Contribution of Extracellular Vesicles in Rebuilding Injured Muscles

Abstract

Skeletal myofibers are injured due to mechanical stresses experienced during physical activity, or due to myofiber fragility caused by genetic diseases. The injured myofiber needs to be repaired or regenerated to restore the loss in muscle tissue function. Myofiber repair and regeneration requires coordinated action of various intercellular signaling factors-including proteins, inflammatory cytokines, miRNAs, and membrane lipids. It is increasingly being recognized release and transmission of these signaling factors involves extracellular vesicle (EV) released by myofibers and other cells in the injured muscle. Intercellular signaling by these EVs alters the phenotype of their target cells either by directly delivering the functional proteins and lipids or by modifying longer-term gene expression. These changes in the target cells activate downstream pathways involved in tissue homeostasis and repair. The EVs are heterogeneous with regards to their size, composition, cargo, location, as well as time-course of genesis and release. These differences impact on the subsequent repair and regeneration of injured skeletal muscles. This review focuses on how intracellular vesicle production, cargo packaging, and secretion by injured muscle, modulates specific reparative, and regenerative processes. Insights into the formation of these vesicles and their signaling properties offer new understandings of the orchestrated response necessary for optimal muscle repair and regeneration.

Keywords: ESCRT; ectosomes; endocytosis; exosomes; injury; miRNA; myogenesis; skeletal muscle.

Figure 1

Vesicular pathways involved in plasma membrane repair. (A) Plasma membrane injury triggers membrane trafficking by Exocytosis—fusion of the intracellular vesicles such as lysosomes with the injured plasma membrane, Endocytosis—internalization of the plasma membrane, and Ectocytosis—shedding of the plasma membrane by way of microvesicles/ectosomes. Exocytosis is aided by calcium-binding membrane proteins such as dysferlin resulting in the release of lysosomal luminal proteins such as acid sphingomyelinase (ASM). The secreted ASM can access the outer and inner leaflets of the injured plasma membrane and hydrolyze the sphingomyelin lipids in these membranes to ceramide. Presence of ceramide in the outer leaflet will facilitate inward curvature and endocytosis, ceramide in the inner leaflet will cause outward curvature and ectocytosis. Both these processes enable removal of damaged membrane lipids from the site of injury by internalizing or shedding these lipids. (B) Ectocytosis is also facilitated by the interaction of proteins such as TSG101, ARRDC1, ESCRT III, and VPS4 as well as rearrangement of cortical actin beneath the membrane which help with vesicle budding and scission (see inset). Membrane shedding is also facilitated by the interaction of membrane lipids (phosphatidylserine) with the Annexin proteins and disassembly/reassembly of the cortical actin cytoskeleton with the help of calpain, Rho A, and Annexin proteins as well as mitochondrial ROS signaling. These latter processes also play a role in facilitating exocytosis and endocytosis indicating a complex set of membrane trafficking events that occur in concert, and failure, or delay in the any of these processes results in the failure of the injured myofiber to repair the plasma membrane injury. (C) Confocal images of live myofibers injured focally (white arrow) in the presence of membrane-impermeable FM 1–43 dye (green). Upon membrane injury, FM dye labels the intracellular membrane as well as all the vesicles secreted by the injured myofibers. Images of the same myofiber taken prior to and 40- or 120-s post injury show the formation of large (500–2,000 nm, red arrow) and small (<500 nm, blue arrow) extracellular vesicles. The released vesicles subsequently traffic away from the site of injury, but remain within the interfiber space (see Video 1). (D) Confocal images of a myofiber in an intact biceps muscle of Lifeact-GFP transgenic mouse showing F-actin response to focal injury (white arrow) by a 10 ms laser pulse. Images of the same fiber were taken just before and 60 s after injury, and show F-actin reorganization and buildup at the injury site.

Figure 2

Multicellular interactions involved in early stages of myofiber regeneration. (1) Myofiber regeneration starts with the initial damage that causes calcium influx into the myofiber leading to release of damaged membrane, as well as breakdown and release of myofibrils and other cellular contents. (2) These and other factors released by the injured myofiber activates and recruits circulating immune cells (neutrophils) to the injury site to commence inflammation and initial phagocytosis of cellular debris. (3) Invading neutrophils secrete pro-inflammatory cytokines that promote inflammatory macrophage enrichment. (4) These cells help clear debris during initial inflammation and also secrete additional cytokines and chemokines that further assist in clearance of cell debris. (5) The inflammatory cells also signal proliferation of cells including the fibroblasts (FAPs) that assist in secretion of growth factors, cytokines, and extracellular matrix-remodeling enzymes (MMPs) that facilitate SC escape from the basal lamina and matrix remodeling required for regeneration. (6) In the final phase of the inflammatory response to injury, macrophages turn pro-regenerative, and together with the other additional cell types activate satellite cell (SC) myogenic program by activating regenerative transcription factors. This tightly orchestrated cellular choreography relies on intercellular communication via secretory factors including EVs that ultimately facilitates regeneration of the lost myofiber.

Figure 3

Biogenesis and selective cargo packaging into EVs. (A) MVB Biogenesis: Spontaneous or injury-triggered endocytosis of the plasma membrane forms (1) endosomes that (2) progress through the endosomal pathway to create the late endosome and the “multivesicular body” (MVB). The MVB membrane contains domains rich in sphingomyelin, cholesterol ceramide, and other specific lipids (lipid raft). (3) Cytosolic neutral sphingomyelinase (nSM), can hydrolyze sphingomyelin on the MVB membrane to generate ceramide that enables spontaneous inward budding of this region of the MVB membrane. (4) This inward budding is also assisted by the assembly of the ESCRT family proteins and is finally cleaved by Vps4, allowing the newly formed vesicles to be released into the MVB lumen as “intraluminal vesicles” (ILVs). (B) RNA Cargo Packaging: (1) Specific cellular stresses, such as cell injury, can trigger miRNA duplex transcription in the cell nucleus and then processed by Drosha. (2) This is followed by cleavage of the miRNA passenger strand by Dicer protein within the cytosol. (3) Based on the sequence of the miRNA, it may preferentially bind the cytosolic protein hnRNPA2B1. (4) These complexes can then associate the miRNA to ceramide at the nascent ILV and packaged into the ILV. (5) Subsequently these miRNA-containing ILVs are secreted as exosomes through the fusion of MVB via membrane-embedded v-SNARE proteins that interact with sarcolemmal t-SNARE proteins. This process helps exosomes functionally traffic miRNA to other cells and tissues. (C) Ectosome Protein Sorting: (1) Post-translational covalent lipid modification of the cysteine residues in the proteins is one of the mechanisms for protein sorting into ectosomes. This process is assisted by a family of lipid modification enzymes collectively known as acyltransferases. (2) Protein-membrane anchoring for stable ectosome packaging is achieved through palmitoylation by the membrane-bound palmitoyltransferase, anchoring the protein to the budding ectosome (see inset for details). (3) The protein containing ectosome can then be released by the cell. (D) Exosome Protein Sorting: For protein sorting into exosomes lipid signaling pathways on the MVB membrane play an important role. (1) Neutral sphingomyelinase hydrolyze sphingomyelin in the MVB membrane to ceramide. (2) Ceramide is subsequently catabolized to sphingosine and sphingosine-1-phosphate (S1P) by cytosolic ceramidase and sphingosine kinase (SphK), respectively. (3) S1P continuously activates S1P receptors on the MVB membrane, stimulating the release of their β and γ subunits. (4) This catalyzes GTPases and actin-mediated sorting pathways for loading proteins into the budding ILVs/exosomes. A parallel pathway not depicted here involves ESCRT complex-mediated sorting and packaging of ubiquitinated proteins on the MVB membrane into ILVs for subsequent degradation or secretion.

Figure 4

Mechanisms for extracellular vesicle uptake by the target cells. Exosomes and ectosomes produced locally or brought in via blood vessels can be taken up by (A) Clathrin-dependent or (B) Caveolin-dependent pathways. (A) Smaller vesicles are preferentially taken up via clathrin-mediated endocytosis. (1) Internalization occurs at a site for EV engagement with the plasma membrane where clathrin-coated vesicle assembly can occur by the deformation of the membrane with the help of the EV and clathrin associated adaptor proteins. (2) Dynamin-2 is recruited to the clathrin-pit where it forms a collar-like structure to pinch off the endosome neck. (3) The internalized endosome subsequently uncoats and the vesicle is shuttled via microtubules to the perinuclear region. (4) There the EV cargo is released. (B) Larger vesicles tend to favor the caveolar endocytosis pathway. Caveolar invaginations in the membrane are created by the oligomerization of Caveolin proteins. (1) Once the EV encounters a caveolae conducive for internalization, it engages with the caveolae. (2) Proteins such as Dynamin-2 and EHD2 are recruited at this site to pinch off the caveolar endosome. (3) Subsequently, the caveolar endosome traffics in a microtubule-independent manner and releases the EV cargo in the cytoplasm. The rate of clathrin-mediated internalization compares to rate of receptor-mediated endocytosis, and is faster than caveolae-mediated endocytosis.