Genes with physiological roles in callipyge muscle hypertrophy

Hui Yu, Purdue University

Abstract

Callipyge sheep is an excellent model to study genes regulation in muscle growth since the up-regulation of DLK1 and/or RTL1 results in extreme postnatal muscle hypertrophy in loin and hindquarters. DLK1 and/or RTL1 are the primary inducers of muscle hypertrophy due to the inheritance model for the callipyge phenotype. The overall aim of this dissertation is to study the physiological pathways responding to the up-regulation of DLK1 and/or RTL1 in the hypertrophied muscles. Microarray analysis of gene expression identified 375 genes that were differentially expressed in callipyge semimembranosus. Twenty-five transcripts were further verified by quantitative PCR in two hypertrophied muscles (semimembranosus and longissimus dorsi) and one non-hypertrophied muscle (supraspinatus ). In order to identify the genes that are consistently co-expressed with DLK1/RTL1, the current study extends the gene expression analysis on these 25 genes in 4 hypertrophied muscles (longissimus dorsi, semimembranosus, semitendinosus and triceps brachii) and 3 non-hypertrophied muscles (supraspinatus, infraspinatus and heart). The quantitative PCR results showed that DLK1 expression was significantly increased in the 4 hypertrophied muscles but not in the 3 non-hypertrophied muscles. However, RTL1 was up-regulated in both hypertrophied and non-hypertrophied muscles, which confirmed that RTL1 is not sufficient to induce muscle hypertrophy but it may act in concert with DLK1 to increase muscle mass in callipyge lambs. Five other genes, including PARK7, DNTTIP1, SLC22A3, METTL21E and PDE4D, were consistently co-expressed with DLK1, herein they were the potential transcriptional target genes responding to DLK1 signaling. Since the hypertrophied muscles have a fiber type shift to a more fast-twitch, glycolytic muscle fibers and less oxidative fibers, DNTTIP1, METTL21E and PARK7 were examined for their effects on MYH promoter activity using luciferase assays. The results showed that both PARK7 and DNTTIP1 had an effect to increase MYH4 and decrease MYH7 luciferase activity, similar to the effects of DLK1. In contrast, METTL21E increase MYH7 and decreased MYH4 luciferase activity. In DLK1-treated myotubes, the mRNA expression of Myh4 and DNTTIP1 were both increased, suggesting DLK1 may regulate Myh4 expression through elevated DNTTIP1 expression. Though PARK7 was not up-regulated in DLK1-treated cells, its increased levels in hypertrophied muscles still indicated its important role in callipyge muscle hypertrophy. PARK7 has been shown to positively regulate PI3K/AKT pathway by suppressing the phosphatase activity of PTEN in mouse fibroblasts. The aim of the second study is to determine whether the elevation of PARK7 was part of the physiological mechanism for muscle hypertrophy in callipyge lambs. Primary myoblasts isolated from Park7 (+/+) and Park7 (-/-) mice were used to examine the effect of differential expression of Park7. The Park7 (+/+) myotubes had significantly larger diameters and more total sacomeric myosin expression than Park7 (-/-) myotubes. IGF1 treatment increased the mRNA abundance of Myh4, Myh7 and Myh8 between 20–40% in Park7 (+/+) myotubes relative to Park7 (-/-). The activity of PI3K/AKT pathway was determined by examination the level of phosphorylation of AKT. The basal level of AKT phosphorylation (S473) was increased in Park7 (+/+) myotubes and this increase was substantially elevated in Park7 (+/+) myotubes at all levels of IGF1 supplementation. The differential levels of AKT phosphorylation were eliminated by the addition of a PTEN inhibitor, suggesting PARK7 inhibited PTEN activity in myotubes. After removal of IGF1, the Park7 (+/+) myotubes could still maintain about 20% higher signal intensity through 12 hours. These combined results suggested PARK7 can positively regulate the PI3K/AKT pathway by inhibition of PTEN phosphatase activity. The elevated expression of PARK7 in the muscles of callipyge lambs would be expected to lead to increased muscle growth in response to the normal IGF1 signaling present in young growing lambs. The results presented in this thesis unveiled the genes consistently co-expressing with DLK1 in callipyge lambs and identified DNTTIP1 as a secondary transcriptional effector gene responding to DLK1 signaling. Furthermore, this thesis suggested a positive role of PARK7 in callipyge muscle hypertrophy.

Degree

Ph.D.

Advisors

Bidwell, Purdue University.

Subject Area

Agriculture|Animal sciences

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