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Extrahepatic tissues which oxidise ketone bodies also have the capacity to accumulate them under particular conditions. We hypothesised that acetyl-CoA accumulation and altered redox status during low-flow ischaemia would support ketone body production in the heart. Combining a Langendorff heart model of low-flow ischaemia/reperfusion with LC-MS/MS, we show that β-hydroxybutyrate (β-OHB) accumulated in the ischaemic heart to 23.9 nmol/gww, and was secreted into the coronary effluent. Sodium oxamate, an LDH inhibitor, increased ischaemic β-OHB levels 5.3-fold, and slowed contractile recovery. Inhibition of HMG-CoA synthase (HMGCS2) with hymeglusin lowered ischaemic β-OHB accumulation by 40%, despite increased flux through Succinyl-CoA:3-oxaloacid CoA transferase (SCOT), resulting in greater contractile recovery. Hymeglusin also protected cardiac mitochondrial respiratory capacity during ischaemia/reperfusion. In conclusion, net ketone generation occurs in the heart under conditions of low-flow ischaemia. The process is driven by flux through both HMGCS2 and SCOT, and impacts on cardiac functional recovery from ischaemia/reperfusion.

Thyroid hormones regulate adult metabolism partly through actions on mitochondrial oxidative phosphorylation (OXPHOS). They also affect neurological development of the brain, but their role in cerebral OXPHOS before birth remains largely unknown, despite the increase in cerebral energy demand during the neonatal period. Thus, this study examined prepartum development of cerebral OXPHOS in hypothyroid fetal sheep. Using respirometry, Complex I (CI), Complex II (CII), and combined CI&CII OXPHOS capacity were measured in the fetal cerebellum and cortex at 128 and 142 days of gestational age (dGA) after surgical thyroidectomy or sham operation at 105 dGA (term ~145 dGA). Mitochondrial electron transfer system (ETS) complexes, mRNA transcripts related to mitochondrial biogenesis and ATP production, and mitochondrial density were quantified using molecular techniques. Cerebral morphology was assessed by immunohistochemistry and stereology. In the cortex, hypothyroidism reduced CI-linked respiration and CI abundance at 128 dGA and 142 dGA, respectively, and caused upregulation of PGC1α (regulator of mitochondrial biogenesis) and thyroid hormone receptor β at 128 dGA and 142 dGA, respectively. In contrast, in the cerebellum, hypothyroidism reduced CI&II- and CII-linked respiration at 128 dGA, with no significant effect on the ETS complexes. In addition, cerebellar glucocorticoid hormone receptor and adenine nucleotide translocase (ANT1) were downregulated at 128 dGA and 142 dGA, respectively. These alterations in mitochondrial function were accompanied by reduced myelination. The findings demonstrate the importance of thyroid hormones in the prepartum maturation of cerebral mitochondria and have implications for the etiology and treatment of the neurodevelopmental abnormalities associated with human prematurity and congenital hypothyroidism.

Prenatal origins of heart disease in the adult offspring are established. However, work in species of developmental milestones comparable to humans is lacking, preventing clinical translation. We combine experiments in vivo with those at the isolated organ, cellular, mitochondrial and molecular levels in sheep and chickens, two species with similar cardiovascular developmental milestones to humans. We studied mitochondria-targeted antioxidant intervention with MitoQ against cardiovascular dysfunction programmed by developmental hypoxia, a common complication in human pregnancy. Experiments in sheep determined MitoQ actions on in vivo fetal and adult cardiovascular function through surgical techniques not possible in humans, while those in chicken embryos isolated effects independent of maternal or placental influences. We show that hypoxia generates mitochondria-derived oxidative stress during development of the cardiovascular system, programming endothelial dysfunction and hypertension in the adult offspring. MitoQ treatment during hypoxic development protects against this cardiovascular risk in the adult offspring via enhanced NO signalling pathways, offering plausible intervention.

Dietary inorganic nitrate prevents aspects of cardiac mitochondrial dysfunction induced by hypoxia, although the mechanism is not completely understood. In both heart and skeletal muscle, nitrate increases fatty acid oxidation capacity, and in the latter case, this involves up-regulation of peroxisome proliferator-activated receptor (PPAR)α expression. Here, we investigated whether dietary nitrate modifies mitochondrial function in the hypoxic heart in a PPARα-dependent manner. Wild-type (WT) mice and mice without PPARα (Ppara-/-) were given water containing 0.7 mM NaCl (control) or 0.7 mM NaNO3 for 35 d. After 7 d, mice were exposed to normoxia or hypoxia (10% O2) for the remainder of the study. Mitochondrial respiratory function and metabolism were assessed in saponin-permeabilized cardiac muscle fibers. Environmental hypoxia suppressed mass-specific mitochondrial respiration and additionally lowered the proportion of respiration supported by fatty acid oxidation by 18% (P < 0.001). This switch away from fatty acid oxidation was reversed by nitrate treatment in hypoxic WT but not Ppara-/- mice, indicating a PPARα-dependent effect. Hypoxia increased hexokinase activity by 33% in all mice, whereas lactate dehydrogenase activity increased by 71% in hypoxic WT but not Ppara-/- mice. Our findings indicate that PPARα plays a key role in mediating cardiac metabolic remodeling in response to both hypoxia and dietary nitrate supplementation.-Horscroft, J. A., O'Brien, K. A., Clark, A. D., Lindsay, R. T., Steel, A. S., Procter, N. E. K., Devaux, J., Frenneaux, M., Harridge, S. D. R., Murray, A. J. Inorganic nitrate, hypoxia, and the regulation of cardiac mitochondrial respiration-probing the role of PPARα.

The increasing global prevalence of diabetes has been accompanied by a rise in diabetes-related conditions. This includes diabetic cardiomyopathy, a progressive form of heart disease that occurs with both insulin-dependent (type-1) and insulin-independent (type-2) diabetes and arises in the absence of hypertension or coronary artery disease. Over time, diabetic cardiomyopathy can develop into overt heart failure. Like other forms of cardiomyopathy, diabetic cardiomyopathy is accompanied by alterations in metabolism which could lead to further progression of the pathology, with metabolic derangement postulated to precede functional changes in the diabetic heart. Moreover in the case of type-2 diabetes, underlying insulin resistance is likely to prevent the canonical substrate switch of the failing heart away from fatty acid oxidation towards increased use of glycolysis. Analytical chemistry techniques, collectively known as metabolomics, are useful tools for investigating the condition. In this article, we provide a comprehensive review of those studies that have employed metabolomic techniques, namely chromatography, mass spectrometry and nuclear magnetic resonance spectroscopy, to profile metabolic remodelling in the diabetic heart of human patients and animal models. These studies collectively demonstrate that glycolysis and glucose oxidation are suppressed in the diabetic myocardium and highlight a complex picture regarding lipid metabolism. The diabetic heart typically shows an increased reliance on fatty acid oxidation, yet triacylglycerols and other lipids accumulate in the diabetic myocardium indicating probable lipotoxicity. The application of lipidomic techniques to the diabetic heart has identified specific lipid species that become enriched and which may in turn act as plasma-borne biomarkers for the condition. Metabolomics is proving to be a powerful approach, allowing a much richer analysis of the metabolic alterations that occur in the diabetic heart. Careful physiological interpretation of metabolomic results will now be key in order to establish which aspects of the metabolic derangement are causal to the progression of diabetic cardiomyopathy and might form the basis for novel therapeutic intervention.

The syndrome of critical illness is a complex physiological stressor that can be triggered by diverse pathologies. It is widely believed that organ dysfunction and death result from bioenergetic failure caused by inadequate cellular oxygen supply. Teleologically, life has evolved to survive in the face of stressors by undergoing a suite of adaptive changes. Adaptation not only comprises alterations in systemic physiology but also involves molecular reprogramming within cells. The concept of cellular adaptation in critically ill patients is a matter of contention in part because medical interventions mask underlying physiology, creating the artificial construct of "chronic critical illness," without which death would be imminent. Thus far, the intensive care armamentarium has not targeted cellular metabolism to preserve a temporary equilibrium but instead attempts to normalize global oxygen and substrate delivery. Here, we review adaptations to hypoxia that have been demonstrated in cellular models and in human conditions associated with hypoxia, including the hypobaric hypoxia of high altitude, the intrauterine low-oxygen environment, and adult myocardial hibernation. Common features include upregulation of glycolytic ATP production, enhancement of respiratory efficiency, downregulation of mitochondrial density, and suppression of energy-consuming processes. We argue that these innate cellular adaptations to hypoxia represent potential avenues for intervention that have thus far remained untapped by intensive care medicine.

More than half a century of skeletal muscle research is continuing at Padua University (Italy) under the auspices of the Interdepartmental Research Centre of Myology (CIR-Myo), the European Journal of Translational Myology (EJTM) and recently also with the support of the A&CM-C Foundation for Translational Myology, Padova, Italy. The Volume 30(1), 2020 of the EJTM opens with the collection of abstracts for the conference “2020 Padua Muscle Days: Mobility Medicine 30 years of Translational Research”. This is an international conference that will be held between March 18-21, 2020 in Euganei Hills and Padova in Italy. The abstracts are excellent examples of translational research and of the multidimensional approaches that are needed to classify and manage (in both the acute and chronic phases) diseases of Mobility that span from neurologic, metabolic and traumatic syndromes to the biological process of aging. One of the typical aim of Physical Medicine and Rehabilitation is indeed to reduce pain and increase mobility enough to enable impaired persons to walk freely, garden, and drive again. The excellent contents of this Collection of Abstracts reflect the high scientific caliber of researchers and clinicians who are eager to present their results at the PaduaMuscleDays. A series of EJTM Communications will also add to this preliminary evidence., Multiple biological pathways contribute to aging. Some prominent pathways and causes are genome instability, immunosenescence, inflammation, the NAD+ salvage pathways, proteostasis, and mitochondrial dysfunction. Altered gene regulation influenced by epigenetics, circadian rhythms, diet and physical activity levels also influences the rate of aging. Our Institute on Aging and collaborators are involved with multiple clinical trials with the goal to target these pathways and to improve physical and cognitive function or slow the rate of decline typically seen during aging. The ultimate goal is to develop interventions that can maintain health and independence of older adults. Trials ongoing and/or completed have utilized compounds shown to extend health-span and/or life span in invertebrates (c. elegans) or vertebrates (mice/rats). The compounds used range from nutritional (nicotinamide riboside, epicatechin, resveratrol, omega 3, fermented papaya) to pharmacological (metformin, aspirin, rapamycin, telmisartin/losartan, testosterone), lifestyle-physiological interventions (calorie restriction, exercise, fasting regimes). These biological compounds and lifestyle interventions target single or multiple pathways to potentially improve mitochondrial function, mitobiogenesis, autophagy, angiogenesis, nitric oxide production, and levels of NAD+ and/or reduce pathways or biomarkers of inflammation or senescence. In most of these trials we collect skeletal muscle, fat, white blood cells and plasma to determine if the compound alters the proposed biological pathway. We will briefly discuss some clinical trials and highlight findings on a recently completed clinical trial using epicatechins a flavonoid, which improved endothelial and mitochondrial function as well as walking speed. We will also highlight the challenges and limitations in conducting clinical trials, such as dose, duration of study, targeting single vs. multiple pathways, combining an intervention with exercise and/or intermittent fasting.1-5, Age-related cognitive decline has become a major public health concern. Even in the absence of frank neurodegenerative disorders, both cognitive and brain function decline as a function of advancing age. At present, there are few effective intervention options for remediating age-related cognitive and neural decline. Fermented Papaya Product has previously shown promise in human and animal models as a potential nutraceutical compound for impacting mitochondrial function, inflammation, and other age-related processes that contribute to cognitive and neural decline.1-3 The current study presents data from a pilot crossover clinical trials in 28 healthy older adults undergoing 8 weeks of treatment on either 9mg/day FPP vs. 9mg/day placebo (table sugar) with a 6-week washout period. Participants underwent multimodal magnetic resonance imaging and spectroscopy before and after each arm, as well as comprehensive neurocognitive assessment. Results demonstrated promising effect sizes on default mode resting state connectivity (Cohen’s d = .44), measures of verbal fluency (d = 1.16) and attention (d = .61), as well as two out of three novel markers of mitochondrial function from 31P magnetic resonance spectroscopy and neuroinflammation from free-water quantification in diffusion weighted imaging (d = .38). While results failed to survive significance after multiple comparison correction, the pattern of effect size improvement in FPP vs. placebo demonstrate significant promise for further investigation in larger Phase II randomized clinical trials. Potential implications for both healthy older adults and adults with neurodegenerative disease will be discussed., Prolonged oxidative stress may play a key role in tumor development. Antioxidants molecules are contained in many foods and seem to have potential role in future anti-tumor strategies. Among the natural antioxidants, the beneficial effect of Fermented Papaya (FPP®) is known. The aim of this study was to investigate the effects of orally administered FPP® in either prevention or treatment of a murine model of melanoma. The tumor growth was analyzed together with the blood levels of both oxidants (ROS) and anti-oxidants (SOD-1 and GSH). The results showed that FPP® controlled tumor growth, reducing the tumor mass of about 3 to 7 times vs untreated mice. The most significant effect was obtained with sublingual administration of FPP® close to the inoculation of melanoma. At the time of the sacrifice, none of mice treated with FPP® had metastases, the subcutaneous tumors were significantly smaller, and amelanotic compared to untreated mice. Moreover, the FPP® anti-tumor effect was consistent with the decrease of total ROS levels and the increase in the blood levels of GSH and SOD-1. This study shows that a potent anti-oxidant treatment through FPP® may contribute to both preventing and inhibiting tumors growth. The results of the above study suggested that FPP® while showing a clear anti-tumor effect it occurred through the in vivo induction of a potent anti-oxidant reaction. In a new set of experiments, we wanted to verify whether FPP had a clear and scientifically solid in vivo anti-aging effect together with the induction of the anti-oxidant reaction. To this purpose we used a mouse model suitable for aging studies (C576J) treating daily each mouse from 4 weeks of life to 10 months with the same dose of IMMUNEAGE dissolved into the daily water as compared to mice receiving only tap water. At the end of the treatment period (10 months) we measured some biological parameters related to aging cell processes: i) the total anti-oxydant capacity in the plasma of mice treated or untreated with FPP®; ii) the telomerase activity in the plasma of mice treated or untreated with FPP®; iii) the telomeres length in the bone marrow and ovaries of mice treated or untreated with FPP®. The results showed that the blood of treated mice, at the end of the treatment period (10 months) had 2-3 folds more anti-oxidant power and telomerase activities than the untreated mice. In the same mice at the sacrifice, we collected both the bone marrow (from the tibias) and the ovaries. We measured the telomere lengths in both cellular preparations. The results showed that daily FPP® assumption induced 3 folds increase in telomeres length in bone marrow and ovary of treated mice as compared to the untreated mice. This suggests that FPP® induce a clear improvement of the aging biomarkers and that the treated mice were for those variables younger than the untreated ones., Increased intake of a diet high in phosphorus (P) or phosphate (Pi) is associated with an increased risk of mortality in patients with chronic kidney disease or cardiovascular disease. Muscle fibers with ectopic calcification are observed in young patients with severe muscle degeneration. Ectopic calcification was also observed in the skeletal and cardiac muscles of mdx mice. In this study, we determined the effects of dietary phosphorus intake on ectopic calcification in skeletal muscle, muscle performance (specific maximal forces and daily running activity), and pathological features during exercise-induced stress in mdx mice. Increased level of dietary P or Pi intake also increased ectopic calcification and decreased muscle performance in mdx mice. On the other hand, decreased level of dietary P or Pi intake decreased ectopic calcification and muscle degeneration and improved muscle performance. When we added calcium absorber in high phosphorus diet, the ectopic calcification decreased significantly. In C2C12 muscle cell cultures, the increased concentration of Pi in culture medium inhibited myogenesis in a dose-dependent manner. Therefore, we propose that phosphorus (P) or phosphate (Pi) is one of the modifiers in regulating muscle degradation/inflammation in vivo and in vitro. In addition, we also found that the long-lasting Vitamin C, ascorbic acid-2 phosphate added into culture medium supported myogenesis even under high Pi- or low temperature-culture conditions. These results suggest that vitamin C may have therapeutic effect on muscle degeneration caused by increased level of Pi or lowered culture temperature., The neuromuscular junctions (NMJ) are specialized synapses between motor neuron and muscle fibers. They are targets for a variety of neuromuscular diseases. Maintenance and remodeling of NMJ are critical to treat these diseases. Different therapeutic strategies for neuromuscular diseases have been developed ranging from gene therapy to antisense-mediated exon skipping and nonsense mutation suppression by small molecules. We are attempting to search for therapeutic potential molecules from natural sources such as several microorganisms and chemical libraries. Among others, natural bioactive molecules, glucocorticoids and vitamin D have been proposed as potential treatments to delay progression in neuromuscular diseases and aging. However, the direct mechanisms for skeletal muscle have not been fully characterized. Recently, using cultured C2C12 myotubes, we showed that glucocorticoids and vitamin D enhance agrin-induced acetylcholine receptors (AChR) clustering compared to agrin alone. However, sex hormones such as estradiol and testosterone are less effective. To elucidate the physiological role of these compounds in agrin-induced AChR clustering, we investigated and will discuss their effects on expression of key molecules involved in AChR cluster formation and maintenance. This work was supported in part by Japan Society for the Promotion of Science, JSPS Grants-in-Aid for Scientific Research, Grant number:17K10089., Japan is facing a “super-aging” society. Seniors over 65 is 28.4% of the population and the life expectancy is the highest in the world, however, the nation’s annual medical cost is now over 42.6 trillion yen. If this trend continues, the Japanese government will not be able to sustain the social services. Therefore, Osato Research Institute has been conducting researches, aimed at reducing medical costs through Preventive Medicine. Our three core activities for achieving this goal are Education, Researches on Fermented Foods, and Improving QOL of senior generation. Firstly, we think the best preventive medicine is education. For example, AIDS is a typical disease that can be prevented by education. We have been working on young people’s education for AIDS prevention with Prof. Montagnier, president of World Foundation AIDS Research and Prevention at UNESCO. Secondly, we have been focusing our attention on “Fermented Food” since traditional fermented foods are considered to be a key to the world's highest longevity of Japanese people. Among our numerous studies, I will present 5 researches on Fermented Papaya Preparation,1-5 which may be a desired candidate for preventive medicine. Finally, we would like to introduce our activities of “Project ORI wine”. We established vineyards to make a place for social activities especially for local retired people. It would give them fun and challenge to live better and keep them productive and healthy. In this way, we hope we could have happy world not only for young but also for senior people., Mitochondria play a crucial role in health and disease, imposing a growing demand for evaluation of mitochondrial function in inherited and preventable degenerative diseases. These are associated with lack of physical exercise and obesity, causing decline of mitochondrial fitness and early aging. High-resolution respirometry (HRR) is based on the state-of-the-art instrument (Oroboros O2k, Innsbruck, Austria) for mitochondrial and cell respiration.1 The O2k combines real-time respirometry with simultaneous measurement of H2O2 production, mt-membrane potential, ADP-ATP exchange, pH, Ca2+, or nitric oxide. HRR can be applied with a wide spectrum of sample preparations (e.g. isolated mitochondria, tissue homogenate, permeabilized fibers, permeabilized cells, and living cells). Optimized substrate-uncoupler-inhibitor titration (SUIT) protocols offer a powerful tool for comprehensive mitochondrial physiology analysis by HRR.2 Peripheral blood mononuclear cells, platelets and permeabilized fibers are relevant models for diagnostic analysis of oxidative phosphorylation (OXPHOS) in clinical applications (e.g. aging, sarcopenia, obesity). Science and particularly mitochondrial respiratory research face a reproducibility crisis.3 Consequently, Oroboros is strongly committed towards reproducibility by promoting: (1) O2k quality control (i.e. calibrations, instrumental background, DatLab software); (2) proficiency tests with reference samples for inter-laboratory harmonization4,5; and (3) training by scientific experts. Supported by the COST Action MitoEAGLE network, we generate reference values for an Open-Access mitochondrial respirometry database, which requires harmonization of nomenclature.6 With a large O2k-Network, the Oroboros Ecosystem operates in the frame of collaborative Open-Innovation (NextGen-O2k with Q-redox sensor, NAD(P)H autofluorescence and PhotoBiology module) to promote quality and reproducibility in mitochondrial physiology. Support. Contribution to European Union Framework Programme Horizon 2020 COST Action CA15203 MitoEAGLE. Supported by project NextGen-O2k which has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 859770., Skeletal muscle plays a significant role in the regulation of whole-body metabolism, and mitochondria are essential organelles for ATP production, thus the studies focused on skeletal muscle mitochondrial activity are necessary to understand the molecular mechanisms controlling the skeletal muscle homeostasis.1 One of the key regulators of mitochondrial metabolism is the second messenger Ca2+. Upon physiological stimuli, skeletal muscle mitochondria rapidly and efficiently accumulate Ca2+ into their matrix via an electrogenic pathway, which relies on the driving force of a steep electrochemical gradient. A large [Ca2+]mt peak occurs dynamically in parallel to agonist-induced [Ca2+]cyt increases, thanks to the activity of the Mitochondrial Calcium Uniporter (MCU), the highly selective channel responsible for mitochondrial Ca2+ accumulation.2 Mitochondrial Ca2+ stimulates aerobic metabolism, tightly regulating three dehydrogenases of the TCA cycle, thus the alteration of mitochondrial Ca2+ homeostasis leads to a dysregulation of metabolism.3 In this talk, we will show an overview of techniques to monitor mitochondrial activity in skeletal muscle, starting with the ex vivo measure of Ca2+ signaling, in terms of cytosolic Ca2+ concentration and mitochondrial Ca2+ uptake, in single isolated fibers thanks to the use of genetically encoded Ca2+ probes.4 Then, to investigate skeletal muscle mitochondrial metabolism we take advantage of genetically encoded indicators for ATP and NADH (Perceval and Peredox probes), and O2 consumption rate measurements.5 Finally, mitophagy, the removal of damaged mitochondria, is a necessary cellular process to maintain healthy muscle homeostasis. Mt-Keima probe is an easy tool to monitor this phenomenon in muscle fibers. Together, all these techniques allow studying many processes that regulate mitochondrial metabolism, a key aspect in the pathophysiology of skeletal muscle., Mitochondria are implicated in atrophy of aging skeletal muscle, yet the link between specific changes in mitochondrial function and aging muscle atrophy has not been established. Amongst many changes occurring with aging, there is a sensitization to mitochondrial permeability transition (MPT) in aged skeletal muscle.1 Building on this observation, we took advantage of heterogeneity in atrophy with aging between hindlimb muscles of the rat, in combination with a muscle cell culture system, to evaluate the hypothesis that MPT is a mechanism of aging muscle atrophy. Our results showed that, exclusive to muscles that atrophy with aging, mitochondria are sensitized to MPT and there is a marked increase in nuclei positive for the mitochondrial-derived protein, apoptosis inducing factor (AIF). Within a given muscle, aged muscle fibers harboring AIF-positive nuclei are severely atrophied relative to normal muscle fibers. In a muscle cell culture system, induction of MPT using doxorubicin is coincident with AIF translocation to myotube nuclei and causes atrophy that can be blocked using inhibitors of MPT (cyclosporin A, bonkrekic acid), inhibiting mitochondrial reactive oxygen species (ROS; using MitoTempo), or knocking down caspase 3 (using shRNA). Together, these results show that: (1) MPT is linked to aging muscle atrophy from the whole muscle to single fiber level; and (2) MPT causes muscle cell atrophy in cell culture in a manner that depends upon both mitochondrial ROS and caspase 3. Our findings thus support the hypothesis that MPT is a mechanism of aging muscle atrophy, FBXL4-related encephalomyopathic mitochondrial DNA depletion syndrome is an autosomal recessive severe, multi-systemic mitochondrial disease with 48 known pathogenic variants. To better understand FBXL4 function and therapies, we characterized novel C. elegans and D. rerio models of FBXL4 disease. This study particularly focuses on analyzing the neuromuscular function of these genetic models using manual and automated assays. We investigated fbxl-1 worms’ neuromuscular function analyzing pharyngeal pump rate, body bends rate and overall motor activity using manual and automated assays. A novel automated video-tracking approach was used to evaluate overall worms’ activity in liquid media. In the latter case, we created a protocol based on the use of the Zebrabox (Viewpoint), automated software created and used for analyzing zebrafish larvae activity and only in this study, applied to worms. Worms’ neuromuscular function was impaired with significantly increased and uncoordinated pharyngeal pumping rate in young adult and decreased rate during aging. Motility was reduced by ~70% compared to N2 control worms. Automated Video-tracking analysis was also used to analyze swim activity in fbxl4sa12470 zebrafish larvae carrying a missense mutation in the FBXL4 allele. Data shows decreased (~50 %) swimming activity in basic condition and after applying stimuli of light on/off cycles. Overall, these data demonstrate that FBXL4 deficiency can be effectively modeled in simple translational animals; new protocols were created for using automated software applications offering robust translational platforms in which therapies can now be modeled to reverse organ-level and behavioral dysfunction directly relevant to the human disease., The neuromuscular junction (NMJ) plays an essential role in enabling the cross-talk between the motoneuron (MN) and skeletal muscle. With progressive age, oxidative damage due to mitochondrial dysfunction, seems to cause a ‘dying-back’ phenomenon, where damage at the axon terminal, causes degeneration of the entire cell.1 The ensuing death of MNs leads to muscle fibre denervation and loss of the entire motor units, a main cause of sarcopenia.2 NMJ degeneration may also arise from changes at the muscle level. In ageing muscle, several phenomena could also cause NMJ damage: mitochondrial dysfunction, increased ROS production, decreased PGC-1alpha expression, known to trigger NMJ changes with ageing.3 Inactivity also promotes muscle fibre denervation as increased neural cell adhesion molecule (N-CAM), marker of denervation/reinnervation, has been found after short periods of bed rest (10-14 days) in young and older men.4,5 In this study, we tested the hypothesis that regular physical activity, such as recreational dancing, would protect against NMJ degeneration while improving functional performance of older individuals. Thirty-one participants aged 60>years (70.4 ± 5.1 years) were recruited for this study after Ethical approval and participants’ informed consent. Among these, 15 (9 female, 6 male) were sedentary (S), while 16 (10 female, 6 male) practiced regular recreational dancing (D) at least 1.5 h/week for a minimum of 1 year. NMJ degeneration was assessed from serum c-terminal agrin fragment (CAF) levels using a commercially available Elisa kit assay. Gait performance was tested with the timed 10m walk test (10WT), dynamic balance (WD) was tested with a stabilometric tablet measuring ankle oscillations, while timed get-up-and go (TUG) was tested as the time taken to get up from a chair, walk 3m and sit back. Muscle size and architecture of the knee extensors were assessed using ultrasonography. Significance was set at p, Stimulation of myogenic cells with vasopressin (AVP), oxytocin (OT), their analogs and antagonists revealed a previously unknown role of the neurohypophyseal hormones (NH) in modulating myogenic differentiation and trophism1 through the activation of complex intracellular signaling, the expression of the MRFs and the stimulation of the hypertrophy pathways2,3. Other studies demonstrated the expression of functional OT receptors (OTR) in human and murine satellite cells (reviewed in2). Extending these studies to in vivo models, we could show a highly stimulated expression of the V1a AVP receptor (V1aR) in post-injury muscle regeneration and assess that the overexpression of V1aR suffices, without exogenous hormone administration, to sustain muscle regeneration2. Furthermore, the administration of AVP rescued the inhibitory effect of TNF in a mouse model of cancer cachexia (reviewed in2). Works conducted in bovine and ovine livestock showed that animals treated with anabolizing steroids displayed dramatically increased OT expression in skeletal muscle, leading to a significant increase of plasma OT level and that the hypertrophying effect of anabolic steroids is mediated by increased OTR signaling (reviewed in 4). Furthermore, in a mouse model of aging, it was shown that reduced circulating OT levels accompany sarcopenia, and that satellite cells obtained from aged mice express reduced levels of OTR5. Efficient regeneration could be restored in aged mice by administering exogenous OT. Taken together, these studies not only confirm that NH target skeletal muscle increasing differentiation and trophism, but also point to muscle as a physiologic source of OT., Non-coding RNAs are emerging as important players in the regulation of several aspects of cellular biology. The expression and therefore the activity of non-coding RNAs is more tissue and cell specific than that of coding RNAs. Therefore, for a better comprehension of their function, it is fundamental to determine their tissue or cell specificity and to identify their subcellular localization. Myofibers are the smallest complete contractile system of skeletal muscle influencing its contraction velocity and metabolism. We compiled a comprehensive catalog of ncRNAs expressed in skeletal muscle, associating the fiber-type specificity and subcellular location to each of them. We demonstrated that many ncRNAs can be involved in the biological processes de-regulated during muscle atrophy. Focusing our attention on a specific long non-coding RNA (Pvt1), activated early during muscle atrophy, we revealed that it impacts on mitochondrial respiration and morphology and affects mito/autophagy, apoptosis and myofiber size in vivo. This work corroborates the importance of long non-coding RNAs in the regulation of metabolism and neuromuscular pathologies and offers a valuable resource to study the metabolism in single cells characterized by pronounced plasticity., One of the consequences of aging is the reduction of the growth hormone/insulin-like growth factor I (GH/IGF-I) axis. For skeletal muscle, this leads to a loss of anabolic and regenerative capacity, which, in part, underlies the onset of sarcopenia. Maintenance of activity in aging can help offset functional deficits, and these benefits are both systemic and specific to skeletal muscle. It is an open question as to the contribution of muscle IGF-I activity to these beneficial adaptations. To address this, we imposed an endurance exercise regimen on a recently developed mouse model with muscle specific inducible deletion of IGF-I (MID mouse), and strain matched controls (CON), comparing their performance to sedentary mice of the same genotypes. After 4 weeks of daily endurance training at 15 m/min for 60 minutes, both MID and CON mice displayed similar and significant improvements in a run-to-exhaustion test. Body composition also displayed a reduction in proportional fat with training. However, in the subsequent 4 weeks of daily endurance training at 18 m/min for 60 minutes, only the CON mice continued to improve in the run-to-exhaustion test, even though there were no differences in the body composition changes in the exercised groups. Over the course of 8 weeks, the sedentary mice progressively gained fat, and by the end of the study, the sedentary MID mice ran for less time in the run-to-exhaustion test compared to sedentary CON mice. Taken together, this suggests that benefits to moderate intensity training can occur in the absence of muscle IGF-I. However, the capacity of muscle to adapt to higher intensity regimens may be limited when actions of local IGF-I signaling are impaired., Skeletal muscle atrophy induced by immobilization is one of the greatest issues in athletes as well in general population. The mechanisms underlying skeletal muscle atrophy, are reasonable known, however, there is no evidence of works that was able to avoid it. A strategy that could preclude the large recovery needed after immobilization would be of great importance. Our group made a research protocol that aims to study the effect of Branched-Chain Amino Acids (BCAA’s) administration to avoid skeletal muscle atrophy. In order to achieve our goals, we use an animal model divided in several groups that includes control, cast-immobilization, exercise animals and animals which have taken BCAA’s. We observed the structure of skeletal muscle, fibre type changes and the satellite cells turnover. To evaluate satellite cells, we used antibodies against Pax-7, Myf-5 and c-met, identification by fluorescence which allows to count the immunoreactive cells and study its evolution and differentiation. As expected, cast immobilization caused atrophy and muscle damage. The satellite cells were activated to regenerate the skeletal muscle, as seen by the increase in Pax-7 and Myf-5 expression with a decrease of c-met, suggesting mobilization of those cells. Visible cell niches in the muscle reinforced these data. The supplement had a partial protective effect when it was taken during immobilization, but it seems to hamper the muscle regeneration, when exercise is performed after immobilization. Thus, our results suggested that BCAA prevent skeletal muscle atrophy by acting over its satellite cells. Partially Supported: FCT, Portugal (Strategic Project UID/NEU/04539/2013), FEDER-COMPETE., According to Bartholomaei Eustachii, Aristotle was the first to describe the complicate interlacement of canaliculi which are in the petrosal bone, identifyng the “coclea”. Aristotle, to explain his idea from an anatomical point of view, compared the inner ear to the strovmboi, i.e. spiral shell (cochlea). Regarding the term labirinto, probably Falloppio was the first to use the term labyrinthus – of egyptian and not greek origin - to describe the inner ear: quum haec cavitas tot habeat meatus et cuniculos, merito labyrinthus dicetur, in quem prospicit fenestra ovalis, clausa a stapede, ecc.». The Anatomic school of Padova gave during the XVI century an enormous contribution to the knowledge of the ear: • Andreas Vesalius 1514-1564 • Andrea Falloppio 1523-1562 • Jeronimus Fabricius ab Acquapendente 1537-1619 • Bartolomeus Eustachi 1514-1574 • Matteo Colombo 1515-1559 • Giovanni Filippo Ingrassia 1510-1580 • Iulius Casserius 1552-1616 One of the major contributions of Vesalius was his suggestion that the organ of hearing should be removed from the skull for investigation. We remember here 3 important contributions: Bartolomeus Eustachi in his work entitled “Epistola de auditus organis” (Eustachi, 1564); Girolamo Fabrici ab Acquapendente De Visione Voce Auditu (1600) and Iulius Casserius De Vocis Auditusque Organis Historia 1601. In 1740, Antonio Valsalva published his anatomical observations on the human auditory system in which he pointed out the importance of the ossicular chain and the oval window for hearing and also observed that the innervation target for the auditory nerve was not the osseous spiral lamina, but was instead the membranous portions of the cochlea and that these areas of sensory epithelium represented, in the opinion of Valsalva, the true receptors of sound. Later, Antonio Scarpa, pupil of Giovanni Battista Morgagni and Marco Antonio Caldani, reported a liquid present within the cochlea's inner membranous compartment, that is, scala media. The Count Alessandro Volta is generally qualified as the first to stimulate the ear with the electricity. Volta, carried out on himself in the late 1790s the first experiment on electrical stimulation of the auditory nerve. Because of the unpleasant sensation experienced by the scientist, any other experiment was carried out over the next half century to study this effect. From the first half of the 18th century onwards, tremendous curiosity about electrical phenomena spread throughout Europe. Machines producing electrostatic electricity were produced, and lectures on electricity attracted members of academia as well as the ruling elite. A field known at the time as “medical electricity” emerged following the electrical researches and the discovery of the effects of electricity on the human body. In 1950, Lundberg performed one of the first recorded attempts to stimulate the auditory nerve with a sinusoidal current during a neurosurgical operation. His patient could only hear noise. In 1957 by Djourno and Eyries provided the first detailed description of the effects of directly stimulating the auditory nerve in deafness. They placed a wire on the auditory nerves that were exposed during an operation for cholesteatoma. When the current was applied to the wire, the patient described generally high-frequency sounds that resembled a “roulette wheel”. The signal generator provided up to 1,000 Hz and the patient gradually developed limited recognition of common words and improved lip-reading capabilities. At this point the clinical application of a cochlear implant started. A cochlear implant is a surgically implanted electronic device that provides a sense of sound to a person who is profoundly deaf or hard of hearing; at now approximately 500,000 people worldwide had received cochlear implants. Padova Audio/Otological group is strongly engaged in the diagnosis and treatment of deafness particularly in childhood and in basic research (Padua Bioacustic lab). Recently an international center was established in Venice: I APPROVE, the International Auditory Processing PROject in Venice, Maximal oxygen consumption (V̇O2max) denotes the reproducible upper limit of oxygen (energy) flux through the respiratory system into skeletal muscle mitochondria that can be reached during intense exercise with a large muscle mass. A high V̇O2max is a key requisite for success in all endurance sports such as cycling, cross-country skiing or running over longer distances. However, V̇O2max has also been strongly and negatively associated with cardiovascular diseases and all-cause mortality. V̇O2max can vary by more than twofold between untrained, sedentary subjects with a heritability value greater than 50%.1 Trainability for an individual's V̇ O2max also varies massively between subjects. Trainability is independent of sedentary V̇O2max with a similarly high heritability as sedentary V̇O2max.2 The high heritability of sedentary V̇O2max and trainability and its importance for athletic performance as well as health has prompted a massive search for its genetic underpinning. Candidate-gene studies, gene-expression studies and genome-wide-association studies (GWAS) have failed to identify a genetic signature of the high V̇ O2max phenotype.3 This may be due to the fact that there are vast multigenetic regulatory networks in skeletal muscle and in other organs that are responsible both for the set-point and the malleability of V̇O2max. Multigenetic phenotypes such as V̇O2max appear to be emergent properties of multiple underlying transcriptomic networks modified by epistasis, the epigenome and the epitranscriptome. It is unclear currently whether an artificial intelligence approach on sufficiently large datasets can make reliable predictions on multigenetic phenotypes such as V̇O2max., In the last decades sparse evidence of the therapeutic potentialities of surface electrical stimulation for the treatment of facial palsy has been published.1,2,3 Our study represents one of the few systematic evaluations of this approach within a homogeneous population suffering from complete unilateral facial paralysis. For this study the stimulation was delivered on the region closest to the upper mouth corner and denervation recovery was monitored for the entire period. Five patients were recruited, undergoing surface electrostimulation for a maximum of 1y, twice a day for 15min (5min pause every 5min stimulation). The parameters set during the first visit were confirmed/adapted every month thereafter. At each visit the patients underwent 3D photos, electromyography, ultrasound,4 magnetic resonance controls,5 and Sunnybrook evaluation, answered to the FaCE and FDI questionnaire. For all the patients effective parameters leading to an utterly Zygomaticus m.-specific stimulation could be found below the discomfort threshold. The required stimulation pulse width decreased with time without requiring a significant amplitude increase to remain effective and specific. Sunnybrook results significantly (p=0.04) improved after 6m stimulation. FaCE (total score) showed a significant (p=0.04) improvement already after 2m, while the social component of the FDI significantly (p=0.04) improved after 7m. Short- (hours) and mid-term (weeks) improvements of muscle tone could also be shown by 3D imaging. Our results showed that surface electrostimulation can be used to deliver a specific response of the Zygomaticus m. without adverse events and without non-specific activation of other facial muscles. Patients were extremely compliant with the treatment protocol and received a significant improvement in their quality of life., Age-related cognitive decline has become a major public health concern. There is currently a paucity of effective interventions to prevent or treat cognitive decline and prevent dementia in the elderly. Decline in working memory is a central facet of the cognitive aging process. Prior research has attempted to use transcranial direct current stimulation (tDCS) to enhance working memory performance in older adults with and without neurodegenerative disease, often in the context of cognitive training paradigms(1, 2). Alone, these methods have shown a degree of promise in remediating age-related working memory decline. In combination, these interventions may prove more effective by pairing two interventions targeting neuroplasticity. However, the neural mechanisms and efficacy of combined cognitive training and tDCS improvements in working memory is not well understood. This talk will discuss data from 3 studies investigating the benefits neural mechanisms of cognitive training paired with tDCS to remediate age-related cognitive decline.1-3 Study 1 was a mechanistic investigation of the impact of tDCS with CT during BOLD fMRI in 14 healthy older adults. Study 2 was a pilot randomized clinical trial of 28 older adults randomized to active tDCS or sham tDCS with cognitive training. Study 3 is an ongoing Phase III RCT investigating the benefits of tDCS with cognitive training in 360 older adults. Neural mechanisms of working memory improvement, efficacy of near transfer from tDCS vs. sham with cognitive training in working memory measures, and future directions of investigation will be discussed., We present preliminary results from the ongoing study entitled “Icelandic AVH TMS” which aim is to study the effectiveness of repetitive transcranial magnetic stimulation (rTMS) treatment for patients with schizophrenia and with persistent auditory verbal hallucinations (AVH) using high-density EEG system (256 channels). The main objectives of this work were to describe P50 and P300 cortical topography pre and post treatment, and to define a robust methodology of signal quantification using high density EEG.1-4 Our results show differences in sensory gating and a stronger response to rare audio stimulus 1 week post treatments. Moreover we show the value of assessing brain electrical activity from high-density EEG (256 channels) analyzing the results in different regions of interest., Reduction in muscle mass and muscle strength together with alterations in the metabolic and cellular machinery of the skeletal muscle are common in type 1 diabetes (T1D)1 Collectively these negative changes have been termed diabetic myopathy.2 Moreover, whole-body fatigue is a frequent complaint in individuals living with T1D but it is unclear if this would depend on muscle alterations per se or on a concomitant impairment in motor units (MU) recruitment capacity. MU recruitment can be now investigated non-invasively using high-density surface electromyography (HD-EMGs) using multi-channels electrodes.3 Sixteen participants, 8 T1D (4M; 33.7 ± 5.2 yrs.; HbA1c 8.1 ± 3.1 %) and 8 healthy (4M; 31.5 ± 5.6 yrs.) volunteered for the study. In one 2-hour visit to the lab participants were tested for maximal isometric strength (MVIC) of knee extensors (KE), sustained KE isometric contractions at 10, 20, 40 and 60 (%MVIC), plus a sustained contraction at 40% MVIC until task failure. During these measures we recorded HD-EMGs from vastus lateralis (VL) muscle with a grid of 64 electrodes. At baseline no differences, between the 2 groups, were observed in MVIC, rate of torque development, and HD-EMGs parameters. In addition, and contrary to our expectations, no differences were observed concerning the fatiguing test either in terms of time to fatigue or of changes in muscle fiber conduction velocity (a marker of MU recruitment). In conclusion, no major differences were detected between the two groups, which could justify the presence of diabetic myopathy (whether neurogenic or myogenic) in our T1D participants., Skeletal muscle is the driver of whole-body aerobic capacity measured by spiroergometry as VO2max/M [mL O2∙min-1∙kg-1]. Obesity is defined as accumulation of excess fat tissue mass, MFE=MF-MF° [kg/x]. MF° is the fat mass [kg] per individual [x] in the healthy reference population at a given height and total body mass M° without overweight.1 Body fat excess, BFE=MFE/M°, is directly related to total body mass excess, BME=ME/M°, where ME=M-M°. In model 1, BFE does not reduce VO2max [mL O2∙min-1∙x-1], but BFE lowers VO2max/M by increasing M. Experimentally, however, VO2max/M declines with BME much steeper than predicted by model 1. The more pronounced loss of ergometric fitness is due to the decline of mitochondrial respiratory capacity per muscle mass, mM,2,3 as a function of BME. Yet this model 2 predicts an even lower VO2max/M at overweight. Finally, model 3 includes the well-known ‘weight-lifting’ effect of obesity on increasing muscle mass with low mitochondrial density, providing a quantitatively complete link between low mitochondrial and whole body aerobic fitness in obesity before onset of sarcopenia. The decline of muscular mitochondrial fitness in overweight states is a biomarker of the systemic mitObesity syndrome: Compromised mitochondrial fitness across metabolically active organs provides the mechanistic link between obesity and comorbidities such as diabetes, cardiovascular and neurodegenerative diseases and various types of cancer bound to redox imbalance, inflammation, oxidative stress and insulin resistance. Today mitObesity is the world-wide leading cause of deaths and early aging, which can be prevented by an active lifestyle and improvement of the quality of life by exercise and caloric balance. Supported by project NextGen-O2k which has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 859770. Contribution to COST Action CA15203 MitoEAGLE funded by the Horizon 2020 Framework Programme of the European Union., By combining the digital holography with the tomography, Nanolive technology is an example of emerging intelligent nanoscopy techniques1. The 3D Cell Explorer microscope guarantees a high spatial-temporal resolution imaging of transparent unlabeled specimens, with the advantage over fluorescence techniques of not requiring sample labeling, thus reduce potential damages to living cells2. This intuitive and affordable technology enables scientists to investigate macro cellular dynamics like cell health, proliferation, movement and function as well as micro organelle dynamics and interactions in a e.g. mitochondrial network label-free characterization.3, Intracellular aggregates of RNA binding proteins and perturbed RNA metabolism are typical features of many human neurological and neuromuscular diseases, including oculopharyngeal muscular dystrophy (OPMD), a rare genetic repeat expansion disease (1). In OPMD, a short abnormal polyalanine expansion in PABPN1 (poly(A)-binding protein nuclear 1) leads to the accumulation of intranuclear tubulofilament aggregates in muscles of patients and ultimately to muscle dysfunction. Symptoms usually appear in the fifth decade of life and we have shown a good genotype phenotype correlation (2). The disease is characterized by weakness of the eyelid and pharyngeal muscles, leading to eyelid drooping and dysphagia. A strong collaboration with clinicians gave us access to a large collection of muscle biopsies from OPMD patients. We characterized PABPN1 nuclear aggregates to evaluate if age and genotype influence their features (submitted). In addition, using mammalian models of OPMD, we assessed the efficacy of the anti-aggregates molecule guanabenz (GA). We showed that aging and genotype of OPMD patients influence the amount, the size, the percentage and the composition of nuclear aggregates. Treatment of cellular and mouse models of OPMD with GA allowed a reduction in the percentage and the size of nuclear aggregates as well as an improvement of the mice muscle phenotype. GA acts through the unfolded protein response to endoplasmic reticulum stress (ER) showing for the first time that ER stress is activated in OPMD. (3) This study suggests that the use of pharmacological molecules modulating ER stress is a promising strategy to treat OPMD., Multiple acyl-CoA dehydrogenase deficiency (MADD) is a rare autosomal recessive lipid storage myopathy, often associated with ETFDH gene mutations.1 This gene encodes electron transfer flavoprotein dehydrogenase, a mitochondrial protein involved in the electron-transfer system.2 In this study we investigated the correlation between ETFDH mutations, expression profile of serum muscle‐specific miRNAs (myomiRs)3 and response to treatment in 2 Italian MADD patients. Patient 1 (53 years) presented late onset of MADD, at the age of 38 years. She carried two different ETFDH missense mutations.4 In patient 2 (54 years) the symptoms appeared at 2 years of age and molecular analysis of ETFDH showed a missense and a splice site variation.5 Muscle biopsies of both patients displayed type I fibers vacuolated and muscle atrophy. Moreover, MRI of patient 2 revealed slight alteration of posterior thigh muscles. Bioinformatic analysis demonstrated that the missense mutations can determine conformational modification of protein structure, decreasing enzyme stability and activity. Serum myomiRs analysis displayed an up-regulation of miR-1, miR-133a, miR-133b and miR-206 expression in the two patients. Treatment led to an improvement of clinical condition in both patients, in particular of muscle vacuolization. In this preliminary study the results seem to indicate that the presence of two ETFDH missense mutations correlate not only with a minor severity of MADD phenotype but also with a partial dysregulation of circulating myomiRs, mainly miR-1 and miR-206. In these cases, various treatments can improve ETFDH stability, resulting in skeletal muscle recovery., Store-operated Ca2+ entry (SOCE) is a ubiquitous cellular Ca2+ influx mechanism, first described in non-excitable cells, that is triggered by depletion of intracellular Ca2+ stores (endoplasmic reticulum, ER). A major breakthrough in the field was the identification of the two essential molecular players in SOCE: STIM1, the Ca2+ sensor in the ER, and Orai1, a Ca2+ permeable channel in the plasma membrane.1,2 SOCE is also well-documented in skeletal muscle4 where it limits muscle fatigue during repetitive stimulation.3 Also in muscle SOCE is mediated by interactions between STIM1 in the SR and Orai1 channels in the PM.4 However, the precise subcellular location of STIM1-Orai1 SOCE complexes in skeletal muscle is still debated. We discovered that exercise in mice drives formation of new junctions between stacks of sarcoplasmic reticulum (SR) cisternae and transverse-tubules (TTs) containing STIM1 and Orai1, two proteins that mediate store-operated Ca2+ entry (SOCE). We proposed that these previously unidentified SR-TT junctions function as Ca2+ Entry Units (CEUs), providing a preferential pathway for rapid reuptake of Ca2+ into the SR during repetitive muscle activity.5 Using electron microscopy and two different functional assays we also studied muscles from mice subjected to an incremental treadmill running and sacrificed within 1hr or after 6-24hrs.6 Data collected indicates that: a) while the number of SR-stacks increased up to at least 6hrs to return to control values only after 24hrs of recovery, the extension of TTs (significantly increased at 1 hr), returned to control values already after 6hrs; b) fatigue resistance of EDL muscles during high-frequency stimulation and Mn2+ quench of Fura-2 fluorescence in FDB fibers were both increased after 1hr, but were not different from control after 6 and 24hrs of recovery. Our work represents a pioneer study that identified exercise-driven dynamic formation of new intracellular structures, a mechanism potentially quite important for the delay of muscle fatigue. As altered SOCE activity contributes to muscle dysfunction in ageing and various myopathies, our findings may also have implications for the understanding of mechanisms involved in muscular dysfunction., The HERG potassium channel is detected as a heteromultimer of 2 alternative splice variants (1A and 1B) in heart and has been shown to be partially responsible for repolarization of the cardiac action potential.1 Both alternative splice variants have been reported in certain cancer cells, but their role in these cells is not clear.2 The HERG1A variant has been detected at low abundance in normal skeletal muscle, but is up-regulated in atrophying skeletal muscle, where it has been shown to increase protein degradation by modulation of both intracellular calcium levels and ubiquitin proteasome proteolysis (UPP).3,4,5 The pathways by which this modulation occurs is not clear. Therefore, we virally transduced C2C12 myotubes with either an adenovirus encoding HERG or an appropriate control virus (n=6). After 48 hours, we extracted total RNA from these cells and reverse transcribed them into cDNA, selecting for coding sequences (i.e., mRNA) by using poly(T) oligomers; the cDNA libraries were sequenced on Illumina’s NovaSeq platform. Sequence quality was assessed using FastQC (v 0.11.7; https://www.bioinformatics.babraham.ac.uk/projects/fastqc) for all samples and quality trimming was done using FASTX-Toolkit (v 0.0.14; http://hannonlab.cshl.edu. fastx_toolkit/) to remove bases with Phred33 score of less than 30. Resulting reads of at least 50 bases were mapped against the reference genome using STAR.6 STAR derived mapping results and annotation file for reference genome were used as input for HTSeq7 (v 0.7.0) to obtain read counts. Counts from all replicates were merged together to produce a read count matrix for all samples and this count matrix was used for downstream differential gene expression analysis (DGEA). DGEA between treatment and control was carried out using ‘R’ (v 3.5.1; http://www.r-project.org/). The results show that HERG does result in numerous changes in gene expression. Limiting results to those with a p, MRI is a powerful non-invasive imaging technique that exploits different contrast mechanisms to provide an unprecedented view into both structure and function. We have used velocity encoded phase contrast technique to map muscle motion in-vivo1 and applied to study age and disuse atrophy related changes. A limitation of this dynamic technique is that it requires of the order of 70 consistent contractions cycles in order to image muscle motion. Thus, it is not easy to extend the MR dynamic studies to senior subjects and to higher % Maximum Voluntary Contractions (%MVC). We have implemented a fairly recent innovation called ‘compressed sensing (CS)’ to dynamic MRI for mapping muscle kinematics during isometric contraction. The implementation of this ‘fast’ technique allowed us to study muscle kinematics in 3D and at three %MVCs in the calf muscle. Eight young and eight senior subjects were imaged using the CS-VEPC sequence and the 3x3 strain and strain rate tensors were calculated in the principle axis and in the muscle fiber basis at 35, 45 and 60 %MVC at much reduced number of contractions cycles. Region of interest measurements are reported for the medial gastrocnemius and in the soleus: principle strains and strain rates were significantly different for 30 and 60% MVC (MG and soleus) and shear strain and shear strain rates were significantly different with age. Surprisingly, the strain and strain rates in the fiber basis were much smaller than in the principal basis and no significant differences were found between %MVCs or in age. The implications of these findings with respect to muscle force loss with age will be discussed., Soft tissue contrast afforded by MRI makes it an ideal candidate for imaging the musculoskeletal system. We will discuss our results using a combination of advanced MR pulse sequences to explore age related changes in tissue composition and tissue microstructure. Tissue composition includes estimation of fat (adipose) fraction, connective tissue/macromolecular fraction and water fraction in different muscles and compartments1. A combination of MRI sequences, IDEAL, UTEs and Magnetization transfer2 contrast sequences were applied to cohorts of ten young and ten senior subjects to determine the intramuscular and intermuscular tissue composition in calf muscle. Initial results show that fat and connective tissue fraction increased while macromolecular fraction (MTsat) decreased with age. Tissue microstructure was extracted from diffusion tensor imaging3,4 using a STEAM-DTI sequence and Random Permeable Barrier Modeling (RPBM)5. DTI studies were performed after IRB approval on a GE 3T scanner on seven young and six senior subjects. Time dependent diffusion was measured at ten values of the mixing time, TM (20 ms to 600 ms). The RPBM fits were made to the time dependence of the average of the secondary and tertiary diffusion eigenvalues λ2 and λ3 (Dᅩ ); the values of Dᅩ were the average over the medial gastrocnemius (MG) muscle segmented from all three slices. Muscle fiber diameters from the fit showed a small increase with age which is surprising as fiber atrophies with age. Fiber membrane permeability increased with age potentially indicating compromised sarcolemma integrity. Initial results of validation with biopsy analysis will also be presented., Chronic inactivity is a main cause of muscle wasting and weakness.1 However, muscle atrophy may not be solely due to a reduction in mechanical loading since muscle denervation and neuromuscular junction damage seem also triggered by inactivity.2,3,4 The identification of biomarkers of muscle atrophy and of neuromuscular degeneration is therefore needed for an early detection of the alterations of the neuromuscular system induced by inactivity. Hence, the aim of the present study was to investigate the onset of muscle atrophy and of neuromuscular alterations during a short-term inactivity period. Ten healthy male volunteers (aged 23±5 years) were recruited for this horizontal 10-day bed rest (BR) study, after Ethical approval and participants’ informed consent. Blood samples were collected every two days for the assessment of NMJ damage, based on serum levels of c-terminal agrin fragment (CAF) measured with a commercially available Elisa kit. Muscle fibre denervation was determined from the expression of neural cell adhesive molecule (N-CAM) in myofibres obtained from vastus lateralis (VL) muscle biopsies collected at baseline, and at 5 and 10 days of BR. Myofiber atrophy was determined from mean fibre cross-sectional area (CSA) measurements on histological sections. Whole muscle atrophy was assessed from changes in muscle architecture (pennation angle and muscle thickness) measured every two days of BR using ultrasonography. Significance was set at p, Over the last 25 years imaging techniques have been extensively used to study the in-vivo muscle and tendon morphology at tissue scale. Whereas Dual-energy X-ray absorptiometry (DXA), computed tomography (CT) and MRI have been widely adopted in health, ageing and clinical populations to track changes in body composition and muscle mass, ultrasound (US) can provide further information about the structure, length, thickness and area/volume of muscles and tendons. Despite these characteristics could also be obtained by MRI, US has the advantage that it can provide the same quantity of information, but during passive joint rotations, isometric or dynamic contractions. Recent advancements in musculoskeletal imaging have also led to the use of US also for the investigation of strain in soft tissues in response to compression (Shear Wave Elastography - SWE). However, some limitations of conventional B-mode US lay on: a) the small field of view used (especially during SWE or the study of in-vivo dynamic changes in muscle and tendon structures) and b) the impossibility of US to provide any in-vivo insights into adaptations at the sarcomeric level. Recent developments in the extended field-of-view US (EFOV) and 3D US techniques may offer further advantages in providing information at a whole-tissue level, providing software-integrated panoramic images and 3D volume reconstructions, allowing to gain insights into regional changes along the muscle-tendon unit. Furthermore, in-vivo microendoscopy has been recently implemented in human skeletal muscle, laying the foundation of new investigations into the remodeling of skeletal muscle “from the micro to the macro”. The purpose of this talk is to present and discuss these advances in imaging techniques for the study of human skeletal muscle in-vivo., Real time Ultrasound (US) imaging is being increasingly utilized by Physical and Rehabilitation Medicine (PRM) specialists to assess abdominal muscle in healthy subjects and in patients with low back pain (LBP).1 The current study set out to evaluate the variability and the intra- and inter-rater reliability of US measurements not only of the thickness of the abdominal muscles but also of the fasciae.2 Three specialists with different levels of training in US measurement techniques followed a standard protocol based on four reference anatomic landmarks to perform US examinations of the abdominal muscles and fasciae of a healthy volunteer in resting and dynamic condition. Each of the specialists measured 17 anatomical structures six times during two sessions (three per session). Their intra-rater reliability was assessed by evaluating the range of relative error and the coefficient of variation (CV). The inter-rater reliability was evaluated using the Kruskal-Wallis test at probability levels of 0.05 and 0.01. There were no significant differences between the measurements that the three raters registered (inter-rater reliability) with the exception of those referring to the anterior fascia of the external oblique muscle (p-value < 0.01), the fascia between the external and internal oblique muscles (p-value< 0.05) and the fascia between the internal oblique and the transversus abdominis muscles (p-value < 0.05).3 Knowledge about the fascial anatomy of the abdominal wall is essential to carrying out accurate US examinations. These findings confirm that US imaging is a reliable, non-invasive, cost-effective instrument for evaluating the abdominal muscles/fasciae.3, Spinal cord injury produces muscle wasting, which is especially severe after complete and permanent damage of lower motor neurons as occurs in complete Cauda Equina Syndrome.1 Even in this worst-case scenario, we have shown that permanently denervated Quadriceps muscle can be rescued by surface electrical stimulation, and a purpose-designed home-based rehabilitation regime, i.e., Home based Functional Electrical Stimulation (hbFES) for human muscles that are completely denervated and degenerating.2,3 Here the aim is to show that the effects are extended to the antagonist muscles and skin of the thighs. Before and after two years of electrical stimulation, the mass and structure of Quadriceps and Hamstrings muscles were quantitated by force measurements.1 Muscle gross cross-sections were evaluated using color computed tomography.4 Biopsies of skeletal muscles,1-3 and of stimulated skin,5 were analyzed by quantitative histology and immuno-histochemistry. The treatment produced: 1) an increase in the cross-sectional area of stimulated muscles; 2) an increase in muscle fiber mean diameter; 3) improvements in ultrastructural organization; and 4) increased force output during electrical stimulation.1-4 The recovery of Quadriceps muscle force was sufficient to allow 25% of the compliant subjects to perform stand-up and step-in place trainings.1-3 Improvements were extended to antagonist hamstrings,5 and skin.6 In conclusion, the cushioning effect provided by recovered tissues is a major clinical benefit. The hope is that new trials may start soon in Europe and beyond to provide stronger evidence of the positive effects of h-bFES for permanently denervated muscles. This will help to extend the results world-wide, and to all persons who are in need of the help they deserve., Patient oriented research in rehabilitation revolves around maintaining, improving or restoring muscle function in conjunction with pain treatment. Based on our findings in treating patients with long-term denervated, degenerated muscle (DDM) after spinal cord injury with electrical stimulation, (Kern et al. 2010)1 our activities in the past were aimed at transferring the knowledge to rehabilitation of elderly people (Kern et al. 2014).2 The current work comprises investigating age-related sarcopenia, a multifactorial disorder which underlying mechanisms are still not fully known. It could be shown, that alterations of mitochondrial Ca2+ homeostasis regulated by mitochondrial calcium uniporter (MCU) affect muscle function. Therefore, improved muscle function and structure are linked to increased protein levels of MCU (Zampieri et al. 2016).3 Future translational research will investigate cellular and molecular mechanisms of aging and physical activity, especially the effect of electrical stimulation training and physical therapy on these mechanisms, because only little research on this topic exists and therefore the evidence for efficacy of these therapy approaches is still missing. Focus of the future clinical research program is the in- and outpatient therapy of orthopedic patients, the remobilization and the rehabilitation of patients after knee and hip replacement surgery. For this reason, standardized assessments, analyzes and documentation will be developed and the results are collected across locations in a data base for in- and outpatient rehabilitation. Overall aim of the program is the evaluation of existing and new approaches in musculoskeletal rehabilitation also concerning the economic aspects with constant or improved quality of treatment. In the next years the following topics will be in focus: i) Underlying cellular and molecular mechanisms of Physical Medicine and Rehabilitation procedures; ii) In- and outpatient rehabilitation after knee and hip replacement surgery; iii) Proof of efficacy and cost-effectivity., Physical inactivity is a global pandemic that not only causes morbidity and mortality, but also represents a major economic burden worldwide. As a longer-term goal, we must strive to integrate physical activity into our everyday lives.1 Increased physical activity, among other things, has an influence on chronic pain of the locomotor system, minimizes the risk of cardiovascular disease, increases self-esteem and cognitive abilities, maintains mobility and autonomy, and thus has direct effects on healthy life years and direct and indirect health expenditure. The Centre of Active Ageing (CAA) project, funded by the EU cross border cooperation program INTERREG, is intended to be in line with the Austrian health targets (R-GZ) for Austria,2 the National Health Program of Slovakia,3 the recommendations of the Lancet Physical Activity Series Executive Committee, and of the WHO.5 Encouraging policymakers to take physical activity seriously, to motivate people, and to create the opportunities to do so on a regular basis. Over the entire project period of three years, more than 1000 people aged 60 years and over will benefit from the offers of the CAAs in the program area. Within the scope of the project, they should carry out a standardized training (10-12 weeks) followed by home training for 12 months. In 2019 the first groups started their “SENIOR aktiv” activity program in Austria. It combines education sessions of training theory and nutrition before and after a 10-week physical activity program for two times a week with alternating strength and gymnastics sessions. The first results show improvements in almost all measured parameters and the subjective feedback of the subjects is consistently positive, When resistance training for the improvement of the neuromuscular function is considered, older adults should base training on submaximal loads (85-80% of their one repetition maximum (1RM); tested or estimated from nRM) and advance to speed-power training regimens (50-75% of 1RM executed explosively). Based on the recent research, the latter is an important supplement facilitating physical ability and functional capacity of older individuals. Recent studies have shown that resistance training stressing performance of repetitions with maximal velocity results in higher performance gains as compared to strength training alone.1 It therefore seems reasonable to include speed-power training into physical conditioning of older adults. As a result, we aim to address this topic in the framework of the current Interreg project Slovakia-Austria, by exposing 60+ subjects to 10-week flywheel speed-power training. This training modality offers innovative solutions for individualised training progression (load, tempo, postural stability), which we will share during with the audience during our conference presentation. Additionally, our plan is to get a better insight into the potential of this kind of resistance training for changing force-velocity-power profile in elderly. This would complement our previously used testing approaches we used in past interventional studies in elderly.2 Namely, improving velocity dominance in the force-velocity profile seems to have significant functional relevance (balance control, gait initiation, change of direction; and prevention of falls as a result).3 Preliminary results on initial methodological considerations and protocols (training and testing) development will be presented, Proper muscle function is controlled by intracellular Ca2+ concentration, which in turn depends on: a) release of Ca2+ from intracellular stores during excitation contraction (EC) coupling, which activates muscle contraction; b) entry of Ca2+ from the extracellular space via store-operated Ca2+ entry (SOCE), a mechanism important to limit muscle fatigue; c) uptake of Ca2+ into the mitochondrial matrix, which stimulates aerobic ATP production; and finally d) sequestration/removal by sarcoplasmic reticulum (SR) and plasma membrane (PM) pumps, which relaxes muscle fibers. Abnormalities in Ca2+ handling underlies many physio-pathological conditions. For instance, reduced SR Ca2+ release has been linked to fatigue and dysfunction in ageing, while excess of SR Ca2+ leak may even underlie life-threatening conditions such as malignant hyperthermia susceptibility (MHS). In the last 15 years we have collected compelling evidence that the proper architecture and function of all those membrane systems involved in Ca2+ handling and aerobic ATP production depends on muscle activity: i) denervation causes disarray of units deputed to EC coupling (calcium release units, CRUs) and mitochondrial apparatuses, while functional electrical stimulation (FES) promotes some rescue of this structural disarray;1 ii) sedentary ageing in mice and humans causes misplacement of mitochondria and partial disarray of CRUs, while long-term training does prevent effectively those changes;2-4 iii) acute exercise promotes functional assembly of SOCE-sites (calcium entry units, CRUs), while post-exercise recovery determines their disassemble;5 iv) sedentary ageing causes accumulation of dysfunctional proteins in tubular aggregates (TAs), while exercise prevents TAs formation (manuscript in preparation). Ca2+ handling is crucial for muscle function and is controlled by diverse membrane systems and intracellular organelles. Our experience collected in different, but complementary, projects: i) indicates the structure and function of intracellular organelles is preserved or rescued by exercise or training; and ii) underlines the importance of physical exercise during ageing to preserve muscle function., The dominant role in muscle regeneration is played by the muscle stem cells known as satellite cells, which reside between the basal lamina and sarcolemma of myofibers. Along with satellite cells, other precursor cells, whose activity is strictly dependent by environmental signals, participate to muscle regeneration.1,2 A tightly regulated interplay between stem cells and other resident cell types, as well as the intimate connection with structural components of the tissue niche, can be responsible for the maintenance of the stem cell pool under steady-state conditions and to guide stem cells activation and differentiation when regenerative signals are provided.1 Because niche factors and components normally control and sustain a physiological stem cell activity and maintenance, the loss of homeostatic input from the niche, as observed under pathological conditions, can deregulate stem cell physiology, critically affecting the ability of muscle tissue to efficiently regenerate and to regain the functional integrity after damage.1-3). Thus, the stem cell niche is not only an anatomical compartment but a complex, integrated network of both cellular and acellular components that provide signals influencing stem cells and muscle homeostasis. The basis of muscle regeneration and the impact of cytokines and growth factors on the physiopathology of skeletal muscle will be discussed.1,4-6, Neuromuscular diseases, ageing, and cancer cachexia are chronic conditions characterized by skeletal muscle wasting (1, 2) resulting from altered muscle metabolism, and catabolic processes. Following muscle pathophysiological changes, proteins are secreted or passively released into systemic circulation. The identification of muscle-derived serum biomarkers can be used to develop a kind of “liquid biopsy” for diagnostic/prognostic approaches and to identify altered muscle physiology. With this aim, we performed combined morphological and biochemical analyses of skeletal muscle biopsies and blood samples obtained from cancer patients, both collected the day of surgery. Samples from non-oncologic patients were also collected as controls. Morphometrical analyses showed a significant reduction of myofiber size in oncologic patients in comparison to controls, and the analyses of myofiber diameter distribution showed a higher prevalence of severely atrophic fibers in oncologic patients, most of them having angulated and flat shaped morphology, typical features of denervation (3). The percentage of in situ Neural cell adhesion molecule (N-CAM) expressing myofibers and serum levels of N-CAM (sN-CAM) and Agrin were also tested, as markers of neuromuscular junction plasticity and remodelling (4, 5). Consistently with morphological observations, in cancer patients with respect to controls, the percentage of N-CAM positive myofibers was significantly higher and circulating levels of sN-CAM and Agrin were also significantly increased. These results correlate morphological changes of skeletal muscle trophism and innervation with serum alterations in NMJ-derived proteins. Moreover, they show that sN-CAM and Agrin are promising and reliable serological biomarkers for neuromuscular junction plasticity and remodeling in conditions characterized by skeletal muscle wasting, Functional electrical stimulation (FES) is used to induce skeletal muscle contractions in persons who are unable or reluctant to move usually by surface neuromuscular electrical stimulation (NMES), also known as transcutaneous electrical nerve stimulation (TENS) in out-patients or hospital. If properly instructed, persons may continue at home with FES-induced training with a daily frequency and for the long duration of time needed to achieve clinically-relevant outcomes. Standing on the results of the EU Project RISE,1 and on those in aging persons,2,3 we are confident that FES may contribute to a better life for an expanding population of aged persons and of early aged patients suffering with mobility impairments due to neuromuscular and metabolic disorders as well as systemic diseases. We will describe in detail results of FES for denervated degenerated muscles (DDM) delivered by large anatomically shaped surface electrodes at the level of the stimulated skin. Indeed h-bFES for DDM is able to recover skin from SCI-induced atrophy and flattening.4,5 Further, we will discuss the value of surface morphometry of neck skin, and of non-invasive blood analyses of circulating myokines (using sweat and mouth fluids6,7 as biological markers of aging progression., Male hypogonadism is a clinical syndrome manifesting with low testosterone (TT) including symptoms like decline in lean mass, muscle strength, increased adiposity, visceral obesity and incidence of insulin resistance and metabolic syndrome. 1 There are only few studies dealing with effect of physical activity on hypogonadal males.2 The aim of the study was to examine the effect of 12-week strength training program on body composition, selected biochemical parameters and physical performance in hypogonadal men. The study compared the effect of resistance training (RT) on hypogonadal patients without hormonal therapy (HP, n=6, 48.41±6.38 yrs, TT= 7.9±1.75 nmol/L) and control group of eugonadal males (EM, n=8, 49.31±5.84 yrs, TT= 15.81±3.99 nmol/L). The subjects performed RT twice a week, the training program consisted of 6 exercises at an intensity from 60-80% of 1RM. Body composition was measured by DXA, muscle strength was measured by predicted dynamic leg press 1RM from multiple repetition maximum, handgrip strength using hand dynamometer. Fasting morning venous blood samples were collected. The parameters analyzed from serum were glucose, total cholesterol, LDL cholesterol, HDL cholesterol, SHBG, insulin, total testosterone, and cortisol. Subjects from both the HP and EM groups significantly decreased relative fat mass by 6.2 % and 4.91% (p>0.05 and p, Central Core Disease (CCD) is a congenital myopathy characterized by presence of amorphous central areas (or cores) lacking glycolytic/oxidative enzymes and mitochondria in skeletal muscle fibers.1 Most CCD families are linked to mutations in ryanodine receptor type-1 (RYR1), the gene encoding for the sarcoplasmic reticulum (SR) Ca2+ release channel of skeletal muscle.2 As no treatments are available for CCD, currently management of patients is essentially based on a physiotherapic approaches. Functional electrical stimulation (FES) is a technique used to deliver low energy electrical impulses to artificially stimulate selected skeletal muscle groups.3-5 Here we tested the efficacy of FES in counteracting muscle loss and improve function in the lower extremities of a 55-year-old female patient which was diagnosed with CCD at the age of 44. Genetic screening of the RyR1 gene identified a missense mutation (c.7354C>T) in exon 46 resulting in an amino acid substitution (p.R2452W) and a duplication (c.12853_12864dup12) in exon 91. The patient was treated with FES for 26 months and subjected before, during, and after training to a series of functional and structural assessments: measurement of maximum isometric force of leg extensor muscles, magnetic resonance imaging, a complete set of functional tests to assess mobility in activities of daily living, and analysis of muscle biopsies by histology electron microscopy. All results point to an improvement of muscle structure and function induced by FES suggesting that this approach could be considered as an additional supportive measure to maintain/improve muscle function and reduce muscle loss in CCD patients., Over the last decade, transcutaneous auricular nerve stimulation (tANS) become established method of VNS and has been verified in various disorders including neurological and psychological trauma, addiction of drugs, inflammation and tinnitus. Several groups examined also effects of tANS on central and peripheral nervous system, effects on behaviour in neuropsychiatric populations and effects on subjets with disorder of cognitive and social functioning. tANS of particular areas at the cymba conchae (CC) require electrodes with high spatial selectivity, low impedance and safe reversible charge delivery to specific populations of receptors through the electrode-tissue interface. Cortisol is a naturally occurring steroid hormone that has many important functions in the body and is released into the bloodstream at times of stress. Cortisol is blamed for anxiety, high blood pressure and stroke. It helps to control blood sugar levels, regulate metabolism, help reduce inflammation and assist with memory formulation. All treatments to correct eventual cortisol imbalance involve medication. The present study was aimed at demonstrating that selective tANS can be potentially used as a method for the induction of Cortisol hormone secretion. tANS was accomplished in a 62-year-old subject with angina pectoris, coronary artery disease, and moderate insomnia. Sites at the CC were selectively stimulated using a silicone plug with four platinum cathodes. For the tANS, current regulated stimulating pulses with an intensity of ic=20 mA, a pulse width of 200 μs, and a frequency of f=25, were used. Results show that in six out of eight samples of the mouth fluids, the cortisol level was significantly lower after tANS that before tANS., The environment is critical when, in order of intrinsic morphological or geographical characteristics, or extrinsic features (caused by human presence or human behavior), it induces strenuous changes (independent of emergency accidents) in the physiological conditions of people who require emergency aid.1 Moreover, in an extrinsic critical environment the possibility to have a not homogenous population to be rescued is greater, consequently we can act only on the variable physical capacities and competence of emergency team, to have more success. This is the purpose of this work that has a background of more than 1200 physical evaluations over 10 years, on Italian Alpine Soldiers.2 Every subject had three evaluations of fitness profile during the year (time 0, after three months of programmed training and after six months). Evaluation and training methods are studied in order to be applied to a large number of subjects. The fitness profile includes: anthropometric assessment, aerobic fitness, muscle endurance, heart rate, diastolic and systolic pressure, respiratory rate, hydration, postural assessment by assessing the spine with spinal mouse system.3 The obtained results have shown that the physical preparation carried out, has improved and made homogeneous the physical capabilities of both the team and individual subjects that make it up. In addition to making the members of the emergency team more conscious, the increase in the level of coordinated physical preparation has objectively improved the safety (for team members) and effectiveness of intervention in the various crisis situations., In daily life situations, if we are exposed to unusual, challenging situations, our body reacts in a special way, called acute stress. This mechanism helps to cope with these situations efficiently. Contrary, exposing the body to stress over a longer time period, called chronic stress, like in working environments, can cause various serious health problems.1 The assessment of electrodermal activity (EDA, skin conductance) might be an easy approach to evaluate individual stress conditions over a longer period of time in an objective way. With the help of cvxEDA,2 the monitored skin conductance,3 can be split into the slow skin conductance level (SCL) and the fast skin conductance response (SCR). 20 volunteers were exposed to a relaxing-challenging-relaxing situation to mimic different stress conditions. The SCR rate proofed to be a reliable measure to evaluate sympathetic arousal. During the relaxation phases, the SCR rate was around 6 min-1and during mathematical challenges, and it raised to 9 min-1(p, The nonlinear trimodal regression analysis (NTRA) method,1,2 based on radiodensitometric computed tomography (CT) distributions,3,4 is here used for building predictive models of cardiovascular health parameters, through tree-based Machine Learning (ML) algorithms. This study reports the use of NTRA parameters for classifying elderly subjects from the AGES-Reykjavik database;5 with coronary heart disease (CHD), cardiovascular disease (CVD), and chronic heart failure (CHF). ML models employing the random forests algorithm yielded the highest classification performance for all analyses, and overall classification scores for all three conditions were excellent: CHD (AUCROC: 0.936); CVD (AUCROC: 0.914); CHF (AUCROC: 0.994). The present work introduces a substantial step forward in the construction of non-invasive, standardized tools for associating adipose, loose connective, and lean tissue changes with cardiovascular health outcomes in elderly individuals., Postural Control is the complex feedback system that allows humans to keep balance and maintain the naturally unstable upright stance. This is the result of an articulate interplay between sensorimotor systems (vision, proprioception, somatosensory, etc.) and the Central Nervous System (CNS)1,2. Lesions to the CNS, pathologies like unilateral vestibular loss (UVL) and disorders like Motion Sickness disrupt the postural feedback system, severely impairing balance. In these adverse postural circumstance, prompt adaptive and habituating processes are activated in the CNS to ensure upright posture and gait. By integrating the traditional dynamic posturography with HD-EEG analysis on a cohort of thirtythree healthy subjects3, we investigate cortical involvement in the phases of adaptation and habituation to a postural control challenge, where the balance disruption is induced through a randomized sequence of vibratory stimuli applied to the gastrocnemius muscles of the participant’s calves Keywords: Balance, cerebral cortex, HD-EEG, kinematics, postural control, power spectral density., During skeletal muscle contraction Ca2+ release units (CRUs), the sites of excitation-contraction coupling (EC) provide Ca2+,1 while mitochondria, the organelles deputed to cellular respiration, produce ATP.2 In fast-twitch fibers from adult mice CRUs and mitochondria are structurally linked by small strands or tethers,3 and interact functionally, as the uptake of Ca2+ into the mitochondrial matrix stimulates the respiratory chain.4 Aging causes separation of mitochondria from CRUs and a cross-talk impairment between the two organelles.5 However, whether this age-related uncoupling is the result of aging per-se or the consequence of reduced muscle activity remains still unclear. Here we tested if muscle activity maintains the correct association of mitochondria to CRUs in a) extensor digitorum longus (EDL) muscles from 2 year old mice, either sedentary or trained for 1 year in wheel cages; and b) EDL muscles from denervated adult mice and rats. We analyzed muscle samples using a combination of structural, biochemical, and functional experimental procedures. The results collected in structural studies indicate that: a) ageing and denervation result in partial uncoupling between CRUs and mitochondria; b) exercise and re-innervation either maintains (in old mice) or restores (in transiently denervated rats) the association between the two organelles. Functional studies support the hypothesis that CRU-mitochondria cross-talk is important for mitochondrial Ca2+ uptake, optimal force generation, and muscle performance. Taken together, our results show that muscle activity maintain/improve proper Association between CRUs and mitochondria, providing a potential tool to counteract muscle function decline in elderly and sedentary persons., Wartenberg introduced the pendulum test in the 1950s as a method to assess spasticity in the clinical setting.1 It has proven to be sensitive to the presence and severity of spasticity.2,3 The pendulum test is based on letting the lower leg swing freely under the influence of gravity while recording joint kinematics. In the presented work, the reflex period, extracted from the pendulum test, will be proposed as a new parameter to measure spasticity. The reflex period is defined as the time period from 50% of the maximum velocity of the leg to the first EMG signal indicating muscle contraction. Data from two separate studies, one on spinal cord injury patients (Halla Kristín Guðfinnsdóttir)4 and the other on stroke patients (Belinda Chenery)5 were analyzed. In both studies a pendulum test with goniometers on the knee joint was performed and simultaneously recording electromyography (EMG) of the quadricpes m. Both studies consisted of 3 trials and data from 4 subjects of each study were analyzed. The EMG data was processed with Matlab R2014b (The MathWorks, Inc.) using the open-source toolbox EEGLab. A 4th order Butterworth high-pass filter with cutoff frequency at 10 Hz and a low-pass filter at 500 Hz was applied along with notch filters at 50 Hz, 100 Hz, 150 Hz and so forth. The data was smoothed with a Gaussian filter prior to the analysis. The mean reflex period of SCI patients was 370 ms with a standard deviation of 72 ms. The mean reflex period of stroke patients was 215 ms with a standard deviation of 54 ms. This suggests a difference between the reflex period of stroke and SCI patients which might be explained by the location of their lesion., The demonstrated expression of endocannabinoids receptors in myofascial tissue suggested the role of fascia as source and modulator of pain.1 It is known that the fibroblasts can modulate the production of the various components of the extracellular matrix according to different stimuli: physical, mechanical, hormonal and pharmacological.2 In this work we isolated the fascial fibroblasts from small samples of human fascia lata of the thigh collected from 3 volunteers patients during orthopedic surgery, 2 males and 1 female. We demonstrated for the first time that the agonist of cannabinoid receptor 2, HU-308, permits the in vitro production of hyaluronan-rich vesicles in only 3-4 hours after the treatment, quickly released in the extracellular environment. The cells treated with the synthetic cannabinoid showed a large number of vesicles near the Golgi apparatus of the cells, and also in the cytoplasmic extensions. We demonstrated by Alcian Blue and Toluidin Blue stainings, immunocytochemistry and Transmission Electron Microscopy that the content of these vesicles was rich in hyaluronan. The hyaluronan is a critical element for the extracellular matrix composition and remodeling,3 and in the deep fascia, it affects the movement of hyaluronan-containing fluid layers within and underlying the deep fascia, facilitating the smooth gliding between these structures during movement.4 So its production induced by the cannabinoids can be able to increase the ability of the collagen bundles inside the fasciae to glide one respect to the other, and consequently to improve the tissue adaptability. Furthermore, the stimulation of the endocannabinoid system could be able to provide an anti-fibrotic activity by suppression of pro-inflammatory cytokines and a relief of the myofascial pain.5 These results can help to understand how the cells of fascia can answer to the endocannabinoid system regulating and remodeling the extracellular matrix formation. This is a first step in the comprehension of how the therapeutic applications of cannabinoids for the treatment of pain can have also a peripheral effect, altering the biosynthesis of extracellular matrix in fasciae and consequently remodeling the tissue and its properties., Muscles generate active forces in each half-sarcomere, the smallest contractile Unit, through the coordinated action of myosin motors, assembled in the thick filament, with actin monomers, assembled in the thin filament. Filaments are then arranged in parallel, forming a main axis in the half-sarcomere along which active forces are directed. However, in the musculoskeletal system these active forces must follow non-linear paths from tendon to tendon. This task is achieved thanks to the ability to transmit active forces between half-sarcomeres, along fibre direction (longitudinal axis) through intra-sarcomeric proteins, and between different fibres, in perpendicular directions, through extra-sarcomeric proteins, generally referred as extra-cellular matrix (ECM).1 This ability is affected by age.2 In this work, we analyzed the passive tension generated in elongated fibres alone and when they are arranged in small bundles young and aged healthy humans.3,4 The mechanical properties of the extracellular passive components in a bundle of fibres were deduced by the subtraction of the passive tension observed in single fibres from the passive tension observed in the bundle itself. ECM-related components of passive force are non-negligible in both cases and of the same order of magnitude of intra-sarcomeric components. However, in young humans the increase in passive tension observed in bundles respect to fibres is smaller than in aged humans. Based on our data, we propose to quantitatively characterize the constitutive parameters of a Hill-type three-elements model incorporated in a finite element (FE) mesh representing a fiber bundle. The characterization can be used in future FE models of whole human muscles., Neutral lipid storage disease with myopathy (NLSDM) is a rare autosomal recessive disorder, associated with mutations of patatin like phospholipase domain containing 2 (PNPLA2) gene.1 PNPLA2 encodes adipose triglyceride lipase, that plays a key role in triacylglycerol breakdown1. NLSDM patients mostly present progressive skeletal myopathy, with both proximal and distal involvement.2 Cardiomyopathy, hepatomegaly and diabetes are often observed. NLSDM clinical severity appears to be highly variable and it is difficult to establish the effects of different PNPLA2 mutations on disease phenotype. Recently, alteration in the expression profile of specific microRNAs involved in skeletal muscle development (myomiRs) and lipid metabolism have been observed in neuromuscular disorders.3,4. In this study, we perform the evaluation by qRT-PCR of some circulating microRNAs levels in serum samples obtained from three NLSDM siblings (two brothers and one sister), carrying two PNPLA2 missense mutations.5 Progressive skeletal myopathy was detected in the two brothers and muscle imaging showed fibro-fatty replacement. The sister presented severe hepatosteatosis and diabetes. The analysis of serum microRNAs revealed that NLSDM patients exhibited an increased amount of myomiRs. In particular, a significant correlation between muscle mass and the level of miR-206 and miR-133a was found. Moreover, an elevation of some microRNAs, mainly expressed in liver, was observed, when there was a hepatic involvement. These results show that the changes of serum muscle and lipid metabolism-specific microRNAs might represent biomarkers of skeletal and hepatic involvement. Moreover, the dysregulation of these small molecules might provide a tool to monitor the progression of NLSDM., Spinal muscular atrophy (SMA) is a common autosomal recessive disorder caused by mutations in the gene for the survival motor neuron 1 (SMN 1), and it leads to progressive muscle weakness. A major goal of disease-modifying therapies is to increase the expression of the SMN protein. Although a number of therapies are under evaluation as potential treatments for SMA, there is a critical lack of a biomarker method for assessing the efficacy of therapeutic interventions, particularly those targeting the upregulation of the SMN protein levels. Sensitive methods for quantifying SMN protein in peripheral blood are needed. Accordingly, we developed an imaging flow cytometry (IFC) method for evaluation of SMN protein using peripheral blood and fibroblasts. First, we demonstrated that IFC successfully identified different expression patterns and subcellular localization patterns of SMN protein in SMA patient-derived fibroblasts1. Second, we tested the sensitivity and utility of IFC in identifying the differences in the expression of SMN protein between SMA patients and normal subjects using cultured Epstein–Barr virus-transformed B cells2. Subsequently, we developed an IFC method for evaluating the functional SMN protein using < 1.5 mL of peripheral blood. IFC is advantageous for the analysis of peripheral blood because of its capacity to analyze heterogeneous cell populations3. IFC analysis can be implemented in future studies to optimize its application as a tool for assessment of the effectiveness of spinal muscular atrophy treatment., Prolonged oxidative stress may play a key role in tumor development. Antioxidants molecules are contained in many foods and seem to have a potential role in future anti-tumor strategies. Among the natural antioxidants the beneficial effect of Fermented Papaya (FPP®) is known. The aim of this study was to investigate the effects of orally administered FPP® in either prevention or treatment of a murine model of melanoma. The tumor growth was analyzed together with the blood levels of both oxidants (ROS) and anti-oxidants (SOD-1 and GSH). The results showed that FPP® controlled tumor growth, reducing the tumor mass of about 3 to 7 times vs untreated mice. The most significant effect was obtained with sublingual administration of FPP® close to the inoculation of melanoma. At the time of the sacrifice none of mice treated with FPP® had metastases and the subcutaneous tumors were significantly smaller and amelanotic, compared to untreated mice. Moreover, the FPP® anti-tumor effect was consistent with the decrease of total ROS levels and the increase in the blood levels of GSH and SOD-1. This study shows that a potent anti-oxidant treatment through FPP® may contribute to both preventing and inhibiting tumors growth. The results of the above study suggested that FPP® while showing a clear anti-tumor effect it occurred though the in vivo induction of a potent anti-oxidant reaction. In a new set of experiments we wanted to verify whether FPP had a clear and scientifically solid in vivo anti-aging effect together with the induction of the anti-oxidant reaction. To this purpose we used a mouse model suitable for aging studies (C576J) treating daily each mouse from 4 weeks of life to 10 months with the same dose of IMMUNEAGE dissolved into the daily water as compared to mice receiving only tap water. At the end of the treatment period (10 months) we measured some biological parameters related to the aging processes of the cells: i) the total anti-oxydant capacity in the plasma of mice treated or untreated with FPP®; ii) the telomerase activity in the plasma of mice treated or untreated with FPP®; iii) the telomeres length in the bone marrow and ovaries of mice treated or untreated with FPP® .The results showed that the blood of treated mice, at the end of the treatment period (10 months) had 2-3 folds more anti-oxidant power and telomerase activities than the untreated mice. In the same mice we obtained both the bone marrow (from the tibias) and the ovaries at the scarify, and from the cellular preparations we measured the telomere lengths in both. The results showed that daily FPP® assumption induced 3 folds increase in telomeres length in bone marrow and ovary of treated mice as compared to the untreated mice. This suggests that FPP® induces a clear improvement of the aging scientific parameters and that the treated mice were younger than the untreated controls., The muscle spindle (MS) plays an important role in proprioception and coordination,1 which is surrounded by a strong capsule of connective tissue.2 A gelatinous fluid rich in glycosaminoglycans fills the space of capsule, probably mainly hyaluronan (HA).3 Age-related proprioceptive and coordinative deficits are unclear, although some hypothesises have been proposed to explain those mechanisms both on the peripheral and CNS changes. Age-related changes of MS were compared in unmature (1month, M); young adults (4M); and old (27 M) C57BL/6J male mice. Hematoxylin Eosin (HE), Sirius-red,4 Van Gieson staining were used to evaluate age-related MS morphology changes The collagen type I antibody, biotin labeled HA binding protein immunostainings,5 were used to monitor age-related changes in collagen type I and HA of MS. The Purple-Jelley HA assay was used to measure age-related changes HA contents. MS is surrounded by the capsule in continuity with the perimysium and epimysium and HA filled the capsule. The capsule of the MS, perimysium, epimysium underwent thicking with aging, primarily consisting of collagen type I. Van Gieson showed that the presence of elastin in the capsule. The amount of HA in Triceps Surae is 27.74g/g in 4M. The presence of the collagen fiber, elastin and HA in the capsule of MS and its continuous with fascia suggested that they may effect the MS function. If their proportion changed, they will influence the function of MS, which properly explain part of the peripheral Neuro-physiological mechanisms of Age-related proprioceptive and coordinative deficits., Muscle structure,1 and performance2 differ between female and males when compared across the same age groups. Masters athletes compete in age groups of five-year divisions, ranging from 35 to more than 100 years of age. Here, our aim is to investigate the gender differences in the rate of age-related decay using the complete series of female and male normalized Masters’ World records. The World record series are lists of up to 16 data points that can be interpolated with polynomial trend-lines with a very high R2, after normalization to allow comparisons of the values among the different sports events.3 Here, gender comparisons were performed for 19 Track and Field specialties using weighted regression analyses. As expected, the aging decline began at 35 years for both women and men. Despite higher values of the male athletes in all the 19 Track and Field Masters world records,4 the rates of aging performance decay were very similar, if not identical.5 This lack of difference is a unique exception to the general rule of gender differences in sports activities, suggesting that neuro-hormonal mechanisms poorly influence the rate of aging muscle power decay. We discuss implications and limitations of our hypothesis that, at least in humans, the rates of age-induced decline are related to fundamental cellular mechanisms, perhaps those that control energy metabolism., The exponential increase of scientific publications in the mitochondrial field shows the growing interest in mitochondria. However, the lack of methodological consistency in many published projects on mitochondrial respiratory function complicates a quantitative inter/intra-laboratory comparison of datasets. This deficiency manifests the need to improve the quality in science.1 In this context, the MitoEAGLE COST Action is a powerful framework committed to evaluate and enhance the reproducibility in mitochondrial physiology as a basis to establish a novel mitochondrial database related to Evolution, Age, Gender, Lifestyle and Environment. Permeabilized muscle fibers are widely used to evaluate mitochondrial function in health and disease.2 Therefore, our main goals are to: 1) compare protocols used in different research laboratories, 2) analyze factors which contribute to experimental variability, 3) define optimal experimental conditions in muscle studies, 4) elaborate guidelines for evaluating mitochondrial function in muscle tissue, 5) establish reference values on mitochondrial respiration, particularly as a test of the skills in preparing high-quality permeabilized muscle fibers, and 6) generate a database. To achieve our aims, two unique studies are currently in progress: 1) 17 international research groups performing independently experiments on respiration in permeabilized fibers of mouse soleus muscle, following the same experimental procedure;3 2) a blinded international study measuring simultaneously in the same laboratory respiration of permeabilized human skeletal fibers by high-resolution respirometry and assessing the effect of different experimental conditions.4 Our results contribute to face the reproducibility crisis and provide the basis for establishing the first database on mitochondrial respiratory parameters in muscle tissues. Support. MitoEAGLE Task Group WG2. Contribution to European Union Framework Programme Horizon 2020 COST Action CA15203 MitoEAGLE., In the search for systemic changes of biomarkers1 in physical activity, within aging and rehabilitation studies, a major goal is to identify methods less invasive than blood sampling. Thus, the clinical use of mouth fluids is increasing.2 The consensus is that the presence of blood in mouth fluid compromises its diagnostic value. However, we have been looking at its’ contamination as a major opportunity for non-invasive serological analyses of systemic biomarkers. A major preliminary result has been obtained by evaluating the presence of serum in mouth fluids of healthy seniors and the eventual change after a modest trauma, i.e., tooth brushing. Seven healthy persons, aged older than 65, provided the fluids for the analyses by drooling saliva into a test tube. After low speed centrifugation, small aliquots of supernatants were frozen in liquid nitrogen and stored at -80°C until use. Aliquots were thawed and used to quantify by the total protein content using the Lowry method. Serum albumin, fibrinogen and lysozyme were determined by colorimetric ELISA (enzyme-linked immunosorbent assay), and hemoglobin content was quantified by use of spectrophotometry. After a preliminary test using colorimetric ELISA, saliva dilutions were adjusted to better determine the content of analytes. The control reference was a pool of sera from age-matched healthy seniors, to judge the quantity of serum in the fluid obtained from mouths of the elderly. Saliva collected from the seven healthy elderly persons before and after tooth-and-gum brushing presented measurable amount of the analytes, including fibrinogen, which is a minor component of the pooled sera. Tooth brushing did not induce a statistically significant difference in analytes’ contents, suggesting that a measurable blood contamination is a frequent event in elderly persons. In conclusion, fibrinogen is a promising reference to quantify serological biomarkers through a non-invasive procedure. Its determination will increase acceptability and frequency of analyses during follow-up in aging and rehabilitation., Older and oldest persons, i.e., early and late octogenarians, spend small amounts of time performing daily physical activity, which can aggravate their limited independence. This may force them to stay in bed, and/or to incur more frequent hospitalizations. All progressive muscle contractile impairments, including those related to advancing age, need permanent management.1,2 Inspired by the proven capability to recover skeletal muscle by home-based functional electrical stimulation (hbFES) and guided by common sense, we suggest to these elderly persons to perform a 10 to 20 min daily routine of 12 easy and safe physical exercises, that will train almost all the main skeletal muscles of the body. Elderly persons can do many of these exercises in bed (full-body in-bed gym), so that if hospitalized they may continue light training. The routine is an extension of the well-established cardiovascular and ventilatory rehabilitation trainings, usually performed under a guidance and supervision of a physiotherapist.5 If properly instructed in an outpatient clinic or during hospitalization, elderly persons may continue these exercises at home, and eventually integrate into their permanent lifestyle.3,4 Monitored arterial blood pressure before and after the daily routine described demonstrates that peripheral resistance decreases in a few minutes by functional hyperemia of the trained body muscles. The exercise intensity ought to be slightly challenging, up to the manifestation of visible sweating. In the long term, this will add systemic resistance to fatiguing arising from strength training against the own weight of an arm, leg and other body parts.2 Continued regularly, home-based full-body in-bed gym can help to maintain independence of the most frail elderly persons (old and oldest), reducing risks of accidental falls, and thus of serious clinical consequences., Scientific literature suggests that fitness characteristics in patients with Multiple Sclerosis (MS) may be compromised. Because nanotechnological devices have shown improvements in different healthcare application,1,2 the purpose of this study was to investigate the use of a nanotechnological device integrated with a postural exercise program (PEP) on cervical range of motion and handgrip strength in patients affected by MS. Seventeen participants with MS were recruited and randomly assigned to the Experimental Group (EG) and the Control Group (CG) including 9 and 8 subjects respectively. All participants carried out a cervical range of motion evaluation using an inertial motion sensor (Moover®;Sensor Medica®) and a handgrip test using an isometric mechanical dynamometer (KernMap model 80K1;Kern®) before and after a PEP (twenty-session/1 hour each, twice/week). A nanotechnological device (Taopatch®;Tao Technologies srls) was applied to the skin of the participants of the EG at the level of the C7 vertebra throughout the period of the PEP. No differences (p>0.05) were found in the EG on the cervical range of motion, while the CG showed a significantly increase on left rotation (p=0.01). Regards the handgrip test, a significantly improvement was found on the non-dominant hand in the CG (p=0.001) and on the non-dominant hand (p=0.04) and on the dominant hand (p=0.01) in the EG. Our preliminary results suggest that the use of a nanotechnological device could influence muscle strength. The results of this pilot study suggest that we should continue testing larger sample of subjects, diversifying also type and stage of the disease. The authors thank the patients and the Italian Association for Multiple Sclerosis (AISM) of Trapani, Italy for collaboration., Athletes have to pay particular attention on the eating patterns and to dietary intake in the hours before exercise, because pre-exercise nutritional strategies can influence not only exercise performance but also other biological responses to exercise and training including bone remodeling. In fact, inadequate nutrition leads to bone injuries. Twenty-eight preadolescent female gymnasts, playing artistic gymnastics at a pre-competitive level, were examined to investigate the effects of two different pre-exercise meals on athletic performance and bone resorption post exercise. Exercise trial were preceded ninety minutes by an isocaloric meal with carbohydrates or proteins. Urine was sampled at four different time points: pre-meal, ninety minutes post-meal/pre-exercise, ninety and hundred and fifty minutes post-exercise. In urine was analyzed the biomarker of bone resorption CTX by using an established enzyme-linked immunosorbent assay technique. Energy Self-Perception Questionnaire (ESPQ) was used to evaluate energy status of the athletes after the performance. Pre-workout supplementation with a carbohydrate-rich meal reduced significantly post-exercise bone resorption, compared with the protein-rich meal as evidenced by the reduction in CTX levels sixty minutes after the end of physical activity. Moreover, the consumption of both pre-workout meal improves performance indifferently. In conclusion the study shows that a carbohydrate rich pre-exercise meal is able to reduce bone resorption after exercise in pre pubertal age gymnasts and improve the athletic performance., The Main objective of the study was to evaluate the effects of treatment with Cannabis-based drugs combined or not with a proprioceptive training, on patients affected by Multiple Sclerosis (MS), a chronic autoimmune demyelinating disease which can have different symptoms; we estimated the possible synergic interaction between therapy and exercise particularly to reduce the collateral adverse effects of the drugs, over all the spasticity, one of the effect of the disease. We enrolled in our study 10 patients randomly assigned in two groups; both was treated with a cannabinoids drug (group 2) but only one group performed a physical activity for 12 weeks two times per week in session of 60 minutes each (group 1). Blood assessment (serum levels of BDNF, proBDNF, interleukine10 and interleukine17) was performed and the results highlighted a changes. As to concern BDNF levels were 111 vs 33 group 1 compare with 82 vs 22 in group 1; whilst proBDNF levels were 190 vs 153 group 1 compare with 121 vs 138 in group 1. About IL’s, we highlighted this results: IL10 (anti-inflammatory) 61,84 vs 75,52 in group 1 compare with 50,64 vs 54,65 in group 2; IL 17 (pro-inflammatory) 31,12 vs 16,87 in group 1 compare with 24,53 vs 9,68 in group 2. The interpretation of the results is still in progress but the changes has occurred; further investigations are needed improving the sample size and the time of the intervention.

Platelet activation plays a key role in normal haemostasis and pathological thrombosis. Platelet activation is rapid; within minutes of stimulation, platelets generate bioactive phospholipids, secrete their granule contents, activate integrins and aggregate together to form a haemostatic plug. These events are dependent on ATP synthesis. Mitochondrial function in platelets from healthy volunteers and patients with a range of diseases indicate an important role for oxygen consumption in oxidative phosphorylation in normal and pathological function. Platelets also consume oxygen during oxidation reactions, such as cyclooxygenase-dependent thromboxane A2 synthesis. In this study, we used high-resolution respirometry to investigate rapid changes in oxygen consumption during platelet activation. We demonstrated a rapid, transient increase in oxygen consumption rate within minutes of platelet stimulation by the physiological activator, thrombin. This was partly inhibited by aspirin and by oligomycin. This shows that high resolution respirometry can provide information regarding rapid and dynamic changes in oxygen consumption during platelet activation.

The Himalayan Sherpas, a human population of Tibetan descent, are highly adapted to life in the hypobaric hypoxia of high altitude. Mechanisms involving enhanced tissue oxygen delivery in comparison to Lowlander populations have been postulated to play a role in such adaptation. Whether differences in tissue oxygen utilization (i.e., metabolic adaptation) underpin this adaptation is not known, however. We sought to address this issue, applying parallel molecular, biochemical, physiological, and genetic approaches to the study of Sherpas and native Lowlanders, studied before and during exposure to hypobaric hypoxia on a gradual ascent to Mount Everest Base Camp (5,300 m). Compared with Lowlanders, Sherpas demonstrated a lower capacity for fatty acid oxidation in skeletal muscle biopsies, along with enhanced efficiency of oxygen utilization, improved muscle energetics, and protection against oxidative stress. This adaptation appeared to be related, in part, to a putatively advantageous allele for the peroxisome proliferator-activated receptor A (PPARA) gene, which was enriched in the Sherpas compared with the Lowlanders. Our findings suggest that metabolic adaptations underpin human evolution to life at high altitude, and could have an impact upon our understanding of human diseases in which hypoxia is a feature.

In adults, glucocorticoids act to match the supply and demand for energy during physiological challenges, partly through actions on tissue mitochondrial oxidative phosphorylation (OXPHOS) capacity. However, little is known about the role of the natural prepartum rise in fetal glucocorticoid concentrations in preparing tissues for the increased postnatal energy demands. This study examined the effect of manipulating cortisol concentrations in fetal sheep during late gestation on mitochondrial OXPHOS capacity of two skeletal muscles with different postnatal locomotive functions. Mitochondrial content, biogenesis markers, respiratory rates and expression of proteins and genes involved in the electron transfer system (ETS) and OXPHOS efficiency were measured in the biceps femoris (BF) and superficial digital flexor (SDF) of fetuses either infused with cortisol before the prepartum rise or adrenalectomised to prevent this increment. Cortisol infusion increased mitochondrial content, biogenesis markers, substrate-specific respiration rates and abundance of ETS complex I and adenine nucleotide translocator (ANT1) in a muscle-specific manner that was more pronounced in the SDF than BF. Adrenalectomy reduced mitochondrial content and expression of PGC1α and ANT1 in both muscles, and ETS complex IV abundance in the SDF near term. Uncoupling protein gene expression was unaffected by cortisol manipulations in both muscles. Gene expression of the myosin heavy chain isoform, MHCIIx, was increased by cortisol infusion and reduced by adrenalectomy in the BF alone. These findings show that cortisol has a muscle-specific role in prepartum maturation of mitochondrial OXPHOS capacity with important implications for the health of neonates born pre-term or after intrauterine glucocorticoid overexposure.

Background Numerous pathologies result in multiple-organ failure, which is thought to be a direct consequence of compromised cellular bioenergetic status. Neither the nature of this phenotype nor its relevance to survival are well understood, limiting the efficacy of modern life-support. Methods To explore the hypothesis that survival from critical illness relates to changes in cellular bioenergetics, we combined assessment of mitochondrial respiration with metabolomic, lipidomic and redox profiling in skeletal muscle and blood, at multiple timepoints, in 21 critically ill patients and 12 reference patients. Results We demonstrate an end-organ cellular phenotype in critical illness, characterized by preserved total energetic capacity, greater coupling efficiency and selectively lower capacity for complex I and fatty acid oxidation (FAO)-supported respiration in skeletal muscle, compared to health. In survivors, complex I capacity at 48 h was 27% lower than in non-survivors (p = 0.01), but tended to increase by day 7, with no such recovery observed in non-survivors. By day 7, survivors’ FAO enzyme activity was double that of non-survivors (p = 0.048), in whom plasma triacylglycerol accumulated. Increases in both cellular oxidative stress and reductive drive were evident in early critical illness compared to health. Initially, non-survivors demonstrated greater plasma total antioxidant capacity but ultimately higher lipid peroxidation compared to survivors. These alterations were mirrored by greater levels of circulating total free thiol and nitrosated species, consistent with greater reductive stress and vascular inflammation, in non-survivors compared to survivors. In contrast, no clear differences in systemic inflammatory markers were observed between the two groups. Conclusion Critical illness is associated with rapid, specific and coordinated alterations in the cellular respiratory machinery, intermediary metabolism and redox response, with different trajectories in survivors and non-survivors. Unravelling the cellular and molecular foundation of human resilience may enable the development of more effective life-support strategies., Graphical abstract Image 1

Mitochondria respond to a range of stimuli and function in energy production and redox homeostasis. However, little is known about the developmental and environmental control of mitochondria in the placenta, an organ vital for fetal growth and pregnancy maintenance in eutherian mammals. Using respirometry and molecular analyses, the present study examined mitochondrial function in the distinct transport and endocrine zones of the mouse placenta during normal pregnancy and maternal inhalation hypoxia. The data show that mitochondria of the two zones adopt different strategies in modulating their respiration, substrate use, biogenesis, density, and efficiency to best support the growth and energy demands of fetoplacental tissues during late gestation in both normal and hypoxic conditions. The findings have important implications for environmentally induced adaptations in mitochondrial function in other tissues and for compromised human pregnancy in which hypoxia and alterations in placental mitochondrial function are associated with poor outcomes like fetal growth restriction.

Ascent to high altitude is associated with physiological responses that counter the stress of hypobaric hypoxia by increasing oxygen delivery and by altering tissue oxygen utilisation via metabolic modulation. At the cellular level, the transcriptional response to hypoxia is mediated by the hypoxia-inducible factor (HIF) pathway and results in promotion of glycolytic capacity and suppression of oxidative metabolism. In Tibetan highlanders, gene variants encoding components of the HIF pathway have undergone selection and are associated with adaptive phenotypic changes, including suppression of erythropoiesis and increased blood lactate levels. In some highland populations, there has also been a selection of variants in PPARA, encoding peroxisome proliferator-activated receptor alpha (PPARα), a transcriptional regulator of fatty acid metabolism. In one such population, the Sherpas, lower muscle PPARA expression is associated with a decreased capacity for fatty acid oxidation, potentially improving the efficiency of oxygen utilisation. In lowlanders ascending to altitude, a similar suppression of fatty acid oxidation occurs, although the underlying molecular mechanism appears to differ along with the consequences. Unlike lowlanders, Sherpas appear to be protected against oxidative stress and the accumulation of intramuscular lipid intermediates at altitude. Moreover, Sherpas are able to defend muscle ATP and phosphocreatine levels in the face of decreased oxygen delivery, possibly due to suppression of ATP demand pathways. The molecular mechanisms allowing Sherpas to successfully live, work and reproduce at altitude may hold the key to novel therapeutic strategies for the treatment of diseases to which hypoxia is a fundamental contributor.

Placental stress has been implicated in the pathophysiology of complications of pregnancy, including growth restriction and pre-eclampsia. Initially, attention focused on oxidative stress, but recently mitochondrial and endoplasmic reticulum stress have been identified. Complex molecular interactions exist among these different forms of stress, making it unlikely that any occurs in isolation. In part, this is due to close physiological connections between the two organelles principally involved, mitochondria and the endoplasmic reticulum (ER), mediated through Ca2+ signalling. Here, we review the involvement of the mitochondria-ER unit in the generation of stress within the trophoblast, and consider consequences for obstetric outcome. Mild stress may induce adaptive responses, including upregulation of antioxidant defences and autophagy, while moderate levels may affect stem cell behaviour and reduce cell proliferation, contributing to the growth-restricted phenotype. High levels of stress can stimulate release of pro-inflammatory cytokines and anti-angiogenic factors, increasing the risk of pre-eclampsia. In addition, chronic stress may promote senescence of the trophoblast, which in other cell types leads to a pro-inflammatory senescence-associated secretory phenotype. Evidence from rodents suggests that a degree of trophoblastic stress develops with increasing gestational age in normal pregnancies. The increase in maternal concentrations of soluble fms-like tyrosine kinase-1 (sFlt-1) and reduction in placental growth factor (PlGF) suggest the same may occur in the human, starting around 30 weeks of pregnancy. Placental malperfusion, or co-existing maternal conditions, such as diabetes, will exacerbate that stress. Amelioration of trophoblastic stress should remain a research priority, but will be difficult due to the complexity of the molecular pathways involved.

Short-term consumption of a high-fat diet impairs exercise capacity in both rats and humans, and increases expression of the mitochondrial uncoupling protein, UCP3, in rodent cardiac and skeletal muscle via activation of the transcription factor, peroxisome proliferator-activated receptor (PPAR). Unlike long-chain fatty acids however, medium-chain fatty acids do not activate PPAR and do not increase muscle UCP3 expression. We therefore investigated exercise performance and cardiac mitochondrial function in rats fed a chow diet (7.5% kcal from fat), a long-chain triglyceride (LCT) rich diet (46% kcal from LCTs) or a medium-chain triglyceride (MCT) rich diet (46% kcal from MCTs). Rats fed the LCT-rich diet for 15 days ran 55% less far than they did at baseline, whereas rats fed the chow or MCT-rich diets neither improved nor worsened in their exercise capacities. Moreover, consumption of an LCT-rich diet increased cardiac UCP3 expression by 35% and decreased oxidative phosphorylation efficiency, whereas consumption of the MCT-rich diet altered neither UCP3 expression nor oxidative phosphorylation efficiency. Our results suggest that the negative effects of short-term high-fat feeding on exercise performance are predominantly mediated by long-chain rather than medium-chain fatty acids, possibly via PPAR-dependent upregulation of UCP3. © 2011 Murray et al; licensee BioMed Central Ltd.

Background Insulin sensitivity in skeletal muscle is associated with metabolic flexibility, including a high capacity to increase fatty acid (FA) oxidation in response to increased lipid supply. Lipid overload, however, can result in incomplete FA oxidation and accumulation of potentially harmful intermediates where mitochondrial tricarboxylic acid cycle capacity cannot keep pace with rates of β-oxidation. Enhancement of muscle FA oxidation in combination with mitochondrial biogenesis is therefore emerging as a strategy to treat metabolic disease. Dietary inorganic nitrate was recently shown to reverse aspects of the metabolic syndrome in rodents by as yet incompletely defined mechanisms. Results Herein, we report that nitrate enhances skeletal muscle FA oxidation in rodents in a dose-dependent manner. We show that nitrate induces FA oxidation through a soluble guanylate cyclase (sGC)/cGMP-mediated PPARβ/δ- and PPARα-dependent mechanism. Enhanced PPARβ/δ and PPARα expression and DNA binding induces expression of FA oxidation enzymes, increasing muscle carnitine and lowering tissue malonyl-CoA concentrations, thereby supporting intra-mitochondrial pathways of FA oxidation and enhancing mitochondrial respiration. At higher doses, nitrate induces mitochondrial biogenesis, further increasing FA oxidation and lowering long-chain FA concentrations. Meanwhile, nitrate did not affect mitochondrial FA oxidation in PPARα−/− mice. In C2C12 myotubes, nitrate increased expression of the PPARα targets Cpt1b, Acadl, Hadh and Ucp3, and enhanced oxidative phosphorylation rates with palmitoyl-carnitine; however, these changes in gene expression and respiration were prevented by inhibition of either sGC or protein kinase G. Elevation of cGMP, via the inhibition of phosphodiesterase 5 by sildenafil, also increased expression of Cpt1b, Acadl and Ucp3, as well as CPT1B protein levels, and further enhanced the effect of nitrate supplementation. Conclusions Nitrate may therefore be effective in the treatment of metabolic disease by inducing FA oxidation in muscle. Electronic supplementary material The online version of this article (doi:10.1186/s12915-015-0221-6) contains supplementary material, which is available to authorized users.

Skeletal muscle undergoes metabolic remodelling in response to environmental hypoxia, yet aspects of this process remain controversial. Broadly, environmental hypoxia has been suggested to induce: (i) a loss of mitochondrial density; (ii) a substrate switch away from fatty acids and towards other substrates such as glucose, amino acids and ketone bodies; and (iii) a shift from aerobic to anaerobic metabolism. There remains a lack of a consensus in these areas, most likely as a consequence of the variations in degree and duration of hypoxic exposure, as well as the broad range of experimental parameters used as markers of metabolic processes. To attempt to resolve some of the controversies, we performed a comprehensive review of the literature pertaining to hypoxia-induced changes in skeletal muscle energy metabolism. We found evidence that mass-specific mitochondrial function is decreased prior to mass-specific mitochondrial density, implicating intra-mitochondrial changes in the response to environmental hypoxia. This loss of oxidative capacity does not appear to be matched by a loss of glycolytic capacity, which on the whole is not altered by environmental hypoxia. Environmental hypoxia does however induce a selective attenuation of fatty acid oxidation, whilst glucose uptake is maintained or increased, perhaps to support glycolysis in the face of a downregulation of oxidative metabolism, optimising the pathways of ATP synthesis for the hypoxic environment. Electronic supplementary material The online version of this article (doi:10.1186/2046-7648-3-19) contains supplementary material, which is available to authorized users.