M. E. Everts

Muscle development of livestock animals: physiology, genetics and meat quality.

Number and size of muscle fibres in relation to meat production The effect of growth and exercise on muscle characteristics in relation to meat quality Nutrition, hormone receptor expression, and gene interactions The impact of minerals and micronutrients on growth control Local and Systemic Regulation of Muscle Growth Proteolytic systems and the regulation of muscle remodeling and breakdown Genome Analysis of QTL for Muscle Tissue Development and Meat Quality Functional genomics and proteomics in relation to muscle tissue Genetic Control of Intramuscular Fat Accretion Post-mortem muscle proteolysis and meat tenderness Water holding capacity of meat

Cervical ripening and parturition in cows are driven by a cascade of pro-inflammatory cytokines.

The final stages of cervical ripening and parturition resemble an inflammatory process. Although the role of cytokines in both spontaneous and experimentally induced parturitions has been described in several small laboratory animals and humans, the involvement of pro-inflammatory and regulatory cytokines in physiologic parturition in cows has not been determined. In this study, the cytokine expression profiles were assessed in bovine cervical tissue at several stages of pregnancy and at parturition. Serial biopsy samples of the cervix were obtained from 10 cows on day 185 and day 275 of pregnancy (which was on average 5.4 days before parturition) and at parturition. Messenger RNA expression levels of interleukin (IL)-1beta, IL-6, IL-8, IL-10 and tumour necrosis factor (TNF)alpha were determined using real-time polymerase chain reaction and the number of neutrophils and eosinophils was estimated by Luna and Sirius Red staining. At parturition, IL-8 expression had increased 430-fold (p < 0.001) when compared with that of the day 185 of pregnancy, large numbers of neutrophils had invaded the cervix while eosinophils remained scarce, IL-1beta had increased eightfold (p < 0.05) and IL-6 had not changed significantly. Additionally, IL-10 was increased by 10-fold (p < 0.001) and TNFalpha decreased by 57% (p < 0.05) when compared with that of the day 185 of pregnancy. The large increase in expression of IL-8, enabling the influx of neutrophils, is indicative of its important role in the final stage of cervical ripening and at parturition. As previous studies have shown that neutrophils excrete matrix metalloproteinases (MMP), this might contribute to softening of the cervix. In contrast, the only slightly increased levels of IL-1, steady concentrations of IL-6 and decreased TNFalpha, the potential consequences of increased IL-10 expression, indicate that final cervical of cows in ripening at term parturition is an inflammatory process influenced by regulatory cytokines.

Treadmill but not wheel running improves fatigue resistance of isolated extensor digitorum longus muscle in mice

Aim:  The present study is the first to compare the physiological impact of either forced treadmill or voluntary wheel running exercise on hindlimb muscle in mice.

MMP-2 expression precedes the final ripening process of the bovine cervix

Collagen is denatured in the gradual cervical ripening process during late pregnancy, already before the onset of final cervical ripening at parturition. Matrix Metallo Proteinases (MMPs) might be responsible for this process. To investigate the presence and potential function of MMPs at the different stages of the ripening process, serial cervical biopsies were obtained from 10 cows at Days 185 and 275 of pregnancy (approximately 5 days before calving), at parturition and at 30 days after parturition. The mRNA and protein expression of MMP‐1, MMP‐2, and MMP‐9 and of the tissue inhibitors of MMPs (TIMP)‐1 and TIMP‐2 were semi‐quantitatively determined using RT‐PCR, respectively, zymography, Westernblot, and ELISA techniques and the localization of MMP‐2 protein and presence of granulocytes by immunohistochemistry and Luna staining. At parturition compared to 185 days pregnancy the MMP‐1 protein expression and the numbers of granulocytes were significantly increased by 3 and 26‐fold respectively. MMP‐2 mRNA and protein expression had already increased 2.5 (P < 0.05) and twofold (P < 0.05) at 5 days before parturition, prior to final ripening. At that time, MMP‐2 was present in smooth muscle cells and extra cellular matrix. TIMP‐1 mRNA expression was significantly increased at parturition and TIMP‐2 mRNA expression peaked at 5 days before parturition. The increased expression of MMP‐2 at 5 days before parturition, suggests that in the cow MMP‐2 is responsible for collagen denaturation in the last part of gradual cervical ripening, while MMP‐1 and MMP‐9 are only active during the final cervical ripening process at parturition. Mol. Reprod. Dev. 75: 1669–1677, 2008.

Myosin heavy chain isoforms in equine gluteus medius muscle: comparison of mRNA and protein expression profiles.

The major structural protein in skeletal muscle, myosin heavy chain (MyHC), is primarily transcriptionally controlled. We compared the expression of MyHC isoforms on the mRNA and protein level in biopsies from the m. gluteus medius from adult untrained horses. In transverse sections, the majority of fibers showed qualitatively identical mRNA and protein expression patterns. However, coexpression of 2a and 2d/x MyHCs was substantially more common at the protein than at the mRNA level, suggesting a fine-tuning of these two genes in normal muscle not subjected to any training protocol. Because transverse sections give a limited sampling of mRNA expression in the case of uneven distribution of transcripts in a muscle fiber, we also analyzed longitudinal sections. We present, for the first time, evidence that expression of MyHC mRNA and protein was equal along the length of the fiber. Hence, mRNA expression is not regulated by differential expression of isoforms by separate myonuclei. It is concluded that the number of protein hybrid fibers in equine gluteus medius muscle is controlled by alteration of the transcription pattern uniformly along the fiber, rather than by simultaneous transcription of genes. The differences with the results in muscle of small animals and humans are discussed.

Exercise-induced hyperkalemia in hypothyroid dogs.

We investigated the effect of hypothyroidism in dogs on (1) the Na+-, K+ -ATPase concentration in skeletal muscle, and (2) potassium (K+) homeostasis at rest and during exercise. Prior to and 1 year after induction of hypothyroidism by surgery and subsequent radiothyroidectomy, the Na+-, K+ -ATPase concentrations were quantified in biopsies of sternothyroid muscles of seven Beagle dogs by measuring [3H]ouabain binding capacity. In addition, plasma K+ concentrations were measured at rest and after treadmill exercise in six hypothyroid and seven euthyroid Beagle dogs. During hypothyroidism, the mean Na+ -, K+ -ATPase concentration in muscle biopsies was 41% lower than during euthyroidism. The mean resting plasma K+ value of the hypothyroid dogs was significantly (14%) higher than that of the euthyroid dogs. In the hypothyroid dogs, plasma K+ concentration increased significantly during exercise, whereas there was no rise in the euthyroid dogs. The rise in plasma K+ concentration could not be ascribed to muscle damage, as plasma creatine kinase concentrations remained within reference range. Also renal K+ retention was an unlikely explanation, as plasma aldosterone concentration and plasma renin activity rather increased than decreased during exercise. In conclusion, hypothyroid dogs tend to develop hyperkalemia during exercise, which for a large part can be explained by the severe reduction of the Na+ -, K+ -ATPase capacity in the skeletal muscle pool.

Reducing chloride conductance prevents hyperkalaemia‐induced loss of twitch force in rat slow‐twitch muscle

Exercise‐induced loss of skeletal muscle K+ can seriously impede muscle performance through membrane depolarization. Thus far, it has been assumed that the negative equilibrium potential and large membrane conductance of Cl− attenuate the loss of force during hyperkalaemia. We questioned this idea because there is some evidence that Cl− itself can exert a depolarizing influence on membrane potential (Vm). With this study we tried to identify the possible roles played by Cl− during hyperkalaemia. Isolated rat soleus muscles were kept at 25 °C and twitch contractions were evoked by current pulses. Reducing [Cl−]o to 5 mm, prior to introducing 12.5 mm Ko, prevented the otherwise occurring loss of force. Reversing the order of introducing these two solutions revealed an additional effect, i.e. the ongoing hyperkalaemia‐related loss of force was sped up tenfold after reducing [Cl−]o. However, hereafter twitch force recovered completely. The recovery of force was absent at [K+]o exceeding 14 mm. In addition, reducing [Cl−]o increased membrane excitability by 24%, as shown by a shift in the relationship between force and current level. Measurements of Vm indicated that the antagonistic effect of reducing [Cl−]o on hyperkalaemia‐induced loss of force was due to low‐Cl−‐induced membrane hyperpolarization. The involvement of specific Cl− conductance was established with 9‐anthracene carboxylic acid (9‐AC). At 100 μm, 9‐AC reduced the loss of force due to hyperkalaemia, while at 200 μm, 9‐AC completely prevented loss of force. To study the role of the Na+−K+−2Cl− cotransporter (NKCC1) in this matter, we added 400 μm of the NKCC inhibitor bumetanide to the incubation medium. This did not affect the hyperkalaemia‐induced loss of force. We conclude that Cl− exerts a permanent depolarizing influence on Vm. This influence of Cl− on Vm, in combination with a large membrane conductance, can apparently have two different effects on hyperkalaemia‐induced loss of force. It might exert a stabilizing influence on force production during short periods of hyperkalaemia, but it can add to the loss of force during prolonged periods of hyperkalaemia.

Differential Expression of Calcineurin and SR Ca2+ Handling Proteins in Equine Muscle Fibers During Early Postnatal Growth

During early postnatal development, the myosin heavy chain (MyHC) expression pattern in equine gluteus medius muscle shows adaptation to movement and load, resulting in a decrease in the number of fast MyHC fibers and an increase in the number of slow MyHC fibers. In the present study we correlated the expression of MyHC isoforms to the expression of sarcoplasmic(endo)reticulum Ca2+-ATPase 1 and 2a (SERCA), phospholamban (PLB), calcineurin A (CnA), and calcineurin B (CnB). Gluteus medius muscle biopsies were taken at 0, 2, 4, and 48 weeks and analyzed using immunofluorescence. Both SERCA isoforms and PLB were expressed in almost all fiber types at birth. From 4 weeks of age onward, SERCA1 was exclusively expressed in fast MyHC fibers and SERCA2a and PLB in slow MyHC fibers. At all time points, CnA and CnB proteins were expressed at a basal level in all fibers, but with a higher expression level in MyHC type 1 fibers. From 4 weeks onward, expression of only CnA was also higher in MyHC type 2a and 2ad fibers. We propose a double function of calcineurin in calcium homeostasis and maintenance of slow MyHC fiber type identity. Although equine muscle is already functional at birth, expression patterns of the monitored proteins still show adaptation, depending on the MyHC fiber type.

Specificity and reversibility of the training effects on the concentration of Na+, K+-ATPase in foal skeletal muscle

The purpose of the present study was to determine whether training and detraining affect the Na+,K+-ATPase concentration in horse skeletal muscles, and whether these effects are specific for the muscles involved in the training programme. Twenty-four Dutch Warmblood foals age 7 days were assigned randomly to 3 groups: Box (box-rest without training), Training (box-rest with training: short-sprint) and Pasture (pasture without training). Exercise regimens were carried out for 5 months and were followed by 6 months of detraining. Five of the foals in each group were subjected to euthanasia at age 5 months and the remaining foals at 11 months. Muscle samples were collected from the deep part of the gluteus medius, semitendinosus and masseter muscles. The Na+,K+-ATPase concentration was quantified by [3H]ouabain binding. In the Training group, the concentration of Na+,K+-ATPase in gluteus medius and semitendinosus muscle, but not in masseter muscle, showed a relative increase of 20% (P<0.05) as compared to Box foals. After detraining for the subsequent 6 months, the concentration of Na+,K+-ATPase in semitendinosus muscle remained the same, while that in gluteus medius muscle was reduced by 10%. It is concluded that: 1) short-sprint training for 5 months induced an increase of the Na+,K+-ATPase concentration in gluteus medius and semitendinosus muscles of the foal. Interestingly, this effect persisted during the 6 months of the detraining period. Whether the higher Na+,K+-ATPase concentration due to training of young foals leads to a better athletic performance when they become mature still needs to be established; 2) the factors that initiate an increase in Na+,K+-ATPase concentration following training are likely to be located in the muscle itself and 3) the training effect may last for several months after returning to normal activity, especially in muscles containing a high percentage of fast-twitch fibres.

Presence of SERCA and Calcineurin during Fetal Development of Porcine Skeletal Muscle

Mechanisms involved in skeletal myofiber differentiation during fetal development of large animals are poorly understood. Studies in small animals suggest that the calcineurin (Cn) pathway is involved in myofiber differentiation. Neural activity is a prerequisite for Cn activity, implying maintenance of sustained low intracellular Ca2+ concentrations. To study the role of Cn in fetal myofiber differentiation, we monitored the temporal and spatial distribution of Cn subunits, sarcoplasmic reticulum Ca2+ ATPase (SERCA), phospholamban (PLB), and myosin heavy chain (MyHC) isoforms in relation to ingrowing nerves in porcine semitendinosus muscle (m. semitendinosus) at 55 and 75 days of gestation (dg) and at term. Immunofluorescence analysis revealed the presence of Cn subunits and SERCA isoforms at all analyzed stages. Cn distribution was not fiber-type specific, but expression became more prominent at term. At 75 dg, differential SERCA2 expression was accompanied by perinuclear PLB in primary fibers. SERCA1 was expressed in all fiber types at all stages. No specific MyHC isoform distribution was seen in relation to neuromuscular contacts, although neuromuscular contacts were present. From these results we speculate that in porcine m. semitendinosus differential SERCA2 expression precedes differential Cn expression. The question whether the Cn pathway is involved in prenatal myofiber differentiation needs further studies.