miRNAS in normal and diseased skeletal muscle

The last 20 years have witnessed major advances in the understanding of muscle diseases and significant inroads are being made to treat muscular dystrophy. However, no curative therapy is currently available for any of the muscular dystrophies, despite the immense progress made using several approaches and only palliative and symptomatic treatment is available for patients. The discovery of miRNAs as new and important regulators of gene expression is expected to broaden our biological understanding of the regulatory mechanism in muscle by adding another dimension of regulation to the diversity and complexity of gene-regulatory networks. As important regulators of muscle development, unravelling the regulatory circuits involved may be challenging, given that a single miRNA can regulate the expression of many mRNA targets. Although the identification of the regulatory targets of miRNAs in muscle is a challenge, it will be critical for placing them in genetic pathways and biological contexts. Therefore, combining informatics, biochemical and genetic approaches will not only expected to reveal the elucidation of the miRNA regulatory network in skeletal muscle and to bring a better knowledge on muscle tissue regulation but will also raise new opportunities for therapeutic intervention in muscular dystrophies by identifying candidate miRNAs as potential targets for clinical application.     

Cloning and characterization of microRNAs from porcine skeletal muscle and adipose tissue

MicroRNAs (miRNAs) are an abundant class of small regulatory RNAs that regulate the stability and translation of cognate mRNAs. Although an increasing number of porcine miRNAs has recently been identified, the full repertoire of miRNAs in pig remains to be elucidated. To identify porcine miRNAs potentially involved in myogenesis and adipogenesis, we constructed small RNA cDNA libraries from skeletal muscle and adipose tissue and identified 89 distinct miRNAs that are conserved in pig, of which 15 were new. Expression analysis of all newly identified and selected known porcine miRNAs revealed that some miRNAs were enriched in a tissue-specific manner, whereas others were expressed ubiquitously in the porcine tissues examined. Our results expand the number of known porcine miRNAs and provide useful information for further investigating the biological functions of miRNAs associated with growth and development of skeletal muscle or adipose tissue in pig.     

MicroRNA调控猪骨骼肌发育的研究进展

骨骼肌的生长发育是影响猪肉产量和品质的重要因素,其受遗传和营养等众多因素的精细调控。MicroRNA (miRNA)是一种长度约为22 nt的非编码RNA,通过与靶基因的mRNA 3′UTR序列结合,调控其转录后的表达发挥作用。近年来,大量的研究表明miRNA参与机体的生长发育、生殖、疾病等多种生命过程。本文对miRNA在猪骨骼肌发育调控中的作用进行了综述,以期为猪的遗传改良提供参考和借鉴。     

猪的骨骼肌生长发育研究进展

瘦肉率对生猪产业来说是一个极其重要的经济指标,而这一指标完全取决于骨骼肌的生长发育。因此,猪骨骼肌生长发育机理的研究是十分必要的。然而,在早期由于各种因素的限制,猪骨骼肌单个基因的研究一直进展缓慢;相反,以小鼠为模型,其骨骼肌单基因的功能研究却取得了较大进展。在这一时期,影响肌决定和肌分化的基因,如MRFs家族和MEF2家族相继被发现,这些基因在猪的肌肉发育中也发挥着同样的作用。然而,这些结果并不能很好地揭示骨骼肌发育过程中复杂的基因间互作关系。随着近年来芯片和测序技术的不断发展,更多人试图从整个转录谱的水平来阐述猪肌肉发育的分子机理,并且也取得了较大的进展。为了对猪骨骼肌生长发育有一个更为清晰的认识,该文将以目前猪骨骼肌生长发育研究结果为基础,同时结合模式动物小鼠骨骼肌单基因的研究成果,对猪的骨骼肌生长发育分子调控机理进行详细的阐述。     

Isolation and characterization of three different forms of arylamidase from rat skeletal muscle

An arylamidase hydrolysing L-leucine-4-nitroanilide was extracted from rat skeletal muscle homogenate and furified by means of anion-exchange chromatography on DEAE-Sephadex A-50 followed by gel filtration on Sephadex G-150 and Sepharose 6B. The enzyme was isolated in the form of three different protein complexes that differ in molecular weight, kinetic data, and sensitivity to metal ions. As studied by SDS-gel electrophoresis and repeated gel chromatography on Sepharose 6B these forms are: 1. a stable monomer (A1) of M_r 122 000; 2. a stable dimer (A2) of M_r 244 000; and 3. a stable polymer (A3) of more than M_r 4·10⁶. The arylamidase was optimally active at pH 7.3 and did not require metal ions. Treatment with 1,10-phenanthroline resulted in complete inactivation, the activity could be restored by the addition of manganous chloride. The sulphhydryl-blocking reagent 4-hydroxymercuribenzoate strongly inactivated the arylamidase, this inhibition could be reversed by the addition of 2-mercaptoethanol. Addition of phenylmethylsulfonyl fluoride had no effect on the enzyme activity. Furthermore, the influence of metal ions as well as the substrate specificity were investigated and compared for all three forms of arylamidase.     

Mapping and expression analyses during porcine foetal muscle development of 12 genes involved in histone modifications

Histone modifications (methylation and demethylation) regulate gene expression and play a role in cell proliferation and differentiation by their actions on chromatin structure. In this context, we studied the temporal expression profiles of genes acting on histone methylation and demethylation during skeletal muscle proliferation and differentiation. Quantitative real-time PCR was used to quantify the mRNA levels of CARM1 , JARID1A , JMJD2A , LSD1 , PRMT2 , PRMT5 , SMYD1 , SMYD2 , SMYD3 , SETDB1 , Suv39h2 and SUZ12 in foetal skeletal muscle. Our results showed that CARM1 , JARID1A , JMJD2A , SMYD1 and SMYD2 were differentially expressed in embryonic muscles of 33 days post-conception (dpc), 65 dpc and 90 dpc. These 12 genes were mapped to porcine chromosomes (SSC) 2q21–24, 5q25, 6q35, 6q12–21, 6p15, 7q21, 3q21–27, 9q26, 10p16, 4q15–16, 10q14–16 and 12p12 respectively. Taking into account the reported QTL mapping results, gene expression analysis and radiation hybrid mapping results, these results suggest that five genes ( CARM1 , JARID1A , JMJD2A , SMYD1 and SMYD2 ) could be good candidate genes for growth and backfat thickness traits.     

Discovery of porcine microRNAs and profiling from skeletal muscle tissues during development

MiRNAs (microRNAs) play critical roles in many important biological processes such as growth and development in mammals. In this study, we identified hundreds of porcine miRNA candidates through in silico prediction and analyzed their expression in developing skeletal muscle using microarray. Microarray screening using RNA samples prepared from a 33-day whole embryo and an extra embryo membrane validated 296 of the predicted candidates. Comparative expression profiling across samples of longissimus muscle collected from 33-day and 65-day post-gestation fetuses, as well as adult pigs, identified 140 differentially expressed miRNAs amongst the age groups investigated. The differentially expressed miRNAs showed seven distinctive types of expression patterns, suggesting possible involvement in certain biological processes. Five of the differentially expressed miRNAs were validated using real-time PCR. In silico analysis of the miRNA-mRNA interaction sites suggested that the potential mRNA targets of the differentially expressed miRNAs may play important roles in muscle growth and development.     

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 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.