Comparison of the small heat shock proteins alphaB-crystallin, MKBP, HSP25, HSP20, and cvHSP in heart and skeletal muscle

Seven members of the small heat shock protein (sHSP) family are exceptional with respect to their constitutive high abundance in muscle tissue. It has been suggested that sHSPs displaying chaperone-like properties may stabilize myofibrillar proteins during stress conditions and prevent them from loss of function. In the present study five sHSPs (alphaB-crystallin, MKBP, HSP25, HSP20, and cvHSP) were investigated with respect to similarities and differences of their expression in heart and skeletal muscle under normal and ischemic conditions. In ischemic heart and skeletal muscle these five sHSPs translocated from cytosol to the Z-/I-area of myofibrils. Myofibrillar binding of all sHSPs was very tight and resisted for the most part extraction with 1 M NaSCN or 1 M urea. MKBP and HSP20 became extracted by 1 M NaSCN to a significant extent indicating that these two sHSPs may bind partially to actin-associated proteins which were completely extracted by this treatment. Ultrastructural localization of alphaB-crystallin showed diffuse distribution of immunogold label throughout the entire I-band in skeletal muscle fibers whereas in cardiomyocytes alphaB-crystallin was preferentially located at the N-line position of the I-band. These observations indicate different myofibrillar binding sites of alphaB-crystallin in cardiomyocytes versus skeletal muscle fibers. Further differences of the properties of sHSPs could be observed regarding fiber type distribution of sHSPs. Thus sHSPs form a complex stress-response system in striated muscle tissue with some common as well as some distinct functions in different muscle types.     

HSP70-related proteins in bovine skeletal muscle

Constitutive expression of HSP70-related proteins was detected in a variety of bovine tissues using a specific antibody. All tissues contained a 73 kilodalton protein. A lower molecular weight form (72 kilodaltons) that co-migrated on two-dimensional gels with the stressed-induced HSP70 was present in high levels in bovine skeletal muscle, but absent from rat skeletal muscle. Two-dimensional gel analysis revealed several isoforms for both the 73 and 72 kilodalton forms. Purification of HSP70-related proteins from bovine skeletal muscle, thymus gland and rat skeletal muscle demonstrated that the antibody recognized all the forms present in the tissue homogenates. The two proteins are similar but distinct as detected by one-dimensional peptide mapping. The lower molecular form was not present in fetal tissue but was detectable in newborn animals, suggesting that the levels are regulated during development.     

Influences of temperature, oxidative stress, and phosphorylation on binding of heat shock proteins in skeletal muscle fibers

Heat shock proteins (HSPs) help maintain cellular function in stressful situations, but the processes controlling their interactions with target proteins are not well defined. This study examined the binding of HSP72, HSP25, and αB-crystallin in skeletal muscle fibers following various stresses. Rat soleus (SOL) and extensor digitorum longus (EDL) muscles were subjected in vitro to heat stress or strongly fatiguing stimulation. Superficial fibers were "skinned" by microdissection and HSP diffusibility assessed from the extent of washout following 10- to 30 min exposure to a physiological intracellular solution. In fibers from nonstressed (control) SOL muscle, >80% of each HSP is readily diffusible. However, after heating a muscle to 40°C for 30 min ～95% of HSP25 and αB-crystallin becomes tightly bound at nonmembranous myofibrillar sites, whereas HSP72 bound at membranous sites only after heat treatment to ≥44°C. The ratio of reduced to oxidized cytoplasmic glutathione (GSH:GSSG) decreased approximately two- and fourfold after heating muscles to 40° and 45°C, respectively. The reducing agent dithiothreitol reversed HSP72 binding in heated muscles but had no effect on the other HSPs. Intense in vitro stimulation of SOL muscles, sufficient to elicit substantial oxidation-related loss of maximum force and approximately fourfold decrease in the GSH:GSSG ratio, had no effect on diffusibility of any of the HSPs. When skinned fibers from heat-treated muscles were bathed with additional exogenous HSP72, total binding increased approximately two- and 10-fold, respectively, in SOL and EDL fibers, possibly reflective of the relative sarco(endo)plasmic reticulum Ca(2+)-ATPase pump densities in the two fiber types. Phosphorylation at Ser59 on αB-crystallin and Ser85 on HSP25 increased with heat treatment but did not appear to determine HSP binding. The findings highlight major differences in the processes controlling binding of HSP72 and the two small HSPs. Binding was not directly related to cytoplasmic oxidative status, but oxidation of cysteine residues influenced HSP72 binding.     

HSP70 heat shock proteins and environmental stress in Antarctic marine organisms: A mini-review

The ability to understand and predict the effects of environmental stress on biodiversity is becoming increasingly important in our changing environment. Antarctic marine species are some of the most stenothermal on the planet and many inhabit the waters off the Antarctic Peninsula which is one of the areas where there is rapid regional climate change. Therefore these animals are highly vulnerable to changing environmental temperatures and clearly we need to understand the complexities of their response, not just at the individual species level, but also the implications for the ecosystem as a whole. Heat shock proteins have a long history of use in studies of organism stress responses and have frequently been proposed as potential universal molecular biomarkers, especially for non-model species. In this mini-review, the heat shock response and heat shock proteins (specifically the HSP70 family) are examined in Antarctic marine species alongside their physiological capabilities and limits to answer a series of questions: do these animals have a heat shock response which includes the expression of HSP70 genes? What is the relationship between their heat shock response and physiological capabilities? Can HSP70 genes be used as molecular biomarkers for these species?     

Accumulation of small heat shock proteins, including mitochondrial HSP22, induced by oxidative stress and adaptive response in tomato cells

Changes in gene expression, by application of H₂O₂, O₂°^–generating agents (methyl viologen, digitonin) and gamma irradiation to tomato suspension cultures, were investigated and compared to the well-described heat shock response. Two-dimensional gel protein mapping analyses gave the first indication that at least small heat shock proteins (smHSP) accumulated in response to application of H₂O₂ and gamma irradiation, but not to O₂°^– generating agents. While some proteins seemed to be induced specifically by each treatment, only part of the heat shock response was observed. On the basis of Northern hybridization experiments performed with four heterologous cDNA, corresponding to classes I–IV of pea smHSP, it could be concluded that significant amounts of class I and II smHSP mRNA are induced by H₂O₂ and by irradiation. Taken together, these results demonstrate that in plants some HSP genes are inducible by oxidative stresses, as in micro-organisms and other eukaryotic cells. HSP22, the main stress protein that accumulates following H₂O₂ action or gamma irradiation, was also purified. Sequence homology of amino terminal and internal sequences, and immunoreactivity with Chenopodium rubrum mitochondrial smHSP antibody, indicated that the protein belongs to the recently discovered class of plant mitochondrial smHSP. Heat shock or a mild H₂O₂ pretreatment was also shown to lead to plant cell protection against oxidative injury. Therefore, the synthesis of these stress proteins can be considered as an adaptive mechanism in which mitochondrial protection could be essential.     

一次性力竭运动致大鼠骨骼肌氧化应激的机制

目的:观察一次性力竭运动对大鼠骨骼肌氧化应激相关酶表达的影响。方法:雄性SD大鼠40只,分为4组(n=10),分别为对照组(C组)、力竭运动组(E组)、运动+PKC抑制剂组(EC组)、运动+NOX抑制剂组(EA组)。三组运动大鼠进行3 d的跑台适应性运动(5 m/min,1次/日,无坡度),然后休息1 d; EC组于运动前1 d和运动前1 h注射PKC抑制剂白屈菜红碱(5 mg/kg),EA组同期注射NADPH氧化酶抑制剂Apocynin(10 mg/kg),C组和E组注射同等剂量生理盐水;三组运动大鼠进行一次性跑台力竭运动,力竭后取大鼠的跖肌,DCF荧光探针检测活性氧(ROS),Western blot分析NOX2、NOX4、3-NT,免疫沉淀分析PKC、NOX2、NOX4。结果:与C组相比,E组的ROS水平、NOX2和NOX4蛋白表达、PKC-NOX2和PKC-NOX4复合物水平、3-NT生成均显著增加(P <0. 01,P <0.05),EC组、EA组ROS无显著差异(P>0.05),EC组NOX4蛋白表达显著增加(P<0.05);与E组相比,EC组和EA...     

Heat shock proteins, HSP25 and HSP70, and apoptosis in developing endocardial cushion of the mouse heart

Formation of the atrioventricular channels and valves from the endocardial cushion occurs through growth and remodeling of the initial endocardial cushion. This process requires balanced coordination of proliferation and apoptosis by still unknown factors. To detect a possible role for the heat shock proteins 25 and 70 (HSP25 and HSP70) as apoptosis-associated proteins and differentiation factors in the development of the endocardial cushion, we analyzed their temporal and regional occurrence during cell proliferation and apoptosis in E11-E17 embryos. The distribution and timing of these events and factors were consistent with the hypothesis that HSP25 is related to myocardial development whereas HSP70 is related to differentiation of the endocardial cushion by cell proliferation and apoptosis.     

Small heat shock proteins are differentially regulated during pollen development and following heat stress in tobacco

In plants small heat shock proteins (sHsp) are abundantly expressed upon heat stress in vegetative tissue, however, sHsp expression is also developmentally induced in pollen. The developmental induction of sHsp has been related to the potential for stress-induced microspore embryogenesis. We investigated the polymorphism among sHsp and their expression during pollen development and after heat stress in tobacco. Real-time RT-PCR was used for quantification of mRNA of two known and nine newly isolated cDNAs representing cytosolic sHsp. At normal temperature most of these genes are not transcribed in vegetative tissues, however, all genes were expressed during pollen development. Low levels of mRNAs were found for sHsp-1A and -1B in early-unicellular stage, increasing four to sevenfold in mature pollen. Nine other genes are up-regulated in unicellular and down-regulated in bicellular pollen; three these genes show stage-specific expression. Western analysis revealed that cytosolic class I and II sHsp are developmentally expressed during all stages of pollen development. Different subsets of cytosolic sHsp genes are expressed in a stage-specific fashion suggesting that certain sHsp genes may play specific roles in early, others during later stages of pollen development. Heat stress results in a relatively weak and incomplete response in pollen: (i) the heat-induced levels of mRNA (excepting sHsp-2B, −3Cand -6) are much lower than in leaves, (ii) several sHsp are not detected after heat stress in pollen, although, they are heat-inducibly expressed in leaves. Application of heat stress, cold, and starvation, which induce microspore embryogenesis, modify mRNA levels and the patterns of 2-D-separated sHsp, but only heat stress enhances the expression of sHsp in microspores. There is no correlation of the expression of specific sHsp with the potential for microspore embryogenesis.Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Taurine supplementation decreases oxidative stress in skeletal muscle after eccentric exercise

Infrequent exercise, typically involving eccentric actions, has been shown to cause oxidative stress and to damage muscle tissue. High taurine levels are present in skeletal muscle and may play a role in cellular defences against free radical‐mediated damage. This study investigates the effects of taurine supplementation on oxidative stress biomarkers after eccentric exercise (EE). Twenty‐four male rats were divided into the following groups (n = 6): control; EE; EE plus taurine (EE + Taurine); EE plus saline (EE + Saline). Taurine was administered as a 1‐ml 300 mg kg^?1 per body weight (BW) day^?1 solution in water by gavage, for 15 consecutive days. Starting on the 14th day of supplementation, the animals were submitted to one 90‐min downhill run session and constant velocity of 1·0 km h^?1. Forty‐eight hours after the exercise session, the animals were killed and the quadriceps muscles were surgically removed. Production of superoxide anion, creatine kinase (CK) levels, lipoperoxidation, carbonylation, total thiol content and antioxidant enzyme were analysed. Taurine supplementation was found to decrease superoxide radical production, CK, lipoperoxidation and carbonylation levels and increased total thiol content in skeletal muscle, but it did not affect antioxidant enzyme activity after EE. The present study suggests that taurine affects skeletal muscle contraction by decreasing oxidative stress, in association with decreased superoxide radical production. Copyright © 2010 John Wiley & Sons, Ltd.     

HSP70 expression in the CNS in response to exercise and heat stress in rats

We havepreviously documented the regional distribution of 70-kDa heat shockprotein (HSP70) in brains of rats made hyperthermic by brief exposureto high-powered microwaves (HPM; 2.06 GHz). We now compare HSP70expression induced by HPM exposure to that induced by exertionaland/or environmental heat stress. Rats were chronicallyimplanted with a temperature probe guide in the hypothalamic region ofthe brain (T_br). After recovery,the following treatment groups were examined: HPM; sham exposed;treadmill exercise at room temperature (24°C; Ex-1); treadmillexercise in a warm environment (34°C; Ex-2); and sedentary groups(Sed-1 and Sed-2), in which ambient temperature was adjusted so thatthe T_br mimicked the T_br in the corresponding exercisegroups. Significant HSP70 expression occurred only in the hyperthermic(Ex-2, Sed-2, and HPM) groups. The pattern of HSP70 expression wassimilar among Ex-2 and Sed-2 rats but differed from that in HPM rats.We conclude that 1) the pattern ofHSP70 expression differs between HPM and nonmicrowave heating, and2) exercise alone was not sufficientto induce central HSP70 expression.

     

Overexpression of inducible 70-kDa heat shock protein in mouse attenuates skeletal muscle damage induced by cryolesioning

Heat shock protein expression is elevated upon exposure to a variety of stresses and limits the extent of stress-induced damage. To investigate the putative role of inducible 70-kDa heat shock protein (HSP70) in skeletal muscle damage and regeneration, soleus and tibialis anterior (TA) muscles from HSP70-overexpressing transgenic mice were subjected to cryolesioning and analyzed after 1, 10, and 21 days. Histological analysis showed that the muscles from both HSP70 and wild-type mice treated with radicicol (a HSP inducer) had decreased necrosis after cryolesioning compared with controls. The decrease in muscle fiber cross-sectional area in both soleus and TA muscles in 10 days postlesioning was attenuated in HSP70 mice compared with wild-type mice. Glutathione peroxidase activity was increased 1 day after cryolesioning in both HSP70 and control mice and remained elevated for up to 21 days. Immunodetection of neuronal cell adhesion molecule (a satellite cell marker) and developmental/neonatal MHC were significantly lower in cryolesioned HSP70-overexpressing mice than in cryolesioned controls. These results suggest that HSP70 protects skeletal muscle against injury and radicicol might be useful as a skeletal muscle protective agent. regeneration; radicicol; transgenic mouse; myoprotection     

Age-related differences in skeletal muscle insulin signaling: the role of stress kinases and heat shock proteins

Aging is associated with an increase in insulin resistance in skeletal muscle, yet the underlying mechanism is not well established. We hypothesize that with aging, a chronic increase in stress kinase activation, coupled with a decrease in oxidative capacity, leads to insulin resistance in skeletal muscle. In aged (24 mo old) and young (3 mo old) Fischer 344 rats, 2-deoxyglucose uptake and insulin signaling [as measured by phosphorylation of insulin receptor substrate-1 (IRS-1), Akt (protein kinase B), and Akt substrate of 160 kDa (AS160)] decreased significantly with age. Activation of, c-Jun NH(2)-terminal kinase (JNK), glycogen serine kinase-3beta (GSK-3beta), and degradation of IkappaBalpha by the upstream inhibitor of kappa B kinase (IKKbeta), as measured by Western blot analysis, were increased with age in both soleus and epitrochlearis (Epi) muscles. However, much higher activation of these kinases in Epi muscles from young rats compared with soleus results in a greater effect of these kinases on insulin signaling in fast-twitch muscle with age. Heat shock protein (HSP) 72 expression and phosphorylation of HSP25 were higher in soleus compared with Epi muscles, and both parameters decreased with age. Age and fiber type differences in cytochrome oxidase activity are consistent with observed changes in HSP expression and activation. Our results demonstrate a significant difference in the ability of slow-twitch and fast-twitch muscles to respond to insulin and regulate glucose with age. A greater constitutive HSP expression and lower stress kinase activation may account for the ability of slow-twitch muscles to preserve the capacity to respond to insulin and maintain glucose homeostasis with age.     

HSP72 as a complementary protection against oxidative stress induced by exercise in the soleus muscle of rats

Given the potential of reactive oxygen species to damage intracellular proteins during subsequent bouts of muscle contractions, it was suggested that, when this production exceeds the antioxidant capacity, the preexisting antioxidant pathways may be complemented by the synthesis of the defense mechanism represented by heat shock proteins (HSPs), stress proteins with the function of repair and maintaining protein folding. To test this hypothesis, we analyzed reactive carbonyl derivatives in plasma and the expression of HSP72 and activities of enzymes from the oxidative and antioxidant defense systems in the soleus muscle of sedentary rats and rats trained by two protocols: continuous and intermittent. We analyzed all three groups at rest and 2 h after acute exercise. After 8 wk of training, the animals from both groups clearly demonstrated higher resistance to exercise. Both trained groups showed significantly higher citrate synthase, catalase, and glutathione reductase activities than the control group (P < 0.01). After acute exercise, catalase and glutathione reductase activities significantly decreased (P < 0.01) and plasma reactive carbonyl derivatives significantly increased (P < 0.05) in the sedentary group, suggesting an oxidative-stress condition as responsible for exhaustion in this group. Finally, after acute exercise, the induction of HSP72 expression occurred only in the sedentary group, suggesting that HSP72 acts as a complementary protective mechanism in exercise-induced oxidative stress.     

Carboxy terminus of heat shock protein (HSP) 70-interacting protein (CHIP) inhibits HSP70 in the heart

Heat shock protein (HSP) 70 plays a critical role in protecting the heart from various stressor-induced cell injuries; the mechanism remains to be further understood. The present study aims to elucidate the effect of a probiotics-derived protein, LGG-derived protein p75 (LGP), in alleviating the ischemia/reperfusion (I/R)-induced heart injury. We treated rats with the I/R with or without preadministration with LGP. The levels of HSP70 and carboxy terminus of HSP70-interacting protein (CHIP) in the heart tissue were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The effect of CHIP on suppression of HSP70 and the effect of LGP on suppression of CHIP were investigated with an I/R rat model and a cell culture model. The results showed that I/R-induced infarction in the heart could be alleviated by pretreatment with LGP. HSP70 was detected in na?ve rat heart tissue extracts. I/R treatment significantly suppressed the level of HSP70 and increased the levels of CHIP in the heart. A complex of CHIP/HSP70 was detected in heart tissue extracts. The addition of recombinant CHIP to culture inhibited HSP70 in heart cells. LGP was bound CHIP in heart cells and prevented the CHIP from binding HSP70. In summary, I/R can suppress HSP70 and increase CHIP in heart cells. CHIP can suppress HSP70 that can be prevented by pretreatment with LGP. The results imply that CHIP may be a potential target in the prevention of I/R-induced heart cell injury.