Ana Isabel Padrão

Physical exercise prior and during treatment reduces sub-chronic doxorubicin-induced mitochondrial toxicity and oxidative stress

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.

Bladder cancer-induced skeletal muscle wasting: disclosing the role of mitochondria plasticity.

Loss of skeletal muscle is a serious consequence of cancer as it leads to weakness and increased risk of death. To better understand the interplay between urothelial carcinoma and skeletal muscle wasting, cancer-induced catabolic profile and its relationship with muscle mitochondria dynamics were evaluated using a rat model of chemically induced urothelial carcinogenesis by the administration of N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN). The histologic signs of non-muscle-invasive bladder tumors observed in BBN animals were related to 17% loss of body weight and high serum levels of IL-1β, TNF-α, TWEAK, C-reactive protein, myostatin and lactate and high urinary MMPs activities, suggesting a catabolic phenotype underlying urothelial carcinoma. The 12% loss of gastrocnemius mass was related to mitochondrial dysfunction, manifested by decreased activity of respiratory chain complexes due to, at least partially, the impairment of protein quality control (PQC) systems involving the mitochondrial proteases paraplegin and Lon. This was paralleled by the accumulation of oxidatively modified mitochondrial proteins. In overall, our data emphasize the relevance of studying the regulation of PQC systems in cancer cachexia aiming to identify therapeutic targets to counteract muscle wasting.

Salivary Proteome and Peptidome Profiling in Type 1 Diabetes Mellitus Using a Quantitative Approach

In the present study, we applied iTRAQ-based quantitative approach to explore the salivary proteome and peptidome profile in selected subjects with type 1 diabetes, with and without microvascular complications, aiming to identify disease-related markers. From a total of 434 distinct proteins, bactericidal/permeability-increasing protein-like 1 and pancreatic adenocarcinoma up-regulated factor were found in higher levels in the saliva of all patients while increased content of other proteins like alpha-2-macroglobulin, defensin alpha 3 neutrophil-specific, leukocyte elastase inhibitor, matrix metalloproteinase-9, neutrophil elastase, plastin-2, protein S100-A8 and protein S100-A9 were related with microvascular complications as retinopathy and nephropathy. Protein-protein interaction network analysis suggests the functional clusters defense, inflammation and response to wounding as the most significantly associated with type 1 diabetes pathogenesis. Peptidome data not only support a diabetes-related higher susceptibility of salivary proteins to proteolysis (mainly of aPRP, bPRP1 and bPRP2), but also evidenced an increased content of some specific protein fragments known to be related with bacterial attachment and the accumulation of phosphopeptides involved in tooth protection. Overall, the salivary protein and peptide profile highlights the importance of the innate immune system in the pathogenesis of type 1 diabetes mellitus and related complications. This study provides an integrated perspective of salivary proteome and peptidome that should be further explored in future studies targeting specific disease markers.

Molecular insights into mitochondrial dysfunction in cancer-related muscle wasting.

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.

OXPHOS susceptibility to oxidative modifications: the role of heart mitochondrial subcellular location.

In cardiac tissue two mitochondria subpopulations, the subsarcolemmal and the intermyofibrillar mitochondria, present different functional emphasis, although limited information exists about the underlying molecular mechanisms. Our study evidenced higher OXPHOS activity of intermyofibrillar compared to subsarcolemmal mitochondria, paralleled by distinct membrane proteins susceptibility to oxidative damage and not to quantitative differences of OXPHOS composition. Indeed, subsarcolemmal subunits of respiratory chain complexes were more prone to carbonylation while intermyofibrillar mitochondria were more susceptible to nitration. Among membrane protein targets to posttranslational modifications, ATP synthase subunits alpha and beta were notoriously more carbonylated in both subpopulations, although more intensely in subsarcolemmal mitochondria. Our data highlight a localization dependence of cardiac mitochondria OXPHOS activity and susceptibility to posttranslational modifications.

Lipidomic characterization of streptozotocin-induced heart mitochondrial dysfunction.

Myocardial mitochondria dysfunction seems to represent an important pathogenic factor underlying cardiomyopathy, a common complication of type 1 diabetes mellitus (T1DM). Despite significant progress in the understanding of the molecular mechanisms of mitochondrial function in the heart, the interplay between phospholipids and membrane proteins of this organelle is still poorly comprehended. Using a well-characterized animal model of T1DM obtained by the administration of streptozotocin, phospholipid profiling of isolated mitochondria was performed using MS-based approaches, which was analyzed together with oxidative phosphorylation (OXPHOS) complexes activities and their susceptibility to oxidation, and the expression of cytochrome c, the uncoupling protein UCP-3 and the mitochondrial transcription factor Tfam. Although in higher amounts, mitochondria from T1DM heart presented lower OXPHOS activity and lower transcription ability. This profile was related to phospholipid (PL) remodeling characterized by higher phosphatidylcholine levels, lower phosphatidylglycerol, phosphatidylinositol and sphingomyelin content, higher amounts of long fatty acyl side chains and increased lipid peroxidation, particularly of cardiolipin (CL). CL peroxidation was paralleled by lower cytochrome c content. Though in higher levels, UCP-3 does not seem to protect heart mitochondrial PL and membrane proteins from the oxidative damage induced by four weeks of hyperglycemia. Taken together, our data suggest that PL remodeling of heart mitochondria is an early event in T1DM pathogenesis and is related with OXPHOS dysfunction.

Intermittent cardiac overload results in adaptive hypertrophy and provides protection against left ventricular acute pressure overload insult

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.

Impaired protein quality control system underlies mitochondrial dysfunction in skeletal muscle of streptozotocin-induced diabetic rats

Hyperglycaemia-related mitochondrial impairment is suggested as a contributor to skeletal muscle dysfunction. Aiming a better understanding of the molecular mechanisms that underlie mitochondrial dysfunction in type 1 diabetic skeletal muscle, the role of the protein quality control system in mitochondria functionality was studied in intermyofibrillar mitochondria that were isolated from gastrocnemius muscle of streptozotocin (STZ)-induced diabetic rats. Hyperglycaemic rats showed more mitochondria but with lower ATP production ability, which was related with increased carbonylated protein levels and lower mitochondrial proteolytic activity assessed by zymography. LC-MS/MS analysis of the zymogram bands with proteolytic activity allowed the identification of an AAA protease, Lon protease; the metalloproteases PreP, LAP-3 and MIP; and cathepsin D. The content and activity of the Lon protease was lower in the STZ animals, as well as the expression of the m-AAA protease paraplegin, evaluated by western blotting. Data indicated that in muscle from diabetic rats the mitochondrial protein quality control system was compromised, which was evidenced by the decreased activity of AAA proteases, and was accompanied by the accumulation of oxidatively modified proteins, thereby causing adverse effects on mitochondrial functionality.

Mitochondria proteome profiling: a comparative analysis between gel- and gel-free approaches.

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.

Lifelong Exercise Training Modulates Cardiac Mitochondrial Phosphoproteome in Rats

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.