Phosphatidylserine directly and positively regulates fusion of myoblasts into myotubes
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.
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2019, International Journal of PharmaceuticsCitation 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).