Expression of heat shock protein 70 in transport-stressed broiler pectoralis major muscle and its relationship with meat quality

Omics research has indicated that heat shock protein 70 (HSP70) is a potential biomarker of meat quality. However, the specific changes and the potential role of HSP70 in postmortem meat quality development need to be further defined. In this study, Arbor Acres broiler chickens (n=126) were randomly categorized into three treatment groups of unstressed control (C), 0.5-h transport (T) and subsequent water shower spray following transport (T/W). Each treatment consisted of six replicates with seven birds each. The birds were transported according to a designed protocol. The pectoralis major (PM) muscles of the transport-stressed broilers were categorized as normal and pale, soft and exudative (PSE)-like muscle samples according to L* and pH_{24 h} values to test the expression and location of HSP70. Results revealed that the activities of plasma creatine kinase and lactate dehydrogenase increased significantly (P<0.05) in normal and PSE-like muscle samples after transportation. The mRNA expression of HSP70 in normal muscle samples increased significantly (P<0.05) compared with that in the controls after stress. The protein expression of HSP70 increased significantly in normal muscle samples and decreased significantly (P<0.05) in PSE-like muscles. Immuno-fluorescence showed that HSP70 was present in the cytoplasm and on surface membranes of PM muscle cells in the normal samples following stress. Meanwhile, HSP70 was present on the surface membranes and extracellular matrix but was barely visible in the cytoplasm of the PSE-like samples. Principal component analysis showed high correlations between HSP70 and meat quality and stress indicators. In conclusion, this research suggests that the variation in HSP70 expression may provide a novel insight into the pathways underlying meat quality development.     

Porcine muscle sensory attributes associate with major changes in gene networks involving CAPZB, ANKRD1, and CTBP2

Principal component analysis of traits related to carcass and meat properties were combined with microarray expression data for the identification of functional networks of genes and biological processes taking place during the conversion of muscle to meat. Principal components (PCs) with high loadings of meat quality traits were derived from phenotypic data of 572 animals of a porcine crossbreed population. Microarray data of 74 M. longissimus dorsi samples were correlated with PC datasets. Lists of significantly correlated genes were analyzed for enrichment of functional annotation groups as defined in the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases as well as the Ingenuity Pathways Analysis library. Ubiquitination, phosphorylation, mitochondrion dysfunction, actin, integrin, platelet-derived growth factor, epidermal growth factor, vascular endothelial growth factor, and Ca signaling pathways are correlated with meat quality. Among the significantly trait-associated genes, CAPZB, ANKRD1, and CTBP2 are promoted as candidate genes for meat quality that provide a link between the highlighted pathways. Knowledge of the relevant biological processes and the differential expression of members of the pathway will provide tools that are predictive for traits related to meat quality and that may also be diagnostic for many muscle defects or damages including muscle atrophy, dystrophy, and hypoxia.     

Regulatory interfaces between the stress protein response and other gene expression programs in the cell

The stress protein response involves the immediate reprogramming of gene expression in cells exposed to proteomic insult leading to massive synthesis of heat shock proteins (HSP). We have examined the outcome when cells are induced to activate two other gene expression programs--the acute inflammatory response and entry of quiescent cells into the cell cycle--and then exposed to protein stress. We find that these responses are mutually antagonistic with, on the one hand, heat shock factor 1 (HSF1) inhibition through the phosphorylation of inhibitory serine residues after inflammatory or mitogenic stimulus and, on the other hand, after stress, HSF1 directly repressing the promoters of genes that mediate acute inflammation and mitogenesis. The expression of the stress protein response during periods of acute protein damage was shown to lead to efficient activation of HSF1 and HSP expression accompanied by repression of other gene expression programs.     

Chronic antioxidant enzyme mimetic treatment differentially modulates hyperthermia-induced liver HSP70 expression with aging.

One postulated mechanism for the reduction in stress tolerance with aging is a decline in the regulation of stress-responsive genes, such as inducible heat shock protein 72 (HSP70). Increased levels of oxidative stress are also associated with aging, but it is unclear what impact a prooxidant environment might have on HSP70 gene expression. This study utilized a superoxide dismutase/catalase mimetic (Eukarion-189) to evaluate the impact of a change in redox environment on age-related HSP70 responses to a physiologically relevant heat challenge. Results demonstrate that liver HSP70 mRNA and protein levels are reduced in old compared with young rats at selected time points over a 48-h recovery period following a heat-stress protocol. While chronic systemic administration of Eukarion-189 suppressed hyperthermia-induced liver HSP70 mRNA expression in both age groups, HSP70 protein accumulation was blunted in old rats but not in their young counterparts. These data suggest that a decline in HSP70 mRNA levels may be responsible for the reduction in HSP70 protein observed in old animals after heat stress. Furthermore, improvements in redox status were associated with reduced HSP70 mRNA levels in both young and old rats, but differential effects were manifested on protein expression, suggesting that HSP70 induction is differentially regulated with aging. These findings highlight the integrated mechanisms of stress protein regulation in eukaryotic organisms responding to environmental stress, which likely involve interactions between a wide range of cellular signals.     

Flash dietary methionine supply over growth requirements in pigs: Multi-facetted effects on skeletal muscle metabolism

Dietary methionine affects protein metabolism, lean gain and growth performance and acts in the control of oxidative stress. When supplied in large excess relative to growth requirements in diets for pigs, positive effects on pork quality traits have been recently reported. This study aimed to decipher the molecular and biochemical mechanisms affected by a dietary methionine supply above growth requirements in the loin muscle of finishing pigs. During the last 14 days before slaughter, crossbred female pigs (n = 15 pigs/diet) were fed a diet supplemented with hydroxy-methionine (Met5; 1.1% of methionine) or not (CONT, 0.22% of methionine). Blood was sampled at slaughter to assess key metabolites. At the same time, free amino acid concentrations and expression or activity levels of genes involved in protein or energy metabolism were measured in the longissimus lumborum muscle (LM). The Met5 pigs exhibited a greater activity of creatine kinase in plasma when compared with CONT pigs. The concentrations of free methionine, alpha-aminobutyric acid, anserine, 3-methyl-histidine, lysine, and proline were greater in the LM of Met5 pigs than in CONT pigs. Expression levels of genes involved in protein synthesis, protein breakdown or autophagy were only scarcely affected by the diet. Among ubiquitin ligases, MURF1, a gene known to target creatine kinase and muscle contractile proteins, and OTUD1 coding for a deubiquitinase protease, were up-regulated in the LM of Met5 pigs. A lower activity of citrate synthase, a reduced expression level of ME1 acting in lipogenesis but a higher expression of PPARD regulating energy metabolism, were also observed in the LM of Met5 pigs compared with CONT pigs. Principal component analysis revealed that expression levels of many studied genes involved in protein and energy metabolism were correlated with meat quality traits across dietary treatments, suggesting that subtle modifications in expression of those genes had cumulative effects on the regulation of processes leading to the muscle transformation into meat. In conclusion, dietary methionine supplementation beyond nutritional requirements in pigs during the last days before slaughter modified the free amino acid profile in muscle and its redox capacities, and slightly affected molecular pathways related to protein breakdown and energy metabolism. These modifications were associated with benefits on pork quality traits.     

Anabolic steroid and gender-dependent modulation of cytosolic HSP70s in fast- and slow-twitch skeletal muscle

Besides their clinical uses, anabolic steroids (AASs) are self-administered by athletes to improve muscle mass and sports performance. The biological basis for their presumed effectiveness at suprapharmacological doses, however, remains uncertain. Since the expression of high levels of some stress proteins (HSPs) has been associated with an increased tolerance to stress and chronic exercise up-regulates HSP72 in skeletal muscle, this investigation was aimed at testing whether the administration of suprapharmacological doses of AASs, either alone or in conjunction with chronic exercise, induced changes in HSP72. Nandrolone decanoate (ND), an estrene derivative, but not stanozolol (ST), a derivative of the androstane series, up-regulated the levels of HSP72 and changed the proportions of various charge variants of the cytosolic HSP70s in sedentary and exercise-trained rats, exclusively in fast-twitch fibres. Since the expression of HSP73-levels in skeletal muscle was dependent on gender but not on muscle type, and that of HSP72-levels was muscle type specific but gender-independent, ND effects on cytosolic HSP70s could not be explained solely by a functional relationship with sex steroids. The reported results indicate that, by up-regulating the expression levels of HSP72 in fast-twitch fibres, nandrolone decanoate could contribute to improving the tolerance of skeletal muscle to high-intensity training.     

模拟失重大鼠心肌与血管组织的热应激诱导HSP70表达

为研究模拟失重是否可以引起大鼠心肌与血管组织HSP70的诱导表达发生改变,用尾部悬吊大鼠模型模拟失重,以研究失重对生理的影响,用Northern杂交与Western印迹分析检测4周模拟失重大鼠热应激后并在室温下恢复1h(SUS-H1)或2h(SUS-H2_心肌,血管组织HSP70表达的变化,结果表明,热应激后,各组大鼠心肌组织的HSP72 mRNA表达的均显著增加,但SUS－H2大鼠心肌组织的表达显著低于CON－H2组;各组大鼠心肌组织HSP72表达也均显著增加,但SUS－H1与SUS－H2大鼠的表达与相应对照组相比,则仅呈降低趋势,其底动脉血管组织的HSP72 mRNA与HSP72诱导表达均显著增高,而在股动脉则两者仅呈降低趋势,上述结果提示,模拟失重可导致大鼠心肌发生类似衰老的心肌改变;身体前,后部血管组织HSP70的诱导表达变化可能与血管的分化性适应方向一致。     

Seasonal variation in expression pattern of genes under HSP70

Heat shock protein 70 (HSP70) is one of the most abundant and best characterized heat shock protein family that consists of highly conserved stress proteins, expressed in response to stress, and plays crucial roles in environmental stress tolerance and adaptation. The present study was conducted to identify major types of genes under the HSP70 family and to quantify their expression pattern in heat- and cold-adapted Indian goats (Capra hircus) with respect to different seasons. Five HSP70 gene homologues to HSPA8, HSPA6, HSPA1A, HSPA1L, and HSPA2 were identified by gene-specific primers. The cDNA sequences showed high similarity to other mammals, and proteins have an estimated molecular weight of around 70 kDa. The expression of HSP70 genes was observed during summer and winter. During summer, the higher expression of HSPA8, HSPA6, and HSPA1A was observed, whereas the expression levels of HSPA1L and HSPA2 were found to be lower. It was also observed that the expression of HSPA1A and HSPA8 was higher during winter in both heat- and cold-adapted goats but downregulates in case of other HSPs. Therefore, both heat and cold stress induced the overexpression of HSP70 genes. An interesting finding that emerged from the study is the higher expression of HSP70 genes in cold-adapted goats during summer and in heat-adapted goats during winter. Altogether, the results indicate that the expression pattern of HSP70 genes is species- and breed-specific, most likely due to variations in thermal tolerance and adaptation to different climatic conditions.     

An integrative proteome analysis of different seedling organs in tolerant and sensitive wheat cultivars under drought stress and recovery

Roots, leaves, and intermediate sections between roots and leaves (ISRL) of wheat seedlings show different physiological functions at the protein level. We performed the first integrative proteomic analysis of different tissues of the drought‐tolerant wheat cultivar Hanxuan 10 (HX‐10) and drought‐sensitive cultivar Chinese Spring (CS) during a simulated drought and recovery. Differentially expressed proteins (DEPs) in the roots (122), ISRLs (146), and leaves (163) showed significant changes in expression in response to drought stress and recovery. Numerous DEPs associated with cell defense and detoxifications were significantly regulated in roots and ISRLs, while in leaves, DEPs related to photosynthesis showed significant changes in expression. A significantly larger number of DEPs related to stress defense were upregulated in HX‐10 than in CS. Expression of six HSPs potentially related to drought tolerance was significantly upregulated under drought conditions, and these proteins were involved in a complex protein–protein interaction network. Further phosphorylation analysis showed that the phosphorylation levels of HSP60, HSP90, and HOP were upregulated in HX‐10 under drought stress. We present an overview of metabolic pathways in wheat seedlings based on abscisic acid signaling and important protein expression patterns.     

A new antioxidant compound H-290/51 attenuates heat shock protein (HSP 72 kD) response, edema and cell injury following acute heat exposure. An experimental study using light and electron microscopy in the rat

Rats exposed to 4 h heat stress at 38°C exhibited upregulation of heat shock protein (HSP 72 kD) expression in several brain regions associated with brain edema and cell injury. Pretreatment with a new anti-oxidant compound H-290/51 (50 mg/kg, per os, 30 min before stress) significantly attenuated HSP expression, brain edema and cell injury. These results suggest that oxidative stress associated with brain edema plays important roles in HSP expression, not reported earlier.     

Genome-wide methylation changes are associated with muscle fiber density and drip loss in male three-yellow chickens

In eukaryotes, DNA methylation is an important epigenetic modification involved in gene expression regulation. Meat quality traits are complicated traits that are controlled by many genes. Changes in the methylation levels of certain genes controlling meat quality traits will inevitably affect their expression levels, thereby affecting meat quality. The objectives of this study were to investigate the differences in the DNA methylation level in pectoral muscle tissues using fluorescent-labeled methylation sensitive amplified polymorphism and their relationships with meat quality traits in three-yellow chickens. The results showed that the differences in the DNA methylation level had a highly significant effect on muscle fiber density and drip loss (P < 0.01). However, no significant correlations were found between the DNA methylation levels and the other investigated traits (P > 0.05).     

Resistance to apoptosis is a mechanism of adaptation of rat stomach to aspirin

Adaptation of the gastric mucosa to nonsteroidal anti-inflammatory drug-induced injury is a well-documented phenomenon, but the mechanisms are not known. We investigated whether changes in stress protein expression and apoptosis play roles in adaptation of rat stomach to aspirin. RT-PCR and Western blotting techniques were used to analyze mRNA and protein expression of HSP72 and HSP90 and cleavage of caspase 3 protein. Apoptosis was detected by the TUNEL method and quantified. HSP72 mRNA and protein expression was unchanged in adapted mucosa, whereas HSP90 mRNA and protein levels decreased. Caspase 3 protein was activated, and the number of apoptotic cells increased in mucosa after one aspirin dose. However, in adapted mucosa after aspirin, activated caspase 3 and the number of apoptotic cells had returned to basal levels. Induction of the stress response was found not to be a mechanism of mucosal adaptation against multiple doses of aspirin. Our results lead us to propose instead that resistance to aspirin-induced apoptosis plays a role in the protective phenomenon of adaptation.     

Comparative Proteomic Analysis of the Hepatic Response to Heat Stress in Muscovy and Pekin Ducks: Insight into Thermal Tolerance Related to Energy Metabolism

The Pekin duck, bred from the mallard (Anas platyrhynchos) in china, is one of the most famous meat duck species in the world. However, it is more sensitive to heat stress than Muscovy duck, which is believed to have originated in South America. With temperature raising, mortality, laying performance, and meat quality of the Pekin duck are severely affected. This study aims to uncover the temperature-dependent proteins of two duck species using comparative proteomic approach. Duck was cultured under 39°C ± 0.5°C for 1 h, and then immediately returned to 20°C for a 3 h recovery period, the liver proteins were extracted and electrophoresed in two-dimensional mode. After analysis of gel images, 61 differentially expressed proteins were detected, 54 were clearly identified by MALDI TOF/TOF MS. Of the 54 differentially expressed protein spots identified, 7 were found in both species, whereas 47 were species specific (25 in Muscovy duck and 22 in Pekin duck). As is well known, chaperone proteins, such as heat shock protein (HSP) 70 and HSP10, were abundantly up-regulated in both species in response to heat stress. However, we also found that several proteins, such as α-enolase, and S-adenosylmethionine synthetase, showed different expression patterns in the 2 duck species. The enriched biological processes were grouped into 3 main categories according to gene ontology analysis: cell death and apoptosis (20.93%), amino acid metabolism (13.95%) and oxidation reduction (20.93%). The mRNA levels of several differentially expressed protein were investigated by real-time RT-PCR. To our knowledge, this study is the first to provide insights into the differential expression of proteins following heat stress in ducks and enables better understanding of possible heat stress response mechanisms in animals.     

Global Changes in the Rat Heart Proteome Induced by Prolonged Morphine Treatment and Withdrawal

Morphine belongs among the most commonly used opioids in medical practice due to its strong analgesic effects. However, sustained administration of morphine leads to the development of tolerance and dependence and may cause long-lasting alterations in nervous tissue. Although proteomic approaches enabled to reveal changes in multiple gene expression in the brain as a consequence of morphine treatment, there is lack of information about the effect of this drug on heart tissue. Here we studied the effect of 10-day morphine exposure and subsequent drug withdrawal (3 or 6 days) on the rat heart proteome. Using the iTRAQ technique, we identified 541 proteins in the cytosol, 595 proteins in the plasma membrane-enriched fraction and 538 proteins in the mitochondria-enriched fraction derived from the left ventricles. Altogether, the expression levels of 237 proteins were altered by morphine treatment or withdrawal. The majority of changes (58 proteins) occurred in the cytosol after a 3-day abstinence period. Significant alterations were found in the expression of heat shock proteins (HSP27, α-B crystallin, HSP70, HSP10 and HSP60), whose levels were markedly up-regulated after morphine treatment or withdrawal. Besides that morphine exposure up-regulated MAPK p38 (isoform CRA_b) which is a well-known up-stream mediator of phosphorylation and activation of HSP27 and α-B crystallin. Whereas there were no alterations in the levels of proteins involved in oxidative stress, several changes were determined in the levels of pro- and anti-apoptotic proteins. These data provide a complex view on quantitative changes in the cardiac proteome induced by morphine treatment or withdrawal and demonstrate great sensitivity of this organ to morphine.     

Prolonged latency to CNS-O2 toxicity induced by heat acclimation in rats is associated with increased antioxidative defenses and metabolic energy preservation

We have previously shown that heat acclimation provides protection against central nervous system oxygen toxicity (CNS-OT). This was well correlated with increased levels of heat shock protein 72 (HSP72). We now examine other antioxidative defenses against CNS-OT that are correlated with heat acclimation. Two groups of male Sprague-Dawley rats were used. The heat-acclimated group (HA) was exposed for 4 wk to 32°C, and the control group (C) was maintained at 24°C. At the end of the acclimation period, rats were exposed to oxygen at 608 kPa. EEG was recorded continuously until appearance of the first electrical discharge. Brain samples were taken from each group after exposure to pressure. Levels of the antioxidant enzymes CuZnSOD, MnSOD, catalase, and glutathione peroxidase, as well as levels of HSP72, were quantified by Western blot. Comparative proteome analysis of the brains of HA and C rats was carried out using two-dimensional electrophoresis and mass spectrometry to define protein spot alterations. Levels of HSP72 and CuZnSOD were higher in HA rats. Levels of the other antioxidant enzymes were not affected significantly by heat acclimation. Differences in the levels of four protein spots identified as α-synuclein, valosin-containing protein, adenylate kinase 1 (AK1), and the mitochondrial H+-ATP synthase α subunit were found between HA and C rats. We conclude that elevation of HSP72, CuZnSOD, AK1, and the mitochondrial H+-ATP synthase α subunit and possible phosphorylation of α-synuclein-all proteins involved in oxidative stress or energy conservation-might contribute to the prolongation of latency to CNS-OT induced by heat acclimation.