Food restriction and refeeding in lambs influence muscle antioxidant status

Compensatory growth, a frequent phenomenon observed in ruminants due to seasonal variation in food availability, affects protein metabolism including protein oxidation. These oxidation processes may have an impact on animal health as well as on meat protein degradation during post mortem aging (ie meat maturation). Sixteen male lambs were randomly divided into four groups. One group was fed ad libitum (C) and one group was food-restricted to 60% of the intake of the C group (R). The last two groups were restricted similarly to the R group and refed either ad libitum (RAL) or similarly to the C group (pair-feeding) (RPF). Muscles samples were taken immediately after slaughter. The present study showed that the restriction/refeeding pattern had no effect on protein oxidation in the muscles studied (longissimus dorsi (LD), semitendinosus (ST) and supraspinatus (SP)). However, total antioxidant capacity decreased after food restriction (-51%, -43%, P < 0.01 for ST and LD muscles, respectively) and re-increased only after ad libitum refeeding. This alteration in the total antioxidant status can partially be explained by the similar pattern of change observed in the glutathione concentration of the muscles (-25%, P < 0.05 for ST muscle and NS for the other muscles). However, none of the concentrations of other water-soluble antioxidants studied (carnosine, anserine, glutathione peroxidase and superoxide dismutase) were altered during compensatory growth. This study showed that an inappropriate feeding level following a nutritional stress induced alterations in the total antioxidant status (particularly that of glutathione), which may have consequences on animal health. Other consequences of a decrease of the animal antioxidant status in vivo could be an alteration of the protein oxidation processes during meat maturation.     

Suboptimal energy balance selectively up-regulates muscle GLUT gene expression but reduces insulin-dependent glucose uptake during postnatal development.

The major facilitative glucose transporters in muscle, GLUT1 (insulin-independent) and GLUT4 (insulin-dependent), are essential for normal growth and metabolism, but factors controlling their expression during postnatal development are poorly understood. We have therefore determined the role of energy status in regulating muscle GLUT gene expression and function in young, growing pigs on a high (H) or low (L) food intake (H =2L) at 35 degrees C or 26 degrees C. RNase protection assays revealed selective up-regulation of GLUT1 and GLUT4 by mild undernutrition 20-24 h after feeding: mRNA levels were elevated in longissimus dorsi (P<0.001) and rhomboideus (P<0.05), but not in diaphragm or cardiac muscles. Assessment of 2-deoxy-glucose uptake in a small isolated muscle, flexor carpi radialis, showed that the 26L group, which had suboptimal energy balance and the greatest GLUT4 expression, had the highest insulin-independent glucose uptake but the lowest insulin-dependent increment: 20% compared with 70% in the other groups. These novel findings are directly relevant to an understanding of mechanisms underlying the development of insulin resistance and demonstrate 1) muscle-specific up-regulation of GLUT gene expression by postnatal undernutrition that is not related simply to myofiber type, but to whole-body function; and 2) that the degree of GLUT up-regulation and the subcellular distribution and function of GLUT proteins are dependent on energy status.     

Myogenin regulates a distinct genetic program in adult muscle stem cells

In contrast to the detailed understanding we have for the regulation of skeletal muscle gene expression in embryos, similar insights into postnatal muscle growth and regeneration are largely inferential or do not directly address gene regulatory mechanisms. Muscle stem cells (satellite cells) are chiefly responsible for providing new muscle during postnatal and adult life. The purpose of this study was to determine the role that the myogenic basic helix-loop-helix regulatory factor myogenin has in postnatal muscle growth and adult muscle stem cell gene expression. We found that myogenin is absolutely required for skeletal muscle development and survival until birth, but it is dispensable for postnatal life. However, Myog deletion after birth led to reduced body size implying a role for myogenin in regulating body homeostasis. Despite a lack of skeletal muscle defects in Myog-deleted mice during postnatal life and the efficient differentiation of cultured Myog-deleted adult muscle stem cells, the loss of myogenin profoundly altered the pattern of gene expression in cultured muscle stem cells and adult skeletal muscle. Remarkably, these changes in gene expression were distinct from those found in Myog-null embryonic skeletal muscle, indicating that myogenin has separate functions during postnatal life.     

Myosin heavy chain isoforms in postnatal muscle development of mice

In this study, using a high-resolution gel electrophoresis technique, we have characterized the myosin heavy chain composition in different skeletal muscle of the mouse during postnatal development. The pattern of myosin heavy chain expression was studied in four hind limb muscles, the diaphragm, the tongue and the masseter. All of these muscles displayed the usual sequential transitions from embryonic to neonatal and to adult myosin heavy chain isoforms but more interestingly these transitions occur with a distinct chronology in the different muscles. In addition, our results demonstrated a transitory pattern of expression for certain adult myosin heavy chain isoforms in the soleus and the tongue. In the soleus muscle IIB and in the tongue IIA myosin heavy chain isoforms were detected only for a short time during postnatal life. Our results demonstrate that muscles of the mouse with different functions are subjected to a distinct programs of myosin isoform transitions during postnatal muscle development. This study describes new data which will help us to understand both postnatal muscle development in transgenic mouse muscles as well as in muscle pathology.     

Contractile properties of the rat external abdominal oblique and diaphragm muscles during development.

We studied the in vitro contractile and fatigue properties of the rat external abdominal oblique (EAO) and costal diaphragm (DIA) muscles during postnatal development. Isometric twitch contraction (CT) and half-relaxation (RT1/2) times were longer in both the EAO and DIA muscles during the early postnatal period and decreased with age. In the first postnatal week, the CT and RT1/2 were longer in the EAO than the DIA muscle. At 14 days of age and thereafter, the CT and RT1/2 were shorter in the EAO than in the DIA muscle. Force-frequency relationships of the EAO and DIA muscles changed during postnatal development such that the relative force (percent maximum) generated at lower frequencies (less than 15 pulses/s) decreased with age. Moreover the relative force generated by the EAO muscle at lower frequencies was greater than that of the DIA muscle during the early postnatal period but less than that of the DIA muscle in adults. The specific force of both the EAO and DIA muscles increased progressively with age. There were no differences in specific force between the EAO and DIA muscles at any age. The fatigability of the EAO and DIA muscles was comparable during the early postnatal period and increased in both muscles with postnatal development. In adults the EAO muscle was more fatigable than the DIA muscle. We conclude that the contractile and fatigue properties of the EAO and DIA muscles undergo significantly different postnatal transitions, which may reflect their functional involvement in sustaining ventilation.     

Genetic Variability and Linkage Disequilibrium Patterns in the Bovine <Emphasis Type="Italic">DNAJA1</Emphasis> Gene

Correlation between expression level of the bovine DNAJA1 gene and meat tenderness was recently found in Charolais longissimus thoracis muscle samples, suggesting that this gene could play an important role in meat tenderness. Here, we report the validation of polymorphisms within the bovine DNAJA1 gene, and the haplotype variability and extent of linkage disequilibrium in the three main French beef breeds (Blonde d’Aquitaine, Charolais, Limousin). Genotyping 18 putative SNPs revealed that 16 SNPs were polymorphic within the breeds tested. Two SNPs were removed from further analyses as one SNP had a low genotyping call rate, while the other SNP was not in Hardy–Weinberg equilibrium. The degree of heterozygosity observed for the remaining 14 SNPs varied between breeds, with Charolais being the breed with the highest genetic variation and Blonde d’Aquitaine the lowest. Linkage disequilibrium and haplotype structure of DNAJA1 were different between breeds. Eighteen different haplotypes, including three shared by all breeds, were discovered, and two to three tag SNPs (depending on the breed) are sufficient to capture all the genetic variability seen in these haplotypes. The results of this study will facilitate the design of optimal future association studies evaluating the role of the DNAJA1 gene in meat tenderness.     

Pre- and Early-Postnatal Nutrition Modify Gene and Protein Expressions of Muscle Energy Metabolism Markers and Phospholipid Fatty Acid Composition in a Muscle Type Specific Manner in Sheep

We previously reported that undernutrition in late fetal life reduced whole-body insulin sensitivity in adult sheep, irrespective of dietary exposure in early postnatal life. Skeletal muscle may play an important role in control of insulin action. We therefore studied a range of putative key muscle determinants of insulin signalling in two types of skeletal muscles (longissimus dorsi (LD) and biceps femoris (BF)) and in the cardiac muscle (ventriculus sinister cordis (VSC)) of sheep from the same experiment. Twin-bearing ewes were fed either 100% (NORM) or 50% (LOW) of their energy and protein requirements during the last trimester of gestation. From day-3 postpartum to 6-months of age (around puberty), twin offspring received a high-carbohydrate-high-fat (HCHF) or a moderate-conventional (CONV) diet, whereafter all males were slaughtered. Females were subsequently raised on a moderate diet and slaughtered at 2-years of age (young adults). The only long-term consequences of fetal undernutrition observed in adult offspring were lower expressions of the insulin responsive glucose transporter 4 (GLUT4) protein and peroxisome proliferator-activated receptor gamma, coactivator 1α (PGC1α) mRNA in BF, but increased PGC1α expression in VSC. Interestingly, the HCHF diet in early postnatal life was associated with somewhat paradoxically increased expressions in LD of a range of genes (but not proteins) related to glucose uptake, insulin signalling and fatty acid oxidation. Except for fatty acid oxidation genes, these changes persisted into adulthood. No persistent expression changes were observed in BF and VSC. The HCHF diet increased phospholipid ratios of n-6/n-3 polyunsaturated fatty acids in all muscles, even in adults fed identical diets for 1½ years. In conclusion, early postnatal, but not late gestation, nutrition had long-term consequences for a number of determinants of insulin action and metabolism in LD. Tissues other than muscle may account for reduced whole body insulin sensitivity in adult LOW sheep.     

Acidic and basic protein contents in Gastrocnemii and pectoralis muscles of chick under denervation and work-induced stress conditions.

Biochemical estimation of acidic and basic proteins of chick gastrocnemii (G. externus, G. medius and G. internus) and pectoralis muscles has been done under normal, denervated and work stress conditions from 1-56 days of postnatal growth. The reciprocal relationship of the two protein groups is clearly established. It is evident that muscle denervation acts as a stimulant for proteosynthetic activities and probably may also be an inhibitory factor for protein degradative reactions. During work overload stress, the rapid growth of muscles has been related to high rate of contractile activity.     

Endocrine and metabolic regulation of muscle growth and body composition in cattle

Muscle metabolism (in interaction with other organs and tissues, including adipose tissue) plays an important role in the control of growth and body composition. Muscle ontogenesis has been described in different genotypes of cattle for myofibres, connective tissue and intramuscular depots. The ontogenesis or the action of putatively important factors controlling muscle development (IGF-II expression, IGF receptors, growth hormone (GH) receptor, myostatin, basic fibroblast growth factor, transforming growth factor-β1, insulin and thyroid hormones) has also been studied on bovine foetal muscle samples and satellite cells. The glucose/insulin axis has been specifically studied in both the bovine adipose tissue and heart. Clearly, cattle, like sheep, are mature species at birth based on their muscle characteristics compared to other mammalian or farm animal species. The different myoblast generations have been well characterised in cattle, including the second generation which is liable to be affected by foetal undernutrition at least in sheep. Interesting genotypes, for example, double-muscled genotype, have been characterised by an altered metabolic and endocrine status associated with a reduced fat mass, specific muscle traits and different foetal characteristics. Finally, the recent development of genomics in cattle has allowed the identification of novel genes controlling muscle development during foetal and postnatal life. Generally, a high muscle growth potential is associated with a reduced fat mass and a switch of muscle fibres towards the glycolytic type. The possibility and the practical consequences of manipulating muscle growth and, hence, body composition by nutritional and hormonal factors are discussed for bovines based on our current biological knowledge.     

Cooperative control of striated muscle mass and metabolism by MuRF1 and MuRF2

The muscle-specific RING finger proteins MuRF1 and MuRF2 have been proposed to regulate protein degradation and gene expression in muscle tissues. We have tested the in vivo roles of MuRF1 and MuRF2 for muscle metabolism by using knockout (KO) mouse models. Single MuRF1 and MuRF2 KO mice are healthy and have normal muscles. Double knockout (dKO) mice obtained by the inactivation of all four MuRF1 and MuRF2 alleles developed extreme cardiac and milder skeletal muscle hypertrophy. Muscle hypertrophy in dKO mice was maintained throughout the murine life span and was associated with chronically activated muscle protein synthesis. During ageing (months 4-18), skeletal muscle mass remained stable, whereas body fat content did not increase in dKO mice as compared with wild-type controls. Other catabolic factors such as MAFbox/atrogin1 were expressed at normal levels and did not respond to or prevent muscle hypertrophy in dKO mice. Thus, combined inhibition of MuRF1/MuRF2 could provide a potent strategy to stimulate striated muscles anabolically and to protect muscles from sarcopenia during ageing.     

Embryonic deregulation of muscle stress signaling pathways leads to altered postnatal stem cell behavior and a failure in postnatal muscle growth

PW1 is a mediator of p53 and TNFalpha signaling pathways previously identified in a screen to isolate muscle stem cell regulators. We generated transgenic mice carrying a C-terminal deleted form of PW1 (DeltaPW1) which blocks p53-mediated cell death and TNFalpha-mediated NFkappaB activation fused to the myogenin promoter. Embryonic/fetal muscle development appears normal during transgene expression, however, postnatal transgenic pups display severe phenotypes including runtism, reduced muscle mass and fiber diameters resembling atrophy. Atrogin-1, a marker of skeletal muscle atrophy, is expressed postnatally in transgenic mice. Electron microscopic analyses of transgenic muscle reveal a marked decrease in quiescent muscle satellite cells suggesting a deregulation of postnatal stem cells. Furthermore, transgenic primary myoblasts show a resistance to the effects of TNFalpha upon differentiation. Taken together, our data support a role for PW1 and related stress pathways in mediating skeletal muscle stem cell behavior which in turn is critical for postnatal muscle growth and homeostasis. In addition, these data reveal that postnatal stem cell behavior is likely specified during early muscle development.     

Chicken heat shock protein HSPB1 increases and interacts with αB-crystallin in aged skeletal muscle

International trading markets of meat require the animal’s age information to prevent cross-contamination of ineligible meat products. Individual livestock age is either evaluated from physiological features or verified by breeding history. However, it remains impossible to perform age verification on meat when a suspicion of error occurred in the importing country. To investigate an age-related protein in skeletal muscle of livestock, we compared protein expression among chicken pectoralis major of different ages. Results indicated that the level of expression of chicken HSPB1, one of the small heat shock proteins, was increased in aged muscles. On the other hand, other heat shock proteins, heat shock factors, and myosin heavy chain isoform did not change the expression levels in aged chicken muscle. In addition, we identified that αB-crystallin interacted with HSPB1 in aged chicken muscle. These results suggest that HSPB1 protein forms complexes with αB-crystallin in aged chicken muscle and suppose to become the candidate of age-related bio-marker for verifying the age of chicken meat.     

Endogenous expression and developmental changes of HSP72 in rat skeletal muscles

The purpose of the present study was to determine whether endogenous factor(s) contributes to the expression of heat shock proteins (HSPs) during the early developmental stages of rat skeletal muscles. HSP72 was expressed in both the soleus and plantaris muscles at embryonic day 22 (E22). On the basis of myosin heavy chain (MHC) immunohistochemistry, HSP72 was specifically expressed in slow type I fibers in both muscles. These slow fibers were observed throughout the entire cross section of the soleus muscle and only in the deep region (close to the bone) of the plantaris muscle. These results indicate that the expression of HSP72 is related to endogenous factors associated with type I fibers, because E22 rats have minimal exogenous influences and the soleus and plantaris muscles of E22 rats have similar metabolic and contractile profiles at this stage of development. We then examined the changes in HSP72 and heat shock cognate (HSC) 73 in the same two muscles from E22 to postnatal day 56 via Western blotting. The level of HSP72 in the soleus muscle gradually increased in parallel with the increment in the type I MHC isoform. Compared with the soleus, only a small amount of HSP72 could be detected in the plantaris muscle throughout the developmental period. For both muscles, HSC73 reached levels observed in adult muscles at postnatal day 3, and these levels were unchanged thereafter. These results indicate that the expression of HSP72, but not HSC73, is influenced by both endogenous and exogenous factors during the embryonic and early developmental periods.     

Expression of the Na(+)/Ca(2+) exchanger in skeletal muscle

The expression of the Na(+)/Ca(2+) exchanger was studied in differentiating muscle fibers in rats. NCX1 and NCX3 isoform (Na(+)/Ca(2+) exchanger isoform) expression was found to be developmentally regulated. NCX1 mRNA and protein levels peaked shortly after birth. Conversely, NCX3 isoform expression was very low in muscles of newborn rats but increased dramatically during the first 2 wk of postnatal life. Immunocytochemical analysis showed that NCX1 was uniformly distributed along the sarcolemmal membrane of undifferentiated rat muscle fibers but formed clusters in T-tubular membranes and sarcolemma of adult muscle. NCX3 appeared to be more uniformly distributed along the sarcolemma and inside myoplasm. In the adult, NCX1 was predominantly expressed in oxidative (type 1 and 2A) fibers of both slow- and fast-twitch muscles, whereas NCX3 was highly expressed in fast glycolytic (2B) fibers. NCX2 was expressed in rat brain but not in skeletal muscle. Developmental changes in NCX1 and NCX3 as well as the distribution of these isoforms at the cellular level and in different fiber types suggest that they may have different physiological roles.     

DNAJA1 positively regulates amino acid-stimulated milk protein and fat synthesis in bovine mammary epithelial cells

Several cellular processes, including the recovery of misfolded proteins, the folding of polypeptide chains, transit of polypeptides across the membrane, construction and disassembly of protein complexes, and modulation of protein control, are carried out by DnaJ homolog subfamily A member 1 (DNAJA1), which belongs to the DnaJ heat-shock protein family. It is unknown if DNAJA1 regulates the production of milk in bovine mammary epithelium cells (BMECs). Methionine and leucine increased DNAJA1 expression and nuclear location, as seen by us. In contrast to DNAJA1 knockdown, overexpression of DNAJA1 boosted the production of milk proteins and fats as well as mammalian target of rapamycin (mTOR) and sterol regulatory element binding protein-1c (SREBP-1c). As a result of amino acids, mTOR and SREBP-1c gene expression are stimulated, and DNAJA1 is a positive regulator of BMECs' amino acid-induced controlled milk protein and fat production.     

Postnatal expression of myosin isoforms in an expiratory muscle--external abdominal oblique.

We studied the postnatal expression of heavy-chain (MHC) and native myosin isoforms in an expiratory abdominal muscle of the rat, the external abdominal oblique (EO). Moreover, we contrasted EO myosin expression with that of the costal diaphragm (DIA) to draw inspiratory vs. expiratory muscle comparisons during development. Examination of MHC gels demonstrated a mature phenotype of slow and adult fast myosin isoforms at an earlier age in the EO (day 60) than in the DIA [day > 115 (adult)]. The mature MHC phenotype of the EO was characterized by a preponderance of MHC 2B, whereas the DIA was characterized by approximately equal portions of MHC slow, MHC 2A, and MHC 2X. During early postnatal development, there was a delay in the expression of MHC 2A in the EO compared with the DIA. However, MHC 2B, expressed later in development in both muscles, was noted in the EO before the DIA. We conclude that 1) the EO mature myosin phenotype is characterized by a preponderance of fast myosin isoforms and 2) the EO and DIA muscles are subject to different temporal patterns of isoform expression during postnatal development.