Target genes of myostatin loss-of-function in muscles of late bovine fetuses

Abstract

Background: Myostatin, a muscle-specific member of the Transforming Growth Factor beta family, negatively regulates muscle development. Double-muscled (DM) cattle have a loss-of-function mutation in their myostatin gene responsible for the hypermuscular phenotype. Thus, these animals are a good model for understanding the mechanisms underpinning muscular hypertrophy. In order to identify individual genes or networks that may be myostatin targets, we looked for genes that were differentially expressed between DM and normal (NM) animals (n = 3 per group) in the semitendinosus muscle (hypertrophied in DM animals) at 260 days of fetal development (when the biochemical differentiation of muscle is intensive). A heterologous microarray (human and murine oligonucleotide sequences) of around 6,000 genes expressed in muscle was used.

Results: Many genes were found to be differentially expressed according to genetic type (some with a more than 5-fold change), and according to the presence of one or two functional myostatin allele(s). They belonged to various functional categories. The genes down-regulated in DM fetuses were mainly those encoding extracellular matrix proteins, slow contractile proteins and ribosomal proteins. The genes up-regulated in DM fetuses were mainly involved in the regulation of transcription, cell cycle/apoptosis, translation or DNA metabolism. These data highlight features indicating that DM muscle is shifted towards a more glycolytic metabolism, and has an altered extracellular matrix composition (e.g. down-regulation of COL1A1 and COL1A2, and up-regulation of COL4A2) and decreased adipocyte differentiation (down-regulation of C1QTNF3). The altered gene expression in the three major muscle compartments (fibers, connective tissue and intramuscular adipose tissue) is consistent with the well-known characteristics of DM cattle. In addition, novel potential targets of the myostatin gene were identified (MB, PLN, troponins, ZFHX1B).

Conclusion: Thus, the myostatin loss-of-function mutation affected several physiological processes involved in the development and determination of the functional characteristics of muscle tissue.

Figure 1

Myostatin expression in the ST muscle of experimental fetuses. Myostatin expression was assessed by quantitative RT-PCR using the Sybergreen method. Values are means ± S.E. for n = 6. The highest crossing point (Ct) corresponds to the lowest expression level. DM: double-muscled fetus; NM: non-double-muscled fetus. DM108 is heterozygous for the Q204X mutation.

Figure 2

Hierarchical clustering and Principal Components Analysis of expression data reveal the influence of heterozygosity for the Q204X mutation and the variability of gene expression among individuals. Four individual arrays were used per animal. Clustering (A, B) and PCA (C) were performed using Genesis and GenANOVA respectively. (A): Hierarchical clustering of genes – each column represents an individual array; (B): Hierarchical clustering of animals' mean (-M) expression data; (C): PCA of animals, each projection is representative of an individual array; DM: double-muscled fetus; NM: non-double-muscled fetus. DM108 is heterozygous for the Q204X mutation.