Use of transcriptome sequencing to explore the effect of CSRP3 on chicken myoblasts

The mechanisms that regulate the specificity and maintenance of chicken muscle fiber types remain largely unknown. In mammals, CSRP3 has been shown to play a vital role in the maintenance of typical muscle structure and function. This study investigated the role that CSRP3 plays in chicken skeletal muscle. First, the antibody against chicken CSRP3 protein was prepared, and the expression levels of the mRNA and protein of the CSRP3 gene in four chicken skeletal muscles with different myofiber compositions were compared. Then the effects of CSRP3 silencing on the expression profile of chicken myoblast transcriptomes were analyzed. The results showed that the expression levels of the mRNA and protein of the CSRP3 gene were both associated with the composition of fiber types in chicken skeletal muscles. A total of 650 genes with at least 1.5-fold differences(Q<0.05) were identified, of which 255 genes were upregulated and 395 genes were downregulated by CSRP3 silencing. Functional enrichment showed that several pathways, including adrenergic signaling in cardiomyocytes, adipocytokine signaling pathway and apelin signaling pathway, were significantly(P<0.05) enriched both in differentially expressed genes and all expressed genes. The co-expressed gene network suggested that CSRP3 silencing caused a compensatory upregulation(Q<0.05) of genes related to the assembly of myofibrils, muscle differentiation, and contraction. Meanwhile, two fast myosin heavy chain genes(MyH1B and MyH1E)were upregulated(Q<0.05) upon CSRP3 silencing. These results suggested that CSRP3 plays a crucial role in chicken myofiber composition, and affects the distribution of chicken myofiber types, probably by regulating the expression of MyH1B and MyH1E.

Implications of vitamin D for flesh quality of grass carp(Ctenopharyngodon idella):antioxidant ability,nutritional value,sensory quality,and myofiber characteristics

Background Muscle represents a unique and complex system with many components and comprises the major edible part of animals.Vitamin D is a critical nutrient for animals and is known to enhance calcium absorption and immune response.In recent years,dietary vitamin D supplementation in livestock has received increased attention due to biological responses including improving shear force in mammalian meat.However,the vitamin D acquisition and myofiber development processes in fish differ from those in mammals,and the effect of vitamin D on fish flesh quality is poorly understood.Here,the influence of dietary vitamin D on fillet quality,antioxidant ability,and myofiber development was examined in grass carp(Ctenopharyngodon idella).Methods A total of 540 healthy grass carp,with an initial average body weight of 257.24±0.63 g,were allotted in 6 experimental groups with 3 replicates each,and respectively fed corresponding diets with 15.2,364.3,782.5,1,167.9,1,573.8,and 1,980.1 IU/kg vitamin D for 70 d.Results Supplementation with 1,167.9 IU/kg vitamin D significantly improved nutritional value and sensory quality of fillets,enhancing crude protein,free amino acid,lipid,and collagen contents;maintaining an ideal pH;and reduc-ing lactate content,shear force,and cooking loss relative to respective values in the control(15.2 IU/kg)group.Average myofiber diameter and the frequency of myofibers>50μm in diameter increased under supplementation with 782.5–1,167.9 IU/kg vitamin D.Levels of oxidative damage biomarkers decreased,and the expression of antioxi-dant enzymes and nuclear factor erythroid 2-related factor 2 signaling molecules was upregulated in the 1,167.9 IU/kg vitamin D treatment compared to respective values in the control group.Furthermore,vitamin D supplementation activated cell differentiation by enhancing the expression of myogenic regulatory factors and myocyte enhancer factors compared to that in the control group.In addition,supplementation with 1,167.9 IU/kg vitamin D improved protein deposition associated with protein synthesis molecule(target of rapamycin)signaling and vitamin D receptor paralogs,along with inhibition of protein degradation(forkhead box protein 1)signaling.Conclusions Overall,the results demonstrated that vitamin D strengthened antioxidant ability and myofiber devel-opment,thereby enhancing nutritional value and sensory quality of fish flesh.These findings suggest that dietary vitamin D supplementation is conducive to the production of nutrient-rich,high quality aquaculture products.

Effect of dietary supplementation with mulberry(Morus alba L.) leaves on the growth performance,meat quality and antioxidative capacity of finishing pigs

The present study was conducted to evaluate the effect of dietary mulberry(Morus alba L.) leaves powder(MLP) supplementation on meat quality of finishing pigs.A total of 40 Duroc×Landrace×Yorkshire pigs(initial body weight of(40.5±0.63) kg) were randomly allotted into two treatments,fed either with control diet or 15% MLP diet for 85 d.The results showed that MLP diet decreased(P≤0.05) average daily gain(ADG) and increased(P<0.05) feed/gain ratio(F/G) in the finishing and whole period.MLP diet also decreased(P<0.05) carcass weight,dressing percentage,last rib and average backfat depth.However,MLP diet increased(P<0.05) intramuscular fat(IMF) content,decreased(P<0.05) shear force,cooking loss and drip loss.In addition,MLP diet increased(P<0.05) total antioxidative capacity,glutathione peroxidase and tended(P<0.10) to increase total superoxide dismutase in serum.In longissimus thoracis,myosin heavy chain(MyHC) I and IIa mRNA levels were increased(P≤0.05) for MLP diet.In conclusion,15% MLP supplementation reduced the growth performance and carcass traits,but improved meat quality of finishing pigs possibly through the change of myofiber characteristics,enhancement of antioxidative capacity and increase of IMF.

circPTPN4 regulates myogenesis via the miR-499-3p/NAMPT axis

Background:Circular RNAs(circ RNAs)are a novel class of endogenous nc RNA,which widely exist in the transcriptomes of different species and tissues.Recent studies indicate important roles for circ RNAs in the regulation of gene expression by acting as competing endogenous RNAs(ce RNAs).However,the specific role of circ RNAs in myogenesis is still poorly understood.In this study,we attempted to systematically identify the circ RNAs involved in myogenesis and analyze the biological functions of circ RNAs in chicken skeletal muscle development.Results:In total,532 circ RNAs were identified as being differentially expressed between pectoralis major(PEM)and soleus(SOL)in 7-week-old Xinghua chicken.Among them,a novel circ RNA(novel_circ_002621),generated by PTPN4 gene,was named circ PTPN4 and identified.circ PTPN4 is highly expressed in skeletal muscle,and its expression level is upregulated during myoblast differentiation.circ PTPN4 facilitates the proliferation and differentiation of myoblast.Moreover,circ PTPN4 suppresses mitochondria biogenesis and activates fast-twitch muscle phenotype.Mechanistically,circ PTPN4 can function as a ce RNA to regulate NAMPT expression by sponging mi R-499-3p,thus participating in AMPK signaling.Conclusions:circ PTPN4 functions as a ce RNA to regulate NAMPT expression by sponging mi R-499-3p,thus promoting the proliferation and differentiation of myoblast,as well as activating fast-twitch muscle phenotype.

Impact of dietary L-arginine supply during early gestation on myofiber development in newborn pigs exposed to intra-uterine crowding

Background: Intra-uterine crowding(IUC) observed in hyperprolific sows impairs myofiber hyperplasia and overall fetal growth. Arginine supplementation(ARG) in gestation diets has been shown to positively affect litter and muscle development. The study objective was to assess whether the effect of ARG on offspring characteristics, with special emphasis on myofiber hyperplasia, differs under IUC conditions from these responses,because in that situation growth retardation is particularly prevalent due to reduced fetal nutrient supply.Unilateral oviduct ligation(OL) was used as a model for an uncrowded and hyperprolificacy(IN) as a model for a crowded intra-uterine environment.Methods: Five OL and five IN sows were fed a diet supplemented daily with either 43 g L-alanine(Ctrl) or 25 g L-arginine from d 14 to 28 of gestation in a cross-over design(two periods: 5^( th) and 6^( th) parity). At farrowing, two male and two female offspring, respectively, with a low and intermediate birth weight(BtW) were selected. After euthanization, the Semitendinosus muscle(STM) was removed and weighed, and the light and dark portions(STM_d and STM_l) were prepared for myofiber histochemistry using ATPase staining and the entire STM for gene expression analysis of myogenesis-related genes using RT-qPCR. In addition, various organs were removed and weighed. Data were analyzed using the MIXED model in SYSTAT.Results: No effect of either IUC or dietary treatment was found in litter characteristics. Offspring of ARG sows displayed a greater muscle area in STM(P < 0.01) as a result of the greater myofiber hyperplasia(P < 0.01). The increase was more distinct in the STM_l(P < 0.05) than in the STM_d(P = 0.131). Offspring of OL sows were heavier at birth(P < 0.01), had a heavier STM(P < 0.05), liver(P < 0.01) and kidney(P < 0.05), but when expressed relative to birth weight, these differences were absent. In addition, IUC had an effect(P < 0.05) on the expression of one of the myogenesis-related genes investigated.Conclusions: Independent from the extent of IUC, ARG improved Bt W, muscle and organ weights and myofiber hyperplasia in offspring.

PGC-1α differentially regulates the mRNA expression profiles of genes related to myofiber type specificity in chicken

Previous studies on mammals showed that peroxisome proliferator-activated receptor gamma coactivator-1α(PGC-1α)played a prominent role in regulating muscle fiber type transition and composition.However,the role of PGC-1αin chicken muscle has seldom been explored.To investigate the effect of PGC-1αon chicken skeletal muscles in this study,the PGC-1αgene was overexpressed or silenced in chicken primary myoblasts by using lentivirus,and then the effects of the PGC-1αgene overexpression and knockdown on the mRNA expression profile of genes related to myofiber type specificity were examined during fiber formation.The results showed that overexpression of PGC-1αfrom proliferation to differentiation was accompanied by the up-regulated expression of Pax7,MyoD,and CnAα,which was significantly(P<0.01)increased after one day of transfection(1 I).The enhancement of MyoG,MEF2 c,and MyHC SM expression lagged,which was improved significantly(P<0.01)after four days of transfection(1 I3 D).Overexpression of PGC-1αdecreased(P<0.01)the MyHC FWM expression after four days of transfection(1 I3 D),and it had no significant impact(P>0.05)on the expression of CnB1,NFATc3,and MyHC FRM during myofiber formation.The effective silence(P<0.01)of PGC-1αby lentivirus mediating short hairpin RNA(shRNA)was detected after four days of transfection(1 I3 D)in cultures,and the lack of its function in chicken primary myoblasts significantly(P<0.01)down-regulated the expression of Pax7,MyoD,CnAα,MyoG,MEF2 c,and MyHC SM,significantly(P<0.01)up-regulated the expression of MyHC FWM,and had no significant impact(P>0.05)on the expression of CnB1,NFATc3,and MyHC FRM.These results indicated that the role of PGC-1αin regulating the fiber type specificity of chicken skeletal muscles might be similar to that in mammals,which interplayed with key genes related to myocyte differentiation and calcineurin signaling pathway.

Myofiber development during embryonic to neonatal development in duck breeds differing in muscle growth rates

Little is known about the muscle developmental patterns during embryonic to neonatal development in ducks.We investigated the developmental patterns in the lateral gastrocnemius muscles of Gaoyou and Jinding ducks differing in their muscle growth rates during the final stages of egg incubation and the first week after hatching.Expression of the MyoD gene was quantified by quantitative real-time PCR（qRT-PCR）.The average cross-sectional area and diameter of the fibers increased from embryonic day 21（E21）,peaking at E27,and then declining slightly 7 d after hatching.The density of the fibers decreased initially but increased after hatching in both breeds and sexes.The within-breed variation in muscle fiber-type composition was greater than the average variation between the breeds.Overall,the percentage of type Ⅰ fibers increased and that of type lIb fibers decreased consistently.However,the percentage of type lla fibers was almost constant as development proceeded in both duck breeds.The profiles of MyoD mRNA expression were similar in both breeds,and a significantly positive relationship was observed between the expression of MyoD and the percentage of type lIb fibers.This study firstly revealed the characteristics of duck muscle development and differences between the two breeds differing in growth rates.Moreover,type lIb fibers might convert to type Ⅰ fibers in the lateral gastrocnemius,while MyoD may potentially function in controlling the muscle fiber phenotype during the secondary myogenesis of muscle development.

Distinct Gene Expression Profile Distinguishes Increased Metabolic Activity in Spontaneously Hyperactive Rats While Sedentary from That Induced by Exercise

The Spontaneously-Running Tokushima Shikoku (SPORTS) strain is an original line derived from Wistar rats, which spontaneously runs >6 km/day on wheels, and has better glucose tolerance and less fat than Wistar rats. However, the molecular mechanism that contributes to the increased metabolic activity in SPORTS rats is unknown. The present study aimed to characterize the gene expression profiles of skeletal muscles in SPORTS rats housed under sedentary (SED) conditions. We found that the expression levels of genes encoding mitochondrial respiratory chain enzymes such as ATP synthase 6 (mt-Atp6) and cytochrome c oxidase subunit 6c (Cox6c), were higher in the soleus (SOL) muscles of SED SPORTS than in SED Wistar rats. The ratio of type IIa myofibers was higher and glucose tolerance was better in SED SPORTS than in Wistar rats that were sedentary and trained daily on treadmills, respectively. We then investigated candidate genes that might contribute to the better glucose tolerance of SED SPORTS rats using DNA microarray analysis. Among 116 upregulated genes in the SOL muscles of SED SPORTS rats, only 19 were also increased in trained Wistar rats. We focused on v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (Erbb3), which was associated with glucose transport in myocytes, and found higher expression levels in the SOL muscles of SED SPORTS than in SED Wistar rats. The SOL muscles of SED SPORTS rats also contained more activity of β-hydroxyacylCoA dehydrogenase, a key enzyme of β-oxidation, indicating enhanced lipid oxidation. These findings suggest that increased metabolic activity in skeletal muscle (especially the SOL muscle) of SPORTS rats is congenital and that gene expression profiles of SPORTS rats and Trained Wistar rats are different.

Myofiber Permeability and Force Production of Rat Muscles Following Eccentric Contractions: The Repeated Bout Effect Depends on the Interval

Past eccentric contraction (ECC)-induced muscle injury reduces the severity of symptoms of subsequent muscle injury;this phenomenon is known as a repeated bout effect (RBE). It has been reported that increases in the duration of the interval between the first and second bouts are linked to weakening of the RBE. However, the histology following the attenuation of the RBE remains unclear. We examined the sustained effects of the second bout with regard to myofiber permeability and muscle force. Sixty-four male rats were randomly assigned to eight groups that varied in the number of exercise sessions and the duration of the interval between the first and second bouts: the non-ECC (Control);the single-injury (Post 1st bout);groups that were allowed to recover for 1, 2, and 4 weeks after a single injury (Pre 2nd bout_1w, Pre 2nd bout_2w, and Pre 2nd bout_4w);and groups that were subjected to second injuries 1, 2, and 4 weeks after the first (Post 2nd bout_1w, Post 2nd bout_2w, and Post 2nd bout_4w). The tibialis anterior was electrically stimulated in each ECC group. Twenty-four hours before muscle sampling, Evans blue dye (EBD) (a marker of myofiber damage) was administered. The maximal isometric contraction tension was measured immediately before sampling. The number of EBD-positive (+) fibers was determined via histological analysis. An RBE was revealed by functional examination at the 1- and 2-week and histological examination at the 1-, 2-, and 4-week time points (P < 0.05). In terms of myofiber permeability, prolongation of the interval before the second bout weakened this effect (P < 0.05). Experiments with 1-, 2-, and 4-week intervals indicated that prolongation of the interval before the second bout weakened the RBE with regard to myofiber permeability.

A new insight on copper:Promotion of collagen synthesis and myofiber growth and development in juvenile grass carp(Ctenopharyngodon idella)

Copper(Cu)is a trace element,essential for fish growth.In the current study,in addition to growth performance,we first explored the effects of Cu on collagen synthesis and myofiber growth and development in juvenile grass carp(Ctenopharyngodon idella).A total of 1080 fish(11.16±0.01 g)were randomly divided into 6 treatments(3 replicates per treatment)to receive five doses of organic Cu which were Cu citrate(CuCit)at 0.99(basal diet),2.19,4.06,6.15,and 8.07 mg/kg,and one dose o inorganic Cu(CuSO_(4)·5H_(2)O at 3.15 mg/kg),for 9 weeks.The results showed appropriate Cu level(4.06 mg kg)enhanced growth performance,improved nutritional Cu status,and downregulated Cu-transporting ATPase 1 mRNA levels in the hepatopancreas,intestine,and muscle of juvenile grass carp.Meanwhile collagen content in fish muscle was increased after Cu intake,which was probably due to the following pathways:(1)activating CTGF/TGF-β1/Smads signaling pathway to regulate collagen transcription;(2upregulating of La ribonucleoprotein domain family 6(LARP6)mRNA levels to regulate translation initiation;(3)increasing proline hydroxylase,lysine hydroxylase,and lysine oxidase activities to regulate posttranslational modifications.In addition,optimal Cu group increased myofiber diameters and the frequency of myofibers with diameter>50μm,which might be associated with upregulation of cyclin B cyclin D,cyclin E,proliferating cell nuclear antigen,myogenic determining factor(MyoD),myogenic factor 5,myogenin(MyoG),myogenic regulatory factor 4 and myosin heavy chain(MyHC)and down regulation of myostatin mRNA levels,increasing protein levels of MyoD,MyoG and MyHC in fish muscle Finally,based on percentage weight gain(PWG),serum ceruloplasmin(Cp)activity and collagen conten in fish muscle,Cu requirements were determined as 4.74,4.37 and 4.62 mg/kg diet(CuCit as Cu source of juvenile grass carp,respectively.Based on PWG and Cp activity,compared to CuSO_(4)·5H_(2)O,the efficacy of CuCit were 131.80%and 115.38%,respectively.Our findings provide new insights into Cu supple mentation to promote muscle growth in fish,and help improve the overall productivity of aquaculture.

The PDZ-Containing Unconventional Myosin XVIIIA Regulates Embryonic Muscle Integrity in Zebrafish

Myosin XVIIIA, or MYO18A, is a unique PDZ domain-containing unconventional myosin and is evolutionarily conserved from Drosophila to vertebrates. Although there is evidence indicating its expression in the somites, whether it regulates muscle function re- mains unclear. We show that the two zebrafish myo18a genes （myo18aa and myo18ab） are predominantly expressed at somite borders during early developmental stages. Knockdown of these genes or overexpression of the MYO18A PDZ domain disrupts myofiber integrity, induces myofiber lesions, and compromises the localization of dystrophin, ^-dystroglycan （^-DG） and laminin at the myotome boundaries. Cell transplantation experiments indicate that myo18a morphant myoblasts fail to form elongated myofibers in the myotomes of wild-type embryos, which can be rescued by the full-length MYO18A protein. These results suggest that MYO18A likely functions in the adhesion process that maintains the stable attachment of myofibers to ECM （extracellular matrix） and muscle integrity during early development.