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

Frese S, Ruebner M, Suhr F, Konou TM, Tappe KA, Toigo M, Jung HH, Henke C, Steigleder R, Strissel PL, Huebner H, Beckmann MW, van der Keylen P, Schoser B, Schiffer T, Frese L, Bloch W, Strick R - PLoS ONE (2015)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cardiovascular Research and Sport Medicine, Department of Molecular and Cellular Sport Medicine, German Sport University Cologne, Am Sportpark Muengersdorf, Cologne, Germany; University Hospital Zurich, Department of Neurology, Frauenklinikstrasse, Zurich, Switzerland; Institute of Human Movement Sciences and Sport, Exercise Physiology, ETH Zurich, Winterthurerstrasse, Zurich, Switzerland.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

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.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).
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pone.0132099.g005: 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.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).

Mentions: In order to investigate, if human muscle demonstrated significant expression of ERV env genes and their receptors, RNA of muscle biopsies from the cyclists was analysed for 22 ERV env genes by qPCR and for the Syncytin-1 receptors SLC1A4 and SLC1A5 by semi-quantitative PCR (Fig 3 and S1 Table). Only Syncytin-1, Syncytin-3, and erv3 showed a significant increase of expression in post-competitive when compared to the pre-competitive season, whereas envFc1 and envFc2 showed a significant decrease (Fig 3 and S1 Table). All other ERV env genes were not significantly changed in expression. Importantly, Syncytin-1 and Syncytin-3 were previously shown as fusogenic proteins participating in cell-cell fusions [5] [54]. The Syncytin-1 cellular receptors SLC1A4 and SLC1A5 demonstrated a significant decrease of expression in the post-competitive compared to the pre-competitive season (Fig 3). Further analysis using IHC of ERV env genes, their receptors, as well as Syncytin-1 transcription factors was performed using biopsies from cyclists in the pre-competitive season and demonstrated different cellular locations (Fig 4). Results showed a homogenous Syncytin-1 protein expression throughout myofibers with enrichment at the membrane or sarcolemma. Interestingly, Syncytin-1 myofiber expression was similar in intensity to term placental control tissue, where Syncytin-1 is considered strongly expressed [55] (Fig 5). The Syncytin-1 receptor SLC1A4 only demonstrated positive expression throughout myofibers, whereas the second receptor SLC1A5 was negative. The other fusogenic env gene Syncytin-2, which was not significantly differentially expressed using qPCR, demonstrated positive expression throughout myofibers, whereas its receptor MFSD2 was mainly enriched at the sarcolemma (Fig 4). We were not able to analyze Syncytin-3 expression due to no currently available antibody. The erv3 env protein showed strong positive protein expression throughout the myofiber, whereas, interestingly envK expression localized at the myonuclei and nuclei of SCs. Examining protein expression of Syncytin-1 transcription factors in myofibers demonstrated a unique pCREB-Ser133 protein expression at the basal lamina of the SCs as well as nuclear SCs and myonuclear expression (Fig 4). In contrast the Syncytin-1 transcription factors PPARγ and RXRα, showed no expression in myofibers supporting no regulatory role. According to the literature, PPARγ was in contrast to PPARδ very lowly expressed in healthy skeletal muscle [56].


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

Frese S, Ruebner M, Suhr F, Konou TM, Tappe KA, Toigo M, Jung HH, Henke C, Steigleder R, Strissel PL, Huebner H, Beckmann MW, van der Keylen P, Schoser B, Schiffer T, Frese L, Bloch W, Strick R - PLoS ONE (2015)

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.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).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4495930&req=5

pone.0132099.g005: 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.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).
Mentions: In order to investigate, if human muscle demonstrated significant expression of ERV env genes and their receptors, RNA of muscle biopsies from the cyclists was analysed for 22 ERV env genes by qPCR and for the Syncytin-1 receptors SLC1A4 and SLC1A5 by semi-quantitative PCR (Fig 3 and S1 Table). Only Syncytin-1, Syncytin-3, and erv3 showed a significant increase of expression in post-competitive when compared to the pre-competitive season, whereas envFc1 and envFc2 showed a significant decrease (Fig 3 and S1 Table). All other ERV env genes were not significantly changed in expression. Importantly, Syncytin-1 and Syncytin-3 were previously shown as fusogenic proteins participating in cell-cell fusions [5] [54]. The Syncytin-1 cellular receptors SLC1A4 and SLC1A5 demonstrated a significant decrease of expression in the post-competitive compared to the pre-competitive season (Fig 3). Further analysis using IHC of ERV env genes, their receptors, as well as Syncytin-1 transcription factors was performed using biopsies from cyclists in the pre-competitive season and demonstrated different cellular locations (Fig 4). Results showed a homogenous Syncytin-1 protein expression throughout myofibers with enrichment at the membrane or sarcolemma. Interestingly, Syncytin-1 myofiber expression was similar in intensity to term placental control tissue, where Syncytin-1 is considered strongly expressed [55] (Fig 5). The Syncytin-1 receptor SLC1A4 only demonstrated positive expression throughout myofibers, whereas the second receptor SLC1A5 was negative. The other fusogenic env gene Syncytin-2, which was not significantly differentially expressed using qPCR, demonstrated positive expression throughout myofibers, whereas its receptor MFSD2 was mainly enriched at the sarcolemma (Fig 4). We were not able to analyze Syncytin-3 expression due to no currently available antibody. The erv3 env protein showed strong positive protein expression throughout the myofiber, whereas, interestingly envK expression localized at the myonuclei and nuclei of SCs. Examining protein expression of Syncytin-1 transcription factors in myofibers demonstrated a unique pCREB-Ser133 protein expression at the basal lamina of the SCs as well as nuclear SCs and myonuclear expression (Fig 4). In contrast the Syncytin-1 transcription factors PPARγ and RXRα, showed no expression in myofibers supporting no regulatory role. According to the literature, PPARγ was in contrast to PPARδ very lowly expressed in healthy skeletal muscle [56].

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cardiovascular Research and Sport Medicine, Department of Molecular and Cellular Sport Medicine, German Sport University Cologne, Am Sportpark Muengersdorf, Cologne, Germany; University Hospital Zurich, Department of Neurology, Frauenklinikstrasse, Zurich, Switzerland; Institute of Human Movement Sciences and Sport, Exercise Physiology, ETH Zurich, Winterthurerstrasse, Zurich, Switzerland.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus