Muscle transcriptomic profiles in pigs with divergent phenotypes for fatness traits

BACKGROUND: Selection for increasing intramuscular fat content would definitively improve the palatability and juiciness of pig meat as well as the sensorial and organoleptic properties of cured products. However, evidences obtained in human and model organisms suggest that high levels of intramuscular fat might alter muscle lipid and carbohydrate metabolism. We have analysed this issue by determining the transcriptomic profiles of Duroc pigs with divergent phenotypes for 13 fatness traits. The strong aptitude of Duroc pigs to have high levels of intramuscular fat makes them a valuable model to analyse the mechanisms that regulate muscle lipid metabolism, an issue with evident implications in the elucidation of the genetic basis of human metabolic diseases such as obesity and insulin resistance. RESULTS: Muscle gene expression profiles of 68 Duroc pigs belonging to two groups (HIGH and LOW) with extreme phenotypes for lipid deposition and composition traits have been analysed. Microarray and quantitative PCR analysis showed that genes related to fatty acid uptake, lipogenesis and triacylglycerol synthesis were upregulated in the muscle tissue of HIGH pigs, which are fatter and have higher amounts of intramuscular fat than their LOW counterparts. Paradoxically, lipolytic genes also showed increased mRNA levels in the HIGH group suggesting the existence of a cycle where triacylglycerols are continuously synthesized and degraded. Several genes related to the insulin-signalling pathway, that is usually impaired in obese humans, were also upregulated. Finally, genes related to antigen-processing and presentation were downregulated in the HIGH group. CONCLUSION: Our data suggest that selection for increasing intramuscular fat content in pigs would lead to a shift but not a disruption of the metabolic homeostasis of muscle cells. Future studies on the post-translational changes affecting protein activity or expression as well as information about protein location within the cell would be needed to to elucidate the effects of lipid deposition on muscle metabolism in pigs.

BDNF promoter I methylation correlates between post-mortem human peripheral and brain tissues.

Several psychiatric disorders have been associated with CpG methylation changes in CG rich promoters of the brain-derived neurotrophic factor (BDNF) mainly by extracting DNA from peripheral blood cells. Whether changes in peripheral DNA methylation can be used as a proxy for brain-specific alterations remains an open question. In this study we aimed to compare DNA methylation levels in BDNF promoter regions in human blood cells, muscle and brain regions using bisulfite-pyrosequencing. We found a significant correlation between the levels of BDNF promoter I methylation measured in quadriceps and vPFC tissues extracted from the same individuals (n = 98, Pearson, r = 0.48, p = 4.5 × 10(-7)). In the hippocampus, BDNF promoter I and IV methylation levels were strongly correlated (Pearson, n = 37, r = 0.74, p = 1.4 × 10(-7)). We found evidence for sex-dependent effect on BDNF promoter methylation levels in the various tissues and blood samples. Taken together, these data indicate a strong intra-individual correlation between peripheral and brain tissue. They also suggest that sex determines methylation patterns in BDNF promoter region across different types of tissue, including muscle, brain, and blood.

Endothelial, but not smooth muscle, peroxisome proliferator-activated receptor β/δ regulates vascular permeability and anaphylaxis.

BACKGROUND: Remodeling of quiescent vessels with increases in permeability, vasodilatation, and edema are hallmarks of inflammatory disorders. Factors involved in this type of remodeling represent potential therapeutic targets. OBJECTIVES: We investigated whether the nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR) β/δ, a regulator of metabolism, fibrosis, and skin homeostasis, is involved in regulation of this type of remodeling. METHODS: Wild-type and various Pparb/d mutant mice were used to monitor dermal acute vascular hyperpermeability (AVH) and passive systemic anaphylaxis-induced hypothermia and edema. PPARβ/δ-dependent kinase activation and remodeling of endothelial cell-cell junctions were addressed by using human endothelial cells. RESULTS: AVH and dilatation of dermal microvessels stimulated by vascular endothelial growth factor A, histamine, and thrombin are severely compromised in PPARβ/δ-deficient mice. Selective deletion of the Pparb/d-encoding gene in endothelial cells in vivo similarly limits dermal AVH and vasodilatation, providing evidence that endothelial PPARβ/δ is the major player in regulating acute dermal microvessel remodeling. Furthermore, endothelial PPARβ/δ regulatory functions are not restricted to the skin vasculature because its deletion in the endothelium, but not in smooth muscle cells, also leads to reduced systemic anaphylaxis, the most severe form of allergic reaction, in which an acute vascular response plays a key role. PPARβ/δ-dependent AVH activation likely involves the activation of mitogen-activated protein kinase and Akt pathways and leads to downstream destabilization of endothelial cell-cell junctions. CONCLUSION: These results unveil not only a novel function of PPARβ/δ as a direct regulator of acute vessel permeability and dilatation but also provide evidence that antagonizing PPARβ/δ represents an important strategy to consider for moderating diseases with altered endothelial integrity, such as acute inflammatory and allergic disorders.

Connexin43 Inhibition Prevents Human Vein Grafts Intimal Hyperplasia.

Venous bypass grafts often fail following arterial implantation due to excessive smooth muscle cells (VSMC) proliferation and consequent intimal hyperplasia (IH). Intercellular communication mediated by Connexins (Cx) regulates differentiation, growth and proliferation in various cell types. Microarray analysis of vein grafts in a model of bilateral rabbit jugular vein graft revealed Cx43 as an early upregulated gene. Additional experiments conducted using an ex-vivo human saphenous veins perfusion system (EVPS) confirmed that Cx43 was rapidly increased in human veins subjected ex-vivo to arterial hemodynamics. Cx43 knock-down by RNA interference, or adenoviral-mediated overexpression, respectively inhibited or stimulated the proliferation of primary human VSMC in vitro. Furthermore, Cx blockade with carbenoxolone or the specific Cx43 inhibitory peptide 43gap26 prevented the burst in myointimal proliferation and IH formation in human saphenous veins. Our data demonstrated that Cx43 controls proliferation and the formation of IH after arterial engraftment.

The Interplay between NF-kappaB and E2F1 Coordinately Regulates Inflammation and Metabolism in Human Cardiac Cells

Pyruvate dehydrogenase kinase 4 (PDK4) inhibition by nuclear factor-κB (NF-κB) is related to a shift towards increased glycolysis during cardiac pathological processes such as cardiac hypertrophy and heart failure. The transcription factors estrogen-related receptor-α (ERRα) and peroxisome proliferator-activated receptor (PPAR) regulate PDK4 expression through the potent transcriptional coactivator PPARγ coactivator-1α (PGC-1α). NF-κB activation in AC16 cardiac cells inhibit ERRα and PPARβ/δ transcriptional activity, resulting in reduced PGC-1α and PDK4 expression, and an enhanced glucose oxidation rate. However, addition of the NF-κB inhibitor parthenolide to these cells prevents the downregulation of PDK4 expression but not ERRα and PPARβ/δ DNA binding activity, thus suggesting that additional transcription factors are regulating PDK4. Interestingly, a recent study has demonstrated that the transcription factor E2F1, which is crucial for cell cycle control, may regulate PDK4 expression. Given that NF-κB may antagonize the transcriptional activity of E2F1 in cardiac myocytes, we sought to study whether inflammatory processes driven by NF-κB can downregulate PDK4 expression in human cardiac AC16 cells through E2F1 inhibition. Protein coimmunoprecipitation indicated that PDK4 downregulation entailed enhanced physical interaction between the p65 subunit of NF-κB and E2F1. Chromatin immunoprecipitation analyses demonstrated that p65 translocation into the nucleus prevented the recruitment of E2F1 to the PDK4 promoter and its subsequent E2F1-dependent gene transcription. Interestingly, the NF-κB inhibitor parthenolide prevented the inhibition of E2F1, while E2F1 overexpression reduced interleukin expression in stimulated cardiac cells. Based on these findings, we propose that NF-κB acts as a molecular switch that regulates E2F1-dependent PDK4 gene transcription.

β-Adrenergic modulation of skeletal muscle contraction: key role of excitation-contraction coupling.

Our aim is to describe the acute effects of catecholamines/β-adrenergic agonists on contraction of non-fatigued skeletal muscle in animals and humans, and explain the mechanisms involved. Adrenaline/β-agonists (0.1-30 μm) generally augment peak force across animal species (positive inotropic effect) and abbreviate relaxation of slow-twitch muscles (positive lusitropic effect). A peak force reduction also occurs in slow-twitch muscles in some conditions. β2 -Adrenoceptor stimulation activates distinct cyclic AMP-dependent protein kinases to phosphorylate multiple target proteins. β-Agonists modulate sarcolemmal processes (increased resting membrane potential and action potential amplitude) via enhanced Na(+) -K(+) pump and Na(+) -K(+) -2Cl(-) cotransporter function, but this does not increase force. Myofibrillar Ca(2+) sensitivity and maximum Ca(2+) -activated force are unchanged. All force potentiation involves amplified myoplasmic Ca(2+) transients consequent to increased Ca(2+) release from sarcoplasmic reticulum (SR). This unequivocally requires phosphorylation of SR Ca(2+) release channels/ryanodine receptors (RyR1) which sensitize the Ca(2+) -induced Ca(2+) release mechanism. Enhanced trans-sarcolemmal Ca(2+) influx through phosphorylated voltage-activated Ca(2+) channels contributes to force potentiation in diaphragm and amphibian muscle, but not mammalian limb muscle. Phosphorylation of phospholamban increases SR Ca(2+) pump activity in slow-twitch fibres but does not augment force; this process accelerates relaxation and may depress force. Greater Ca(2+) loading of SR may assist force potentiation in fast-twitch muscle. Some human studies show no significant force potentiation which appears to be related to the β-agonist concentration used. Indeed high-dose β-agonists (∼0.1 μm) enhance SR Ca(2+) -release rates, maximum voluntary contraction strength and peak Wingate power in trained humans. The combined findings can explain how adrenaline/β-agonists influence muscle performance during exercise/stress in humans.

The nitric oxide pathway in pig isolated calyceal smooth muscle.

In pig and humans, whose kidneys have a multi-calyceal collecting system, the initiation of ureteral peristalsis takes place in the renal calyces. In the pig and human ureter, recent evidence suggests that nitric oxide (NO) is an inhibitory mediator that may be involved in the regulation of peristalsis. This study was designed to assess whether the NO synthase/NO/cyclic GMP pathway modulates the motility of pig isolated calyceal smooth muscle. Immunohistochemistry revealed a moderate overall innervation of the smooth muscle layer, and no neuronal or inducible NO synthase (NOS) immunoreactivities. Endothelial NOS immunoreactivities were observed in the urothelium and vascular endothelium, and numerous cyclic GMP-immunoreactive (-IR) calyceal smooth muscle cells were found. As measured by monitoring the conversion of L-arginine to L-citrulline, Ca(2+)-dependent NOS activity was moderate. Assessment of functional effects was performed in tissue baths and showed that NO and SIN-1 decreased spontaneous and induced contractions of isolated preparations in a concentration-dependent manner. In strips exposed to NO, there was a 10-fold increase of the cyclic GMP levels compared with control preparations (P < 0.01). It is concluded that a non-neuronal NOS/NO/cyclic GMP pathway is present in pig calyces, where it may influence motility. The demonstration of cyclic GMP-IR smooth muscle cells suggests that NO acts directly on these cells. This NOS/NO/cyclic GMP pathway may be a target for drugs inhibiting peristalsis of mammalian upper urinary tract. Neurourol. Urodynam. 18:673-685, 1999.

Prostaglandin E2 Prevents Hyperosmolar-Induced Human Mast Cell Activation through Prostanoid Receptors EP2 and EP4.

Background: Mast cells play a critical role in allergic and inflammatory diseases, including exercise-induced bronchoconstriction (EIB) in asthma. The mechanism underlying EIB is probably related to increased airway fluid osmolarity that activates mast cells to the release inflammatory mediators. These mediators then act on bronchial smooth muscle tocause bronchoconstriction. In parallel, protective substances such as prostaglandin E2 (PGE2) are probably also released and could explain the refractory period observed in patients with EIB. Objective: This study aimed to evaluate the protective effect of PGE2 on osmotically activated mast cells, as a model of exercise-induced bronchoconstriction. Methods: We used LAD2, HMC-1, CD34-positive, and human lung mast cell lines. Cells underwent a mannitol challenge, and the effects of PGE2 and prostanoid receptor (EP) antagonists for EP14 were assayed on the activated mast cells. Betahexosaminidase release, protein phosphorylation, and calcium mobilization were assessed. Results: Mannitol both induced mast cell degranulation and activated phosphatidyl inositide 3-kinase and mitogenactivated protein kinase (MAPK) pathways, thereby causing de novo eicosanoid and cytokine synthesis. The addition of PGE2 significantly reduced mannitol-induced degranulation through EP2 and EP4 receptors, as measured by betahexosaminidase release, and consequently calcium influx. Extracellular-signal-regulated kinase 1/2, c-Jun N-terminal kinase,and p38 phosphorylation were diminished when compared with mannitol activation alone. Conclusions: Our data show a protective role for the PGE2 receptors EP2 and EP4 following osmotic changes, through the reduction of human mast cell activity caused by calcium influx impairment and MAP kinase inhibition.

The Regulation of Skeletal and Cardiac Muscle Blood Flow in Humans. Studies by positron emission tomography with special reference to exercise, adenosine and nitric oxide

Virtually every cell and organ in the human body is dependent on a proper oxygen supply. This is taken care of by the cardiovascular system that supplies tissues with oxygen precisely according to their metabolic needs. Physical exercise is one of the most demanding challenges the human circulatory system can face. During exercise skeletal muscle blood flow can easily increase some 20-fold and its proper distribution to and within muscles is of importance for optimal oxygen delivery. The local regulation of skeletal muscle blood flow during exercise remains little understood, but adenosine and nitric oxide may take part in this process. In addition to acute exercise, long-term vigorous physical conditioning also induces changes in the cardiovasculature, which leads to improved maximal physical performance. The changes are largely central, such as structural and functional changes in the heart. The function and reserve of the heart’s own vasculature can be studied by adenosine infusion, which according to animal studies evokes vasodilation via it’s a_2A receptors. This has, however, never been addressed in humans in vivo and also studies in endurance athletes have shown inconsistent results regarding the effects of sport training on myocardial blood flow. This study was performed on healthy young adults and endurance athletes and local skeletal and cardiac muscle blod flow was measured by positron emission tomography. In the heart, myocardial blood flow reserve and adenosine A_2A receptor density, and in skeletal muscle, oxygen extraction and consumption was also measured. The role of adenosine in the control of skeletal muscle blood flow during exercise, and its vasodilator effects, were addressed by infusing competitive inhibitors and adenosine into the femoral artery. The formation of skeletal muscle nitric oxide was also inhibited by a drug, with and without prostanoid blockade. As a result and conclusion, it can be said that skeletal muscle blood flow heterogeneity decreases with increasing exercise intensity most likely due to increased vascular unit recruitment, but exercise hyperemia is a very complex phenomenon that cannot be mimicked by pharmacological infusions, and no single regulator factor (e.g. adenosine or nitric oxide) accounts for a significant part of exercise-induced muscle hyperemia. However, in the present study it was observed for the first time in humans that nitric oxide is not only important regulator of the basal level of muscle blood flow, but also oxygen consumption, and together with prostanoids affects muscle blood flow and oxygen consumption during exercise. Finally, even vigorous endurance training does not seem to lead to supranormal myocardial blood flow reserve, and also other receptors than A_2A mediate the vasodilator effects of adenosine. In respect to cardiac work, atheletes heart seems to be luxuriously perfused at rest, which may result from reduced oxygen extraction or impaired efficiency due to pronouncedly enhanced myocardial mass developed to excel in strenuous exercise.

Brains and guts in human evolution: The Expensive Tissue Hypothesis

The brain is a very expensive organ in metabolic terms. Each unit of brain tissue requires over 22 times the amount of metabolic energy as an equivalent unit of muscle tissue. There is no correlation across mammals, however, between the relative size of the brain and the relative basal metabolic rate. The Expensive Tissue Hypothesis explains this apparent paradox by looking at the metabolic cost of the brain in the context of the costs of other metabolically expensive organs in the body. The results show that the increase in brain size in humans is balanced by an equivalent reduction in the size of the gastro-intestinal tract. In other words, the increased energetic demands of a relatively large brain are balanced by the reduced energy demands of a relatively small gastro-intestinal tract. This relationship also seems to be true in non-human primates. The size of the gastro-intestinal tract is dependent on both body size and the quality of the diet. It is argued that humans (and other primates) could not have developed a relatively large brain without also adopting a high quality diet that would have permitted a reduction in the relative size of the gastro-intestinal tract. Dietary change is therefore viewed as a 'prime releaser' in brain evolution. It is argued that a high quality diet is necessary for the evolution of a relatively large brain. However, the change to such a high quality diet, which involved an increased proportion of animal based products, need not have been one of the 'prime movers' in brain evolution. In this context, and based on the archaeological and palaeoanthropological record, the factors most probably surrounding the evolution of the human brain are discussed.

Cyanobacterial toxins in Portugal: effects on aquatic animals and risk for human health

Toxic cyanobacteria are common in Portuguese freshwaters and the most common toxins are microcystins. The occurrence of microcystin-LR (MCYST-LR) has been reported since 1990 and a significant number of water reservoirs that are used for drinking water attain high levels of this toxin. Aquatic animals that live in eutrophic freshwater ecosystems may be killed by microcystins but in many cases the toxicity is sublethal and so the animals can survive long enough to accumulate the toxins and transfer them along the food chain. Among these, edible mollusks, fish and crayfish are especially important because they are harvested and sold for human consumption. Mussels that live in estuarine waters and rivers where toxic blooms occur may accumulate toxins without many significant acute toxic effects. In this study data are presented in order to understand the dynamics of the accumulation and depuration of MCYST-LR in mussels. The toxin is readily accumulated and persists in the shellfish for several days after contact. In the crayfish the toxin is accumulated mainly in the gut but is also cleared very slowly. In carps, although the levels of the toxins found in naturally caught specimens were not very high, some toxin was found in the muscle and not only in the viscera. This raises the problem of the toxin accumulation by fish and possible transfer through the food chain. The data gathered from these experiments and from naturally caught specimens are analyzed in terms of risk for human consumption. The occurrence of microcystins in tap water and the incidence of toxic cyanobacteria in fresh water beaches in Portugal are reported. The Portuguese National Monitoring Program of cyanobacteria is mentioned and its implications are discussed.

Aerobic exercise training in heart failure: impact on sympathetic hyperactivity and cardiac and skeletal muscle function

Heart failure is a common endpoint for many forms of cardiovascular disease and a significant cause of morbidity and mortality. Chronic neurohumoral excitation (i.e., sympathetic hyperactivity) has been considered to be a hallmark of heart failure and is associated with a poor prognosis, cardiac dysfunction and remodeling, and skeletal myopathy. Aerobic exercise training is efficient in counteracting sympathetic hyperactivity and its toxic effects on cardiac and skeletal muscles. In this review, we describe the effects of aerobic exercise training on sympathetic hyperactivity, skeletal myopathy, as well as cardiac function and remodeling in human and animal heart failure. We also discuss the mechanisms underlying the effects of aerobic exercise training.

Functional and morphological effects of resistance exercise on disuse-induced skeletal muscle atrophy

Abstract The reduction of skeletal muscle loss in pathological states, such as muscle disuse, has considerable effects in terms of rehabilitation and quality of life. Since there is no currently effective and safe treatment available for skeletal muscle atrophy, the search for new alternatives is necessary. Resistance exercise (RE) seems to be an important tool in the treatment of disuse-induced skeletal muscle atrophy by promoting positive functional (strength and power) and structural (hypertrophy and phenotypic changes) adaptive responses. Human and animal studies using different types of resistance exercise (flywheel, vascular occlusion, dynamic, isometric, and eccentric) have obtained results of great importance. However, since RE is a complex phenomenon, lack of strict control of its variables (volume, frequency, intensity, muscle action, rest intervals) limits the interpretation of the impact of the manipulation on skeletal muscle remodeling and function under disuse. The aim of this review is to critically describe the functional and morphological role of resistance exercise in disuse-induced skeletal muscle atrophy with emphasis on the principles of training.

The effect of elevated muscle fluid volume on indices of muscle damage following an acute bout of eccentric exercise

The primary purpose of the current investigation was to develop an elevated muscle fluid level using a human in-vivo model. The secondary purpose was to determine if an increased muscle fluid content could alter the acute muscle damage response following a bout of eccentric exercise. Eight healthy, recreationally active males participated in a cross-over design involving two randomly assigned trials. A hydration trial (HYD) consisting of a two hour infusion of a hypotonic (0.45%) saline at a rate of 20mL/minVl .73m"^ and a control trial (CON), separated by four weeks. Following the infusion (HYD) or rest period (CON), participants completed a single leg isokinetic eccentric exercise protocol of the quadriceps, consisting of 10 sets of 10 repetitions with a one minute rest between each set. Muscle biopsies were collected prior to the exercise, immediately following and at three hours post exercise. Muscle analysis included determination of wet-dry ratios and quantification of muscle damage using toluidine blue staining and light microscopy. Blood samples were collected prior to, immediately post, three and 24 hours post exercise to determine changes in creatine kinase (CK), lactate dehydrogenase (LD), interleukin-6 (IL-6) and Creactive protein (CRP) levels. Results demonstrated an increased muscle fluid volume in the HYD condition following the infusion when compared to the CON condition. Isometric peak torque was significantly reduced following the exercise in both the HYD and CON conditions. There were no significant differences in the number of areas of muscle damage at any of the time points in either condition, with no differences between conditions. CK levels were significantly greater 24hour post exercise compared to pre, immediately and three hours post similarly in both conditions. LD in the HYD condition followed a similar trend as CK with 24 hour levels higher than pre, immediately post and three hours post and LD levels were significantly greater 24 hours post compared to pre levels in the CON condition, with no differences between conditions. A significant main effect for time was observed for CRP (p<0.05) for time, such that CRP levels increased consistently at each subsequent time point. However, CRP and IL-6 levels were not different at any of the measured time points when comparing the two conditions. Although the current investigation was able to successfully increase muscle fluid volume and an increased CK, LD and CRP were observed, no muscle damage was observed following the eccentric exercise protocol in the CON or HYD conditions. Therefore, the hypotonic infusion used in the HYD condition proved to be a viable method to acutely increase muscle fluid content in in-vivo human skeletal muscle.

3D movement and muscle activity patterns in a violin bowing task

Objective: Overuse injuries in violinists are a problem that has been primarily analyzed through the use of questionnaires. Simultaneous 3D motion analysis and EMG to measure muscle activity has been suggested as a quantitative technique to explore this problem by identifying movement patterns and muscular demands which may predispose violinists to overuse injuries. This multi-disciplinary analysis technique has, so far, had limited use in the music world. The purpose of this study was to use it to characterize the demands of a violin bowing task. Subjects: Twelve injury-free violinists volunteered for the study. The subjects were assigned to a novice or expert group based on playing experience, as determined by questionnaire. Design and Settings: Muscle activity and movement patterns were assessed while violinists played five bowing cycles (one bowing cycle = one down-bow + one up-bow) on each string (G, D, A, E), at a pulse of 4 beats per bow and 100 beats per minute. Measurements: An upper extremity model created using coordinate data from markers placed on the right acromion process, lateral epicondyle of the humerus and ulnar styloid was used to determine minimum and maximum joint angles, ranges of motion (ROM) and angular velocities at the shoulder and elbow of the bowing arm. Muscle activity in right anterior deltoid, biceps brachii and triceps brachii was assessed during maximal voluntary contractions (MVC) and during the playing task. Data were analysed for significant differences across the strings and between experience groups. Results: Elbow flexion/extension ROM was similar across strings for both groups. Shoulder flexion/extension ROM increaslarger for the experts. Angular velocity changes mirrored changes in ROM. Deltoid was the most active of the muscles assessed (20% MVC) and displayed a pattern of constant activation to maintain shoulder abduction. Biceps and triceps were less active (4 - 12% MVC) and showed a more periodic 'on and off pattern. Novices' muscle activity was higher in all cases. Experts' muscle activity showed a consistent pattern across strings, whereas the novices were more irregular. The agonist-antagonist roles of biceps and triceps during the bowing motion were clearly defined in the expert group, but not as apparent in the novice group. Conclusions: Bowing movement appears to be controlled by the shoulder rather than the elbow as shoulder ROM changed across strings while elbow ROM remained the same. Shoulder injuries are probably due to repetition as the muscle activity required for the movement is small. Experts require a smaller amount of muscle activity to perform the movement, possibly due to more efficient muscle activation patterns as a result of practice. This quantitative multidisciplinary approach to analysing violinists' movements can contribute to fuller understanding of both playing demands and injury mechanisms .