Mitochondrial Adaptations in Elderly and Young Men Skeletal Muscle Following 2 Weeks of Bed Rest and Rehabilitation

The aim of the study was to evaluate the expression levels of proteins related to mitochondrial biogenesis regulation and bioenergetics in vastus lateralis muscle biopsies from 16 elderly and 7 young people subjected to 14 days of bed-rest, causing atrophy, and subsequent 14 days of exercise training. Based on quantitative immunoblot analyses, in both groups a reduction of two key regulators of mitochondrial biogenesis/remodeling and activity, namely PGC-1alpha and Sirt3, was revealed during bedrest,with a subsequent up-regulation after rehabilitation, indicating an involvement of PGC-1alpha-Sirt3 axis in response to the treatments. A difference was observed comparing the young and elderly subjects as, for both proteins, the abundance in the elderly was more affected by immobility and less responsive to exercise. The expression levels of TOM20 and Citrate Synthase, assayed as markers of outer mitochondrial membrane and mitochondrial mass, showed a noticeable sensitivity in the elderly group, where they were affected by bed-rest and rehabilitation recalling the pattern of PGC-1alpha. TOM20 and CS remained unchanged in young subjects. Single OXPHOScomplexes showed peculiar patterns, which were in some cases dissimilar from PGC 1alpha, and suggest different influences on protein biogenesis and degradation. Overall,exercise was capable to counteract the effect of immobility, when present, except for complex V, which was markedly downregulated by bed-rest, but remained unaffected after rehabilitation, maybe as result of greater extent of degradation processes over biogenesis. Phosphorylation extent of AMPK, and its upstream activator LKB1, did not change after bed-rest and rehabilitation in either young or elderly subjects, suggestingthat the activation of energy-sensing LKB1-AMPK signaling pathway was \u201cmissed\u201d due to its transient nature, or was not triggered under our conditions. Our study demonstrates that, as far as the expression of various proteins related to mitochondrial biogenesis/remodeling, adaptations to bed-rest and rehabilitation in the two populations were different. The impact of bed-rest was greater in the elderly subjects, where the pattern (decrease after bed rest and recovery following rehabilitation) was accompanied by changes of mitochondrial mass. Modifications of protein abundance were matched with data obtained from gene expression analyses of four public human datasets focusing on related genes

Mitochondrial Adaptations in Elderly and Young Men Skeletal Muscle Following 2 Weeks of Bed Rest and Rehabilitation

The aim of the study was to evaluate the expression levels of proteins related to mitochondrial biogenesis regulation and bioenergetics in vastus lateralis muscle biopsies from 16 elderly and 7 young people subjected to 14 days of bed-rest, causing atrophy, and subsequent 14 days of exercise training. Based on quantitative immunoblot analyses, in both groups a reduction of two key regulators of mitochondrial biogenesis/remodeling and activity, namely PGC-1α and Sirt3, was revealed during bed-rest, with a subsequent up-regulation after rehabilitation, indicating an involvement of PGC-1α-Sirt3 axis in response to the treatments. A difference was observed comparing the young and elderly subjects as, for both proteins, the abundance in the elderly was more affected by immobility and less responsive to exercise. The expression levels of TOM20 and Citrate Synthase, assayed as markers of outer mitochondrial membrane and mitochondrial mass, showed a noticeable sensitivity in the elderly group, where they were affected by bed-rest and rehabilitation recalling the pattern of PGC-1α. TOM20 and CS remained unchanged in young subjects. Single OXPHOS complexes showed peculiar patterns, which were in some cases dissimilar from PGC-1α, and suggest different influences on protein biogenesis and degradation. Overall, exercise was capable to counteract the effect of immobility, when present, except for complex V, which was markedly downregulated by bed-rest, but remained unaffected after rehabilitation, maybe as result of greater extent of degradation processes over biogenesis. Phosphorylation extent of AMPK, and its upstream activator LKB1, did not change after bed-rest and rehabilitation in either young or elderly subjects, suggesting that the activation of energy-sensing LKB1-AMPK signaling pathway was “missed” due to its transient nature, or was not triggered under our conditions. Our study demonstrates that, as far as the expression of various proteins related to mitochondrial biogenesis/remodeling, adaptations to bed-rest and rehabilitation in the two populations were different. The impact of bed-rest was greater in the elderly subjects, where the pattern (decrease after bed rest and recovery following rehabilitation) was accompanied by changes of mitochondrial mass. Modifications of protein abundance were matched with data obtained from gene expression analyses of four public human datasets focusing on related genes

Mitochondrial oxidative phosphorylation plasticity/adaptation triggered by disturbances and stresses and targeted by therapies

Mitochondrion is an important organelle for cells survival. In fact, it is responsible for many processes such as cellular metabolism, i.e. oxidative phosphorylation for ATP production, energy homeostasis and regulation of apoptosis and autophagy. Mitochondrion, due to this role, needs to be \u201cplastic\u201d in order to respond and adapt quickly to any perturbation and change of conditions in the different tissues of the human body. The induction of mitochondria biogenesis is required to meet different energetic demands under stress conditions. Thus, mitochondrial plasticity is the mechanism that controls modification in conditions of cellular stress or in response to environmental stimuli like exercise, caloric restriction, cold exposure, oxidative stress, cell division and renewal, and differentiation. Recently, mitochondrial modulation has become also a topic of interest as a therapeutic target. The master regulator gene of mitochondrial biogenesis is PGC1\u3b1 that, through nuclear transcription factors and subsequent metabolic sensors and other signalling proteins, is capable to modulate mitochondrial abundance, activity and oxidative phosphorylation as a consequence of energy homeostasis unbalance. Mitochondrial plasticity during the last few years was extensively studied in skeletal muscle models, due to its fast adaptation in exercise and rest condition, but also in cancer cachexia, ageing and heart disease. Also in cancer, mitochondrial adaptations have become a fundamental topic, in particular to understand the underling pathogenic mechanism of disease progression, to identify prognostic factors and to design adjuvant therapies targeting mitochondria. In this frame, this PhD Thesis investigates the role and adaptations of mitochondria in different pathophysiological models of skeletal muscle and brain tumors. The expression of some key proteins of the signalling pathways involved in mitochondrial biogenesis regulation, such as PGC1\u3b1, LKB1-AMPK an energy sensing axis, Sirt3 a regulator of mitochondrial enzymes functionality, are investigated together with the OXPHOS complexes, HSP60, CS and TOM20 as mitochondrial mass markers. The first model is aimed at testing the expression levels of the protein panel in skeletal muscle biopsies from a cohort of 16 elderly and 7 young people subjected to immobility (bed-rest) causing hypotrophy and subsequent rehabilitation via exercise training. Based on quantitative immunoblot data, there is a down-regulation of PGC1\u3b1, Sirt3 and OXPHOS complexes II, III and IV occurring during bed-rest with a subsequent up-regulation after rehabilitation in both groups. AMPK and LKB1 do not change during bed-rest and rehabilitation in elderly and young subjects suggesting that there is not energetic impairment. According to the down-regulation of OXPHOS biogenesis during bed-rest there is the up-regulation of GAPDH evocative of a metabolic shift during hypotrophy from oxidative phosphorylation towards glycolysis, which is reversed by exercise training. OXPHOS complex V is down-regulated in both groups during bed-rest, but after rehabilitation the complex expression does not increase, maybe due to an imbalance between protein biogenesis and degradation. It is tempting to speculate that exercise could regulate complex V activity, as a compensatory response, through deacetylation mediated by Sirt3, which is up-regulated after rehabilitation. CS and TOM20 present the same pattern: in elderly subjects there is a down-regulation during immobility that is counteracted by exercise training, whereas young subjects present a similar pattern but differences do not reach statistical significance. In conclusion, immobility is effective in down-regulation of mitochondria-related protein expression and training protocol counteracts this effect. The pattern is similar in both elderly and young subjects, with some differences for PGC1\u3b1, and Sirt3 appearing less responsive to rehabilitation in elderly. Training is a fundamental tool to recover from immobility periods but also to maintain muscle tonicity as a non-pharmacologic therapeutic treatment for chronic heart failure patients (CHF). In the second model is studied the effect of aerobic exercise training (2 months) on mitochondrial respiration in skeletal muscle of CHF transgenic (Tg\u3b1q*44) mice, focusing also on the impact of CHF on skeletal muscle of sedentary mice. Oxidative phosphorylation and electron transport system capacity of biopsies from soleus muscle is assayed by high-resolution respirometry. Sedentary CHF mice exhibit in comparison to wild type an impaired complex I \u2013 State 3 respiration and ADP-stimulated respiration sustained by Complex I+II, in contrast to rotenone insensitive electron transport system respiration that is unchanged. This suggests an inactivation of complex I rather than an impairment of OXPHOS biogenesis in soleus muscle, also confirmed by unchanged value of PGC1\u3b1 expression. Exercise training improves exercise performance, but it does not affect mitochondrial respiration. Factors \u201cupstream\u201d of mitochondria are likely mainly responsible for the functional improvement. The third model focuses on the study of ATP synthase Inhibitory Factor 1 as prognostic marker in low-grade astrocytomas (LGA). 19 pairs of surgical specimens of LGA are evaluated for the tumor border zone in which IF1 abundance is significantly lower than in the tumoral zone. Immunohistochemistry analyses of 68 specimens by Tissue-MicroArrays prove a weak association of IF1 with NF-kB p65-subunit and consolidated radiologic indexes of tumor infiltration and resection. Kaplan\u2013Meier estimation of patients overall survival indicates that IF1 may serve as a prognostic marker. Intriguingly, IF1 expression significantly increases in lesions with suspected first signs of anaplastic transformation (LGA*) as showed, in accordance, by immunofluorescence (12 specimens), immunohistochemistry (11) and immunoblot (9) analyses. Finally, immunoblot analyses provide a picture of mitochondrial and glycolytic markers, suggesting no improvement of glycolysis and little changes in mitochondrial mass. On the contrary, OXPHOS complexes show a significant upregulation in LGA*. IF1 expression levels could be proposed as a biomarker of OS in LGA, rare tumors with a good prognosis, which could nonetheless evolve in anaplastic lesions and are still without an adjuvant therap

Mitochondrial energy metabolism and signalling in human glioblastoma cell lines with different PTEN gene status

Glioblastomas epidemiology and aggressiveness demand for a well characterization of biochemical mechanisms of the cells. The discovery of oxidative tumours related to chemoresistance is changing the prevalent view of dysfunctional mitochondria in cancer cells. Thus, glioblastomas metabolism is now an area of intense research, wherein was documented a high heterogeneity in energy metabolism and in particular in mitochondrial OxPhos. We report results gained by investigating mitochondrial OxPhos and bioenergetics, in a model of three human glioblastoma cell lines characterized by a different PTEN gene status. Functional data are analysed in relation to the expression levels of some main transcription factors and signalling proteins, which can be involved in the regulation of mitochondrial biogenesis and activity. Collectively, our observations indicate for the three cell lines a similar bioenergetic phenotype maintaining a certain degree of mitochondrial oxidative activity, with some difference for PTEN-wild type SF767 cells respect to PTEN-deleted A172 and U87MG characterized by a loss-of-function point mutation of PTEN. SF767 has lower ATP content and higher ADP/ATP ratio, higher AMPK activating-phosphorylation evoking energy impairment, higher OxPhos complexes and PGC1\u3b1-Sirt3-p53 protein abundance, in line with a higher respiration. Finally, SF767 shows a similar mitochondrial energy supply, but higher non-phosphorylating respiration linked to dissipation of protonmotive force. Intriguingly, it is now widely accepted that a regulated mitochondrial proton leak attenuate ROS generation and in tumours may be at the base of pro-survival advantage and chemoresistance

Exercise training in Tgαq*44 mice during the progression of chronic heart failure:cardiac vs. peripheral (soleus muscle) impairments to oxidative metabolism

Cardiac function, skeletal (soleus) muscle oxidative metabolism and the effects of exercise training were evaluated in a transgenic murine model (Tgαq*44) of chronic heart failure (CHF) during the critical period between the occurrence of an impairment of cardiac function and the stage at which overt cardiac failure ensues (i.e. from 10 to 12 months of age). Forty-eight Tgαq*44 mice and 43 wild-type (WT) FVB controls were randomly assigned to control groups and to groups undergoing 2 months of intense exercise training (spontaneous running on a instrumented wheel). In mice evaluated at the beginning and at the end of training we determined: exercise performance (mean distance covered daily on the wheel); cardiac function in vivo (by magnetic resonance imaging); soleus mitochondrial respiration ex vivo (by high-resolution respirometry); muscle phenotype (myosin heavy chain [MHC] isoforms content; citrate synthase [CS] activity) and variables related to the energy status of muscle fibers (p-AMPK/AMPK) and mitochondrial biogenesis and function (PGC-1α). In the untrained Tgαq*44 mice functional impairments of exercise performance, cardiac function and soleus muscle mitochondrial respiration were observed. The impairment of mitochondrial respiration was related to the function of complex I of the respiratory chain, and it was not associated with differences in CS activity, MHC isoforms, p-AMPK/AMPK and PGC-1α levels. Exercise training improved exercise performance and cardiac function, but it did not affect mitochondrial respiration, even in the presence of an increased % of type 1 MHC isoforms. Factors “upstream” of mitochondria were likely mainly responsible for the improved exercise performance