Stac3 is a novel regulator of skeletal muscle development in mice.

The goal of this study was to identify novel factors that mediate skeletal muscle development or function. We began the study by searching the gene expression databases for genes that have no known functions but are preferentially expressed in skeletal muscle. This search led to the identification of the Src homology three (SH3) domain and cysteine rich (C1) domain 3 (Stac3) gene. We experimentally confirmed that Stac3 mRNA was predominantly expressed in skeletal muscle. We determined if Stac3 plays a role in skeletal muscle development or function by generating Stac3 knockout mice. All Stac3 homozygous mutant mice were found dead at birth, were never seen move, and had a curved body and dropping forelimbs. These mice had marked abnormalities in skeletal muscles throughout the body, including central location of myonuclei, decreased number but increased cross-sectional area of myofibers, decreased number and size of myofibrils, disarrayed myofibrils, and streaming Z-lines. These phenotypes demonstrate that the Stac3 gene plays a critical role in skeletal muscle development and function in mice

Generation and analyses of <i>Stac3</i> mutant mice.

<p><b>A</b>, Schematic representation of the targeted <i>Stac3</i> allele. The wild-type (WT) <i>Stac3</i> allele has 12 exons, with exon 3 containing the translation start codon. The targeted allele is inserted with a trapping cassette between exons 1 and 2, and this insertion is expected to disrupt the generation of normal <i>Stac3</i> mRNA. Arrows indicate locations of PCR primers for genotyping. <b>B</b>, Genotypes and life of newborn offspring from intercrossing mice heterozygous for the targeted <i>Stac3</i> allele. “+/+”, “+/−”, and “−/−” indicate wild-type mice, and mice heterozygous and homozygous for the targeted <i>Stac3</i> allele, respectively. A total of 10 litters were included in this analysis. C, Images of a <i>Stac3</i> homozygous mutant mouse and a wild-type littermate. Note the curved body shape and dropping forelimbs in the mutant. <b>D,</b> Genotyping by PCR. See panel A for the locations of two pairs of primers. <b>E</b>, Validation of diminished expression of <i>Stac3</i> mRNA in <i>Stac3</i>^−/− mice by standard RT-PCR. Shown is a representative image. <b>F</b>, Real-time RT-PCR validation of diminished expression of <i>Stac3</i> mRNA in <i>Stac3</i>^−/− mice. Data are expressed as mean ± SEM (n = 4). ** <i>P</i><0.01 <i>vs.</i> “+/+”. <b>G</b>, LacZ staining of E13 wild-type and <i>Stac3</i>^+/− embryos. Shown are representative images. The image on the right highlights the stained somites in an E13 <i>Stac3</i>^+/− embryo.</p

Skeletal muscle-predominant expression of <i>Stac3</i> mRNA in mice.

<p>Tissues from three adult C57BL/6 male mice were analyzed by real-time RT-PCR. In this analysis, <i>18s</i> rRNA was used as an internal control. Data are expressed as mean ± SEM (n = 3). Bars not sharing the same letter labels are different (<i>P</i><0.05). The y-axis is displayed on a log₁₀ scale. M: molecular ladder; SOL: soleus; EDL: extensor digitorum longus; TA: tibialis anterior; GAS: gastrocnemius.</p

Quantitative analyses of extensor digitorum longus (EDL) muscle in newborn <i>Stac3</i>^−/− mice and wild-type and heterozygous littermates.

<p><b>A</b>, Percentage of myofibers that had centrally located nuclei. <b>B</b>, Total number of myosin heavy chain (MyHC)-positive myofibers at the widest girth of EDL muscle. <b>C</b>, Average cross-sectional area of myofibers in EDL muscle. <b>D</b>, Frequency distribution of cross-sectional areas (CSA) of myofibers in EDL muscle. Data are expressed as mean ± SEM (n = 3 to 7). * <i>P</i><0.05 and ** <i>P</i><0.01 <i>vs.</i> wild-type or heterozygous mice.</p

Histological analyses of skeletal muscles in newborn <i>Stac3</i> homozygous mutant mice (−/−) and wild-type (+/+) littermates.

<p><b>A–H</b>, Hematoxylin and eosin staining of diaphragm, tongue, tibialis anterior (TA), and extensor digitorum longus (EDL) muscles. Note the difference in the location of myonuclei between the two genotypes. <b>I</b> and <b>J</b>, Immunohistochemical staining of EDL muscle for myosin heavy chain protein (red) and nuclei (blue). Shown are representative images taken from matched areas. Scale bars  = 50 μm for micrographs A, B, I, and J. Scale bars  = 25 μm for micrographs C-H.</p

Representative electron micrographs of extensor digitorum longus (EDL) muscle of newborn <i>Stac3</i>^−/− mice and <i>Stac3</i>^+/+ littermates.

<p>Note the differences in the location of myonuclei and the size and arrangement of myofibrils between <i>Stac3</i> homozygous mutant and wild-type muscles. Arrows point to myonuclei (micrographs A and B), and arrowheads indicate Z-lines (micrographs C and D). Scale bars  = 10 µm for micrographs A and B; Scale bars  = 500 nm for micrographs C and D.</p

Energy dense, protein restricted diet increases adiposity and perturbs metabolism in young, genetically lean pigs.

Animal models of obesity and metabolic dysregulation during growth (or childhood) are lacking. Our objective was to increase adiposity and induce metabolic syndrome in young, genetically lean pigs. Pre-pubertal female pigs, age 35 d, were fed a high-energy diet (HED; n = 12), containing 15% tallow, 35% refined sugars and 9.1-12.9% crude protein, or a control corn-based diet (n = 11) with 12.2-19.2% crude protein for 16 wk. Initially, HED pigs self-regulated energy intake similar to controls, but by wk 5, consumed more (P<0.001) energy per kg body weight. At wk 15, pigs were subjected to an oral glucose tolerance test (OGTT); blood glucose increased (P<0.05) in control pigs and returned to baseline levels within 60 min. HED pigs were hyperglycemic at time 0, and blood glucose did not return to baseline (P = 0.01), even 4 h post-challenge. During OGTT, glucose area under the curve (AUC) was higher and insulin AUC was lower in HED pigs compared to controls (P = 0.001). Chronic HED intake increased (P<0.05) subcutaneous, intramuscular, and perirenal fat deposition, and induced hyperglycemia, hypoinsulinemia, and low-density lipoprotein hypercholesterolemia. A subset of HED pigs (n = 7) was transitioned back to a control diet for an additional six weeks. These pigs were subjected to an additional OGTT at 22 wk. Glucose AUC and insulin AUC did not improve, supporting that dietary intervention was not sufficient to recover glucose tolerance or insulin production. These data suggest a HED may be used to increase adiposity and disrupt glucose homeostasis in young, growing pigs

Effect of chronic dietary treatment on carcass characteristics¹.

1<p>Data are least-square means per treatment, CON, n = 11; HED, n = 5; INT, n = 7.</p>2<p>High-energy diet fed over a 16 wk period.</p>3<p>Intervention at the end of HED. Pigs were transitioned to a control diet for a 6-wk period.</p>4<p>Pooled SE of treatment groups.</p>5<p>Data are body weight-corrected.</p>6<p>Calculated percent fat on carcass. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072320#pone.0072320-Schinckel1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072320#pone.0072320-Wagner1" target="_blank">[27]</a>.</p>a,b,c<p>Means in a row without a common superscript differ, <i>P</i><0.05.</p>x,y,z<p>Means in a row without a common superscript differ, <i>P</i><0.10.</p

Growth traits of pigs fed control (CON) or high-energy diet (HED).

<p>Pigs were fed control (CON, n = 11) or high energy diets (HED; n = 12) containing 15% fat and 35% refined sugars for 16 weeks Adjusted ultrasonic <i>Longissimus dorsi</i> (LD) muscle depth (A), adjusted ultrasonic subcutaneous (USubQ) fat depth (B), USubQ fat deposition adjusted by LD depth (C). USubQ fat and LD depth are adjusted by BW. Data are presented as LS means ± SE; asterisks indicate significant differences from control within week at <i>P</i><0.05.</p

Changes in BW (A) and metabolizable energy intake (B) during a 16 wk dietary treatment.

<p>Pigs were fed control (CON, n = 11) or high energy diets (HED; n = 12) containing 15% fat and 35% refined sugars for 16 weeks. Data are presented as LS means ± SE; asterisks indicate significant differences at <i>P</i><0.05.</p