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

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.肌发生（Myogenesis）被定义为肌肉的生长、分化与修复过程，其中成肌细胞融合为多核肌纤维是其核心特征之一。人体中其他天然细胞融合事件可见于骨吸收破骨细胞与胎盘合体滋养层细胞，目前尚未发现适用于所有这类天然细胞融合过程的统一基因调控机制。 鉴于人类内源性逆转录病毒（human endogenous retrovirus, ERV）包膜基因参与破骨细胞与合体滋养层细胞的细胞融合过程，本研究对竞技自行车运动员的骨骼肌活检样本展开分析，以探究肌肉特异性特征及ERV包膜基因的表达情况。对比赛季后与赛季前的骨骼肌活检样本，可见肌细胞核数/每毫米肌纤维显著升高2.25倍，肌纤维面积/每个细胞核显著降低2.38倍，而卫星细胞（satellite cells, SCs）数量显著降低3.1倍。本研究推测，在赛季前阶段卫星细胞发生增殖，而在赛季阶段则伴随细胞融合过程的增强。 对22种包膜基因的肌肉活检样本表达分析显示，推定的肌细胞融合相关基因Syncytin-1与Syncytin-3的表达显著升高，非融合性基因erv3的表达同样出现上调。免疫组织化学分析结果表明，Syncytin-1主要定位于表达肌球蛋白重链亚型的肌纤维肌膜。Syncytin-1的细胞受体SLC1A4与SLC1A5在赛季后肌肉组织中的表达量较赛季前显著降低，且仅SLC1A4蛋白表达遍布整个肌纤维。Erv3蛋白在整个肌纤维中均呈强表达，而envK1-7则定位于卫星细胞核与肌细胞核。 Syncytin-1的转录因子PPARγ与RXRα在肌纤维中未检测到蛋白表达，而Syncytin-1的激活因子pCREB-Ser133则富集于卫星细胞核与肌细胞核。在增殖或活跃融合状态的人原代成肌细胞培养体系中，Syncytin-1、Syncytin-3、SLC1A4、PAX7的基因调控模式，以及MyoD1与肌细胞生成素（myogenin）的表达特征均得到验证，且与自行车运动员的骨骼肌活检样本结果一致。体外实验中，用抗Syncytin-1抗体处理成肌细胞可阻断细胞融合过程。本研究结果证实，人类内源性逆转录病毒包膜蛋白，尤其是Syncytin-1，在肌管细胞融合过程中发挥功能性作用。