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Callipyge sheep exhibit extreme postnatal muscle hypertrophy in the loin and hindquarters as a result of a single nucleotide polymorphism (SNP) in the imprinted DLK1-DIO3 domain on ovine chromosome 18. The callipyge SNP up-regulates the expression of surrounding transcripts when inherited in cis without altering their allele-specific imprinting status. The callipyge phenotype exhibits polar overdominant inheritance since only paternal heterozygous animals have muscle hypertrophy. Two studies were conducted profiling gene expression in lamb muscles to determine the down-stream effects of over-expression of paternal allele-specific DLK1 and RTL1 as well as maternal allele-specific MEG3, RTL1AS and MEG8, using Affymetrix bovine expression arrays. A total of 375 transcripts were differentially expressed in callipyge muscle and 25 transcripts were subsequently validated by quantitative PCR. The muscle-specific expression patterns of most genes were similar to DLK1 and included genes that are transcriptional repressors or affect feedback mechanisms in beta-adrenergic and growth factor signaling pathways. One gene, phosphodiesterase 7A had an expression pattern similar to RTL1 expression indicating a biological activity for RTL1 in muscle. Only transcripts that localize to the DLK1-DIO3 domain were affected by inheritance of a maternal callipyge allele. Callipyge sheep are a unique model to study over expression of both paternal allele-specific genes and maternal allele-specific non-coding RNA with an accessible and nonlethal phenotype. This study has identified a number of genes that are regulated by DLK1 and RTL1 expression and exert control on postnatal skeletal muscle growth. The genes identified in this model are primary candidates for naturally regulating postnatal muscle growth in all meat animal species, and may serve as targets to ameliorate muscle atrophy conditions including myopathic diseases and age-related sarcopenia.

The callipyge mutation causes postnatal muscle hypertrophy in heterozygous lambs that inherit a paternal callipyge allele (+/CLPG). Our hypothesis was that the up-regulation of one or both of the affected paternally expressed genes (DLK1 or PEG11) initiates changes in biochemical and physiological pathways in skeletal muscle to induce hypertrophy. The goal of this study was to identify changes in gene expression during the onset of muscle hypertrophy to identify the pathways that are involved in the expression of the callipyge phenotype. Gene expression was analysed in longissimus dorsi total RNA from lambs at 10, 20, and 30 days of age using the Affymetrix Bovine Expression Array. An average of 40.6% of probe sets on the array was detected in sheep muscle. Data were normalized and analysed using a two-way anova for genotype and age effects with a false discovery rate of 0.10. From the anova, 13 genes were significant for the effect of genotype and 13 were significant for effect of age (P < 0.10). No significant age-by-genotype interactions were detected (P > 0.10). Of the 13 genes indicating an effect of genotype, quantitative PCR assays were developed for all of them and tested on a larger group of animals from 10 to 200 days of age. Nine genes had significantly elevated transcript levels in callipyge lambs. These genes included phosphofructokinase, a putative methyltransferase protein, a cAMP phosphodiesterase, and the transcription factor DNTTIP1.

Calpains are non-lysosomal proteases involved in myofibrillar protein degradation. To facilitate studying the expression of the porcine calpain genes and their influence on protein accretion, we have cloned partial cDNAs for mu- and m-calpain from porcine skeletal muscle via PCR amplification. A 289 bp fragment for mu-calpain and a 629 bp fragment for m-calpain were cloned into the EcoRV site of pBluescript II KS+ vector. The nucleotide sequence for porcine mu-calpain and m-calpain were 92% and 90% identical to corresponding regions of rabbit mu- and m-calpain, respectively. The deduced amino acid sequences for both mu- and m-calpain share 94% identity with respective rabbit mu- and m-calpains. Isoform specificity was validated by Southern hybridization of mu- and m-calpain probes with cloned mu- and m-calpain fragments and Northern hybridization with pig muscle mRNA. These clones will be used to evaluate the role of calpain expression in muscle hypertrophy.