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

Effects of Voluntary Physical Activity and Endurance Training on Cardiac Mitochondrial Function of Rats Sub-Chronically Treated with Doxorubicin Dissertation submitted to the Faculty of Sports, University of Porto to obtain the 2nd cycle in Physical Activity for Elderly, under the decree-law no. 74/2006 of 24 March Supervisors. Professor Doutor António Ascensão Professor Doutor José Magalhães Diogo Nuno Mariani Felix Porto, June 2013 Mariani, D. (2013). Effects of Voluntary Physical Activity and Endurance Training on Cardiac Mitochondrial Function of Rats Sub-Chronically Treated with Doxorubicin. Porto: D. Mariani. Master thesis presented to the Faculty of Sport, University of Porto. KEY-WORDS: EXERCISE; HEART; BIOENERGETICS; MITOCHONDRIAL FUNCTION; DOXORUBICIN. FUNDING The present work was supported by a research grant from the FCT (PTDC/DTP/DES/1071/2012 – FCOMP-01-0124-FEDER-028617) and from IJUP (PP_IJUP2011 253) to António Ascensão; from Research Centre In Physical Activity, Health And Leisure (CIAFEL) I&D UNIT (PESTOE/SAU/UI0617/2011). DEDICATÓRIA Ao papá, à mamã, ao Gonçalo e à Ely. AGRADECIMENTOS Agora que finalizo o mestrado, gostaria de expressar os meus profundos e sinceros agradecimentos: Aos Professores José Magalhães e António Ascensão, pela disponibilidade sempre demonstrada na orientação deste estudo, pelos sábios ensinamentos que me transmitiram, pelo incentivo, por estarem sempre presentes, pela paciência, amabilidade e pelos momentos de carinho e amizade sempre demonstrados. Ao Centro de Investigação em Atividade Física, Saúde e Lazer (CIAFEL) por todo o apoio e colaboração prestados na realização deste estudo. A todo o grupo de trabalho em especial à Inês, à Estela e à Mané, que me acompanharam em todos os momentos do protocolo experimental deste estudo, pela disponibilidade, pelo incentivo e pela amizade. Inês, obrigada por todas as explicações, pela paciência e por estares sempre disponível! E a todas as pessoas e amigos que direta ou indiretamente contribuíram para a concretização deste trabalho. ix Table of Contents! 1.!Introduction .................................................................................................................... 1! 2. State of art ........................................................................................................................ 3! 2.1Age effects on cardiac function ................................................................................. 3! 2.1.1 Aging effects on cardiac mitochondria function ....................................................... 8! 2.2. Exercise and cardioprotection ................................................................................. 16! 2.2.1 Exercise and cardiac mitochondrial adaptations ................................................... 18! 2.2.1.1. Morphological and biochemical adaptations ...................................................... 18! 2.2.1.2. Mitochondrial biogenesis ................................................................................... 20! 2.2.1.3. Oxidative stress and antioxidant capacity ......................................................... 21! 2.2.1.4. Cell death pathways .......................................................................................... 24! 2.3. Doxorubicin: therapeutic agent vs. cardiotoxicity .................................................... 25! 2.3.1 Cardiac mitochondrial toxicity induced by DOX ..................................................... 27! 2.3.1.1 Morphological evidences .................................................................................... 28! 2.3.1.2 Increased oxidative stress .................................................................................. 29! 2.3.1.3 Increased susceptibility to apoptosis .................................................................. 30! 2.4. Exercise as a therapeutic and preventive strategy against DOX-induced cardiotoxicity. .................................................................................................................. 31! 2.4.1 Acute exercise ....................................................................................................... 31! 2.4.2 Chronic exercise .................................................................................................... 32! 3. Aim ................................................................................................................................. 37! 4. Materials and methods ................................................................................................... 39! 4.1 Reagents .................................................................................................................. 39! 4.2 Animals ..................................................................................................................... 39! 4.3 Exercise protocols .................................................................................................... 40! 4.3.1 Endurance training protocol ................................................................................... 40! xvii Abbreviations and Symbols VO2max Maximal Oxygen Uptake A.M. Before Midday AIF Apoptosis Inducing Factor AMPK Adenosine Monophosphate-Activated Protein Kinase ANT Adenosine Nucleotide Translocase ATP Adenosine Triphosphate Ca2+ Calcium Ion CAT Catalase CHF Congestive Heart Failure CS Citrate Synthase Cyc D Cyclophilin D DNA Deoxyribonucleic Acid DOX Doxorubicin DTNB Beta Dystrobrevin E Exercise ER Endoplasmic Reticulum FW Free Wheel G Glutamate GPX Glutathione Peroxidase GSH Glutathione h Hours H2O2 Hydrogen Peroxide HSP Heat Shock Proteins IFM Intermyofibrillar Mitochondria IR Ischemia Reperfusion KCl Potassium Chloride Kg Kilogram KH2PO4 Monopotassium Phosphate m Metro M Malate xviii MDA Malondialdehyde MFN Mitofusin mg Milligram min Minute ml Milliliter mM Millimolar MnSOD Manganese Superoxide Dismutase MPTP Mitocohndrial Permeability Transition Pore mtDNA Mitochondrial Deoxyribonucleic Acid MΩ Megaohm NaCl Sodium Chloride NADH Reduced Nicotinamide Adenine Dinucleotide NADPH Reduced Nicotinamide Adenine Dinucleotide Phosphate nmol Nanomol NO Nitric Oxide NS Non-Significant O2 Oxygen O2Superoxide Radical ºC Degree Celsius OH. Hydroxyl Radical PGC Proliferator-Activated Receptor Gamma RCR Respiratory Control Ratio RNA Ribonucleic Acid ROS Reactive Oxygen Species SAL Saline SEM Standard Error Of The Mean -SH Sulfhydryl groups SOD Superoxide Dismutase SSM Subsarcolemmal Mitochondria T Treatment t Time TM Treadmill TNF Tumor Necrosis Factor xix TPP+ Tetraphenylphosphonium VDAC Voltage Dependent Anion Channel (A-VO2)diff Arteriovenous oxygen difference Δᴪ Transmembrane Electrical Potential 1 1. Introduction Doxorubicin (DOX, or adriamycin) is a highly effective antibiotic used to treat several types of malignancies. Unfortunately, the clinical use of DOX is limited by the occurrence of a dose-related cardiac toxicity that results in lifethreatening cardiomyopathy. DOX-induced cardiomyocyte dysfunction is associated with increased levels of oxidative damage involving mitochondrial bioenergetics collapse in the process (Wallace 2007). Actually, sub-chronic DOX treated rats reveal defects on heart mitochondrial function, which are accompanied by compromised mitochondrial electron transport chain activity and increased oxidative stress and damage (Berthiaume, Oliveira et al. 2005, Santos, Moreno et al. 2002). Among the strategies advised to counteract the cardiac side effects associated with DOX treatment, physical exercise has been studied and recommended as a non-pharmacological tool against myocardial injury (Ascensao, Ferreira et al. 2007, Ascensao, Lumini-Oliveira et al. 2011, Ascensao, Oliveira et al. 2012). Previous work has suggested that the advantage of both acute (Ascensao, Lumini-Oliveira et al. 2010, Wonders, Hydock et al. 2008) and chronic exercise models (Ascensao, Ferreira et al. 2006, Ascensao, Magalhaes et al. 2005, Ascensao, Magalhaes et al. 2005, Chicco, Hydock et al. 2006, Chicco, Schneider et al. 2005, 2006) on the preconditioning of DOX-treated rats include the protection of cardiac tissue and mitochondria against induced impairments. Moreover, recent studies investigating the effects of exercise performed during and following models of late-onset cardiotoxicity caused by DOX provide evidence of exercise-induced cardioprotection in both adult and juvenile rat models (Hayward, Lien et al. 2012, Hydock, Lien et al. 2012). However, the cellular and molecular mechanisms underlying this protective phenotype induced by exercise are still elusive. In particular, whether perturbations in heart mitochondrial oxidative phosphorylation capacity and pro-oxidant redox modifications associated with cumulative DOX administration are modulated by long-term physical exercise performed during and after treatments is yet unknown. 2 We therefore aimed to analyze the effects of two types of long-term exercise with distinct characteristics, performed before and during the overall DOX treatment, on cardiac mitochondrial bioenergetics. Heart mitochondrial respiratory parameters associated with oxygen consumption, transmembrane electrical potential and osmotic swelling during mitochondrial permeability transition pore (MPTP) induction as well as markers of oxidative stress (sulfhydryl groups (-SH) and malondialdehyde (MDA) contents) were determined. 3 2. State of art 2.1Age effects on cardiac function Aging can be characterized as a time dependent decline of maximal functionality that affects tissues and organs of the whole body (Figueiredo, Mota et al. 2008). The average human life span has markedly increased in modern society, a fact largely attributed to advances in medical and therapeutic sciences that have successfully reduced the severity of several diseased conditions (Chaudhary, El-Sikhry et al. 2011). However, elderly individuals continue to suffer the greatest burden from cardiovascular disease, including coronary heart disease that remains the leading cause of death in industrialized countries (Wei 2004). That can be understood as the result of several morphophysiological, structural and functional alterations, which we will further address in detail (Table 1). The aging-induced increase on vascular stiffness, septum and myocardium thickness and fibrosis, is associated, among others, with: i) decreased myocyte number; ii) increasing cardiomyocyte size with alterations in calcium (Ca2+) homeostasis and iii) increased collagen fibers deposition, leading to cardiac diastolic dysfunction, increased afterload, and loss of arterial and heart compliance (Strait and Lakatta 2012). Importantly, these physiological-related impairments along with unchanged cavity size and increased left ventricle hypertrophy are the major characteristic of aging heart (Chaudhary, El-Sikhry et al. 2011). In fact, Dai et al (2009) reported that aging left ventricle mass index increased by around 75% compared to a young adult group, indicating the increase prevalence of left ventricular pathological hypertrophy with age. It was also reported reduction in diastolic function, as well as worsening of myocardial performance index (Dai, Santana et al. 2009). Furthermore, the conduction system also undergo some structural alterations leading to heart dysfunction, including fat accumulation around sinoatrial node, which creates a partial or complete separation of the node from atrial tissue (Strait and Lakatta 2012), a marked decreased in peacemakers number cells 4 (Wei 2004) and an increased calcification in conduction system leading ultimately to atrioventricular conduction lock. Slow and prolonged systole and diastole are other aging-related heart features usually related to an aberrant Ca2+ handling (Strait and Lakatta 2012). The decreased diastolic function, which falls linearly with aging at a rate of about 6–7% per decade (Gates, Tanaka et al. 2003), is associated to a decrease in diastolic filling reported at rest and under stress conditions, as some daily tasks that promote an acute increase of physical demands. In addition, the degeneration of cardiac valvular apparatus is commonly reported, being valvular annular dilation found in the majority of older persons and is also associated with concomitant coronary artery calcification (Wei 2004). Table 1. Effects of aging on cardiovascular system Morphophysiological changes Structural Changes Functional Changes Reference Heart and Vascular system ↓ Cardiomyocytes number ↑ Cardiomyocytes size ↑ Collagen deposition ↑ Fibers deposition ↓ Cardiomyocytes function ↑ Septum and Myocardium stiffness ↓ Heart compliance ↑ Vascular thickness ↓ Vascular compliance ↑ Systolic pressure ↓ Diastolic function ↓ max heart rate ↓ max cardiac output ↑ Afterload Prolonged systole and diastole Hotta and Uchida (2010), Reynolds (2004), Strait and Lakatta (2012), T. Shioi (2012), Taylor, Cable et al. (2004) Conduction system ↑ Fibrosis and fat accumulation (SA node) ↓ Peacemakers cells number ↑ Conduction system calcification ↓ Ventricular compliance ↑ Heart rhythm disturbances ↑ Risk atrioventricular conduction lock ↑ Risk atrial arrhythmias, ↑ Risk atrial fibrillation ↑ Risk tachycardia ↑ Ventricular arrhythmias Hotta and Uchida (2010), Strait and Lakatta (2012) Valvular system ↑ Valvular annular dilatation Coronary artery calcification Reynolds (2004) (↑) – increase; (↓) – decrease ↓ VO2 max 5 Overall, these aging related heart morphophysiological, structural and functional alterations result in increased systolic pressure, one major risk factors for development of atherosclerosis, hypertension and stroke, and arterial fibrillation (North and Sinclair 2012). As a consequence of all those age-associated alterations, cardiac function declines maximal functionality, being that, under basal conditions this decline may not be detected (Wessells and Bodmer 2007). The most used standard index of cardiorespiratory fitness is the maximal oxygen consumption (VO2max), which declines approximately 10% per decade starting at 20 to 30 years old (Souza 2012). VO2max can be estimated by the Fick’s equation: Equation 1: VO2max=Q×(A-VO2)diff Q is cardiac output, and is the product of max heart rate and stroke volume, (AVO2)diff is the arteriovenous difference of oxygen. As it is observed in figure 1, based on Fick´s equation, aerobic exercise capacity depends on cardiac output and arteriovenous difference (Seeley, Stephens et al. 2005). Reductions in peak heart rate, peripheral oxygen utilization and stroke volume appear to mediate the age-associated decline in VO2max. Actually, impairments in cardiac filling and increased afterload leads to a decrease in heart rate, in a stress situation (Taylor, Cable et al. 2004) being the major responsible for much of the age-associated decrease in maximal cardiac output. Reductions in muscle oxygen delivery and therefore (A-VO2)diff are mainly due to reduced and maldistribution of cardiac output. Also, a decline in skeletal muscle oxidative capacity with aging is reported, due in part to mitochondrial dysfunction, which appears to play a particularly important role in old age, where skeletal muscle VO2max is observed to decline by approximately 50% even under conditions of similar oxygen delivery as young adult muscle (Betik and Hepple 2008). 12 Besides nucleus, mitochondria are the only organelles in animal cells that possess their own DNA, the mtDNA, as well as related transcriptional and translational synthesis machinery. mtDNA is localized in matrix with physical proximity to the mitochondrial respiratory chain (Bratic and Trifunovic 2010). However, mitochondria cannot be synthesized de novo, instead they replicate in the cytosolic compartment through a process of division (for refs see Chaudhary, El-Sikhry et al. 2011). Importantly, the proteins that are encoded by mtDNA are vital for normal mitochondrial function and mtDNA does not have the protein protection as nuclear DNA and has less effective repair mechanisms (Desler, Marcker et al. 2011). Therefore, mutations in mtDNA through oxidative stress affect the expression and integrity of oxidative phosphorylation complexes and can cause mitochondrial dysfunction and increased ROS production (Wallace 2010). So, it is notable the interrelationship between oxidative phosphorylation complexes, ROS levels, mtDNA mutations and ultimately cell death. Furthermore, mutations in mtDNA and the resultant decline in mitochondrial activity observed in aged tissues are responsible for the increased generation of ROS, which in turn, will further negatively impact mitochondria causing further mtDNA damage. This “vicious cycle” concept postulated that accumulation of mtDNA mutations is exponential and associated with massive increase in ROS production (Lenaz 1998). Being mitochondria strategic organelles essential for cell function and homeostasis, providing energy to the cell (Chaudhary, El-Sikhry et al. 2011), they must undergo some dynamic mechanisms. Mitochondrial turnover and death can occur via several processes that are suggested to be interrelated namely apoptosis, necrosis, autophagy and related dynamics of organelles. In this dynamic network biogenesis, fusion and fission are closely associated mechanisms (Chen and Knowlton 2011). In fact, apoptosis is mediated by two pathways: the extrinsic and the intrinsic pathways, and both have been described in cardiac myocytes (Whelan, Kaplinskiy et al. 2010). The extrinsic apoptotic pathway can be triggered by Fas ligand or tumor necrosis factor (TNF)-α, which are expressed in cardiac 13 myocytes and have been implicated in cardiovascular pathology (Whelan, Kaplinskiy et al. 2010). Intrinsic pathways involve the participation of endoplasmic reticulum (ER) and/or mitochondria (Kroemer, Galluzzi et al. 2007). Impairment in mitochondrial integrity, dynamics or metabolic activity may result in a range of deleterious effects to the cell, such as reduced ATP production, elevated cytosolic Ca2+, increased ROS release, release of proapoptotic factors as cytocrome c or caspases activation and Bax translocation, triggering cell death (Chen and Knowlton 2011). In mammalian cells, apoptosis is regulated by a variety of factors that are essentially either pro-life or pro-death (Goldspink, Burniston et al. 2003). Quantitation of the expression of genes involved in the apoptotic pathway might represent a good index of the probability for a cell to undergo apoptosis. As the number of Bax-expressing cells dramatically increases in left ventricular hypertrophy and left ventricular dysfunction (Condorelli, Morisco et al. 1999, Green and Reed 1998), it is suggested that mitochondrion is the primary organelle mediating the intrinsic apoptotic pathway in these conditions (Chiong, Wang et al. 2011) Also, if excessive Ca2+ enters to mitochondria and enhanced oxidative stress conditions are present, a phenomenon known as permeability transition may occur (Ascensao, Lumini-Oliveira et al. 2011). The mitochondrial permeability transition is characterized by the loss of the impermeability of the mitochondrial membranes and it is suggested that this condition is mediated by the formation and opening of protein complex-like pores in the inner mitochondrial membrane, the mitochondrial permeability transition pore (MPTP) (for refs see Ascensao, Lumini-Oliveira et al. 2011). Increased pro-oxidant generation causing oxidative stress is one condition that augmented the susceptibility for the opening of these pores and the release of pro-apoptotic proteins within mitochondria as cytochrome c, SMAC/DIABLO and the apoptosis inducing factor (AIF), which will activate the caspase-related apoptotic pathways. It is suggested that the release of these proteins is dependent on the formation and opening of MPTP that cross the inner and the outer membranes leading to the loss of 14 mitochondrial membrane potential (Δᴪ), increased mitochondrial osmotic swelling and rupture of the outer mitochondrial membrane, which leads to death (Ascensao, Lumini-Oliveira et al. 2011). It is believed that the structure and regulation of this multi-protein complex comprises the outer membrane voltagedependent anion channel (VDAC) as well as the inner membrane adenine nucleotide translocator (ANT) and cyclophilin D (Cyc D). Myocyte loss has been shown to occur in the aged rat heart and to precede the occurrence of ventricular dysfunction (Anversa, Hiler et al. 1986), being apoptotic cardiomyocyte death present under different conditions in humans (Haunstetter and Izumo 1998, Narula, Haider et al. 1996, Olivetti, Abbi et al. 1997). Among the excess of biological phenomena affected by aging, the malfunction and decrease of biogenesis of mitochondrial biogenesis seems to exert some of the most potent effects on the organism (Lopez-Lluch, Irusta et al. 2008). If biogenesis is affected, it is reasonable to expect that mitochondrial turnover must be slower and the accumulation of modified lipids, proteins and DNA must also increase, further aggravating the conditions resulting on deficient activity of aged mitochondria (Lopez-Lluch, Irusta et al. 2008). The precise reason for the decrease in the rate of mitochondrial biogenesis during aging is currently unknown. However, it seems that both, extraand intra-cellular regulatory factors of mitochondrial biogenesis are implicated. Specifically, peroxisome proliferator-activated receptor gamma coactivator (PGC1-α) has been shown to act as a common intracellular mediator during mitochondrial biogenesis induced by hormonal factors (Weitzel, Iwen et al. 2003), and adenosine monophosphate-activated protein kinase (AMPK) an intracellular regulator of mitochondrial biogenesis, which activity appears to be one of the main factors associated with deficient mitochondrial biogenesis (Reznick, Zong et al. 2007). PGC family members have gained particular interest because of their ability to drive virtually all mechanisms of mitochondrial biogenesis in the heart, including mitochondrial number, mitochondrial respiration, expression of oxidative phosphorylation and fatty acids oxidation genes, and ROS levels (Lehman, Barger et al. 2000). Decreased PGC-1α expression has been linked to the development of heart failure in mouse models (for refs see Moslehi, DePinho et 15 al. 2012), and decline in mitochondrial biogenesis and mitochondrial protein quality control in cardiac muscle was found in aging (Koltai, Hart et al. 2012) In addition, its well known that mitochondria are dynamic organelles that constantly undergo fission and fusion and it has been found to be vibrant organelles that continuously divide and fuse within the cell and have functions extending beyond energy production, including cell signaling (Liesa, Palacin et al. 2009). Disruption of fission and/or fusion can also lead to cellular dysfunction and to apoptosis. This dynamic mechanism is regulated by proteins controlling fission, such as hFis1 and Drp1, and fusion, such as mitofusin 1 and 2 (MFN1 and MFN2) and OPA1. The correct function of these proteins seems to be critical for normal mitochondrial activity, and their deregulation is associated with several pathologic conditions (Lopez-Lluch, Irusta et al. 2008). Indeed, the impairments on fission-related protein hFis1 has been associated with the process of senescence in mammalian cell cultures (Lee, Jeong et al. 2007). Moreover, depletion of hFis1 by RNA interference (RNAi) induces dramatic changes in mitochondrial structure, including the enlargement and flattening of the organelle. Futrthermore, elimination of any of the mitochondrial fusion proteins as MFN1, MFN2 or OPA1, induces mitochondrial fragmentation, as expected, being that down-regulation of Opa1 expression in cells by RNAi results in spontaneous apoptosis (for refs see Chen and Knowlton 2011). Overall, defects in the mitochondrial fission/fusion machinery and so loss of the symmetry between fusion and fission (Hoppins, Edlich et al. 2011) may contribute to the decline in mitochondrial function during aging. However, several fundamental questions remain to be answered (Bossy-Wetzel, Barsoum et al. 2003). In the next section, the roles of physical exercise as a strategy to improve cardiac function in adult and old subjects as well as the mitochondrial-mediated mechanisms associated with exercise-induced cardioprotection will be addressed. 16 2.2. Exercise and cardioprotection Cardiac damage is a major contributor to morbidity and mortality in industrialized countries; so it becomes important to develop strategies that result in cardioprotective phenotype. In this regard, several approaches have been investigated and physical activity has been shown to be an important countermeasure to protect against myocardial injuries (Bowles, Farrar et al. 1992, Harris and Starnes 2001, Powers, Demirel et al. 1998, Powers, Quindry et al. 2004). In fact, cardiorespiratory fitness is inversely related to cardiovascular and all-cause mortality and it has crucial role preventing heart injury (Kokkinos, Myers et al. 2010). Some reports had postulated exerciseinduced benefits based on decreases of some risk factors to develop cardiac and myocardium impairments such as bodymass index, body weight, waist circumference, abdominal and visceral fat and consequently insulin resistance, triglyceride levels, blood pressure and, in general, metabolic syndrome-related parameters (for refs see Golbidi and Laher 2012). Therefore, chronic aerobic exercise is able, not only, to improve cardiovascular function in young healthy subjects, but also, and most importantly, in older people and those with cardiovascular risk factors (Hambrecht, Fiehn et al. 1998). The study of the mechanisms responsible for exercise-induced cardioprotection has been ongoing for over decades and morphological and biochemical/molecular alterations have been considered as putative mechanisms of exercise-induced cardioprotection. Those include morphological adaptations of heart and coronary arteries, induction of myocardial HSPs, increased myocardial cyclooxygenase-2 activity, elevated ER stress proteins, nitric oxide production, improved function of sarcolemmal and/or mitochondrial adenosine triphosphate (ATP)-sensitive potassium channels and increased myocardial antioxidant capacity (for refs see Kavazis 2009). For instance, exercise induces vascular remodeling and so, morphological alterations in coronary arteries through angiogenesis and arteriogenesis (Leung, Yung et al. 2008). Here, nitric oxide (NO) assumes important roles due its anti-inflammatory, vasodilator and platelet inhibitory effects (Landmesser and 17 Drexler 2005). Also, NO protects against inschemia-reprefusion (IR) injury in such a way that the heart responds to ischemia using nitric oxide species in a harmonized manner and these mechanisms could be based on inhibition of Ca2+ influx into myocytes, antagonism of β-adrenegic stimulation, reduction in cardiac oxygen consumption and ability to increase the expression of HSP70 (for refs see Golbidi and Laher 2011). HSPs protect cell against oxidative injury and apoptosis (Polla, Kantengwa et al. 1996) and furthermore, enhance recovery from acute myocardial cellular injury protecting heart for subsequent injury (for refs see Powers, Locke et al. 2001) by promoting restoration of dysfunctional enzymes and preventing aggregation of severely denatured proteins. The majority of evidences indicate that members of the 70-kDa family are the cytoprotective proteins most responsible for cell protection (for refs see Powers, Locke et al. 2001). The expression of HSP70 in cardiomyocytes is associated with increased cell survival and protection against ischemic damage and its now well established that acute and chronic exercise are able to induce increases in the expression of HSP70 (Kregel and Moseley 1996, Powers, Demirel et al. 1998), although in a temperature-dependent “fashion”. The improved function of sarcolemmal ATP sensitive potassium channels (Powers, Quindry et al. 2008) and elevated ER stress proteins are other important exercise-induced cardioprotection-related alterations and both have special relevance during a cardiac insult (Golbidi and Laher 2012). The ER stress proteins help cellular homeostasis by maintaining intracellular Ca2+ regulation and protein folding during IR injury (Logue, Gustafsson et al. 2005). Considering the importance of mitochondrial machinery in the maintenance of cardiac function, mitochondrial-mediated mechanisms have also been associated with exercise induced cardioprotection phenomenon. This topic will be further discussed in the next sections. 18 2.2.1 Exercise and cardiac mitochondrial adaptations As previously mentioned, mitochondrial adaptations may play a critical role in exercise-induced protection against cardiovascular impairments. The mechanisms behind this phenomenon remain unclear, however it may be related to morphological and biochemical adaptations including biogenesis, antioxidant production or resistance to cell death pathways (Ascensao, Ferreira et al. 2007, Kavazis, Alvarez et al. 2009, Kavazis, McClung et al. 2008). Those will be briefly addressed in the following section. 2.2.1.1. Morphological and biochemical adaptations Heart is a highly oxidative organ, with a low rate of cell growth and slow turnover of proteins; so, it would be expected that heart might have a limited ability to adapt to acute and/or chronic conditions. Although there are scarcely addressed outcomes about exercise-induced cardiac mitochondrial morphological adaptations in healthy hearts, the same cannot be stated regarding hearts under deleterious conditions. In fact, under deleterious conditions as DOX treatment, diabetes, or aging, morphological impairments on heart mitochondria have been reported (Ascensao, Oliveira et al. 2012, Searls, Smirnova et al. 2004). Despite this fact, in a study where trained animals were submitted to an endurance swimming training program, while the non-trained were not engaged in any exercise program Ascensao et al. (2006) reported that endurance swimming training per se caused notable changes in myocardial structure seen as an apparent increased glycogen content, intercalated discs showing a notorious scalloped appearance and evident signs of mitochondria biogenesis with elevated number of encroached mitochondria per fiber area, probably resulting in an increased volume/density of mitochondria. Also, mitochondria division, mild and focal loss of cristae density and organization as minimal degradation by-products, were also present in non-treated trained hearts. Another study developed by Searls et al. (2004) 19 with type I diabetes rats found in type I diabetes rats that 9 weeks of moderate exercise is able to reverse some of the phenotype of diabetic cardiomyopathy. Specifically, the mitochondrial quality, cytoplasmic area, and collagen crosssectional area returned toward non-diabetic values with exercise. Mitochondrial biochemical adaptations to exercise were also reported and several approaches have been trying to prove the exercise-induced modifications of some parameters involved in mitochondrial bioenergetics. Judge et al. (2005) reported that wheel running had no effect on mitochondrial protein yield, rates of oxygen consumption (states 4 and 3) or RCR. This was not entirely surprising given that other studies have shown that, unlike skeletal muscle, oxidative capacity of cardiac muscle is not increased in response to treadmill training, an exercise protocol that is typically much more intense than voluntary wheel running (for refs see Judge, Jang et al. 2005). Also, Starnes et al. (2007) reported that endurance training has no impact on mitochondrial oxidative phosphorylation, being that all oxidative phosphorylation parameters were similar in endurance trained and sedentary rats. Because the heart is highly oxidative, it is not expected to be as responsive to exercise-induced increases in oxidative capacity as skeletal muscle. Although, several reports suggested that exercise per se is able to improve mitochondrial protein yield, rates of oxygen consumption as well as cardiac function (for refs see Ascensao, Ferreira et al. 2007, Ascensao, Magalhaes et al. 2006, Chicco, Schneider et al. 2006, Powers, Lennon et al. 2002). Moreover, Kavasis et al. (2009) reported that the abundance of several proteins involved in bioenergetics was altered following endurance exercise in both SS and IMF mitochondria. Most specifically, the protein levels of several proteins involved in β-oxidation of fatty acids were increased following repeated bouts of endurance exercise. It is of especial concern as during the development of heart disease or genetic defects in mitochondrial fatty acid β-oxidation the myocardial energy source switches from fatty acid β-oxidation to glycolysis. Also, there are some controversial reports about endurance exercise benefits against Ca2+-induced mitochondrial dysfunction. In fact, while Starnes et al. (2007) found that endurance exercise training had no influence when mitochondria was challenged with identical Ca2+ 20 concentrations, being that mitochondria from trained and sedentary animals displayed similar declines in ATP production, French et al. (2008) found that exercise-induced protection against IR injury, in part, by attenuating IR-induced oxidative modification of important Ca2+-handling proteins and preventing subsequent calpain activation. In this study, MnSOD may had an important role as, the results support that exercise-induced increases in myocardial MnSOD activity attenuate the oxidation and degradation of Ca2+-handling proteins, preventing calpain activation during IR. It has special concern because calpain activation within the myocardium has been directly linked with IR-induced necrotic and apoptotic cardiac myocyte death (French, Hamilton et al. 2008). Furthermore, several reports suggest that exercise per se is able to enhance cardiac mitochondria Ca2+ uptake capacity without MPTP induction (Ascensao, Lumini-Oliveira et al. 2011, Kavazis, McClung et al. 2008, Marcil, Bourduas et al. 2006). Therefore, exercise may induce beneficial morphological and biochemical adaptations in mitochondria, which can be translated in a cardiac phenotype more functional and protected against deleterious stimuli. 2.2.1.2. Mitochondrial biogenesis Due to the high-energy demand of the heart, a decline in mitochondrial function can result in a deterioration of cardiac performance (Li, Muhlfeld et al. 2011) and can lead to a wide variety of pathophysiological conditions. According to some reports, the malfunction of mitochondria and the decrease of mitochondrial biogenesis, together with increased oxidative damage, seem to exert some of the most deleterious effects on the organism (Guarente 2008, Lopez-Lluch, Irusta et al. 2008). Fortunately, although controversial, heart seems to have the ability to alter some gene expression profile and phenotype to produce new and more functional mitochondria (Lee and Wei 2007). Regular exercise or increased energy demand are important stimuli that lead to increased mitochondria biogenesis (for refs see Lopez-Lluch, Irusta et al. 2008). In fact, increases in cytosolic Ca2+ levels induced by exercise stimulates 21 calmodulin kinase which then promotes PGC1-α expression (Wu, Kanatous et al. 2002) that has been shown to be a major regulator of mitochondrial biogenesis (Rodgers, Lerin et al. 2005). Also, Geng et al. (2010) showed that in skeletal muscle-specific PGC-1α knockout mice, exercise-induced mitochondrial biogenesis and angiogenesis were significantly attenuated. However, exerciseinduced mitochondrial biogenesis on cardiac muscle is still not fully illusive. In fact, although a significant amount of experimental data on mechanisms involved in exercise-induced mitochondrial biogenesis has been obtained in skeletal muscle, less information is available about the heart. In this regard, Li et al. (2011) reported no changes in mitochondrial biogenesis on left ventricle after 3 months of endurance exercise. Actually, in contrast to skeletal muscle in which adaptive changes in volume fraction of mitochondria can readily occur, this is not so frequent in cardiac muscle (Hood, Balaban et al. 1994). So, the exercise-induced benefits on heart mitochondria biogenesis are still controversial. 2.2.1.3. Oxidative stress and antioxidant capacity With exercise performance, increased O2 consumption creates favorable conditions for increased generation of ROS, an inevitable consequence that may increase oxidative stress at the organelle, cell, and tissue (Ji, Leeuwenburgh et al. 1998). However, if these stimuli are repeated over time, it may have a strong modulating effect on various defense systems in cardiac cells (Powers, Lennon et al. 2002). Also, in some deleterious conditions that lead to ROS production, as IR-induced myocardial injury, DOX administration or aging, physical exercise as been reported to induce benefic countermeasures. In fact, IR-induced myocardial injury is manifested due to the complex interaction of numerous factors but ROS generated by mitochondria during IR injury are believed to play key role in this process (for refs see Honda, Korge et al. 2005). During IR, mitochondrial ROS generation can lead to increased general oxidative stress and consequently Ca2+ overload, which could be of 28 Unfortunately, mitochondria have been identified as primary DOX target organelles, and their involvement is evidenced by the results of many studies reporting functional and morphological alterations, as will be briefly described. Indeed, it has been suggested that cardiomyocyte dysfunction induced by DOX treatment is related to increased levels of ROS-induced damage and apoptotic cellular death, involving mitochondria in the process (Jung and Reszka 2001) culminating in the disruption of major functions (Jung and Reszka 2001). The next sections will briefly describe some DOX-induced mitochondrial dysfunctions such as morphological evidences, increased oxidative stress and susceptibility to apoptosis. 2.3.1.1 Morphological evidences As a consequence of acute or chronic DOX treatment, ultrastructural alterations in rat cardiomyocytes have been reported (Ascensao, Ferreira et al. 2007, Ascensao, Oliveira et al. 2012, Yilmaz, Atessahin et al. 2006, Zhou, Starkov et al. 2001). These include nuclear swelling associated with disruption of nuclear membrane structure, a marked interstitial and cellular edema, perinuclear vacuolation, disorganization and degeneration of the myocardium, loss of myofibrils, distension of the sarcoplasmic reticulum, slight enlargement of the Ttubules and myofibrillar damage and loss (for refs see Carvalho, Santos et al. 2009, Zhou, Starkov et al. 2001). Also, in mice cardiomyocytes, DOX-induced acute alterations in mitochondria were observed, such as vacuolization, myelin deposition, disruption of membrane and organelle degeneration with cristae degeneration, intramitochondrial vacuoles as well as myelin figures (Ascensao, Ferreira et al. 2007, Ascensao, Oliveira et al. 2012, Sardao, Oliveira et al. 2009). These ultrastructural alterations are dose-dependent being that higher DOX concentrations promote more profound cellular alterations (Carvalho, Santos et 29 al. 2009) and those ultrastructural injuries are not repaired after cessation of DOX treatment becoming even more extensive. 2.3.1.2 Increased oxidative stress One of the most known DOX-induced side effects is the increased oxidative stress and it seems to be an event that occurs both acutely and chronically (Ascensao, Oliveira et al. 2012, Wallace 2003). It has been suggested that DOX accumulation in mitochondria leads to a redox cycling by complex I of the mitochondrial respiratory chain, where single electrons are transferred to DOX (Ascensao, Oliveira et al. 2012). As previously mentioned, the possible but controversial existence of a specific NADH dehydrogenase able to start this redox cycling may have an important role. DOX enters mitochondria and reacts with mitochondrial complex I to form semiquinone radical intermediates, which is a short-lived metabolite and can react with O2 producing ROS (for refs see Carvalho, Santos et al. 2009). Then, ROS can react with mitochondrial biomolecules in the vicinity, which include lipids, proteins and nuclei acids. Furthermore, increased cardiac oxidative stress associated with DOX toxicity leads to the depletion of reducing equivalents, impairment in oxidative phosphorylation with consequent decline in ATP, and interference with cellular Ca2+ homeostasis (Wallace 2003). Also, DOX is known to react with mitochondrial mtDNA forming adducts that interfere with proteins expression, lipid oxidation and normal mitochondrial function, which in turn further increases ROS production (Sardao, Pereira et al. 2008). This “vicious cycle” postulated that accumulation of mtDNA mutations is exponential and associated with massive increase in ROS production (Lenaz 1998). As previously described, heart mitochondria are important target of DOX, accumulating the drug at relatively high concentrations. So, it is not surprising that these organelles are especially susceptible to DOX-induced oxidative damage, and at the same time, they are also important sources of DOX-induced ROS (Ascensao, Oliveira et al. 2012). On other hand, studies show an upregulation of antioxidant defenses, 30 suggesting an adaptive response of cells to oxidative unbalance promoted by DOX (Yilmaz, Atessahin et al. 2006). Interestingly, the antioxidant capacity seems to increase significantly following DOX treatment in young but not in old Fischer suggesting an increase in DOX-induced oxidative damage with age (Pritsos and Ma 2000). Despite this fact, is notable that, although the heart has relatively low antioxidant capacity, it shows an upregulation as an adaptive response to this oxidative unbalance. Moreover, as a response to this cellular stress, HSP 60 and HSP 70 increased in hearts and mitochondria from DOX treated animals and cells (Ascensao, Magalhaes et al. 2005, Kavazis, Smuder et al. 2010). 2.3.1.3 Increased susceptibility to apoptosis Another important DOX-induced alteration on cell is the increased susceptibility to trigger apoptosis. DOX toxicity leads to impairments on cellular Ca2+ homeostasis, which leads to increased susceptibility to the MPTP opening (Oliveira and Wallace 2006) that is characterized by the loss of the impermeability that characterizes the inner mitochondrial membrane, as it was previously described. This complex process is mediated by the formation and opening of protein complex-like pores, the MPTP (Ascensao, Lumini-Oliveira et al. 2011, Lumini-Oliveira, Magalhaes et al. 2011). As already mentioned, one of the consequences of the MPTP induction, besides the disturbance of cell and mitochondrial Ca2+ homeostasis, is the release of cytochrome c and some others pro-apoptotic proteins, with consequent initiation of apoptotic cascades. Also, the increased oxidative stress induced by DOX, is able to interfere with mitochondrial functionality, which leads to apoptosis. Indeed, DOX-induced cardiomyocytes apoptosis has been suggested to occur both acutely and chronically (Carvalho, Santos et al. 2009). As previously mentioned, its important to develop a strategy that results in a cardioprotective phenotype against DOX-induced impairments. In this regard, physical exercise in its various forms has been shown to be an effective 31 intervention that can counteract the acute and chronic deleterious insults for the myocardium, which includes DOX treatment. The following sections will briefly discuss this issue. 2.4. Exercise as a therapeutic and preventive strategy against DOXinduced cardiotoxicity. Exercise has been considered the most effective strategy to promote a healthy lifestyle and its benefits against DOX-related impairments are evident (Ascensao, Lumini-Oliveira et al. 2011, Ascensao, Magalhaes et al. 2005, Ascensao, Oliveira et al. 2012, Chicco, Schneider et al. 2005, Emter and Bowles 2008, Hayward, Lien et al. 2012, Kavazis, Smuder et al. 2010, Powers, Lennon et al. 2002, Wonders, Hydock et al. 2008). Although the most studied form of exercise in DOX treated animals is the chronic exercise, acute exercise also seems to provide beneficial effects. Mitochondrial adaptations may play critical role in exercise-induced protection against DOX-induced cardiac impairments (Ascensao, Ferreira et al. 2007, Ascensao, Oliveira et al. 2012). The effects of exercise on DOX-treated mitochondria are highlighted in table 2. 2.4.1 Acute exercise Acute exercise broadly refers to a single bout of exercise performed only once and it was already proved to be effective against DOX-induced impairments. In fact, Wonders et al. (2008) reported that an acute bout of treadmill running performed 24 hours prior to DOX injection attenuated the hemodynamic impairment observed after acute DOX administration and reduced left ventricular lipid peroxidation. In addition, Ascensao et al. (2011) showed that an acute bout of treadmill exercise protects against cardiac mitochondrial dysfunction, preserving mitochondrial phosphorylation capacity and attenuating DOX-induced decreased tolerance to MPTP induction. In the same study, it was 32 observed that acute exercise prevented the decreased cardiac mitochondrial function detected as impaired state 3, phosphorylative lag-phase and maximal transmembrane potential. Also, acute exercise prevented the inhibitory effects of DOX treatment on the activity of cardiac mitochondrial respiratory chain complexes I and V, and on increased caspase-3 and -9 activities. Furthermore, it has also been described that acute exercise may contribute to diminished free radical production (for refs see Ascensao, Oliveira et al. 2012). However, further research is needed to clarify the exact mechanisms by which an acute exercise induces a protective phenotype in DOX-treated cardiac mitochondria. 2.4.2 Chronic exercise Unlike acute exercise, chronic exercise has been hardly studied and the results consistently demonstrate that is able to antagonize DOX-induced cardiac impairments (Ascensao, Magalhaes et al. 2005, Chicco, Schneider et al. 2005, Hayward, Lien et al. 2012, Hydock, Lien et al. 2008, Kavazis, Smuder et al. 2010). Those alterations induced by chronic exercise can be seen at morphological, functional and biochemical levels and the role of mitochondria is pivotal in this process (Ascensao, Oliveira et al. 2012, Kavazis, Smuder et al. 2010). Ascensao et al. (2005) reported the involvement of mitochondria in cardioprotection afforded by endurance training against DOX treatment, demonstrating prevention of acute DOX-induced mitochondrial alterations regarding oxidative stress, respiration, and Ca2+ loading capacity. At ultrastructural level, it has been reported that cardiac alterations induced by DOX treatment are also attenuated by previous chronic exercise (Ascensao, Magalhaes et al. 2005). In fact, when compared with saline (SAL) group, sedentary (SED) DOX-treated animals showed myocardial damage (Ascensão, Magalhães et al. 2006). The observed morphological alterations consisted of mitochondrial damage with extensive degeneration and loss of cristae, swelling 33 and abnormal size and shape, intramitochondrial vacuoles and notorious myelin figures that probably resulted in the formation of secondary lysosomes. All of these alterations were attenuated in trained animals treated with DOX (Ascensão, Magalhães et al. 2006). At functional level, different authors reported the protective effects of chronic exercise. In fact, Hydock et al. (2008) suggested that exercise training in rats before DOX treatment attenuated DOX-induced cardiac dysfunction, through the maintenance of fractional shortening, developed pressure and contractility. Also, Chicco et al. (2005) reported that both low intensity exercise training and an endurance training protocol with gradually increased intensity attenuated the adverse effects of DOX by preventing DOX-induced decline in cardiac function through maintenance of left ventricular diastolic pressure, rate of left ventricular pressure development and rate of left ventricular relaxation. At biochemical level, as depicted on table 2, several authors had proved the beneficial effects of chronic exercise. Importantly, some of the most studied biochemical parameters associated with exercise and DOX are the antioxidant capacity, oxidative stress markers and apoptotic susceptibility (for refs see Ascensao, Oliveira et al. 2012). As previous referred, HSP have an important role as antioxidants molecules contributing to normal cellular integrity and are overexpressed after endurance training. However, Chicco et al (2006) and Kavasis et al. (2010) also showed that exercise had no influence on HSP or that it is not determinant on cardioprotection being that exercise in cold vs. normal temperatures may also display other types of differences regarding alteration of mitochondrial physiology, besides alteration in the expression of HSPs. Furthermore, the upregulation of mitochondrial manganese superoxide dismutase (MnSOD) seems contribute for cardioprotection. In fact, as mitochondrial DOX toxicity has been largely attributed to increased oxidative stress, increased antioxidant activity may be important to explain how endurance training counteracts some of DOX-induced myocardial damage. Chicco et al. (2006) associated low-intensity treadmill exercise training-induced cardioprotection to the inhibition of apoptotic signaling and the increased activity 34 of GPX. Also, the effect of training on preventing activation of cardiac apoptotic pathways has been described being that, training decreased the susceptibility of appearance of apoptotic markers in the hearts of DOX-treated animals, as increased mitochondrial Bax, Bax-to-Bcl2 ratio and tissue caspase 3 activity (Ascensao, Magalhaes et al. 2005). In fact, it has been suggested that chronic exercise stimulation may also afford protection against the increased susceptibility to the MPTP as the deleterious effects of Ca2+ on heart mitochondrial respiration of DOX-treated animals were attenuated in trained group treated with DOX (Ascensao, Magalhaes et al. 2005). As previous described, MPTP is related to oxidative damage and therefore, it is possible that increased resistance of cardiac mitochondria from trained animals to the MPTP can be related to increased antioxidant defenses. Accordingly, the higher levels of reduced sulfhydryl groups in trained mitochondria than in sedentary groups may be indicative of enhanced antioxidant capacity and/or of more elevated sulfhydryl-donors, such as GSH, in mitochondria from trained animals (for refs see Ascensao, Oliveira et al. 2012). However, further studies are necessary in order to better understand this issue. Table 2. Summary of some described mitochondrial-related alterations associated with DOX-induced cardiotoxicity and the modulation effect afforded by physical exercise against DOX (adapted from Ascensao, Oliveira et al. 2012). DOX effect Exercise effect against DOX ROS production ↑ ↓ Oxidative damage markers Lipid peroxidation ↑ ↓ Protein oxidation ↑ ↓ DNA oxidation ↑ Aconitase activity ↓ or = ↑ Apoptotic signaling 35 (↑) – increase; (↓) – decrease; (=) – no alterations Despite the extensive number of studies on this topic, the effects of both endurance treadmill training and voluntary free-wheel running activity performed Bax-Bcl-2 ratio ↑ ↓ Cytochrome c release ↑ Caspase 9 activation ↑ ↓ Respiratory endpoints State 3 ↓ ↑ or = State 4 ↑ or = or ↓ = or ↓ RCR ↓ = or ↑ ADP/O ratio = or ↓ Uncoupled respiration ↓ ↑ Creatine-stimulated respiration ↓ Maximal ΔΨ ↓ or = ↑ Ca2+-induced MTPT ↑ ↓ ANT content and functioning ↓ Mitochondrial chaperones ↑ ↑ Mitochondrial antioxidants Thiols ↓ ↑ Vitamin E ↓ or = Enzymes = or ↑ ↑ Coenzyme Q isoenzymes = ETC complex activity Complex I ↓ ↑ Complex II ↓ Complex III = Complex IV = or ↓ Complex V ↓ ↑ 36 before and during sub-chronic DOX treatment schedule on cardiac mitochondrial bioenergetics are yet to be elucidated. This is of particular importance in the context of exercise-induced protection against DOX-related cardiac mitochondriopathy, as cancer patients undergoing DOX treatment may be advised to exercise for many reasons including to counteract physical fatigue and to improve performance, and also to mitigate cardiac damage as a result of chemotherapy. This master sports science course in which this work is inserted is the context of physical activity and elderly. Despite developed with adult rats, the present work can contribute to extend the knowledge in this particular area representing preliminary findings in adult population and considering that DOX-based chemotherapeutic treatments against several types of malignances are more prevalent with increasing age. 37 3. Aim The aim of the present study was to analyze the effect of two types of physical exercise (treadmill endurance training (TM) and free-wheel voluntary physical activity (FW)) against heart mitochondrial dysfunction induced by sub-chronic treatment of DOX. We can define as specific purposes of this work the analysis of the adaptations induced by both types of exercise on heart mitochondria from DOX treated animals on: • Mitochondrial respiratory function; • Mitochondrial electrical transmembrane potential; • MPTP susceptibility; • Mitochondrial oxidative damage. 44 4.9 Mitochondrial osmotic swelling during MPTP induction Mitochondrial osmotic volume changes were followed by monitoring the classic decrease of absorbance at 540 nm with a Jasco V-630 spectrophotometer. Swelling amplitude and rate of decreased absorbance upon Ca2+ addition were considered as MPTP susceptibility indexes. The reaction was continuously stirred and the temperature was maintained at 25 ºC. The assays were performed in 1 ml of reaction medium containing 200 mM sucrose, 10 mM HEPES, 5 mM KH2PO4, 10 µM EGTA , pH 7.4, supplemented with 1.5 µM rotenone, 8 mM succinate and a single pulse of 80 nmol of Ca2+ with 0.5 mg/ml protein. Control trials were performed by using 1 µM of cyclosporin-A, the selective MPTP inhibitor (Broekemeier, Dempsey et al. 1989). 4.10 Mitochondrial oxidative damage Before analysis, mitochondrial membranes were disrupted by several freeze– thawing cycles to allow free access to substrates. The extent of lipid peroxidation in heart mitochondria was determined by measuring MDA contents by colorimetric assay, according to a modified procedure described previously (Buege and Aust 1978). Suspended mitochondria were centrifuged at 12,000 xg for 10 min and re-suspended in 150 µL of a medium containing 175 mM KCl and 10 mM Tris-HCl, pH 7.4. Subsequently, mitochondria from the six groups were mixed with 2 volumes of trichloroacetic acid (10%) and 2 volumes of thiobarbituric acid (1%). The mixtures were heated at 80–90 ºC for 10 min and re-cooled in ice for 10 min before centrifugation (4,000 xg for 10 min). The supernatants were collected and the absorbance measured at 535 nm. The amount of MDA content formed was expressed as nanomoles of MDA per milligram of protein (ε535=1.56 x 10−5 M−1 cm−1) The basal mitochondrial content of oxidative modified -SH groups, including GSH and other -SH containing proteins, was quantified by spectrophotometric measurement according to Hu (1990). Briefly, a mitochondrial suspension 45 containing 5 mg/mL protein was mixed with 0.25 M Tris buffer pH 8.2 and 10 mM DTNB and the volume was adjusted to 1 mL with absolute methanol. Subsequently, the samples were incubated for 30 min in the dark at room temperature and centrifuged at 3000 xg for 10 min. The colorimetric assay of supernatant was performed at 414 nm against a blank test. Total -SH content was expressed in nanomoles per milligrams of mitochondrial protein (ε414=13.6 mM−1 cm−1). 4.11 Soleus citrate synthase activity Soleus CS activity was measured using the method proposed by Coore et al. (1971). The principle of assay was to initiate the reaction of acetyl-CoA with oxaloacetate and link the release of CoA-SH to 5,5-dithiobis (2-nitrobenzoate) at 412 nm. 4.12 Statistical analysis All data are expressed as the mean±SEM (Standard Error of the Mean). Statistical analyses were performed using GraphPad Prism (version 6.0) or Statistical Package for the Social Sciences (SPSS version 21.0). Three-way repeated-measures ANOVA for body weight and distance cover by exercised groups to verify the effect of exercise and treatment over time. Two-way analysis of variance ANOVA were used to examine possible effect of treatment and/or exercise. To determine specific group differences, the two-way ANOVA were followed by Bonferroni post-hoc tests. In all cases, the significance level was set at p≤0.05. 47 5. Results 5.1. Characterization of animals and exercise protocols Body weight alterations and distances covered by the animals during the entire protocol are shown in figure 3. No significant differences in the mean body weight of the animals from the beginning of the protocol until the 5th week, when sub-chronical DOX treatment was initiated, were found. Body weights of DOX treated animals were lower than SAL counterparts at the end of the protocol (DOX+SED vs. SAL+SED; DOX+TM vs. SAL+TM; DOX+FW vs. SAL+FW; p≤0.05). No differences in body weight between exercised groups, were found. TM and FW decreased body weight at 12th and 9th week, respectively (SAL+TM and SAL+FW vs. SAL+SED; p≤0.05). DOX treatment combined with TM decreased body weight from the 5th week (DOX+TM vs. DOX+SED; p≤0.05), whereas no significant differences were found between FW and SED treated groups (Figure 3A). After the 5th week DOX treated groups consumed less food than their SAL counterparts (data non shown, p≤0.05). Water consumption increased in FW groups (SAL and DOX) compared with SAL+SED and DOX+SED groups (data non shown, p≤0.05). Figure 3. Effect of exercise and DOX treatment on (A) body mass over time and (B) distance covered per day by TM and FW groups during the 12 wks of protocol. Significant differences (p≤0.05) are mentioned in the text. Significant (p≤0.05) effects of Exercise (E), Treatment (T), time (t) or their interaction (E x T x t) are shown; Non Significant (NS, p>0.05). 48 As can be seen in Figure 3B, voluntary running distance decreased significantly in DOX+FW after the 5th week and remained lower until the end of the protocol (p≤0.05). Animals from TM group ran at the same velocity throughout the 8 weeks of the protocol. Running velocity and distance covered diminished in DOX+TM group at 11th and 12th week compared to SED+TM (p≤0.05). Body, heart absolute weights, heart weight and femur length to body weight ratios, mitochondrial protein yielding as well as the activity of soleus citrate synthase in the six groups are shown in Table 4. Final body, heart weight and ratio of heart weight to body weight significantly decreased with DOX treatment (SAL+SED vs DOX+SED). Both chronic exercise types decreased final body weight, increased heart weight and the heart to body ratio (SAL+TM and SAL+FW vs SAL+SED). DOX treatment combined with TM and FW exercise induced a significant increase in heart weight and heart weight to body weight ratio compared with their DOX+SED counterparts. No significant differences were observed between groups regarding the Initial body weight, femur length to body weight ratio and yield of mitochondria isolation. TM induced a significant increase in the activity of soleus citrate synthase in both SAL and DOX treated animals (SAL+SED vs SAL+TM and DOX+SED vs DOX+TM). 49 Table 4. Animal data and yield of mitochondrial protein isolation SAL+SED SAL+TM SAL+FW DOX+SED DOX+TM DOX+FW P* Initial body weight (g) 207±3.91a 214±3.76a 212±1.55a 209±4.68a 207±4.63a 209±2.60a NS Final body weight (g) 598±10.58a 522±9.87b 498±5.98b 438±6.22c 426±10.79c 429±16.95c ExT Heart weight (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 Heart weight/body weight (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 Femur length/body weight (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 Mitochondrial protein yielding (mg protein/g tissue) 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 citrate synthase activity (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). Different letters are significantly different (p≤0.05). * Significant (p≤0.05) effects of Exercise (E), Treatment (T), or their interaction (E x T) are shown; Non Significant (NS, p> 0.05). 50 5.2 Heart mitochondrial oxygen consumption Mitochondrial respiratory activity in both SAL and DOX treated groups was measured to identify exercise-dependent effects (Figure 4). DOX treatment decreased heart mitochondrial respiration during state 3 and increased state 4 in SED animals (DOX+SED vs SAL+SED). Importantly, TM and FW exercise per se increased State 3 respiration in both SAL and DOX (SAL+TM and SAL+FW vs. SAL+DOX; DOX+TM and DOX+FW vs. DOX+SED). The coupling between oxygen consumption and ADP phosphorylation (RCR) was significantly affected by DOX treatment (DOX+SED vs. SAL+SED). TM significantly increased RCR in both SAL and DOX groups (SAL+TM vs. SAL+SED; DOX+TM vs. DOX+SED). FW increased RCR in DOX treated animals (DOX+FW vs. DOX+SED). Also, both TM and FW increased ADP/O in DOX group (DOX+SED vs. DOX+TM and DOX+FW). 51 Figure 4. Effect of exercise and DOX treatment on (A) state 3 of heart mitochondrial respiration, (B) state 4 of heart mitochondria respiration, (C) RCR and (D) ADP/O. Data are means±SEM for heart mitochondria (0.5 mg/mL protein) obtained from different mitochondrial preparations for each experimental group. Oxidative phosphorylation was measured polarographically at 30ºC in a total volume of 0.75 mL. Respiration medium and other experimental details are provided in methods. RCR, respiratory control ratio (state 3/state 4); ADP/O, number of nmol ADP phosphorylated by atom of oxygen consumed. Different letters are significantly different (P≤0.05). Significant (p≤0.05) effects of Exercise (E), Treatment (T), or their interaction (E x T) are shown; Non Significant (NS, p>0.05). 5.3 Heart mitochondrial transmembrane electric potential Heart mitochondrial variations in maximal ∆ψ and during ADP phosphorylation were determined using G/M as substrates. DOX treatment significantly affected the maximal ∆ψ, repolarization and ADP lag-phase (Figure 5). FW but not TM, increased maximal ∆ψ and repolarization, whereas both types of exercise decreased the ADP lag phase (SAL+TM and SAL+FW vs. SAL+SED). Both exercise protocols were able to counteract the DOX harmful effect normalizing maximal ∆ψ, repolarization and ADP lag phase. 52 Figure 5. Effect of exercise and DOX treatment on heart mitochondria ∆ψ fluctuations (A) maximal energization, (B) ADP-induced depolarization, (C) repolarization and (D) ADP phosphorylation lag phase. Data are mean±SEM for heart mitochondria (0.5 mg/mL protein) obtained from different mitochondrial preparations for each experimental group. Figure shows the average response of maximal mitochondrial membrane potential developed with glutamate (5 mM) plus malate (2.5 mM), the decrease in membrane potential after ADP addition (depolarization), the repolarization value after ADP phosphorylation, and the lag phase. Mitochondrial transmembrane potential was measured using a TPP+-selective electrode at 30ºC in a total volume of 1 mL. Reaction medium and other experimental details are provided in methods. Different letters are significantly different (p≤0.05). * Significant (p≤0.05) effects of Exercise (E), Treatment (T), or their interaction (E x T) are shown; Non Significant (NS, p>0.05) 5.4 Mitochondrial osmotic swelling during MPTP induction The effects of both types of exercise training and DOX treatment on in vitro susceptibility to Ca2+-induced MPTP opening were investigated. The addition of Ca2+ on mitochondria suspension resulted in a decrease in absorbance with three distinct phases. Initially, an increase in absorbance was observed, which most likely results from the formation of opaque Ca2+ crystals inside mitochondria (Andreyev, Fahy et al. 1998). Upon MPTP opening, a decrease of 53 absorbance with a slow followed by a fast kinetic rate is usually observed in cardiac mitochondria. Incubation of mitochondrial suspension with cyclosporine A, a specific MPTP inhibitor (Broekemeier, Dempsey et al. 1989), limits the absorbance decrease after Ca2+ addition, which demonstrate the association with MPTP opening. Figure 6 shows different end-points measured from the recordings obtained, namely (A) swelling amplitude (the difference between the initial and the final absorbance value) and (B) the average swelling rate. The results demonstrate that DOX treatment significantly increased susceptibility to Ca2+-induced MPTP opening (DOX + SED vs. SAL + SED). Heart mitochondria isolated from SAL+TM group, but not SAL+FW were less susceptible to Ca2+-induced MPTP opening (SAL + TM vs. SAL + SED). Both types of exercise were able to mitigate DOX-induced increased susceptibility to MPTP opening (DOX + TM and DOX + FW vs. DOX + SED). Figure 6. Effect of exercise and DOX treatment on heart mitochondria to Ca2+-induced MPTP (A) Swelling amplitude; (B) Average swelling rate. Data are mean ± SEM. The absorbance of mitochondrial suspension was followed at 540 nm. Mitochondria were incubated as described in methods. A 80 nmol of Ca2+ pulse (160 nmol/mg protein) was added to 0.5 mg of mitochondrial protein in order to attain the cyclosporin Asensitive swelling, indicating that the decreased optical density corresponding to the increased swelling was due to MPTP opening. Different letters are significantly different (p≤0.05). * Significant (p≤0.05) effects of Exercise (E), Treatment (T), or their interaction (E x T) are shown; Non Significant (NS, p>0.05) 60 increased functionality of the phosphorylative system in general, and the ETC in particular, induced by both exercise protocols may have some implications in Ca2+ uptake capacity. However, to better understand this phenomenon, further studies need to be addressed. 6.3 Oxidative stress markers Prevailing hypotheses suggest that myocardial oxidative stress is a primary event in DOX-induced cardiotoxicity and it is believed to initiate several of the deleterious cellular events reported following DOX treatment (Zucchi and Danesi 2003). In fact, at present the principal mechanism of DOX-induced cardiotoxicity is believed to be increased mitochondrial oxidant production leading to protease activation and induction of apoptosis (Ascensão, Magalhães et al. 2006, Ascensao, Magalhaes et al. 2005, Chicco, Hydock et al. 2006, Chicco, Schneider et al. 2005). Accordingly, oxidative injury of fatty acids at subcellular level measured by increased levels of lipid peroxidation products has been frequently reported following DOX exposure (for refs see Chicco, Schneider et al. 2005). The present results show that TM, but not FW per se was able to decrease MDA level and increase -SH groups. In accordance to previous reports, DOX induced a significant decrease in -SH, indicating increased disulfide linkages from both proteins and GSH. As polyunsaturated fatty acids are considered highly susceptible to ROS attack, the increased oxidative stress caused by DOX led to peroxidative modification of lipid membranes affecting membrane integrity and permeability, which leads to decoupled mitochondria, altering normal mitochondrial respiratory function. Myocardial antioxidant enzymes defend the heart against the damaging effects of ROS and have been hypothesized to play an important role in exerciseinduced resistance to oxidative stress (Powers, Lennon et al. 2002) and in the attenuation of DOX cardiotoxicity (Singal, Iliskovic et al. 1997). In particular, 61 some studies suggested that the presence of myocardial SOD might be important for the prevention of DOX cardiotoxicity (Ascensao, Lumini-Oliveira et al. 2011, Sarvazyan, Askari et al. 1995, Yen, Oberley et al. 1996). This is reasonable, as SOD dismutates superoxide into H2O2, thereby providing the first line of defense against DOX-induced oxidative stress. Furthermore, increasing evidence suggest that myocardial HSP72 induction plays a pivotal role in exercise-induced cardioprotection against oxidative stress (Powers, Lennon et al. 2002, Powers, Locke et al. 2001, Taylor and Starnes 2003). 6.4 Meaning for exercise-induced cardioprotection in aging Considering the present results in the context of exercise-induced cardioprotection in advanced age, they can be interpreted as preliminary. Indeed, it can be carefully speculated that the observed protective phenotype caused by both chronic models of exercise against DOX can also be observed in aged rats. In fact, Quindry et al. (2005) reported that aged rats submitted to exercise training ameliorate cardiac hemodynamic response with significant improvements in the apoptotic levels and signaling caused by IR injury. Furthermore, the authors observed that trained old rats increased MnSOD activity, which can be interpreted as a sign of cardiac mitochondrial adaptations induced by chronic exercise in old rats compared to their young counterparts. Similar results were found by Starnes et al. (2003), which suggest that, although observing cardioprotective protein phenotype alterations with age, exercise can enhance cardioprotection regardless of elderly. Furthermore, physical exercise has the ability to positively modulate some gene expression associated with improved heart function in aged rats. In fact, heart is known for its ability to produce energy from fatty acids because of its important β-oxidation equipment, which capacity is reduced with age (Starnes, Beyer et al. 1983). Confirming the potential beneficial effects of physical exercise on cardiac metabolism in elderly, Iemitsu et al. (2002) reported that exercise training improved the aging-induced decreased expression of peroxisome proliferator-activated receptor, which regulates genes related to fatty acid metabolism in the heart. Giving those 62 alterations reported in aged hearts, it is possible to speculate that the results of the present work could also be observed in aged rats; affording protection and mitigating the deleterious consequences associated with sub-chronic DOX treatment schedules. 63 7. Conclusion In summary, the data from the present work provide additional support about the effect of two types of physical exercise (treadmill endurance training and free-wheel voluntary physical activity) against heart mitochondrial dysfunction induced by sub-chronic treatment of Doxorubicin (DOX). Our results showed us that: • Regarding mitochondrial respiratory function both types of exercise reverted the effects induced by DOX on state 3, RCR and ADP/O. Interestingly, free wheel voluntary physical activity was more efficient at counteracting DOX-induced defects on RCR; • Both types of exercise were able to counteract DOX-induced impairments in mitochondrial transmembrane endpoints. Importantly, free wheel voluntary physical activity was also more efficient at normalizing DOX-induced increases in lag phase; • Regarding mitochondrial osmotic swelling during MPTP induction, both exercise protocols reverted DOX-induced impairments. In fact, both types of exercise mitigated DOX-induced increases in swelling amplitude and average swelling rate. However, once again free wheel voluntary physical activity was more efficient at counteracting Ca2+-induced MPTP induction • Exercise protocols were able to revert DOX-induced increases in MDA content and decrease in sulfhydryl groups. 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