Skeletal muscle HSP72 and norepinephrine response to static magnetic field in rat

Summary. The present work was undertaken in order to investigate the noradrenergic system and skeletal muscle heat shock protein 72 (HSP72) response to static magnetic field (MF) in male rats. At thermoneutrality (25°C), the exposition of rats 1 hour/day for 5 consecutive days to MF of 128 mT (m tesla) induced an increase in norepinephrine content in gastrocnemius muscle (+25%, p < 0.05) but had no effect at 67 mT (+1%, p > 0.05), indicating a stimulatory effect of sub-acute MF exposure on the noradrenergic system activity. Moreover, exposed rats to MF displayed a non-significant increase of HSP72 levels in gastrocnemius muscles (+29%, p > 0.05). The results indicate that noradrenergic systems in rat’s gastrocnemius muscles are affected by MF exposure. Interestingly, sub-acute exposure insufficiency increased HSP72 levels in gastrocnemius muscles.     

Differential responses of L5 and rat primary muscle cells to factors in rat brain extract

Crude brain extract (100,000 g supernate from newborn or fetal rat brain homogenate) was studied for its effects on the number and distribution of acetylcholine receptors (AChRs) on myotubes of the L5 cloned myogenic cell line and compared to that of rat primary cultures. Gamma counting, light autoradiography and scanning electron microscopic autoradiography were used. We found that the L5 cells responded to the brain extract with an increase in the average AChR site density (2-5-fold) and with an increase in AChR clustering. Clustering was manifested by both an increase in the number of AChR clusters and in the ratio of receptor site density within clusters relative to that between clusters. The increase in average AChR site density was shown to be due to an increase in the rate of AChR insertion into the surface membrane with little change in the rate of receptor degradation. As also previously reported, the rat myotubes had a similar clustering response but only a very slight (approximately 1.2-fold) increase in average AChR site density. The surface area of myotubes was also increased slightly (approximately 1.2-1.3-fold) by the brain extract. Autoradiography viewed by scanning EM was found to be very useful in illustrating the shape and distribution of the receptor clusters. After the brain extract was fractionated on Sephadex G-200, the fractions with greatest clustering activity could be separated from those causing predominantly an increase in receptor site density. Increased receptor site density was primarily produced by the low molecular weight fractions (less than 12 kD), whereas the strongest (but not exclusive) effect on clustering was produced by the high molecular weight fractions (greater than 140 kD). Furthermore, the two cell types assayed had different sensitivity to the different factors. L5 cells responded to both the high and low molecular weight factors while rat primary cells are sensitive primarily to the high molecular weight factors.     

Distribution of motor nerve sproutings in the mouse gastrocnemius muscle after partial denervation

The distribution and frequency of motor nerve sproutings in the mouse gastrocnemius muscle were examined after a partial denervation. Terminal and nodal sproutings could be seen for a period of 7–28 days after transecting one of the two muscular nerves in the medial head. They grew out not only from the endplates and terminal nerves,but also from the preterminal nerve regions of both the intra-and extramuscular nerve. The terminal sproutings originated from endplates close to the denervated portion and subsequently they were seen farther away in the medial head. Upon regeneration of the originally transected nerve the terminal sproutings were withdrawn in the reverse sequence, i.e., first from the region farthest from the denervated area and last from the region adjacent to the denervation. The frequency of terminal sproutings was constant in the area immediately bordering the denervated portion (11–15% of the total number of enplates). In other areas more distant from the denervated portion, however, it showed a gradient which varied with the time course. The higher the frequency of terminal sproutings, the more actively each endplate prroduced two of three terminal sproutings without significant difference in length.     

Induction of heat shock (stress) protein 70 and its mRNA in the normal and light-damaged rat retina after whole body hyperthermia

In situ hybridization and immunocytochemistry were used to investigate the distribution of the 70 kDa heat shock or stress protein (hsp70) and its mRNA in specific layers of the retina of adult rats at 0, 4, 18, and 48 or 50 hr after a brief whole body hyperthermic treatment. Induction of hsp70 mRNA was noted in the photoreceptor layer of the retina within 4 hr after hyperthermia. Pronounced accumulation of inducible hsp70 immunoreactivity was observed in cytoplasmic extensions of the photoreceptor cells, especially the inner segment zone which attained peak levels at the 18 hr time point. Selective destruction of photoreceptors by light damage prior to hyperthermia inhibited the post-hyperthermic rise in newly synthesized retinal hsp70. Our results suggest that the photoreceptor cell layer is the primary site of synthesis of hsp70 in the rat retina and that the greatest increase in hsp70 immunoreactivity following such a hyperthermic stress occurs in that layer. This stress response of the photoreceptors is discussed in relation to their location and function in the retina. © 1994 Wiley-Liss, Inc.     

Hippocampal 72-kDa heat shock protein expression varies according to mice learning performance independently from chronic exposure to stress

The possibility that the inducible 72-kDa heat shock protein (hsp72) is involved in learning-related plasticity mechanisms was investigated in two inbred strains of mice that show spontaneous differences in spatial learning performance as well as an opposite reactivity to stress. Induction of hsp72 after radial maze training was measured by immunoblotting in the hippocampus of C57BL/6 (C57) and DBA/2 (DBA) inbred mice exposed or nonexposed to chronic acoustic stress. In agreement with previous studies, inter-strain differences in radial maze performance were found in nonstressed mice with C57 mice showing the higher scores. Chronic acoustic stress, however, impaired performance in the high-learner C57 strain and improved performance in the low-learner DBA strain. Western blot analysis revealed that post-training expression of hsp72 was low in the condition each strain was showing the higher-performance (nonstressed C57 and stressed DBA) and high in the condition each strain was showing the lower performance (stressed C57 and nonstressed DBA). These findings indicate that expression of hsp72 in the hippocampus varies as a function of the learning performance independently from exposure to chronic acoustic stress.     

Distinct patterns of plasticity in prefrontal cortex neurons that encode slow and fast responses to stress

The prefrontal cortex (PFC) has been implicated in cognitive and affective responses to acute and chronic stress; however, direct evidence for the reactivity or adaptability of PFC neurons to stress is lacking. We followed the unit activity of medial PFC (mPFC) neurons in awake rats during two consecutive exposures to restraint stress or to a non-aversive novel object. The majority (75%) of mPFC neurons had significant responses to the initial restraint that was differentiated into one of three temporal patterns: (i) phasic increase in firing rate during the restraint period, (ii) slow onset increase in firing rate that was sustained for > 2 h after restraint, and (iii) brief bi-phasic responses to initiation and termination of restraint. Exposure to a novel object elicited an exposure-locked phasic response in 40% of the neurons. None of the neurons displayed the sustained activation that was prominent after restraint. A second exposure to the object no longer elicited this phasic response while neurons in the three restraint-responsive groups modified their firing rate during the second restraint in a manner that was specific to their pattern of response to the first restraint. These findings demonstrate that whereas some mPFC neurons respond phasically to novel stimuli irrespective of their aversive nature, a separate population of PFC neurons responds to a stressful stimulus with a sustained increase in firing rate that persists in the absence of that stimulus. These neurons may be a substrate for adaptive responses that are necessary for behavioral modification.     

Statin-associated changes in skeletal muscle function and stress response after novel or accustomed exercise

Introduction: The most common side effect of statins, myopathy, is more likely in exercisers. We investigated the interaction of statin treatment with novel vs. accustomed exercise on muscle function, heat shock protein (Hsp) expression, and caspase activation. Methods: Mice received daily cerivastatin or saline for 2 weeks, with/without wheel running (RW) (novel/sedentary). Accustomed groups completed 2 weeks of RW before statins. At 4 weeks, plantarflexor isometric force, Hsp25, αB‐crystallin, caspase‐3 and ‐9, and plasma creatine kinase (CK) were quantified. Results: Statins reduced force in sedentary and novel groups, compared with saline, by 15% and 27%, respectively. Muscle fatigability increased 21% and 30% with statins compared with saline in sedentary and novel groups, respectively. Accustomed exercise prevented statin‐associated force loss and increased fatigability. CK did not correlate with functional outcomes. RW increased Hsp protein in all groups. Conclusion: Our results suggest that exercise prior to statin treatment can protect against decrements in muscle function. Muscle Nerve 2011     

Partial transformation from fast to slow muscle fibers induced by deafferentation of capsaicin-sensitive muscle afferents

Mechanical and histochemical characteristics of the lateral gastrocnemius (LG) muscle of the rat were examined 21 days after capsaicin injection into the LG muscle. The capsaicin caused a decrease in generation rate of twitch and tetanic tension and an increase in fatigue resistance of LG muscle. The histochemical muscle fiber profile evaluated by myosin adenosine triphosphatase and reduced nicotinamide adenine dinucleotide tetrazolium reductase methods showed an increase of type I and IIC fibers and a decrease of the type IIB in whole muscle, and a decrease of the IIA, IIX fibers in the red part accompanied by their increase in the white part. Therefore the capsaicin treatment, which selectively eliminated fibers belonging to the III and IV groups of muscle afferents, induced muscle fiber transformation from fast contracting fatiguing fibers to slowly contracting nonfatiguing ones. © 1997 John Wiley & Sons, Inc. Muscle Nerve 20: 1404–1413, 1997     

Transcriptional malfunctioning of heat shock protein gene expression in spinocerebellar ataxias

Among the various dominantly-inherited spinocerebellar ataxias (SCAs), at least seven of them belong to the polyglutamine disease group and are caused by glutamine-coding CAG triplet repeat expansion. The expanded coding CAG repeat translates into a polyglutamine stretch in the disease protein, which leads to late-onset and progressive neurodegeneration. Expanded polyglutamine adopts a misfolded protein conformation, and is itself a cellular stressor which induces robust heat shock response (HSR). Under polyglutamine stress, heat shock proteins (Hsps) are produced in neurons to assist refolding and/or promote the degradation of misfolded proteins. Along with the progressive nature of polyglutamine degeneration, a gradual decline of HSR in degenerating neurons was observed. Such kind of reduction can be observed in a large family of hsp gene expression, including hsp22, 26, 27, and 70. This underscores an intimate relationship between the inducibility of hsp gene expression and the disease progression. In this review, we describe the current understandings of hsp gene dysregulation in polyglutamine disease.     

Temporomandibular joint-evoked responses by spinomedullary neurons and masseter muscle are enhanced after repeated psychophysical stress

Psychological stress is a risk factor for the development of musculoskeletal pain of the head and neck; however, the basis for this relationship remains uncertain. This study tested the hypothesis that psychophysical stress alone was sufficient to alter the encoding properties of spinomedullary dorsal horn neurons and masseter muscle activity in male rats. Repeated forced swim conditioning increased markedly both the background firing rate and temporomandibular joint (TMJ)-evoked activity of neurons in deep dorsal horn, while neurons in superficial laminae were less affected. Stress also increased the responses to stimulation of facial skin overlying the TMJ of neurons in deep and superficial dorsal horn. TMJ-evoked masseter muscle activity was enhanced significantly in stressed rats, an effect that was reduced by prior blockade of the spinomedullary junction region. These data indicated that repeated psychophysical stress induced widespread effects on the properties of medullary dorsal horn neurons and masseter muscle activity. The effects of stress were seen preferentially on neurons in deep dorsal horn and included enhanced responses to chemosensory input from the TMJ and mechanical input from overlying facial skin. The stress-induced elevation in TMJ-evoked masseter muscle activity matched well with the changes seen in dorsal horn neurons. It is concluded that the spinomedullary junction region plays a critical role in the integration of psychophysical stress and sensory information relevant for nociception involving deep craniofacial tissues.     

Differential response of heat shock proteins to hindlimb unloading and reloading in the soleus

Hindlimb unloading (HU) results in oxidative stress, skeletal muscle atrophy, and increased damage upon reloading. Heat shock proteins (HSPs) protect against oxidative stress. However, it is unknown whether HSPs are depressed with long-term unloading (28 days) or reloading. We tested the hypotheses that long-term HU would depress Hsp70 and Hsp25 pathways, whereas reloading would allow recovery in the soleus. Adult Sprague-Dawley rats were divided into three groups: controls; HU for 28 days; and HU + 7 days of reloading (HU-R). Soleus mass decreased with HU, and did not recover to control values with reloading. Hsp70 decreased with HU (-78.5%) and did not recover with HU-R (-81.4%). Upstream heat shock factor-1 was depressed with HU and HU-R. Hsp25 was reduced with HU, but recovered with reloading. Downstream of Hsp25, NADP-specific isocitrate dehydrogenase and glutathione peroxidase decreased with unloading, but only NADP-specific isocitrate dehydrogenase recovered with HU-R. Lipid peroxidation increased in both HU and HU-R. These data indicate that prolonged unloading and subsequent reloading results in complex, differential regulation of Hsp70 and Hsp25 pathways in the rat soleus muscle. Thus dysregulation and uncoupling of the Hsp70 and Hsp25 pathways may lead not only to muscle atrophy with prolonged unloading, but also impaired recovery of muscle mass during early reloading.     

Differential expression and induction of small heat shock proteins in rat brain and cultured hippocampal neurons

The so-called stress response involving up-regulation of heat shock proteins (Hsps) is a powerful mechanism of cells to deal with harmful conditions to which they are exposed throughout life, such as hyperthermia, hypoxia, or oxidative stress. Some members of the group of small Hsps (sHsps) seem to play a neuroprotective role in the brain. Here we analyzed the expression of all 11 sHsps in the rat brain by using RNA in situ hybridization and quantitative real-time RT-PCR. Additionally, we investigated sHsps in cultured neurons exposed to heat shock. We found seven sHsps to be expressed in the rat brain, with HspB5 (αB-crystallin), HspB6 (Hsp20), and HspB11 (Hsp16.2) showing the highest expression levels (4-24% of reference genes) followed by HspB1 (Hsp25) and HspB8 (Hsp22; 0.1-2% of reference genes), all being widely expressed in the brain areas investigated. HspB2 (MKBP) and HspB3, however, showed selective expression in only some regions (B2: cortex and hippocampus, B3: cortex and cerebellum). Whereas HspB5 was expressed mainly in the white matter, HspB6 showed the greatest expression in the cerebellar cortex, and HspB11 was widely distributed over the whole brain. In cultured hippocampal neurons, heat shock led to an increase of HspB1 and HspB8 mRNA and additionally HspB5 protein. Our data indicate that the sHsps induced by heat shock, HspB1, B5, and B8, might be especially involved in neuroprotection under stress conditions. The other sHsps showing constant neuronal expression may play a constitutive role or may be up-regulated and important in types of stresses other than heat shock.     

阿尔茨海默病大鼠海马热休克蛋白70表达变化的研究

目的:双侧海马注射Aβ1-42建立大鼠阿尔茨海默病(AD)模型,研究其海马热休克蛋白70(HSP70)表达的变化并探讨与AD病理进程可能存在的联系。方法:48只SD大鼠随机分为模型组与生理盐水对照组,双侧海马分别注射Aβ1-42,分别于术后1天、7天、14天、21天在Y迷宫行为学测试后处死大鼠,采用RT-PCR和Western-blot方法,观察各组大鼠海马HSP70表达的变化。结果:术后14天、21天模型组学习记忆能力较对照组和术前明显减退(P<0.05)。术后模型组大鼠海马HSP70表达逐渐减少,术后7天(mRNA:0.34±0.05;蛋白:0.29±0.03)、14天(mRNA:0.32±0.06;蛋白:0.23±0.04)与对照组比较差异有统计学意义(P<0.05),至术后21天,模型组HSP70表达减少最为明显(mRNA:0.29±0.04;蛋白:0.11±0.03)(P<0.01)。结论:大鼠海马注射Aβ1-42导致HSP70表达减少,HSP70表达减少可能参与了AD的病理发展过程。     

Differential effects of NT-3 on reinnervation of the fast extensor digitorum longus (EDL) and the slow soleus muscle of rat

Previous studies of gastrocnemius muscle reinnervation showed specific normalization of the proportion and diameter of fast type 2b muscle fibres following NT-3 delivery to the proximal stump of the cut sciatic nerve. Here, we investigate if normalization was related to greater improvement of muscle reinnervation of fast (extensor digitorum longus; EDL) than slow (soleus) motor units. NT-3-impregnated (NT-3 group) or plain fibronectin (FN group) mats were inserted into a sciatic nerve gap. Neuromuscular junctions (NMJs) labelled with TRITC-alpha-bungarotoxin were colabelled with calcitonin gene-related peptide (CGRP) or 4E2 antisera and imaged using confocal microscopy. CGRP and 4E2 were used as markers for newly reinnervated and structurally mature NMJs, respectively. At 40 days postsurgery, denervated NMJs in EDL and soleus muscles of both groups presented a 50% decrease of surface area due to decreased width. At day 80 in EDL, more NMJs were reinnervated by CGRP-immunoreactive terminals in the NT-3 (7.1%) than in the FN group (4.2%); there was no difference between groups for soleus. At 120 days, 4E2-immunoreactive NMJs were more numerous in EDL of the NT-3 (40.0%) than in the FN group (7.3%), unlike in soleus (NT-3, 1. 6%; FN, 1.8%), and presented a partial size recovery. These results indicate that NT-3 preferentially improves reinnervation of fast muscles over slow muscle, although the mechanism of this improvement is still unclear.     

Localization of ingensin in rat central nervous system and skeletal muscle

A high molecular weight, fatty acid- and SDS-sensitive protease named ingensin was purified from rat brain in this study. The enzyme purified from rat brain has the same biochemical properties as those purified from other tissues, e.g., porcine skeletal muscle, human placenta, and rat liver in our laboratory, and rat skeletal muscle and bovine pituitary gland in other laboratories, independently. Immunoblot bands were detected in the same positions as those in the case of ingensin from rat liver. In addition, its topographical distribution was studied in rat brain and muscle by means of the immunohistochemical method. The cytoplasm of motor neurons of the spinal cord, pyramidal cells, and granular cells of the hippocampus, Purkinje cells, and glial cells were stained. Axons were stained. The cytoplasm of muscle was also stained, especially that of type 2 fibers.     

缺血预处理模型大鼠心肌组织小分子热休克蛋白的表达*

背景：小分子热休克蛋白,尤其是热休克蛋白27和α-B晶体蛋白高表达量对于心肌组织缺血再灌注的保护作用已比较明确。但是在老龄大鼠中小分子热休克蛋白是否仍具有这种保护作用迄今仍无报道。 目的：观察热休克蛋白27和α-B晶体蛋白在青年及老年大鼠心肌缺血预处理时表达的变化。 方法：24月龄老龄大鼠及两三月龄青年大鼠在体心脏经缺血5 min、再灌注5 min(反复3次)预处理后,于0,5,15,45,60 min取左心室缺血的前壁与非缺血的后壁心肌组织分别匀浆,分离上清及沉淀蛋白,用Western Blot检测热休克蛋白27和α-B晶体蛋白在预处理不同时间可溶性蛋白及不溶性蛋白含量的改变;采用RT-PCR方法观察缺血预处理对青年及老龄大鼠热休克蛋白27和α-B晶体蛋白mRNA在不同时间点表达的改变情况,采用免疫荧光观察缺血预处理后不同时间点热休克蛋白27和α-B晶体蛋白的移位情况。 结果与结论：老龄大鼠在缺血预处理后热休克蛋白27和α-B 晶体蛋白表达量增加,表明老龄大鼠仍具有正常的基因转录及蛋白合成的能力;与青年大鼠比较,老龄大鼠缺血预处理后小分子热休克蛋白移位能力降低,从而减弱了与各相应蛋白结合的能力,限制其保护作用的发挥。提示老年大鼠心肌组织中小分子热休克蛋白丧失移位能力,可能是老年大鼠缺血预处理保护作用减弱的重要原因之一。     

Contrast of time courses of changes in muscular potentials to prolonged stimulation at 5 Hz in rat medial gastrocnemius and soleus muscles

Time courses of changes in muscular potentials to repetitive stimulation at 5 Hz for 10 min were compared between rat medial gastrocnemius (MG) and soleus (SOL) muscles. Stimuli were applied to a sciatic nerve near the entrance of the MG and lateral gastrocnemius (LG) muscles and its exit segment from the LG to the SOL muscles. Muscular potentials were generally evoked in the form of a biphasic wave at the MG muscle and were always of a simple biphasic pattern at the SOL muscle. It was found that, due to prolonged stimulation, muscular potentials were rapidly and notably reduced in the MG muscle, whereas they were gradually facilitated in the SOL muscle. This contrast was similar to differences in the time course of changes in muscular potentials under these conditions in the MG muscle of dystrophic and littermate normal mice.     

Neuroprotective effect of heat shock protein 70 Inhibition of Tau protein expression

BACKGROUND： Previous studies have shown that heat shock protein 70 （HSP70） has neuroprotective effects by decreasing phosphorylation of Tau protein, thereby reducing the expression of Tau protein and proper aggregation. OBJECTIVE： To observe and verify expressional changes of HSP70 and Tau in retinal ganglion cells following stretch injury to the right optic nerve in rats, and to determine the effect of heat stress pretreatment on HSP70 and Tau protein expressions. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Laboratory of Neurology Research Institute of the First Affiliated Hospital of Chongqing Medical University from March to June 2006. MATERIALS： Instant SABC immunohistochemistry kit, as well as mouse anti-HSP70 and rabbit anti-Tau polyclonal antibodies, were purchased from Wuhan Boster Bioengineering Limited, China. METHODS： A total of 57 male, Wistar rats were randomly assigned to 4 groups： control （n = 3）; 150-180 g stretch force was induced in the right optic nerves in stretch-only group （n = 18） to establish optic nerve stretch injury model; heat stress was applied to 18 animals in heat-stress treatment group; 18 rats in the heat-stress pretreatment plus stretch group were subjected to identical stretch injury as stretch-only group after 24-hour heat-stress pretreatment. According to sacrifice time, the groups were assigned to 6 subgroups at different time points of 4, 8, and 16 hours, and 1,3, and 5 days, with 3 rats in each subgroup. No treatment was performed in the control group except anesthesia. MAIN OUTCOME MEASURES： Morphological changes of optic nerves and retinal ganglion cells following stretch injury were observed by light microscopy following hematoxylin-eosin staining. HSP70 and Tau protein expression levels were observed in retinal ganglial cells from each group using im munohistochemistry. RESULTS： （1） Compared with the control group, morphological axonal and retinal ganglial cell changes, as well as a decreased number of retinal ganglial cells, were identified in the stretch-only group （P 〈 0.01）. Pathological damage in optical nerve and retinal ganglial cells were not remarkable in the heat-stress pretreatment plus stretch group, with no statistical difference in the number of retinal ganglial cells compared with the control group （P 〉 0.05）. （2） Compared with the control group significantly increased HSP70 expression in retinal ganglial cells occurred in the stretch-only, heat-stress treatment, and heat-stress pretreatment plus stretch groups （P 〈 0.05 or P 〈 0.01 ）. The peak of HSP70 expression was earlier in the heat-stress pretreatment plus stretch group compared with the stretch-only and heat-stress treatment groups, and was expressed over a longer period of time compared with the heat-stress treatment group. Compared with the control group, Tau expression in the retinal ganglial cells rapidly increased 4-16 hours following stretch injury in the stretch-only group （P 〈 0.05 or P 〈 0.01）, and obviously decreased in the heat-stress pretreatment plus stretch group （P 〈 0.05 or P 〈 0.01 ）. CONCLUSION： Tau expression increased following stretch injury, with an earlier expression peak than HSP70, which indicated that stretch injury-induced HSP70 expression was not strong or quick enough to sufficiently protect the nerve. A much more enhanced HSP70 expression, with an earlier peak and longer expression period, was observed in rats subjected to stretch injury following heat stress, which demonstrated that HSP70 exhibited neuroprotective functions by reducing abnormal aggregation of Tau.