Phosphorylation of AKT (S473) protein in <i>Park7</i> (+/+) and <i>Park7</i> (−/−) myotubes.

<p>Myoblasts were induced to differentiation for 72 hours and treated with IGF1 at the indicated concentrations for 15 minutes. There was increased phosphorylation of AKT (P-AKT) in <i>Park7</i> (+/+) myotubes relative to total AKT (AKT) in myotubes in fusion medium (5% horse serum). There was a substantial increase in AKT phosphorylation in <i>Park7</i> (+/+) myotubes relative to <i>Park7</i> (−/−) myotubes at all IGF1 concentrations. α-Tubulin was the control for protein loading.</p

Sustained activity of AKT phosphorylation (S473) in <i>Park7</i> (+/+) and <i>Park7</i> (−/−) myotubes after IGF1 stimulation.

<p>Mature myotubes were stimulated with IGF1 (12.5 ng/mL) for 15 minutes. Total protein was extracted at the indicated time after removal of IGF1. A: Western blot analysis showed a higher level of AKT phosphorylation in <i>Park7</i> (+/+) relative to <i>Park7</i> (−/−) myotubes. PARK7 blotting confirmed the cell genotypes and α-tubulin blotting was used as a loading control. B: Analysis of the signal intensity for P-AKT/total AKT ratio (adj. signal intensity) showed the <i>Park7</i> (+/+) myotubes had higher AKT phosphorylation without added IGF1 and had a 3 fold higher level of P-AKT/total AKT than <i>Park7</i> (−/−) myotubes at 45 minutes after IGF1 treatment. The decline in AKT phosphorylation was similar between the genotypes after 90 minutes with <i>Park7</i> (+/+) myotubes generally maintaining approximately 20% higher signal intensity. This experimental design was replicated twice on individual blots with similar results. The signal intensity data was derived from the blot shown in A.</p

Park7 Expression Influences Myotube Size and Myosin Expression in Muscle

<div><p>Callipyge sheep exhibit postnatal muscle hypertrophy due to the up-regulation of <i>DLK1</i> and/or <i>RTL1</i>. The up-regulation of <i>PARK7</i> was identified in hypertrophied muscles by microarray analysis and further validated by quantitative PCR. The expression of PARK7 in hypertrophied muscle of callipyge lambs was confirmed to be up-regulated at the protein level. PARK7 was previously identified to positively regulate PI3K/AKT pathway by suppressing the phosphatase activity of PTEN in mouse fibroblasts. The purpose of this study was to investigate the effects of PARK7 in muscle growth and protein accretion in response to IGF1. Primary myoblasts isolated from <i>Park7</i> (+/+) and <i>Park7</i> (−/−) mice were used to examine the effect of differential expression of <i>Park7</i>. The <i>Park7</i> (+/+) myotubes had significantly larger diameters and more total sarcomeric myosin expression than <i>Park7</i> (−/−) myotubes. IGF1 treatment increased the mRNA abundance of <i>Myh4, Myh7 and Myh8</i> between 20-40% in <i>Park7</i> (+/+) myotubes relative to <i>Park7</i> (−<i>/</i>−). The level of AKT phosphorylation was increased in <i>Park7</i> (+/+) myotubes at all levels of IGF1 supplementation. After removal of IGF1, the <i>Park7</i> (+/+) myotubes maintained higher AKT phosphorylation through 3 hours. PARK7 positively regulates the PI3K/AKT pathway by inhibition of PTEN phosphatase activity in skeletal muscle. The increased PARK7 expression can increase protein synthesis and result in myotube hypertrophy. These results support the hypothesis that elevated expression of <i>PARK7</i> in callipyge muscle would increase levels of AKT activity to cause hypertrophy in response to the normal IGF1 signaling in rapidly growing lambs. Increasing expression of PARK7 could be a novel mechanism to increase protein accretion and muscle growth in livestock or help improve muscle mass with disease or aging.</p></div

Detection of total sarcomere myosin expression in myotubes by ELISA.

<p>There were significant effects for genotype and IGF1 treatment on total myosin, but there was no significant interaction. The <i>Park7</i> (+/+) myotubes had significantly more total sarcomere myosin expression than <i>Park7</i> (−/−) myotubes and addition of IGF1 at all concentrations induced significant higher sarcomere myosin compared to no added IGF1.</p

PARK7 protein expression in <i>vastus lateralis</i>.

<p>PARK7 protein expression was not detectable in <i>vastus lateralis</i> in <i>Park7</i> (−/−) mice in two replicated experiments of two animals per genotype. α-Tubulin was used as a control to show equal protein loading.</p

Comparison of myotube size between <i>Park7</i> (−/−) and <i>Park7</i> (+/+) genotypes.

<p>A: Primary myoblasts were induced to differentiate for 72 hours and stained with an antibody for total myosin heavy chain (red: MF20) and DNA (blue: DAPI). B: No significant differences were detected between the two genotypes for fusion index. C: The distributions of myotube diameters for the two genotypes are shown. The mean diameter for <i>Park7</i> (+/+; 1.70±0.24) myotubes were significantly larger (P<.0001) than <i>Park7</i> (−/−; 1.05±0.025) myotubes.</p

PARK7 protein expression level in sheep skeletal muscles.

<p>A: There were higher levels of PARK7 and DLK1 proteins in the semimembranosus (SM) of callipyge lambs (+/<i>C</i>) relative to normal lambs (+/+), but no difference in PARK7 or DLK1 protein levels in the supraspinatus (SS). B: Quantitative analysis of PARK7 protein expression was significantly higher in the SM of callipyge lambs. α-Tubulin was the control for protein loading. Data shown are mean signal intensity ± SE for the PARK7 without normalization using three animals per genotype. The p-values for comparisons between genotypes for each muscle are shown.</p

Diagrammatic representation of the organization of imprinted genes located at the telomeric end of ovine chromosome 18.

<p>(A) Representation of the approximate 1 Mbp region from <i>BEGAIN</i> to <i>DIO3</i>. The core imprinted genes affected by the callipyge mutation are colored while imprinted genes unaffected by the mutation are shown in grey. Paternally expressed genes are shaded blue and maternally expressed genes are shaded pink. The direction of transcription of each gene is indicated by the arrow in the gene symbol. Introns are not shown. The red asterisk denotes the position of the callipyge point mutation (<i>CLPG</i>). The precise lengths of the maternally expressed genes, which all produce non-coding RNAs, are unclear. The diagram is based on that deduced by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008638#pone.0008638-Georges1" target="_blank">[53]</a> supplemented with annotation for a miRNA cluster (<i>MIRG</i>) deduced by comparative sequence analyses with the orthologous murine and human sequence regions. (B) Representations of the <i>PEG11</i> and <i>PEG11as</i> genes. A large black arrow denotes the direction of transcription of each gene. Small arrows show the relative positions of PCR primers. The region of <i>PEG11</i> expressed as a recombinant protein (rPEG11) is also shown. The precise length of the <i>PEG11as</i> gene is unclear but it extends beyond the <i>PEG11</i> gene in both directions (represented by broken lines).</p

Ovine PEG11 peptides detected by mass spectrometry¹.

1<p>Data from the seven different peptide fragments identified by LC-MALDI-MS/MS and LC-ESI-MS/MS are tabulated with their corresponding theoretical and experimental peptide masses (Da), as well as the difference in mass (Δm).</p

Conserved regions of the ovine <i>PEG11</i> gene correspond with antisense miRNA.

<p>The <i>PEG11</i> open reading frame is shown in blue while the locations of antisense miRNA (mir-431, mir-433, mir-127, mir-432 and mir-136) are shown in red. The black arrow denotes the direction of transcription of <i>PEG11</i>. The graph shows the extent of <i>PEG11</i> gene conservation using the UCSC PhastCons Conserved Elements 17-way Vertebrate Multiz Alignment and Conservation tool <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008638#pone.0008638-Kent1" target="_blank">[34]</a>.</p