RESUMEN
Doxorubicin (DOX) is a topoisomerase II inhibitor used in cancer therapy. Despite its efficacy, DOX causes serious adverse effects, such as short- and long-term cardiotoxicity. This work aimed to assess the short- and long-term cardiotoxicity of DOX and the role of inflammation and antioxidant defenses on that cardiotoxicity in a mice model. Adult CD-1 male mice received a cumulative dose of 9.0 mg/kg of DOX (2 biweekly intraperitoneal injections (ip), for 3 weeks). One week (1W) or 5 months (5M) after the last DOX administration, the heart was collected. One week after DOX, a significant increase in p62, tumor necrosis factor receptor (TNFR) 2, glutathione peroxidase 1, catalase, inducible nitric oxide synthase (iNOS) cardiac expression, and a trend towards an increase in interleukin (IL)-6, TNFR1, and B-cell lymphoma 2 associated X (Bax) expression was observed. Moreover, DOX induced a decrease on nuclear factor erythroid-2 related factor 2 (Nrf2) cardiac expression. In both 1W and 5M, DOX led to a high density of infiltrating M1 macrophages, but only the 1W-DOX group had a significantly higher number of nuclear factor κB (NF-κB) p65 immunopositive cells. As late effects (5M), an increase in Nrf2, myeloperoxidase, IL-33, tumor necrosis factor-α (TNF-α), superoxide dismutase 2 (SOD2) expression, and a trend towards increased catalase expression were observed. Moreover, B-cell lymphoma 2 (Bcl-2), cyclooxygenase-2 (COX-2), and carbonylated proteins expression decreased, and a trend towards decreased p38 mitogen-activated protein kinase (MAPK) expression were seen. Our study demonstrated that DOX induces adverse outcome pathways related to inflammation and oxidative stress, although activating different time-dependent response mechanisms.
Asunto(s)
Cardiotoxicidad , Factor 2 Relacionado con NF-E2 , Ratones , Masculino , Animales , Cardiotoxicidad/etiología , Cardiotoxicidad/metabolismo , Cardiotoxicidad/patología , Factor 2 Relacionado con NF-E2/metabolismo , Catalasa/metabolismo , Cardiooncología , Doxorrubicina/efectos adversos , Estrés Oxidativo , Interleucina-6/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Inflamación/tratamiento farmacológico , ApoptosisRESUMEN
Doxorubicin (DOX) is a topoisomerase II inhibitor commonly used in the treatment of several types of cancer. Despite its efficacy, DOX can potentially cause fatal adverse effects, like cardiotoxicity. This work aimed to assess the role of inflammation in DOX-treated infant and adult mice and its possible link to underlying cardiotoxicity. Two groups of CD-1 male mice of different ages (infants or adults) were subjected to biweekly DOX administrations, to reach a cumulative dose of 18.0 mg/kg, which corresponds approximately in humans to 100.6 mg/m2 for infants and 108.9 mg/m2 for adults a clinically relevant dose in humans. The classic plasmatic markers of cardiotoxicity increased, and that damage was confirmed by histopathological findings in both groups, although it was higher in adults. Moreover, in DOX-treated adults, an increase of cardiac fibrosis was observed, which was accompanied by an increase in specific inflammatory parameters, namely, macrophage M1 and nuclear factor kappa B (NF-κB) p65 subunit, with a trend toward increased levels of the tumor necrosis factor receptor 2 (TNFR2). On the other hand, the levels of myeloperoxidase (MPO) and interleukin (IL)-6 significantly decreased in DOX-treated adult animals. In infants, a significant increase in cardiac protein carbonylation and in the levels of nuclear factor erythroid-2 related factor 2 (Nrf2) was observed. In both groups, no differences were found in the levels of tumor necrosis factor (TNF-α), IL-1ß, p38 mitogen-activated protein kinase (p38 MAPK) or NF-κB p52 subunit. In conclusion, using a clinically relevant dose of DOX, our study demonstrated that cardiac effects are associated not only with the intensity of the inflammatory response but also with redox response. Adult mice seemed to be more prone to DOX-induced cardiotoxicity by mechanisms related to inflammation, while infant mice seem to be protected from the damage caused by DOX, possibly by activating such antioxidant defenses as Nrf2.
Asunto(s)
Cardiotoxicidad , Animales , Doxorrubicina , Ratones , FN-kappa B , Estrés OxidativoRESUMEN
Mitoxantrone (MTX) is a pharmaceutical drug used in the treatment of several cancers and refractory multiple sclerosis (MS). Despite its therapeutic value, adverse effects may be severe, namely the frequently reported cardiotoxicity, whose mechanisms need further research. This work aimed to assess if inflammation or oxidative stress-related pathways participate in the cardiotoxicity of MTX, using the mouse as an animal model, at two different age periods (infant or adult mice) using two therapeutic relevant cumulative doses. Histopathology findings showed that MTX caused higher cardiac toxicity in adults. In MTX-treated adults, at the highest dose, noradrenaline cardiac levels decreased, whereas at the lowest cumulative dose, protein carbonylation increased and the expression of nuclear factor kappa B (NF-κB) p65 subunit and of M1 macrophage marker increased. Moreover, MTX-treated adult mice had enhanced expression of NF-κB p52 and tumour necrosis factor (TNF-α), while decreasing interleukin-6 (IL-6). Moreover, while catalase expression significantly increased in both adult and infant mice treated with the lowest MTX cumulative dose, the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glutathione peroxidase only significantly increased in infant animals. Nevertheless, the ratio of GAPDH to ATP synthase subunit beta decreased in adult animals. In conclusion, clinically relevant doses of MTX caused dissimilar responses in adult and infant mice, being that inflammation may be an important trigger to MTX-induced cardiotoxicity.
RESUMEN
Aerobic exercise training induces a unique cardioprotective phenotype, but it is becoming clear that it does not promote the same structural, functional, and molecular adaptations in both ventricles. In the present study, we aimed to better characterize and compare the molecular pathways involved in the exercise-induced remodeling of both ventricles. Female Sprague-Dawley rats were randomly assigned to control and exercise groups. Animals in the exercise group were submitted to low-intensity treadmill exercise for 54 weeks. After the experimental period, biventricular hemodynamic analysis was performed and right and left ventricles were harvested for morphological and biochemical analyses. Data showed that long-term low-intensity exercise training improves cardiac function, especially left ventricular diastolic function; however, the expression of connexin-43, CCAAT-enhancer binding protein ß, and c-kit did not change in none of the ventricles. In the right ventricle, long-term exercise training induced an increase of manganese superoxide dismutase and sirtuin 3 protein expression, suggestive of improved antioxidant capacity. Our results also support that long-term aerobic exercise training imposes greater metabolic remodeling to the right ventricle, mainly by increasing mitochondrial ability to produce ATP, with no association to estrogen-related receptor α regulation.
Asunto(s)
Ventrículos Cardíacos/metabolismo , Miocardio/metabolismo , Condicionamiento Físico Animal/fisiología , Función Ventricular Izquierda/fisiología , Adaptación Fisiológica , Animales , Femenino , Hemodinámica , Estrés Oxidativo/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Cancer associated body wasting is the cause of physical disability, reduced tolerance to anticancer therapy and reduced survival of cancer patients and, similarly to cancer, its incidence is increasing. There is no cure for this clinical condition, and the pathophysiological process involved is largely unknown. Exercise training appears as the gold standard non-pharmacological therapy for the management of this wasting syndrome. Herein we used a lipidomics approach based on liquid chromatography coupled with high-resolution mass spectrometry (LC-HR-MS) to study the effect of exercise in the modulation of phospholipids profile of mitochondria isolated from gastrocnemius muscle of a pre-clinical model of urothelial carcinoma-related body wasting (BBN induced), submitted to 13 weeks of treadmill exercise after diagnosis. Multivariate analysis showed a close relationship between the BBN exercise group and both control groups (control sedentary and control exercise), while the BBN sedentary group was significantly separated from the control groups and the BBN exercise group. Univariate statistical analysis revealed differences mainly in phosphatidylserine (PS) and cardiolipin (CL), although some differences were also observed in phosphatidylinositol (PI, LPI) and phosphatidylcholine (PC) phospholipids. PS with shorter fatty acyl chains were up-regulated in the BBN sedentary group, while the other species of PS with longer FA and a higher degree of unsaturation were down-regulated, but the BBN exercise group was mostly similar to control groups. Remarkably, exercise training prevented these alterations and had a positive impact on the ability of mitochondria to produce ATP, restoring the healthy phospholipid profile. The remodelling of mitochondria phospholipid profile in rats with urothelial carcinoma allowed confirming the importance of the lipid metabolism in mitochondria dysfunction in cancer-induced skeletal muscle remodelling. The regulation of phospholipid biosynthetic pathways observed in the BBN exercise group supported the current perspective that exercise is an adequate therapeutic approach for the management of cancer-related muscle remodeling.
Asunto(s)
Mitocondrias Musculares/metabolismo , Fosfolípidos/metabolismo , Neoplasias Urológicas/fisiopatología , Animales , Caquexia/metabolismo , Caquexia/prevención & control , Modelos Animales de Enfermedad , Lipidómica/métodos , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Condicionamiento Físico Animal , Ratas WistarRESUMEN
Despite the importance attributed to exercise training in the breast cancer (BC) continuum, the underlying mechanisms modulating tumor behavior are unknown. We evaluated the effects of long-term moderate-exercise in the development of mammary tumors, and studied the microenvironment of infiltrative lesions, the amount of connective tissue, and balance between cellular proliferation/death.Fifty Sprague-Dawley rats, randomly assigned into four groups: two control groups (sedentary and exercised) and two models of BC groups (sedentary and exercised) induced by N-methyl-N-nitrosoureia (MNU), were sacrificed after 35 weeks of moderate-exercise, and all perceptible tumors were removed for histological and immunohistochemistry analysis.The median number of infiltrative-lesions per animal was lower in the MNU exercised animals (p=0.02). More than one histological pattern was identified, and papillary carcinoma was the most frequent in both groups. Within infiltrative-lesions, the number of immunopositive cells per µm2 of Ki67 was lower in exercised animals (p=0.002). This presents increased cell death per µm2 (p=0.019). Tumors from sedentary animals had a higher expression of collagen deposition (p=0.027).Long-term moderate-exercise has beneficial effects in tumor development with a diminished prevalence of malignancy. Within infiltrative-lesions, moderate-exercise improves the balance between cell-proliferation and cell-death with decreased connective tissue that suggests lower tumor aggressiveness.
Asunto(s)
Neoplasias Mamarias Experimentales/terapia , Condicionamiento Físico Animal , Microambiente Tumoral , Animales , Carcinoma Papilar/terapia , Muerte Celular , Proliferación Celular , Femenino , Inmunohistoquímica , Invasividad Neoplásica , Ratas , Ratas Sprague-DawleyRESUMEN
Limiting cancer-induced cardiac damage has become an increasingly important issue to improve survival rates and quality of life. Exercise training has been shown to reduce cardiovascular complications in several diseases; however, its therapeutic role against cardiovascular consequences of cancer is in its infancy. In order to add new insights on the potential therapeutic effect of exercise training on cancer-related cardiac dysfunction, we used an animal model of urothelial carcinoma submitted to 13 weeks of treadmill exercise after 20 weeks of exposure to the carcinogenic N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN). Data showed that 13 weeks of treadmill exercise reverted cancer-induced cardiomyocytes atrophy and fibrosis, improved cardiac oxidative capacity given by citrate synthase activity and MnSOD content, and increased the levels of the mitochondrial biogenesis markers PGC-1α and mtTFA. Moreover, exercise training reverted cancer-induced decrease of cardiac c-kit levels suggesting enhanced regenerative ability of heart. These cardiac adaptations to exercise were related to a lower incidence of malignant urothelial lesions and less signs of inflammation. Taken together, data from the present study support the beneficial effect of exercise training when started after cancer diagnosis, envisioning the improvement of the cardiovascular function.
Asunto(s)
Remodelación Atrial , Condicionamiento Físico Animal , Neoplasias Urológicas/patología , Animales , Modelos Animales de Enfermedad , Fibrosis/prevención & control , Masculino , Atrofia Muscular/complicaciones , Atrofia Muscular/prevención & control , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Ratas , Ratas Wistar , Regeneración , Neoplasias Urológicas/metabolismo , Neoplasias Urológicas/fisiopatologíaRESUMEN
The use of the chemotherapeutic drug doxorubicin (DOX) is limited by its toxicity in several organs such as testes. So, we analyzed the effect of endurance treadmill exercise training (EX) performed before sub-chronic DOX treatment on sperm count and motility, testes markers of oxidative damage and apoptosis. Tissue profiling of proteins more susceptible to oxidation was made to identify the molecular pathways regulated by oxidative modifications, as nitration and carbonylation. Twenty-four adult male rats were divided into four groups (n=6/group): sedentary saline (SED+SAL), sedentary sub-chronically injected with DOX (2mg-kg-1 per week, during 7 weeks; SED+DOX), 12 weeks trained saline (EX+SAL) and trained treated with DOX (EX+DOX). DOX treatment started 5 weeks after the beginning of the exercise program. Testes caspase-3, -8 and -9, as well as aconitase activities, the content of malondialdehyde (MDA), sulfhydryl groups (-SH), carbonyl and nitrotyrosine derivatives were determined. Modified proteins were identified by 2D-Western blot followed by MALDI-TOF/TOF mass spectrometry, and bioinformatic analysis was performed to assess the biological processes regulated by these chemical modifications. The decreased sperm motility induced by DOX was not modified by exercise. Significant increases in MDA content in SED+DOX and in caspase-3 and -9 activities in EX+DOX were found. Despite no significant differences in the levels of carbonylated and nitrated proteins, exercise modulated testis proteome susceptibility to oxidation in DOX-treated group, with less modified proteins identified. Zinc finger Ran-binding domain-containing protein 2 (ZRAB2) and AN1-type zinc finger protein 3 (ZFAN3) were among the proteins found oxidativelly modified. Although no marked alterations in testes oxidative damage were noticed, proteomic analysis of oxidativelly modified proteins highlighted the protective role of exercise against oxidative damage of some proteins involved in metabolism and stress response against DOX.
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Doxorrubicina/toxicidad , Regulación de la Expresión Génica/efectos de los fármacos , Condicionamiento Físico Animal , Proteoma/efectos de los fármacos , Testículo/efectos de los fármacos , Testículo/metabolismo , Animales , Antibióticos Antineoplásicos/toxicidad , Apoptosis , Electroforesis en Gel Bidimensional , Masculino , Estrés Oxidativo , Distribución Aleatoria , Ratas , Espectrometría de Masas en Tándem , Transcriptoma/efectos de los fármacosRESUMEN
Exercise training has been recommended as a nonpharmacological strategy for the prevention and attenuation of skeletal muscle atrophy in distinct pathophysiological conditions. Despite the well-established phenotypic alterations, the molecular mechanisms underlying exercise-induced skeletal muscle remodeling are poorly characterized. Proteomics based on mass spectrometry have been successfully applied for the characterization of skeletal muscle proteome, representing a pivotal approach for the wide characterization of the molecular networks that lead to skeletal muscle remodeling. Nevertheless, few studies were performed to characterize the exercise-induced proteome remodeling of skeletal muscle, with only six research papers focused on the cross-talk between exercise and pathophysiological conditions. In order to add new insights on the impact of distinct exercise programs on skeletal muscle proteome, molecular network analysis was performed with bioinformatics tools. This analysis highlighted an exercise-related proteome signature characterized by the up-regulation of the capacity for ATP generation, oxygen delivery, antioxidant capacity and regulation of mitochondrial protein synthesis. Chronic endurance training up-regulates the tricarboxylic acid cycle and oxidative phosphorylation system, whereas the release of calcium ion into cytosol and amino acid metabolism are the biological processes up-regulated by a single bout of exercise. Other issues as exercise intensity, load, mode and regimen as well as muscle type also influence the exercise-induced proteome signature. The comprehensive analysis of the molecular networks modulated by exercise training in health and disease, taking in consideration all these variables, might not only support the therapeutic effect of exercise but also highlight novel targets for the development of enhanced pharmacological strategies.
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Ejercicio Físico/fisiología , Músculo Esquelético/metabolismo , Resistencia Física/fisiología , Proteómica/métodos , Ciclo del Ácido Cítrico/fisiología , Humanos , Espectrometría de Masas , Fosforilación OxidativaRESUMEN
Clinical studies suggest that aerobic exercise can exert beneficial effects in pulmonary arterial hypertension (PAH), but the underlying mechanisms are largely unknown. We compared the impact of early or late aerobic exercise training on right ventricular function, remodeling and survival in experimental PAH. Male Wistar rats were submitted to normal cage activity (SED), exercise training in early (EarlyEX) and in late stage (LateEX) of PAH induced by monocrotaline (MCT, 60 mg/kg). Both exercise interventions resulted in improved cardiac function despite persistent right pressure-overload, increased exercise tolerance and survival, with greater benefits in EarlyEX+MCT. This was accompanied by improvements in the markers of cardiac remodeling (SERCA2a), neurohumoral activation (lower endothelin-1, brain natriuretic peptide and preserved vascular endothelial growth factor mRNA), metabolism and mitochondrial oxidative stress in both exercise interventions. EarlyEX+MCT provided additional improvements in fibrosis, tumor necrosis factor-alpha/interleukin-10 and brain natriuretic peptide mRNA, and beta/alpha myosin heavy chain protein expression. The present study demonstrates important cardioprotective effects of aerobic exercise in experimental PAH, with greater benefits obtained when exercise training is initiated at an early stage of the disease.
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Hipertensión Pulmonar/terapia , Condicionamiento Físico Animal , Función Ventricular Derecha , Remodelación Ventricular , Animales , Biomarcadores/sangre , Tolerancia al Ejercicio , Hipertensión Pulmonar/inducido químicamente , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/fisiopatología , Masculino , Monocrotalina , Distribución Aleatoria , Ratas WistarRESUMEN
The present study aimed to test whether a chronic intermittent workload could induce an adaptive cardiac phenotype Chronic intermittent workload induced features of adaptive hypertrophy This was paralleled by protection against acute pressure overload insult The heart may adapt favourably to balanced demands, regardless of the nature of the stimuli. The present study aimed to test whether submitting the healthy heart to intermittent and tolerable amounts of workload, independently of its nature, could result in an adaptive cardiac phenotype. Male Wistar rats were subjected to treadmill running (Ex) (n = 20), intermittent cardiac overload with dobutamine (ITO) (2 mg kg(-1) , s.c.; n = 20) or placebo administration (Cont) (n = 20) for 5 days week(-1) for 8 weeks. Animals were then killed for histological and biochemical analysis or subjected to left ventricular haemodynamic evaluation under baseline conditions, in response to isovolumetric contractions and to sustained LV acute pressure overload (35% increase in peak systolic pressure maintained for 2 h). Baseline cardiac function was enhanced only in Ex, whereas the response to isovolumetric heartbeats was improved in both ITO and Ex. By contrast to the Cont group, in which rats developed diastolic dysfunction with sustained acute pressure overload, ITO and Ex showed increased tolerance to this stress test. Both ITO and Ex developed cardiomyocyte hypertrophy without fibrosis, no overexpression of osteopontin-1 or ß-myosin heavy chain, and increased expression of sarcoplasmic reticulum Ca(2+) protein. Regarding hypertrophic pathways, ITO and Ex showed activation of the protein kinase B/mammalian target of rapamycin pathway but not calcineurin. Mitochondrial complex IV and V activities were also increased in ITO and Ex. Chronic submission to controlled intermittent cardiac overload, independently of its nature, results in an adaptive cardiac phenotype. Features of the cardiac overload, such as the duration and magnitude of the stimuli, may play a role in the development of an adaptive or maladaptive phenotype.
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Adaptación Fisiológica , Cardiomegalia , Animales , Presión Sanguínea , Cardiomegalia/patología , Cardiomegalia/fisiopatología , Cardiotónicos/farmacología , Dobutamina/farmacología , Corazón/fisiología , Masculino , Mitocondrias Cardíacas/fisiología , Miocardio/patología , Tamaño de los Órganos , Fenotipo , Condicionamiento Físico Animal/fisiología , Ratas Wistar , Carrera/fisiologíaRESUMEN
Strategies to prevent tumour burden-induced cardiac remodelling that might progress to heart failure are necessary to improve patients' health outcomes and tolerability to cancer therapies. Exercise has been suggested as a measure to prevent cardiac damage; however, its effectiveness on regulating cardiac remodelling secondary to cancer was never addressed. Using an animal model of mammary tumorigenesis, we studied the impact of 35weeks of endurance training on heart, focusing on the signalling pathways modulated by pro-inflammatory and wasting cytokines. The cardiac fibrosis and myofiber disorganization induced by tumour burden was paralleled by the increase of myostatin and TWEAK with the activation of signalling pathways involving Smad-3, NF-κB, TRAF-6 and atrogin-1. The activation of Akt/mTOR was observed in heart from rats with tumours, for which contributed the extracellular matrix. Endurance training prevented the increase of serum and cardiac TWEAK promoted by cancer, as well as the activation of NF-κB, TRAF6, atrogin-1 and p70S6K in heart. Data highlight the impact of exercise in the modulation of signalling pathways activated by wasting cytokines and the resulting outcomes on heart adaptation. Future studies focused on the cellular pathways underlying cardiac remodelling will assist in the development of exercise programs targeting cancer-related cardiac alterations.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Caquexia/complicaciones , Caquexia/fisiopatología , Corazón/fisiopatología , Neoplasias Mamarias Animales/complicaciones , Proteínas de la Membrana/metabolismo , Miostatina/metabolismo , Condicionamiento Físico Animal , Factores de Necrosis Tumoral/metabolismo , Adaptación Fisiológica , Animales , Caquexia/metabolismo , Caquexia/patología , Citocina TWEAK , Femenino , Corazón/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Urea/química , Urea/farmacologíaRESUMEN
Doxorubicin (DOX) is an anti-cancer agent whose clinical usage results in a cumulative and dose-dependent cardiotoxicity. We have previously shown that exercise performed prior to DOX treatment reduces the resulting cardiac(mito) toxicity. We sought to determine the effects on cardiac mitochondrial toxicity of two distinct chronic exercise models (endurance treadmill training-TM and voluntary free-wheel activity-FW) when used prior and during DOX treatment. Male-young Sprague-Dawley rats were divided into six groups (n=6 per group): SAL+SED (saline sedentary), SAL+TM (12-weeks TM), SAL+FW (12-weeks FW), DOX+SED (7-weeks of chronic DOX treatment 2mg/kg per week), DOX+TM and DOX+FW. DOX administration started 5weeks after the beginning of the exercise protocol. Heart mitochondrial ultrastructural alterations, mitochondrial function (oxygen consumption and membrane potential), semi-quantification of oxidative phosphorylation (OXPHOS) proteins and their in-gel activity, as well as proteins involved in mitochondrial oxidative stress (SIRT3, p66shc and UCP2), biogenesis (PGC1α and TFAM), acetylation and markers for oxidative damage (carbonyl groups, MDA,SH, aconitase, Mn-SOD activity) were evaluated. DOX treatment resulted in ultrastructural and functional alterations and decreased OXPHOS. Moreover, DOX decreased complex I activity and content, mitochondrial biogenesis (TFAM), increased acetylation and oxidative stress. TM and FW prevented DOX-induced alteration in OXPHOS, the increase in oxidative stress, the decrease in complex V activity and in complex I activity and content. DOX-induced decreases in TFAM and SIRT3 content were prevented by TM only. Both chronic models of physical exercise performed before and during the course of sub-chronic DOX treatment translated into an improved mitochondrial bioenergetic fitness, which may result in part from the prevention of mitochondrial oxidative stress and damage.
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Antibióticos Antineoplásicos/efectos adversos , Cardiotoxinas/efectos adversos , Doxorrubicina/efectos adversos , Estrés Oxidativo , Condicionamiento Físico Animal , Animales , Antibióticos Antineoplásicos/administración & dosificación , Cardiotoxinas/administración & dosificación , Doxorrubicina/administración & dosificación , Metabolismo Energético , Humanos , Masculino , Ratas Sprague-DawleyRESUMEN
Alterations in muscle mitochondrial bioenergetics during cancer cachexia were previously suggested; however, the underlying mechanisms are not known. So, the goal of this study was to evaluate mitochondrial phospholipid remodeling in cancer-related muscle wasting and its repercussions to respiratory chain activity and fiber susceptibility to apoptosis. An animal model of urothelial carcinoma induced by exposition to N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) and characterized by significant body weight loss due to skeletal muscle mass decrease was used. Morphological evidences of muscle atrophy were associated to decreased respiratory chain activity and increased expression of mitochondrial UCP3, which altogether highlight the lower ability of wasted muscle to produce ATP. Lipidomic analysis of isolated mitochondria revealed a significant decrease of phosphatidic acid, phosphatidylglycerol and cardiolipin in BBN mitochondria, counteracted by increased phosphatidylcholine levels. Besides the impact on membrane fluidity, this phospholipid remodeling seems to justify, at least in part, the lower oxidative phosphorylation activity observed in mitochondria from wasted muscle and their increased susceptibility to apoptosis. Curiously, no evidences of lipid peroxidation were observed but proteins from BBN mitochondria, particularly the metabolic ones, seem more prone to carbonylation with the consequent implications in mitochondria functionality. Overall, data suggest that bladder cancer negatively impacts skeletal muscle activity specifically by affecting mitochondrial phospholipid dynamics and its interaction with proteins, ultimately leading to the dysfunction of this organelle. The regulation of phospholipid biosynthetic pathways might be seen as potential therapeutic targets for the management of cancer-related muscle wasting.
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Metabolismo Energético/genética , Atrofia Muscular/metabolismo , Estrés Oxidativo/genética , Neoplasias de la Vejiga Urinaria/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Apoptosis/genética , Butilhidroxibutilnitrosamina/toxicidad , Humanos , Canales Iónicos/metabolismo , Peroxidación de Lípido/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Proteínas Mitocondriales/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Atrofia Muscular/etiología , Atrofia Muscular/patología , Proteína Desacopladora 3 , Neoplasias de la Vejiga Urinaria/inducido químicamente , Neoplasias de la Vejiga Urinaria/complicaciones , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Moderate physical activity has traditionally been associated with the improvement of cardiac function and, consequently, with the extension of life span. Mitochondria play a key role in the adaptation of heart muscle to exercise-related metabolic demands. In order to disclose the molecular mechanisms underlying the beneficial effect of lifelong physical activity in cardiac function, we performed label-free quantitative mass spectrometry-based proteomics of Sprague-Dawley rat heart mitochondrial proteome and phosphoproteome. Our data revealed that 54 weeks of moderate treadmill exercise modulates the abundance of proteins involved in the generation of precursor metabolites and cellular respiration, suggesting an increase in carbohydrate oxidation-based metabolism. Moreover, from the 1335 phosphopeptides identified in this study, 6 phosphosites were exclusively assigned to heart mitochondria from sedentary rats and 17 to exercised animals, corresponding to 6 and 16 proteins, respectively. Most proteins exhibiting significant alterations in specific phosphorylation sites were involved in metabolism. Analysis of the acquired data led to the identification of several kinases potentially modulated by exercise training, which were selected for further validation. Indeed, higher protein abundance levels of RAF and p38 in mitochondria were confirmed to be modulated by sustained exercise. Our work describes the plasticity of heart mitochondria in response to long exercise programs manifested by the reprogramming of phosphoproteome and provides evidence for the kinases involved in the regulation of metabolic pathways and mitochondrial maintenance.
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Proteínas Mitocondriales/análisis , Miocardio/metabolismo , Fosfoproteínas/análisis , Condicionamiento Físico Animal/fisiología , Proteoma/análisis , Animales , Femenino , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilación , Mapas de Interacción de Proteínas/fisiología , Proteínas Quinasas/análisis , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Proteoma/química , Proteoma/metabolismo , Proteómica , Ratas , Ratas Sprague-DawleyRESUMEN
Diabetes mellitus is a chronic metabolic disease with multiple complications, and its successful management requires early diagnosis, to allow timely interventions. Here, we have comprehensively analyzed the proteome changes in urine of type 1 diabetic subjects with and without complications such as retinopathy and nephropathy. gel electrophoresis combined to liquid chromatography-tandem mass spectrometry (GeLC-MS/MS) analysis of midstream urine highlighted the mechanisms involved in disease pathogenesis as, for instance wound healing and blood coagulation in all diabetics or altered ganglioside metabolism in retinopathy, and also some urinary proteins with potential diagnosis value. From these, gelsolin and antithrombin-III appear as promising diagnosis markers for type 1 diabetes mellitus (T1DM), whereas ephrin type-B receptor 4 and vitamin K-dependent protein Z seem to be promising markers for advanced T1DM disease state presenting retinopathy and nephropathy (T1DM-R + N). Data also suggest urinary ganglioside GM2 activator and beta-hexosaminidase subunit beta as potential urinary markers of retinopathy in diabetics. Taken together, the present exploratory urinary proteomic analysis might be seen as an important starting point for studies targeting specific urinary proteins aimed at the implementation of new biomarkers for the early detection of T1DM-related microvascular complications.
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Biomarcadores/orina , Complicaciones de la Diabetes/orina , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/orina , Proteinuria , Proteómica , Nefropatías Diabéticas/orina , Retinopatía Diabética/orina , Electroforesis en Gel de Poliacrilamida , Femenino , Cromatografía de Gases y Espectrometría de Masas , Humanos , Masculino , Proteínas/química , Proteínas/genética , UrinálisisRESUMEN
Mitochondrial proteomics emerged aiming to disclose the dynamics of mitochondria under various pathophysiological conditions. In the present study we investigated the relative merits of gel-based (2DE and SDS-LC) and gel-free (2D-LC) protein separation approaches and protein identification algorithms (Mascot and Paragon) in the proteome profiling of mitochondria isolated from cultured fibroblasts, a sample traditionally used for diagnosis purposes. Combining data retrieved from 2DE, 2D-LC and SDS-LC and search methods, a total of 696 non-redundant proteins were identified. An overlap of only 19% between the proteins identified by the three different methods was observed when Mascot and Paragon were used. Regarding protein ID, a consistency in the number of identified proteins per sample was noticed for 2DE approach. Independent of the methodological approach chosen, it was noticed that the predominance in mitochondria of hydrophilic proteins with 20-50 kDa and pI 5-6 and 8-9; however, 2D-LC and SDS-LC allowed the enrichment of proteins with a mass below 30 kDa and of basic proteins with pI values above 8. In conclusion, data from the present study highlight the power of integrating different separation technologies and protein identification algorithms.
Asunto(s)
Algoritmos , Fibroblastos/química , Mitocondrias/química , Proteínas Mitocondriales/aislamiento & purificación , Proteoma/aislamiento & purificación , Cromatografía Liquida , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Humanos , Punto Isoeléctrico , Peso Molecular , Cultivo Primario de Células , Proteómica/métodosRESUMEN
With mitochondrion garnering more attention for its inextricable involvement in pathophysiological conditions, it seems imperative to understand the means by which the molecular pathways harbored in this organelle are regulated. Protein phosphorylation has been considered a central event in cellular signaling and, more recently, in the modulation of mitochondrial activity. Efforts have been made to understand the molecular mechanisms by which protein phosphorylation regulates mitochondrial signaling. With the advances in mass-spectrometry-based proteomics, there is a substantial hope and expectation in the increased knowledge of protein phosphorylation profile and its mode of regulation. On the basis of phosphorylation profiles, attempts have been made to disclose the kinases involved and how they control the molecular processes in mitochondria and, consequently, the cellular outcomes. Still, few studies have focused on mitochondrial phosphoproteome profiling, particularly in diseases. The present study reviews current data on protein phosphorylation profiling in mitochondria, the potential kinases involved and how pathophysiological conditions modulate the mitochondrial phosphoproteome. To integrate data from distinct research papers, we performed network analysis, with bioinformatic tools like Cytoscape, String, and PANTHER taking into consideration variables such as tissue specificity, biological processes, molecular functions, and pathophysiological conditions. For instance, data retrieved from these analyses evidence some homology in the mitochondrial phosphoproteome among liver and heart, with proteins from transport and oxidative phosphorylation clusters particularly susceptible to phosphorylation. A distinct profile was noticed for adipocytes, with proteins form metabolic processes, namely, triglycerides metabolism, as the main targets of phosphorylation. Regarding disease conditions, more phosphorylated proteins were observed in diabetics with some distinct phosphoproteins identified in type 2 prediabetic states and early type 2 diabetes mellitus. Heart-failure-related phosphorylated proteins are in much lower amount and are mainly involved in transport and metabolism. Nevertheless, technical considerations related to mitochondria isolation and protein separation should be considered in data comparison among different proteomic studies. Data from the present review will certainly open new perspectives of protein phosphorylation in mitochondria and will help to envisage future studies targeting the underlying regulatory mechanisms.