Phosphatidylserine directly and positively regulates fusion of myoblasts into myotubes

Abstract

Cell membrane consists of various lipids such as phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). Among them, PS is a molecular marker of apoptosis, because it is located to the inner leaflet of plasma membrane generally but it is moved to the outer leaflet during programmed cell death. The process of apoptosis has been implicated in the fusion of muscle progenitor cells, myoblasts, into myotubes. However, it remained unclear whether PS regulates muscle cell differentiation directly. In this paper, localization of PS to the outer leaflet of plasma membrane in proliferating primary myoblasts and during fusion of these myoblasts into myotubes is validated using Annexin V. Moreover, we show the presence of PS clusters at the cell-cell contact points, suggesting the importance of membrane ruffling and PS exposure for the myogenic cell fusion. Confirming this conclusion, experimentally constructed PS, but not PC liposomes dramatically enhance the formation of myotubes from myoblasts, thus demonstrating a direct positive effect of PS on the muscle cell fusion. In contrast, myoblasts exposed to PC liposomes produce long myotubes with low numbers of myonuclei. Moreover, pharmacological masking of PS on the myoblast surface inhibits fusion of these cells into myotubes in a dose-dependent manner.

Figure 1

PS was exposed on the outer leaflet of the plasma membrane and was enriched at the cell-cell contact regions during myoblast differentiation into myotubes. H₂O₂ treated myoblasts were used as a positive control for apoptosis and for PS translocation (a, b). Apoptotic cell nuclei were detected with PI (red). The myoblasts were cultured in GM (c, d) and DM (e, f) for 48 hours and then stained using Annexin V (green). Scale bar, 20 µm.

Figure 2

PS treated myoblasts form robust myotubes. (A) PS 100%, PS:PC (50%:50%) or PC 100% liposomes were added to myoblasts that were cultured in DM for 48 hours and then fixed with 70% EtOH for 24 hours and stained with anti-eMyHC specific antibody (red). Nuclei were stained with Hoechst. Scale bar, 100 µm. (B) The fusion index was calculated by quantifying the percent of myonuclei in myotubes that have more than 2 nuclei, out of the total cell nuclei number. (C, D) The length and the width of the de-novo formed myotubes that have more than 2 nuclei were quantified. *p= 0.0006; **p= 0.0004 (B). *p= 0.0388; **p=0.0398 (C). *p=0.0248; **p=0.0024 (D).

Figure 3

Myoblast fusion index is decreased by masking PS with Annexin V or PS-specific antibody. (A) Annexin V or (B) PS-specific antibody was added to myoblasts in DM for 48 hours. De-novo myotubes were detected by immunostaining with eMyHC-specific antibody. Scale bar, 100 µm. The percent of myonuclei in myotubes out of total cell nuclei number was quantified for Annexin V (C) and for PS-specific antibody (D). *p=0.0002; **p=0.0005.