Heat shock pretreatment enhances porcine myoblasts survival after autotransplantation in intact skeletal muscle

Myoblast transplantation (MT) is a cell-based gene therapy treatment, representing a potential treatment for Duchenne muscular dystrophy (DMD), cardiac failure and muscle trauma. The rapid and massive death of transplanted cells after MT is considered as a major hurdle which limits the efficacy of MT treatment. Heat shock proteins (HSPs) are overexpressed when cells undergo various insults. HSPs have been described to protect cells in vivo and in vitro against diverse insults. The aim of our study is to investigate whether HSP overexpression could increase myoblast survival after autotransplantation in pig intact skeletal muscle. HSP expression was induced by warming the cells at 42°C for 1 h. HSP70 expression was quantified by Western blot and flow cytometry 24 h after the treatment. To investigate the myogenic characteristics of myoblasts, desmin and CD56 were analysed by Western blot and flow cytometry; and the fusion index was measured. We also quantified cell survival after autologous transplantation in pig intact skeletal muscle and followed cell integration. Results showed that heat shock treatment of myoblasts induced a significative overexpression of the HSP70 (P < 0.01) without loss of their myogenic characteristics as assessed by FACS and fusion index. In vivo (n=7), the myoblast survival rate was not significantly different at 24 h between heat shock treated and nontreated cells (67.69% ± 8.35% versus 58.79% ± 8.35%, P > 0.05). However, the myoblast survival rate in the heat shocked cells increased by twofold at 48 h (53.32% ± 8.22% versus 28.27% ± 6.32%, P < 0.01) and more than threefold at 120 h (26.33% ± 5.54% versus 8.79% ± 2.51%, P < 0.01). Histological analysis showed the presence of non-heat shocked and heat shocked donor myoblasts fused with host myoblasts. These results suggested that heat shock pretreatment increased the HSP70 expression in porcine myoblasts, and improved the survival rate after autologous transplantation. Therefore, heat shock pretreatment of myoblast in vitro is a simple and effective way to enhance cell survival after transplantation in pig. It might represent a potential method to overcome the limitations of MT treatment.     

Heat shock proteins of chicken lens

The presence of heat shock proteins HSP-40, HSP-70, and HSc-70 in adult and embryonic chicken lenses were determined. The epithelium, cortex, and nucleus of adult chicken lens were separated and tested for the presence of heat shock proteins (hsps) by western blot, using specific antibodies for HSP-40, HSP-70, and HSc-70. Water soluble (WSF) and water insoluble fractions (WIF) of embryonic chicken lenses were isolated and tested for the presence of HSP-40, HSP-70, and HSc-70 by immunoblot. Embryonic chicken lens sections were also analyzed for the presence of heat shock proteins by immunofluorescence technique. Data obtained from these experiments revealed that HSP-40, HSP-70, and HSc-70 are present in all areas of both adult and embryonic chicken lens. Presence of hsps protein in the deep cortex and nucleus is intriguing as no detectable metabolic activities are reported in this area. However it can be proposed that hsps HSP-40, HSP-70, and HSc-70 can interact with protein of these areas and protect them from stress induced denaturation.     

人肝癌细胞BEL-7402中热休克蛋白70相伴甲胎蛋白的实验研究

为研究人肝癌细胞BEL-7402中热休克蛋白70(HSP70)与甲胎蛋白(AFP)的相互作用,采用免疫化学和免疫荧光检测HSP70和AFP在肝癌细胞中的表达和定位.HSP70与AFP的相互关系通过免疫共沉淀和蛋白印迹杂交进行分析.结果免疫化学显示人肝癌细胞BEL-7402中存在高水平的HSP70和AFP共表达,均定位于细胞浆.AFP存在于HSP70单抗的免疫沉淀中,而HSP70则存在于AFP单抗的免疫沉淀中.结果表明人肝癌细胞BEL-7402中HSP70与AFP相伴.两者之间的相互关系研究将成为探讨肝癌的发生和免疫治疗的新途径.     

Glycosphingolipids during skeletal muscle cell differentiation: Comparison of normal and fusion-defective myoblasts

The regulation of glycosphingolipid (GSL) synthesis in culture by fusion-competent (E63) myoblasts and fusion-defective (fu-1) cells was examined. Upon reaching confluency E63 cells fused to form multinucleated myotubes and demonstrated many characteristics of developing skeletal muscle including induction of creatine kinase activity and a shift in creatine kinase isozymes to the MM isoform. The fu-1 cells displayed none of these characteristics, despite the fact that both cells were cloned from the same parental myoblast line (rat L8). There was a transient increase in the synthesis of total neutral GSLs by E63 cells at the time of membrane fusion. In contrast, neutral GSL synthesis by fu-1 cells gradually decreased with time in culture. The major GSLs synthesized by both cell types were lactosylceramide and ganghoside GM3, with more complex structures being observed with prolonged time in culture. Several glycosyltransferase activities were assayed at varying times in culture. Generally, the changes in activities fell into three groups. One group was maximally activated at the end of the culture period (GalT-3, GalNAcT-1 and GalT-6). Another group was maximally activated during the time of active membrane fusion (GlcT and SAT-1). A third group was maximally activated at the time of cell contact and the beginning of membrane fusion (GlcNAcT-1 and GalT-2). In terms of the times of maximal activation there were few differences between E63 and fu-1 cells, with one notable exception. The activity of GalT-2 (lactosylceramide synthase) in E63 cells increased dramatically upon contact and the beginning of membrane fusion, whereas there were no changes in GalT-2 activity in fu-1 cells during time in culture. These results support our hypothesis that membrane glycosphingolipids play an important role in the differentiation of skeletal muscle cells.Abbreviations GSL glycosphingolipid - CK creatine kinase - HPTLC high performance thin layer chromatography - PMSF phenylmethylsulfonyl fluoride - CTH ceramide trihexoside (GbOse₃Cer) - GlcCer glycosylceramide - LacC N-acetylglucosamine - NeuNAc N-acetylneuraminic acid (sialic acid)     

The microRNA expression profile in porcine skeletal muscle is changed by constant heat stress

Heat stress has profound effects on animal performance and muscle function, and microRNAs (miRNAs) play a critical role in muscle development and stress responses. To characterize the changes in miRNAs in skeletal muscle responding to heat stress, the miRNA expression profiles of longissimus dorsi muscles of pigs raised under constant heat stress (30 °C; n = 8) or control temperature (22 °C; n = 8) for 21 days were analyzed by Illumina deep sequencing. A total of 58 differentially expressed miRNAs were identified with 30 down‐regulated and 28 up‐regulated, and 63 differentially expressed target genes were predicted by miRNA–mRNA joint analysis. GO and KEGG analyses showed that the genes regulated by differentially expressed miRNAs were enriched in glucose metabolism, cytoskeletal structure and function and stress response. Real‐time PCR showed that the mRNA levels of PDK4, HSP90 and DES were significantly increased, whereas those of SCD and LDHA significantly decreased by heat exposure. The protein levels of CALM1, DES and HIF1α were also significantly increased by constant heat. These results demonstrated that the change in miRNA expression in porcine longissimus dorsi muscle underlies the changes in muscle structure and metabolism in porcine skeletal muscle affected by constant heat stress.     

Heat shock factor 1 is a key regulator of the stress response in Chlamydomonas

We report here on the characterization of heat shock factor 1 (HSF1), encoded by one of two HSF genes identified in the genome of Chlamydomonas reinhardtii. Chlamydomonas HSF1 shares features characteristic of class A HSFs of higher plants. HSF1 is weakly expressed under non-stress conditions and rapidly induced by heat shock. Heat shock also resulted in hyperphosphorylation of HSF1, and the extent of phosphorylation correlated with the degree of induction of heat shock genes, suggesting a role for phosphorylation in HSF1 activation. HSF1, like HSFs in yeasts, forms high-molecular-weight complexes, presumably trimers, under non-stress, stress and recovery conditions. Immunoprecipitation of HSF1 under these conditions led to the identification of cytosolic HSP70A as a protein constitutively interacting with HSF1. Strains in which HSF1 was strongly under-expressed by RNAi were highly sensitive to heat stress. 14C-labelling of nuclear-encoded proteins under heat stress revealed that synthesis of members of the HSP100, HSP90, HSP70, HSP60 and small HSP families in the HSF1-RNAi strains was dramatically reduced or completely abolished. This correlated with a complete loss of HSP gene induction at the RNA level. These data suggest that HSF1 is a key regulator of the stress response in Chlamydomonas.     

Aldehyde dehydrogenase activity promotes survival of human muscle precursor cells

Aldehyde dehydrogenases (ALDH) are a family of enzymes that efficiently detoxify aldehydic products generated by reactive oxygen species and might therefore participate in cell survival. Because ALDH activity has been used to identify normal and malignant cells with stem cell properties, we asked whether human myogenic precursor cells (myoblasts) could be identified and isolated based on their levels of ALDH activity. Human muscle explant‐derived cells were incubated with ALDEFLUOR, a fluorescent substrate for ALDH, and we determined by flow cytometry the level of enzyme activity. We found that ALDH activity positively correlated with the myoblast‐CD56⁺ fraction in those cells, but, we also observed heterogeneity of ALDH activity levels within CD56‐purified myoblasts. Using lentiviral mediated expression of shRNA we demonstrated that ALDH activity was associated with expression of Aldh1a1 protein. Surprisingly, ALDH activity and Aldh1a1 expression levels were very low in mouse, rat, rabbit and non‐human primate myoblasts. Using different approaches, from pharmacological inhibition of ALDH activity by diethylaminobenzaldehyde, an inhibitor of class I ALDH, to cell fractionation by flow cytometry using the ALDEFLUOR assay, we characterized human myoblasts expressing low or high levels of ALDH. We correlated high ALDH activity ex vivo to resistance to hydrogen peroxide (H₂O₂)‐induced cytotoxic effect and in vivo to improved cell viability when human myoblasts were transplanted into host muscle of immune deficient scid mice. Therefore detection of ALDH activity, as a purification strategy, could allow non‐toxic and efficient isolation of a fraction of human myoblasts resistant to cytotoxic damage.     

In vitro culture and induced differentiation of sheep skeletal muscle satellite cells

Skeletal muscle satellite cells are adult muscle-derived stem cells receiving increasing attention. Sheep satellite cells have a greater similarity to human satellite cells with regard to metabolism, life span, proliferation and differentiation, than satellite cells of the rat and mouse. We have used 2-step enzymatic digestion and differential adhesion methods to isolate and purify sheep skeletal muscle satellite cells, identified the cells and induced differentiation to examine their pluripotency. The most efficient method for the isolation of sheep skeletal muscle satellite cells was the type I collagenase and trypsin 2-step digestion method, with the best conditions for in vitro culture being in medium containing 20% FBS+10% horse serum. Immunofluorescence staining showed that satellite cells expressed Desmin, α-Sarcomeric Actinin, MyoD1, Myf5 and PAX7. After myogenic induction, multinucleated myotubes formed, as indicated by the expression of MyoG and fast muscle myosin. After osteogenic induction, cells expressed Osteocalcin, with Alizarin Red and ALP (alkaline phosphatase) staining results both being positive. After adipogenic induction, cells expressed PPARγ2 (peroxisome-proliferator-activated receptor γ2) and clear lipid droplets were present around the cells, with Oil Red-O staining giving a positive result. In summary, a successful system has been established for the isolation, purification and identification of sheep skeletal muscle satellite cells.     

Heat shock protein expression enhances heat tolerance of reptile embryos

The role of heat shock proteins (HSPs) in heat tolerance has been demonstrated in cultured cells and animal tissues, but rarely in whole organisms because of methodological difficulties associated with gene manipulation. By comparing HSP70 expression patterns among representative species of reptiles and birds, and by determining the effect of HSP70 overexpression on embryonic development and hatchling traits, we have identified the role of HSP70 in the heat tolerance of amniote embryos. Consistent with their thermal environment, and high incubation temperatures and heat tolerance, the embryos of birds have higher onset and maximum temperatures for induced HSP70 than do reptiles, and turtles have higher onset and maximum temperatures than do lizards. Interestingly, the trade-off between benefits and costs of HSP70 overexpression occurred between life-history stages: when turtle embryos developed at extreme high temperatures, HSP70 overexpression generated benefits by enhancing embryo heat tolerance and hatching success, but subsequently imposed costs by decreasing heat tolerance of surviving hatchlings. Taken together, the correlative and causal links between HSP70 and heat tolerance provide, to our knowledge, the first unequivocal evidence that HSP70 promotes thermal tolerance of embryos in oviparous amniotes.     

Heat shock protein expression in fish

Heat shock proteins (HSP) are a family of proteins expressed in response to a wide range of biotic and abiotic stressors. They are thus also referred to as stress proteins. Their extraordinarily high degree of identity at the amino acid sequence level and the fact that this cellular stress response has been described in nearly all organisms studied, make this group of proteins unique. We provide a brief historical overview of HSP research, as a background to summarizing what is known about HSP expression in fish. The expression of HSPs in fish has been described in cell lines, primary cultures of various cells, and in the tissues of whole organisms. Collectively, the data show that the expression of HSPs are affected in a wide variety of fish cells and tissues, in response both to biological stressors such as infectious pathogens, as well as to abiotic stressors such as heat and cold shock, and environmental contaminants. HSP research in fish is in its early stages and many studies are describing the expression of proteins in response to various stressors. Several studies have contributed to our understanding of the molecular nature and the molecular biology of HSPs in fish. Recent studies have shown a relationship between HSP expression and the generalized stress response in fish, but further research is needed to clarify the complex relationships between stress hormones and the cellular HSP response. In general, the HSP response seems to be related to the sensing of the stressor and the subsequent cellular effects which may adapt the cells to cope with the stressors. Consequently, such data may be of central importance in understanding the significance of HSP expression to the whole organism. We conclude with sections on laboratory methods used in HSP research and on potential applications of this knowledge in biomonitoring.     

Heat shock protein synthesis of the cyanobacterium Synechocystis PCC 6803: purification of the GroEL-related chaperonin

Synechocystis PCC 6803 cells could be induced to synthesize four major HSPs with apparent molecular sizes of 70, 64, 15 and 14 kDa. Heat stress at 42.5 °C appeared to be the optimum temperature for HSP formation in cells grown at 30 °C.The relative rate of synthesis of HSP70 and HSP15 reached a maximum at 30 min after the temperature shift-up whereas the capability of cells to accumulate HSP64 and HSP14 continued through 2 h.The two most abundant HSPs, HSP70 and HSP64, were recognized on western blots by antibodies raised against authentic DnaK and GroEL from Escherichia coli. To furnish sufficient evidence for the assumption that HSP64 is a GroEL-related chaperonin, this protein was purified to homogeneity. There was a 76% sequence identity between the amino acid sequence of HSP64 and the corresponding protein in Synechococcus PCC 7942. Moreover, the purified HSP64 cross-reacted to anti-E. coli GroEL antibody. To our knowledge, this is the first report about the purification and partial protein sequencing of a cyanobacterial chaperonin.     

Heat shock protein responses to aging and proteotoxicity in the olfactory bulb

The olfactory bulb is one of the most vulnerable brain regions in age‐related proteinopathies. Proteinopathic stress is mitigated by the heat shock protein (Hsp) family of chaperones. Here, we describe age‐related decreases in Hsc70 in the olfactory bulb of the female rat and higher levels of Hsp70 and Hsp25 in middle and old age than at 2–4 months. To model proteotoxic and oxidative stress in the olfactory bulb, primary olfactory bulb cultures were treated with the proteasome inhibitors lactacystin and MG132 or the pro‐oxidant paraquat. Toxin‐induced increases were observed in Hsp70, Hsp25, and Hsp32. To determine the functional consequences of the increase in Hsp70, we attenuated Hsp70 activity with two mechanistically distinct inhibitors. The Hsp70 inhibitors greatly potentiated the toxicity of sublethal lactacystin or MG132 but not of paraquat. Although ubiquitinated protein levels were unchanged with aging in vivo or with sublethal MG132 in vitro, there was a large, synergistic increase in ubiquitinated proteins when proteasome and Hsp70 functions were simultaneously inhibited. Our study suggests that olfactory bulb cells rely heavily on Hsp70 chaperones to maintain homeostasis during mild proteotoxic, but not oxidative insults, and that Hsp70 prevents the accrual of ubiquitinated proteins in these cells.

     

Restriction fragment length polymorphisms at the heat shock protein HSP70 gene(s) in pigs*

Pigs from a population consisting of eight US breeds or strains and three Chinese breeds were examined by restriction fragment length polymorphism (RFLP) analysis of the heat shock protein HSP70 gene(s). Limited polymorphisms with PstI and PvuII restriction enzymes were observed, but there were no polymorphisms with BomIII and BglI.     

Kinetics of myoblast proliferation show that resident satellite cells are competent to fully regenerate skeletal muscle fibers

The satellite cell compartment provides skeletal muscle with a remarkable capacity for regeneration. Here, we have used isolated myofibers to investigate the activation and proliferative potential of satellite cells. We have previously shown that satellite cells are heterogeneous: the majority express Myf5 and M-cadherin protein, presumably reflecting commitment to myogenesis, while a minority is negative for both. Although MyoD is rarely detected in quiescent satellite cells, over 98% of satellite cells contain MyoD within 24 h of stimulation. Significantly, MyoD is only observed in cells that are already expressing Myf5. In contrast, a minority population does not activate by the criteria of Myf5 or MyoD expression. Following the synchronous activation of the myogenic regulatory factor+ve satellite cells, their daughter myoblasts proliferate with a doubling time of approximately 17 h, irrespective of the fiber type (type I, IIa, or IIb) from which they originate. Although fast myofibers have fewer associated satellite cells than slow, and accordingly produce fewer myoblasts, each myofiber phenotype is associated with a complement of satellite cells that has sufficient proliferative potential to fully regenerate the parent myofiber within 4 days. This time course is similar to that observed in vivo following acute injury and indicates that cells other than satellite cells are not required for complete myofiber regeneration.     

Dynamic vacuolation in skeletal muscle fibres after fatigue

Vacuoles develop after fatiguing stimulation in frog skeletal muscle fibres. Experiments on isolated Xenopus muscle fibres show that this vacuolation is a dynamic process that reaches its maximum about 20 min after the end of fatiguing stimulation and then recedes. Fatigue-induced vacuoles originate from the t-tubular system. Recent data indicate that vacuoles are formed because of lactate accumulation in the t-tubules resulting in increased osmotic pressure and subsequent water influx. There is no obligatory connection between the presence of vacuoles and force depression, which is another common feature during the recovery from fatigue. Nevertheless, extensive vacuolation may exaggerate this force depression.