The changes in body temperature,oxygen consumption,CO2 production and muscle protein turnover rate by selection for body size in Japanese quail,Coturnix coturnix japonica

• 1.1. Body temperature, oxygen consumption, CO₂ production and muscle protein degradation rate were measured in the three quail lines selected for body size, a random bred line (RR) and two lines selected for large (LL) or small (SS) body size.
• 2.2. The body temperature at 15 weeks of age was highest for small body size line and lowest for large body size line.
• 3.3. The body temperature, oxygen consumption and CO₂ production of females were significantly higher than that of males.
• 4.4. The fractional degradation rate of muscle protein of SS, RR and LL lines were measured as 2.4, 1.6 and 1.2% per day in male, and 2.6, 1.7 and 1.4% per day in female.

     

SMAD7, an antagonist of TGF-beta signaling,is a candidate of prenatal skeletal muscle development and weaning weight in pigs

SMAD7 promotes and enhances skeletal muscle differentiation by inhibiting transforming growth factor beta (TGF-β)/activin signaling and bone morphogenetic protein (BMP) pathways. However, its function, the mechanism regulating its translation, and its association with production meat traits remain unclear in pigs. In this study, we explored SMAD7 gene spatio-temporal and tissue distribution, conducted a single nucleotide polymorphism association analysis, and examined regulation of its expression during skeletal muscle development. We found that SMAD7 was positively related to TGF-β pathway genes and mainly expressed in prenatal developing muscle, and dual luciferase and western blot assays demonstrated that SMAD7 expression was regulated by miRNA-21 at the protein level via inhibition of mRNA translation. Finally, the association analysis showed that a single nucleotide mutation (Exon 4_28816;C/A) was significantly associated with the weaning weight of piglets among Yorkshire pigs. These data indicate that SMAD7 plays a potentially important role in mammalian prenatal skeletal muscle development and is a candidate gene for promoting greater weaning weight in pig breeding.     

Calpain and calpastatin in myoblast differentiation and fusion: Effects of inhibitors

Myoblast differentiation and fusion to multinucleated muscle cells can be studied in myoblasts grown in culture. Calpain (Ca²⁺-activated thiol protease) induced proteolysis has been suggested to play a role in myoblast fusion. We previously showed that calpastatin (the endogenous inhibitor of calpain) plays a role in cell membrane fusion. Using the red cell as a model, we found that red cell fusion required calpain activation and that fusibility depended on the ratio of cell calpain to calpastatin. We found recently that calpastatin diminishes markedly in myoblasts during myoblast differentiation just prior to the start of fusion, allowing calpain activation at that stage; calpastatin reappears at a later stage (myotube formation). In the present study, the myoblast fusion inhibitors TGF-β, EGTA and calpeptin (an inhibitor of cysteine proteases) were used to probe the relation of calpastatin to myoblast fusion. Rat L8 myoblasts were induced to differentiate and fuse in serum-poor medium containing insulin. TGF-β and EGTA prevented the diminution of calpastatin. Calpeptin inhibited fusion without preventing diminution of calpastatin, by inhibiting calpain activity directly. Protein levels of μ-calpain and m-calpain did not change significantly in fusing myoblasts, nor in the inhibited, non-fusing myoblasts. The results indicate that calpastatin level is modulated by certain growth and differentiation factors and that its continuous presence results in the inhibition of myoblast fusion.     

Variation among lines selected for body size in the fractional rate of degradation of protein and acid protease activity in the muscle of quail (Coturnix coturnix japonica)

Fractional rates (%/day) of degradation of muscle protein were determined by measuring the output of NT-methylhistidine (NT-MH) in the excreta at 2 and 10 weeks of age in three lines of quail, a random-bred line and two lines selected for body size, one for increased and the other for decreased size. In all lines, fractional rates of degradation of muscle protein at 2 weeks of age were higher than those at 10 weeks of age. The fractional rate of degradation at 2 weeks of age was highest for the RR line, 9.1-9.2%/day. However, at 10 weeks of age, the rank order changed, and the RR line showed the lowest rate, 1.8-1.9%/day. The SS line (5.8-6.2%/day at 2 weeks and 5.8-5.9%/day at 10 weeks of age) was significantly higher than the LL line (4.1-4.2%/day at 2 weeks and 2.1-2.2%/day at 10 weeks of age). Acid protease activities in supernatants of homogenized muscle of the three lines of quail at 2 and 10 weeks of age were measured. In all lines, the acid protease activities in supernatant of homogenized muscle decreased from 2 to 10 weeks of age. At 2 weeks, the protease activity of the RR line was significantly higher than that of the LL and SS lines, which did not differ significantly. However, at 10 weeks of age, the SS line had higher activity in both sexes than the LL and RR lines. The results suggest that selection for body size brings about significant changes in both fractional degradation rate and acid protease activity in the muscle.     

CNN3 Is Regulated by microRNA-1 during Muscle Development in Pigs

The calponin 3 (CNN3) gene has important functions involved in skeletal muscle development. MicroRNAs (miRNAs) play critical role in myogenesis by influencing the mRNA stability or protein translation of target gene. Based on paired microRNA and mRNA profiling in the prenatal skeletal muscle of pigs, our previous study suggested that CNN3 was differentially expressed and a potential target for miR-1. To further understand the biological function and regulation mechanism of CNN3, we performed co-expression analysis of CNN3 and miR-1 in developmental skeletal muscle tissues (16 stages) from Tongcheng (a Chinese domestic breed, obese-type) and Landrace (a Western, lean-type) pigs, respectively. Subsequently, dual luciferase and western blot assays were carried out. During skeletal muscle development, we observe a significantly negative expression correlation between the miR-1 and CNN3 at mRNA level. Our dual luciferase and western blot results suggested that the CNN3 gene was regulated by miR-1. We identified four single nucleotide polymorphisms (SNPs) contained within the CNN3 gene. Association analysis indicated that these CNN3 SNPs are significantly associated with birth weight (BW) and the 21-day weaning weight of the piglets examined. These facts indicate that CNN3 is a candidate gene associated with growth traits and regulated by miR-1 during skeletal muscle development in pigs.     

Synthesis and investigation of dihydroxychalcones as calpain and cathepsin inhibitors

In order to identify potential calpain and cathepsin inhibitors we prepared 12 dihydroxychalcone analogues and tested their ability to inhibit μ-calpain, m-calpain, cathepsins B and L. In the calpain inhibition test, compound 10 exhibited the most active inhibitory activity against m-calpain with an IC₅₀ value of 25.25 ± 0.901 μM. With respect to inhibition of cathepsins B and L, compound 13 exhibited the most potent inhibitory activity on cathepsin L and moderate inhibitory activity on cathepsin B with IC₅₀ values of 2.80 ± 0.100 and 11.47 ± 0.087 μM, respectively. Our results suggest the possibility of developing dual calpain and cathepsin inhibitors by properly modulating structures and/or combining the essential aspects of the functional group effective for specific calpain and cathepsin inhibition.     

Analysis of polymorphisms in the insulin-like growth factor 1 receptor (IGF1R) gene from Japanese quail selected for body weight

Insulin-like growth factor 1 receptor ( IGF1R ) is essential for the signalling of growth. In this study, we performed single nucleotide polymorphism (SNP) detection in the Japanese quail IGF1R coding region and an association study between SNPs and body weight in two lines (SS and LL) selected for large and small body weight. Of 21 SNPs obtained, a SNP at position AB292766:c.2293G>A led to the replacement of a valine with an isoleucine (V765I). The two lines were fixed for alternate alleles, with allele encoding valine fixed in the LL line. A significant effect of the SNP genotype was found on 10-week body weight ( P  < 0.01) and on 4- to 10-week and 6- to 10-week average daily gain ( P  < 0.05) in the F₂ family obtained from lines LL and SS. In six populations maintained in Japan or France, the frequency of allele encoding valine was higher than the allele encoding isoleucine.     

Sepsis stimulates calpain activity in skeletal muscle by decreasing calpastatin activity but does not activate caspase-3

We examined the influence of sepsis on the expression and activity of the calpain and caspase systems in skeletal muscle. Sepsis was induced in rats by cecal ligation and puncture (CLP). Control rats were sham operated. Calpain activity was determined by measuring the calcium-dependent hydrolysis of casein and by casein zymography. The activity of the endogenous calpain inhibitor calpastatin was measured by determining the inhibitory effect on calpain activity in muscle extracts. Protein levels of mu- and m-calpain and calpastatin were determined by Western blotting, and calpastatin mRNA was measured by real-time PCR. Caspase-3 activity was determined by measuring the hydrolysis of the fluorogenic caspase-3 substrate Ac-DEVD-AMC and by determining protein and mRNA expression for caspase-3 by Western blotting and real-time PCR, respectively. In addition, the role of calpains and caspase-3 in sepsis-induced muscle protein breakdown was determined by measuring protein breakdown rates in the presence of specific inhibitors. Sepsis resulted in increased muscle calpain activity caused by reduced calpastatin activity. In contrast, caspase-3 activity, mRNA levels, and activated caspase-3 29-kDa fragment were not altered in muscle from septic rats. Sepsis-induced muscle proteolysis was blocked by the calpain inhibitor calpeptin but was not influenced by the caspase-3 inhibitor Ac-DEVD-CHO. The results suggest that sepsis-induced muscle wasting is associated with increased calpain activity, secondary to reduced calpastatin activity, and that caspase-3 activity is not involved in the catabolic response to sepsis.     

Identification of μ-, m-calpains and calpastatin and capture of μ-calpain activation in endothelial cells

The presence of the calpain-calpastatin system in human umbilical vein endothelial cells (HUVEC) was investigated by means of ion exchange chromatography, Western blot analysis, and Northern blot analysis. On DEAE anion exchange chromatography, calpain and calpastatin activities were eluted at approximately 0.30 M and 0.15-0.25 M NaCl, respectively. For half-maximal activity, the protease required 800 μM Ca²⁺, comparable to the Ca²⁺ requirement of m-calpain. By Western blot analysis, the large subunit of μ-calpain (80 kDa) was found to be eluted with calpastatin (110 kDa). Both the large subunit of m-calpain (80 kDa) and calpastatin were detected in the respective active fractions. By Northern blot analysis, mRNAs for large subunits of μ- and m-calpains were detected in single bands, each corresponding to approximately 3.5 Kb. Calpastatin mRNA was observed in two bands corresponding to approximately 3.8 and 2.6 Kb. Furthermore, the activation of μ-calpain in HUVEC by a calcium ionophore was examined, using an antibody specifically recognizing an autolytic intermediate form of μ-calpain large subunit (78 kDa). Both talin and filamin of HUVEC were proteolyzed in a calcium-dependent manner, and the reactions were inhibited by calpeptin, a cell-permeable calpain specific inhibitor. Proteolysis of the cytoskeleton was preceded by the appearance of the autolytic intermediate form of μ-calpain, while the fully autolyzed postautolysis form of μ-calpain (76 kDa) remained below detectable levels at all time points examined. These results indicate that the calpain-calpastatin system is present in human endothelial cells and that μ-calpain may be involved in endothelial cell function mediated by Ca²⁺ via the limited proteolysis of various proteins. J. Cell. Biochem. 66:197-209, 1997. © 1997 Wiley-Liss, Inc.     

猪肌肉组织Col3a1基因表达的发育性变化及其对肌内胶原特性的影响

以30—90妇体重莱芜猪和40—100kg体重鲁莱黑猪共84头去势公猪为试验对象（每组6头）,采用相对定量RT-PCR方法,以β-actin作为内标,研究肌肉中编码Ⅲ型胶原的Col3al基因表达的发育性变化及其对肌肉中胶原蛋白含量和性质（溶解度）的影响。结果表明：研究的两个品种猪肌肉中Col3al基因表达的发育性变化基本一致,即随体重的增加,肌肉中Col3al mRNA表达呈逐渐增加趋势,但莱芜猪和鲁莱黑猪分别在70妇和80妇体重组表达量略有下降。总体上莱芜猪肌肉组织Col3al mRNA表达丰度显著高于鲁莱黑猪（P〈0．05）。相关分析表明,莱芜猪肌肉组织Col3al mRNA表达的发育性变化与总胶原和不溶性胶原含量呈极显著正相关（P〈0．01）,与胶原溶解度呈极显著负相关（P〈0．01）。鲁莱黑猪肌肉组织Col3al mRNA表达的发育性变化与不溶性胶原和胶原溶解度分别呈显著正相关和负相关妒〈0．05）。研究结果提示：猪肌肉组织中Col3al基因表达具有明显的体重发育和品种特征,其mRNA表达对于肌内胶原的含量和性质有重要影响。     

TRPM7 Activates m-Calpain by Stress-Dependent Stimulation of p38 MAPK and c-Jun N-Terminal Kinase

TRPM7 is a Ca²⁺-permeant and Mg²⁺-permeant ion channel in possession of its own kinase domain. In a previous study, we showed that overexpression of the channel-kinase in HEK-293 cells produced cell rounding and loss of adhesion, which was dependent on the Ca²⁺-dependent protease m-calpain. The TRPM7-elicited change in cell morphology was channel-dependent and occurred without any significant increase in cytosolic Ca²⁺. Here we demonstrate that overexpression of TRPM7 increased levels of cellular reactive oxygen species (ROS) and nitric oxide, causing the activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Application of inhibitors of p38 MAPK and JNK blocked TRPM7-induced cell rounding and activation of m-calpain, without affecting the phosphorylation state of the protease. Overexpression of TRPM7 increased intracellular Mg²⁺; however, when the concentration of either external Ca²⁺ or Mg²⁺ was increased to favor the permeation of one divalent cation over the other, a similar increase in cell rounding and calpain activity was detected, indicating that TRPM7-mediated activation of m-calpain is not dependent on the nature of the divalent conducted by the channel. Application of inhibitors of nitric oxide synthase and mitochondrial-derived ROS reduced TRPM7-induced increases in nitric oxide and ROS production, blocked the change in cell morphology, and reduced cellular calpain activity. Collectively, our data reveal that excessive TRPM7 channel activity causes oxidative and nitrosative stresses, producing cell rounding mediated by p38 MAPK/JNK-dependent activation of m-calpain.     

Cardiac high molecular weight calmodulin-binding protein is homologous to calpastatin I and calpastatin II

Calpastatin is an endogenous inhibitor of calpain, which has been implicated in various physiological and pathological processes. In the present study we determined the molecular and inhibitory properties of HMWCaMBP, calpastatin I, and calpastatin II. Western blot analysis with antibodies raised against either full length HMWCaMBP or internal peptides that are common to all isoforms showed that all three homologs have common antigenic epitopes. However, additional Western blot analysis with N-terminal specific antibodies showed that all three proteins are different at the N-terminal end. HMWCaMBP is clearly different from two other homologues, calpastatin I and II, at the N-terminal end. In addition, HMWCaMBP also showed the same affinities for m-calpain as calpastatin I and calpastatin II. Our findings suggest that HMWCaMBP is the homolog of calpastatin and may be a CaM-binding form of calpastatin.     

Effect of exercise training on calpain systems in lean and obese Zucker rats

Exercise training plays a major role in the improving physiology of diabetes. Herein we aimed to investigate the influence of exercise upon the calcium-dependent calpain-isoform expressions of lean or obese Zucker rats, a model of obesity and type II diabetes (NIDDM). Five-month-old rats were divided: (1) obese sedentary (OS, n=7); (2) obese exercise (OE, n=7); (3) lean sedentary (LS, n=7); (4) lean exercise (LE, n=7). After 2-month exercise (treadmill running), the body weight (BW) and expression of calpain 10, μ-calpain, and m-calpain in skeletal muscles were determined by RT-PCR, using β-actin as internal standard. We found exercise is useful for BW lossing, especially in the obese rats. The BW difference between OS and OE rats (69 g vs. 18.2 g) was more significantly than that between LS and LE rats (41.8 g vs. 28.7g). The calpain 10 expression of LS rats (0.965) was lower than that of LE rats (1.006), whereas those of OS and OE were comparable. The μ- or m-calpain expressions of sedentary groups (OS, LS) was significantly higher than those of exercise groups (OE, LE). The μ-calpain expression (1.13/0.92) and m-calpain expression (1.01/0.99) of OS/LS rats was significantly higher than those of OE/LE rats [1.07/0.9 (μ-calpain); 0.97/0.95 (m-calpain)]. We concluded that the μ- or m-calpains in skeletal muscle are regulated by exercise in both lean and obese Zucker rats. Exercise and BW controlling might improve the physiopathology of obesity and diabetes. Both μ- or m-calpains might become useful markers for prognoses of diabetes.     

Effects of GHRP-2 and Cysteamine Administration on Growth Performance,Somatotropic Axis Hormone and Muscle Protein Deposition in Yaks (Bos grunniens) with Growth Retardation

The objective of this study was to investigate the effects of growth hormone-releasing peptide-2 (GHRP-2) and cysteamine (CS) administration on growth performance in yaks with growth retardation and try to elucidate its regulatory mechanisms. Trial 1, thirty-six 1-year-old Qinghai high plateau yaks (body weight 38–83.2 kg) were randomly chosen for body weight and jugular blood samples collection. The relationship between body weight and serum GHRH (P < 0.05, R = 0.45), GH (P < 0.05, R = 0.47), IGF-1 (P < 0.05, R = 0.62) was significantly correlated in yaks colonies with lighter body weights. Trial 2, fifteen 1-year-old Qinghai high plateau yaks with growth retardation (average body weight 54.8 ± 8.24 kg) were randomly selected and assigned to negative control group (NG), GHRP-2 injection group (GG) and cysteamine feeding group (CG), with 5 yaks per group. Another five 1-year-old Qinghai high plateau yaks with normal growth performance (average body weight 75.3 ± 2.43 kg) were selected as positive control group (PG). The average daily gain (ADG) of the GG and CG were significantly higher than those in the PG and NG (P < 0.05). Both GHRP-2 and CS administration significantly enhanced the myofiber diameter and area of skeletal muscle (P<0.05). GHRP-2 significantly enhanced the serum GH and IGF-1 levels (P < 0.05), and up-regulated GHR, IGF-1 and IGF-1R mRNA expression in the liver and skeletal muscle (P < 0.05), enhanced the mRNA expression of PI₃K, AKt and mTOR in the skeletal muscle (P<0.05). CS significantly reduced the serum SS levels and the hypothalamus SS mRNA expression (P < 0.05), and enhanced GHR and IGF-1 mRNA expression in the liver (P < 0.05), decreased the mRNA expression of muscle atrophy F-box (Atrogin-1) and muscle ring finger 1 (MuRF1) mRNA (P < 0.05). Conclusions: Growth retardation in yaks was primarily due to somatotropic axis hormones secretion deficiency. Both GHRP-2 and CS administration can accelerate growth performance and GH, IGF-1 secretion in yaks with growth retardation. GHRP-2 enhanced muscle protein deposition mainly by up-regulated the protein synthesis pathways, whereas CS worked mainly by down-regulated the ubiquitin-proteasome pathway.     

Genetic studies on the muscle protein turnover rate of coturnix quail

The validation of the urinary excretion of N-methylhistidine (N-MH) by quail as an index of the muscle protein turnover rate was tested using the criterion of the rate of recovery of radioactivity in urine following an intraperitoneal dose of l-[3-¹⁴C]methylhistidine. A genetic study on muscle protein turnover in quail was conducted using three genetically diverse lines (LL, large body size; SS, small body size; RR, random-bred control line) selected for body size. When l-[3-¹⁴C]methylhistidine was administered to 20-week-old male and female coturnix quail by direct intraperitoneal injection, approximately 90% of the l-[3-¹⁴C]methylhistidine was recovered by 96 hr postinjection. Recoveries were low in the egg and muscle. These results show that N-MH released from myofibrillar protein is not reutilized and the excretion of N-MH is a satisfactory index of muscle protein breakdown. In all lines, the amount of urinary N-MH excretion and fractional synthesis (K_s) and degradation (K_d) rates at the high growing period were higher than those at the low growing period. The K_s and K_d are significantly different among selected lines at both 3 and 6 weeks of age. At 3 weeks of age, the fractional rate of synthesis of the LL line (13.2%/day) was higher than that of the RR line (11.5%/day), whereas the SS (8.1%/day) was lower than that of the RR line (11.5%/day). The fractional rates of degradation of both the LL line (4.1%/day) and the SS line (5.6%/day) were lower than that of the RR line (7.0%/day) at 3 weeks of age. From these results, it was recognized that selection for body size gave rise to the changes in the muscle protein turnover rate.     

Muscle fiber characteristics of pectoralis major muscle as related to muscle mass in different Japanese quail lines

The objectives of this study were to investigate the muscle fiber characteristics of the pectoralis major muscle, and its relation to growth performance in the random bred control (RBC) and heavy weight (HW) Japanese quail lines at 42 days of age. The HW line had greater body (232.0 v. 100.2 g, P < 0.001) and pectoralis major muscle (19.0 v. 6.2 g, P < 0.001) weights than the RBC line. Color differences were observed between the superficial and deep regions of the pectoralis major muscle, with the superficial region showing a higher value of lightness than the deep region of the RBC or HW lines (P < 0.001). The percentage of the superficial region in the pectoralis major muscle was higher in the HW line compared with the RBC line (46.2% v. 38.0%, P = 0.017). There were no significant differences in the total fiber number in the superficial and deep regions between the two quail lines (P = 0.718). The HW quail line showed a larger mean fiber cross-sectional area (CSA; 375.5 v. 176.6 μm², P < 0.001) and type IIA fiber CSA (243.7 v. 131.9 μm², P < 0.001) than the RBC quail line. The HW line also had greater CSA percentage (60.2% v. 34.2%, P < 0.001) and number percentage (41.6% v. 14.2%, P < 0.001) of type IIB fibers, although there were no significant differences in type IIB fiber CSA between the RBC and HW lines (P = 0.219). Therefore, greater body and muscle weights of the HW line are caused by differences in muscle fiber characteristics, especially the proportion of type IIB fiber and the CSA of type IIA fiber, compared with the RBC line. The results of this study suggest that muscle fiber hypertrophy has more impact on body and muscle weights of the different quail lines than muscle fiber hyperplasia.     

Fiber type-specific expression of major proteolytic systems in fast- to slow-transforming rabbit muscle

The present study investigatesthe role of two major proteolytic systems in transforming rabbit andrat muscles. The fast-to-slow transformation of rabbit muscle bychronic low-frequency stimulation (CLFS) induces fast-to-slowtransitions of intact, mature fibers and replacement of degeneratingfibers by newly formed slow fibers. Ubiquitination, an indicator of theATP-dependent proteasome system, and calpain activity were measured inhomogenates of control and stimulated extensor digitorum longusmuscles. Calpain activity increased similarly (~2-fold) in stimulatedrat and rabbit muscles. CLFS had no effect on protein ubiquitination inrat muscle but led to elevations in ubiquitin protein conjugates inrabbit muscle. Immunohistochemistry was used to study the distributionof µ-calpain and m-calpain and of ubiquitinated proteins in myosinheavy chain-based fiber types. The findings suggest that bothproteolytic systems are involved in fiber transformation andreplacement. Transforming mature fibers displayed increases inµ-calpain and accumulation of ubiquitin protein conjugates. Themajority of these fibers were identified as type IIA. Enhancedubiquitination was also observed in degenerating and necrotic fibers.Such fibers additionally displayed elevated m-calpain levels.Conversely, p94, the skeletal muscle-specific calpain, decayed rapidlyafter stimulation onset and was hardly detectable after 4 days of CLFS.