Long-Term Endurance Exercise in Humans Stimulates Cell Fusion of Myoblasts along with Fusogenic Endogenous Retroviral Genes In Vivo

Myogenesis is defined as growth, differentiation and repair of muscles where cell fusion of myoblasts to multinucleated myofibers is one major characteristic. Other cell fusion events in humans are found with bone resorbing osteoclasts and placental syncytiotrophoblasts. No unifying gene regulation for natural cell fusions has been found. We analyzed skeletal muscle biopsies of competitive cyclists for muscle-specific attributes and expression of human endogenous retrovirus (ERV) envelope genes due to their involvement in cell fusion of osteoclasts and syncytiotrophoblasts. Comparing muscle biopsies from post- with the pre-competitive seasons a significant 2.25-fold increase of myonuclei/mm fiber, a 2.38-fold decrease of fiber area/nucleus and a 3.1-fold decrease of satellite cells (SCs) occurred. We propose that during the pre-competitive season SC proliferation occurred following with increased cell fusion during the competitive season. Expression of twenty-two envelope genes of muscle biopsies demonstrated a significant increase of putative muscle-cell fusogenic genes Syncytin-1 and Syncytin-3, but also for the non-fusogenic erv3. Immunohistochemistry analyses showed that Syncytin-1 mainly localized to the sarcolemma of myofibers positive for myosin heavy-chain isotypes. Cellular receptors SLC1A4 and SLC1A5 of Syncytin-1 showed significant decrease of expression in post-competitive muscles compared with the pre-competitive season, but only SLC1A4 protein expression localized throughout the myofiber. Erv3 protein was strongly expressed throughout the myofiber, whereas envK1-7 localized to SC nuclei and myonuclei. Syncytin-1 transcription factors, PPARγ and RXRα, showed no protein expression in the myofiber, whereas the pCREB-Ser133 activator of Syncytin-1 was enriched to SC nuclei and myonuclei. Syncytin-1, Syncytin-3, SLC1A4 and PAX7 gene regulations along with MyoD1 and myogenin were verified during proliferating or actively-fusing human primary myoblast cell cultures, resembling muscle biopsies of cyclists. Myoblast treatment with anti-Synycytin-1 abrogated cell fusion in vitro. Our findings support functional roles for ERV envelope proteins, especially Syncytin-1, contributing to cell fusion of myotubes.status: publishe

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

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

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