Phosphatidylserine directly and positively regulates fusion of myoblasts into myotubes

https://doi.org/10.1016/j.bbrc.2011.08.128Get rights and content

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.

Highlights

PS broadly and persistently trans-locates to the outer leaflet of plasma membrane during myoblast fusion into myotubes. ► Robust myotubes are formed when PS liposomes are added exogenously. ► PS increases the width of de novo myotubes and the numbers of myonuclei, but not the myotube length. ► Annexin V or PS antibody inhibits myotube formation by masking exposed PS.

Introduction

The cell plasma membrane largely consists of a phospholipid bilayer containing phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE) [1], [2]. PS is a hallmark of apoptosis because in normal healthy cells it is localized to the inner lipid layer, or leaflet, of the plasma membrane, but during early apoptosis PS localizes to the outer leaflet by phospholipid(s) scramblase [3], [4]. The exposed PS recruits various immune cells and in particular is one of the ‘eat me’ signals present on the surface of apoptotic cells signaling macrophage engulfment [5].

During muscle maintenance and repair, muscle stem or satellite, cells activate, proliferate and give rise to myoblasts, which can both proliferate and differentiate into myotubes. Myotubes are the final product of the muscle lineage, where each cell is post-mitotic and multinucleated and is produced by the fusion of many myoblasts [6]. Some growth factors [7], [8], [9], macrophages [10], [11], and leukocytes in general [12] can control muscle regeneration and specifically, cell fusion. However, the molecular nature of fusogen(s) remains unknown and it is yet to be determined what specific molecules in the plasma membrane of myoblasts play a crucial role during fusion of these cells into myotubes or myofibers. To date, several molecules that are thought to control myoblast fusion, such as caveolin-3 [13], [14], myoferin [15] and nephrin [16], were reported to cluster at the cell membrane, specifically localizing at the cell–cell contact regions during myotube formation. However, none of these molecules were demonstrated to directly regulate the membrane fusion process. Additionally, damaging stimuli that are known to promote apoptosis as well as direct experimental induction of apoptosis have been shown to enhance myotube formation [17], [18]. However, no molecular mechanism connecting apoptosis with myoblast fusion was revealed, until now.

In this paper, we demonstrate that one of the key membrane-bound regulators of myoblast fusion into myotubes is PS, which provides the first molecular identification of a fusogen and gives an explanation as to why induction of apoptosis promotes terminal muscle differentiation.

Section snippets

Cell culture of mouse primary myoblasts

Primary myoblasts were isolated from young (2–4 month) C 57/Bl6 mice (Jackson Lab., ME) as described [20]. Briefly, tibialis and gastrocnemius muscles were dissociated into myofibers by a digest for 1 h at 37 °C in DMEM with 250 units/ml Collagenase Type II (Sigma–Aldrich, MO), containing 1% Penicillin–Streptomycin. Digested muscles were washed with PBS two times and triturated into myofibers in Ham’s F10 (Mediatech, VA), 20% Bovine growth serum (BGS; Hyclone, IL) and 1% Penicillin–streptomycin. In

Phosphatidylserine is enriched at cell–cell contact regions during myoblast differentiation into myotubes

To determine whether phosphatidylserine (PS) is exposed on the outer leaflet of the plasma membrane during fusion of primary myoblast into de novo myotubes, mitogenic growth medium (GM) was replaced by the mitogen-low differentiation medium (DM) where myoblasts normally form myotubes by 48 h, and these cell cultures were stained by Alexa Fluor 488 conjugated-Annexin V (Fig. 1). H2O2 treated myoblasts were used as a positive control for PS translocation and Annexin V staining, as well as for

Discussion

Phosphatidylserine (PS) is a hallmark of eukaryotic cell apoptosis and its translocation from the inner to outer leaflet of the membrane is well known [3], [4]. While previous work suggested that some membrane bound proteins may regulate myoblast fusion and that apoptosis generally promotes myotube formation, the molecular regulator of this event remained unknown and the mechanism by which apoptosis promotes myotube generation was undefined. This work is the first to provide a molecular

Acknowledgments

We would like to thank Dr. Michael Conboy for helpful discussion and critical reading of the manuscript. This work was supported by NIH/NIA AG 027252 and CIRM RN1-00532 grants to IMC.

References (25)

  • M.S. Bretscher

    Asymmetrical lipid bilayer structure for biological membranes

    Nature-New Biology

    (1972)
  • V.A. Fadok et al.

    Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages

    Journal of Immunology

    (1992)
  • Cited by (58)

    • Regulation of the myoblast fusion reaction for muscle development, regeneration, and adaptations

      2022, Experimental Cell Research
      Citation Excerpt :

      The best candidates for the initiation of the signaling cascade is phosphatidylserine (PS) activation of the brain-specific angiogenesis inhibitor (Bai) family of proteins. In normal cells, PS is in the inner leaflet of the plasma membrane but flipping to the outside is proposed as a trigger for fusion of myoblasts and other cell types that fuse [40–42]. Bai3 is a GPCR that promotes myocyte fusion in the chick and mouse and functions to link cell surface signals to actin nucleation pathways by recruiting ELMO/DOCK to the membrane, which activates Rac1 resulting in cytoskeletal rearrangements [43].

    • Mechanisms regulating myoblast fusion: A multilevel interplay

      2020, Seminars in Cell and Developmental Biology
    • Uptake and intracellular distribution of different types of nanoparticles in primary human myoblasts and myotubes

      2019, International Journal of Pharmaceutics
      Citation Excerpt :

      Our liposomes contain DSPC, a derivative of phosphatidylcholine (PC). It has been reported that PC-liposomes decrease primary murine myoblasts fusion and myotube elongation; however the uptake and the intracellular distribution of PC-liposomes were not evaluated (Jeong and Conboy 2011). The internalization of liposomes has been demonstrated also in C2C12 by flow cytometry and an increased uptake/association with the cells was reported for polydopamine (PDA)-coated liposomes (van der Westen et al., 2012).

    View all citing articles on Scopus
    View full text