Joan Ramon Torrella

Modulation of cardiac mitochondrial permeability transition and apoptotic signaling by endurance training and intermittent hypobaric hypoxia

BACKGROUND Modulation of the mitochondrial permeability transition pore (MPTP) and inhibition of the apoptotic signaling are critically associated with the cardioprotective phenotypes afforded by both intermittent hypobaric-hypoxia (IHH) and endurance-training (ET). We recently proposed that IHH and ET improve cardiac function and basic mitochondrial capacity, although without showing addictive effects. Here we investigate whether a combination of IHH and ET alters cardiac mitochondrial vulnerability to MPTP and related apoptotic signaling. METHODS Male Wistar rats were divided into normoxic-sedentary (NS), normoxic-exercised (NE, 1h/day/5 week treadmill-running), hypoxic-sedentary (HS, 6000 m, 5h/day/5 weeks) and hypoxic-exercised (HE) to study susceptibility to calcium-induced cardiac MPTP opening. Mitochondrial cyclophilin D (CypD), adenine nucleotide translocator (ANT), Bax and Bcl-2 protein contents were semi-quantified by Western blotting. Cardiac caspase 3-, 8- and 9-like activities were measured. Mitochondrial aconitase and superoxide dismutase (MnSOD) activity and malondialdehyde (MDA) and sulphydryl group (-SH) content were determined. RESULTS Susceptibility to MPTP decreased in NE and HS vs. NS and even further in HE. The ANT content increased in HE vs. NS. Bcl-2/Bax ratio increased in NE and HS compared to NS. Decreased activities in tissue caspase 3-like (HE vs. NS) and caspase 9-like (HS and HE vs. NS) were observed. Mitochondrial aconitase increased in NE and HS vs. NS. No alterations between groups were observed for caspase 8-like activity, MnSOD, CypD, MDA and -SH. CONCLUSIONS Data confirm that IHH and ET modulate cardiac mitochondria to a protective phenotype characterized by decreased MPTP induction and apoptotic signaling, although without visible addictive effects as initially hypothesized.

COMPARATIVE SKELETAL MUSCLE FIBRE MORPHOMETRY AMONG WILD BIRDS WITH DIFFERENT LOCOMOTOR BEHAVIOUR

Six muscles of the mallard duck (Anas platyrhynchos), the common coot (Fulica atra) and the yellow‐legged gull (Larus cachinnans) were analysed morphometrically, with special emphasis on their functional implications and physiological needs. Oxidative fibres always had significantly smaller size than anaerobic fibres, although no differences in the number of capillaries per fibre were found. This resulted in greater capillary counts per unit of fibre area and perimeter in oxidative than anaerobic fibres, which indicates that the greater demand for oxygen supply may be achieved by decreasing the size of the muscle fibre rather than by increasing the number of associated capillaries. Fast oxidative fibres of the pectoralis and the triceps of the gull had greater sizes than the fast oxidative fibres of the mallard and the coot, which correlates with the difference in energetic demands between flapping and gliding flight. Greater fibre cross‐sectional areas and perimeters seem suited to afford the long‐lasting activity with low metabolic demands required during gliding. By contrast, mallards and coots attain a high oxidative metabolism, during sustained flapping flight, by reducing fibre size at the expense of a diminished ability for force generation. Between‐species comparisons of the hindlimb muscles only yielded differences for the anaerobic fibres of the gastrocnemius, as an important adaptive response to force generation during burst locomotion. The need to manage sustained swimming abilities effectively may result in similar FOG fibre morphometry of the hindlimb muscles studied, indicating that a compromise between the oxygen flux to the muscle cell and the development of power is highly optimised in oxidative fibres of the bird species studied.

Swimming-induced exercise promotes hypertrophy and vascularization of fast skeletal muscle fibres and activation of myogenic and angiogenic transcriptional programs in adult zebrafish

BackgroundThe adult skeletal muscle is a plastic tissue with a remarkable ability to adapt to different levels of activity by altering its excitability, its contractile and metabolic phenotype and its mass. We previously reported on the potential of adult zebrafish as a tractable experimental model for exercise physiology, established its optimal swimming speed and showed that swimming-induced contractile activity potentiated somatic growth. Given that the underlying exercise-induced transcriptional mechanisms regulating muscle mass in vertebrates are not fully understood, here we investigated the cellular and molecular adaptive mechanisms taking place in fast skeletal muscle of adult zebrafish in response to swimming.ResultsFish were trained at low swimming speed (0.1 m/s; non-exercised) or at their optimal swimming speed (0.4 m/s; exercised). A significant increase in fibre cross-sectional area (1.290 ± 88 vs. 1.665 ± 106 μm2) and vascularization (298 ± 23 vs. 458 ± 38 capillaries/mm2) was found in exercised over non-exercised fish. Gene expression profiling by microarray analysis evidenced the activation of a series of complex transcriptional networks of extracellular and intracellular signaling molecules and pathways involved in the regulation of muscle mass (e.g. IGF-1/PI3K/mTOR, BMP, MSTN), myogenesis and satellite cell activation (e.g. PAX3, FGF, Notch, Wnt, MEF2, Hh, EphrinB2) and angiogenesis (e.g. VEGF, HIF, Notch, EphrinB2, KLF2), some of which had not been previously associated with exercise-induced contractile activity.ConclusionsThe results from the present study show that exercise-induced contractile activity in adult zebrafish promotes a coordinated adaptive response in fast muscle that leads to increased muscle mass by hypertrophy and increased vascularization by angiogenesis. We propose that these phenotypic adaptations are the result of extensive transcriptional changes induced by exercise. Analysis of the transcriptional networks that are activated in response to exercise in the adult zebrafish fast muscle resulted in the identification of key signaling pathways and factors for the regulation of skeletal muscle mass, myogenesis and angiogenesis that have been remarkably conserved during evolution from fish to mammals. These results further support the validity of the adult zebrafish as an exercise model to decipher the complex molecular and cellular mechanisms governing skeletal muscle mass and function in vertebrates.

Capillarity and fibre types in locomotory muscles of wild mallard ducks (Anas platyrhynchos)

Six locomotory muscles from wild mallard ducks (Anas platyrhynchos) were analysed by histochemical methods. Special care was taken in sample procedure in order to describe the heterogeneity found throughout each muscle. Capillarity and fibre-type distributions were correlated to the functional implications and physiological needs of each muscle. Comparisons between our results and similar previous reports on dabbling and diving ducks are also discussed. Muscles from the leg presented the most heterogeneous fibre-type distributions, which are correlated to the wide range of terrestrial and aquatic locomotory performances shown by these animals. More specialized muscles such as pectoralis, used almost exclusively for flapping flight, had more homogeneous fibretype distributions, whereas muscles from the wing presented a high proportion of glycolytic fibres probably recruited during non-steady flapping flight. Deep muscle pectoralis zones and parts of the gastrocnemius which are closer to the bone are remarkable for their high capillarity indices and oxidative capacities, which suggests that these parts are recruited during sustained flapping flight and swimming. However, two different strategies for achieving these high oxygen needs are evident, indicating that the fibre cross-sectional area plays an important role in the modulation of the oxygen supply to the muscle cells.

A histochemical ATPase method for the demonstration of the muscle capillary network.

A histochemical method for demonstration of the capillaries in skeletal muscle of birds is proposed. The present method, which is a modification of a previously reported myosin ATPase technique used for simultaneous staining of capillaries and fiber types, provides an accurate count of capillaries associated with different fiber types in avian skeletal muscles. We have applied the original and the modified method to serial adjacent sections of certain skeletal muscles and our results show that after the application of the original technique: (a) in muscles having dark Type II fibers, these fibers produce a masking effect on their adjacent capillaries; (b) a consistent and significant undercounting in capillary densities can be seen even in muscles having no dark Type II fibers; and (c) the staining quality and capillary count are substantially improved with the use of the modified method. We attribute the better results obtained with our modification to differences in thermosensitivity of ATPase activity from the capillary endothelium and of the myofibers. A mathematical treatment is therefore proposed to correct the values of capillary count obtained with the original method.

Oxidative Stress Status in Rats After Intermittent Exposure to Hypobaric Hypoxia

OBJECTIVE Programs of intermittent hypobaric hypoxia (IHH) exposure are used to raise hemoglobin concentration and erythrocyte mass. Although acclimation response increases blood oxygen transport capacity leading to a VO(2max) increase, the effects of reactive oxygen species (ROS) might determine the behavior of erythrocytes and plasma, thus causing a worse peripheral blood flow. The goals of the study were to establish the hematological changes and to discern whether an IHH protocol modifies the antioxidant/pro-oxidant balance in laboratory rats. METHODS Male rats were subjected to an IHH program consisting of a daily 4-hour session for 5 days/week until completing 22 days of hypoxia exposure in a hypobaric chamber at a simulated altitude of 5000 m. Blood samples were taken at the end of the exposure period (H) and at 20 (P20) and 40 (P40) days after the end of the program, and compared to control (C), maintained at sea-level pressure. Hematological parameters were measured together with several oxidative stress indicators: plasma thiobarbituric acid reactive substances (TBARS) and erythrocyte catalase (CAT) and superoxide dismutase (SOD). RESULTS Red blood cell (RBC) count, hemoglobin concentration and hematocrit were higher in H group as compared to all the other groups (p < 0.001). However, there were no significant differences between the 4 groups in any of the oxidative stress-related parameters. CONCLUSIONS The absence of significant differences between groups indicates that our IHH program has little impact on the general redox status, even in the laboratory rat, which is more sensitive to hypoxia than humans. We conclude that IHH does not increase oxidative stress.

Skeletal muscle capillarization and fiber types in urban and homing pigeons (Columba livia)

1. Fiber types, capillary supply and other morphometrical parameters were analysed in pectoral, gastrocnemius and pronator muscles of homing and urban pigeons. 2. The two kinds of birds were analysed before and after a restrainment period of at least 5 months. 3. Only slight differences in fiber type frequencies were noted between urban and homing pigeons in control conditions. 4. The effect of restrainment on the different parameters studied was unclear in gastrocnemius and pronator muscles and negligible in M. pectoralis. 5. Mean diffusion distances for oxygen from capillaries were smaller in oxidative fibers; also, vascularization indexes were higher for these fiber types. 6. The contribution of each fiber type to total sectional ara of muscle remains stable in spite of fiber type frequencies heterogeneity.

Physical exercise prevents and mitigates non-alcoholic steatohepatitis-induced liver mitochondrial structural and bioenergetics impairments.

Exercise is considered a non-pharmacological tool against several lifestyle disorders in which mitochondrial dysfunction is involved. The present study aimed to analyze the preventive (voluntary physical activity-VPA) and therapeutic (endurance training-ET) role of exercise against non-alcoholic steatohepatitis (NASH)-induced liver mitochondrial dysfunction. Sixty male Sprague-Dawley rats were divided into standard-diet sedentary (SS, n=20), standard-diet VPA (SVPA, n=10), high-fat diet sedentary (HS, n=20) and high-fat diet VPA (HVPA, n=10). After 9weeks of diet-treatment, half of SS and HS animals were engaged in an ET program (SET and HET) for 8weeks, 5days/week and 60min/day. Liver mitochondrial oxygen consumption and transmembrane-electric potential (ΔΨ) were evaluated in the presence of glutamate-malate (G/M), palmitoyl-malate (P/M) and succinate (S/R). Mitochondrial enzymes activity, lipid and protein oxidation, oxidative phosphorylation (OXPHOS) subunits, cytochrome c, adenine nucleotide translocator (ANT) and uncoupling protein-2 (UCP2) content were assessed. HS groups show the histological features of NASH in parallel with decreased ΔΨ and respiratory control (RCR) and ADP/O ratios (G/M and P/M). A state 3 decrease (G/M and S/R), FCCP-induced uncoupling respiration (S/R) and ANT content were also observed. Both exercise types counteracted oxygen consumption (RCR, ADP/O and FCCP-uncoupling state) impairments and improved ΔΨ (lag-phase). In conclusion, exercise prevented or reverted (VPA and ET, respectively) the bioenergetic impairment induced by NASH, but only ET positively remodeled NASH-induced liver structural damage and abnormal mitochondria. It is possible that alterations in inner membrane integrity and fatty acid oxidation may be related to the observed phenotypes induced by exercise.

Endurance training and chronic intermittent hypoxia modulate in vitro salicylate-induced hepatic mitochondrial dysfunction.

Mitochondrial function is modulated by multiple approaches including physical activity, which can afford cross-tolerance against a variety of insults. We therefore aimed to analyze the effects of endurance-training (ET) and chronic-intermittent hypobaric-hypoxia (IHH) on liver mitochondrial bioenergetics and whether these effects translate into benefits against in vitro salicylate mitochondrial toxicity. Twenty-eight young-adult male rats were divided into normoxic-sedentary (NS), normoxic-exercised (NE), hypoxic-sedentary (HS) and hypoxic-exercised (HE). ET consisted of 1h/days of treadmill running and IHH of simulated atmospheric pressure of 49.3 kPa 5h/days during 5weeks. Liver mitochondrial oxygen consumption, transmembrane-electric potential (ΔΨ) and permeability transition pore induction (MPTP) were evaluated in the presence and absence of salicylate. Aconitase, MnSOD, caspase-3 and 8 activities, SH, MDA, SIRT3, Cyp D, HSP70, and OXPHOS subunit contents were assessed. ET and IHH decreased basal mitochondrial state-3 and state-4 respiration, although no alterations were observed in ΔΨ endpoints evaluated in control mitochondria. In the presence of salicylate, ET and IHH decreased state-4 and lag-phase of ADP-phosphorylation. Moreover, ADP-lag phase in hypoxic was further lower than in normoxic groups. Neither ET nor IHH altered the susceptibility to calcium-induced MPTP. IHH lowered MnSOD and increased aconitase activities. ET and IHH decreased caspase 8 activity whereas no effect was observed on caspase 3. The levels of SIRT3 increased with ET and IHH and Cyp D decreased with IHH. Data suggest that ET and IHH do not alter general basal liver mitochondrial function, but may attenuate some adverse effects of salicylate.

Exercise modulates liver cellular and mitochondrial proteins related to quality control signaling

AIMS The effects of exercise on cardiac and skeletal muscle, including the increase on mitochondrial function, dynamics, biogenesis and autophagy signaling are well described. However, these same effects on liver mitochondria, important in the context of hepatocyte ability to mitigate drug-induced injury and obesity-related disorders, are not fully understood. Therefore, the effects of two distinct chronic exercise models (endurance training--ET and voluntary physical activity--VPA) on liver cellular and mitochondrial quality control were analyzed. MAIN METHODS Eighteen male-adult Sprague-Dawley rats were divided into sedentary (SED), ET (12-week treadmill) and VPA (12-week voluntary free wheel). Liver mitochondrial alterations were evaluated by semi-quantification of proteins involved in oxidative stress (SIRT3, p66shc, p66(Ser36)), biogenesis (citrate synthase, PGC-1α and mtTFA), dynamics (MFN1, OPA1 and DRP1) and auto(mito)phagy (Beclin-1, Bcl-2, LC3II/LC3I, p62, Parkin and PINK) signaling. Liver ultrastructural alterations were also evaluated. KEY FINDINGS Both exercise models induced beneficial alterations on liver mitochondrial morphology and increased mitochondrial biogenesis (PGC-1α and mtTFA), autophagy-related proteins (Beclin-1, LC3-II, LC3II/LC3I), and DRP1 and SIRT3 proteins. Increased citrate synthase activity and OPA1, p62 and Parkin content as well as decreased PINK protein levels were only observed after ET. VPA decreased OPA1, Beclin-1/Bcl-2, Parkin and p66(Ser36). Mitochondrial density and circularity increased in both exercised groups. SIGNIFICANCE Both chronic exercise models increased proteins related with mitochondrial biogenesis and alteration proteins involved in mitochondrial dynamics and autophagy signaling, suggesting that exercise can induce liver mitochondrial adaptive remodeling and hepatocyte renewal.