Disuse Atrophy Elevates Skeletal Muscle CD63 Expression and Ex Vivo Release of Extracellular Vesicles in a Muscle‐Specific Manner

Abstract

Extracellular vesicles (EVs) are recognized as mediators of intercellular communication during skeletal muscle adaptation to various stimuli. The role and behavior of skeletal-muscle derived EVs during disuse atrophy, however, has not been well characterized. The purpose of this study was to determine how disuse atrophy alters the release of muscle-derived EVs and well-known factors associated with EV production in rat and human muscle. Adult 10 month old male Fisher 344/BN rats underwent control, weight-bearing conditions (WB; n=7) or hindlimb suspension (HS) for either 4 hours (4h HS; n=4), 24 hours (24h HS; n=4), or 7 days (7d HS; n=5). Muscle-derived EVs were collected by excising the soleus muscle and incubating it in continuously gassed (95% O2/5% CO2) Kreb's Henseleit Buffer (118.5mM NaCl, 1.2mMMgSO4, 4.7mM KCl, 1.2mM KH2PO4, 25mM NaHCO3, 2.5mM CaCl2; pH 7.4) supplemented with 5mM glucose at 37OC for 1 hour. The soleus muscle was chosen for ex vivo experiments since it atrophies extensively and can be excised without disrupting the myofibers. EV abundance in the Kreb's buffer was measured using a Zetaview® Nanoparticle Tracking Analyzer. Skeletal muscle biopsies of the vastus lateralis (VL) were obtained from human subjects before and after 7 days of unilateral lower limb suspension (ULLS). RT-qPCR analysis was used to measure mRNA expression of factors in the soleus (rat), gastrocnemius (rat), and VL (human) muscles that are associated with EV production, including CD63, CD9, RAB27A, RAB27B, SMPD3, ARF6, CTTN, ANXA2, ISG15, and ARRDC1. We further examined CD63 protein expression via immunohistochemical (IHC) analysis. Muscle-derived EV release/mg muscle was 28% higher from 4h HS and 66% and 60% higher from 24h and 7d HS muscle, respectively, compared with WB (p<0.05 for both 4h HS and 24h HS). In addition, CD63, which is both a marker for and can mediate production of EVs, mRNA expression was positively correlated with ex-vivo skeletal muscle EV release (R2=0.22, P=0.03). Interestingly, CD63 mRNA expression, as well as expression of other EV factors, did not follow the same pattern of changes in the gastrocnemius suggesting muscle-specific changes in EV biology with disuse atrophy. Most EV factors had lower mRNA expression in the 4h HS and 24h HS groups with only RAB27A and RAB27B remaining lower expression in the 7dHS gastrocnemius muscle. However, CD63 expression was significantly increased in the VL from human subjects after disuse atrophy similar to the soleus from rats. IHC analysis of the soleus showed that the presence of CD63 protein was exclusive to a subset of non-myocyte cells within skeletal muscle. Altogether, our data demonstrate that disuse atrophy can increase CD63 expression and muscle EV release, although this may be in a muscle-specific or fiber type-specific manner. In addition, the majority of the change in EV production with disuse atrophy may be dictated by non-myocyte cells since there is no detection of CD63 in myocytes and CD63 protein is in very high abundance only in a subset of cells residing in the muscle interstitial space.