Schematic model showing the links between the significant changes of muscle-specific attributes with the expression of ERV env genes, their receptors and muscle specific genes relating to cell fusion occurring <i>in vivo</i> (biopsies from cyclists at the pre- and post- competitive seasons) and <i>in vitro</i>.

<p>The top represents the muscle differentiation in cyclists from pre- (PRE) to post-competitive season (POST), whereas the bottom symbolizes the myoblast cultures proliferating in growth media (GM) or differentiating to myotubes in differentiation media (DM). Additionally, since SCs and myonuclei showed positive expression for protein kinase A activated pCREB-Ser133 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132099#pone.0132099.g004" target="_blank">Fig 4</a>) and treatment of primary myoblast cultures with the cAMP stimulator Forskolin did not promote myoblast cell fusion (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132099#pone.0132099.g006" target="_blank">Fig 6</a>), we predict that cAMP may be important for regulating SCs. SC = satellite cells; MP = muscle progenitors; MT = myotubes; PRE = pre-competition; POST = post-competition; GM = growth media; DM = differentiation media; arrow up = significantly up-regulated and arrow down = significantly down-regulated.</p

Immunofluorescence of human primary myoblasts in culture with GM and DM with or without anti-Syncytin-1 (-/+ Ab) for 4 days and analysis by confocal microscopy for nuclei (Hoechst 33342), F-actin (Phalloidin Alexa488), membrane (wheat germ agglutinin Alexa594) and the merge with all.

<p>Note the multinucleated myofibre in DM. DM composite of 96 z-stacks and GM of 17 z-stacks. Bars = 50μm.</p

Muscle cross-sections of cyclists after pre-competitive season show consecutive tissue sections with immuno-localization of MyHC-I and MyHC-IIA as well as the fusogenic ERVW-1 env protein Syncytin-1.

<p>For comparison of Syncytin-1 protein expression with muscle cells the far right picture shows a positive control of Syncytin-1 immunolocalization on normal third trimester placental tissues [left = extra villous trophoblasts (EVT); right = syncytiotrophoblast (SCT)]. The graph represents a semi-quantitative analysis of Syncytin-1 protein signal intensity measured using ImageJ. The Syncytin-1 expression was then correlated with the fiber types, including MyHC-I (set to 100%), MyHC-IIA and MyHC-IIX. Note that the upper panels show IHC and the lower panels show magnifications of the squares. Color code represents fiber type in the lower panels: red = MyHC-I, green = MyHC-IIA and difference of both is marked black = MyHC-IIX. Bars = 100μm. *** = statistically significant (p< 0.005).</p

Gene expression profiles of Syncytin-1, -2, -3, and receptor SLC1A4 as well as different muscle specific genes by qPCR and PCR of human primary myoblasts grown for 1, 2 and 4 days in growth medium (GM) and differentiation medium (DM).

<p>The value of each gene for day 1 in GM was set as 1. Diamond: Significances (P<0.05) comparing GM and DM at different days; *: significant differences (P<0.05) between Forskolin and no Forskolin addition.</p

Myonuclear and satellite cell (SC) characteristics in human vastus lateralis muscle before (PRE) and after (POST) competitive-season.

<p>Data presented as mean ± SEM. POST significantly different from PRE as</p><p>** <i>P</i> < 0.01 and</p><p>*** <i>P</i> < 0.001.</p><p>Myonuclear and satellite cell (SC) characteristics in human vastus lateralis muscle before (PRE) and after (POST) competitive-season.</p

Serial cryocut muscle cross-sections from cyclists after the pre-competitive season demonstrate immuno-localization of different <i>ERV</i> env genes and their receptors, transcription factors (pCREB-Ser133, PPARγ and RXRα).

<p>An antibody recognizing keratins from skin was used as a negative control for skeletal muscle, as well as a negative (neg.) control without primary antibodies. Bars = 25μm.</p

Immunostaining of serial cryocut cross-sections in <i>vastus lateralis</i> muscle of cyclist after the pre-competitive season.

<p>(A) Muscle fibers are shown, where one area is viewed at a higher magnification (white box) in (B) and (C); (D, E, F) co-immunolocalization of Laminin (green), PAX7 (red) and myonuclei (arrowhead) counterstained with DRAQ5 (blue); the marked area in (A-C) represents the same area as shown in (D–F); SCs (arrows) are indicated. (C) Note that PAX7 positive SC is located between the sarcolemma and the basal lamina of the muscle fibre. Bars: 50 μm (A), 10 μm (B) and 5 μm (C–F).</p

Gene expression profiles of ERV env genes and two receptors SLC1A4 and SLC1A5 after qPCR of cyclists after pre- (PRE) and post-competitive season (POST).

<p>Statistical significant genes are in red (PRE = 1-fold) and green (POST = fold). * = P<0.05 and ** = P<0.005.</p