Functional evaluation of muscle oxidative metabolism in metabolic myophaties. A cross-talk between exercise physiology and clinical medicine

Metabolic myopathies are a heterogeneous group of diseases characterized by genetically determined defects which impair skeletal muscle energy production and/or function. These diseases include errors of glycogen metabolism, lipid metabolism, and mitochondrial respiratory chain. The most common clinical features are muscle weakness, pain, easy fatigability, cramping and, sometimes myoglobinuria due to muscle fiber necrosis. Exercise intolerance is another hallmark of clinical features of metabolic myopathies. As a result, patients note undue fatigue and dyspnea during low levels of exertion, including moderate activities of daily living. Symptoms are usually first experienced in childhood or early adulthood, however, late-onset is well recognized. The diagnostic process of these diseases usually begins with a careful medical history, a physical and neurological examination to assess reflexes, strength and the distribution of weakness. Creatine kinase is an extremely useful laboratory test for the evaluation of patients with a suspected myopathy and electromyography may be used to rule out a number of other neuromuscular disorders that cause similar patterns of weakness. As for exercise testing, the six minute walking test and the forearm ischaemic lactate test have traditionally been employed to screen for suspected disorders of glycogen metabolism; however, they have been superseded by improved biochemical and genetic techniques. Confirmation of the diagnosis usually requires muscle biopsy and/or molecular genetic testing. Functional evaluation of oxidative metabolism during exercise provides information regarding the physiological responses required by the cardiovascular and respiratory systems to meet the metabolic demands of the skeletal muscle. Moreover, the study of the physiological adjustments to exercise in patients is of extreme interest also from a “basic science” point of view, allowing to investigate aspects related to the regulation and integration of physiological and bioenergetic responses. Although exercise testing is widely utilized by cardiology, pulmonary, and sports medicine clinicians as a means to assess heart failure, respiratory disease, or athletic capacity, only few neurologists utilize a comprehensive assessment of oxidative metabolism to clarify the etiology of exercise intolerance and unexplained dyspnea among patients with metabolic myopathies. In previous studies our group applied on mitochondrial myopathies (MM) and McArdle’s disease (McA) patients two non−invasive methods of functional evaluation specifically aimed at oxidative metabolism at the skeletal muscle level. The variables of functional evaluation that we investigated were: A) Skeletal muscle oxygenation indices during exercise, obtained by near−infrared spectroscopy (NIRS) and taken as estimates of the capacity of O2 extraction; B) Kinetics of adjustment of pulmonary O2 uptake (V’O2 kinetics) during the transition from rest to exercise. We demonstrated that these methods allow to identify and quantify, in MM and in McA, the metabolic impairment. Moreover, these studies represent a typical example of “translational medicine”, in which methods and tools developed over the years in the exercise physiology laboratory are taken to the bed of the patient. In this thesis will be reported data of four studies in which the above−mentioned tools of functional evaluation of muscle oxidative metabolism were utilized, with specific purposes, on patients with metabolic myopathies. In the first study we evaluated, during a 24-month follow-up, cardiovascular and metabolic responses to exercise of a 50-yr-old patient with glycogen storage disease type II (Pompe disease)undergoing enzyme replacement therapy (ERT). At the same constant-workload submaximal exercise, rate of perceived exertion, pulmonary ventilation, and heart rate were lower during ERT versus pre-treatment, suggesting an increased exercise tolerance. Peak oxygen uptake increased by approximately 35% after 1 month of treatment and did not significantly change thereafter. Also, peak cardiac output significantly increased during ERT, whereas peak skeletal muscle fractional O2 extraction was unchanged compared with pre-treatment. Thus, this case report suggest that ERT may increase peak exercise capacity and exercise tolerance at submaximal workloads in patients with glycogen storage disease type II after 1 month of therapy, without no further changes occurring up to 24 months. In the second study, we followed the same approach of the case study previously mentioned and we evaluated the effects of 12-month of ERT on physiological variables related to exercise tolerance of four patients with Pompe disease. Patients performed an incremental exercise on a cycle ergometer, up to voluntary exhaustion, before and after 12 months of ERT. Peak workload and oxygen uptake values significantly increased after ERT whereas the observed increases of both peak cardiac output and the NIRS-determined peak skeletal muscle fractional O2 extraction were not statistically significant. These findings suggest that in glycogen storage disease type II patients enzyme replacement therapy is associated with a mild improvement of exercise tolerance. Since exercise training could improve exercise tolerance, motor function and muscle strength, counteracting the general deconditioning typical of chronic diseases, in the future may be interesting to evaluate if exercise training could be helpful in increasing ERT clinical efficacy, improving patients' muscle function and ameliorating their quality of life. A new study based upon a collaboration between neurologists and exercise physiologists has now started and it should give the opportunity to better investigate crucial issues related to patients’ follow-up and treatment. In the third study we evaluated in McArdle’s (McA) patients whether a first bout of exercise determines a sudden decrease in heart rate (HR) and an improved exercise tolerance (the so-called “second-wind” phenomenon) during a second bout, separated by the first by a few minutes of recovery. A second-wind phenomenon (marked decrease in heart rate and in the rating of perceived exertion) was indeed observed in McA patients during the second of two consecutive 6-min constant-work rate submaximal exercises. The second wind was associated with changes of physiological variables, suggesting an enhanced skeletal muscle oxidative metabolism: enhanced O2 extraction; signs of better matching between intramuscular O2 delivery and O2 utilization; disappearance of the “slow component” of pulmonary VO2 kinetics. The second wind was not described in McA patients after a longer (18-min) recovery period or in patients affected by a mitochondrial myopathy who have similar exercise intolerance. Besides being of interest from a basic science point of view (elucidating the mechanisms responsible for the second wind in McA patients), results of the present study are of interest also from a clinical point of view, since they identify a method (a warm up moderate-intensity exercise, carried out a few minutes before performing a task) capable of significantly increasing exercise tolerance in these patients. Finally, still unpublished data of another study are presented in this thesis, demonstrating the utility of non−invasive functional evaluation methods utilized by physiologists in the follow−up of patients as well as in the evaluation of the effects of therapies and/or rehabilitation intervention (i.e. exercise training). Since at present the therapeutic interventions available for metabolic myopathies patients are very limited and evidence from the literature suggests that moderate−intensity aerobic exercise training represents a safe intervention, the variables of functional evaluation determined by the above−mentioned tools were utilized to evaluate, in MM and McA patients, the effects of a program of moderate−intensity aerobic exercise training carried out by the patients at their home. Peak O2 uptake, variable evaluating maximal aerobic power, and peak skeletal muscle (vastus lateralis) fractional O2 extraction, as estimated by near-infrared spectroscopy (NIRS), increased significantly with training both in MM and in McA. Thus, training induced an increase of exercise tolerance at least in part due to a reduction of the impaired fractional O2 extraction by skeletal muscles. Moreover, training significantly speeded the V’O2 kinetics, even though only in the patients who had presented, before training, markedly slow V’O2 kinetics (i.e. sign of the most pronounced metabolic impairment). Surprisingly, the improvements in exercise tolerance obtained by the training program did not determine an increase in the habitual level of physical activity evaluated a couple of months after the termination of the training program. Overall, the results of the studies reported in this thesis demonstrate that, within a translational approach, a combination of traditional and more innovative functional evaluation methods can effectively detect the functional improvements of patients with metabolic myopathies following pharmacological and/or exercise interventions, yielding insights also on the mechanisms of the improvements at the pathophysiological level. Thus, functional evaluation of oxidative metabolism by non−invasive methods could be usefully employed in the diagnostic process of metabolic myopathies, in the follow−up of patients, and in the evaluation of the effects of therapies and/or rehabilitation interventions. Moreover, the analysis of the physiological and bioenergetics adaptations to exercise in patients with metabolic myopathies represents an interesting model to investigate and elucidate, in vivo, the regulation of basic physiological processes.openDottorato di ricerca in Scienze biomediche e biotecnologicheopenPorcelli, Simon

Training Effects on ROS Production Determined by Electron Paramagnetic Resonance in Master Swimmers

Acute exercise induces an increase in Reactive Oxygen Species (ROS) production dependent on exercise intensity with highest ROS amount generated by strenuous exercise. However, chronic repetition of exercise, that is, exercise training, may reduce exercise-induced oxidative stress. Aim of this study was to evaluate the effects of 6-weeks high-intensity discontinuous training (HIDT), characterized by repeated variations of intensity and changes of redox potential, on ROS production and antioxidant capacity in sixteen master swimmers. Time course changes of ROS generation were assessed by Electron Paramagnetic Resonance in capillary blood by a microinvasive approach. An incremental arm-ergometer exercise (IE) until exhaustion was carried out at both before (PRE) and after (POST) training (Trg) period. A significant (P<0.01) increase of ROS production from REST to the END of IE in PRE Trg (2.82±0.66 versus 3.28±0.66 µmol·min−1) was observed. HIDT increased peak oxygen consumption (36.1±4.3 versus 40.6±5.7 mL·kg−1·min−1 PRE and POST Trg, resp.) and the antioxidant capacity (+13%) while it significantly decreased the ROS production both at REST (−20%) and after IE (−25%). The observed link between ROS production, adaptive antioxidant defense mechanisms, and peak oxygen consumption provides new insight into the correlation between ROS response pathways and muscle metabolic function

Skeletal muscle oxidative function in vivo and ex vivo in athletes with marked hypertrophy from resistance training

Oxidative function during exercise was evaluated in 11 young athletes with marked skeletal muscle hypertrophy induced by long-term resistance training (RTA, body mass 102.67.3 kg, meanSD) and 11 controls (CTRL, body mass 77.86.0). Pulmonary O2 uptake (V\u27O2) and vastus lateralis muscle fractional O2 extraction (by near-infrared spectroscopy) were determined during an incremental cycle ergometer (CE) and one-leg knee-extension (KE) exercise. Mitochondrial respiration was evaluated ex vivo by high-resolution respirometry in permeabilized vastus lateralis fibers obtained by biopsy. Quadriceps femoris muscle cross sectional area, volume (determined by magnetic resonance imaging) and strength were greater in RTA vs. CTRL (by ~40%, ~33% and ~20%, respectively). V\u27O2peak during CE was higher in RTA vs. CTRL (4.050.64 L min-1 vs. 3.560.30)no difference between groups was observed during KE. The O2 cost of CE exercise was not different between groups. When divided per muscle mass (for CE) or quadriceps muscle mass (for KE) V\u27O2peak was lower (by 15-20%) in RTA vs. CTRL. Vastus lateralis fractional O2 extraction was lower in RTA vs. CTRL at all work rates, both during CE and KE. RTA had higher ADP-stimulated mitochondrial respiration (56.723.7 pmolO2s-1mg-1 ww) vs. CTRL (35.710.2), and a tighter coupling of oxidative phosphorylation. In RTA the greater muscle mass and maximal force, and the enhanced mitochondrial respiration seem to compensate for the hypertrophy-induced impaired peripheral O2 diffusion. The net results are an enhanced whole body oxidative function at peak exercise, and unchanged efficiency and O2 cost at submaximal exercise, despite a much greater body mas

Metabolic Escape Routes of Cancer Stem Cells and Therapeutic Opportunities

Although improvement in early diagnosis and treatment ameliorated life expectancy of cancer patients, metastatic disease still lacks effective therapeutic approaches. Resistance to anticancer therapies stems from the refractoriness of a subpopulation of cancer cells—termed cancer stem cells (CSCs)—which is endowed with tumor initiation and metastasis formation potential. CSCs are heterogeneous and diverge by phenotypic, functional and metabolic perspectives. Intrinsic as well as extrinsic stimuli dictated by the tumor microenvironment (TME)have critical roles in determining cell metabolic reprogramming from glycolytic toward an oxidative phenotype and vice versa, allowing cancer cells to thrive in adverse milieus. Crosstalk between cancer cells and the surrounding microenvironment occurs through the interchange of metabolites, miRNAs and exosomes that drive cancer cells metabolic adaptation. Herein, we identify the metabolic nodes of CSCs and discuss the latest advances in targeting metabolic demands of both CSCs and stromal cells with the scope of improving current therapies and preventing cancer progression

Grape seed extracts modify the outcome of oxaliplatin in colon cancer cells by interfering with cellular mechanisms of drug cytotoxicity

Grape seed extracts are commonly utilized as dietary supplements for their antioxidant properties, even from cancer patients. However, whether these natural extracts interfere with chemotherapeutics utilized in colon cancer treatment is still poorly investigated. The cytotoxicity of extracts from Italia and Palieri cultivars either alone or in combination with oxaliplatin was evaluated in colon cancer cells. Grape seed extracts displayed anti-proliferative activity depending on the concentration utilized through apoptosis induction. In combination, they affected the activation of Erk1/2 and counteracted the intrinsic and the extrinsic pathway of apoptosis, the DNA damage and the generation of ROS induced by oxaliplatin. Noteworthy grape seed extracts strongly enhanced the uptake of oxaliplatin into all cells, by affecting the cell transport system of platinum. The addition of these natural extracts to oxaliplatin strongly reduced the cellular response to oxaliplatin and allowed a huge accumulation of platinum into cells. Here, we shed light on the chemical biology underlying the combination of grape seed extracts and oxaliplatin, demonstrating that they might be detrimental to oxaliplatin effectiveness in colon cancer therap

Detecting Iron Oxidation States in Liquids with the VOXES Bragg Spectrometer

Determining the oxidation states of metals assumes great importance in various applications because a variation in the oxidation number can drastically influence the material properties. As an example, this becomes evident in edible liquids like wine and oil, where a change in the oxidation states of the contained metals can significantly modify both the overall quality and taste. To this end, here we present the MITIQO project, which aims to identify oxidation states of metals in edible liquids utilizing X-ray emission with Bragg spectroscopy. This is achieved using the VOXES crystal spectrometer, developed at INFN National Laboratories of Frascati (LNF), employing mosaic crystal (HAPG) in the Von Hamos configuration. This combination allow us to work with effective source sizes of up to a few millimeters and improves the typical low efficiency of Bragg spectroscopy, a crucial aspect when studying liquids with low metal concentration. Here we showcase the concept behind MITIQO, for a liquid solution containing oxidized iron. We performed several high-resolution emission spectra measurements, for the liquid and for different powdered samples containing oxidized and pure iron. By looking at the spectral features of the iron's K$\beta$ emission lineshape, we were able to obtain, for a liquid, a result consistent with the oxidized iron powders and successfully quantifying the effect of oxidation

A multi-domain ontology on healthy ageing for the characterization of older adults status and behaviour

Ageing is a multi-factorial physiological process and the development of novel IoT systems, tools and devices, specifically targeted to older people, must be based on a holistic framework built on robust scientific knowledge in different health domains. Furthermore, interoperability must be guaranteed using standardized frameworks or approaches. These aspects still largely lack in the specific literature. The main aim of the paper is to develop a new ontology (the NESTORE ontology) to extend the available ontologies provided by universAAL-IoT (uAAL-IoT). The ontology is based on a multidomain healthy ageing holistic model, structuring well-assessed scientific knowledge, specifically targeted to healthy older adults aged between 65 and 75. The tool is intended to support, and standardize heterogeneous data about ageing in compliance with the uAAL-IoT framework. The NESTORE ontology covers all the relevant concepts to represent 3 significant domains of ageing: (1) Physiological Status and Physical Activity Behaviour; (2) Nutrition; and (3) Cognitive and Mental Status and Social Behaviour. In total, 12 sub-ontologies were modelled with more than 60 classes and sub-classes referenced among them by using more than 100 relations and around 20 enumerations. The proposed ontology increases the uAAL collection by 40%. NESTORE ontology provides innovation both in terms of semantic content and technological approach. The thorough use of this ontology can support the development of a decision support system, to promote healthy ageing, with the capacity to do dynamic multi-scale modelling of user-specific data based on the semantic annotations of users’ profile

Near-infrared spectroscopy estimation of combined skeletal muscle oxidative capacity and O2 diffusion capacity in humans

The final steps of the O2 cascade during exercise depend on the product of the microvascular-tointramyocyte PO2 difference and muscle O2 diffusing capacity (DmO2). Non-invasive methods to determine DmO2 in humans are currently unavailable. Muscle oxygen uptake (mVO2) recovery rate constant (k), measured by near-infrared spectroscopy (NIRS) using intermittent arterial occlusions, is associated with muscle oxidative capacity in vivo. We reasoned that k would be limited by DmO2 when muscle oxygenation is low (kLOW), and hypothesized that: i) k in well-oxygenated muscle (kHIGH) is associated with maximal O2 flux in fiber bundles; and ii) Δk (kHIGH-kLOW) is associated with capillary density (CD). Vastus lateralis k was measured in 12 participants using NIRS after moderate exercise. The timing and duration of arterial occlusions were manipulated to maintain tissue saturation index (TSI) within a 10% range either below (LOW) or above (HIGH) half-maximal desaturation, assessed during sustained arterial occlusion. Maximal O2 flux in phosphorylating state was 37.7±10.6 pmol·s−1·mg−1 (~5.8 ml·min−1·100g−1). CD ranged 348 to 586 mm-2. kHIGH was greater than kLOW (3.15±0.45 vs 1.56±0.79 min-1, p\u3c0.001). Maximal O2 flux was correlated with kHIGH (r=0.80, p=0.002) but not kLOW (r=-0.10, p=0.755). Δk ranged -0.26 to -2.55 min-1, and correlated with CD (r=- 0.68, p=0.015). mVO2 k reflects muscle oxidative capacity only in well-oxygenated muscle. Δk, the difference in k between well- and poorly-oxygenated muscle, was associated with CD, a mediator of DmO2. Assessment of muscle k and Δk using NIRS provides a non-invasive window on muscle oxidative and O2 diffusing capacity

Messing Up the Cancer Stem Cell Chemoresistance Mechanisms Supported by Tumor Microenvironment

Despite the recent advances in cancer patient management and in the development of targeted therapies, systemic chemotherapy is currently used as a first-line treatment for many cancer types. After an initial partial response, patients become refractory to standard therapy fostering rapid tumor progression. Compelling evidence highlights that the resistance to chemotherapeutic regimens is a peculiarity of a subpopulation of cancer cells within tumor mass, known as cancer stem cells (CSCs). This cellular compartment is endowed with tumor-initiating and metastasis formation capabilities. CSC chemoresistance is sustained by a plethora of grow factors and cytokines released by neighboring tumor microenvironment (TME), which is mainly composed by adipocytes, cancer-associated fibroblasts (CAFs), immune and endothelial cells. TME strengthens CSC refractoriness to standard and targeted therapies by enhancing survival signaling pathways, DNA repair machinery, expression of drug efflux transporters and antiapoptotic proteins. In the last years many efforts have been made to understand CSCTME crosstalk and develop therapeutic strategy halting this interplay. Here, we report the combinatorial approaches, which perturb the interaction network between CSCs and the different component of TME