Chronic binge alcohol‐mediated alterations in myotube EV profile affect recipient myotube miRNA expression

Abstract

The average age of people living with HIV (PLWH) is now over 50 years, and this increased life expectancy is associated with increased prevalence of comorbid conditions including myopathy. At‐risk alcohol use is twice as prevalent in PLWH as the general population, and loss of muscle mass is a concern in PLWH and those with at‐risk alcohol use. Our group has previously shown that chronic binge alcohol (CBA) in SIV‐infected macaques decreases myoblast differentiation and is partly mediated by altered microRNA (miR) profile. miRs transported between cells in extracellular vesicles (EVs) mediate numerous physiologic and pathologic cellular responses and can alter the function of target cells through miR delivery. We tested the hypothesis that EVs derived from myotubes isolated from CBA‐administered female macaques would alter miR expression in recipient myotubes. Seven SIV‐infected female rhesus macaques received either CBA (2.5 g/kg/day, N=4) or water (VEH, N=3) for 14.5 months. Three months following the initiation of CBA or VEH, the animals were vaginally inoculated with SIVmac251. Antiretroviral therapy was initiated 2.5 months later. Animals were sacrificed 9 months later, and primary cells were derived from skeletal muscle tissue 24 hours following the last dose of alcohol (blood alcohol=0 mM). EVs were isolated by ultracentrifugation from the conditioned cell culture supernatant of three 10 cm plates of differentiated primary myoblasts (i.e. myotubes). EVs were transferred to naïve (prior to SIV or CBA) recipient macaque myotubes in serum‐free media and incubated for 48 hours. Following incubation, cells were washed, and RNA extracted to determine relative expression of miRs 133a, 133b, 206, 16, and 23a. EVs derived from CBA myotubes increased expression of miR‐206 in target myotubes (p<0.05, Cohen d=2.68). There was no difference in the expression of other measured miRs; however, there were large effect sizes for CBA‐derived EVs to increase miR‐133a and miR‐23a expression (Cohen d=1.13 and 1.51, respectively) in recipient myotubes. These results indicate that CBA‐mediated alterations in myotube EV profile affect recipient myotube miRNA expression. Ongoing studies will determine if this altered recipient miR expression reflects altered EV miR expression and/or host cell miR expression and determine the functional relevance of increased miR‐206 on myotube differentiation and hypertrophy.