Studies on Calpain Expression during Differentiation of Rat Satellite Cells in Primary Cultures in the Presence of Heparin or a Mimic Compound

We have synthesized dextran derivatives called RGTAs (for regenerating agents) that were designed to mimic some of the properties of heparin or heparan sulfate to interact with and protect heparin binding growth factors. Some of these growth factors have been described to be involved in myogenesis control. In previous studies, we have shown that muscle regeneration in adults could be greatly enhanced in vivo by treatment with RGTA. Since muscle regeneration occurs through the activation of satellite cells, in the present study we have used primary cultures of rat satellite cells and treated them with the heparan sulfate analogue RGTA or heparin in order to stimulate their growth and differentiation. We also studied the effect of these substances on calpain (calcium-activated neutral proteases) expression in these cultures. Indeed, several reports, principally based on fetal myoblast cultures or myogenic cell lines, have suggested that calpains might be involved in myoblast fusion during myogenic differentiation. We therefore studied the expression of microcalpain (mu-calpain), millicalpain (m-calpain), and calpain 3 in the course of differentiation of these satellite cell cultures in the absence or in the presence of heparin or of a mimic compound (the RGTA RG1282). RGTA and heparin were shown to have a dual effect on satellite cell proliferation and differentiation: RGTA stimulated proliferation with a maximum dose effect at 1 microgam/ml. Heparin used at concentrations similar to those of RGTA was less efficient at stimulating proliferation. Both substances were shown, however, to induce precocious and enhanced differentiation of satellite cells. We showed by quantitative RT-PCR analysis that mu-calpain, m-calpain, and calpain 3 mRNAs were expressed in satellite cell cultures in proliferating myoblasts (day 3) and differentiating cultures (days 7 and 12). The level of mu-calpain mRNA was increased by a factor of 3 during differentiation of satellite cells, whereas the level of m-calpain mRNAs was slightly increased at day 12 only, and calpain 3 mRNA was slightly reduced in these differentiating cultures. Interestingly enough, RGTA and heparin, which both strongly increased differentiation, reduced the expression of the mu- and m-calpains and slightly increased that of calpain 3 in differentiating cultures. These results showed that there was no correlation between the extent of myoblast differentiation and the level of calpain expression in satellite cells grown in primary cultures and underscored the differences between these adult cells and fetal myoblasts.     

Epistasis between calpain 1 and its inhibitor calpastatin within breeds of cattle

The calpain gene family and its inhibitors have diverse effects, many related to protein turnover, which appear to affect a range of phenotypes such as diabetes, exercise-induced muscle injury, and pathological events associated with degenerative neural diseases in humans, fertility, longevity, and postmortem effects on meat tenderness in livestock species. The calpains are inhibited by calpastatin, which binds directly to calpain. Here we report the direct measurement of epistatic interactions of causative mutations for quantitative trait loci (QTL) at calpain 1 (CAPN1), located on chromosome 29, with causative mutations for QTL variation at calpastatin (CAST), located on chromosome 7, in cattle. First we identified potential causative mutations at CAST and then genotyped these along with putative causative mutations at CAPN1 in >1500 cattle of seven breeds. The maximum allele substitution effect on the phenotype of the CAPN1:c.947G>C single nucleotide polymorphism (SNP) was 0.14 sigma(p) (P = 0.0003) and of the CAST:c.155C>T SNP was also 0.14 sigma(p) (P = 0.0011) when measured across breeds. We found significant epistasis between SNPs at CAPN1 and CAST in both taurine and zebu derived breeds. There were more additive x dominance components of epistasis than additive x additive and dominance x dominance components combined. A minority of breed comparisons did not show epistasis, suggesting that genetic variation at other genes may influence the degree of epistasis found in this system.     

Characterization of the calcium-dependent proteolytic system in a mouse muscle cell line

Many studies have demonstrated that the calcium-dependent proteolytic system (calpains and calpastatin) is involved in myoblast differentiation. It is also known that myogenic differentiation can be studied in vitro. In the present experiments, using a mouse muscle cell line (C₂C₁₂) we have analyzed both the sequences of appearance and the expression profiles of calpains 1, 2, 3 and calpastatin during the course of myoblast differentiation. Our results mainly show that the expression of ubiquitous calpains (calpain 1 and 2) and muscle-specific calpain (calpain 3) at the mRNAs level as well as at the protein level do not change significantly all along this biological process. In the same time, the specific inhibitor of ubiquitous calpains, calpastatin, presents a stable expression at mRNAs level as well as protein level, all along myoblast to myotube transition. A comparison with other myogenic cells is presented.     

Calpain Activation Promotes BACE1 Expression,Amyloid Precursor Protein Processing,and Amyloid Plaque Formation in a Transgenic Mouse Model of Alzheimer Disease

Abnormal activation of calpain is implicated in synaptic dysfunction and participates in neuronal death in Alzheimer disease (AD) and other neurological disorders. Pharmacological inhibition of calpain has been shown to improve memory and synaptic transmission in the mouse model of AD. However, the role and mechanism of calpain in AD progression remain elusive. Here we demonstrate a role of calpain in the neuropathology in amyloid precursor protein (APP) and presenilin 1 (PS1) double-transgenic mice, an established mouse model of AD. We found that overexpression of endogenous calpain inhibitor calpastatin (CAST) under the control of the calcium/calmodulin-dependent protein kinase II promoter in APP/PS1 mice caused a remarkable decrease of amyloid plaque burdens and prevented Tau phosphorylation and the loss of synapses. Furthermore, CAST overexpression prevented the decrease in the phosphorylation of the memory-related molecules CREB and ERK in the brain of APP/PS1 mice and improved spatial learning and memory. Interestingly, treatment of cultured primary neurons with amyloid-β (Aβ) peptides caused an increase in the level of β-site APP-cleaving enzyme 1 (BACE1), the key enzyme responsible for APP processing and Aβ production. This effect was inhibited by CAST overexpression. Consistently, overexpression of calpain in heterologous APP expressing cells up-regulated the level of BACE1 and increased Aβ production. Finally, CAST transgene prevented the increase of BACE1 in APP/PS1 mice. Thus, calpain activation plays an important role in APP processing and plaque formation, probably by regulating the expression of BACE1.     

Short communication: Single nucleotide polymorphisms in an intron of the ovine calpastatin gene

Abstract

Calpastatin is the specific inhibitor of the ubiquitous calcium‐dependent proteases μ‐calpain and m‐calpain. Enzyme assay data from sheep and cattle inversely correlates post‐mortem muscle calpastatin levels with ultimate meat tenderness. Genetic markers of meat quality may therefore be found linked to the calpastatin gene (CAST). A three‐allele system detected by polymerase chain reaction ‐ single strand conformational polymorphism (PCR‐SSCP) has been observed in the ovine CAST. The three allele amplimers have been fully nucleotide sequenced and their differences in terms of single nucleotide polymorphism (SNPs) in the intron region of the amplimer are reported and compared to a consensus sequence of the orthologous region of the cattle CAST. A PCR‐RFLP for more rapid CAST genotyping of all three ovine alleles was also developed.     

绵羊CAST基因2型和4型转录本的克隆及特性分析

钙蛋白酶抑制蛋白(Calpastatin, CAST)是一种内源性的需要Ca²⁺激活的钙蛋白酶抑制剂, 在肌肉组织的蛋白质降解过程中起重要的调节作用。文章利用牛^CAST基因的mRNA序列, 通过逆转录RT-PCR首次克隆获得绵羊CAST基因2型转录本和4型转录本的部分cDNA序列, 并对序列进行生物信息学分析。CAST基因2型转录本的扩增片段为4 385 bp, 完整的开放阅读框为2 361 bp, 编码786个氨基酸; CAST基因4型转录本的扩增片段为1 467 bp, 完整的开放阅读框为1 317 bp, 编码438个氨基酸。CASTⅡ型蛋白序列存在4个保守结构域, CASTⅣ型蛋白序列存在3个保守结构域; 两者的二级结构均以螺旋为主, 富含疏水区域, 其氨基酸序列存在多个磷酸化位点以及蛋白激酶C(Protein kinase C, PKC)的磷酸化位点。通过RT-PCR分析CAST基因2型转录本和4型转录本的组织表达谱, 结果表明CAST基因2型转录本在所检测的10个组织中均表达, CAST基因4型转录本仅在睾丸组织中表达。     

Decreased expression of calpain and calpastatin mRNA during development is highly correlated with muscle protein accumulation in neonatal pigs

It is well known that rapid gain of muscle mass in neonatal pigs is highly related to protein synthesis. However, the role of protein degradation in muscle gain of the neonatal period has not been well established. Calpains and their endogenous inhibitors, calpastatins, play a significant role in early-stage myofibrillar protein degradation. To investigate the role of calpain–calpastatin system in muscle protein accumulation, we studied the expressions of their mRNA in muscle tissue sampled at days 1, 4, 6, 12, 20 and 28 from a total of 36 neonatal pigs. The steady-state mRNA levels of calpains 1A, 2 and 3A, calpastatin types 1, 2 and 3, obtained by quantitative real-time PCR analysis, decreased by 2–4 folds at the age of 4 to 6 days compared to 1-day-old piglets. Then, the relatively low expression level was maintained through 28 days of age. Expressions of calpains 1A, 3A and calpastatin type 1 were significantly correlated with the measurements of muscle protein accumulations such as muscle protein content and RNA/protein ratio. Expressions of calpain 1A, calpastatin types 1 and 3 were negatively correlated with birth weight and fractional rate of growth. The levels of calpains 1A and 2 mRNA were correspondent to their protease activities. In conclusion, decreased levels of calpain and calpastatin expressions over development in neonatal pigs are associated with high protein accumulations, suggesting that dramatic muscle growth during the neonatal period may be partially controlled by down-regulated calpain–calpastatin system.     

Age‐related loss of nitric oxide synthase in skeletal muscle causes reductions in calpain S‐nitrosylation that increase myofibril degradation and sarcopenia

Sarcopenia, the age‐related loss of muscle mass, is a highly‐debilitating consequence of aging. In this investigation, we show sarcopenia is greatly reduced by muscle‐specific overexpression of calpastatin, the endogenous inhibitor of calcium‐dependent proteases (calpains). Further, we show that calpain cleavage of specific structural and regulatory proteins in myofibrils is prevented by covalent modification of calpain by nitric oxide (NO) through S‐nitrosylation. We find that calpain in adult, non‐sarcopenic muscles is S‐nitrosylated but that aging leads to loss of S‐nitrosylation, suggesting that reduced S‐nitrosylation during aging leads to increased calpain‐mediated proteolysis of myofibrils. Further, our data show that muscle aging is accompanied by loss of neuronal nitric oxide synthase (nNOS), the primary source of muscle NO, and that expression of a muscle‐specific nNOS transgene restores calpain S‐nitrosylation in aging muscle and prevents sarcopenia. Together, the findings show that in vivo reduction of calpain S‐nitrosylation in muscle may be an important component of sarcopenia, indicating that modulation of NO can provide a therapeutic strategy to slow muscle loss during old age.     

Effect of beta-agonists on expression of calpain and calpastatin activity in skeletal muscle.

Administration of beta-adrenergic agonists to domestic species can lead to skeletal muscle hypertrophy, probably by reducing the rate of myofibrillar protein breakdown. Myofibrillar breakdown is associated with the calcium-dependent proteinase system (calpains I,II and calpastatin) whose activity also changes during beta-agonist treatment. A number of growth trials using the agonists cimaterol and clenbuterol with cattle, sheep, chicken and rat are reported which suggest a general mechanism whereby beta-agonists reduce calpain I activity, but increase calpain II and calpastatin activity in skeletal muscle. Parallel changes in specific mRNAs indicate that changes in gene expression or stabilisation of mRNA could in part explain the changes in activity.     

草鱼(♀)×赤眼鳟(♂)F1及其亲本CAST基因cDNA全长克隆与结构差异

钙蛋白酶抑制蛋白(Calpastatin, CAST)在肌肉生长和肉质特征形成中发挥重要作用。为探究草鱼(Ctenopharyngodon idellus)(♀)×赤眼鳟(Squaliobarbus curriculus)(♂)正交F1的肉质相关分子基础,通过RACE(Rapid-amplification of cDNA ends)技术分别克隆了草鱼(♀)×赤眼鳟(♂) F1及其亲本的CAST基因cDNA全长,并利用生物信息学方法分析比较了三种鱼的CAST结构差异。结果表明:草鱼(♀)、赤眼鳟(♂)及草鱼(♀)×赤眼鳟(♂) F1的CAST基因cDNA全长分别为3036、3165和3086 bp,编码901、893和904个氨基酸;预测蛋白质分子量分别为93.72、92.77和94.02 kD;推测的理论等电点分别为5.92、6.01和6.02。草鱼(♀)×赤眼鳟(♂) F1 CAST与草鱼(♀)和赤眼鳟(♂)核苷酸序列相似性分别为94.52%和90%。三种CAST蛋白均包括4个含有典型七肽的钙蛋白酶抑制结构域。草鱼(♀)、赤眼鳟(♂)和F1 CAST氨基酸残基中分别存在73、82和...     

Calpastatin, an endogenous calpain-inhibitor protein, regulates the cleavage of the Cdk5 activator p35 to p25

Cyclin-dependent kinase 5 (Cdk5) is a Ser/Thr kinase that is activated by binding to its regulatory subunit, p35. The calpain-mediated cleavage of p35 to p25 and the resulting aberrant activity and neurotoxicity of Cdk5 have been implicated in neurological disorders, such as Alzheimer's disease. To gain further insight into the molecular mechanisms underlying the pathological function of Cdk5, we investigated the role of the calpain inhibitor protein calpastatin (CAST), in controlling the aberrant production of p25. For this purpose, brain tissue from wild-type, CAST-over-expressing (transgenic), and CAST knockout mice were analyzed. Cleavage of p35 to p25 was increased in extracts from CAST knockout mice, compared with wild-type. Conversely, generation of p25 was not detected in brain lysates from CAST-over-expressing mice. CAST expression was 5-fold higher in mouse cerebellum than cerebral cortex. Accordingly, p25 production was lower in the cerebellum than the cerebral cortex. Furthermore, the Ca(2+) -dependent degradation of p35 by proteasome was evident when calpain was inhibited. Taken together, these results suggest that CAST is a crucial regulator of calpain activity, the production of p25, and, hence, the deregulation of Cdk5. Therefore, impairment of CAST expression and its associated mechanisms may contribute to the pathogenesis of neurodegenerative disorders.     

Dexamethasone and BCAA Failed to Modulate Muscle Mass and mTOR Signaling in GH-Deficient Rats

Branched-chain amino acids (BCAAs) and IGF-I, the secretion of which is stimulated by growth hormone (GH), prevent muscle atrophy. mTOR plays a pivotal role in the protective actions of BCAA and IGF-1. The pathway by which BCAA activates mTOR is different from that of IGF-1, which suggests that BCAA and GH work independently. We tried to examine whether BCAA exerts a protective effect against dexamethasone (Dex)-induced muscle atrophy independently of GH using GH-deficient spontaneous dwarf rats (SDRs). Unexpectedly, Dex did not induce muscle atrophy assessed by the measurement of cross-sectional area (CSA) of the muscle fibers and did not increase atrogin-1, MuRF1 and REDD1 expressions, which are activated during protein degradation. Glucocorticoid (GR) mRNA levels were higher in SDRs compared to GH-treated SDRs, indicating that the low expression of GR is not the reason of the defect of Dex’s action in SDRs. BCAA did not stimulate the phosphorylation of p70S6K or 4E-BP1, which stimulate protein synthesis. BCAA did not decrease the mRNA level of atrogin-1 or MuRF1. These findings suggested that Dex failed to modulate muscle mass and that BCAA was unable to activate mTOR in SDRs because these phosphorylations of p70S6K and 4E-BP1 and the reductions of these mRNAs are regulated by mTOR. In contrast, after GH supplementation, these responses to Dex were normalized and muscle fiber CSA was decreased by Dex. BCAA prevented the Dex-induced decrease in CSA. BCAA increased the phosphorylation of p70S6K and decreased the Dex-induced elevations of atrogin-1 and Bnip3 mRNAs. However, the amount of mTORC1 components including mTOR was not decreased in the SDRs compared to the normal rats. These findings suggest that GH increases mTORC1 activity but not its content to recover the action of BCAA in SDRs and that GH is required for actions of Dex and BCAA in muscles.     

Ceramide Selectively Decreases Tau Levels in Differentiated PC12 Cells Through Modulation of Calpain I

Abstract: Ceramide has been recently proposed to be a signal mediator in several important physiological processes including apoptosis, cellular growth, and differentiation. Because the microtubule-associated protein tau plays an important role in the establishment and maintenance of neuronal morphology, the effects of ceramide on tau were examined. Treatment of differentiated PC12 cells with the cell-permeable ceramide derivative N-acetylsphingosine (C2) resulted in a significant reduction in tau levels. Significant decreases in tau levels were also observed when the cells were treated with another ceramide derivative, N-hexanoylsphingosine (C6). In addition, C2 treatment increased the levels of a calpain-derived spectrin breakdown product but did not alter the levels of two cytoskeletal proteins, α-actin and α-tubulin. Because both tau and spectrin are proteolyzed in vitro by the calcium-activated cysteine protease calpain, the effects of ceramide analogues on the activity of this protease were examined. Treatment of PC12 cells with C2 enhanced calcium-stimulated proteolytic activity significantly, as revealed by monitoring the hydrolysis of the membrane-permeable calpain-selective fluorescence probe N-succinyl-l -leucyl-l -leucyl-l -valyl-l -tyrosine-7-amido-4-methylcoumarin. This activity increase was not due to a direct effect of C2 on calpains, because C2 did not alter the activities of purified calpain I or II. In addition, C2 treatment of PC12 cells resulted in a significant increase in the levels of calpain I and, to a lesser extent, the levels of calpastatin (an endogenous calpain inhibitor protein), whereas the levels of calpain II were not changed. Moreover, treatment of the cells with the synthetic calpain-specific inhibitor N-carbobenzoxy-l -leucyl-l -leucyl-l -tyrosine diazomethyl ketone blocked the C2-induced decreases in tau levels. These results indicate that tau levels are regulated in response to a physiological factor and, thus, have implications for ceramide-mediated changes in normal and pathological neuronal processes.     

Calpain Translocation during Muscle Fiber Necrosis and Regeneration in Dystrophin-Deficient Mice

Previous studies have shown that calpains are autolytically cleaved during the disease process of mdx dystrophy, a mouse model for Duchenne muscular dystrophy, indicating that calpains may be activated and play a role in proteolysis that occurs in muscular dystrophy (J. Biol. Chem.270(18), 10909–10914, 1995). In the present study, we investigated the location of calpain in dystrophic muscle fibers over the course of mdx dystrophy, to relate the protease distribution to its state of activation, and to determine whether calpain translocation was an early event in mdx dystrophy. Immunolabeling of healthy, fully differentiated muscle fibers showed calpain present throughout the cytosol, but more concentrated near the plasma membrane. However, degenerating mdx fibers did not contain higher concentrations of calpain at the plasma membrane and showed only a homogeneous, cytosolic distribution. Calpain distribution was similarly diffuse in young myotubes and regenerating fibers with increased cytosolic concentration in early myotubes. Calpain distribution in adult mdx tissue was similar to that occurring in healthy, fully differentiated fibers, although adult mdx fibers displayed higher concentrations of membrane-associated calpain than those observed in C57 controls. The association of calpain with the plasma membrane was verified by immunoblots of isolated sarcolemmal membrane from adult mdx and control muscle which showed calpain present predominantly in the cytosol along with some membrane association. Thus, changes in calpain distribution coincide with changes in enzymatic cleavage over the course of mdx dystrophy shown previously. Furthermore, the stages of pathology at which calpain cleavage is least coincides with those stages when calpain is most concentrated at the cell membrane, suggesting that calpain is retained in an inactive form at the plasma membrane.