 2005, Haywa d, Lien e al. 2012, Hydock, Lien e al. 2008, Ka azis, Smude e al. 2010). Those al e a ions induced by ch onic exe cise can be seen a mo phological, unc ional and biochemical le els and he ole o mi ochond ia is pi o al in his p ocess (Ascensao, Oli ei a e al. 2012, Ka azis, Smude e al. 2010). Ascensao e al. (2005) epo ed he in ol emen o mi ochond ia in ca diop o ec ion a o ded by endu ance aining agains DOX ea men , demons a ing p e en ion o acu e DOX-induced mi ochond ial al e a ions ega ding oxida i e s ess, espi a ion, and Ca2+ loading capaci y. A ul as uc u al le el, i has been epo ed ha ca diac al e a ions induced by DOX ea men a e also a enua ed by p e ious ch onic exe cise (Ascensao, Magalhaes e al. 2005). In ac , when compa ed wi h saline (SAL) g oup, seden a y (SED) DOX- ea ed animals showed myoca dial damage (Ascensão, Magalhães e al. 2006). The obse ed mo phological al e a ions consis ed o mi ochond ial damage wi h ex ensi e degene a ion and loss o c is ae, swelling 33 and abno mal size and shape, in ami ochond ial acuoles and no o ious myelin igu es ha p obably esul ed in he o ma ion o seconda y lysosomes. All o hese al e a ions we e a enua ed in ained animals ea ed wi h DOX (Ascensão, Magalhães e al. 2006). A unc ional le el, di e en au ho s epo ed he p o ec i e e ec s o ch onic exe cise. In ac , Hydock e al. (2008) sugges ed ha exe cise aining in a s be o e DOX ea men a enua ed DOX-induced ca diac dys unc ion, h ough he main enance o ac ional sho ening, de eloped p essu e and con ac ili y. Also, Chicco e al. (2005) epo ed ha bo h low in ensi y exe cise aining and an endu ance aining p o ocol wi h g adually inc eased in ensi y a enua ed he ad e se e ec s o DOX by p e en ing DOX-induced decline in ca diac unc ion h ough main enance o le en icula dias olic p essu e, a e o le en icula p essu e de elopmen and a e o le en icula elaxa ion. A biochemical le el, as depic ed on able 2, se e al au ho s had p o ed he bene icial e ec s o ch onic exe cise. Impo an ly, some o he mos s udied biochemical pa ame e s associa ed wi h exe cise and DOX a e he an ioxidan capaci y, oxida i e s ess ma ke s and apop o ic suscep ibili y ( o e s see Ascensao, Oli ei a e al. 2012). As p e ious e e ed, HSP ha e an impo an ole as an ioxidan s molecules con ibu ing o no mal cellula in eg i y and a e o e exp essed a e endu ance aining. Howe e , Chicco e al (2006) and Ka asis e al. (2010) also showed ha exe cise had no in luence on HSP o ha i is no de e minan on ca diop o ec ion being ha exe cise in cold s. no mal empe a u es may also display o he ypes o di e ences ega ding al e a ion o mi ochond ial physiology, besides al e a ion in he exp ession o HSPs. Fu he mo e, he up egula ion o mi ochond ial manganese supe oxide dismu ase (MnSOD) seems con ibu e o ca diop o ec ion. In ac , as mi ochond ial DOX oxici y has been la gely a ibu ed o inc eased oxida i e s ess, inc eased an ioxidan ac i i y may be impo an o explain how endu ance aining coun e ac s some o DOX-induced myoca dial damage. Chicco e al. (2006) associa ed low-in ensi y eadmill exe cise aining-induced ca diop o ec ion o he inhibi ion o apop o ic signaling and he inc eased ac i i y 34 o GPX. Also, he e ec o aining on p e en ing ac i a ion o ca diac apop o ic pa hways has been desc ibed being ha , aining dec eased he suscep ibili y o appea ance o apop o ic ma ke s in he hea s o DOX- ea ed animals, as inc eased mi ochond ial Bax, Bax- o-Bcl2 a io and issue caspase 3 ac i i y (Ascensao, Magalhaes e al. 2005). In ac , i has been sugges ed ha ch onic exe cise s imula ion may also a o d p o ec ion agains he inc eased suscep ibili y o he MPTP as he dele e ious e ec s o Ca2+ on hea mi ochond ial espi a ion o DOX- ea ed animals we e a enua ed in ained g oup ea ed wi h DOX (Ascensao, Magalhaes e al. 2005). As p e ious desc ibed, MPTP is ela ed o oxida i e damage and he e o e, i is possible ha inc eased esis ance o ca diac mi ochond ia om ained animals o he MPTP can be ela ed o inc eased an ioxidan de enses. Acco dingly, he highe le els o educed sul hyd yl g oups in ained mi ochond ia han in seden a y g oups may be indica i e o enhanced an ioxidan capaci y and/o o mo e ele a ed sul hyd yl-dono s, such as GSH, in mi ochond ia om ained animals ( o e s see Ascensao, Oli ei a e al. 2012). Howe e , u he s udies a e necessa y in o de o be e unde s and his issue. Table 2. Summa y o some desc ibed mi ochond ial- ela ed al e a ions associa ed wi h DOX-induced ca dio oxici y and he modula ion e ec a o ded by physical exe cise agains DOX (adap ed om Ascensao, Oli ei a e al. 2012). DOX e ec Exe cise e ec agains DOX ROS p oduc ion ↑ ↓ Oxida i e damage ma ke s Lipid pe oxida ion ↑ ↓ P o ein oxida ion ↑ ↓ DNA oxida ion ↑ Aconi ase ac i i y ↓ o = ↑ Apop o ic signaling 35 (↑) – inc ease; (↓) – dec ease; (=) – no al e a ions Despi e he ex ensi e numbe o s udies on his opic, he e ec s o bo h endu ance eadmill aining and olun a y ee-wheel unning ac i i y pe o med Bax-Bcl-2 a io ↑ ↓ Cy och ome c elease ↑ Caspase 9 ac i a ion ↑ ↓ Respi a o y endpoin s S a e 3 ↓ ↑ o = S a e 4 ↑ o = o ↓ = o ↓ RCR ↓ = o ↑ ADP/O a io = o ↓ Uncoupled espi a ion ↓ ↑ C ea ine-s imula ed espi a ion ↓ Maximal ΔΨ ↓ o = ↑ Ca2+-induced MTPT ↑ ↓ ANT con en and unc ioning ↓ Mi ochond ial chape ones ↑ ↑ Mi ochond ial an ioxidan s Thiols ↓ ↑ Vi amin E ↓ o = Enzymes = o ↑ ↑ Coenzyme Q isoenzymes = ETC complex ac i i y Complex I ↓ ↑ Complex II ↓ Complex III = Complex IV = o ↓ Complex V ↓ ↑ 36 be o e and du ing sub-ch onic DOX ea men schedule on ca diac mi ochond ial bioene ge ics a e ye o be elucida ed. This is o pa icula impo ance in he con ex o exe cise-induced p o ec ion agains DOX- ela ed ca diac mi ochond iopa hy, as cance pa ien s unde going DOX ea men may be ad ised o exe cise o many easons including o coun e ac physical a igue and o imp o e pe o mance, and also o mi iga e ca diac damage as a esul o chemo he apy. This mas e spo s science cou se in which his wo k is inse ed is he con ex o physical ac i i y and elde ly. Despi e de eloped wi h adul a s, he p esen wo k can con ibu e o ex end he knowledge in his pa icula a ea ep esen ing p elimina y indings in adul popula ion and conside ing ha DOX-based chemo he apeu ic ea men s agains se e al ypes o malignances a e mo e p e alen wi h inc easing age. 37 3. Aim The aim o he p esen s udy was o analyze he e ec o wo ypes o physical exe cise ( eadmill endu ance aining (TM) and ee-wheel olun a y physical ac i i y (FW)) agains hea mi ochond ial dys unc ion induced by sub-ch onic ea men o DOX. We can de ine as speci ic pu poses o his wo k he analysis o he adap a ions induced by bo h ypes o exe cise on hea mi ochond ia om DOX ea ed animals on: • Mi ochond ial espi a o y unc ion; • Mi ochond ial elec ical ansmemb ane po en ial; • MPTP suscep ibili y; • Mi ochond ial oxida i e damage. 44 4.9 Mi ochond ial osmo ic swelling du ing MPTP induc ion Mi ochond ial osmo ic olume changes we e ollowed by moni o ing he classic dec ease o abso bance a 540 nm wi h a Jasco V-630 spec opho ome e . Swelling ampli ude and a e o dec eased abso bance upon Ca2+ addi ion we e conside ed as MPTP suscep ibili y indexes. The eac ion was con inuously s i ed and he empe a u e was main ained a 25 ºC. The assays we e pe o med in 1 ml o eac ion medium con aining 200 mM suc ose, 10 mM HEPES, 5 mM KH2PO4, 10 µM EGTA , pH 7.4, supplemen ed wi h 1.5 µM o enone, 8 mM succina e and a single pulse o 80 nmol o Ca2+ wi h 0.5 mg/ml p o ein. Con ol ials we e pe o med by using 1 µM o cyclospo in-A, he selec i e MPTP inhibi o (B oekemeie , Dempsey e al. 1989). 4.10 Mi ochond ial oxida i e damage Be o e analysis, mi ochond ial memb anes we e dis up ed by se e al eeze– hawing cycles o allow ee access o subs a es. The ex en o lipid pe oxida ion in hea mi ochond ia was de e mined by measu ing MDA con en s by colo ime ic assay, acco ding o a modi ied p ocedu e desc ibed p e iously (Buege and Aus 1978). Suspended mi ochond ia we e cen i uged a 12,000 xg o 10 min and e-suspended in 150 µL o a medium con aining 175 mM KCl and 10 mM T is-HCl, pH 7.4. Subsequen ly, mi ochond ia om he six g oups we e mixed wi h 2 olumes o ichlo oace ic acid (10%) and 2 olumes o hioba bi u ic acid (1%). The mix u es we e hea ed a 80–90 ºC o 10 min and e-cooled in ice o 10 min be o e cen i uga ion (4,000 xg o 10 min). The supe na an s we e collec ed and he abso bance measu ed a 535 nm. The amoun o MDA con en o med was exp essed as nanomoles o MDA pe millig am o p o ein (ε535=1.56 x 10−5 M−1 cm−1) The basal mi ochond ial con en o oxida i e modi ied -SH g oups, including GSH and o he -SH con aining p o eins, was quan i ied by spec opho ome ic measu emen acco ding o Hu (1990). B ie ly, a mi ochond ial suspension 45 con aining 5 mg/mL p o ein was mixed wi h 0.25 M T is bu e pH 8.2 and 10 mM DTNB and he olume was adjus ed o 1 mL wi h absolu e me hanol. Subsequen ly, he samples we e incuba ed o 30 min in he da k a oom empe a u e and cen i uged a 3000 xg o 10 min. The colo ime ic assay o supe na an was pe o med a 414 nm agains a blank es . To al -SH con en was exp essed in nanomoles pe millig ams o mi ochond ial p o ein (ε414=13.6 mM−1 cm−1). 4.11 Soleus ci a e syn hase ac i i y Soleus CS ac i i y was measu ed using he me hod p oposed by Coo e e al. (1971). The p inciple o assay was o ini ia e he eac ion o ace yl-CoA wi h oxaloace a e and link he elease o CoA-SH o 5,5-di hiobis (2-ni obenzoa e) a 412 nm. 4.12 S a is ical analysis All da a a e exp essed as he mean±SEM (S anda d E o o he Mean). S a is ical analyses we e pe o med using G aphPad P ism ( e sion 6.0) o S a is ical Package o he Social Sciences (SPSS e sion 21.0). Th ee-way epea ed-measu es ANOVA o body weigh and dis ance co e by exe cised g oups o e i y he e ec o exe cise and ea men o e ime. Two-way analysis o a iance ANOVA we e used o examine possible e ec o ea men and/o exe cise. To de e mine speci ic g oup di e ences, he wo-way ANOVA we e ollowed by Bon e oni pos -hoc es s. In all cases, he signi icance le el was se a p≤0.05. 47 5. Resul s 5.1. Cha ac e iza ion o animals and exe cise p o ocols Body weigh al e a ions and dis ances co e ed by he animals du ing he en i e p o ocol a e shown in igu e 3. No signi ican di e ences in he mean body weigh o he animals om he beginning o he p o ocol un il he 5 h week, when sub-ch onical DOX ea men was ini ia ed, we e ound. Body weigh s o DOX ea ed animals we e lowe han SAL coun e pa s a he end o he p o ocol (DOX+SED s. SAL+SED; DOX+TM s. SAL+TM; DOX+FW s. SAL+FW; p≤0.05). No di e ences in body weigh be ween exe cised g oups, we e ound. TM and FW dec eased body weigh a 12 h and 9 h week, espec i ely (SAL+TM and SAL+FW s. SAL+SED; p≤0.05). DOX ea men combined wi h TM dec eased body weigh om he 5 h week (DOX+TM s. DOX+SED; p≤0.05), whe eas no signi ican di e ences we e ound be ween FW and SED ea ed g oups (Figu e 3A). A e he 5 h week DOX ea ed g oups consumed less ood han hei SAL coun e pa s (da a non shown, p≤0.05). Wa e consump ion inc eased in FW g oups (SAL and DOX) compa ed wi h SAL+SED and DOX+SED g oups (da a non shown, p≤0.05). Figu e 3. E ec o exe cise and DOX ea men on (A) body mass o e ime and (B) dis ance co e ed pe day by TM and FW g oups du ing he 12 wks o p o ocol. Signi ican di e ences (p≤0.05) a e men ioned in he ex . Signi ican (p≤0.05) e ec s o Exe cise (E), T ea men (T), ime ( ) o hei in e ac ion (E x T x ) a e shown; Non Signi ican (NS, p>0.05). 48 As can be seen in Figu e 3B, olun a y unning dis ance dec eased signi ican ly in DOX+FW a e he 5 h week and emained lowe un il he end o he p o ocol (p≤0.05). Animals om TM g oup an a he same eloci y h oughou he 8 weeks o he p o ocol. Running eloci y and dis ance co e ed diminished in DOX+TM g oup a 11 h and 12 h week compa ed o SED+TM (p≤0.05). Body, hea absolu e weigh s, hea weigh and emu leng h o body weigh a ios, mi ochond ial p o ein yielding as well as he ac i i y o soleus ci a e syn hase in he six g oups a e shown in Table 4. Final body, hea weigh and a io o hea weigh o body weigh signi ican ly dec eased wi h DOX ea men (SAL+SED s DOX+SED). Bo h ch onic exe cise ypes dec eased inal body weigh , inc eased hea weigh and he hea o body a io (SAL+TM and SAL+FW s SAL+SED). DOX ea men combined wi h TM and FW exe cise induced a signi ican inc ease in hea weigh and hea weigh o body weigh a io compa ed wi h hei DOX+SED coun e pa s. No signi ican di e ences we e obse ed be ween g oups ega ding he Ini ial body weigh , emu leng h o body weigh a io and yield o mi ochond ia isola ion. TM induced a signi ican inc ease in he ac i i y o soleus ci a e syn hase in bo h SAL and DOX ea ed animals (SAL+SED s SAL+TM and DOX+SED s DOX+TM). 49 Table 4. Animal da a and yield o mi ochond ial p o ein isola ion SAL+SED SAL+TM SAL+FW DOX+SED DOX+TM DOX+FW P* Ini ial body weigh (g) 207±3.91a 214±3.76a 212±1.55a 209±4.68a 207±4.63a 209±2.60a NS Final body weigh (g) 598±10.58a 522±9.87b 498±5.98b 438±6.22c 426±10.79c 429±16.95c ExT Hea weigh (g) 1.44±0.03a 1.92±0.08b 1.85±0.10b 1.10±0.03c 1.45±0.05a 1.55±0.11a E, T Hea weigh /body weigh (mg.g−1) 2.32±0.08a 3.53±0.10bc 3.68±0.12c 2.62±0.11d 3.34±0.08b 3.34±0.12b ExT Femu leng h/body weigh (mm.g-1) 0.08±0.00a 0.08±0.00a 0.09±0.00a 0.08±0.01a 0.09±0.00a 0.09±0.00a E, T Mi ochond ial p o ein yielding (mg p o ein/g issue) 18.58±0.62a 15.15±1.22a 15.01±1.50a 16.96±0.83a 18.60±4.68a 17.84±1.57a NS soleus ci a e syn hase ac i i y (nmol. min-1.mg-1) 10.94±2.07a 23.34±1.79b 11.22±2.65a 8.69±1.15a 22.22±1.97b 10.27±1.32a E, T Values (mean ± SEM). Di e en le e s a e signi ican ly di e en (p≤0.05). * Signi ican (p≤0.05) e ec s o Exe cise (E), T ea men (T), o hei in e ac ion (E x T) a e shown; Non Signi ican (NS, p> 0.05). 50 5.2 Hea mi ochond ial oxygen consump ion Mi ochond ial espi a o y ac i i y in bo h SAL and DOX ea ed g oups was measu ed o iden i y exe cise-dependen e ec s (Figu e 4). DOX ea men dec eased hea mi ochond ial espi a ion du ing s a e 3 and inc eased s a e 4 in SED animals (DOX+SED s SAL+SED). Impo an ly, TM and FW exe cise pe se inc eased S a e 3 espi a ion in bo h SAL and DOX (SAL+TM and SAL+FW s. SAL+DOX; DOX+TM and DOX+FW s. DOX+SED). The coupling be ween oxygen consump ion and ADP phospho yla ion (RCR) was signi ican ly a ec ed by DOX ea men (DOX+SED s. SAL+SED). TM signi ican ly inc eased RCR in bo h SAL and DOX g oups (SAL+TM s. SAL+SED; DOX+TM s. DOX+SED). FW inc eased RCR in DOX ea ed animals (DOX+FW s. DOX+SED). Also, bo h TM and FW inc eased ADP/O in DOX g oup (DOX+SED s. DOX+TM and DOX+FW). 51 Figu e 4. E ec o exe cise and DOX ea men on (A) s a e 3 o hea mi ochond ial espi a ion, (B) s a e 4 o hea mi ochond ia espi a ion, (C) RCR and (D) ADP/O. Da a a e means±SEM o hea mi ochond ia (0.5 mg/mL p o ein) ob ained om di e en mi ochond ial p epa a ions o each expe imen al g oup. Oxida i e phospho yla ion was measu ed pola og aphically a 30ºC in a o al olume o 0.75 mL. Respi a ion medium and o he expe imen al de ails a e p o ided in me hods. RCR, espi a o y con ol a io (s a e 3/s a e 4); ADP/O, numbe o nmol ADP phospho yla ed by a om o oxygen consumed. Di e en le e s a e signi ican ly di e en (P≤0.05). Signi ican (p≤0.05) e ec s o Exe cise (E), T ea men (T), o hei in e ac ion (E x T) a e shown; Non Signi ican (NS, p>0.05). 5.3 Hea mi ochond ial ansmemb ane elec ic po en ial Hea mi ochond ial a ia ions in maximal ∆ψ and du ing ADP phospho yla ion we e de e mined using G/M as subs a es. DOX ea men signi ican ly a ec ed he maximal ∆ψ, epola iza ion and ADP lag-phase (Figu e 5). FW bu no TM, inc eased maximal ∆ψ and epola iza ion, whe eas bo h ypes o exe cise dec eased he ADP lag phase (SAL+TM and SAL+FW s. SAL+SED). Bo h exe cise p o ocols we e able o coun e ac he DOX ha m ul e ec no malizing maximal ∆ψ, epola iza ion and ADP lag phase. 52 Figu e 5. E ec o exe cise and DOX ea men on hea mi ochond ia ∆ψ luc ua ions (A) maximal ene giza ion, (B) ADP-induced depola iza ion, (C) epola iza ion and (D) ADP phospho yla ion lag phase. Da a a e mean±SEM o hea mi ochond ia (0.5 mg/mL p o ein) ob ained om di e en mi ochond ial p epa a ions o each expe imen al g oup. Figu e shows he a e age esponse o maximal mi ochond ial memb ane po en ial de eloped wi h glu ama e (5 mM) plus mala e (2.5 mM), he dec ease in memb ane po en ial a e ADP addi ion (depola iza ion), he epola iza ion alue a e ADP phospho yla ion, and he lag phase. Mi ochond ial ansmemb ane po en ial was measu ed using a TPP+-selec i e elec ode a 30ºC in a o al olume o 1 mL. Reac ion medium and o he expe imen al de ails a e p o ided in me hods. Di e en le e s a e signi ican ly di e en (p≤0.05). * Signi ican (p≤0.05) e ec s o Exe cise (E), T ea men (T), o hei in e ac ion (E x T) a e shown; Non Signi ican (NS, p>0.05) 5.4 Mi ochond ial osmo ic swelling du ing MPTP induc ion The e ec s o bo h ypes o exe cise aining and DOX ea men on in i o suscep ibili y o Ca2+-induced MPTP opening we e in es iga ed. The addi ion o Ca2+ on mi ochond ia suspension esul ed in a dec ease in abso bance wi h h ee dis inc phases. Ini ially, an inc ease in abso bance was obse ed, which mos likely esul s om he o ma ion o opaque Ca2+ c ys als inside mi ochond ia (And eye , Fahy e al. 1998). Upon MPTP opening, a dec ease o 53 abso bance wi h a slow ollowed by a as kine ic a e is usually obse ed in ca diac mi ochond ia. Incuba ion o mi ochond ial suspension wi h cyclospo ine A, a speci ic MPTP inhibi o (B oekemeie , Dempsey e al. 1989), limi s he abso bance dec ease a e Ca2+ addi ion, which demons a e he associa ion wi h MPTP opening. Figu e 6 shows di e en end-poin s measu ed om he eco dings ob ained, namely (A) swelling ampli ude ( he di e ence be ween he ini ial and he inal abso bance alue) and (B) he a e age swelling a e. The esul s demons a e ha DOX ea men signi ican ly inc eased suscep ibili y o Ca2+-induced MPTP opening (DOX + SED s. SAL + SED). Hea mi ochond ia isola ed om SAL+TM g oup, bu no SAL+FW we e less suscep ible o Ca2+-induced MPTP opening (SAL + TM s. SAL + SED). Bo h ypes o exe cise we e able o mi iga e DOX-induced inc eased suscep ibili y o MPTP opening (DOX + TM and DOX + FW s. DOX + SED). Figu e 6. E ec o exe cise and DOX ea men on hea mi ochond ia o Ca2+-induced MPTP (A) Swelling ampli ude; (B) A e age swelling a e. Da a a e mean ± SEM. The abso bance o mi ochond ial suspension was ollowed a 540 nm. Mi ochond ia we e incuba ed as desc ibed in me hods. A 80 nmol o Ca2+ pulse (160 nmol/mg p o ein) was added o 0.5 mg o mi ochond ial p o ein in o de o a ain he cyclospo in A- sensi i e swelling, indica ing ha he dec eased op ical densi y co esponding o he inc eased swelling was due o MPTP opening. Di e en le e s a e signi ican ly di e en (p≤0.05). * Signi ican (p≤0.05) e ec s o Exe cise (E), T ea men (T), o hei in e ac ion (E x T) a e shown; Non Signi ican (NS, p>0.05) 60 inc eased unc ionali y o he phospho yla i e sys em in gene al, and he ETC in pa icula , induced by bo h exe cise p o ocols may ha e some implica ions in Ca2+ up ake capaci y. Howe e , o be e unde s and his phenomenon, u he s udies need o be add essed. 6.3 Oxida i e s ess ma ke s P e ailing hypo heses sugges ha myoca dial oxida i e s ess is a p ima y e en in DOX-induced ca dio oxici y and i is belie ed o ini ia e se e al o he dele e ious cellula e en s epo ed ollowing DOX ea men (Zucchi and Danesi 2003). In ac , a p esen he p incipal mechanism o DOX-induced ca dio oxici y is belie ed o be inc eased mi ochond ial oxidan p oduc ion leading o p o ease ac i a ion and induc ion o apop osis (Ascensão, Magalhães e al. 2006, Ascensao, Magalhaes e al. 2005, Chicco, Hydock e al. 2006, Chicco, Schneide e al. 2005). Acco dingly, oxida i e inju y o a y acids a subcellula le el measu ed by inc eased le els o lipid pe oxida ion p oduc s has been equen ly epo ed ollowing DOX exposu e ( o e s see Chicco, Schneide e al. 2005). The p esen esul s show ha TM, bu no FW pe se was able o dec ease MDA le el and inc ease -SH g oups. In acco dance o p e ious epo s, DOX induced a signi ican dec ease in -SH, indica ing inc eased disul ide linkages om bo h p o eins and GSH. As polyunsa u a ed a y acids a e conside ed highly suscep ible o ROS a ack, he inc eased oxida i e s ess caused by DOX led o pe oxida i e modi ica ion o lipid memb anes a ec ing memb ane in eg i y and pe meabili y, which leads o decoupled mi ochond ia, al e ing no mal mi ochond ial espi a o y unc ion. Myoca dial an ioxidan enzymes de end he hea agains he damaging e ec s o ROS and ha e been hypo hesized o play an impo an ole in exe cise- induced esis ance o oxida i e s ess (Powe s, Lennon e al. 2002) and in he a enua ion o DOX ca dio oxici y (Singal, Ilisko ic e al. 1997). In pa icula , 61 some s udies sugges ed ha he p esence o myoca dial SOD migh be impo an o he p e en ion o DOX ca dio oxici y (Ascensao, Lumini-Oli ei a e al. 2011, Sa azyan, Aska i e al. 1995, Yen, Obe ley e al. 1996). This is easonable, as SOD dismu a es supe oxide in o H2O2, he eby p o iding he i s line o de ense agains DOX-induced oxida i e s ess. Fu he mo e, inc easing e idence sugges ha myoca dial HSP72 induc ion plays a pi o al ole in exe cise-induced ca diop o ec ion agains oxida i e s ess (Powe s, Lennon e al. 2002, Powe s, Locke e al. 2001, Taylo and S a nes 2003). 6.4 Meaning o exe cise-induced ca diop o ec ion in aging Conside ing he p esen esul s in he con ex o exe cise-induced ca diop o ec ion in ad anced age, hey can be in e p e ed as p elimina y. Indeed, i can be ca e ully specula ed ha he obse ed p o ec i e pheno ype caused by bo h ch onic models o exe cise agains DOX can also be obse ed in aged a s. In ac , Quind y e al. (2005) epo ed ha aged a s submi ed o exe cise aining amelio a e ca diac hemodynamic esponse wi h signi ican imp o emen s in he apop o ic le els and signaling caused by IR inju y. Fu he mo e, he au ho s obse ed ha ained old a s inc eased MnSOD ac i i y, which can be in e p e ed as a sign o ca diac mi ochond ial adap a ions induced by ch onic exe cise in old a s compa ed o hei young coun e pa s. Simila esul s we e ound by S a nes e al. (2003), which sugges ha , al hough obse ing ca diop o ec i e p o ein pheno ype al e a ions wi h age, exe cise can enhance ca diop o ec ion ega dless o elde ly. Fu he mo e, physical exe cise has he abili y o posi i ely modula e some gene exp ession associa ed wi h imp o ed hea unc ion in aged a s. In ac , hea is known o i s abili y o p oduce ene gy om a y acids because o i s impo an β-oxida ion equipmen , which capaci y is educed wi h age (S a nes, Beye e al. 1983). Con i ming he po en ial bene icial e ec s o physical exe cise on ca diac me abolism in elde ly, Iemi su e al. (2002) epo ed ha exe cise aining imp o ed he aging-induced dec eased exp ession o pe oxisome p oli e a o -ac i a ed ecep o , which egula es genes ela ed o a y acid me abolism in he hea . Gi ing hose 62 al e a ions epo ed in aged hea s, i is possible o specula e ha he esul s o he p esen wo k could also be obse ed in aged a s; a o ding p o ec ion and mi iga ing he dele e ious consequences associa ed wi h sub-ch onic DOX ea men schedules. 63 7. Conclusion In summa y, he da a om he p esen wo k p o ide addi ional suppo abou he e ec o wo ypes o physical exe cise ( eadmill endu ance aining and ee-wheel olun a y physical ac i i y) agains hea mi ochond ial dys unc ion induced by sub-ch onic ea men o Doxo ubicin (DOX). Ou esul s showed us ha : • Rega ding mi ochond ial espi a o y unc ion bo h ypes o exe cise e e ed he e ec s induced by DOX on s a e 3, RCR and ADP/O. In e es ingly, ee wheel olun a y physical ac i i y was mo e e icien a coun e ac ing DOX-induced de ec s on RCR; • Bo h ypes o exe cise we e able o coun e ac DOX-induced impai men s in mi ochond ial ansmemb ane endpoin s. Impo an ly, ee wheel olun a y physical ac i i y was also mo e e icien a no malizing DOX-induced inc eases in lag phase; • Rega ding mi ochond ial osmo ic swelling du ing MPTP induc ion, bo h exe cise p o ocols e e ed DOX-induced impai men s. In ac , bo h ypes o exe cise mi iga ed DOX-induced inc eases in swelling ampli ude and a e age swelling a e. Howe e , once again ee wheel olun a y physical ac i i y was mo e e icien a coun e ac ing Ca2+-induced MPTP induc ion • Exe cise p o ocols we e able o e e DOX-induced inc eases in MDA con en and dec ease in sul hyd yl g oups. 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