Factor 1 inducible por hipoxia y enfermedad pulmonar obstructiva crónica: respuestas epigenéticas al ejercicio físico. Revisión sistemática / Hypoxia-inducible factor 1 and chronic obstructive pulmonary disease: Epigenetic responses to physical exercise. Systematic review

Antecedentes: El ejercicio físico es una de las principales recomendaciones para la mejora de la calidad de vida de las personas con enfermedad pulmonar obstructiva crónica (EPOC). El ejercicio genera múltiples reacciones moleculares que favorecen el ciclo y funcionamiento celular a partir de estímulos que modulan la transcripción y traducción de genes mediados por el factor 1 inducible por hipoxia (HIF-1). Objetivo: Determinar el conocimiento actual sobre las respuestas epigenéticas mediadas por HIF-1 y su comportamiento posterior a la práctica del ejercicio físico en personas con EPOC. Métodos: Búsqueda de artículos en las bases de datos Science Direct, PubMed, Scielo y PEDro. Se emplearon los términos de búsqueda Chronic obstructive pulmonary disease; Hypoxia; Hypoxia-inducible factor 1; Epigenomics; Exercise. Criterios de inclusión: publicaciones que presentaran las respuestas biomoleculares reguladas por HIF-1 en personas con EPOC. Criterios de exclusión: publicaciones de personas con diagnóstico de anemia, enfermedad hepática, enfermedad renal o antecedentes de hemorragia digestiva. Resultados: Se identificaron 75 estudios, de los cuales se seleccionaron 40 con posterioridad al cumplimiento de criterios de inclusión y exclusión. Se encontraron diferencias sobre las modificaciones postranscripcionales para la expresión de genes que favorecen al transporte hematológico de O2, el incremento del metabolismo oxidativo y el favorecimiento de la angiogénesis en el tejido muscular en personas con EPOC posterior a la práctica de ejercicio físico. Conclusiones: Las investigaciones en torno a las respuestas del HIF-1 en personas con EPOC son contradictorias. Se encuentran estudios que estipulan una retroalimentación positiva del HIF-1 para la expresión o silenciamiento de genes susceptibles al O2 y otros que plantean retroalimentación negativa. Es necesario realizar más estudios sobre las respuestas mediadas por HIF-1 en personas con EPOC y que realizan ejercicio físico.

Background: Physical exercise is one of the main recommendations for improving the quality of life of people with chronic obstructive pulmonary disease (COPD). Exercise generates multiple molecular reactions that promote cell cycle and function from stimuli that modulate the transcription and translation of genes mediated by hypoxia-inducible factor 1 (HIF-1). Objective: To determine the current knowledge about the epigenetic responses mediated by HIF-1 and their behavior after the practice of physical exercise in people with COPD. Methods: Search for articles in the Science Direct, PubMed, Scielo and PEDro databases. Se search terms Chronic Obstructive Pulmonary Disease; Hypoxia; Hypoxia-Inducible Factor 1; Epigenomics; Exercise. Inclusion criteria: publications presenting the biomolecular responses regulated by HIF-1 in people with COPD. Exclusion criteria: publications of people with a diagnosis of anemia, liver disease, kidney disease or a history of gastrointestinal bleeding. Results: Seventy-five studies were identified, of which 40 were selected after meeting the inclusion and exclusion criteria. Differences were found on the post-transcriptional modifications for the expression of genes that favor the hematological transport of O2, the increase in oxidative metabolism and the favoring of angiogenesis in muscle tissue in people with COPD after practicing physical exercise. Conclusions: Research on HIF-1 responses in people with COPD is conflicting. There are studies that stipulate a positive feedback of HIF-1 for the expression or silencing of genes susceptible to O2 and others that pose negative feedback. More studies are needed on HIF-1-mediated responses in people with COPD and those who engage in physical exercise.

A field tool for the aerobic power evaluation of middle-aged female recreational runners.

This study used time to exhaustion (TTE) to predict V̇O2max in female recreational master runners. Forty-two middle-aged women (mean = 40.5 ± 5.9 years) who had trained for recreational running performed two Université de Montréal Track Tests in the facilities of the University of Barcelona (Spain). The first was performed on a treadmill (t), the second, on an athletics track (field: f). After measuring TTE and V̇O2max on the treadmill, a first-order equation was obtained to estimate fV̇O2max from fTTE. No significant difference was observed between the estimated fV̇O2max (46.5 ± 2.9 mL·kg-1·min-1) and the measured tV̇O2max (46.2 ± 5.3), with a mean value of the absolute differences of less than 8% of the tV̇O2max average. High agreement between the two V̇O2max values was also evident, as shown by the low bias of the differences and the Bland-Altman plot. The equation obtained is of interest to evaluate performance in middle-aged female recreational runners. It will allow coaches and runners to set running paces for training and could be used in training routines to determine improvements after a training program. Moreover, these tools could be used in the field to assess the physical fitness of middle-aged women, in efforts to preserve their health and physical function.

Exercise and Doxorubicin Treatment Modulate Cardiac Mitochondrial Quality Control Signaling.

The cross-tolerance effect of exercise against heart mitochondrial-mediated quality control, remodeling and death-related mechanisms associated with sub-chronic Doxorubicin (DOX) treatment is yet unknown. We therefore analyzed the effects of two distinct chronic exercise models (endurance treadmill training-TM and voluntary free wheel activity-FW) performed during the course of the sub-chronic DOX treatment on mitochondrial susceptibility to permeability transition pore (mPTP), apoptotic and autophagic signaling and mitochondrial dynamics. Male Sprague-Dawley rats were divided into six groups (n = 6 per group): saline sedentary (SAL + SED), SAL + TM (12-weeks treadmill), SAL + FW (12-weeks voluntary free-wheel), DOX + SED [7-weeks sub-chronic DOX treatment (2 mg kg-1 week-1)], DOX + TM and DOX + FW. Apoptotic signaling and mPTP regulation were followed by measuring caspase 3, 8 and 9 activities, Bax, Bcl2, CypD, ANT, and cophilin expression. Mitochondrial dynamics (Mfn1, Mfn2, OPA1 and DRP1) and auto(mito)phagy (LC3, Beclin1, Pink1, Parkin and p62)-related proteins were semi-quantified. DOX treatment results in augmented mPTP susceptibility and apoptotic signaling (caspases 3, 8 and 9 and Bax/Bcl2 ratio). Moreover, DOX decreased the expression of fusion-related proteins (Mfn1, Mfn2, OPA1), increased DRP1 and the activation of auto(mito)phagy signaling. TM and FW prevented DOX-increased mPTP susceptibility and apoptotic signaling, alterations in mitochondrial dynamics and inhibits DOX-induced increases in auto(mito)phagy signaling. Collectively, our results suggest that both used chronic exercise models performed before and during the course of sub-chronic DOX treatment limit cardiac mitochondrial-driven apoptotic signaling and regulate alterations in mitochondrial dynamics and auto(mito)phagy in DOX-treated animals.

Intermittent hypobaric hypoxia combined with aerobic exercise improves muscle morphofunctional recovery after eccentric exercise to exhaustion in trained rats.

Unaccustomed eccentric exercise leads to muscle morphological and functional alterations, including microvasculature damage, the repair of which is modulated by hypoxia. We present the effects of intermittent hypobaric hypoxia and exercise on recovery from eccentric exercise-induced muscle damage (EEIMD). Soleus muscles from trained rats were excised before (CTRL) and 1, 3, 7, and 14 days after a double session of EEIMD protocol. A recovery treatment consisting of one of the following protocols was applied 1 day after the EEIMD: passive normobaric recovery (PNR), a 4-h daily exposure to passive hypobaric hypoxia at 4,000 m (PHR), or hypobaric hypoxia exposure followed by aerobic exercise (AHR). EEIMD produced an increase in the percentage of abnormal fibers compared with CTRL, and it affected the microvasculature by decreasing capillary density (CD, capillaries per mm2) and the capillary-to-fiber ratio (CF). After 14 days, AHR exhibited CD and CF values similar to those of CTRL animals (789 and 3.30 vs. 746 and 3.06) and significantly higher than PNR (575 and 2.62) and PHR (630 and 2.92). Furthermore, VEGF expression showed a significant 43% increase in AHR when compared with PNR. Moreover, after 14 days, the muscle fibers in AHR had a more oxidative phenotype than the other groups, with significantly smaller cross-sectional areas (AHR, 3,745; PNR, 4,502; and PHR, 4,790 µm2), higher citrate synthase activity (AHR, 14.8; PNR, 13.1; and PHR, 12 µmol·min-1·mg-1) and a significant 27% increment in PGC-1α levels compared with PNR. Our data show that hypoxia combined with exercise attenuates or reverses the morphofunctional alterations induced by EEIMD.NEW & NOTEWORTHY Our study provides new insights into the use of intermittent hypobaric hypoxia combined with exercise as a strategy to recover muscle damage induced by eccentric exercise. We analyzed the effects of hypobaric exposure combined with aerobic exercise on histopathological features of muscle damage, fiber morphofunctionality, capillarization, angiogenesis, and the oxidative capacity of damaged soleus muscle. Most of these parameters were improved after a 2-wk protocol of intermittent hypobaric hypoxia combined with aerobic exercise.

Sildenafil does not Improve Exercise Capacity under Acute Hypoxia Exposure.

The increase in pulmonary arterial pressure (PAP) due to hypoxic pulmonary vasoconstriction (HPV) could be a limiting factor for physical performance during hypoxic exposure. Sildenafil has been shown to reduce PAP in situations of moderate or severe hypoxia, and consequently its role as an ergogenic aid and even a possible doping substance must be considered. We performed a double-blind crossover study to determine the effects of sildenafil on cardiovascular, respiratory and metabolic parameters in normoxia and during acute exposure to hypobaric hypoxia (4 000 m) at rest and during maximal and submaximal (60% VO2 max) exercise tests. One hour before testing started, sildenafil (100 mg) or a placebo was orally administered to 11 volunteers. In normoxic conditions, sildenafil did not affect performance. Similarly, no significant differences were found in cardiovascular and respiratory parameters in hypoxic conditions at rest or during exercise. The use of sildenafil to improve physical performance in non-acclimatized subjects is not supported by our data.

A semiquantitative scoring tool to evaluate eccentric exercise-induced muscle damage in trained rats.

Unaccustomed eccentric exercise is a well-documented cause of exercise-induced muscle damage. However, in trained subjects muscle injury involves only light or moderate tissue damage. Since trained rats are widely used as a model for skeletal muscle injury, here we propose a semiquantitative scoring tool to evaluate muscle damage in trained rats. Twenty male Sprague-Dawley rats were trained fortwo weeks following a two-week preconditioning period, and randomly divided into two groups: control rats (CTL; n=5) and rats with eccentric exercise-induced muscle damage (INJ; n=15). Injured rats were sacrificed at three time points: 1, 3 and 7 days post injury (n=5 each). Transverse sections from the right soleus were cut (10 µm) and stained with haematoxylin-eosin. Samples were evaluated by two groups of observers (four researchers experienced in skeletal muscle histopathology and four inexperienced) using the proposed tool, which consisted of six items organised in three domains: abnormal fibre morphology, necrotic/(re)degenerating fibres (muscle fibre domain), endomysial and perimysial infiltration (inflammatory state domain) and endomysium and perimysium distension (interstitial compartment domain). We observed the expected time course in the six evaluated items. Furthermore, agreement among observers was evaluated by measuring the Intraclass Correlation Coefficient (ICC). Within the experienced group, items from the muscle fibre and interstitial compartment domains showed good agreement and the two items from the infiltration compartment domain showed excellent agreement. in conclusion, the proposed tool allowed quick and correct evaluation of light to moderate muscle damage in trained rats with good agreement between observers.

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.

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.

Synergistic impact of endurance training and intermittent hypobaric hypoxia on cardiac function and mitochondrial energetic and signaling.

BACKGROUND: Intermittent hypobaric-hypoxia (IHH) and endurance-training (ET) are cardioprotective strategies against stress-stimuli. Mitochondrial modulation appears to be an important step of the process. This study aimed to analyze whether a combination of these approaches provides additive or synergistic effects improving heart-mitochondrial and cardiac-function. METHODS: Two-sets of rats were divided into normoxic-sedentary (NS), normoxic-exercised (NE, 1 h/day/5 weeks treadmill-running), hypoxic-sedentary (HS, 6000 m, 5h/day/5 weeks) and hypoxic-exercised (HE) to study overall cardiac and mitochondrial function. In vitro cardiac mitochondrial oxygen consumption and transmembrane potential were evaluated. OXPHOS subunits and ANT protein content were semi-quantified by Western blotting. HIF-1α, VEGF, VEGF-R1 VEGF-R2, BNP, SERCA2a and PLB expressions were measured by qRT-PCR and cardiac function was characterized by echocardiography and hemodynamic parameters. RESULTS: Respiratory control ratio (RCR) increased in NE, HS and HE vs. NS. Susceptibility to anoxia/reoxygenation-induced dysfunction decreased in NE, HS and HE vs. NS. HS decreased mitochondrial complex-I and -II subunits; however HE completely reverted the decreased content in complex-II subunits. ANT increased in HE. HE presented normalized ventricular-arterial coupling (Ea) and BNP myocardial levels and significantly improved myocardial performance as evaluated by increased cardiac output and normalization of the Tei index vs. HS CONCLUSION: Data demonstrates that IHH and ET confer cardiac mitochondria with a more resistant phenotype although without visible addictive effects at least under basal conditions. It is suggested that the combination of both strategies, although not additive, results into improved cardiac function.

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.

Hemorheology and oxygen transport in vertebrates. A role in thermoregulation?

We studied the effect of temperature on blood rheology in three vertebrate species with different thermoregulation and erythrocyte characteristics. Higher fibrinogen proportion to total plasma protein was found in turtles (20%) than in pigeons (5.6%) and rats (4.2%). Higher plasma viscosity at room temperature than at homeotherm body temperature was observed in rats (1.69 mPa x s at 20 degrees C vs. 1.33 mPa x s at 37 degrees C), pigeons (3.40 mPa x s at 20 degrees C vs. 1.75 mPa x s at 40 degrees C), and turtles (1.74 mPa x s at 20 degrees C vs. 1.32 mPa x s at 37 degrees C). This fact allow us to hypothesize that thermal changes in protein structure may account for an adjustment of the plasma viscosity. Blood viscosity was dependent on shear rate, temperature and hematocrit in the three species. A different behaviour in apparent and relative viscosities between rat and pigeon at environmental temperature was found. Moreover, the blood oxygen transport capacity seems more affected by a reduction of temperature in rats than in pigeons. Both findings indicate a greater influence of temperature on mammalian erythrocyte than on nucleated red cells, possibly as a consequence of differences in thermal sensitivity and mechanical stability between them. A comparison between the three species revealed that apparent blood viscosity measured at homeotherm physiological temperature was linearly related to the hematocrit level of each species. However, when measured at environmental temperature, rat blood showed a higher apparent viscosity than those found in species with non-nucleated red cells, thus indicating a higher impact of temperature decrease on blood viscosity in mammals. This suggest that regional hypothermia caused by cold exposure may affect mammalian blood rheological behaviour in a higher extent than in other vertebrate species having nucleated red cells and, consequently, influencing circulatory function and oxygen transport.

Capillarity, fibre types and fibre morphometry in different sampling sites across and along the tibialis anterior muscle of the rat.

Capillarity, fibre types, fibre cross-sectional areas and perimeters were studied along and across the rat tibialis anterior muscle. The muscle was sectioned at three different levels (proximal, equatorial and distal) choosing five sampling fields for measurements at each level (from anterior to posterior and lateral to medial zones). Significant differences were found in the percentage of fibre types and capillarity between different fields of the same muscle section. Slow oxidative fibres were confined to the posterior muscle zone with a maximum of 3.7%. The posterior fields also had a greater percentage of fast oxidative glycolytic fibres at proximal (72.3%) and equatorial (61.3%) levels, but a lower value at the distal level (44.8%) and lower capillary density counts in total cross-section means (758 vs. 1,069 capillaries/mm(2) in equatorial and 1,035 capillaries/mm(2) at proximal levels). The uneven distribution of both fast fibre types and the different degrees of capillarisation along the muscle are statistically significant and may be due to different biomechanical performances along the rat tibialis anterior. Fibre size was significantly larger at the distal level, but no morphometric differences were found across the section of the same level. At the distal level, the mean total fibre area of fast glycolytic fibres (5,130 microm(2)) and fast oxidative glycolytic fibres (2,493 microm(2)) contrasted with values at the proximal (fast glycolytic: 4,070 microm(2), fast oxidative glycolytic: 1,970 microm(2)) and equatorial (fast glycolytic: 3,535 microm(2), fast oxidative glycolytic: 1,714 microm(2)) levels. The differences along and across the muscle show the need to design a standardised procedure for sample location when performing comparative studies of morphofunctional adaptive changes in skeletal muscle. A significant difference between individuals (animals) in all parameters was evident and should be taken into consideration when analysing the variability: the factor 'animal' should be considered in multiway ANOVAs, especially when low sample sizes are used.

Capillarity and fibre types in locomotory muscles of wild yellow-legged gulls (Larus cachinnans).

This study analyzes the capillarity and fibre-type distribution of six locomotory muscles of gulls. The morphological basis and the oxygen supply characteristics of the skeletal muscle of a species with a marked pattern of gliding flight are established, thus contributing to a better understanding of the physiology of a kind of flight with low energetic requirements. The four wing muscles studied (scapulotriceps, pectoralis, scapulohumeralis, and extensor metacarpi) exhibited higher percentages of fast oxidative glycolytic fibres (>70%) and lower percentages of slow oxidative fibres (<16%) than the muscles involved in nonflight locomotion (gastrocnemius and iliotibialis). Capillary densities ranged from 816 to 1,233 capillaries mm(-2), having the highest value in the pectoralis. In this muscle, the fast oxidative glycolytic fibres had moderate staining for succinate dehydrogenase and relatively large fibre sizes, as deduced from the low fibre densities (589-665 fibres mm(-2)). All these findings are seen as an adaptive response for gliding, when the wing is held outstretched by isometric contractions. The leg muscles studied included a considerable population of slow oxidative fibres (>14% in many regions), which suggests that they are adapted to postural activities. Regional variations in the relative distributions of fibre types in muscle gastrocnemius may reflect different functional demands placed on this muscle during terrestrial and aquatic locomotion. The predominance of oxidative fibres and capillary densities under 1,000 capillaries mm(-2) in leg muscles is probably a consequence of an adaptation for slow swimming and maintenance of the posture on land rather than for other locomotory capabilities, such as endurance or sprint activities.

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.

Capillarity and fiber types in locomotory muscles of wild common coots, Fulica atra.

Six locomotory muscles of wild common coots, Fulica atra, were analyzed histochemically. Capillarity and fiber-type distributions were correlated to the functional implications and physiological needs of each muscle. Leg muscles exhibit three unevenly distributed fiber types, a pattern that reflects the great variety of terrestrial and aquatic locomotory performances that coots are able to develop. Aerobic zones are presumably recruited during steady swimming and diving, while regions with anaerobic characteristics may be used for bursts of activity such as sprint swimming or during take off, when coots run along the water's surface. Fiber types and capillarization in wing muscles have a marked oxidative trend. High wing beat frequencies, short and broad wings, and the long distance migrations that these birds perform indicate that the presence of high numbers of oxidative fibers and the well developed capillary supply are needed for enhanced oxygen uptake. The pectoralis muscle, except in its deep part, has exclusively fast oxidative fibers with a very high staining intensity for succinate dehydrogenase assay as compared to the same fiber type of other muscles. Its predominant role in flapping flight justifies these characteristics that are typical of fibers with high aerobic metabolism. The deep part of the pectoralis muscle presents a low proportion of an unusual slow anaerobic fiber type. These fibers could play a role during feeding dives when the bird presses the air out of the feathers by tightening the wings against the body. A linear relationship between capillary and fiber densities in all coot muscles studied reflects an adjustment between fiber diameter and vascularization in order to obtain the oxygen for mitochondrial supply. This strategy seems a suitable way to cope with the rigid aerobic constraints that flying and diving impose upon the coot's physiology. J. Morphol. 237:147-164, 1998. © 1998 Wiley-Liss, Inc.

Innervation distribution pattern, nerve ending structure, and fiber types in pigeon skeletal muscle.

Four fiber types have been characterized in different pigeon skeletal muscles according to their innervation pattern (nerve ending structure and innervation distribution) and histochemical properties (SDH and m-ATPase activities). All fast fibers, types IIA and IIB, present aggregated distribution of their nerve endings with "en plaque" structures and very low innervation frequencies. The two kinds of slow fibers recognized are multiple innervated and present higher innervation frequencies. However, type I fibers have nerve terminals in small knobs with uniform localization, whereas type III fibers present "en grappe" nerve endings, which tend to be randomly distributed. Fiber type composition of skeletal muscles has been found closely related to their biomechanical function. Fast fibers are predominant in muscles with an active role in locomotive movements, whereas slow fibers are mainly or exclusively located in postural muscles.

A combined myosin ATPase and acetylcholinesterase histochemical method for the demonstration of fibre types and their innervation pattern in skeletal muscle.

An improved, combined staining method for myofibrillar ATPase (m-ATPase) and for acetylcholinesterase activity is described. This method allows the observations, on the same slide, of the classical histochemical m-ATPase profile following the Brooke and Kaiser technique and the neuromuscular junction morphology. Thus the pattern of innervation, nerve ending structure and number of nerve endings along the fibres is shown simultaneously for the basic differentiation between slow and fast fibres. The use of acidic and alkaline preincubation allows better visualization of endplate morphology and avoids the masking effect of a positive m-ATPase reaction. The technique has been validated on skeletal muscles from avian and mammalian species.

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.

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.